Metabiotics: Boris A. Shenderov Alexander V. Sinitsa Mikhail M. Zakharchenko Christine Lang

Boris
A. Shenderov
Alexander V. Sinitsa
Mikhail M. Zakharchenko
Christine Lang
METABIOTICS
PRESENT STATE, CHALLENGES AND
PERSPECTIVES
METABIOTICS
Boris A. Shenderov • Alexander V. Sinitsa
Mikhail M. Zakharchenko • Christine Lang
METABIOTICS
PRESENT STATE, CHALLENGES
AND PERSPECTIVES
Boris A. Shenderov Alexander V. Sinitsa
Research Laboratory for Design Kraft Ltd.
& Implementation of Personalized St. Petersburg, Russia
Nutrition-Related Product & Diets
K.G. Razumovsky University of Technology Christine Lang
& Management MBCC Group
Moscow, Russia Berlin, Germany
Mikhail M. Zakharchenko
Kraft Ltd.
St. Petersburg, Russia
ISBN 978-3-030-34166-4 ISBN 978-3-030-34167-1 (eBook)

https://doi.org/10.1007/978-3-030-34167-1
© Springer Nature Switzerland AG 2020

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Preface
This book presents in a concise form the latest data on symbiotic microbiota which
is directly or indirectly involved in the evolvement and performance of human phys-
iological functions, biochemical, behavioral, regulatory, and signaling reactions, in
maintaining energy, immune, and neurohormone homeostasis. Multifaceted contri-
bution of this microbiota in health support is ensured by endogenous production of
multiple low-molecular-weight microbial compounds, many of which bear similar-
ity to biologically active molecules of cells of human tissues and organs in their
chemical structure, pharmacological and signaling activity, or which can be found
in food products. Their lack or disproportionately large amount results in imbalance
of mitochondrial, microbial, and cell metabolites and may become a predictor and
probably the main factor of metabolic diseases. This leads to the conclusion that
microbial low-molecular-weight bioactive compounds are the most important
human health regulators in all periods of life and the risk of many chronic metabolic
diseases depends on adequate diet and balanced condition of digestive tract micro-
biota. The most common approach to preserving human symbiotic microbiocenoses
consists in using probiotics and prebiotics. Unfortunately, beneficial effects of pro-
biotics on the base of live microorganisms are often short-term, uncertain, or entirely
absent; the application of traditional probiotics may also cause various side effects.
The book offers a fundamentally new approach to the prevention of chronic defi-
ciency of biologically and pharmacologically active low-molecular-weight micro-
bial compounds by way of introduction into preventive and clinical medicine of
functional nutrition products (metabiotics) engineered on the basis of cell structural
components, metabolites, and signaling molecules of probiotic microbial strains
with a determined (known) chemical structure. Metabiotics are capable of optimiz-
ing host-specific physiological functions, metabolic, epigenetic, informational,
regulator, transport, and/or behavior reactions connected with the activity of symbi-
otic microbiota. They can act as therapeutics or as nutritional supplements to tradi-
tional functional foods. In the future, as is the case with antibiotics, synthetic (and/
or semisynthetic) metabiotics may be expected to appear, which will be artificially
created as analogues or improved versions of natural biologically active compounds
formed by symbiotic microorganisms. It should be borne in mind that many issues
v
vi Preface
related to understanding the interaction between metabiotics and host organism

remain open. This publication is intended for broad audience including doctors of
all categories, dieticians, biochemists, food technologists, teachers, and students of
higher educational institutions or departments specializing in medicine, biology,
and foods, as well as for all those interested in drug-free healthcare methods.
UDC 615
LBC 52.64
Shenderov BА, Sinitsa AV, Zakharchenko MM, Lang C
Metabiotics: Present State, Challenges, and Perspectives
Moscow, Russia Boris A. Shenderov

St. Petersburg, Russia Alexander V. Sinitsa
St. Petersburg, Russia Mikhail M. Zakharchenko
Berlin, Germany Christine Lang
Contents
Introduction�� 1
The Composition and Functions of Human Gut Symbiotic
Microbiota�� 5
Contemporary Views on Biotechnological Potential of Symbiotic
Microorganisms�� 11
The Digestive Function of Human Gut Microbiota�� 13
Metabolic Relationship Between the Host and Its Gut Microbiota�� 15
Factors and Agents that Modify the Composition and Functions
of Symbiotic Microbiota; Diagnostic Methods for Microecological
Imbalance and its Consequences�� 23
Contemporary Microecological Strategies of Gut Microbiota
Modulation for Human Health Preservation, Restoration
and Improvement�� 27
Molecular Language of Symbiotic Gut Microorganisms �� 33
Drawbacks and Negative Consequences of Traditional Probiotics
Based on Live Microorganisms�� 43
Metabiotics: New Stage of the Probiotic Concept Development �� 49
Methods and Techniques Used for Obtaining and Identifying
of Microbial Low Molecular Weight Cellular Compounds,
Metabolites and Signaling Molecules �� 53
Classification of Metabiotics and their Brief Description�� 57
Some of the Best-known Metabiotics on the Market
of Microecological Products�� 59
vii
viii Contents
Cellular Metabiotics and Metabolite Metabiotics�� 63

Cellular Metabiotics�� 63
Helicobacter and Stomach Health�� 64
Probiotics in Helicobacter Therapy�� 64
The Helinorm/Pylopass Strategy�� 65
The Pylopass Strain�� 66
Characteristics of Pylopass�� 67
Cell Walls Exhibit the Co-Aggregation Sites �� 68
The Power of Metabiotic Formulation �� 68
Clinical Evaluation �� 69
Outlook�� 71
Metabolite Metabiotics�� 71
Clinical Assessment�� 74
Prospects in the Field of Intended-Use Metabiotics Creation�� 77
Some New Targets and Approaches to the Construction
of Intended-Use Metabiotics �� 79
Modulators of QS-Regulation �� 79
Modulators of Immunity �� 79
Modulators of Energy Metabolism �� 81
Modulators of Antioxidant Status �� 82
Modulators of Intercellular Information Exchange�� 83
Modulators of Neuropsychic and Social Behavior �� 85
Modulators of Epigenome Regulation�� 85
Metabiotics on the Base of Microbial Gas Molecules�� 87
Hybrid Multicomponent Microbial Molecules as a Base
for Metabiotics�� 87
Other Contemporary Examples of the Techniques Needed
to Create, Maintain and Correct Human Microbial Ecology�� 90
Conclusion�� 93
Bibliography �� 103
Index�� 119
About the Authors
Boris A. Shenderov is a physician, doctor of Medical Science (1976), professor of

Microbiology (1979), active member of New York Academy of Sciences (1996).
Also, he is professor in Scientific Research Laboratory “Design and Implementation
of Personal Nutrition Products and Rations,” K.G. Razumovsky Moscow State
University of Technologies and Management, and in Interdisciplinary Neurology
Department, Institute of Interdisciplinary Medicine, Moscow, Russia. His main
research interests include medical microbial ecology, genetics, epigenetics, metabo-
lomics, probiotics, functional foods, and biotechnology. He is an author of more
than 500 scientific papers, including 12 books, and 30 invention patents. He was a
councilor of Executive Board of International Society of Microbial Ecology and
Disease (SOMED) (2008–2013) and a member of Executive Board and President of
International Association for Gnotobiology (IAG) (2008–2014); Russian Society
for Epidemiology, Microbiology, and Parasitology after I.I. Mechnikov; and
Editorial Boards of five Russian science journals and international journal Microbial
Ecology in Health and Disease.
e-mail: shenderof@yandex.ru (professional)
Alexander V. Sinitsa is president of “Kraft” Group, Saint Petersburg, Russia, and

candidate of Technical Sciences (1987). His main scientific interests include probiot-
ics, prebiotics, metabiotics (postbiotics), functional foods, and sports nutrition. He is
an author and coauthor of more than 100 scientific papers. He heads a company, one
of the leaders in the development of metabiotic products in Russia.
e-mail: prezident@kkraft.ru (professional)
Mikhail M. Zakharchenko is a physician, candidate of Medical Science (2003),

and head of R&D Department of “Kraft” LLC, Saint Petersburg, Russia. His main
scientific interests include microbiome, probiotics, prebiotics, metabiotics (postbi-
otics), and functional foods. He is an author and coauthor of more than 100 scien-
tific papers and a member of Executive Board of Scientific Conference “Prenosology”
(2005–2019) and Editorial Board of Russian Science Journal Prenosology and
ix
x About the Authors
Healthy Lifestyle (2007–2019). He leads the development of new products at

“Kraft.”
e-mail: m.zaharchenko@kkraft.ru (professional)
Christine Lang is an entrepreneur in biotechnology. She acts as a consultant for

the German government for bioeconomy topics and co-chairs the German
Bioeconomy Council. She is an associate professor of Microbiology and Molecular
Genetics at Technical University of Berlin and vice president of the German
Association of General and Applied Microbiology (VAAM). In 2001, she founded
the company Organobalance GmbH and established it as an R&D facility to develop
novel microbial active ingredients and strains for food, feed, personal care, and
pharma applications. Since September 2016, Organobalance is a part of Novozymes
A/S. Till June 2018, she was the company’s CEO and now serves as a consultant for
Novozymes A/S. Christine Lang is co-founder and partner of BELANO medical
AG focused on production and sales of microbiotic products for human health.
e-mail: christine.lang@mybioconsulting.de (professional)
Introduction
All diseases start in the gastrointestinal tract.

(Hippocrates 460–370 BC)
Multiple and numerous associations of microorganisms
inhabiting human gastrointestinal tract determine, to a great
extent, our physical and spiritual health.
(Mechnikov 1908)
Microbes rule the world. We must listen closely to their
language and when we learn more, we will be capable of a
better and more harmonic interaction with them. The presence
of active microbial compounds in the gut has physiological and
pathophysiological consequences for the host.
(Midtvedt 2008)
Microorganisms are starters of the nascence and subsequent evolution of all biologi-
cal life varieties on our planet including humans. A contemporary view of a human
organism presents it as a superorganism, a dynamic symbiotic community of vari-
ous prokaryotic and eukaryotic cells (Vakhitov and Sitkin 2014, Shenderov 2008,
Shenderov 2014b, Ugolev 1991, Caselli et al. 2011, Dietert and Dietert 2015,
Lederberg 2000). The microbial component of this community includes bacteria
(which dominate), Eukarya and Archaea (up to 1013–14 cells totally). Microbiome/
microbiota (a term introduced into scientific literature by Joshua Lederberg) repre-
sents the totality of commensal, symbiotic and pathogenic microorganisms inhabit-
ing human body (Lederberg and McCray 2001). According to the latest data, the
human organism contains around 3 × 1013 eukaryotic cells and 3.9 × 1013 microor-
ganisms colonizing human body; it means that quantitatively, the content of human
cells and microorganisms is quite similar (Sender et al. 2016). As regards bacterial
content, symbiotic microbiota in the digestive tract reaches its maximum in the
large intestine (up to 1012 CFU/g); this is the most densely inhabited bio-ecosystem
on our planet. Human microbiota has a distinctly individual character on the genus
© Springer Nature Switzerland AG 2020 1

B. A. Shenderov et al., METABIOTICS,
https://doi.org/10.1007/978-3-030-34167-1_1
2 Introduction
and species levels and — especially — on the strain levels (Gilbert et al. 2016,
Meisel and Grice 2017, Mimee et al. 2016, Sonnenburg and Backhed 2016).
Multiple and numerous intestinal microorganisms are the main adaptive compo-
nent of the human superorganism determining close mutually beneficial relations
between the metagenome, meta-epigenome and intercellular information exchange
of all its participants. It ensures adaptation of the human organism to ever-changing
environmental factors, maintaining its energetic, metabolic and immune homeosta-
sis. Moreover, the digestive tract, skin and vaginal microbiota has diverse effects on
the development and functioning of the nervous and the hormone systems, on social
and psychical behavior. The structure of symbiotic microbial population in women
prior to, during and after pregnancy influences the development of the fetus, the
genesis and succession of fetal and infant microbiota, and even the course of preg-
nancy of their children and their children’s offsprings. The connection has been
traced between the microbiota of pregnant and breastfeeding women and
oligosaccharide content in breast milk; the quantitative and qualitative composition
of milk oligosaccharides determines, to a large extent, the development and content
of fetal microbiota and that of newborn babies and infants, and in the long run — the
health of teenagers and adults (Charbonneau et al. 2016). It means that indigenous
microbiota of all mammals including man is a complex, indispensable, peculiar to
them extracorporeal organ, which plays a fundamental role in maintaining their
health and reducing the risk of various metabolic diseases. Practically all functions,
metabolic and signaling reactions in the cells of human organs and tissues are in
accord with the content and functions of its symbiotic microbiota. It is beneficial for
both the whole organism and its components. The effects of symbiotic (probiotic)
microorganisms are realized through the production of multiple low molecular
weight compounds of different nature and chemical structure (Shenderov 2013c,
Shenderov 2014b, Cani 2018, Shenderov 2011a, Shenderov 2013a, Sonnenburg and
Backhed 2016).
The “host – its normal microbiota” ecosystem bears innate self-regulatory ele-
ments and is capable to withstand – at least, within bounds – environmental changes
and abrupt fluctuations in microbial populations density. Unfortunately, many bio-
logical and abiotic factors and agents are capable of damaging natural symbiotic
microbiocenoses and their interaction with eukaryotic cells of human tissues and
organs, which is a predisposing risk factor for not only traditional infectious dis-
eases but also for many somatic metabolic diseases associated with microecological
imbalance. Many various therapeutics, dietary supplements, functional foods have
been developed and are used in order to preserve and restore human microbial ecol-
ogy. The most popular among them are those based on the use of specially matched
probiotic strains of lactobacilli, bifidobacteria and some other microorganisms, as
well as soluble dietary fibers stimulating their growth. The efficiency of traditional
microecological therapeutics (probiotics, prebiotics, synbiotics) that dominate now-
adays and are used for maintaining and restoring human symbiotic microbiota is
determined by the origin of probiotic cultures (hetero-, homo- or autoprobiotics),
the kind of species and strain they belong to, the state of probiotic bacteria (live,
killed, cell fragments, their metabolites or signaling molecules), the probiotic
Introduction 3
manufacturing technologies, the method and duration of use, the quantity of viable
probiotic microorganisms, the user’s health status, physical and chemical conditions
in different biotopes of his gastrointestinal tract, the state of gut microbiota, the
combination of all the above-mentioned and other factors. Given that, probiotics
display their direct or indirect, specific or nonspecific effects — both on a local level
and on the systemic level, as a result of a long-term or transitory colonization of the
host organism.
Ample data have been published concerning probiotics based on live organisms
highlighting their health-promoting effects in case of acute and chronic, localized
and systemic diseases (various types of diarrhea, allergy, inflammatory pathologic
conditions in the gut, hypercholesterolemia, malignancies etc.) (Shenderov 2014b,
Bomba et al. 2012, Pflughoeft and Versalovic 2012, Shenderov 2011b, Sonnenburg
and Backhed 2016). In this book, we have analyzed the materials published over the
last 10 years both in Russia and globally regarding positive effects, drawbacks and
adverse consequences of ample use of probiotics on the base of live microorganisms
in medicine and veterinary practice. The concept of probiotics is being revised now.
It is giving way to the concept of metabiotics whose main constituent is represented
by cell components, metabolites and signaling molecules of probiotic cultures. The
presented materials summarize the data available in scientific literature on the such
microbial molecules, new approaches are considered to constructing safe and effec-
tive microecological therapeutics on their basis, and new data is presented on some
of the best understood molecular mechanisms of metabiotics’ positive effects on
human organism.
The Composition and Functions of Human
Gut Symbiotic Microbiota
From the contemporary perspective, the human organism should be viewed as the
most complex “superorganism”, a symbiotic community of eukaryotic, prokaryotic
cells including archaebacteria, and viruses (Ugolev 1991, Lederberg 2000). The
microbial component of this community is represented by the aggregate of sets of
microbiocenoses characterized by a definite composition and occupying the respec-
tive biotope in the human organism which is open to the environment (skin, naso-
pharynx, mouth cavity, respiratory and gastrointestinal tracts, genitourinary system
mucosa). In any microbiocenosis one can distinguish widespread species, the so-
called characteristic or dominating (core) species (autochthonous, indigenous sym-
biotic microbiota) and additional or accidental species (transitory allochthonous
microbiota). The number of characteristic species is relatively not too big, but to
make up for it, they are always well-represented. The “metagenome” of this “super-
organism” consists of the Homo sapiens genes proper and the genes of microorgan-
isms colonizing human body. The genome of every human being is quite stable
(except for the changes in genes related to the immune system, the metabolism of
various dietary substrates or xenobiotic destruction, neoplasms); the microbiome,
on the other hand, undergoes rather profound changes in the course of a lifetime
(Gilbert et al. 2016).
The last twenty years have seen a sharp increase in the information flow on the
important role played by symbiotic microbiota in maintaining human physical and
mental health. The example of gut microbiocenosis shows the existence of a com-
plex ramified and multi-tier system for cooperation between populations of micro-
organisms inhabiting the gut, as well as between these microorganisms and
eukaryotic cells of the gut and human organism as a whole. Deficiency or excess of
a certain substrate, metabolite or signaling molecules serves as a signal for increased
growth, death or disfunction of the corresponding chain of the complex system
called “human superorganism.” In course of evolution, symbiotic microorganisms
and cells of plants and animals including humans had been transforming into an
increasingly interconnected whole. For greater efficiency, they had been specializ-
ing as regards the habitat and functional activity. This kind of integration has made

https://doi.org/10.1007/978-3-030-34167-1_2
6 The Composition and Functions of Human Gut Symbiotic Microbiota
it possible to consider symbiotic and commensal microbiota as a peculiar extracor-

poreal human organ, which works concertedly for the benefit of the whole system
of the host organism where it is localized.
Russian academician A. M. Ugolev (1991) was the first to formulate the notion
of endo-ecology of humans and other higher organisms, having pointed out that
from the trophic and metabolic viewpoint, mammals represent a most complex sys-
tem consisting of the dominating multicellular organism and specific bacterial cul-
tures maintaining complex symbiotic relations. All the necessary plastic, energetic,
metabolic and signaling regulatory compounds are released from food products due
to cavitary, membrane, intracellular digestion, as well as in course of their synthesis
by gut microorganisms. Regulatory substances (the so-called exohormones) may be
present in food or be produced from it under the effect of digestive enzymes of the
microorganism and a multitude of enzymes of various bacteria colonizing its skin
and mucosae. Such exohormones are necessary for functioning of the organism as a
whole, not only the digestive system.
In subsequent years, a number of amendments were introduced into A. M. Ugolev’s
view of human superorganism, the most important of which is about humans repre-
senting a community of prokaryotic cells rather than eukaryotic cells (Sitkin et al.
2015, Shenderov 2014b, Dietert and Dietert 2015, Lederberg 2000). This conclusion
is based on the fact that various kinds of cells of specifically human tissues and
organs do not exceed 500 in number; nevertheless, the total number of these cells
ranges between 5 and 10 trillion. On the contrary, the number of various species of
bacteria inhabiting the human organism may reach 10 thousand, and the number of
strains may be as much as 50 thousand; the total bacterial content is within hundreds
of trillions, and with viruses included it exceeds quadrillion. The number of genes in
human chromosomes is in the range of about 25,000, while the human microbiome
includes up to 10 million genes. The replacement of all eukaryotic cells in the human
being requires not less than 20–25 years. During this time, all symbiotic bacteria can
be replaced at least five or six times, which is indicative of high adaptive capacity of
a human being as a superorganism. Up to 80% of all man’s energy is produced in
mitochondria (the most ancient microbial endosymbionts of eukaryotic cells), 20%
of energy is given by gut microorganisms. Due to the metabolic activity of gut bac-
teria, over 90% of energy is generated for the epithelial cells of the digestive tract
(Shenderov 2016a). Over 70% of prokaryotic organisms are obligatory anaerobes;
and now only the representatives of a little more than 1500 bacterial species can be
cultivated. The representatives of 50– 60% of bacterial species in the gut produce
spores, which is believed to help obligatory anaerobe bacteria persist and get trans-
mitted to other hosts (Braune and Blaut 2016, Ilinskaya et al. 2017).
Long-term studies of gut microbiota composition in one and the same persons
have shown that 60% of strains of dominant bacterial species are preserved in this
biotope for not less than 5 years (Faith et al. 2013, Nicholson et al. 2012). Such
long-term individual stability of gut or skin microbiota which allows for identifying
up to 80% of people by their microbiota content has given grounds to speak about
the existence of unique microbial fingerprints (Franzosa et al. 2015). The large
intestine is predominately inhabited by such phylum representatives as Bacteroides
The Composition and Functions of Human Gut Symbiotic Microbiota 7
Table 1 The composition of digestive tract microbiome in healthy people (Shenderov 2014b,
Shenderov et al. 2018a, Bik et al. 2018, Blum 2017, Chernevskaya and Beloborodova 2018, Falony
et al. 2016, Meisel and Grice 2017, Yadav et al. 2018, Zhernakova et al. 2016)
Phylum Genera
Firmicutes Blautia, Butyrivibrio, Clostridium, Coprococcus, Dorea, Eubacterium,
Faecalibacterium, Roseburia, Ruminococcus, Subdoligranulum; Bacillus,
Lactobacillus, Streptococcus
Bacteroides Bacteroides, Prevotella, Aliistipes
Actinobacteria Bifidobacterium, Collinsella
Fusobacteria Fusobacterium
Proteobacteria Desulfovibrio, Bilophila, Esherichia
Verrucomicrobia Akkermansia
Melainabacteria Cyanobacteria
Euryarchaeota Methanobacteriales, Metanomassiliicoccales
Fungi Saccharomyces, Candida, Cladosporum
Viruses Eukaryotic viruses, Bacteriophages
and Firmicutes followed by Actinobacteria, Proteobacteria, Fusobacteria,

Spirochaetes, Verrucomicrobia, Lentisphaerae and Archea (Table 1).
The ratio between these phyla changes throughout life. For example, in infants,
adults and people older than 75 the ratio between the representatives of Firmicutes
and Bacteroides in the large intestine content is 0.4, 10.9 and 0.6 respectively. Only
18– 20 bacterial species are ever-present in the majority of adult people. The repre-
sentatives of 14 gut microbial genera (core microbiota) are found in the majority of
4000 screened inhabitants of Eastern and Western Europe; given that, the represen-
tatives of other 664 genera are barely identified and are found with different inci-
dence in adults (Falony et al. 2016). Human microbiota has a distinctly individual
character and varies both on the level of the genus and species and on the strain
level. Intraspecies differences among strains can reach 25% of their genome and
more. Bacterial content (CFU/g) and the number of species among particular indi-
viduals can vary 12–2200-fold. Fecal bacteria differ in viability: 30% of them are
dead cells; 50% are live cells, and 20% are damaged cells with a low capacity to
form colonies on growth media (Shenderov 2014b, Carding et al. 2015, Charbonneau
et al. 2016, Gilbert et al. 2016, Sonnenburg and Backhed 2016, Suvorov 2013, The
Human Microbiome Project Consortium 2012). Also, over 1200 species of viruses,
mostly bacteriophages, are present in the gut. Over recent years, increasingly more
attention is paid to fungi, protozoans and helminths in the framework of research
into human microbiome (Proal et al. 2017). Feeding habits, natural surroundings,
immune and genetic peculiarities of the host are the decisive factors for the compo-
sition and functions of indigenous microbiota of human digestive tract and its meta-
bolic phenotype (Donaldson et al. 2016, Nicholson et al. 2012, Voreades et al. 2014,
Yadav et al. 2018). According to the proportion of microbial phyla representatives
(Bacteroides, Prevotella and Ruminococcus) in the large intestine microbiota, the
majority of people can be divided into three “enterotypes.” Russian urban and rural
8 The Composition and Functions of Human Gut Symbiotic Microbiota
people differ in the number and content of bacterial “enterotypes” in their digestive
tract (Vakhitov and Sitkin 2014, Sitkin et al. 2015).
Owing to a well-coordinated work of symbiosis-regulating systems that have
taken shape in course of the evolution, the genes of eukaryotic cells and microbi-
omes of many thousands of symbiotic microorganisms are in a close and consistent
interaction. In the long run, this is what determines the composition and quantitative
content of the corresponding prokaryotic and eukaryotic cells in particular biotopes
of mammals including humans, which prevents their inevitable competition for
similar fixation places and dietary substrates, regulates the exchange of metabolites,
signal molecules, genetic information transmission etc. Symbiotic microorganisms
are a source of a multitude of endogenous mono- and multiple-factor signaling mol-
ecules that ensure health or risk of human diseases from the moment of birth till
extreme old age (Sitkin et al. 2015, Chervinets et al. 2018, Shenderov 2014b,
Carding et al. 2015, Gilbert et al. 2016, Lagier et al. 2012, Lyu and Hsu 2018,
Sonnenburg and Backhed 2016). For the most part, symbiotic microorganisms exist
in human and animal organisms as microcolonies fixed to particular receptors and
enclosed in a biofilm, which provides a glove-like cover to the skin and mucosae
lining up the cavities open to environmental influence in healthy humans and ani-
mals. Apart from microorganisms, this biofilm contains exopolysaccharides of
microbial origin varying in composition, as well as mucin produced by mucosal
goblet cells. From the functional perspective, biofilm is often compared to placenta.
While the latter regulates the relations between the fetus and the mother’s organism,
the biofilm plays the same role in the relations between the host organism and the
environment. Dietary peculiarities by 57% determine structural changes in gut
microbiota composition; genetic predisposition is only by 12% responsible for such
peculiarities of microbiota. There are dozens some population groups among people
that differ greatly as regards their diets (innate inhabitants of tropical and subtropi-
cal zones, deserts, highlands, northern territories, strict vegetarians, people of the
so-called “western pattern diet”). Similarly, commensal and symbiotic microbiota
of the digestive tract in the representatives of these ethnic groups is characterized by
considerable or even critical differences (Shenderov 2014b, Sonnenburg and
Backhed 2016).
In all multi-cellular organisms including humans, symbiotic microorganisms are
actively involved in physiological functions, biochemical, behavioral and signaling
reactions, in maintaining health and in development of various, primarily metabolic,
diseases. Immune, metabolic and signaling functions of symbiotic microbiota simi-
lar to those of adults, take shape by the age of 2 or 3 years, developing to the full
extent by the age between 12 and 14. Therewith, metabolic changes coincide with
the evolution and maturation of gut symbiotic microbiota primarily on the strain
level, which is confirmed by studying and comparing the profiles of bacterial prod-
ucts of protein and energy metabolism in different human and animal body fluids
(Nicholson et al. 2012). Under standard conditions, normal microbiota effects
directly or indirectly practically all vitally important human processes and functions
(morphokinetic action; regulation of gas composition in the cavities and water-salt
exchange; participation in the metabolism of proteins, lipids and carbohydrates;
The Composition and Functions of Human Gut Symbiotic Microbiota 9
supplying the body with energy, in the recirculation of bile acids and other
macromolecules; immunogenic and detoxication functions; mutagenic/antimuta-
genic and oxidative/antioxidative activities; regulation of behavioral reactions; the
storage of genetic material; production of low molecular weight compounds of dif-
ferent chemical nature, with a broad spectrum of biological, pharmacological and
signaling activity; regulation of metagenome stability; replication and phenotypic
gene expression, modification of genomic and post-translational reactions of pro-
karyotic and eukaryotic cells, programmed death of eukaryotic cells (apoptosis);
involvement in information exchange between prokaryotic and/or eukaryotic cells
of the host, in disease etiopathogenesis and functioning as starter cultures when
creating various biotechnological products) (Gao et al. 2013; Shenderov 2014b,
Cani and Delzenne 2007, Dorrestein et al. 2014, Freitas et al. 2003, Maguire and
Maguire 2019, Lyu and Hsu 2018, Mimee et al. 2016, Nicholson et al. 2012, Yadav
et al. 2018).
Contemporary Views on Biotechnological
Potential of Symbiotic Microorganisms
Microorganisms are essentially the main “workhorses” of contemporary biotech-

nology. It should be noted that a large proportion of microorganism strains existing
in nature, including many representatives of anaerobe symbiotic bacteria in the
human gut, are difficult and often impossible to cultivate in ordinary microbiologi-
cal laboratories (Browne et al. 2016). As many as 1000–10,000 prokaryotic micro-
organism species are present in 1 g of soil. Out of 23,000 microbial metabolites
known to date (42% of them are produced by fungi and 32% by actinomycetes),
many are used already in the form of antibiotics, anti-cancer agents, immunosup-
pressors, hypocholesterolemic, antivirus, deworming drugs, nutraceutics, dietary
and technical exopolysaccharides, surfactants, herbicides, various enzymes, amino
acids, vitamins, vaccines etc. Suffice it to say that over 15,000 various natural and
synthetic and semi-synthetic antibiotics created on their base are known by now,
and 150 of them are commercially available. To date, over 1000 antimicrobial pep-
tides have been isolated, with bacteriocins being a common example. The represen-
tatives of the Myxobacteria genus alone form over 300 different antimicrobial
compounds. Presently, the world’s market of antibiotics exceeds USD 35 billion
(Guilherme et al. 2006). The annual production of the glutamic acid by using micro-
bial starter cultures exceeds 2.2 million tons, lysine — 1.3 million tons, threonine —
77,000 tons, phenylalanine — 16,000 tons, В12 vitamin — 12 tons. The production
of the polylactic acid of microbial origin does not differ in its volumes from poly-
propylene and reaches 140,000 tons a year; the main characteristic feature of the
polylactic acid is its capability of quick biodegradation.
In the last 50–75 years, secondary metabolites of microbial origin allowed
humankind to achieve a practically double increase in average life expectancy. The
overwhelming majority of microbial biotechnological products manufactured
worldwide were mostly based on the cultures of microorganisms isolated from soils
and waterbodies. In the last decade, representatives of symbiotic microbiota of
plants, lower and higher animals including humans came into use for biotechnologi-
cal purposes (Chervinets et al. 2018). It became the revolution in contemporary
biotechnology. Thus, it turned out that symbiotic bacteria of sponges alone make up

https://doi.org/10.1007/978-3-030-34167-1_3
12 Contemporary Views on Biotechnological Potential of Symbiotic Microorganisms
to 40% of their biomass. The symbiotic bacteria of these protozoans form up to 400
different secondary metabolites with marked pharmacological and other effects; a
number of antineoplastic drugs have been formulated on their basis, two of which
have been introduced into clinical practice. It should be remembered that the skin
and mucosa of an adult carries up to 1.5–3 kg of commensal and symbiotic micro-
organisms belonging to over 1500 genera and including up to 40–70 thousand dif-
ferent strains.
In 2011, the world’s market of traditional probiotics and probiotic fermented
milk products on the base of symbiotic lactic acid bacteria of animal origin and
prebiotics was valued at USD 27.9 billion, in 2015 it was valued at USD 30 billion;
it is expected to reach USD 44.9 billion in 2018, with a yearly growth between 2 and
5% (Buriti et al. 2016). According to Elisa Fernandez, director of the International
Probiotics Association (IPA), the world’s probiotics market is expected to reach
EUR 53 billion by 2023 (www.internationalprobiotics.org). It should be borne in
mind that a number of commensal bacteria (representatives of Ruminococcus,
Eubacterium, Roseburia, Faecalibacterium, Akkermansia spp. etc.) that do not
belong to traditional probiotic bacteria, can also produce various bioactive metabo-
lites capable of beneficial effects on the human organism (Engevik and Versalovic
2017). Probiotic strains commercially available on present-day markets are just the
first generation of biotechnological products. In the perspective, symbiotic microor-
ganisms of mammals will become the base for starter cultures in the production of
the newer, safer and more effective therapeutics that restore or improve the host
organism microecology (Chervinets et al. 2018, Shenderov et al. 2018a).
The Digestive Function of Human Gut
Microbiota
According to the contemporary data, the daily needs of the human organism required
for construction and functioning of millions of simple and complex molecular enti-
ties amount to over 20 thousand of different macro- and micronutrients (Shenderov
2018). Around 70% of the total microbiota inhabiting the human organism is local-
ized on its digestive tract mucosa. A. M. Ugolev deserves credit for providing con-
vincing evidence of symbiotic microorganisms actively participating in the
metabolization of complex compounds (polysaccharides, phenolic and other com-
ponents) coming with food. In recent years, the importance of the digestive function
of symbiotic gut bacteria has been gaining further confirmation (Bengmark 1998;
Bik et al. 2018, Sonnenburg and Backhed 2016, Yadav et al. 2018). By digesting
endogenous sources, gut microbiota returns all the necessary components. It has
been proven that over 400 or 450 grams of endogenous substrates and those coming
with food undergo daily microbial metabolization in the digestive tract of adults,
which makes up as much as one third of the total mass of the consumable food
products (Table 1).
Form the chemical perspective, human “superorganism” is supposed to consist
of not less than 2.5 million molecules including around one million proteins (Wang
et al. 2006), 300 thousand lipids (Sampath and Ntambi 2005) and hundreds of thou-
sands of other simple and complex compounds (Engevik and Versalovic 2017).
Unlike symbiotic microbiota and its microbiome in people living in industrial coun-
tries, the metabolome of ethnic groups has not been extensively characterized until
now, which is explained by its extreme diversity (Dorrestein et al. 2014). Formerly,
food products were believed to be the only basic substrates, co-substrates and co-
factors for the synthesis of these compounds. Notably, complex dietary compounds
undergo destruction under the influence of the digestive tract enzymes; the resulting
compounds of simple chemical structure are absorbed in the gastrointestinal tract
and are used by cells for the synthesis of energy and the required structural and
signaling molecules. They are either ingested with food products or produced
endogenously by various cells of the microorganism, or result from the metabolic
activity of symbiotic microbiota (gut microbiota in the first place). Modern diet

https://doi.org/10.1007/978-3-030-34167-1_4
14 The Digestive Function of Human Gut Microbiota
Table 1 Dietary and endogenous substrates metabolized daily by gut microbiota in adults
(calculated data) (Shenderov 2017)
Substrates Daily amount
Mucosal fluid (in the first place, mucopolysaccharides of the Up to 1000 ml (20–25 g
nasopharynx and gut mucous layer) of solids)
Saliva Up to 1.5 l (20–35 g of
solids)
Gastric juice Up to 2.5 l (20–25 g of
solids)
Bile 0.5–1.0 l (20–25 g of
solids)
Pancreatic juice Up to 1.0 l (10–15 g of
solids)
Small intestine juice Up to 2.5 l (30–40 g of
solids)
Large intestine juice 50–70 ml (1.5–2 g of
solids)
Intake of microorganisms with food, water, inhaled air Up to 1011–12 cells
(2.5–3 g)
Dead cells of the digestive tract symbiotic microbiota 50–150 g
Desquamated cells of dead epithelium of the digestive tract 10–50 × 107 cells (up to
10 g)
The quantity of endogenous substrates metabolized by gut Up to 200–250 g on the
microorganisms average
The quantity of non-digestible dietary substrates metabolized by gut Up to 100–120 g on the
microorganisms average
Total quantity of dietary and endogenous substrates daily metabolized Up to 400–450 g
by microbes
c annot provide the intake of even a half of this amount. In case of dietary intake
deficiency, it is compensated by microbial formation of bioactive compounds from
various sources of dietary, endogenous and microbial origin.
Gut microbiota is most actively involved in dietary metabolism by processing
dietary and endogenous substrates and forming low molecular weight nutrients,
regulatory and signaling molecules required for the life sustaining activity of the
host and its microbiota. Gut bacteria are capable of splitting various ingested vege-
table components (polyphenols, polysaccharides, oligosaccharides etc.) into bio-
logically active molecules which take an active part in different human functions
and reactions (Volkov et al. 2007, Moreira et al. 2017; Panyushin 2012). The amount
of macro- and microelements (mg/kg) contained in raw biomass of fecal microor-
ganisms in adults exceeds daily requirement by 5–50 times (Shenderov 2008). It is
this daily recirculation that helps to partially or fully cover chronic deficiency in
many critical nutrients and other biologically active substances of dietary and
microbial nature.
Metabolic Relationship Between the Host
and Its Gut Microbiota
Symbiotic microorganisms ever-present in the organisms of adult people form over

25 thousand different biologically and pharmacologically active compounds. With
regard to potential biological effects, the most extensively studied are short-chain
fatty and bile acids, choline metabolites, the derivatives of phenol, benzene and
phenyl, indole derivatives, vitamins, polyamines, lipids, enzymes and other pro-
teins, amino acids (Beloborodova et al. 2011, Shenderov et al. 2010, Carding et al.
2015, Chernevskaya and Beloborodova 2018, Clarke et al. 2014, Engevik and
Versalovic 2017, Ilinskaya et al. 2017, Maguire and Maguire 2019, Nicholson et al.
2012, Neis et al. 2015, Shaikh and Sreeja 2017), catecholamines and other neuro-
modulators (Oleskin et al. 2016, Clarke et al. 2014, El Aidy et al. 2016, Ilinskaya
et al. 2017, Maguire and Maguire 2019, Oleskin et al. 2017), gas molecules
(Shenderov 2015, Carding et al. 2015) and many others (Chervinets et al. 2018,
Carding et al. 2015, Engevik and Versalovic 2017, Nicholson et al. 2012). In healthy
people, gut symbiotic microorganisms are the source of these microbial bioactive
molecules. In the process of evolution, humans have been selecting and retaining
the kinds of microorganisms that produced substances best of all corresponding to
the healthy organism in their physical, chemical and biological characteristics
(valency, isotope condition, structural, stereoisomeric shape of the molecule, solu-
bility, dispersity, oxidation state, half-life, safety and other parameters) (Shenderov
2014b, Bik et al. 2018, Shenderov 2011b, Sonnenburg and Backhed 2016, Wilson
and Nicholson 2017). The presence in the digestive tract of various nutrients and
diverse components of gut symbiotic microbiota may considerably alter the inten-
sity of effects of probiotic bacterial metabolites or even completely eliminate their
action. Violation of homeostasis of these molecules can often be a risk factor for
different diseases (Shenderov 2014b, Beloborodova and Osipov 2000, Clarke et al.
2014, Maguire and Maguire 2019).
There is a considerable chemical and functional resemblance between many
metabolic and signaling molecules synthesized by the cells of mammals, indige-
nous and probiotic microorganisms as well as micronutrients of food products.
These low molecular weight compounds of dietary, endogenous and microbial

https://doi.org/10.1007/978-3-030-34167-1_5
16 Metabolic Relationship Between the Host and Its Gut Microbiota
o rigin often act as regulators of intra- and interpopulation interaction (nervous,

humoral, immune, metabolic, information and epigenetic) of all macroorganism
cells, as well as between the cells of host and its symbiotic microbiota. Nondigestible
or harmful substances of dietary and/or microbial origin are either digested by
endogenous microbial enzymes or excreted with feces and urine. Gut microbiota is
also capable of accumulating different macro- and microelements in the amounts
sufficient to meet human daily demand in these elements for a period between 3 and
50 days (for example, B, Mg, Se, Zn) and even 25–190 days (for example, Co, Cu,
Mn, Si) (Shenderov 2008, 2013, Nicholson et al. 2012, Shenderov and Midtvedt
2014, Sonnenburg and Backhed 2016). The metagenome of gut microorganisms is
100 times as large as the metagenome of eukaryotic cells of organs and tissues in the
human gastrointestinal tract. Many biochemical metabolic pathways are absent in
humans and are only provided by the genome of gut microbiota. Recent studies of
the genomes of over 700 different representatives of gut microbiota have allowed to
identify the genes that encode over 3200 unique chemical reactions (Bik et al.
2018). Primarily, gut microorganisms are associated with the degradation of dietary
fibers, proteins and peptides, deconjugation and fermentation of non-digestible
complex polysaccharides and oligosaccharides, with glycophospholipids and
deconjugation and decarboxidizing of bile acids, the biosynthesis of vitamins (K
and В vitamins), isoprenoids, polyphenols, cholesterol lowering, the metabolism of
amino acids and xenobiotics with the production of biologically active molecules
which take an active part in various human functions and reactions. For instance,
daily energetic contribution of symbiotic gut microorganisms to its total energy
content is comparable to the contribution of daily dietary intake through food com-
ponents that are currently used for calorie calculation (Volkov et al. 2007, Panyushin
2012, Braune and Blaut 2016, Yadav et al. 2018).
In its distal part, human digestive tract is a natural bioreactor where symbiotic
and commensal microorganisms actively destruct and metabolize non-digestible
dietary fibers (resistant starches, cellulose, polysaccharides, oligosaccharides, pec-
tins, unabsorbed sugars such as raffinose, lactose, stachyose etc.). Gut bacteria
(Bacteroides, Ruminococcus spp etc.) produce a large complex of various hydro-
lytic enzymes that degrade the above-mentioned macromolecules. Bacteroides,
Bifidobacterium, Propionibacterium, Eubacterium, Lactobacillus, Clostridium,
Roseburia, Prevotella and other anaerobe microorganisms split the developing oli-
gosaccharides into short-chain fatty acids and different gases (hydrogen, carbon
dioxide, methane and others). Propionic and butyric acids can also be formed from
amino acids and peptides through fermentation by the representatives of the
Bacteroides and Firmicutes Phyla (Chervinets et al. 2018, Shenderov 2013b, 2015,
Clarke et al. 2014, Hall and Versalovic 2018, Maguire and Maguire 2019, Shaikh
and Sreeja 2017, Yadav et al. 2018). Gut microbiota takes part in degradation and
modification of nitrogen-containing substances (different proteins, urea, nitrates
etc.), lipids, nucleic acids, glycosides, amino sugars, chitins, organic acids, epithe-
lial and microbial cells and other components getting into this section of the diges-
tive tract. Bacteroides, Clostridium, Propionibacterium, Fusobacterium,
Lactobacillus, Streptococcus and other microorganisms contained in the large
Metabolic Relationship Between the Host and Its Gut Microbiota 17
intestine actively participate in the proteolysis of endogenous and exogenous pro-

teins. Proteins of intestinal mucosa, desquamated epithelium, as well as enzymes of
pancreatic and other intestinal juices are the endogenous sources of proteins needed
for this purpose. Complex proteins are split by various microbial peptidases, prote-
ases and endopeptidases. By participating in transamination, decarboxylation,
deamination and dehydrogenation reactions, different gut microorganisms
(Clostridium, Bacteroides, Bifidobacterium, Lactobacillus, Peptostreptococcus
etc.) are capable of metabolizing aromatic amino acids (tyrosine, phenylacetic acid,
tryptophan) along with the formation of noticeable amounts of indole, indolepropi-
onic acid, tryptamine, kynurenine, serotonin, ammonium, pyruvate and other com-
pounds. To date, over 85 species of indole-producing bacteria have been identified
in the human gut (Hall and Versalovic 2018, Maguire and Maguire 2019, Shaikh
and Sreeja 2017, Yadav et al. 2018, Zelante et al. 2013). The resulting free amino
acids and short peptides are used as substrates and nutrients for subsequent cata-
bolic processes or else undergo fermentation forming branched fatty acids (2-methyl
butyrate, isobutyrate, isovalerate), organic acids, various gases (hydrogen, carbon
dioxide) and small amounts of phenols, amines, indoles and ammonia (Neis et al.
2015, Shaikh and Sreeja, 2017, Yadav et al. 2018).
Gut bacteria in the human organism synthesize a broad spectrum of vitamins
(niacin, biotin, riboflavin, thiamine, pantothenic acid, folate, pyridoxine, cobala-
min, as well as vitamin K). B vitamins are primarily synthesized by the representa-
tives of Bacteroides. It was found that riboflavin and niacin are synthesized by the
representatives of 166 and 162 bacterial groups, respectively. Riboflavin and biotin
are mainly synthesized by Proteobacteria, Fusobacteria and Bacteroides species,
as well as by the Actinobacteria and Firmicutes species (Said 2013, Shaikh and
Sreeja 2017, Yadav et al. 2018). Over the past few years, the research focus has been
increasingly on the role of gut bacteria in polyphenol metabolism. To date, over
6000 species of various herbal phenols have already been identified, and their daily
dietary intake can amount to 800 mg. Ingested polyphenol compounds belong to
flavonoids, phenolic acids, stilbenes, lignans and other groups. It is important to
note that the overwhelming majority of these herbal compounds reach the large
intestine cavity without getting absorbed in the small intestine (Pérez-Jiménez et al.
2011, Yadav et al. 2018). Polyphenol degradation to different metabolites is observed
in Bacteroides, Clostridium, Eggerthella, Slakia (Braune and Blaut 2016). When
polyphenols are hydrolyzed by microbial enzymes, the resulting monomers and
aglycons subsequently undergo decarboxylation and phenol ring splitting whereby
hydroxyphenyl propionic acid and hydroxyphenyl acetic acid are formed. Different
bacteria were noted to vary both in phenol compounds metabolism and in adsorp-
tion and excretion of intermediate and final metabolic products (Braune and Blaut
2016, Quartieri et al. 2016, Tomas-Barberan et al. 2014, Tomás-Barberán et al.
2017, Yadav et al. 2018).
Bile acids that are prerequisite for lipid metabolism are constantly being synthe-
sized in the liver (lithocholic acid and deoxycholic acid). Gut bacteria in the diges-
tive tract can metabolize both the side chain of these molecules and the steroid
nucleus of these acids. Over thirty different bile acids circulate in the human
o rganism. This variety is ensured by the metabolic activity of gut bacteroides, bifi-
dobacteria, clostridia, lactobacilli and other microorganisms carrying the genes of
an enzyme — hydrolase of salts of these acids. It is owing to breaking the bond
between a bile acid and a conjugated amino acid that microbial detoxication of bile
acids is achieved. Microbial degradation can modify bile acids through epimeriza-
tion of its hydroxyl group (Clarke et al. 2014, García-Cañaveras et al. 2012, Yadav
et al. 2018). Gut microbiota as well as enzymes of the host organism can degrade
choline. Notably, the microbial conversion of choline results in the formation of
trimethylamine, which is transformed through the action of liver enzymes into tri-
methylamine N-oxide whose accumulation is associated with cardiovascular dis-
eases in humans (Clarke et al. 2014, Wang et al. 2011).
Also, digestive tract microbiota plays an important role in sulfur metabolism in
the human organism. Hydrogen sulfide formation by gut bacteria is due to both
bacterial enzymes participation in the fermentation of sulfur-containing amino acids
(cystein, cystine, methionine, taurine, sulfur-containing mucin) and microbial (for
instance, by the representatives of Desulfovibrio) utilization of sulfate (sulfite) ions.
Presently, 60 genera and 220 species of gut microorganisms are known which can
reduce sulfate-containing compounds (Shenderov 2015, Yadav et al. 2018).
Discussing the digestive and other functional roles played by gut microbiota in the
human organism, it should be noted that it is crucial for water and mineral homeo-
stasis. Just enough to remind about the amount of macro- and microelements con-
tained in the raw biomass of fecal microorganisms in adults, which exceeds their
daily requirement in healthy people by 5–50 times (Shenderov 2008).
Metabolic, signaling, transport and other functions of the representatives of
indigenous microbiota are more important than the quantitative content of microor-
ganisms belonging to various species in a biotope (Oleskin et al. 2016, Shenderov
2014b, 2016, 2017, Bik et al. 2018, Blum 2017, Carding et al. 2015, Engevik and
Versalovic 2017, Falony et al. 2016, Gilbert et al. 2016, Nicholson et al. 2012,
Shenderov 2011b). Metabolome analysis of feces and the content of different diges-
tive tract sections, urine, blood plasma, cerebrospinal fluid in mammals including
humans, germfree and conventional mice and other experimental animals, as well as
the juxtaposition of co-metabolomes of other animals and their microbiota have
shown that metabolism in mammals is closely connected with their gut microbiota
(Nicholson et al. 2005, Wikoff et al. 2009). Metabolome studies at the end of the
previous (Midtvedt 1985, Shenderov et al. 1989, Shenderov et al. 1990, Tamm et al.
1987) and the beginning of this century (Berer et al. 2011, Clarke et al. 2014,
Marcobal et al. 2013, Nicholson et al. 2012, Zierer et al. 2018, Trugnan et al. 2002),
capable of detecting a great number of low molecular weight metabolites, have
revealed that the concentrations of proteins, carbohydrates, peptides, short-chain
fatty and other organic acids, cations, anions and other low molecular weight com-
pounds in the feces of germfree and conventional mice and humans are essentially
different (Table 1). In animals, intragastral administration of different antibiotics
(lincomycin, ampicillin, azlocillin, cefalexin, tetracycline, rifampicin, sizomicin,
pefloxacin) in therapeutic concentrations for 7 days led to considerable changes in
the fecal excretion of amino acids, short-chain fatty acids (SCFA) and other organic
Table 1 Fecal excretion of some nitrogen and carbonic containing compounds in rats and humans
(Shenderov et al. 1990)
Concentration or relative distribution, %
Rats
Compound and method of its determination Conventional Germfree Humans
– total proteins, mg/g (Lowry method) 30.9 ± 7.4 80.2 ± 6.6 21.01 ± 3.8
– total aminoacid pool, μg/g (Aminoacid 708 ± 60 28,000 ± 3800 5656 ± 1800
analyzer “Biotronik IC-5000”)
ASP 6.6 ± 1.2 3.2 ± 0.7 9.5 ± 1.8
THR 3.6 ± 0.8 10.7 ± 2.8 7.2 ± 0.6
SER 4.2 ± 0.9 11.1 ± 2.7 5.3 ± 0.3
GLU 44.9 ± 4.3 11.8 ± 2.4 24.6 ± 2.2
ALA 8.9 ± 1.4 12.2 ± 2.7 9.9 ± 1.2
LYS 7.4 ± 2.4 5.5 ± 1.4 9.3 ± 1.0
GLY 7.2 ± 1.3 30.8 ± 2.4 4.9 ± 0.3
VAL 5.5 ± 1.6 8.8 ± 1.9 7.4 ± 0.7
MET 1.7 ± 0.7 5.8 ± 1.2 4.6 ± 0.3
ILE 6.6 ± 1.4 6.1 ± 1.9 7.1 ± 0.6
LEU 5.7 ± 1.2 8.6 ± 1.6 9.2 ± 1.4
NH3, μg/g 114.4 ± 38.9 80.0 ± 18.0 1118 ± 700
– total carbohydrates, μg/g (reaction with 6900 ± 2200 23,300 ± 2800 12,500 ± 1500
phenol and H2SO4)
– concentration of total SCFAs, μg/g 6000 ± 1400 200 ± 40 9400 ± 2500
(gas chromatography “LHM-80”, USSR)
acetic acid 85.1 ± 3.4 100 65.0 ± 2.5
propionic acid 9.0 ± 1.9 – 14.0 ± 1.3
i-butyric acid 0.7 ± 0.1 – 2.0 ± 0.2
n- butyric acid 2.7 ± 0.6 – 11.0 ± 2.1
i-valeric acid 0.8 ± 0.2 – 3.0 ± 0.5
n-valeric acid 1.0 ± 0.3 – 3.0 ± 0.6
caproic acid – – 2.0 ± 0.2
– the concentration of some carbonic acids,
μg/g (ion exchange chromatography
“Dionex” model 10, USA)
formic acid 1500 ± 300 1800 ± 400 traces∗
succinic acid 3700 ± 400 2000 ± 500 Traces
α-ketoglutaric acid 8.0 ± 1.0 – 8.5 ± 1.9
lactic acid 6.0 ± 1.0 50.0 ± 11.0 12.1 ± 1.6
pyruvic acid 150.0 ± 12.0 310.0 ± 19.0 Traces
trace: < 2.5 μg/g
*
acids 5 days after antimicrobial drugs withdrawal (Table 2). At the same time, mor-
phological changes were observed in the biofilm that covers the digestive tract
mucosa; antagonistic activity with respect to opportunistic gram-negative bacteria
in in vitro tests was decreasing, and colonization resistance of the digestive tract to
externally introduced similar opportunistic pathogenic bacteria was also impaired
Table 2 Influence of antibiotics on fecal excretion (Shenderov et al. 1990)

A: of some compounds in Wistar rats (5 days after end of antibiotic intake)
Antibiotics (mg/kg Ammonia % from total pool aminoacids
orally for 7 days) nitrogen, mg% GLU ALA ASP VAL, LEU,
ILE
Lincomycin (180) 3.0 ± 0.6 63.0 ± 5.7 8.0 ± 1.5 5.0 ± 0.9 9.0 ± 2.5
Ampicillin (180) 3.3 ± 1.1 42.0 ± 2.4 20.0 ± 3.8 6.0 ± 1.9 11.0 ± 2.8
Azlocillin (1350) 3.6 ± 0.8 19.0 ± 1.1 17.0 ± 2.9 5.0 ± 1.3 22.0 ± 4.9
Cefalexin (180) 3.0 ± 0.7 48.0 ± 2.7 12.0 ± 1.9 8.0 ± 1.7 8.0 ± 2.0
Tetracycline (90) 1.0 ± 0.2 30.0 ± 3.1 12.0 ± 2.1 2.0 ± 0.3 18.0 ± 6.3
Rifampicin (40.5) 1.5 ± 0.4 20.0 ± 4.5 14.0 ± 1.4 4.0 ± 0.4 11.0 ± 4.1
Sizomicin (12.6) 1.0 ± 0.2 31.0 ± 4.2 10.0 ± 2.5 8.0 ± 1.0 22.0 ± 7.9
Peflacin (90) 1.0 ± 0.2 34.0 ± 2.3 13.0 ± 1.8 7.0 ± 1.8 13.0 ± 3.3
Without antibiotics 3.0 ± 0.5 42.0 ± 4.5 6.2 ± 1.0 4.0 ± 0.5 8.1 ± 1.5
B: of some SCFAs in Wistar rats (5 days after end of antibiotic intake)
Antibiotics (mg/kg Relative distribution (%) of SCFAs in faeces
orally for 7 days) acetic acid propionic n-butyric n-valeric acid
acid acid
Lincomycin (180) 90.0 ± 5.0 7.0 ± 3.0 3.0 ± 0.8 traces
Ampicillin (180) 89.0 ± 6.0 6.5 ± 2.0 4.5 ± 1.0 traces
Azlocillin (1350) 75.0 ± 5.0 20.0 ± 4.9 5.0 ± 1.7 traces
Cefalexin (180) 83.0 ± 5.0 14.0 ± 5.3 2.2 ± 0.9 0.8 ± 0.07
Tetracycline (90) 90.0 ± 6.0 7.2 ± 2.3 2.0 ± 0.5 0.8 ± 0.1
Rifampicin (40.5) 87.0 ± 6.0 11.0 ± 3.0 1.6 ± 0.8 0.4 ± 0.03
Sizomicin (12.6) 82.0 ± 7.0 15.0 ± 4.0 2.5 ± 0.7 0.9 ± 0.2
Peflacin (90) 84.0 ± 4.0 12.0 ± 2.0 3.5 ± 0.8 0.8 ± 0.1
Without antibiotics 84.0 ± 5.0 12.0 ± 3.0 3.0 ± 1.0 1.0 ± 0.5
C: of some carbonic acids in Wistar rats (5 days after end of antibiotic intake)
Antibiotics (mg/kg Ratio:
orally for 7 days) Pyruvic acid Lactic acid α-ketoglutaric acid
Lincomycin (180) 10 18 23
Ampicillin (180) 10 35 26
Azlocillin (1350) 10 32 30
Cefalexin (180) 10 34 19
Tetracycline (90) 10 2 20
Rifamicin (40.5) 10 35 20
Sizomicin (12.6) 10 33 27
Peflacin (90) 10 41 34
Ampicillin (180) 10 35 26
Without antibiotics 10 0.5 0.3
(Shenderov et al. 1990). Feeding mice with foodstuff containing particular compo-
nents has helped modify the spectrum and quantitative content of metabolites in
animal urine and feces; it has led the researchers to conclude: diet plays a crucial
role in changing gut microbiota and host metabolism functionality (Marcobal
et al. 2013).
In recent studies of gut microbiome in 786 persons, mono- and dizygotic twins
aged 55 to 65 and predominantly female (Zierer et al. 2018), over 1116 metabolites
(amino acids, nucleotides, sugars, vitamins, fatty acids, other compounds and prod-
ucts of their intermediate metabolism) have been identified in blood and fecal sam-
ples. Among them, 469 similar metabolites have been simultaneously detected both
in fecal and blood samples; 647 metabolites were unique for the feces of study
participants. Over 36% of all compounds detected in blood were microbial in origin
(Hood 2012); for example, 1 to 20% of lysine and threonine contained in blood
plasma of an adult is synthesized by gut microbiota. The microbial origin of 40% of
metabolites in human blood was also demonstrated by other researchers
(Beloborodova and Osipov 2000). It was found that the peak of molecular microbial
markers in blood serum is proportional to the biomass of specific microorganisms.
In light of this, Russian researchers (Beloborodova and Osipov 2000) offered a
theory of homeostasis of low molecular weight microbial molecules which was for
the time innovative. According to the theory, their content in biological fluids is the
most important regulatory mechanism of symbiotic relationship between the host
and his microbiota; violation of homeostasis of these molecules is a risk factor for
various diseases. They can boost or inhibit the activity of different microorganism
cells, or be indifferent to it. In a recent publication, it has been suggested that “over-
feeding” leads to an increased activity and changed functionality of the microbiota,
thus severely disturbing host-microbe interactions and leading to dysbiosis and dis-
ease development (Lachnit et al. 2019).
Low molecular weight microbial compounds can act as metabolic molecules,
precursors of co-factors of bioactive compounds, signaling molecules and mole-
cules at the same time having metabolic and signaling activity. Their effects can be
displayed on the molecular level (DNA and chromatin structure; RNA interference;
post-translational modification of gene products) and on the cell level (on cell sur-
faces and membranes, in mitochondria and ribosomes), inside cell cytoplasm, in
intercellular space, in tissues, organs, physiological systems, as well as on the level
of the whole organism (Carding et al. 2015, Shenderov 2011a). By origin, most
metabolites found in human biological fluids are not related to the metabolic activ-
ity of its own cells; symbiotic microbiota is largely responsible for human metabo-
lome composition. Until now, the total number of potential gut bacteria metabolites
remains impossible to determine; they include molecules of dietary, endogenous
and microbial origin. One can only speculate that there are thousands and millions
of metabolites of microbial origin or those produced as a result of microbial trans-
formation of substrates and metabolites of different nature. In different persons,
many microbial metabolites are structurally and functionally similar; but some of
them are unique and can be found only in a few individuals. In any case, when esti-
mating human metabolome, it should be borne in mind that microbial metabolites,
as well as those microbially transformable, can act both as health promoting factors
and as agents involved in disease processes (Dorrestein et al. 2014, Trugnan et al.
2002). It should be noted that the molecular mechanisms through which symbiotic
microorganisms influence different physiological functions, biochemical and
behavior reactions of the human organism, as well as how they interact among
themselves and with host cells, — all this so far remains largely unclear.
Factors and Agents that Modify
the Composition and Functions
of Symbiotic Microbiota; Diagnostic
Methods for Microecological Imbalance
and its Consequences
According to the latest data, human microbiome variability is only by 10% related
to individual genetic traits; microbiome differences between individuals are largely
associated with the effects of various endogenous and exogenous factors: diet in the
first place (Blum 2017; Falony et al. 2016; Zhernakova et al. 2016). Out of 69 evalu-
ated factors, various therapeutics (over 10% varieties) are playing the most impor-
tant role in modification of gut microbiota composition (Falony et al. 2016).
Starvation, low physical activity, diets with increased levels of sugar, fat, with low
dietary fiber content, processing aids and heavy metal salts contained in foods, alco-
hol consumption, pesticide and radiation exposure, the influence of space flights,
surgeries, bacterial and viral infections, other factors and agents or their combina-
tions can reversibly or irreversibly alter human microbial ecology (Shenderov
2014b; Carding et al. 2015; Dietert and Dietert 2015; Maguire and Maguire 2019;
Pflughoeft and Versalovic 2012; Shenderov 2011b; Sonnenburg and Backhed 2016).
Of all pharmaceuticals, antibiotics have the most pronounced negative effect on
human indigenous microbiota. Many immunosuppressors, antihistamines in phar-
macological concentrations also inhibit the growth of bifidobacteria, lactobacilli,
enterococci, Escherichia coli and other commensal and symbiotic gut microorgan-
isms. Also, microecological disorders are caused by the administration of local
anesthetics, absorbents, nauseants, enveloping agents, laxatives, expectorants, cho-
leretics and other therapeutics. Certain colorants, nitrites, nitrates and some hor-
mones can be potential dysbiotic agents (Shenderov 2014b; Maguire and Maguire
2019). According to Swedish researchers’ data (Bengmark 2013), half of the 2000
pharmaceuticals registered in this country can cause side effects in the human diges-
tive tract (nausea, vomiting, diarrhea, constipation etc.) associated with microbiota
imbalance in this system. In a context where the compensation abilities of the host-
microbiota system are exceeded by negative effects on microbial ecology in length
and intensity, microecological disorders (dysbioses) develop, as well as the imbal-
ance of systems controlling intra- and interpopulation symbiotic relationship
between the host and its microbiota and, consequently, the risks of numerous
diseases. Thus, the negative stress effects of a multitude of biogenous and abiotic

https://doi.org/10.1007/978-3-030-34167-1_6
24 Factors and Agents that Modify the Composition and Functions of Symbiotic…
factors conflict with adaptive capabilities of modern humans and lead to a consid-
erable unbalancing of those gut microbiota functions that are connected with
maintaining dietary, metabolic, epigenetic, neurohormonal and immune homeo-
stasis (Shenderov 2008; Shenderov 2016a).
According to contemporary views, chronic diseases and premature ageing in
humans are associated with the imbalance of energy metabolism and redox pro-
cesses in tissues, accelerated cell ageing, imbalance of cell proliferation and apop-
tosis, diminishing of stem cell pool, chronic inflammation, loss of proteostasis,
mitochondrial disfunction, telomere shortening, microecological imbalance in gas-
trointestinal tract, nutritional disorders and violation of intrapopulation and intercel-
lular relations between eukaryotic and prokaryotic cells, genome instability,
epigenetic changes in gene expression, modification of information signaling path-
ways on the cell surfaces and membranes and inside the cells (Shenderov 2013c;
Shenderov 2016a; Dietert and Dietert 2015; Kanherkar et al. 2014; López-Otín
et al. 2013; Suvorov 2013). The degree of manifestation of these cellular and molec-
ular parameters, as well as a relative “balance” of pathophysiological processes are
responsible for the phenotypic manifestation of ageing, of a definite pathological
syndrome or a particular chronic disease (Shenderov 2008; Shenderov 2016a;
Bomba et al. 2012; Hanahan and Weinberg 2011; López-Otín et al. 2013; Shenderov
and Midtvedt 2014; Sonnenburg and Backhed 2016).
A profound microecological imbalance of natural microbiocenoses in all spheres
of our planet is the key factor of health deterioration in the majority of its inhabit-
ants. The question arises of preserving not only human life on the Earth, but the
whole existing variety of living organisms (Shenderov 2008). Profound and long-
term changes in the structure and quantitative content of human indigenous micro-
biota often induce the risk of gastrointestinal diseases (diarrhea, constipation,
colitis, irritable bowel syndrome, gastritis, gastric ulcer, other chronic diseases),
neurodegenerative diseases (Alzheimer’s disease, Parkinson’s disease, Huntington’s
disease, Amyotrophic lateral sclerosis, Friedreich’s ataxia), metabolic syndrome
(atherosclerosis, Diabetes mellitus type 2, obesity, gout), autoimmune disorders
(multiple sclerosis, Diabetes mellitus type 1, systemic lupus erythematosus), behav-
ior and mental disorders (autism, schizophrenia, chronic fatigue syndrome), muscu-
loskeletal disorders (fibromyalgia, skeletal muscle hypertrophy/atrophy, nonspecific
arthritis and arthrosis), kidney stone disease and gallstone disease, bronchial asthma,
atopic dermatitis, psoriasis, neoplasms, menstrual disorders, infertility, preterm
birth, neonatal anemia, cachexia, transplant against host syndrome, opportunistic
endo- and superinfections of different localizations (Shenderov 2014b; Shenderov
2016a; Carding et al. 2015; Frank et al. 2011; Gilbert et al. 2016; Maguire and
Maguire 2019; Sonnenburg and Backhed 2016). As any given manifestations of
microecological imbalance are currently found in a considerable number of people
living in countries with developed economies, it becomes clear how important it is
to stop further destruction of human microbial ecology and living environment. It
explains the need for still greater attention of a wide range of experts to the research
into normal and pathological human microbial ecology as the primary target for
many physical, chemical and biological agents. Along with this, the available data
Factors and Agents that Modify the Composition and Functions of Symbiotic… 25
Table 1 Methods and technologies used for the characteristics of human microbiota components
and functions
Item No. Methods and technologies
1 Different kinds of microscopy
2 Classic bacteriological method
3 Molecular and genetic methods
4 Different kinds of chromatography and chromatography–mass
spectrometry
5 Application of specific biochips
6 Use of germfree animals and different gnotobiotes
7 OMIC-technologies
allows us to think that maintaining and restoring normal human microbiota should
be considered as one of the most vital scientific experimental problems for health
preservation both in individuals and in the whole human population.
Assessment of symbiotic microbiota and detection of violations in its structure
and functions under exposure to various stress factors and agents, as well as moni-
toring of their restoration in case of prescription of microecological agents, is cur-
rently done by (Bezrodny and Shenderov 2016; Shenderov 2012; Shenderov 2015;
Berer et al. 2011; Gilbert et al. 2016; Karczewski and Snyder 2018; Meisel and
Grice 2017; O’Flaherty and Kleenhammer 2011; Sonnenburg and Backhed 2016)
both traditional tests and post-genomic OMIC-technologies (Table 1). The best-
known among the latest techniques are the methods based on the research into
molecular genomics, transcriptomics, proteomics, metabolomics, epigenomics,
phenomics and bioinformatics.
Contemporary Microecological Strategies
of Gut Microbiota Modulation for Human
Health Preservation, Restoration
and Improvement
For health preservation and reducing the risk of premature ageing and related meta-
bolic diseases, human diet should include various traditional, organic and functional
foods as well as therapeutics that support alimentary and other functions of gut
microbiota. To create or restore the impaired human biocenoses, various targeted
microecological techniques and methods are used (Table 1), with a focus on either
prevention or treatment. In the longer term, these approaches can be used in micro-
ecological engineering of pregnant and breastfeeding mothers, as well as infants for
shaping their targeted microbial ecology. It should be remembered that though the
responses of the host-microbiota system to the implementation of different dietary
and microecological agents are on the whole predictable, they can vary consider-
ably from individual to individual as far as their focus, efficiency and distinct mani-
festations are concerned (Shenderov 2014b; Lenoir-Wijnkoop et al. 2007; Shenderov
2011b; Sonnenburg and Backhed 2016). Since 1950s, over 150 different microeco-
logical therapeutics have been developed and produced commercially for preven-
tion and treatment of diseases related to the imbalance of symbiotic microbiota. To
preserve and restore human microbial ecology, a wide range of microecological
therapeutics are used (probiotics, symbiotics, combiotics, prebiotics, synbiotics,
virobiotics (including phagobiotics), genetically engineered probiotics, metabiot-
ics), as well as the technique of transplantation of large intestine microbiota.
The term “probiotics“is of Greek origin and means “for life” and was introduced
into the scientific literature in the 50s of the last century by the German nutritionist
Werner Kollath in contrast to the word “antibiotics“. Most foreign experts in this
field understand the term “probiotics” as living microorganisms that, when admin-
istered in an adequate amount, have a positive effect on the health of the host (Reid
et al. 2011). In Russia, this term often refers to living microorganisms or substances
of any origin, which in the natural way of appointment can have beneficial effects
on physiological functions, metabolic and behavioral reactions of the body by opti-
mizing its microecological status (Shenderov 2001; Shenderov 2008; Shenderov
2011b). Symbiotics are probiotics that include two or more strains of live probiotic
microorganisms that provide additional, more often stimulating effects (a typical

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28 Contemporary Microecological Strategies of Gut Microbiota Modulation for Human…
Table 1 Principles and techniques for correction of the digestive tract microbial ecology
Item
no. Principles and techniques
1 The use of functional foods including those containing poly- and oligosaccharides of
plant and microbial origin designed to the restoration of human nutritive and microbial
statuses.
2 Optimization of рН and redox potential in the digestive tract by prescribing sorbents,
antioxidants, detoxication agents etc.
3 Prescription of immunostimulants of different genesis that enhance secretory
immunoglobulin production and improve other mechanisms of local and systemic
immunity
4 Injections of anti-adhesive antibodies and lectins that block the adhesive capability of
potential pathogenic microorganisms
5 Selective decontamination of the digestive tract through the use of specially chosen
non-absorbing antibiotics, bacteriophages, antimicrobial peptides and other compounds
6 Optimization of pregnant and breastfeeding women microbiota, newborns inoculation
with certain microorganisms and their complexes, prescription to children and adults of
probiotics, prebiotics, symbiotics, synbiotics on the base of the representatives of normal
gut microbiota contained in pharmacopeial drugs, dietary supplements or special-purpose
food products
7 The application of therapeutics contributing to selective reproduction of “human-
friendly” anaerobe representatives of symbiotic microbiota
8 Enrichment of food products with dietary supplements, structural components,
metabolites and signaling molecules isolated from known strains of probiotic
microorganisms
example of the Russian symbiotic is “Bifikol”, which includes E. coli, removing

excess oxygen from the intestinal lumen, and bifidobacteria, which in these condi-
tions multiply more intensively, producing increased amounts of biologically active
substances (for example, vitamins that stimulate the growth of intestinal microor-
ganisms). Prebiotics - a variety of non-digestible carbon-containing compounds,
mainly plant polysaccharides and oligosaccharides, which have a positive effect on
the host, selectively stimulating the growth and activity of representatives of the
intestinal microbiota, relating primarily to lactobacilli and bifidobacteria. Synbiotics
are probiotic complex products containing live probiotic microorganisms and prebi-
otic substances that stimulate their growth. Combiotic are variants of synbiotic
agents, which in addition to probiotic microorganisms and prebiotics, added other
functional food ingredients (eg, vitamin and mineral premixes, phenolic or other
plant compounds, etc.). To date, the demand has been the greatest for prebiotics and
synbiotics highly diverse in composition (Tables 2 and 3) (Chervinets et al. 2018;
Shenderov 2001; Shenderov 2014b; Dore et al. 2013; Lenoir-Wijnkoop et al. 2007;
Maguire and Maguire 2019; Mimee et al. 2016; Reid et al. 2011; Roberfroid et al.
2010; Shenderov 2011b; Shenderov 2013a; Sonnenburg and Backhed 2016;
Suvorov 2013; Venema and Carmo 2015; Vyas and Ranganathan 2012).
Probiotic effectiveness of microorganisms is determined by their adaptive and
probiotic potentials (Lebeer et al. 2008; Lebeer et al. 2018; Shenderov 2011b).
Adaptive potential includes the resistance of probiotic bacteria to physical and
Contemporary Microecological Strategies of Gut Microbiota Modulation for Human… 29
Table 2 The list of certain probiotics commercially available in Russia

Name Composition
Acilact Three strains of Lactobacillus acidophilus
Bactisubtil, strains of bacilli (B. cereus)
Sporobacterin
Bilactin strains of Enterococcus faecium М-3185 and E. faecium М
Biovestin, strains of B. bifidum, B. аdolescеntis, L. plantarum
Biovestin-lacto
Biosporin strains of B. subtilis and B. licheniformis
Bifidin strain of B. adolescеntis МС
Bifidumbacterin strain of B. bifidum N1, sorbed by coal
forte
Bifilin strain of B. adolescеntis
Bifilong strains of B. bifidum and B. longum
Bificol strains of E. coli and B. bifidum
Bifiform strains of bifidobacteria (B. longum) and enterococci (E. faecium)
Gastropharm strain of L. bulgaricus LB-51
Colibacterin strain of E. coli M-17
Lactobacterin strain of L. plantarum 8PA-3
Linex strains of L. acidophilus, В. infantis and Е. faecalis
Normoflorin L and strain of lactobacilli or bifidobacteria
B
Polybacterin strains of B. bifidum ЛВА-3, B. longum B379M, B. breve 79–119,
B. adolescеntis ГО-13, L. acidophilus NK-1, L. plantarum 8PA-3,
L. fermentum 90-T-4
Florin forte sorbed by activated charcoal B. bifidum 1 and L. plantarum 8PA-3
Enterol yeast cells (Saccharomyces boulardii)
chemical stresses (рН value, oxidative and osmotic stresses), the activity of adaptive
metabolism (the capability of utilization of carbohydrate and other substrates), as
well as the capability for adhesion and its activity (the amount and type of surface
mucin- and fibronectin-binding proteins, exopolysaccharides, lipoteichoic acids
etc.). The probiotic potential is connected with the production by live microorgan-
isms of a multitude of low molecular weight compounds different in chemical ori-
gin (autoinducers, chemokines, modulins, effectors, substrates, co-substrates,
enzymes, co-factors, metabolites, signaling molecules), similar to those of endoge-
nous or dietary origin, as well as microbial compounds eliminating (or suppressing)
the growth of pathogenic and opportunistic microorganisms in the digestive tract of
the user of these therapeutics and/or positively interacting with his autochthonous
microbiota and gut epithelium, improving local and systemic immunity, activating
metabolic and other processes, localized or taking place outside the gastrointestinal
tract (Shenderov et al. 2018a; Bik et al. 2018; Engevik and Versalovic 2017;
Shenderov 2011b). Recent studies of the metabolomic genome of over 700 different
representatives of gut microbiota allowed for identifying the genes encoding over
3200 unique chemical reactions (Bik et al. 2018).
30 Contemporary Microecological Strategies of Gut Microbiota Modulation for Human…
Table 3 The list of prebiotics, synbiotics and combiotics commercially available in Russia
Name Composition
Astrolin 80% of purified inulin isolated from Jerusalem artichoke tubers
Bilaminolact E. faecium L-3 + bifidobacteria + plant extracts
Laminolact E. faecium L-3 in combination with plant extracts
Bioflor E. coli M-17 + extracts of soybeans, vegetables and propolis
Bifilis bifidobacteria (B. bifidum) + lysozyme
Bifistim B. bifidum, B. breve, В. infantis, B. longum, B. adolescentis + vitamins
(B1, B2, В3; В5, B6, B12, H, C, E, А, D3, folic acid) + cellulose, pectin +
Са hydrophosphate + fructose
Vitaflor lactobacilli, vitamin С, baker’s yeast autolyzate, β-carotene
Duphalac, disaccharide consisting of fructose and galactose
Lactusan, lactulose
Normospectrum B. bifidum 1 and 791, B. longum В379М and J-3, B. adolescеntis GО-13,
(multispectrum) L. plantarum 8PA-3, L. acidophilus NK-1 and K3Ш24, L. casei KHM-12,
vitamins (B1, B2, В3; В5, B6, B12, H, C, E, folic acid), minerals (Zn, Se),
inulin
Primadophilus В. infantis, B. longum, L. casei subsp. rhamnosus and maltodextrin
Rekicen-RD, wheat of rye bran with wine yeast sorbed on them
Fervital, Eubicor
Stimbifid inulin, oligofructose, vitamin and mineral premix (Zn, Se, vitamins B1,
B2, B3, B5, B6, B12, H, C, E, folic acid)
With the growth of the market of therapeutics that improve microbial ecology,
new approaches to their construction and use were being developed and known
methods were being modified. The mode of action of traditional probiotics and
synbiotics was being detailed, additional targets for known and new probiotics were
being located (the capability of antioxidative, anti-inflammatory, anti-mutagenic
activity, the ability to influence mental status, epigenotype, quorum-sensing regula-
tion etc.). Probiotic strains were being selected not only among traditional symbi-
otic bacteria (Lactobacilli, Bifidobacteria, Enterococci, Escherichia, Bacilli), but
also among anaerobe kinds of bacteria (for example, Bacteroides) and the kind of
microorganisms that are always present, if sparingly, in one biotope or another.
Attempts were being made to create symbiotic probiotics on the base of strains
simultaneously participating in the realization of a number of particular physiologi-
cal functions, biochemical and behavioral reactions; it was attempted to enrich the
composition of probiotics and synbiotics with nanoparticles containing certain low
molecular weight compounds or their groups (“functional” probiotics).
Probiotics are differentiated into those intended for the majority of the human
population or for specific cohorts including autoprobiotics for personal use.
Moreover, probiotics on the base of genetically engineered strains have been worked
out by artificial introduction into their genome of genetic determinants responsible
for the synthesis of bacteriocins, antimicrobial enzymes, immunoglobulins, inter-
ferons and other compounds that enhance adaptive and/or probiotic potential of
probiotic microorganisms (Shenderov 2014b; Browne et al. 2016; Maguire and
Maguire 2019; Mimee et al. 2016; Sonnenburg and Backhed 2016).
Contemporary Microecological Strategies of Gut Microbiota Modulation for Human… 31
Probiotics on the base of live symbiotic bacteria have quite a long history of
consumption; but until now it remains unclear why a few hundred milligrams or
even grams of probiotic bacteria can cause positive changes in physiological func-
tions, biochemical and signaling reactions and in human health on the whole, when
an adult’s large intestine alone contains between 1.5– 3 kg of live microorganisms
of hundreds and even thousands of different species (Bengmark 2001). In the same
way, it is difficult to answer the question why live probiotic microorganisms, which
are foreign for people consuming them for a long time, do not cause an immediate
rejection immune response or allergic reactions, as is often the case with respect to
dozens and hundreds of billions of other foreign microorganisms that are daily fed
into any person’s digestive tract with food, water, inhaled air or as a result of contact
with live and inanimate objects of the environment.
Molecular Language of Symbiotic Gut
Microorganisms
In the 1990s, it was found that various microorganisms, their cell fragments, colloid
particles, food starch granules and other compounds similar in size to bacterial cells
or smaller can easily penetrate intestinal mucosa. Owing to contemporary chroma-
tography and mass spectrometry methods, many similar low molecular weight par-
ticles and molecules are easily detectable in blood, urine and other human biological
fluids. Their appearance in these fluids can be detected as early as a few minutes
after oral administration of any given indicator compounds or microorganisms
(Freter and Nader de Macias 1995; Osipov and Verkhovtseva 2011). It was shown
clinically and experimentally that effects of probiotics on human and animal organ-
isms arise in case of administration not only of live but also of killed (by heating,
radiation etc.) microorganisms, as well as their cell-free extracts, purified cell walls,
supernatants of culture fluids for bifidobacteria and lactobacilli (Lee et al. 2002;
Reading and Sperandio 2006). The results of such studies have convincingly dem-
onstrated that the translocation of probiotic bacteria, their cell fragments, metabo-
lites and signaling molecules, as well as low molecular weight compounds formed
as a result of microbial degradation of epithelial cells, saliva, intestinal juices, food
substrates from the intestinal lumen is a common physiological process. Biologically
active compounds (autoinducers) associated with the metabolic activity of symbi-
otic (probiotic) microorganisms can potentially modify and take part practically in
any physiological function, metabolic, signaling, behavioral reaction, in intra- and
intercellular information exchange. Given that, the mechanisms of their effects may
be different in different symbiotic and probiotic microorganisms. Autoinducers
(chemokines, modulins) of microbial nature allow symbiotic microorganisms to
discern the environment, interact with each other and with host cells. These mole-
cules trigger a cascade of processes in prokaryotic and eukaryotic cells, when their
quantity reaches a certain level (“quorum”). Autoinducers interact with cell recep-
tors, with regulatory proteins recognizing them and, in the long run, activate (induce)
the work (expression) of the corresponding genes in the DNA of microbial, mito-
chondrial and eukaryotic host cells. Thanks to autoinducers, microorganisms and
cells of macroorganism exchange the information and coordinate their activity. That

https://doi.org/10.1007/978-3-030-34167-1_8
34 Molecular Language of Symbiotic Gut Microorganisms
is why such signaling molecules are viewed in scientific literature as “words” in the
molecular information “language”.
Numerous research in the past two decades has shown that symbiotic (probiotic)
microorganisms synthesize and discern a broad range of autoinducers of diverse
chemical nature (Vakhitov et al. 2005; Shenderov 2008; Shenderov 2009; Carding
et al. 2015; Engevik and Versalovic 2017; Lebeer et al. 2018; Schauder and Bassler
2001; Shenderov 2013a; Taga and Bassler 2003). The best known among them are
volatile and other organic acids, lactones, peptide pheromones, furanones and other
autoinducers involved in realization of the quorum-sensing phenomenon, proteins,
adenosine triphosphate and other compounds produced under stress, various pro-
teins, peptides and amino acids, various simplest metabolites of microbial cells
(CH4, H2S, NO, CO, H2, H2O2 etc.), nucleic acids, nucleotides, nucleosides, vita-
mins (mostly B vitamins, biotin, folic and pantothenic acids, vitamin K), short- and
long-chain fatty acids, amino acids, amines, polyamines, hormone-like substances,
neurotransmitters, regulatory molecules of different chemical nature involved in the
quorum sensing signaling, polysaccharides, oligosaccharides, various surface pro-
teins (pili, fimbriae, flagella etc.), mucins, peptidoglycans, lipoteichoic acids,
numerous bioactive peptides, glycopeptides, lipopolysaccharides, antimicrobial
compounds of different chemical structure, lectins, biosurfactants, pigments etc.
For example, it is known that the representatives of commensal and symbiotic bac-
teria produce over two dozens of various antimicrobial substances alone (lactic,
acetic, butyric, benzoic and other organic acids, hydrogen peroxide, carbon dioxide,
nitrogen oxide, diacetyl, bacteriocins, microcins, antibiotics, defensin-like peptides,
lysozyme, biosurfactants, lectins etc.) (Chervinets et al. 2018; Shenderov 2017;
Shenderov and Lakhtin 2004; Shenderov et al. 2018a; Aguilar-Toaláa et al. 2018;
Clarke et al. 2014; Engevik and Versalovic 2017; Gu and Li 2016; Lebeer et al.
2018; Oleskin et al. 2017; Shaikh and Sreeja 2017; Shenderov 2011a; Shenderov
2013a; Singh et al. 2018). It should be noted that molecular mechanisms through
which symbiotic microorganisms influence various physiological functions, bio-
chemical and behavioral reactions of the human organism, as well as how they
interact with each other and host cells — all this remains until now in many respects
unclear. The main “energetically significant” metabolites of symbiotic microbiota
in the digestive tract are represented by fatty acids (lactate, acetate, propionate,
butyrate, succinate), alcohols and gases (hydrogen, methane) (Panyushin 2012;
Aguilar-Toaláa et al. 2018; Shaikh and Sreeja 2017; Singh et al. 2018). Apart from
energy synthesis, in the process of complex microbial transformation including oxi-
dation, reduction, the formation of chelate complexes, various stereoisomeric forms,
isotope fractions, other physical and chemical modifications, lots of low molecular
weight compounds are formed from the above dietary and endogenous substrates in
the best available form for host cells, which are then absorbed and incorporated in
enzymes and other physiologically active substances.
Microorganisms of the digestive tract are the most important source of microbial
metabolites, as they secrete various terminal metabolites and/or their intermediate
products throughout their life (Carding et al. 2015; Dorrestein et al. 2014; Engevik
and Versalovic 2017; Gilbert et al. 2016; Nicholson et al. 2012; Singh et al. 2018;
Molecular Language of Symbiotic Gut Microorganisms 35
Lyu and Hsu 2018). Some microbial metabolites are the result of work of the genes
obtained by microorganisms in course of horizontal or vertical transfer of genetic
material which is ever-present in microbial populations of the digestive tract; others
are formed as a consequence of microbial transformation of substrates formed by
host cells. Microbial transformation of exogenous substances ingested from the
environment (pharmaceuticals, other organic compounds including food colorants,
thickeners, many xenobiotics of household chemicals) can also be accompanied by
the formation of products of their microbial modification with their entering intesti-
nal lumen. By now, hundreds and even thousands of low molecular weight com-
pounds had been identified that are formed as a result of metabolic activity of gut
microbiota or being low molecular components of microbial cells. Many com-
pounds of microbial origin are present in healthy and ill people, both in intestinal
lumen and in biological fluids outside it, as substrates, co-substrates, enzymes or
co-factors of metabolic reactions in the macroorganism, that is, molecules involved
in the regulation of intra- and interpopulation interactions between prokaryotic and
eukaryotic cells, as well as between the host and its microbiota. They may activate,
inhibit or be indifferent with respect to various prokaryotic and eukaryotic host cells
(Shenderov 2013c; Shenderov 2017; Shenderov et al. 2018a; Gilbert et al. 2016;
Nicholson et al. 2012; Shenderov 2011a; Sonnenburg and Backhed 2016).
Metabolome studies of intestinal content, liver, blood plasma, urine, fecal extracts
have shown that any given effects of symbiotic (probiotic) microorganisms on a live
organism are related to a mixture of various microbial compounds rather than one
low molecular compound in particular (Lebeer et al. 2018; Martin et al. 2008;
Sonnenburg and Backhed 2016). In this respect, the chemical dialogue between
symbiont microbes and host cells includes both signal exchange via low molecular
metabolites, proteins and peptides, and indirectly through neurohormonal, immune
and epigenetic mechanisms (El Aidy et al. 2016; Maguire and Maguire 2019;
Shenderov 2016b). Grown on milk base or soymilk, germfree filtrates of various
probiotic strains of lactobacilli, bifidobacteria and other symbiotic microorganisms
contained peptides, glycoproteins, enzymes, exopolysaccharides, peptidoglycans,
SCFA and other low molecular metabolites that inactivated different mutagens, car-
cinogens and prevented metastatic spreading of tumor cells (Aguilar-Toaláa et al.
2018; Dzutsev et al. 2017; Shaikh and Sreeja 2017; Sharma and Shukla 2016).
Microbial molecules interact with surface, membrane, cytoplasmic, mitochon-
drial and nuclear receptors of epithelial cells, causing induction of different genes,
maintaining genome and microgenome stability, modulating epigenome program of
development and realization, ensuring intra- and intercellular information exchange
and interaction between microorganisms and the host. Bioactive molecules synthe-
sized by the representatives of indigenous microbiota lead to both local and sys-
temic effects through neuroendocrine, immune, metabolic and epigenetic
mechanisms. Many microbial metabolites influence metabolism by interacting with
specific receptors on host cell membranes and nuclear structures (Oleskin and
Shenderov 2016; Shenderov 2008; Bourdichon et al. 2012; Cani 2018; Carding
et al. 2015; Clarke et al. 2014; Engevik and Versalovic 2017; Galland 2014; Gilbert
et al. 2016; Nicholson et al. 2012; Paul et al. 2015; Sampson and Mazmanitan 2015;
Holmes et al. 2011; Lebeer et al. 2018; Shenderov 2011a; Shenderov and Midtvedt
2014; Stilling et al. 2014; Singh et al. 2018). As previously stated, digestive tract
microorganisms actively metabolize complex substrates ingested with food or
endogenous in origin (components of saliva, digestive juices, desquamated epithe-
lium, dead microorganisms etc.). Microbial transformation of various substrates
results in the formation of many hundreds (possibly, thousands) of low molecular
biologically active functional ingredients (Shenderov 2009; Carding et al. 2015;
Chilloux et al. 2016; Corthesy et al. 2007; Engevik and Versalovic 2017; Shenderov
2011b; Sonnenburg and Backhed 2016). In accordance with the latest data (Aguilar-
Toaláa et al. 2018; Clarke et al. 2014; Lebeer et al. 2018; Lyte 2013; Maguire and
Maguire 2019; Lyu and Hsu 2018; Oleskin et al. 2017; Singh et al. 2018), gut
microbiota is proposed to be viewed as the most important structural and functional
component of a single neuroendocrine and immune organ. Candidate gut microbi-
ota hormones are SCFA (acetate, butyrate, propionate), neurotransmitters (sero-
tonin, dopamine, noradrenaline, GABA), precursors of neuroactive compounds
(tryptophan, kynurenine, L-DOPA), secondary bile acids, host metabolites (trimeth-
ylamine), cortisol, digestive tract hormones (ghrelin, leptin, glucagon-like peptide
1, PYY).
To better understand the importance of this complex function of gut microbiota
in the physiology and pathophysiology of hormonal-neuro-immune axis of the
human organism, let us have a closer look at its work. In the organisms of mammals,
apart from specialized hormone-producing glands (the adrenal glands, thyroid
gland, hypophysis etc.), the brain, the spinal cord and immune organs (the thymus,
lymph gland, bone marrow etc.), diffuse regulatory endocrine system (APUD-
system) is also found in many organs and tissues. The majority of its cells are pres-
ent in the gastrointestinal tract, producing various signal agents (hormones) that
regulate digestive juice secretion, appetite, mood, vascular tone and other mammal
functions (Yaglov and Yaglova 2012; Demeure 1993; O’Callaghan et al. 2016). By
now, over 20 gastrointestinal hormones have been identified (bombesin, gastrin,
histamine, ghrelin, glucagon, galanin, catecholamine, leptin, motilin, melatonin,
peptide YY, secretin, serotonin, somatostatin, substance Р, beta-endorphin, enkeph-
alin, enteroglucagon etc.) that are formed by diffusely distributed cells (APUD
cells) of the APUD system localized throughout the whole digestive tract length.
Many of the previously specified tissue mediators and hormones are of peptide
nature, they are used locally and are also fed to systemic blood circulation; as
regards their chemical composition and functions, they often bear similarity to brain
neurotransmitters and compounds produced by gut microbiota representatives
(Oleskin et al. 2017; Singh et al. 2018; Tan et al. 2015; Tsang et al. 2013).
Also, gut microorganisms play a fundamental role in the functioning of mammal
immune system. When the performance of the “symbiotic microbiota – immunity”
system is optimal, humans can successfully resist pathogenic microorganisms and
support the work of the regulatory mechanisms that ensure tolerance to harmless
antigens. Microbial metabolites and microbial cell components produced in the gut
are necessary for keeping immunologic balance (Aguilar-Toaláa et al. 2018; Engevik
and Versalovic 2017; Forbes et al. 2016; Laino et al. 2016; Oleskin et al. 2017).
Among the metabolites of symbiotic bacteria having effect on immune composition

in mammals, the best studied are SCFA formed in course of microbial fermentation
of undigestible dietary fibers (Shenderov 2013b; Engevik and Versalovic 2017).
Microbial SCFA modulate immune responses through the activation of chemoat-
traction receptors GPR41 and GPR43, localized in different immune cells, and/or
through inhibiting the activity of intracellular histone deacetylases. Depending on
the type and concentration, SCFA can decrease neutrophil adhesion and chemo-
taxis, suppress immune cell infiltration from blood stream to the inflammation site.
Also, these acids regulate the production of a range of leukocytic cytokines (TNF-α,
IL-2, IL-6, IL-10), eicosanoids and chemokines (MCP-1, CINC-2). Acetate and
butyrate modify the activity of neutrophils and macrophages that get involved in
inflammation processes through the induction in epithelial and immune killer cells
of the corresponding cytokines that take part in cell migration, leukocyte chemo-
taxis and suppression of adhesion molecule formation. These SCFA inhibit the
activity of sirtuins and NF-κB involved in attenuation of immune response to LPS
of gram-negative bacteria (Beloborodova 2012; Golovenko et al. 2011; Shenderov
2013b; Canani et al. 2011), suppress T-cell proliferation and activation (Meijer et al.
2010), reduce antibody synthesis and content in peripheral blood (Erofeev et al.
2012; Faith et al. 2014), cause apoptosis in lymphocytes, macrophages and neutro-
phils (Shenderov 2013b).
It is assumed that some SCFA (butyrate, propionate, but not acetate) and other
metabolic products of symbiotic microorganisms can modify the balance between
pro- and anti-inflammatory immune mechanisms by enhancing the egress and activ-
ity of extrathymic (intestinal) anti-inflammatory regulatory T cells (Tregs) and by
affecting dendritic cells (DC) involved in the process of their formation. SCFA
effect on Tregs is attributed to the change in epigenetic gene regulation in immune
cells caused by these organic acids (Faith et al. 2014; Shaikh and Sreeja 2017).
Detailed research into the mechanisms of microbial activation of the immune sys-
tem has shown that immune response modulation is achieved by not only SCFA
produced by host microbiota cells, but also by their various components (skeletons,
bacterial liposaccharides, peptidoglycans, surface polysaccharides and proteins,
nucleic acids, peptides and their complexes). Surface polysaccharide and protein
structures of probiotic lactobacilli elicit anti-inflammatory TNF-α immune response
in peripheral blood mononuclear cells, while anti-inflammatory immune effects are
caused by the joint action of microbial metabolites and surface components of these
bacteria. LPS of E. coli is by 100 to 1000 times as effective in stimulating the syn-
thesis of cytokines (TNF-α, IL-6, IL-10) by peripheral blood monocytes as peptido-
glycans; the latter, in turn, were 10 to 100 times more effective than microbial
lipoteichoic acid.
Metabolites and surface structures (cell walls, peptidoglycans, surface polysac-
charides) of probiotic bacteria reveal their anti-inflammatory effect through
increased induction of IL-10 synthesis along with other cytokines (Ashraf et al.
2014; Laino et al. 2016). The representatives of gut commensal and symbiotic
microbiota (clostridia, bacteroides, bifidobacteria, propionibacteria, lactobacilli
etc.), their components and metabolites interact with human immune system via
ligand-receptor link with Toll- and NOD-like cell receptor families. The crucial role
belongs to TLR receptors; they are localized in macrophages, neutrophils, dendritic
cells, endothelial and other cells. TLR1, TLR2, TLR6 recognize microbial lipopro-
teins, TLR4 — lipopolysaccharides (LPS), TLR5 — flagellin, TLR7, TLR8 —
single-chain RNAs, TLR9 — non-methylated DNA. TLR3, TLR7, TLR8 and TLR9
are involved in viral infection recognition. TLR5 and, less markedly, TLR2 and
TLR4 play the leading role in the formation of symbiotic microbiota structure in the
large intestine (Pang and Iwasaki 2012). As a result of various microbial ligand
interaction with these receptors, signals are induced that are crucial for immune
response to inflammatory effectors and for the activation of cell and innate immu-
nity humoral components (Laino et al. 2016; Lebeer et al. 2018; Pang and
Iwasaki 2012).
Low molecular peptides released from microbial cells in course of their life-
sustaining activity can stimulate the development and functions of T helper (inducer)
phenotype lymphatic cells (Buffie and Pamer 2013; Shaikh and Sreeja 2017).
Reacting with Toll-like receptor 5, bacterial flagellins (protein component of micro-
bial flagella) take part in the activation of gut mucosal immunity, in the chemotaxis
of both pathogenic and symbiotic bacteria. DC localized in the gut mucus layer
respond to bacterial flagellin effect with adhesion to host tissue cells and rapid pro-
duction of chemokines, antimicrobial peptides and cytokines involved in immune
response initiation. In response to flagellin effect, CD103+ DC produce IL-23 and
partly induce IL-22 cytokine synthesis by innate lymphoid cells (ILCs), which stim-
ulates protective immune mechanisms of the host related to epithelial cells
(Kinnebrew et al. 2012; Shaikh and Sreeja 2017; Singh et al. 2018).
The interaction of bacterial DNA containing non-methylated cytosine phosphate
guanosine dinucleotides (CpG) with Toll-R9 allows symbiont microbes to act as a
local immune response adjuvant. Peptidoglycans of symbiotic (probiotic) bacteria
that pass in considerable amounts from intestinal lumen into blood contribute to
enhanced immune response of bone marrow neutrophils with respect to pneumo-
cocci and staphylococci. The presence of polysaccharide А (PSA) Bacteroides fra-
gilis in a free state stimulated Tregs differentiation, IL-10 production and protected
mice from experimental colitis caused by Helicobacter hepaticus; it was connected
with PSA capability to increase the number of Tregs producing this interleukin.
Skeleton P-CWS (the structure of cell wall skeleton consisting of peptidoglycane,
isopentadecanoic acid and acidic polysaccharide) isolated from Propionibacterium
acnes had anti-tumor activity due to the activation of macrophage cytotoxic func-
tions. LPS of gram-negative symbiotic bacteria in vitro stimulated DNA synthesis
in В cells in absence of Т cells and macrophages. Probiotic strains of lactobacilli
inactivated by heating triggered different immune responses, which is attributed to
microbial pro- and anti-inflammatory mediator ratio (surface and secreted proteins
and metabolites) capable to induce cytokine secretion as a result of NF-κB activa-
tion in monocytic cells. Cell-free filtrate of these lactobacilli cultural fluid inhibited
the production of pro-inflammatory cytokines, signaling immune cells, tumor
necrosis factor (TNF). One of the possible lactobacilli metabolites capable of sup-
pressing TNF production in human myeloid cells is histamine, which is formed in
course of microbial transformation of L-histidine amino acid (Engevik and

Versalovic 2017; Ganesh and Versalovic 2015; Lebeer et al. 2018; Shaikh and Sreeja
2017). Surface components of probiotic lactobacilli cell walls induced cytokine pro-
duction by type 1 and type 2 helper Т cells in healthy donor blood monocytes.
TGF-β secretion increased most markedly, while IL-10 and TNF-α secretion
increased to lesser extent. In the same model, cell-free filtrates of cultural fluid of 17
lactic acid bacteria strains including probiotic cultures helped increase the produc-
tion of IL-10 and decreased the formation of IL-2, TNF-α and IL-4; under such
conditions the production of IL-12, IFN-γ and TGF-β stopped completely. Based on
these data, the authors (Aguilar-Toaláa et al. 2018; Ashraf et al. 2014; Engevik and
Versalovic 2017; Legent and Norris 2009) came to the conclusion that anti-
inflammatory TNF-α immune responses are primarily related to the effect of probi-
otic lactobacilli surface structures on peripheral blood mononuclear cells, while
anti-inflammatory immune effects are caused by joint action of microbial metabo-
lites and bacterial surface components. Moreover, symbiotic (probiotic) bacteria
produce various bacteriocins, microcins and other antimicrobial molecules that can
indirectly modulate the immune functions by suppressing the growth and virulent
potential of pathogenic and opportunistic microorganisms having intruded into the
macroorganism (Engevik and Versalovic 2017; Lebeer et al. 2018; Mimee et al.
2016; Shaikh and Sreeja 2017; Singh et al. 2018).
Various probiotic microorganisms, their structural components (peptidoglycans,
exopolysaccharides, LPS, nucleic acids, S-layer proteins etc.) and metabolites (vari-
ous peptides, SCFA, homoserine lactones, dopamine, serotonin, histamine etc.)
have specific receptors and targets in different immune system components and
cells. Immunologic effects of probiotics and their low molecular effectors can differ
in mammals and humans, in a healthy organism and in immune-compromised peo-
ple with inflammatory manifestations (Engevik and Versalovic 2017; Laino et al.
2016; Nakagawa and Miyazaki 2016; Oleskin and Shenderov 2016). Low molecu-
lar weight compounds and cell components linked to cell walls, membranes and
other microbial organelles of probiotic bacteria released during bacteriolysis,
metabolites and signaling molecules (exopolysaccharides, membrane fragments,
lectins, glutathione peroxidase, superoxide dismutase, nicotinamide adenine dinu-
cleotide oxidase and peroxidase, organic acids, uronic acid etc.) can exhibit antioxi-
dant and signaling activity (Shenderov and Lakhtin 2004; Aguilar-Toaláa et al.
2018; Engevik and Versalovic 2017; Lakhtin et al. 2007; Shaikh and Sreeja 2017;
Singh et al. 2018; Wang et al. 2017). Also, the representatives of symbiotic gut
microbiota form a multitude of various low molecular neuromediators capable of
modifying the total pool of these regulators of nervous system functions in the
human organism (Table 1).
Along with various eukaryotic human cells, the digestive tract microbiota takes
an active part in the formation of the total pool of gas neurotransmitters (nitrogen
oxide, carbon monoxide, hydrogen sulfide, ammonia, hydrogen) (Shenderov 2015;
Nicolson et al. 2016; Oleskin and Shenderov 2016), oxytocin, vasopressin, neuro-
peptides, brain-derived neurotrophic factor and other hormones and neuroactive
compounds in mammal organisms (Sampson and Mazmanitan 2015).
Table 1 Some neuromediators formed by the representatives of human symbiotic microbiota

(Oleskin et al. 2016; Carabotti et al. 2015; Clarke et al. 2014; Cryan and Dinan 2015; Engevik and
Versalovic 2017; Lebeer et al. 2018; Maguire and Maguire 2019; Oleskin et al. 2014; Oleskin and
Shenderov 2016; Sampson and Mazmanitan 2015; Singh et al. 2018)
Neuromediators Microorganisms
Serotonin lactococci, lactobacilli, streptococci, E. coli,
Morganella, Hafnia
C-dihydroxyphenylalanine (DOPA) lactobacilli
Dopamine / noradrenaline bacilli, lactobacilli, E. coli, staphylococci, Hafnia
Noradrenaline bacilli, E. coli, Serratia, proteidae
Acetylcholine lactobacilli
GABA lactobacilli, bifidobacteria
Histamine lactococci, lactobacilli, streptococci, Morganella,
Klebsiella, Hafnia
Tryptamine clostridia, ruminicocci
Tyramine lactobacilli, enterococci
Aspartate, glutamate, glycine, taurine, lactobacilli
tryptophan
Butyrate, acetate, propionate, GABA lactobacilli, bacteroides, clostridia, bifidobacteria,
Roseburia, eubacteria
Over the course of many decades, the dominating opinion was that health, lon-
gevity and disease risk are related to mutative changes in DNA linear structure. In
the modern view, the genetic system should be supplemented with the epigenetic
one, which regulates activation and silencing of microbial and eukaryotic cell genes
in response to exogenous or endogenous influence of different effectors. Epigenetic
changes arise as a result of covalent attachment/detachment of different chemical
groups (methyl, acetyl, phosphate etc.) to DNA, histones and other proteins.
Epigenetic alterations are observed 100 times as often as traditional ones. Epigenetic
changes in DNA and chromatin are reversible, but they can persist over several cell
generations. Throughout the life of an individual the spectrum of “active and silent
genes” may change.
There are many factors and agents (Fig. 1) that can interfere with epigenome
regulation of gene expression and post-translational modification of gene products
(Kanherkar et al. 2014). Among different modulators of epigenetic mechanisms
responsible for the expression of chromosomal, mitochondrial and microbial genes
throughout the life of an individual, symbiotic microorganisms have an important
role to play (Shenderov 2013c; Paul et al. 2015; Shenderov 2016b; Singh et al.
2018). It is known that epigenetic biochemical machinery is closely connected with
energy processes in the organism. According to the latest data, mitochondria and
membranes of symbiotic microorganisms should be considered as a single meta-
bolically active collective “organ” responsible for energy synthesis in the host
organism and production of compounds regulating the expression of nuclear, mito-
chondrial and microbial genes. Energetic and other processes in mitochondria and
their functional analogs (inner bacterial membranes) require a multitudes of
Microbiome Genetic
Xenobiotics status
Nutritional
Diseases
status
Epigenome
of the
person Medical
Stresses impacts
Physical climatic and Physical

activity geographical factors
factors
Fig. 1 Exogenous and endogenous factors involved in the formation of human epigenome
enzymes and their co-factors: vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, K1,
K2), non-vitamin substrates (NAD, NADH, NADP+, NADPH, ATF, cytidine tri-
phosphate, S-adenosyl methionine, 3’phosphoadenosine-5’phosphosulfate, gluta-
thione, coenzyme B, coenzyme M, coenzyme Q10, co-factor F-430, heme,
alpha-lipoic acid, methanofuran, molybdopterin/molybdenum co-factor, pyrrolo-
quinoline quinone, tetrahydrobiopterin, tetrahydromethanopterin), minerals (Ca,
Cu, Fe++, Fe+++, Mg, Mn, Mo, Ni, Se, Zn), amino acids (arginine, lysine, methionine,
cysteine, β-alanine, serine, threonine, histidine, tryptophan, aspartic acid, carnitine),
organic acids, Krebs cycle participants, certain nucleotides (for instance, pyrimi-
dine), microRNA etc., which can be of microbial, dietary or endogenous origin
(Shenderov 2016b; Shenderov and Midtvedt 2014).
Food products and symbiotic microorganisms have their effects on energy
metabolism, human behavioral, metabolic and signaling reactions by supplying the
organism with dietary substrates, co-factors, as well as through neuroendocrinal,
metabolic, epigenetic and immune mechanisms. Long-term and profound func-
tional impairment of the bacterial axis – the mitochondrion – epigenetics is accom-
panied with insufficient production of adenosine triphosphate and delayed delivery
of this energy source to cells and tissues, sharp increase in the cells of high-reactive
free radicals of oxygen, nitrogen and peroxidation products, antioxidant defense
deficiency, disturbance of apoptosis and other cell development cycles, changes in
signal transduction, proteostasis, cellular senescence, telomere length shortening,
diminishing of stem cell pool, modification of balance of the compounds involved
in the regulation of gene expression processes in mitochondrial, bacterial and
nuclear DNA, in post-translational modification of the end products of these genes.
It follows from the above that low molecular components of food and functional
ingredients of symbiotic microbiota can be viewed as key players in the operation
of energetic and epigenetic biochemical machinery (Shenderov 2008; Shenderov

2015; Shenderov 2016a; Bhat and Kapila 2017; El Aidy et al. 2016; Galland 2014;
Sampson and Mazmanitan 2015; Shenderov and Midtvedt 2014; Singh et al. 2018;
Stilling et al. 2014). Epigenetic changes that arise in mammal organisms throughout
the life of animals and humans can be passed on to successive generations resulting
in a growing disposition toward stress response defects and the development of a
multitude of chronic metabolic diseases (Shenderov 2016a; Shenderov 2016b).
When discussing the importance of symbiotic (probiotic) microorganisms in the
formation of human metabolome, it should be mentioned that the microbiome con-
tains a cluster of genes responsible for the synthesis of a number of specialized
microbial metabolites (polyketides, sterols, isoprenoids, nonribosomal peptides,
other natural products) whose functions are currently underexplored; it is beyond
dispute that such microbial molecules can potentially take part in a number of
important immune, metabolic and regulatory reactions of the macroorganism. It
will suffice to mention that such a special microbial metabolite as rapamycin is cur-
rently being clinically tested as means of treatment and prophylaxis in case of dis-
eases connected with impairment of immune system functions. Undoubtedly, in
perspective, the number of similar microbial metabolites with specific targets will
only be growing (Dorrestein et al. 2014). In structure and function, microbial com-
pounds often represent analogues or homologues of dietary substances and/or pro-
duced endogenously by host eukaryotic cells. It is safe to say that low molecular
weight compounds serve as global (universal) regulators of intra- and interpopula-
tion informational communication of any living organisms irrespective of evolu-
tionary organization level (Lebeer et al. 2008; Lebeer et al. 2018; Shenderov 2011a;
Shenderov 2013a).
Drawbacks and Negative Consequences
of Traditional Probiotics Based on Live
Microorganisms
The growing market of pharmaceuticals, dietary supplements, and particularly

food products based on live probiotic microorganisms, is evidence of their suffi-
cient prophylactic and, if less marked, therapeutic effects. However, it is currently
impossible to precisely determine the optimum quantity of bacteria required for
beneficial probiotic effects on human health; in many cases exact data are lacking
on probiotic effect mechanisms and targets. It turned out that positive effects of
live probiotic microorganisms may be short-term, uncertain or even absent in case
of long use. The absence of clear health-promoting effect of prescribed probiotics
on the base of live organisms is usually explained by low concentrations of biologi-
cally active microbial compounds in places of their application (Reid et al. 2011).
In the past few years, experimental and clinical studies have also shown the diffi-
culty, and sometimes even impossibility, of constructing industrially manufactured
probiotics for targeted maintaining optimum levels of indigenous microbiota by
way of simple selection of a particular live probiotic strain or even their group
(Shenderov 2011a).
In the last 10–15 years, our knowledge regarding the safety of probiotics on the
base of live microorganisms has changed considerably. And though the use of such
probiotics for over 50 years has shown that they are quite safe as a means of disease
prevention and treatment and as food products, it turned out that some probiotic
bacterial strains can be harmful even if they belong to Lactobacillus or
Bifidobacterium genera whose representatives have no traditionally known genes
of pathogenicity. The accumulated documentary evidence shows that lactobacilli
and bifidobacteria used in the production of fermented milk products or probiotics
can act (if rarely) as agents of opportunistic infections (endocarditis, sepsis, bacte-
raemia, pneumonia, abdominal abscesses, meningitis, urological infections), espe-
cially in patients receiving antibiotic treatment or those severely immune
compromised (Bourdichon et al. 2012; Cannon et al. 2005; Ohishi et al. 2010; Van
Reenen and Dicks 2011; Yazdankhah et al. 2009). The same bacteria may be
responsible for allergic or autoimmune pathologies (Berer et al. 2011; Guilherme

https://doi.org/10.1007/978-3-030-34167-1_9
44 Drawbacks and Negative Consequences of Traditional Probiotics Based on Live…
et al. 2006; Kiseleva et al. 2011; Prangli et al. 2010). Symbiotic microorganisms
including probiotic strains may increase platelet aggregation aggravating clinical
manifestations of hemolytic uremic syndrome (Van Reenen and Dicks 2011;
Yazdankhah et al. 2009); some of them may be a source of toxic biogenic amines
(Bourdichon et al. 2012). For example, in the process of microbial transformation
of phosphatidylcholine contained in red meat and cheese, choline reacts to form
trimethylamine which oxidizes to trimethylamine -N-oxide — a compound that
takes part in atherosclerotic plaque formation; it also plays a role in thrombosis
development, certain hepatic lesions and colon cancer (Gilbert et al. 2016;
Sonnenburg and Backhed 2016).
The majority of strains used as starter cultures in the production of probiotics
have been selected according to such characteristic as high antagonistic activity
(against bacteria or fungi). Once in the gastrointestinal tract or on female genital
mucosa, these probiotic microorganisms can suppress the growth and development
of symbiotic gut and vaginal microbiota and modify its metabolic activity
(Glushanova and Shenderov 2005; Shenderov and Glushanova 2006; Yazdankhah
et al. 2009). Furthermore, some gut bacteria can transform orally administered
pharmaceuticals, modifying their activity and/or transforming inactive pro-drug
forms into a pharmacologically active drug (Clayton et al. 2009). Unfortunately,
we have not found any data in literature on in vitro and in vivo research into the
capability of probiotic microorganisms to interfere with drug bioaccessibility and
pharmacological activity; but such effects of probiotics cannot be excluded. The
situation becomes even more uncertain if probiotic strains belong to Enterococcus,
Streptococcus, Escherichia, Bacillus, Coprococcus, Bacteroides or other genera
containing several strains or created on the basis of genetically modified microor-
ganisms (Shenderov 2013a).
Data is available that certain probiotics (Lactobacillus rhamnosus GG strain) can
induce additional expression of 400 new genes in the gut which are involved in
immune response and inflammation, cell growth and differentiation, apoptosis,
intracellular information exchange and adhesion processes (Di Caro et al. 2005).
Oral administration of a probiotic containing live E. faecalis bacteria changed the
activity of 42 human genes involved in cell cycle regulation, apoptosis and intermi-
crobial exchange (Cancer-causing gut bacteria exposed 2018). Moreover, it was
found that when passing the digestive tract, some silent genes of probiotic microor-
ganisms can also get activated; unfortunately, many newly formed bacterial prod-
ucts have never been extensively characterized from both the chemical and the
functional perspective (Bron et al. 2004; Di Caro et al. 2005; Yuan et al. 2008). So,
these were the conditions for the induction of 72 genes of a probiotic strain L. plan-
tarum WSFS1 to occur. Nine genes among them are responsible for the transport
and enzymatic fermentation of sugars, another nine — for the synthesis of amino
acids, nucleotides, vitamins and other co-factors, four — for the synthesis of surface
proteins controlling the synthesis to the specific immune factors of the host; one
more process that took place in this probiotic Lactobacillus strain was the induction
of expression of 46 more genes; chemical and protein products of these genes had
Drawbacks and Negative Consequences of Traditional Probiotics Based on Live… 45
Table 1 Some ecological and social consequences of the use of probiotics on the base of live
microorganisms and fecal content transplantation (Tkachenko 2014; Chernevskaya and
Beloborodova 2018; Shen et al. 2018; Shenderov 2007)
Item
No. Some consequences of probiotic use
1 Spreading of drug-resistant microorganisms
2 Decreased effectiveness of chemotherapy and chemoprophylaxis; increased cost of
treatment for many diseases
3 Selection of microorganism strains with atypical biological characteristics
4 Formation of new unconventional microbial associations
5 Changes in pharmacokinetics and biotransformation medicines and food nutrients
6 Changes in etiological structure of infectious diseases
7 Broadened spectrum of diseases related to microbial factor
8 Increased number of people with low resistance to infectious agents
9 Increased number of people with altered mental and behavioral responses
not been identified (Bron et al. 2004). Thus, the expression of known or silent genes
in microbial and/or eukaryotic cells when consuming live probiotic microorganisms
may have an undesired or even unpredictable effect on human health. In the last
10 years, evidence was gathered (Table 1) showing that lactic acid and other
probiotic bacteria are involved in horizontal gene transfer of antibiotic resistance
genes, traditional and new pathogenicity factors and genes controlling other func-
tions (hemolysis, D-galactose, DNA-methyltransferases, sirtuines, glucuronidase,
acetaldehyde, pks genes etc.) by way of transformational, transduction and conjugal
DNA transfer. Gene transfer and recombination processes related to the administra-
tion of live probiotic microorganisms can involve far-reaching potentially negative
health and environmental effects (Bourdichon et al. 2012; Sommer et al. 2010; Van
Reenen and Dicks 2011).
Various experimental animal models, as well as clinical observations have shown
that live probiotic microorganisms can modify the functions of the NF-κB signaling
system which regulates the activity of genes of synthesis of anti-inflammatory inter-
leukins, hemokinins and cytokines (especially, IL-6, IL-8, adhesive and other mol-
ecules). They also lead to impaired regeneration of liver and gut cell lesions, cause
telomere disfunction which results in increased risk of premature ageing. Moreover,
it was found that DNA-methyltransferases of E. coli can cause potential damage to
epigenetic integrity of cell genomes. In the process of chromosomal DNA sections
methylation, DNA-specific microbial DNAses can cause cell death resulting from
microbial cleavage of eukaryotic cell DNA at sites where methyl groups are added
(Ishikawa et al. 2011). There are indications that some E. coli strains (including
probiotic strain E. coli Nissle 1917) possess a set of genes (pks island) that are
responsible for the production of double-strand breaks in eukaryotic host cell
DNA. Bacteria containing the pks genes induce in eukaryotic cells a process
(referred by the term “megalocytosis”) in which the cell body and nucleus become
enlarged and mitosis stops. A break in a double-strand DNA of eukaryotic cells
occurs after their four-hour contact with bacteria carrying pks islands. Over 30% of
E. coli strains isolated from healthy adult feces had such pks islands.
The speed of DNA damage of eukaryotic cells in the gut depends on the number
of E. coli contacting host cells. The exposure of the latter to 100 or more bacteria, a
great number of lesions develop in DNA structure of epithelial intestinal cells. Such
DNA breaks caused by microbial peptide-polyketide genotoxins formed by certain
symbiotic (probiotic) bacteria can trigger various disorders in an animal organism
including development of neoplasms (Arthur et al. 2012; Nougayrede et al. 2006).
Live cells of E. faecalis inside the intestinal tract of mammals release substantial
amounts of extracellular superoxide, hydroxyl radicals and hydrogen peroxide dur-
ing carbohydrate fermentation and via autoxidation of membrane dimethylmena-
quinone. These oxidants can damage DNA of enterocytes and facilitate the
development of sporadic adenomatous polyps and colorectal cancer (Huyckel et al.
2003; Cancer-causing gut bacteria exposed 2018). Ethanol and its metabolite acet-
aldehyde are classified as class I carcinogens. Acetaldehyde concentrations between
100 and 500 μМ can cause mutagenic damage in the structure of eukaryotic cell
DNA. Many symbiotic microorganisms including lactic acid bacteria used as starter
cultures in the production of fermented food products and in probiotics can convert
ethanol and/or glucose to carcinogenic acetaldehyde. The levels of this compound
contained in foods or in the gut can exceed the values required for the manifestation
of acetaldehyde’s mutagenic effect. Moreover, as live probiotic microorganisms can
colonize the digestive tract for quite a long time and produce considerable amounts
of acetaldehyde locally, they can be potentially more harmful for health than tradi-
tional dairy lactic acid bacteria (Helminen et al. 2011; Nummi et al. 2011). Thus,
probiotics on the base of various microorganisms have a wide range of potential
drawbacks. This calls for more thorough research into the efficiency and safety of
probiotics – both already produced commercially and being developed. It is espe-
cially important when it comes to consuming such probiotics by pregnant women,
neonates, infants, as well as people with various immunodeficiencies. Recently pub-
lished data have presented convincing evidence that transitory colonization of preg-
nant germfree mice led to considerable changes in the functions of small intestine
immune cells in newborn mice (Charbonneau et al. 2016). It follows that the
attempts to use probiotics produced by traditional technologies for manipulating
microecological, immune, metabolic and epigenetic status in pregnant and breast-
feeding women may have a considerable influence on neonatal development of their
offsprings (Charbonneau et al. 2016; Jurk et al. 2014; Shenderov 2013a; Yazdankhah
et al. 2009).
The analysis of the published data (Green et al. 2017; Sanders et al. 2016) allows
to arrive at a conclusion that currently available knowledge related to the effective-
ness and safety of traditional probiotics made with live microorganisms are not
enough for reliable assessment of their ecological and clinical risk in short- and
long-term perspective. The above mentioned shortcomings of live probiotic micro-
organisms had led European Food Safety Authority (EFSA) to conclude on 4
Drawbacks and Negative Consequences of Traditional Probiotics Based on Live… 47
400
350
300
250
200
150
100
50
Fig. 1 The number of publications on clinical trials of probiotics according to PubMed.gov
December 2012 that a ban is necessary for using designations (labels) on products
containing live probiotic microorganisms which state that using these foods has
positive health effects. From the perspective of EFSA experts, there is not sufficient
clinical proof for their health benefits (Katan 2012). A similar ban was imposed by
Food & Drug Administration (FDA) of the USA in 2014 (Frequently Asked
Questions About Medical Foods 2016). In course of the workshop “Science and
Regulation of Live Microbiome based Products: no headway on regulatory issues”
held by the FDA Center for Biologics Evalution and Research and Nutritional
Institute of Allergy and Infectious Diseases” (Rockville, USA, September, 2018) it
was stated that many issues related to safety of probiotic products call for extra deci-
sions. In some US hospitals, it is forbidden to buy, store, prescribe and distribute
probiotics to prevent contamination of wards and patients with live probiotic micro-
organisms. In recent years, there has been a tendency of reduce the number of pub-
lications devoted to clinical studies of probiotics (Fig. 1).
As an alternative to traditional probiotics, prebiotics, dietary fibers and other
compounds had been offered which selectively stimulate the growth and/or activity
of one or a limited number of representatives of genus (genera)/species in gut
microbiota having positive effects on the host organism (Gibson and Roberfroid
1995). By now, the prebiotic range has grown dramatically to include several doz-
ens of alcohols, oligosaccharides, polysaccharides, enzymes, peptides, plant and
microbial extracts and other compounds (Table 2). Their production volumes have
reached hundreds of thousands tons annually.
Table 2 Basic types of prebiotic substances (Doronin and Shenderov 2002; Shenderov 2008;
Shenderov 2014b; Maguire and Maguire 2019; Roberfroid et al. 2010)
Item
No. Substances
1 Monosaccharides, alcohols (xylitol, melibiose, sorbitol, raffinose etc.)
2 Oligosaccharides (lactulose, galacto-oligosaccharides, fructo-oligosaccharides, soybean
oligosaccharides — stachyose etc.)
3 Polysaccharides (pectin, inulin, chitosan, pullulan etc.)
4 Enzymes (microbial galactosidases, proteases in Saccharomycetes etc.)
5 Peptides (of soybean, milk etc.)
6 Amino acids (valine, arginine, cysteine, glutamic acid etc.)
7 Antioxidants (vitamins А, С, Е, carotenes, glutathione, ubiquinone, selenium salts etc.)
8 Unsaturated fatty acids (omega-3 etc.)
9 Organic acids (butyric, propionic, acetic etc.)
10 Plant and microbial extracts (carrot, potato, tomato, rice, garlic, yeast etc.)
11 Other (lecithin, para-aminobenzoic acid, lysozyme, lactoferrin, gluconic acid, starch
syrup etc.)
12 Prebiotics on the base of microbial-origin polysaccharides
Metabiotics: New Stage of the Probiotic
Concept Development
The knowledge of molecular language of symbiotic (probiotic) microorganisms

allows for a more intensive and targeted development of the next generation of pro-
biotics and functional foods. In our opinion, the development of a traditional probi-
otic concept includes the creation of therapeutics, dietary supplements and functional
foods that can be denoted by the common term of “metabiotics” (Shenderov 2007;
Shenderov 2009; Shenderov 2011a; Shenderov 2011b). We suggest to understand
by the term all microecological products designed to preserve and restore the con-
tent and functions of symbiotic microbiota in humans, animals and plants and based
on water soluble components of cells, metabolites and signaling molecules secreted
or released at the time of microbial cell destruction in known or potential probiotic
strains of microorganisms, as well as synthetic (and/or semisynthetic) metabiotics
which will be artificially constructed as analogues or improved copies of natural
biologically active compounds formed by symbiotic microorganisms. Metabiotics
have a known chemical structure and their application allows to optimize organism-
specific epigenetic, physiological functions, regulatory, metabolic and/or behavioral
reactions related to the activity of host indigenous microbiota. This new group of
microecological products should be equal to or surpass the first-generation probiot-
ics in their therapeutic and prophylactic efficiency, while being safer than traditional
probiotics.
It is worth noting that the idea of using symbiotic (probiotic) microorganisms as
starter cultures for the production of low molecular weight bioactive compounds
was discussed in scientific literature by many researchers. For such microbial prod-
ucts, apart from the term “metabiotics” suggested by us over 20 years ago (Shenderov
et al. 1997; Shenderov 2007), such terms were used as “killed by heating probiot-
ics” (Indriyani et al. 2012), “metabolite probiotics” (Vakhitov and Sitkin 2014;
Vakhitov et al. 2005), “postbiotics” (Neish 2009; Tsilingiri and Rescigno 2012),
biological drugs (Sonnenburg and Fischbach 2011), “pharmabiotics” (Caselli et al.
2011; Sobol 2017), “paraprobiotics” (Almada et al. 2016). Over the past few years,
functional products and pharmaceuticals for metabolite therapy of diseases related
to human microbiota imbalance have been most frequently denoted in clinical and

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50 Metabiotics: New Stage of the Probiotic Concept Development
scientific literature by the term “metabiotics” (Ardatskaya 2015; Ploskireva 2016;

Shenderov 2017; Shaikh and Sreeja 2017; Sharma and Shukla 2016; Shenderov
2011a; Shenderov 2011b; Singh et al. 2018), “postbiotics” (Aguilar-Toaláa et al.
2018; Maguire and Maguire 2019; Sobol 2017) and “pharmabiotics” (Sobol 2017).
Known and new potential strains of symbiotic (probiotic) microorganisms may
become a source of hundreds or even thousands of low molecular weight bioactive
compounds (bacteriocins and other antimicrobial molecules, short-chain and other
fatty acids, biosurfactants, polysaccharides, peptidoglycans, teichoic acids, lipo-
and glycoproteins, vitamins, antioxidants, nucleic acids; proteins including enzymes
and lectins; peptides with different functional activity, amino acids, growth and
coagulation factors, inducers of defensin-like molecules, various signaling and
transport molecules, plasmalogens, phenol-containing compounds, co-factors etc.)
(Shenderov et al. 2010; Aguilar-Toaláa et al. 2018; Almada et al. 2016; Caselli et al.
2011; Engevik and Versalovic 2017; Holmes et al. 2011; Lebeer et al. 2008, 2010;
Shenderov 2013a; Singh et al. 2018; Sonnenburg and Fischbach 2011). Various
strains of symbiotic (probiotic) microorganisms can form sets of low molecular
weight compounds, and are thus candidates for the construction of general-purpose
or specific metabiotics. Real effectiveness and safety of these microbial molecules
and metabiotics developed on their basis will largely depend on the physical and
chemical characteristics of microbial bioactive components, their interrelationship
and relations with other components at the sphere of application, which can both
intensify and inhibit their activity, competition in the struggle for specific transport
proteins or absorption site. The physiological state of the consumer or his/her dis-
ease, diet, prescribed medicine can considerably modify the effectiveness and safety
of microbial biologically active substances (metabiotics). Some groups of microbial
low molecular weight compounds formed by probiotic microorganisms (SCFA,
other organic acids, various proteins, peptides, amino acids, nucleic acids, nucleo-
tides, microbial polysaccharides, peptidoglycans, vitamins, antioxidants; various
co-factors — coenzyme А, coenzyme Q; various transport and signaling molecules
including gas and other neurotransmitters) are currently being used in industrial
production of metabiotics (Shenderov 2011a; Shenderov 2013a; Singh et al. 2018).
For some metabolites produced by symbiotic (probiotic) bacteria, targets and
effects have been set in prokaryotic and eukaryotic cells. Low molecular weight
microbial compounds formed from endogenous and exogenous substrates can either
pass freely through the wall and membranes of various eukaryotic and microbial
cells, or else they need specific receptors and transporters for penetration. They can
act as substrates, co-substrates, metabolites in different biochemical and signaling
reactions; some of them possess either metabolic or only signaling activity.
Depending on their application site, these bioactive molecules are divided into those
with molecular-level effects (gene replication and expression, transcription and
translation of genetic information; post-translational effects), cell-level effects (on
the surface, in cell membranes; biosynthesis of protein and energy in mitochondria
and ribosomes), inside cell hyaloplasm (in location sites of the nucleus, organelles
and cell inclusions), in extracellular matrix (at location sites of capillaries, synapses
of nerve endings, performance of metabolic reactions, extracellular information
Metabiotics: New Stage of the Probiotic Concept Development 51
exchange etc.), in tissues, organs, physiological systems and the organism on the
whole (Aguilar-Toaláa et al. 2018; Engevik and Versalovic 2017; Shenderov 2011a;
Singh et al. 2018). The targets for microbial bioactive compounds in mammal
organisms may be represented by the metagenome and meta-epigenome of eukary-
otic and microbial cells, epigenetic control of particular gene expression and post-
translational modification of gene products, intra- and intercellular information
exchange among microbial populations between the host and its symbiotic micro-
biota, including quorum-sensing regulation, metabolic and behavioral reactions,
growth and development of the superorganism and its health on the whole.
Methods and Techniques Used
for Obtaining and Identifying of Microbial
Low Molecular Weight Cellular
Compounds, Metabolites and Signaling
Molecules
Different techniques and analytical technologies are used for identification of com-
pounds potentially suitable for constructing various metabiotics. Their choice
depends on analytical purposes, qualitative and/or quantitative characteristics of
microbial complexes and molecules under study. Table 1 shows some techniques
used for microbial cell destruction, removal of live microorganisms and extraction
of low molecular weight biologically active compounds’.
Modern techniques and methods of metabolomics allow to identify low molecu-
lar weight structural cellular compounds, metabolites and signaling molecules in
source raw materials (cellular microbial suspensions and low molecular weight
compounds in cultural fluid purified from viable microbial cells), in ready-made
products (metabiotics of various form and nature), in biological fluids (saliva, blood,
urine, cerebrospinal fluid), as well as in human tissues and intestinal content. Human
gut microbiota possesses metabolic activity comparable with that of the liver (Beger
et al. 2016; Yadav et al. 2018). In the last 15–20 years, numerous analytical tech-
niques and technologies had been developed for metabolome profile study and eval-
uation in different samples, which found their way into practice (Table 2). In
countries with advanced industrial economies (the USA, UK etc.) phenotype cen-
ters have been established and are running, capable of annually analyzing many
hundreds of thousands of various biological material samples and simultaneously
identifying dozens, hundreds and thousands of low molecular weight molecules.
Various kits have been developed and find an increasingly greater application for
the analysis of antibiotics, specific nutrients, enzymes, co-factors and other mole-
cules involved in any given metabolite pathway of prokaryotic and eukaryotic cells.
The Biocrates Life Sciences company sells test systems for measurement and iden-
tification of a limited range of metabolites, amino acids, biogenic amines, 150 lipids
and 16 bile acids (Beger et al. 2016; Biocrates Life Sciences. http://www.biocrates.
com/products/research-products). The combination of a great number of technolo-
gies and platforms allows to evaluate a wide range of metabolite profiles in different
biological materials.

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54 Methods and Techniques Used for Obtaining and Identifying of Microbial Low…
Table 1 The techniques of removal of live starter microorganisms, destruction of microbial

suspensions, isolation of complex low molecular weight biologically active compounds, microbial
metabolites and signaling molecules (Aguilar-Toaláa et al. 2018; Almada et al. 2016; Birmpa et al.
2013; Diels and Michiels 2016; Efaq et al. 2015)
Item
No. Techniques
1 Mechanical destruction
2 Frequent freezing and thawing
3 Use of enzymes with specific activity against microbial cell walls, membranes,
intracellular structures
4 Microwave radiation
5 Supercritical water extraction
6 Ionizing radiation
7 Ultraviolet radiation
8 High hydrostatic pressure
9 Ultrasound damaging
10 Cell damaging by chemical agents (for example, exposure to acids)
11 Centrifugation at different rotor revolutions
12 Different kinds of dialysis, the use of ion exchange resins
13 Different techniques of ultrafiltration of whole and destroyed cell suspensions
14 Inactivation of microorganism suspensions and extraction of cell components and
metabolite compounds using liquid carbon dioxide
Thermal effects on microbial cell suspensions (60°С, 30 min; 80°С, 20 min; 95°С, 5 min; 100°С,
5 min)
Table 2 Some metabolome technologies used for content evaluation and identification of low
molecular weight compounds that define the metabolome of the corresponding biological sample
(Aguilar-Toaláa et al. 2018; Aurich and Thiele 2016; Beger et al. 2016; Braune and Blaut 2016;
Nicholson et al. 2012; Yadav et al. 2018)
Item
No. Technologies
1 Gas chromatography
2 Liquid chromatography
3 Mass-spectrometry
4 Nuclear magnetic resonance spectroscopy
5 High-efficiency liquid chromatography with subsequently obtained mass spectrum in
negative and positive modes of operation
6 High-efficiency liquid chromatography with tandem high-resolution mass-selective
detection with electrospray ionization
7 High-efficiency high-resolution liquid chromatography with tandem high-resolution
mass-selective detection
8 Pulsed Field Gel Electrophoresis — PFGE
9 Capillary electrophoresis with mass-spectrometry-based detection
10 Two-dimensional gel electrophoresis
Methods and Techniques Used for Obtaining and Identifying of Microbial Low… 55
Low molecular weight bioactive compounds formed by commensal and symbi-

otic microorganisms of gut microbiota can easily penetrate the walls and mem-
branes of eukaryotic and prokaryotic cells in the human organism; alternatively,
they need preliminary interaction with specific receptors on the cell surface.
According to effects on the corresponding targets, these bioactive microbial mole-
cules may be divided into metabolic molecules, purely signaling molecules or mol-
ecules having at the same time specific structural, metabolic, signaling or another
biological and pharmacological activity. As signaling molecules, they can activate,
silence or modify the corresponding genes through the specific interaction with
DNA receptors of human cells different in functionality by using kinases and/or
other enzymes and other cell systems (Shenderov 2011a).
Low molecular weight bioactive compounds of symbiotic (probiotic) and com-
mensal microorganisms display their effects on different levels of the organism:
1. Molecular level (gene replication and expression, transcription, translation of
genetic information and its epigenomic modification).
2. Cellular level (surfaces, membranes, post-translational effects, energy and pro-
tein biosynthesis in mitochondria, ribosomes, exosomes).
3. Intracellular compartment — gyaloplasme (the location of nucleus, organelles,
exosomes, various inclusions, ion channels).
4. Intercellular matrix (the location of capillary, neural synapses, metabolic reac-
tions, synthesis and accumulation of hormonally active cell peptides, intercellu-
lar information exchange etc.).
5. Tissues, organs, physiological and metabolic systems.
6. Host organism as a whole.
The analysis of scientific literature data (Beloborodova 2012; Vakhitov et al.
2005; Sitkin et al. 2015; Aguilar-Toaláa et al. 2018; Dorrestein et al. 2014; Engevik
and Versalovic 2017; Lebeer et al. 2018; Maguire and Maguire 2019; Singh et al.
2018), as well as our own experimental studies and clinical observations (Volkov
et al. 2007; Vorobeichikov et al. 2008; Oleskin and Shenderov 2016; Shenderov
2013a; Shenderov 2015a; Shenderov and Lakhtin 2004; Shenderov et al. 2010;
Shenderov et al. 2018a; Francavilla et al. 2008) have shown that, as a rule, in vitro
cultural fluids of probiotic bacteria, as well as biological fluids (blood, urine, saliva
etc.) of people receiving various probiotics orally, had very low levels of the quan-
titative content of target active microbial compounds. Unfortunately, traditional
models and technologies used to determine the targets for application of such
microbial bioactive compounds in nano- and micromolar concentrations and to
objectively assess their beneficial health effects or negative side effects yield little
information. New model systems sensitive to low concentrations of metabiotics
should be found and tested.
Classification of Metabiotics and their
Brief Description
Commercially available metabiotics can be divided according to their composition

into dietary supplements, functional and personal foods, therapeutics, cosmetic
products (liquid, dry, pastes, creams etc.); pharmaceuticals on the base of low
molecular weight components related to structural components of microbial cells
(Cellular metabiotics), low molecular weight metabolites and signaling microbial
molecules (Metabolite metabiotics), metabiotics at the same time containing both
microbial cell components and their metabolites and signaling molecules; hybrid
multicomponent microbial components and metabolic and signaling molecules;
metabolite formulations made with low molecular weight microbial components
isolated from several microorganism strains or species; as well as hybrid multicom-
ponent low molecular weight microbial and plant components and molecules. Also,
semi-synthetic and synthetic metabiotics are currently being developed and launched
on the food market. Metabiotics may be used both independently and as nutritional
supplements to various known microecological therapeutics (Table 1) (Shenderov
2017; Shenderov et al. 2018a; Shenderov 2011a; Shenderov 2013a).
Depending on the nutritional base used for growing starter strains of symbiotic
microorganisms, the obtained metabiotics may be differentiated into those of milk,
plant and combined origin. Metabiotics may also be differentiated by method of extrac-
tion, inactivation of starter culture cells, delivery to the host organism, evaluation of
stability and activity, the use of different technologies for their biological activity evalu-
ation (in in vitro tests, on different kinds of experimental animals, in clinical trials).

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58 Classification of Metabiotics and their Brief Description
Table 1 Potential forms of № Forms of metabiotics

metabiotics
1 Metaprobiotics
2 Metaprebiotics
3 Metasynbiotics
4 Metanutritive supplements
5 Metabiotics
Some of the Best-known Metabiotics
on the Market of Microecological Products
In the last decade, some metabiotics have been launched on the markets of a number
of countries including the Russian Federation, whose production includes natural
(or artificial) bioactive molecules similar or identical to those formed by the repre-
sentatives of normal human microbiota. They have proven their therapeutic and
prophylactic efficiency in some infectious and metabolic diseases (Table 1).
In addition, other metabiotics are available on the international market. Among
them, of special note are pharmaceuticals which are essentially E. coli glycoprotein
with anorectic activity (Tsuda et al. 1992); microbial complex of polysaccharide/
glycopeptide of a Lactobacillus сasei strain with hypotensive effect (Beider and
Flambard 2003; Sawada et al. 1990); tripeptides (Val-Pro-Pro and Ile-Pro-Pro) of
Lactobacillus helveticus strains which inhibit angiotensin-converting enzyme and
mildly lowering blood pressure (Nakamura 2004; Nakamura et al. 2005). In the
years to come, the metabiotics market will see the appearance of therapeutics and
functional foods that contain proteins, peptides, adhesins (Lebeer et al. 2008; Lebeer
et al. 2010), biosurfactants (Lakhtin et al. 2010), lectins (Shenderov and Lakhtin
2004; Lakhtin et al. 2007), nucleic acids and other cell wall components (Caselli
et al. 2011) of lactobacilli, bifidobacteria, enterococci, as well as other symbiotic
(probiotic) bacteria.
The manufacturers of metabiotics launched on the market as pharmaceuticals,
dietary supplements or as functional food components are registered in accordance
with national laws controlling the quality and safety of pharmaceuticals and food
products. One of the key aspects in serial production of metabiotics on the base of
symbiotic (probiotic) microorganisms of human origin for different applications,
apart from isolation, taxonomic identification, research into positive target features
and safety, is supplying biotechnological factories with standard high-quality starter
cultures, or the so-called starter cultures for direct inoculation (SCDI), stable in
their biological and technical properties. Traditional starters need preliminary incu-
bation followed by a series of starter culture re-inoculations at the factory’s prem-
ises for obtaining the required quantity of microbial cells prior to inoculating them

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60 Some of the Best-known Metabiotics on the Market of Microecological Products
Table 1 Some of the best-known metabiotics (Belousova et al. 2005; Shenderov et al. 2018a;
Aguilar-Toaláa et al. 2018; Shenderov 2013a; Singh et al. 2018)
Name Contents and properties
Hylak forte Liquid pharmaceutical containing no microbial cells and intended for oral
(Germany) administration. It includes metabiotic products produced by the following
strains: Escherichia coli DSM 4087 (25 g / 100 ml), Streptococcus faecalis
DSM 4086 (12.5 g / 100 ml), Lactobacillus acidophilus DSM 4149 (12.5 g /
100 ml) and Lactobacillus helveticus DSM 4183 (50 g / 100 ml). The positive
health effects of this metabiotic in children and adults is related to the
restoration of gut microbiota content and functions, water-salt balance,
acid-alkaline balance, normalization of vitamin B and K levels in
microorganism, supplying enterocytes and immune intestinal cells with
energy. The positive effects of this sterile liquid pharmaceutical are explained
by the presence of volatile fatty acids, lactic acid and some unidentified low
molecular microbial substances (Belousova et al. 2005; Kocharovec 2018;
Rudkowski and Bromirska 1991)
Bactistatin Lyophilized cultural fluid of Bacillus subtilis, containing no live bacteria and
(Russia) including a complex of various low molecular weight compounds and
metabolites and natural sorbent zeolite (http://www.kkraft.ru)
Colibiogen Filtrate of Escherichia coli (strain Laves 1931) cultural fluid, containing no
(Switzerland) proteins and including peptides, amino acids, polysaccharides and fatty acids
(Zihler et al. 2009)
Pro-Symbioflor Microbe-free lysate of bacteria and cultural fluid of Enterococcus faecalis,
(Germany) E.coli (Klein et al. 2013)
Helinorm Inactivated cells of probiotic bacteria L.reuteri that link specifically to
(Germany/ H. pylori and remove these bacteria naturally from the organism;
Russia) recommended for gastritis, gastric ulcer and cancer prevention (Hunt et al.
2011) (http://www.kkraft.ru/en/#bs4)
CytoFlora Microbe-free cell wall lysates of Bifidobacterium bifidum, B. infantis,
(USA) B. longum, Streptococcus thermophiles, Lactobacillus reuteri, L. salivarius,
L. casei, L. bulgaris (Bioray. CytoFlora®. http://www.bioray.com/cytoflora/)
Del-Immune V Peptidoglycans and DNA fragments of Lactobacillus rhamnosus (Del-
(USA) Immune V. http://www.delimmune.com/research/)
Daigo (Japan) A mixture of bioregulator peptides extracted from cultural fluids of 16 strains
of various lactobacilli L.curvatus, L.casei, L.acidophilus, L.plantarum,
L.fermentum, L.salivarius, L.brevis, L.rhamnosus after cultivating them on
soymilk for a year (Popova et al. 2016)
Complex Cell-free filtrates of probiotic Lactobacillus acidophilus NK-1 and
functional foods Bifidobacterium longum В 379 M grown on hydrolyzed milk medium and
fortifier (Russia) purified from live bacteria by microfiltration; contains В vitamins, amino
acids, macro- and microelements and organic acids. It is a complex fortifier,
and the products resulting from its application are characterized by metabolic
activity and positive effect on the human organism. Used as main or auxiliary
component in production of various kinds of drinks, in the native or powder
form or as a concentrate (Shenderov et al. 2010)
”New Class of Obtained by long fermentation of raw materials (vegetables, herbs, fruit,
Pharmabiotic” berries, animal products) by lactobacilli (three strains) and Streptococcus
(Russia) thermophilus with marked proteolytic activity. After removal of unfermented
sediment by centrifugation, peptides, amino acids, vitamins, microelements,
proteases and volatile organic acids get accumulated in the liquid part. The
resulting РР has multiple beneficial effects on human health (Sobol 2017;
Sobol and Sobol 2012)
Some of the Best-known Metabiotics on the Market of Microecological Products 61
into industrial bulk containers. If quality standards are not properly applied, as a
result of multiple re-inoculations, numerous microbial clones can appear in the
original culture, which may be essentially different from the originally claimed
starter strain as regards their technological and designated properties. One solution
might be to introduce SCDIs i.e. bulk starters produced at specialized laboratories
in the form of dry or frozen biomass of the producing strain; after delivery to the
corresponding biotechnological factories, SCDIs are immediately inoculated into
industrial bulk containers.
It will be promising to construct combined therapeutics (metabiotics + other
known microecological substances), which will be launched in the market either as
dietary supplements in their own right, or as new groups of functional foods forti-
fied with these components (Shenderov 2013a; Sonnenburg and Backhed 2016).
Cellular Metabiotics and Metabolite
Metabiotics
To provide a clearer picture of what modern techniques and technologies are used
when developing metabiotics and launching them on the market of therapeutics and
functional foods, let us describe how metabiotics were developed on the base of cell
walls of the Lactobacillus reuteri DSM 17648 (Pylopass/Helinorm) and metabolites
and signaling molecules on the base of the Bacillus subtilis No.3 probiotic strain
(Bactistatin).
Cellular Metabiotics
Cellular metabiotics contains in its composition non-living probiotic cells. We will

describe cellular metabiotics using the example of Pylopass/Helinorm.
Pylopass/Helinorm is a metabiotic product designed to control the Helicobacter
pylori load in the stomach. Helicobacter pylori are gram-negative bacteria ever present
in the stomach of humans and some mammals (for example, the dog, the cat). More
than 50% of the population globally is infected with the bacterium Helicobacter pylori.
H. pylori is a major component in the causation of gastritis and H. pylori infec-
tion can also lead to gastric cancers and ulcers (Suerbaum and Michetti 2002).
Today’s strategy to manage H. pylori is based on antibiotics treatment with a regime
called “triple therapy”, which comprises two antibiotics and a proton pump inhibi-
tor (Malfertheiner et al. 2017). This often leads to side effects and negative effects
on our microbiota. The regime’s efficiency is also becoming compromised by
increasing rates of antibiotic resistance.
The probiotic strain Lactobacillus reuteri DSM17648 was developed as a meta-
biotic (a non-viable microbial preparation with health-beneficial properties) formu-
lation (Pylopass) to manage stomach and gut health.
The chapter is written by Christine Lang (Technical University Berlin, Germany) and Kimmo
Makinen (Novozymes S/A, Denmark).

https://doi.org/10.1007/978-3-030-34167-1_14
64 Cellular Metabiotics and Metabolite Metabiotics
Pylopass is a lactic acid bacterium of the species Lactobacillus reuteri which was
specifically selected and developed to recognize Helicobacter pylori in the stomach
and to co-aggregate with this bacterium. After binding, Helicobacter’s motility is
impaired and the co-aggregated Lactobacillus–Helicobacter complex is naturally
removed by being flushed out of the stomach. This leads to a significant reduction
of H. pylori load and offers a novel regimen to control H. pylori load in the stomach
after triple eradication therapy and as a prophylactic supplement.
Helicobacter and Stomach Health
Contrary to the long-held belief that germs do not survive in the stomach,
Helicobacter pylori (H. pylori) is found in the stomachs of more than 50% of the
population worldwide. H. pylori is a Gram-negative microaerophilic, spiral bacte-
rium that colonizes the gastric mucosa. It is able to produce the enzyme urease and
thereby degrade urea to ammonia, which buffers against the acidic environment of
the stomach. This mechanism, together with the bacterium’s motility and ability to
adhere to the gastric mucosa, provides for its survival in the acidic environment of
the stomach. H. pylori also colonizes the mucosal lining of the stomach.
H. pylori is generally acquired during childhood and can persist for the lifetime
of the host. The presence and the role of H. pylori has been implicated in a number
of gastrointestinal disorders and imbalances. Infection by H. pylori may lead to an
inflammatory response, increased secretion of gastric acid, and type-B gastritis
(Zarrilli et al. 1999; Viganò et al. 2018). Given the normal barrier function of the
stomach’s mucosal lining, infection by H. pylori often leads to a minor concomitant
inflammation. In case of chronic manifestation, malignant tumors may result, as has
been demonstrated for MALT-lymphoma. The classical anti-H. pylori protocol con-
sists of a proton pump inhibitor (PPI) and two antimicrobial agents. The Maastricht/
Florence Consensus report, which outlines the diagnostic guidelines and treatment
strategies for those with H. pylori (Malfertheiner et al. 2017) advises individuals
with certain risk factors to undergo eradication therapy. In particular, it is recom-
mended that those with functional dyspepsia, undergo the “test and treat” strategy.
However, there remains a lack of options for persons who are either asymptomatic
or experience only mild gastrointestinal symptoms. Alternative anti-H. pylori proto-
cols are being searched for.
Probiotics in Helicobacter Therapy
The use of probiotics, as monotherapy or synergistic therapy (in combination with

antibiotics), is being researched as an alternative way of controlling H. pylori infec-
tion and reducing side effects of antibiotic treatment (Lesbros-Pantoflickova et al.
2007; Wang et al. 2017b).
Cellular Metabiotics 65
Mechanisms by which probiotics work in this application include suppressive

effects against gastrointestinal inflammation and against H. pylori (Khoder
et al. 2016).
Several potential mechanisms by which probiotics may influence H. pylori have
been discussed. Non-immunological barriers represent the first line of defense
against pathogenic bacteria. Probiotics may strengthen this barrier by producing
antimicrobial substances, stimulating mucin production, and stabilizing the gut
mucosal barrier. Antimicrobial activity of probiotics could be due not only to a
direct effect on H. pylori but also to the inhibition of its urease activity. In addition,
probiotics might enhance the production of prostaglandins, growth factors and anti-
inflammatory cytokines (Cain and Karpa 2011). Another possible mechanism is
displacement of H. pylori through competitive binding to adhesion receptors of
H. pylori (Mukai et al. 2002).
Previous studies on the effect of probiotics on H. pylori have been done with a
broad range of probiotic strains on diverse endpoints. A study applying L. casei
reported a non-significant suppressive effect seen as decrease of urease activity
(Cats et al. 2003). Using live cells of a L. brevis strain, a reduction of UBT values
has been reported, and a correlation to decreased polyamine synthesis was proposed
(Linsalata et al. 2004). In case of L. johnsonii La1, consumption of whey-based
supernatant of L. johnsonii La1 culture was found to induce a marked decrease in
Helicobacter in infected subjects as measured by breath test analysis, either in com-
bination with Omeprazol or alone (Michetti et al. 1999). Subsequent studies showed
that La1 given in a fermented milk beverage reduced H. pylori load in adults and in
children as measured by the UBT (Cruchet et al. 2003). The species L. reuteri has
been named in several studies to be active in Helicobacter management and in
reducing side effects of standard treatments (Lionetti et al. 2006; Francavilla et al.
2008; Scaccianoce et al. 2008).
Emara et al. (2014) used a L. reuteri preparation (a mixture of strains L. reuteri
DSM17938 and L. reuteri ATCC PTA6475 in combination with a triple therapy. The
Lactobacillus supplementation increased the Gastrointestinal Symptom Rating
Scale (GSRS) score significantly, but did not improve H. pylori eradication rate.
However, successful in vivo studies demonstrating the effects of probiotics on
H. pylori gastritis are limited. This could be due to the adverse physiological condi-
tions of the stomach, such as an acidic environment, gastric enzymes, bile acids and
mechanical stress that reduce the survival and metabolic activity of probiotics.
The Helinorm/Pylopass Strategy
Reducing the amount of H. pylori in the stomach by selective bacterial-bacterial cell

interaction has been proposed as an effective and novel strategy for combating this
stomach bacterium.
While H. pylori resides in the mucus where it is present in its motile form, mucus
is constantly produced by the epithelium and shed into the stomach lumen. This
continuously releases planktonic H. pylori cells into the stomach. The probiotic
strain used in the Helinorm formulation, L. reuteri DSM 17648 (Pylopass), specifi-
cally captures such H. pylori cells. Pylopass has been carefully selected to enable a
rapid and sensitive and highly selective trapping of Helicobacter pylori cells in the
stomach lumen. Once bound, co-aggregates are flushed from the stomach by natural
bowel movement. L. reuteri DSM17648 has been developed to specifically co-
aggregate with Helicobacter pylori under gastric conditions.
Reducing the amount of H. pylori in the stomach by selective bacterial-bacterial
surface interaction represents an alternative method for managing the stomach
pathogen.
The Pylopass Strain
The strain L. reuteri DSM17648 was selected by laboratory screening assays among
Lactobacilli strains of a large culture collection (ORGANOBALANCE GmbH,
Berlin, Germany).
Among 700 Lactobacillus strains, only 8 were found to co-aggregate with spiral
forms of H. pylori DSM21031. One of these strains — L. reuteri DSM17648 — was
analysed in depth and used for product development.
Fluorescence images of co-aggregates of cells stained with HI or CFDA (Fig. 1),
then allowed to co-aggregate (Fig. 1B), demonstrated that both DSM 17648 and
H. pylori participated in the aggregation but did not auto-aggregate.
Quantification of co-aggregate formation between L. reuteri DSM17648 and
H. pylori DSM21031 by flow cytometry revealed that one Lactobacillus cell binds
2–3 Helicobacter cells.
Co-aggregates can be seen visually as flocking structures, whereas no such struc-
tures are present in controls (Fig. 2). Co-aggregation between H. pylori and L. reuteri
DSM17648 occurs within seconds.
Fig. 1 Co-aggregation between Helicobacter pylori DSM 21031 and Lactobacillus reuteri DSM
17648 incubated in artificial stomach juice (pH 4). Phase contrast microscopy with fluorescence
filter. Magnification 1000×. H. pylori was stained with hexidium iodide (A), L. reuteri was stained
with CFDA (C). Co-aggregate shows clumping of both strains (B). To confirm that both species
were present in the aggregates, cells were stained separately using either hexidium iodide or car-
boxyfluorescein diacetate succinimidyl ester. Both DSM17648 and H. pylori DSM21031 partici-
pate in the aggregation
Fig. 2 Co-aggregation is
macroscopically visible.
Co-aggregation of
Helicobacter pylori DSM
21031 T and L. reuteri
DSM 17648 incubated in
artificial stomach juice
pH 4/ PBS (B). No
co-aggregation was
observed when single
strains were analysed (A:
H. pylori only, C: L. reuteri
DSM 17648 only)
Pylopass specifically co-aggregates with H. pylori under in vitro conditions and

in artificial gastric juice, but not with other bacteria of the commensal intesti-
nal flora.
Numerous other L. reuteri strains were tested in the screening process for co-
aggregation with H. pylori. None of them formed co-aggregates with H. pylori,
underlining that the effect exhibited by Pylopass is unique and specific.
This specific binding masks the surface structures of H. pylori and interferes
with Helicobacter motility. The aggregated pathogens no longer adhere to the gas-
tric mucosa and are flushed out of the stomach.
Characteristics of Pylopass
Expression of co-aggregation activity is dependent on the growth phase of L. reuteri

DSM17648, and it is manifest at entry into stationary growth and during stationary
phase (Fig. 3). It is dependent on a L. reuteri DSM17648 cell surface factor that is
present at the end of the exponential growth phase and during stationary phase.
Co-aggregation takes place in the presence of sugar (sucrose, lactose, glucose,
fructose, maltose, isomaltose, sorbitol). It occurs at comparable efficiency at room
temperature and at 37 °C, and co-aggregation activity is observed over a wide pH
range (pH 2.0 – corresponding to empty stomach conditions - up to pH 8, including
typical pH values after meals). No pure cultures evidenced auto-aggregation within
this pH range. Slightly smaller co-aggregates are formed at pH 2 compared to pH 8
in vitro. Thus, aggregation of H. pylori by L. reuteri DSM17648 occurs at pH values
and conditions encountered in the human stomach.
Fig. 3 Relationship between growth phase and co-aggregation activity of Lactobacillus reuteri
DSM 17648
To understand if proteins are involved in binding, the susceptibility to protease

inactivation of the co-aggregation determinants on the surfaces of both DSM17648
and of H. pylori was tested after treatment with protease Strep. griseus Type XIV,
proteinase K, trypsin, or pepsin. Incubating L. reuteri DSM17648 with any protease
before co-aggregation reduced binding to H. pylori DSM21031 by 30%, but did not
eliminate it completely. H. pylori required pretreatment with the protease pepsin (as
is naturally present in gastric fluids) to be fully active in co-aggregation with
L. reuteri DSM17648.
Cell Walls Exhibit the Co-Aggregation Sites
SEM images of co-aggregates were prepared to analyse cellular sites of the attach-
ment (Fig. 4).
Clearly, single cells of L. reuteri DSM 17648 bind several H. pylori cells result-
ing in a cross-linking of the co-aggregates; and the binding sites on DSM 17648
appear evenly distributed over its surface, binding sites on H. pylori do not seem to
be exposed on the flagella.
The Power of Metabiotic Formulation
Metabiotics are ‘non-viable’ bacterial products or metabolic byproducts from pro-

biotic microorganisms that have biological activity in the host. Interestingly, no
viable cells of Pylopass are required for the specific action towards H. pylori. The
co-aggregation activity is preserved during lyophilisation or spray-drying of whole
cells of L. reuteri DSM17648 and persists in non-viable cells (Table 1). Spray-dried
or lyophilised L. reuteri DSM17648 cells induce co-aggregate formation with the
same sensitivity as untreated cells.
Fig. 4 (а) Scanning electron microscopy of co-aggregates consisting of L. reuteri DSM17648

(blue) and Helicobacter pylori (red), 1.800× magnification (b) Scanning electron microscopy of
co-aggregates consisting of L. reuteri DSM17648 (blue) and Helicobacter pylori (red), 11.000×
magnification
Table 1 Co-aggregation activity is present in non-viable cells of L. reuteri DSM 17648

Values culture lyophilised cells non-viable cells∗
Colony forming units [ml−1 or mg−1] 2,0 × 109 3,0 × 107 0
Co-aggregation score +++ +++ +++
Cells were spray-dried and subsequently treated at 40 °C for 24 h
*
It can be assumed that binding is due to specific surface molecules on the

L. reuteri DSM17648 cells which are strain-specific and are resistant to such pro-
cess steps. Such surface molecules might include lipoteichoic acid and carbohy-
drate structures.
L. reuteri DSM17648 is active as non-viable cell preparation, which greatly
reduces any potential side effects of living cultures and helps ensure stable activity
in consumer products such as supplements and in medicinal formulations.
Clinical Evaluation
It is hypothesized that Lactobacillus reuteri DSM17648 interferes with mobility of

H. pylori and its adherence to the gastric mucosa by entangling H. pylori cells into
aggregates and masking H. pylori surface sites which are ordinarily available for
binding to human gastric epithelium. Once bound, co-aggregates will be flushed
from the stomach by natural bowel movement. This novel anti-H. pylori activity has
not been described previously as a mode-of-action for probiotic treatment of
Helicobacter infections.
The effectiveness of the metabiotic formulation Pylopass was shown in vivo.
Lactobacillus reuteri DSM17648 (Pylopass) was used in several clinical studies
to demonstrate that it effectively reduces the amount of H. pylori in the stomach

in the absence of antibiotic treatment (Holz et al. 2015; Mehling and Busjahn 2013).
In a study with a placebo controlled design, the effect of using Pylopass for
2 weeks was analysed in a group of persons that were carriers of H. pylori.
Participants of the study were people aged 18 years or older that had been diagnosed
as H. pylori positive using the standard 13C urea breath test (UBT, Helicobacter
Test INFAI®, Dd ≥ 4‰). The test is based on measuring urease activity of H. pylori
cells. The test’s specificity (98,5%) and sensitivity (97,9%) are well comparable to
traditional invasive detection methods, such as endoscopy or biopsies. The breath
test measures directly the status of H. pylori colonisation and it is well suited to
detect reduction or eradication of the bacterium. As a result, the amount and the
activity of H. pylori-urease is quantified. This reflects the degree of colonisation of
the stomach with H. pylori. The active ingredient in the study was freeze-fried
L. reuteri DSM17648 taken orally as a tablet, with 5 × 109 cells each, and a daily
dose of 4 tablets. A significant reduction of H. pylori load as a result of Pylopass
application was detected by following intra-individual changes over time, i.e. in one
person during supplementation period. Placebo supplementation did not result in
significant changes in UBT values. At the end of the supplementation period, most
persons having ingested Pylopass were found to have a significantly reduced
H. pylori load (Fig. 5).
This protective effect lasted for several weeks after the supplementation period.
Clinical studies have also been performed in Ireland (Buckley et al. 2018), Russia (in
press), and Pakistan (unpublished) and all of them showed the same effect: H. pylori
Fig. 5 Absolute 13C UBT values of individuals before and after treatment with verum and
placebo
Metabolite Metabiotics 71
load is significantly reduced and symptoms accompanying Helicobacter pylori

infection are reduced. Adverse side effects were not observed in any participant.
Outlook
Processing probiotic strains to metabiotic formulations is a potent strategy to pro-

vide high quality active ingredients in a stable product. Knowing the mode of action
of a product answers the scientific and consumer needs and paves the way for devel-
oping highly effective and safe novel bioactives.
Metabolite Metabiotics
Metabolite metabiotics contains in its composition metabolites produced by probi-

otic strains. We will describe metabolite metabiotics using the example of Bactistatin.
Among the wide range of probiotics used worldwide for treatment and preven-
tion of diseases associated with microecological digestive tract disorders, a promi-
nent role is played by probiotics on the base of live spore-forming gram-positive
bacteria belonging to Bacillus subtilis. The representatives of this species are among
the most ancient microorganisms on our planet; that is why humans and other com-
plex eukaryotic organisms are very well adapted to these commensal microorgan-
isms. Isolated as a pure culture over 100 years ago, these bacilli are extensively
represented in samples of soil, water, plant (especially on the base of soybeans) and
dairy food products. In healthy people, B.subtilis is ever-present in the gastrointes-
tinal tract in the amount of 105–7 CFU/g, which is comparable with the quantitative
content of lactic acid bacteria (for instance, lactobacilli) in the large intestine. In
case of limiting food substrates in culture medium, B. subtilis produces spores.
Multi-year research had shown that these commensal bacteria are safe for humans,
which enabled the regulatory bodies in the USA (FDA), European Union (EFSA),
Russia (GosSanEpidNadzor) and in other countries to consider B. subtilis strains to
be safe (GRAS) for use as probiotic dietary supplements. B. subtilis appeal for pro-
biotics production is explained not only by their safety characteristics, but also by
their marked biotechnological potential (methodologies of their genetic and molec-
ular taxonomic identification have long since been developed and tested; they have
high growth and productivity rates in aerobic and anaerobic conditions in different
cheap growth substrates, high rate and broad spectrum of production of enzymes,
primary and secondary metabolites, antimicrobial substances, other proteins and
low molecular substances; spore production allows for stable and long-term preser-
vation of the genetic characteristics of starter cultures). Different Bacillus species
are among the most powerful producers of bioactive secondary metabolites (up to
800 compounds) (Ilinskaya et al. 2017). Overproduction of antimicrobial peptides
and enzymes and proven biotechnologies are already in widespread commercial use.
The above-mentioned characteristics allowed for considering the strains of these

species as “the most perfect multifunctional probiotic bacteria” (Olmos and
Paniagua-Michel 2014; Sorokulova 2013; Suva et al. 2016). Broad use of probiotics
on the base of live B. subtilis strains for people, animals, birds and aquacultures
have enabled to set multiple targets and mechanisms of their beneficial effect on live
eukaryotic organisms. Bacillar probiotics form SCFA (acetic, propionic, butyric
and, less markedly, isobutyrate, isovalerate), synthesize antimicrobial substances
that suppress the growth of pathogenic and opportunistic microorganisms, reversing
the symptoms of diarrhea, secrete various amylolytic, lipolytic, cellulolytic and pro-
teolytic enzymes which are stable in acidic environment, stimulate the immunity,
inhibit intestinal inflammation, normalize the growth of gut microbiota and stimu-
late the propagation of gut symbiotic lactobacilli and bifidobacteria. The probiotic
effect of B. subtilis is attributed to a quorum-sensing peptide, that takes part in com-
munication, proliferation and sporulation of these bacteria. Also, it displays anti-
inflammatory effects by modulating the synthesis of IL-4, IL-6, IL-10 cytokines and
producing the synthesis of anti-shock peptides (Ilinskaya et al. 2017). The research
into B.subtilis immune stimulating activity have revealed the ability of these bacilli
and their spores to sustain gut barrier, to promote inborn and adaptive immunity,
activation and development of lymphoid tissue, to stimulate the maturation of
antigen-presenting cells (phagocytes with macrophage and dendritic phenotypes),
to stimulate cytokine production by macrophages and the formation of IgA, IgG and
IFN-γ (Sebastian and Keerthi 2014; Sorokulova 2013; Quintana et al. 2014).
The potential ability of B.subtilis to normalize the intestinal inflammation pro-
cesses are attributed to their capability to inhibit the growth of pathogenic bacteria
in the digestive tract and increase the levels of bifidobacteria and lactobacilli
(Lefevre et al. 2015; Suva et al. 2016). Antimicrobial agents formed by various
B.subtilis strains include compounds differing in their chemical composition (vari-
ous peptides, bacteriocins, antibiotics, lytic enzymes, organic acids; vitamins, folate
and biotin in the first place, and others); the spectrum of their effects includes a
broad range of microorganisms. Bacillar antimicrobial peptides can form mem-
brane channels and destroy the cell wall of pathogenic and opportunistic microor-
ganisms, including antibiotic-resistant ones. They have antibacterial, antimycotic,
antiprotozoal and antiviral action, as well as immunosuppressive and antitumor
effects, they can inhibit quorum-sensing and the related virulence and biofilm for-
mation. Also, bacilli produce a lot of ribosomally and nonribosomally connected
similar peptides (over 20 lipoproteins, glycopeptides, cyclic peptides, bacteriocins
and antibiotics). They are synthesized from almost 300 different precursors. Then
they can be modified by methylation, acetylation, glycosylation, formation of het-
erocyclic rings (Ilinskaya et al. 2017; Perez et al. 2017; Phister et al. 2004;
Sorokulova 2013; Sumi et al. 2015; Suva et al. 2016). Bacilli excrete amino acids
and vitamins into the culture medium; bacillar probiotics administration reduces the
levels of serum cholesterol and triglycerides in model animals.
The B. subtilis pentapeptide can activate the key ways to preserve gut epithe-
lial cells. Bacilli are involved in the metabolism of food components, xenobiotics
and therapeutics, sustaining intestinal homeostasis in healthy people. All the
above- mentioned biological characteristics of B. subtilis are strain-specific

(Ilinskaya et al. 2017; Sorokulova 2013). Moreover, bacilli produce extracellular
vesicles, spherically-shaped membrane-like structures, in the composition of
which proteins, lipids, nucleic acids and metabolites are found that show biologi-
cal activity. These vesicles take part in the interaction of these bacteria both with
other microorganisms and with the host cells (Kim et al. 2016).
Within a period from 1994 to 2006, by using the Вacillus subtillis strain No. 3
(deposited with the All-Russian Collection of Microorganisms, VKPM V-2335),
Russian researchers had developed the technology for generation of the Bactistatin
dietary supplement which is a germfree filtrate of cultural fluid of the B. subtilis
strain No. 3 grown by pour plate method in liquid culture medium on the base of soy
protein hydrolysate. Bactistatin contains a complex of low molecular weight com-
pounds (total carbon and nitrogen contents are 9.6 g/l and 2.2 g/l, respectively)
immobilized on zeolite in admixture with soy flour hydrolysate; it comes in powder-
containing capsules. The zeolites of Holinsky deposits are micropore silicate miner-
als containing ions of sodium, potassium and calcium, easily exchanging among
themselves and with the surrounding substrate. The acid hydrolysate of soy flour
with maximum protein breakdown level provided high bacilli biomass yields.
Bactistatin examination has shown that a liter of freeze-dehydrated supplement con-
tains low molecular weight compounds with antimicrobial activity (such as bacili-
sin, subtilin, iturin, globicin, mycosubtilin; bacillin, obutin, endosubtilisin), enzymes
splitting carbohydrates, fats and proteins (such as nitrate reductase, α-amilase, pyru-
vate kinase, ribonuclease, aminopeptidase, subtilopeptidase), lysozyme, catalase,
polypeptides, peptidoglycans, polysaccharides, soybean components etc. (Volkov
2008; Volkov et al. 2006; 2007; Vorobeichikov et al. 2008).
In subsequent metabolome studies of germfree cultural fluids of this strain of
probiotic Bacillus subtilis with the use of high-efficiency liquid chromatography;
high-efficiency liquid chromatography with tandem mass-selective detection; high-
efficiency liquid chromatography with tandem high-resolution mass-selective
detection with electrospray ionization; high-efficiency high-resolution liquid chro-
matography with tandem high-resolution mass-selective detection; gas chromatog-
raphy; gas chromate-mass-spectrometry — over 100 other low molecular weight
metabolites had been tested including В vitamins (В5, В2, В1, В6), С vitamins and
РР (nicotinic acid), free amino acids (alanine, arginine, asparagine, aspartic acid,
valine, hydroxyproline, histidine, glycine, glutamine, glutamic acid, isoleucine,
lysine, methionine, leucine, ornithine, proline, serine, tyrosine, threonine, trypto-
phan, phenylalanine, cystine, citrulline), short-chain (acetic, butyric, propionic),
free (16) and esterified (20) fatty acids, metabolites of aromatic amino acids – tryp-
tophan and polyphenols.
The following acids had the highest levels in cell-free concentrate of Bacillus
subtilis (strain No. 3) cultural fluid: palmitic, stearic, myristic, palmitoleic, pen-
tadecanoic, lauric, capric, margaric and lignoceric acids. Bactistatin also includes
18 chemical elements (Na, Mg, К, Ca, Mn, Fe, Co, Cu, Zn, Se, Cr, Mo etc.).
Additionally, amikacin А and B (dehydroisocoumarin derivatives) have been found
in Bactistatin, having antibacterial, antimycotic, antitumor and antiviral effects,
phengicins А, В and С (cyclic lipopeptides with antimicrobial activity), methyl-

butanoic, lactic, hydroxisocaproic, oxalic, phosphoric, phenylacetic, hydrocin-
namic, phenyl-lactic, 3-hydroxyphenil-lactic, 4- hydroxyphenil-lactic acids,
methacryloyl-glycine, phenol class compounds (phenol, catechol (1,2-dihydroxy-
benzene, pyrocatechol, hydroquinone, 3-methylcatechol), peptides (DL-alanyl-L-
leucine) and cyclo(leucylprolyl) (L-Phe-D-Pro lactam), unidentified cyclic dipeptide
(3- benziohexahydropyrrol[1,2-a]pyrazine-1,4-dione (Cyclo(D-phenylalanine-L-
prolyl) and other three unidentified cyclic dipeptides, some identified nitrogen
compounds (caprolactam, phenylethylamine, phenylalanine, thymine, uracil, dihy-
drouracil, indole, tyramine, 5-hydroxi-1Н-indole, 9H-purin-6-ol, 13-docosenamide,
erucic acid amide), alcohols and glycols (ethylene glycol, propylene glycol, methy-
onol, benzyl alcohol, heptaethylene glycol, nonaethylene glycol, decaethylene gly-
col). The above mentioned low molecular weight compounds found among
Bactistatin components can potentially be involved – independently or in combina-
tion with other nutrients and microbial bioactive molecules – in aerobic respiration,
in energetic and redox reactions, get embedded within a multitude of biochemical
natural compounds, in complex aggregates of lipids, proteins and carbohydrates, in
cell membranes, mitochondria and other subcellular structures.
Clinical Assessment
Clinical trials of Bactistatin with various animal models and in humans have shown
that oral administration of Bactistatin promotes the intestinal digestion of carbohy-
drates, lipids and proteins, reduces diarrhea syndrome, has marked immunomodu-
lating and detoxicating effects. Bactistatin prescription normalizes the microbial
ecology of the digestive tract, restores gut barrier function, stimulates the growth
of symbiotic lactobacilli and bifidobacteria, inhibits the propagation of pathogenic
and opportunistic bacteria, fungi and some viruses. Also, Bactistatin restores many
other clinical symptoms of gastrointestinal disorders in animals and humans caused
by short- and/or long-term exposure of gut symbiotic microbiota to biological and
chemical factors and agents (Ardatskaya 2015, Ardatskaya et al. 2015; Volkov
2008; Volkov et al. 2006; 2007; Minushkin et al. 2006; Shenderov et al. 2018a). It
is not improbable that multiple low molecular weight compounds of the Bactistatin
metabiotic can act as molecules that normalize acid-alkaline balance in cells and
tissues, as signals that remodel membranes, mitochondria and other intracellular
organelles involved in metabolic processes, in the development of brain cells and
endocrine APUD system, as regulators of intercellular ion channels modulation, as
epigenetic components that help accomplish gene expression in the metagenome
and meta-epigenome of the organism, post-translational protein modification and
other biochemical reactions in live organisms that remain not quite clear until now
(Oleskin et al. 2016; Shenderov 2017; Shenderov 2018; Shenderov et al. 2018a).
Table 2 New complex metabiotics on the base of B. subtilis

Composition Indications for use
Metabiotic B. subtilis + coenzyme Q10 + resveratrol Health of cardiovascular system,
metabolic syndrome
Metabiotic B. subtilis + polysaccharide complex Immune boosting
(β-glucan and mannan)
Metabiotic B. subtilis + hyaluronic acid + zinc + Skin health
vitamins С and Е
Metabiotic B. subtilis + taurine + plant hepatoprotector Liver and hepatobiliary system health
(Phylantus amarus)
Metabiotic B. subtilis + multivitamins + multi-minerals Complex nutritive support
Modification of this product is aimed at creation of combined therapeutics incor-

porating targeted active ingredients. This approach allows for creating metabiotics
that contribute to targeted health support with respect to particular systems of the
organism (Table 2).
Prospects in the Field of Intended-Use
Metabiotics Creation
The analysis of contemporary literature bears evidence that the development of

therapeutics intended for optimum formation, preservation and restoration of human
symbiotic microbiota and their practical implementation are among the critical
ways of preventive and regenerative medicine development. On the one hand, search
is ongoing for new application targets of traditional probiotics made on the base of
live probiotic strains and their detailed analysis, on the other hand, biologically
active microbial molecules or their complexes are being isolated and identified
which determine their application targets, efficiency and possible negative conse-
quences of using these probiotics. New potential probiotic strains capable of modi-
fying microelement, antioxidant, anti-inflammatory, psychic statuses, epigenotype,
quorum-sensing signaling are selected among both dominant microorganism spe-
cies and among species represented on the skin and mucosa in low amounts
(Shenderov 2014b). Supposedly, knowing the range and concentration of low
molecular weight biologically active compounds formed by known or potential pro-
biotic strains will help construct new effective metabiotics and set their optimum
frequency, doses and method of administration. The selected strains of human ori-
gin with unique functional or biochemical activity can become perspective starter
cultures for large-scale biotechnological production of microbial bioactive special-
purpose substances. Their use as metabiotics in their own right or as fortifiers of
known traditional probiotics, dietary supplements and functional foods will help
develop and implement more efficient microecological products for symbiotic
microbiota reservation and restoration in the majority of the population, in specific
population groups and in individuals. It should also be remembered that beneficial
effects of the prospective metabiotics depend to a great degree on the composition
of the growth medium, time and conditions for initial starter culture incubation,
methods of lysis of microbial cells, isolation, purification and (optionally) encapsu-
lation of the corresponding low molecular weight components of a microbial cell,
their metabolites and signaling molecules.
More intensive and detailed research is needed into the genome, epigenome and
metabolome of known and potential probiotic strains in order to identify the genes

https://doi.org/10.1007/978-3-030-34167-1_15
78 Prospects in the Field of Intended-Use Metabiotics Creation
associated with the synthesis of particular biologically active compounds. It will

allow for developing improved technologies for production of microbial compounds
with increased biological and pharmacological activity and a marked technological,
financial and economic appeal. Practical implementation of the “metabiotics” con-
cept will give an option of supplying biotechnology not only with strains belonging
to traditional probiotic species (bifidobacteria, lactobacilli, streptococci, leuco-
nostocs, pediococci, propioni bacteria, E. coli, enterococci, bacilli, saccharomyce-
tes and other rarer species of probiotic microorganisms), but also with dozens and
hundreds of strains belonging to other species of dominating phyla of symbiotic
microorganisms (Bacteroides, Firmicutes, Proteobacteria, Actinobacteria, Archae)
(Shenderov 2017; Shenderov et al. 2018a; Engevik and Versalovic 2017). Broadening
the spectrum of low molecular weight microbial components and metabolites (anti-
microbial agents, regulators of energy metabolism in the host’s mitochondria, com-
pounds that reduce triglyceride levels, low-density lipoprotein cholesterol, immune
response modifiers, antidepressants, memory boosters etc.) found in symbiotic
microorganisms that dominate in the human organism ecosystem is conductive to a
sharp increase in the range of produced metabiotics. They can become safer and
more effective than those already created on the base of bioactive compounds syn-
thesized by traditional probiotic strains of bifidobacteria, lactobacilli, E. coli, bacilli
and yeasts (Dorrestein et al. 2014; Shenderov 2013c; Sonnenburg and Fischbach
2011; Sonnenburg and Backhed 2016; Wikoff et al. 2009).
Establishing databases of individual low molecular weight biologically and
pharmacologically active compounds or their groups formed by representatives of
various species of human symbiotic microorganisms can play a key role in the cre-
ation of new intended-use metabiotics, and subsequently — synthetic and semi-
synthetic metabiotics. The example of such synthetic metabiotic is Actoflor C (the
complex of 12 organic and amino acids) produced in Russia (Vakhitov and Sitkin
2014). In the recent years, new gene clusters have been found in the representatives
of gut microbiota, which are responsible for the production of new specialized
metabolites (such as polyketides, sterols and isoprenoids, nonribosomally synthe-
sized peptides etc.), whose functions are almost completely unknown (Dorrestein
et al. 2014). Expanding our knowledge in microbial metabolites and signaling mol-
ecules contained in biological fluids of healthy people belonging to different ethnic
groups may also be of great diagnostic value and can become a pre-requisite for the
construction of express test systems for metabolome assessment in healthy and ill
people (Beloborodova and Osipov 2000; Engevik and Versalovic 2017; Gilbert
et al. 2016; Holmes et al. 2011; Li et al. 2008; Nicholson et al. 2005; Nicholson
et al. 2012; Shenderov 2013a).
Some New Targets and Approaches
to the Construction of Intended-Use
Metabiotics
Tables 1, 2, 3, 4, 5, 6, 7 and 8 show some new targets and approaches to the use of
low molecular microbial molecules for the development and production of essen-
tially new metabiotics in order to prevent and treat common conditions associated
with microecological imbalance in humans. These approaches, if realized, will
enable to promptly and efficiently use the potential of the basic representatives of
human symbiotic microbiota when creating targeted metabiotics.
Modulators of QS-Regulation
Quorum sensing (QS) is a cell-to cell communication allowing bacteria to sensing

small diffusible signaling molecules. QS microbial intercellular signaling coordi-
nate gene regulation in signal bacterium species within a community as well as
across kingdoms and controls various types of cooperative social behaviours (bio-
film formation, virulence traits, metabolic demands, antibiotic resistance, host-
microbe interactions etc). In the long term, these microbial bioactive molecules are
likely to become – individually or in combination – the base for potential QS meta-
biotics (Table 2).
Modulators of Immunity
Probiotic microorganisms actively and in various ways affect local and systemic
immunity, participate in maintaining colonization resistance of the skin and mucous
membranes, induce oral tolerance, reduce the frequency and risk of allergic disor-
ders, inhibit the growth of pathogenic and opportunistic bacteria, produce various
antibiotics, bacteriocins and other antimicrobial compounds, increase the activity of
phagocytic cells and natural killer cells, stimulate the production of IgA, inhibit the

https://doi.org/10.1007/978-3-030-34167-1_16
80 Some New Targets and Approaches to the Construction of Intended-Use Metabiotics
Table 1 Some approaches to the construction of intended-use metabiotics (Shenderov 2017;

Shenderov et al. 2018a; Shenderov 2011a; Shenderov 2011b)
Item
No. Approaches
1 Modulators of quorum-sensing regulation (QS-metabiotics)
2 Modulators of immune (immuno-metabiotics), antioxidant (antioxi-metabiotics),
neuropsychic (psycho-metabiotics) statuses
3 Modulators of energy metabolism in mitochondria and gut microbiota
(energy-metabiotics)
4 Modulators of epigenome regulation of phenotypic gene expression and post-
translational effects (epigeno-metabiotics)
5 Modulators of intracellular information exchange in the populations of prokaryotic,
eukaryotic cells and between the host cells and bacteria (info-metabiotics)
6 Modulators sustaining the genome and microbiome stability, preventing large intestine
and skin neoplasms and other targets
Table 2 Some microbial low molecular weight compounds capable of modulating quorum-
sensing regulation of microbial communities, cells of eukaryotic tissues, and probably sub-cell
organelles (Shenderov et al. 2018a; Engevik and Versalovic 2017; Shenderov 2011a; Shenderov
2011b; Thompson et al. 2015; Singh et al. 2018; Verbeke et al. 2017)
Item
No. Compounds
1 Inhibitors of protein synthesis (for example, antibiotics that suppress protein synthesis
on ribosomal level; different microbial peptides)
2 Receptor-ligand relationship antagonists (for example, microbial fatty acids trans-
isomers; bacteriocins)
3 Inhibitors of acyl-HSL signaling (for example, microbial halogenated furanones)
4 Inhibitors of histidine kinase
5 Enzymes that degrade QS-autoinducers (for example, microbial acylases, lactonases,
specific proteases similar to serpins of bifidobacteria)
6 Synthetic analogues of microbial autoinducers imitating signaling molecules
7 Micronutrients of microbial, herbal or animal origin, interfering QS regulation (for
example, peptides, lactones, lectins, polyphenols)
proliferation of lymphocytes, modifies the profile and the number of synthesized

cytokinins/chemokinins, affect the differentiation and maturation of dendrid cells,
stimulate and change the nature of Th1 ITH2 responses, synthesize the production
of vitamins B and K, exopolysaccharides, bifidogenic factors, conjugated linoleic
acid, regulate the synthesis of various intestinal epithelial cells antimicrobial pep-
tides (defensins) and other substances involved in the implementation of immune
effects. Low-molecular metabolites, surface and deep structures of probiotic bacte-
rial strains (Table 3) have specific receptors and targets in various parts and cells of
the immune system.
Modulators of Energy Metabolism 81
Table 3 Low molecular weight antimicrobial compounds and effector structural components
related to probiotic (symbiotic) microorganisms, as the base for potential immuno-metabiotics
(Chervinets et al. 2018; Shenderov et al. 2018a; Blander et al. 2017; Engevik and Versalovic 2017;
Kamada and Nunez 2014; Karczewski et al. 2014; Laino et al. 2016; Lebeer et al. 2018; Proal et al.
2017; Singh et al. 2018; Zhernakova et al. 2016)
Item
No. Compounds
1 Antimicrobial effect (lactic, acetic, propionic, butyric, benzoic, other organic acids,
hydrogen peroxide, carbon dioxide, nitrogen oxide, diacetyl, bacteriocins, microcins,
bacteriocin-like antibiotics, defensin-like peptides, enzymes with antimicrobial effect
(lysozyme), biosurfactants, polyamines, lectins etc.)
2 Structural components (surface S-proteins of fimbriae, peptidoglycans, lipoteichoic acids,
exopolysaccharides, LPS, nucleic acids etc.) and microbial cell metabolites (various
peptides, proteins, DNA, rich in СрG loci, SCFA, homoserine lactones, dopamine, serotonin,
metabolites of histamine and tryptophan etc.) of probiotic microorganisms that interact with
specific receptors and targets in different components and cells of the immune system.
Table 4 Modulators of energy metabolism in mitochondria and gut microbiota as a base for
potential energy-metabiotics (Shenderov 2018; Engevik and Versalovic 2017; Sharma and Shukla
2016; Wallace 2010; Wallace and Redinbo 2013; Singh et al. 2018)
Item
No. Compounds
1 Vitamins (B1, B2, B3, B5, B6, B7, B9, B12, C, K1, K2)
2 Non-vitamin substrates (NAD, NADH, NADP+, NADPH, ATF, cytidine triphosphate,
S-adenosyl methionine, 3’phosphoadenosine-5′-phosphosulphate, glutathione, coenzyme
B, coenzyme M, coenzyme Q10, co-factor F-430, heme, alpha lipoic acid, methanofuran,
molibdopterin/molybdenum co-factor, pyrroloquinoline quinone, tetrahydrobiopterin,
tetrahydromethanopterin)
3 Minerals (Ca, Cu, Fe++, Fe+++, Mg, Mn, Mo, Ni, Se, Zn)
4 Amino acids (arginine, lysin, methionine, cysteine, β-alanine, serine, threonine, histidine,
tryptophan, aspartic acid, carnitine)
5 Organic acids, Krebs cycle participants
6 Some nucleotides (for example, pyrimidine), microRNA etc.
7 Combinations of the listed low molecular weight microbial and herbal compounds
Modulators of Energy Metabolism
Energy-related processes in mitochondria and their functional analogues (inner

membranes) in bacteria need a multitude of enzymes and co-factors (Table 4).
Table 5 Antioxidant activity of low molecular microbial compounds as a base for creating
potential antioxi-metabiotics (Shenderov et al. 2018a; Engevik and Versalovic 2017; Minushkin
et al. 2006; Jones and Neish 2017; Mishra et al. 2015; Prosekov et al. 2015; Wang et al. 2017a)
Item
No. Mechanisms of antioxidant activity
1 Binding of metal ions involved in the host’s oxidative reactions (for example, Fe2+, Cu2+)
2 Microbial antioxidant enzymes (superoxide dismutases-Fe-SOD, Mn-SOD; catalase etc.)
3 Microbial structural components and metabolites with antioxidant effect (proteins,
peptides, polysaccharides, glutathione, butyric acid, folate, vitamin В12, thiamine),
reducing oxidative stress due to directly or indirectly increased synthesis of the host
antioxidative enzymes
4 Microbial compounds involved in the regulation of host signaling antioxidant pathways
(Nrf2-Keap1-ARER; NF-κB, protein kinase MAPK, PKS) engaged in maintaining
redox-potential of the organism
5 Microbial components modulating the host cell regulation of oxidative free radicals
synthesis
6 Microbial compounds (for example, organic acids, bacteriocins, biosurfactants) that
restore the host gut microbiota and that, on the contrary, inhibit proliferation of
pathogenic and opportunistic microorganisms and the related intestinal endotoxemia,
metabolic disorders and oxidative stress
Modulators of Antioxidant Status
Oxidant/antioxidant system regulates the processes of free radical oxidation. The

main active forms of oxygen (ROS), capable of damaging the membranes, nucleic
acids and other vital cell structures, are superoxide radicals, hydrogen peroxide,
hydroxyl (free) radicals, singlet oxygen forms, ions NO2-. The level of ROS
formed in the body during various physiological processes is controlled by a mul-
tistage system of endogenous and exogenous bioantioxidants, which support free
radical oxidative processes in physiological conditions and protect cell structures
from destruction. Imbalance between oxidation reactions associated with ROS
and reactions of their neutralization leads to uncontrolled increase in ROS con-
centration and development of oxidative stress, which leads, first of all, to disrup-
tion of the structure and functions of the membranes of various cells. Normally,
the content of ROS in the body is effectively regulated by a system of antiradical
and antiperoxide protection, which includes both enzyme and non-enzyme sys-
tems. Symbiotic microbiota is also actively involved in the fight against oxidative
stress of various origins. Oxidative stress and imbalance of antioxidant defense
mechanisms (the amount and activity of enzyme and non-enzyme antioxidants
and antioxidant systems of symbiotic microbiota) play an important role in the
pathogenesis of many metabolic diseases (chronic fatigue syndrome, metabolic
syndrome, premature aging and others). To reduce the risk of such diseases, it is
recommended to additionally introduce microbial and food antioxidants in the
form of various functional foods (Table 5).
Modulators of Intercellular Information Exchange 83
Table 6 Neuromodulation effects and targets of microbial ingredients as a base for potential psycho-
metabiotics (Oleskin and Shenderov 2016; Shenderov 2016a; Shenderov et al. 2018a; Engevik and
Versalovic 2017; El Aidy et al. 2016; Lebeer et al. 2018; Maguire and Maguire 2019; Oleskin et al.
2017; Singh et al. 2018)
Item
No. Effects and targets
1 Trimethylamine is involved in social communication (attractive smell)
2 Bile acids. Dysbioses accompanied by bile acid metabolism disorders caused by gut
microorganisms, often result in the risk of hepatic encephalopathy
3 Amino acids (aspartate, glutamate, glycine, taurine, tryptophan)
4 Phenol and phenol derivatives. Their increased concentrations in urine samples are found
in children with autism and schizophrenic patients. They also inhibit dopamine
transformation into noradrenaline
5 Indole is a microbial metabolite formed from tryptophan. Indolepropionic acid protects
neurons from oxidative damage and death caused by beta amyloid protein effects in
people with Alzheimer’s disease
6 Tryptamine is a neuropeptide stimulating the release of serotonin. Low levels of
tryptamine are found in patients with severe depression
7 Vitamins В12, K, biotin, cobalamin, folates, nicotinic and pantothenic acids, pyridoxine,
riboflavin, thiamine are co-factors of metabolic reactions that take part in the synthesis of
neurotransmitters and the realization of neurohormones or neuropsychic responses and
functions
8 Short-chain fatty acids (SCFA). Propionate influences social behavior, changes brain cell
phospholipid composition, as well as neuropeptide levels (serotonin, glutamate,
dopamine). Butyrate, gamma-aminobutyrate or gamma-aminobutyric acid (GABA)
influence energy homeostasis, nerve cell functions, mood and behavior. Acetate is the
basic substrate for the synthesis of acetyl-CoA, it enhances histone acetylation, influences
long-term memory, consolidation and neuroprtotection/regeneration. SCFA modify the
number of methyl group donors by interfering with the metabolism of monocarbon
compounds thus potentially influencing DNA and histone methylation
9 Spermine is a polyamine that slows down age-related memory impairment
10 Microbial hormones and transmitters (serotonin, C-dihydroxyphenylalanine — DOPA,
dopamine/noradrenaline, acetylcholine, histamine, tryptamine), acetylcholine,
catecholamines, serotonin, histamine and others involved in в the formation of a pool of
these compounds and stimulating epithelial, gut enteroendocrine cells and the synthesis
of peptide YY, neuropeptide Y, cholecystokinin, glucagon-like peptide-1,2 and substance
P in the human organism
Modulators of Intercellular Information Exchange
The relationship between the host and its microbiota is regulated by constant mutual
exchange of signaling information (Oleskin et al. 2016; Atkinson and William 2009;
Engevik and Versalovic 2017; Schauder and Bassler 2001) which also includes the
production by symbiotic microorganisms of SCFA, nitrogen oxide and other gas
microbial molecules, glutamate, beta-alanine, biotin, surface proteins, peptides, poly-
saccharides, lectins, peptidoglycans, lipoteichoic acid, glycopeptides, flagellins and
their complexes, lipopolysaccharides and other low molecular weight compounds that
Table 7 Modulation of epigenetic regulation of host gene expression as a target for selecting
potential epigeno-metabiotics (Shenderov et al. 2018a; Bhat and Kapila 2017; Feil and Fraga
2012; Roda et al. 2007; Shenderov 2016b; Singh et al. 2018; Wallace 2010)
Item
No. Mechanisms of epigenetic regulation
1 Microbial substrates, co-factors interfering with epigenetic regulation (enzymes, organic
acids and amino acids, vitamins, lectins, other coenzymes)
2 Microbial inhibitors and activators of general-purpose epigenetic signaling (volatile fatty
acids, gaseous molecules, lectins)
3 Microbial activators and inhibitors having a specific effect on particular enzymatic
participants of epigenetic machinery (methyltransferases, demethylases,
acetyltransferases, sirtuins, ribosyltransferases, hydrolases, phosphotransferases, kinases,
BirA ligase, synthetases, nucleases, DNA and RNA ligases) — Butyrate, microbial
derivatives of plant polyphenols
4 Microbial antagonists of receptor-ligand relationship (fatty acid trans isomers,
carbohydrate L-isomers, amino acid D-isomers, lectins)
5 Microbial proteases that degrade enzymes, effectors or receptors involved in epigenetic
processes (acylases, lactonases, bifidobacterial serpins)
6 Semi- and fully synthetic analogues of different modifiers of epigenetic mechanisms
Table 8 Best-known natural bioactive compounds of dietary or microbial origin involved in

epigenomic processes (Shenderov 2013a; Shenderov 2013c; Shenderov et al. 2018a; Bhat and
Kapila 2017; Cyr et al. 2013; Feil and Fraga 2012; Jimenez-Chillaron et al. 2012; Li and Tollefsbol
2010; Mischke and Plosch 2016; Remely et al. 2015; Said 2013; Shenderov 2016b; Shenderov and
Aleshkin 2012)
Item
No. Compounds
1 Mono-, di-, oligo- and polysaccharides
2 DNA, RNA, adenine, cytosine, guanine, NAD, ATP
3 Polyphenols and carotenoids (epicatechin, genistein, quercetin, hesperidin, luteolin,
garcinol, isocyanates, curcumin, resveratrol, coumarin)
4 Acetyl-coenzyme А, coenzyme Q10, S-adenosyl methionine
5 Melatonin, glutathione
6 Acetaldehyde, ethyl alcohol
7 Selenium, magnesium, potassium, zinc, iodine, cobalt, iron, calcium, manganese, copper
8 Pyruvate, citrate, lactate, α-ketoglutarate, succinate, formate
9 Spermidine
10 Folate, B1, B2, B12, B6, C, E, D3, biotin, pantothenate, nicotinic, orotic acids, choline,
alpha-lipoic acid
11 Proteins, peptides, arginine, lysine, methionine, cysteine, α-alanine, valine, leucine,
serine, threonine, glycine, histidine, tryptophan, aspartic acid, glutamate, acetyl-L-
carnitine, carnosine
12 Phospholipids, EPA, DHA
13 Glucosamine
14 Butyrate, propionate, acetate, caprylic acid
15 Sulforaphane, cysteines of cruciferous vegetables
16 Betaine
17 Allyl-mercaptans of garlic
Modulators of Epigenome Regulation 85
can modify the growth, regeneration and apoptosis of gut epithelial and immune cells,
the motility, fermentative activity and structure of intestinal mucosa, the structure of
Toll- and other receptors, induce the synthesis of regulatory signals, antimicrobial
peptides, enhance or inhibit gut vascularization, receptor activity in the enteric ner-
vous system, to prevent and restore mucosal lesions, gut barrier permeability
(Shenderov 2015a; Corthesy et al. 2007; Lebeer et al. 2008; Maguire and Maguire
2019; Marco et al. 2006; Ng et al. 2009; Shenderov 2011a; Shenderov 2013c). The
presented information allows for saying that there is a prospective possibility of creat-
ing one more line of metabiotic therapeutics – “meta-informobiotics.”
Modulators of Neuropsychic and Social Behavior
Over recent years, quite a few publications have appeared demonstrating that cer-
tain low molecular weight compounds formed by symbiotic (probiotic) microorgan-
isms can modify behavioral responses in humans, animals and insects (Engevik and
Versalovic 2017; Foster et al. 2016; Maguire and Maguire 2019; Oleskin et al.
2017). It is known that over 400 microbial metabolites pass from the gut to the brain
under normal and pathological conditions (Chernevskaya and Beloborodova 2018).
The obtained knowledge opens up the perspectives of effective microbial products
used as meta-psychobiotics both in clinical studies and in medicine and veterinary
(Oleskin et al. 2016; Shenderov 2015b; Oleskin and Shenderov 2016). For produc-
tion of meta-psychobiotics in the nearest future, it is recommended to use symbiotic
(probiotic) bacterial strains that form low molecular weight substances with certain
neurotransmitter activity or its whole complex (Table 6).
Modulators of Epigenome Regulation
Previously, it was thought that human health and chronic metabolic diseases are
predominantly driven by acquired genetic mutations. From the modern point of
view, the genetic system should be further supplemented with an epigenetic system
responsible for switching genes on or off in response to various endogenous and
exogenous factors and agents. Epigenetic changes are inherited changes that are not
related to changes in the sequence of nucleotides in the DNA. It is assumed that
epimutations occur 100 times more often than genetic mutations. These epigenetic
changes are both adaptive and risk factors for various diseases. It has recently been
proposal that exogenous, commensal and symbiotic microorganisms can participate
in various epigenetic mechanisms relevant to human health and disease. Owing to
low molecular weight microbial structural, metabolic and signalic molecules are
able to sense environmental factors, interact with corresponding cell surface, mem-
brane, cytoplasm and nucleic acid receptors and modify epigenomic regulation of
gene expression and/or alterate the information exchange in numerous bacterial
and bacteria-host systems. The design of microbial epigenetic-based remedies and
functional foods (prepared on the base of probiotic living bacteria, their structural
compounds, metabolites and/or signal molecules) could be a very important scien-
tific-applied approach for the treatment and prophylaxis of epigenetic-associated
human diseases. Potential reversibility of epigenetic changes allows for developing
metabiotics (Bhat and Kapila 2017; Feil and Fraga 2012; Remely et al. 2015;
Shenderov and Aleshkin 2012) which can activate or inhibit the corresponding epi-
genetic processes and signal transduction (Tables 6 and 7).
Low molecular weight compounds of microbial origin have different effects on
epigenetic mechanisms involved in the regulation of gene expression with regard to
genes responsible for the formation and activity of various enzymes and other mol-
ecules, their absorption, expansion, metabolism and excretion, for the functioning of
mitochondria, ribosomes, signaling molecules, ion channels and other molecular for-
mations in the host eukaryotic and microbial cells (Bhat and Kapila 2017; Shenderov
2016b). Moreover, in the process of degradation and metabolization of various
dietary components, gut microbiota is capable of endogenous formation of epige-
netically active compounds that modulate the work of cellular epigenetic machinery.
For example, secondary metabolites of plant polyphenols having aromatic rings and
one or many hydroxyl groups and found in vegetables, fruits, green tea, red wine.
Plant polyphenols play an important part in the life of plants protecting them from
photosynthetic stress, various reactive oxidants. Over 80% of natural polyphenols
undergo microbial degradation with the formation of metabolites that differ in their
activity, including the modulation of epigenetic cell processes. The resulting second-
ary metabolites not only show antioxidant properties in patients with cancer, neurode-
generative diseases, chronic inflammatory bowel disorders, but also directly influence
enzymes, other proteins, receptors and signaling cell pathways. It was found that ben-
eficial effects of such secondary metabolites on mammal organisms is realized through
the modulation of NF-kB expression, chromatin remodeling by changing the fermen-
tative activity of deacetylases (HDAC) and DNA- methyltransferases (DNMT)
involved in epigenomic gene expression. The example of such plant polyphenols that
have undergone microbial modification are curcumin, genistein, resveratrol etc.
(Russell et al. 2013). Food components (butyrate, sulforaphane, curcumin) act upon
histone-acetyltransferase (HAT) and HDAC activities. NAD+, S-adenosyl methionine
and 2-oxoglutarate interfere with the processes of DNA and histone methylation,
acetylation and ribosylation (Cyr et al. 2013). Water soluble В vitamins (biotin, nia-
cin, pantothenic acid) cause various histone modifications. For example, biotin is a
substrate for histone biotinylation, niacin — for histone rybosylation. Resveratrol,
butyrate, sulforaphane and diallyl sulfate inhibit HDAC activity, while curcumin
inhibits the activity of histone acetyltransferases (Choi and Friso 2010).
Introduction into preventive medicine of active microbial metabiotics – individu-
ally or in combination with specially chosen herbal compounds – will help to effec-
tively deal with pathologic conditions resulting from epigenomic or informational
disorders (atherosclerosis, diabetes mellitus type 2, hypertensive disease, cancer,
Alzheimer’s and Parkinson’s diseases, other neurodegenerative, metabolic and
autoimmune chronic conditions).
Hybrid Multicomponent Microbial Molecules as a Base for Metabiotics 87
Metabiotics on the Base of Microbial Gas Molecules
Components and metabolites of anaerobe and archaebacteria involved in the synthe-

sis and modulation by microbial and eukaryotic cells of various gas molecules (NО,
СО, Н2Ѕ, Н2, СН4, NH3 etc.) may be viewed as potential metabiotics. Many of these
gas molecules represent very ancient signaling molecules. They are formed in the
organism by way of microbial transformation of different compounds, displaying
their effects via neuroendocrine, immunological and biochemical responses, modu-
lating redox-signaling, regulating the activity of ion channels and ion transporters in
different cells, interfering practically with all physiological functions, biochemical
reactions and behavior, influencing the stability of metagenome, epigenome, pheno-
typic gene expression and post-translational modification of cell products. For
example, methane can change intestinal motility; constipations which are not infre-
quently caused by impaired microbial methane production, often precede the devel-
opment of neurodegenerative diseases and cancers (Shenderov 2015a; Althaus et al.
2013; Nicolson et al. 2016; Oleskin and Shenderov 2016; Savidge 2015). The first
research into the problem have shown that ill and healthy people prescribed milk or
water enriched with higher concentrations of molecular hydrogen experienced
marked positive effect at various pathological conditions and increased exercise
(Nicolson et al. 2016).
ybrid Multicomponent Microbial Molecules as a Base

H
for Metabiotics
Multi-year research into the metabolome of commensal and symbiotic microorgan-

isms have shown that low molecular weight compounds of microbial origin can
interact with different receptors of mammal microorganisms and eukaryotic cells
including humans, at the same time modifying several different functions and bio-
chemical reactions in individual bacteria, in their symbiotic associations, as well as
in mammal organisms. Such polymodal effectors with respect to different human
bacteria, cells, tissues and organs can be exemplified by microbial SCFA (butyrate,
acetate, propionic acid etc.). In low concentrations, representatives of this group of
microbial lipids can simultaneously suppress and optimize the growth of various
microorganisms in the digestive tract, modify the differentiation, proliferation or
apoptosis of intestinal epithelial cells, regulate the barrier function of intestinal epi-
thelium, take part in carbohydrate, lipid, energy, water-salt and electrolyte
metabolism of both individual cells and the whole organism; some fatty acids or
their complex interfere with redox balance, with the synthesis of LuxS proteins and
quorum-sensing regulation, gene expression and other cell processes both in in vitro
experiments on tissue cultures and on conventional and gnotobiotic animal models.
In physiological concentrations, butyrate synthesized by symbiotic (probiotic)
bacteria takes part in energy homeostasis of intestinal epithelial cells and neurons in
the brain, changes mood and behavior. Acetate is the key substrate for acetyl-CoA
synthesis, it is involved in epigenetic processes of histone acetylation, it influences
long-term memory, cell consolidation and neuroprotection/regeneration in brain tis-
sues. Propionic acid of microbial origin can regulate human social behavior, alterate
cell phospholipid composition, neuropeptide levels (serotonin, glutamate, dopa-
mine) in cerebral vessels.
Many SCFAs interfere with the metabolism of mono-carbohydrate compounds
responsible for methylation of DNA and nerve cell histones (Shenderov 2013b;
Shenderov 2017; Engevik and Versalovic 2017; Mischke and Plosch 2016;
Shenderov 2016b). Now the information is available that helps explain how indi-
vidual microbial low molecular weight bioactive compounds or their groups can
simultaneously cause different effects in prokaryotic and eukaryotic cells. It is
known that the structure of bacteria and eukaryotic cells is essentially different. In
mammals, cell organelles (the nucleus, mitochondria, ribosomes, exosomes etc.)
are separated from each other by own membrane formations; the integration, coor-
dination and regulation of these organelles work in the cell cytoplasm is realized
through special information regulatory network structures using a multitude of sig-
naling mediators (ion channel proteins, various kinases, transport proteins, organic
acids etc.). By contrast, bacteria and archaea have no differentiated cell organelles:
their role is functionally played by special formations (so-called hyperstructures)
which feature temporary, complex assemblies of different molecules and macro-
molecules with different specific functions. Such hyperstructures in bacteria and
archaea are defined as functionally dependent structures (FDS); they only exist in a
bacterial cell until functionally needed. FDS may include sets of different low
molecular structures, co-factors, proteins, peptides, organic acids, signaling and
other molecules which interact with one another, often getting simultaneously
involved in several particular functions and metabolic reactions. During the bacte-
rial cell development cycle, in case of inducer liquidation, FDS fall into separate
molecules, which subsequently can get involved into the same or new microbial
hyperstructures responsible for the corresponding function and/or metabolic reac-
tion and generation of the final metabolic product.
A multitude of FDS are simultaneously formed, function or disintegrate in the
cells of bacteria and archaea. By now, functionally dependent structures have been
identified in bacteria and archaea, which are responsible for the detection and trans-
portation of food substrates and co-factors, synthesis and functioning of cell mem-
branes, formation of cytoskeleton, synthesis of various enzymes and signaling
molecules, transcription, translation and degradation of DNA and RNA, for chro-
mosomal segregation, cell division, metabolism, polyphosphate synthesis, viru-
lence, lipopolysaccharide synthesis, chemotaxis and other functions and biochemical
reactions of microbial cells (Ghisian et al. 2018; Legent and Norris 2009; Norris
et al. 2016). Through the example of E. coli and bacilli it was revealed that their
membrane FDS may simultaneously include a multitude of various molecules (for
instance, over 1000 membrane proteins alone have been identified) involved and
coordinating the processes related to bacterial cell growth and division. For exam-
ple, when glucose, which is a source of energy for bacterial cells (E. coli), is added
Hybrid Multicomponent Microbial Molecules as a Base for Metabiotics 89
into the culture medium, transcription occurs in many genes involved in import and
synthesis of polyamines, inorganic phosphate and ions of magnesium, which ensure
the functions of membrane hyperstructure of these bacteria. When bacilli enter sta-
tionary phase, the FDS of bacterial membranes show increased levels of micronutri-
ents involved in the utilization of glycerol, ribose, lactate, nucleoside, succinate,
fumarate and zinc; at the same time, quantitative decrease is observed in proteins
transporting malate, citrate, hydroxamate of iron and hydroxymethyl-thiamine/thia-
mine. Upon completion of these processes, at the time of bacterial cell division, the
membrane breaks down and a new membrane FDS appears which may differ by its
component molecules, considering the particular factors of the medium surrounding
the bacterial cell.
The affinity of molecules and ions inside and between hyperstructures ensures
the work and coordination of all microbial FDS (Matsumoto et al. 2015; Thellier
et al. 2006). Prokaryotic organisms can form both the simplest hyperstructures
(sometimes consisting of two or three elements), and complex FDS assemblies,
capable of generating particular metabolic signals (Thellier et al. 2006). Separate
molecules, their complexes and even whole microbial FDS can be passed among
various cells of one species and also among the representatives of other species (for
example, among B. subtilis, E. coli, S. aureus). Intercellular molecular exchange
occurs via cell pores (nanotubes), which contributes to the formation of new FDS
assemblies in different bacterial cells and even in whole consortia of symbiotic bac-
teria in any given ecological niche (Buffie and Pamer 2013). Bacterial FDS (some-
times of a gigantic size) and their individual molecular components and complexes
formed by combining dozens to thousands of simple molecules, may subsequently
be transmitted and get involved in the work of metabolic and signaling processes in
bacterial and archaeal FDS, and also, supposedly, in the formation and functioning
of organelles in mammal and plant cells (Table 9) (Shenderov 2017; Browne et al.
2016; Buffie and Pamer 2013).
For targeted delivery of complex hybolites into human cells, the use of cell exo-
somes or cationic lipid complexes (lipoplexes) is recommended.
Table 9 Examples of complex hybrid metabolites (hybolites) with targeted effects on the human
organism (Shenderov 2017; Shenderov 2018; Legent and Norris 2009; Norris et al. 2016)
Item
No. Hybolites
1 Artificially constructed hibolite on the base of microbial cardiolipin (phospholipid of
bacterial membrane) and bacterial transport protein (flotillin-1) that regulates
normalization of functioning of human cell membrane channels
2 Complex hybolite on the base of microbial D-isomers of leucine, methionine, tyrosine
and tryptophan isolated from B. subtilis which inhibits staphylococci and P. aeruginosa
biofilm formation
3 Complex hybolite on the base of serine, threonine, sarcosine and phosphocholine of
bacterial origin having a potential anticancer effect
ther Contemporary Examples of the Techniques Needed

O
to Create, Maintain and Correct Human Microbial Ecology
In the future, microecological techniques of symbiotic microbiota correction will be

developing and improving. The perspectives are opening for the construction of new
metabolomic dietary supplements and functional foods intended to support the
genome and microbiome stability (metabolomic biotics), low molecular weight
modulators of information exchange between bacteria and the host cells (informo-
biotics), complex modulators of development mechanisms for colorectal and skin
neoplasms and their metastatic spreading into different human tissues and organs.
Work will continue to create metabiotics on the base of microbial low molecular
weight compounds in combination with different low molecular weight plant com-
ponents (for instance, bioflavonoids). New generations of biotherapeutics with
pharmaceutic activity are being created on the base of live commensal anaerobe
bacteria of human origin. Such metabiotics will serve as fortifiers in functional
foods, as a part of the formulation of metaprobiotics, meta-autoprobiotics, meta-
prebiotics, meta-synbiotics, synthetic metabiotics, and as pharmaceuticals and cos-
metic products. In some countries, national cryogenic banks of human
microbiocenoses will be established for long-term preservation of individual micro-
biota (Vakhitov et al. 2005; Volkov et al. 2007; Shenderov 2015b; Shenderov 2016a;
Shenderov et al. 2018a).
Among new approaches and techniques of symbiotic microbiota correction some
new technologies should be mentioned that are not directly connected with the con-
cept of metabiotics. Of these, for over twenty years the attention has been focused
on the so-called autoprobiotics, whose development requires the arrangement of
cryogenic banks of individual symbiotic microbiocenoses of the digestive tract in
healthy people in different countries and regions of our planet. The importance of
such banks is determined by a great ethnic diversity of the human population, dietic
peculiarities and the composition of symbiotic microbiota in its individual represen-
tatives, as well as by ecological and geographical peculiarities in their places of resi-
dence. Symbiotic associations of microorganisms preserved for long periods at low
temperatures (from −80 to −196°С) can serve as a base for autoprobiotics for
individual use or for microecological engineering (Shenderov 2003; Shenderov
2011b; Shenderov et al. 1996; Shenderov et al. 2018b; Suvorov et al. 2018).
Collections of people’s natural individual microbiocenoses allows for long-term
preservation of biodiversity of microbiota in the human population currently living
on our planet, and for manufacturing autoprobiotics by using autostrains of indige-
nous microbiota designed for people from whom these microbiocenoses had been
isolated. If required, the preserved microbiocenoses can become a base for prepar-
ing starter cultures for manufacturing special-purpose probiotics/metabiotics for
correction of changes in a particular person’s metabolome that serve as potential
disease risk markers or indicators of the already developed pathological condition.
A person’s epigenetic program is formed in the first thousand days of his/her life;
the programming process is influenced by numerous factors and agents — dietary
Other Contemporary Examples of the Techniques Needed to Create, Maintain… 91
and microbial compounds, in the first place. In this respect, it is doubtlessly rational
to artificially form a mother’s and a child’s symbiotic microbiota (microecological
engineering) (Shenderov 2007; Shenderov et al. 1996). Cryogenically preserved
microbiocenoses can be used for microecological engineering – primarily, for preg-
nant and breastfeeding women, for neonates and infants in the first two years after
birth – for controlled shaping of an individual’s microbial ecology. In cases when
using autoprobiotics in this category of people is impossible or impracticable, they
will be substituted by individual special-purpose metabiotics (Shenderov 2003;
Shenderov 2007; Shenderov 2011b; Shenderov and Midtvedt 2014). In the USA
and in some Western European countries, in Japan and in Australia, chronic inflam-
matory diseases of the digestive tract (primarily, in the treatment of people with
ulcerative colitis caused by Clostridium difficile) are treated by using the technology
of colonic microbiota transplantation right at the patient’s bedside. The technology
consists in taking healthy people’s fecal material and its peroral (via a special plastic
tube) or intrarectal (via an enema) administration to patients with severe clinical
manifestations of colitis. Usually, the number of such procedures does not exceed
10 within a period of two or three weeks. Though over 10,000 fecal transplants have
been performed worldwide, they are increasingly criticized due to frequent compli-
cations following such procedures (Kazerouni et al. 2015). Negative consequences
of the proposed fecal transplant technology are quite predictable, as in the vast
majority of cases the procedure of transferring diluted feces fails to take into account
strictly individual nature of each person’s gut symbiotic microbiota (Shenderov
2014b). Another reason for failure is the fact that it is quite impossible today to
identify a priori a “healthy” donor. Cryogenic banks created for preserving people’s
microbiocenoses in early childhood, as well as complex autoprobiotics, are the
technologies that could solve all the problems emerging today in case of traditional
fecal matter transplantation (Shenderov et al. 2018a, Shenderov et al. 2018b).
To date, over 500 genes have been identified in microorganisms including sym-
biotic ones that determine the synthesis of toxins and other pathogenicity factors;
also, around 200 genes are known which are responsible for resistance to different
antimicrobial compounds including antibiotics and other pharmaceuticals with anti-
microbial effects (antihistamines, antidepressants etc.). In this connection, the use
of live probiotics may be an important condition responsible for spreading
transmissible genes of virulence and antibiotic resistance among the human popula-
tion. Some scientific communities are discussing the opportunity of probiotic cre-
ation by using synthetic biology techniques. This molecular technology comprises
in detailed examination of strains of symbiotic microbiota representatives for the
presence in potential starter cultures of the following genes: of antibiotic resistance,
pathogenicity or those responsible for somatic cell malignization, as well as genes
responsible for compatibility with indigenous microbiota and immunological toler-
ance. After detailed examination of the genomes of potential starter cultures by
using the required modern molecular genetic technologies, it is suggested to con-
struct synthetic probiotic strains devoid of genes responsible for the synthesis of
compounds with potentially harmful health and environmental effects.
Simultaneously, these cultures may take genes that will be introduced to determine
the synthesis of bioactive compounds potentially beneficial for human health.

Actually, what this means is the development of new microbiological cultures
including those that have never existed in nature (Shenderov 2014b). The search for
new locally active potential antimicrobial compounds (phagobiotics, bacteriocins,
other antimicrobial peptides, new versions of antibiotics etc.) should be viewed as
one more line for correction of unbalanced microecology of the digestive tract. Its
implementation can also be based on microorganism strains kept in cryogenic banks
of healthy people’s individual microbiocenoses (Shenderov 2014b; Shenderov et al.
2014; Shenderov et al. 2018a; Shenderov et al. 2018b; Sanshez et al. 2015; Venema
and Carmo 2015).
Conclusion
As regards cell quantitative content, a human being is essentially a more prokaryotic

than eukaryotic organism. Great success has been achieved in the characterization
of microorganisms inhabiting the human being, all their genetic elements have been
explored in detail by using leading-edge molecular sequencing techniques. The
state of “host-its microbiota” system is one of the leading factors that determine the
growth, development and health of a human being; the importance of the microbi-
ome in the pathogenesis, phenotype, prognosis of many diseases has been estab-
lished. Symbiotic (indigenous) microorganisms inhabiting his/her skin and mucosa
are a source of various low molecular weight compounds involved in environmental
perception, in bacterial information intra- and intercellular exchange both among
themselves and with the host cells, in the regulation of physiological functions,
biochemical and behavioral responses. Different strains form their own strain-
specific range of similar substrates, co-factors, enzymes and regulatory molecules
which bear chemical and functional similarity with those produced by the host’s
eukaryotic cells and numerous micronutrients contained in food products. It means
that many compounds of microbial origin should be viewed as universal low molec-
ular weight substrates and co-factors of epigenetic, metabolic, immune, neurohor-
monal reactions, diffuse regulatory endocrine system (APUD system) and the
participants of intra- and interpopulation communication between bacteria and the
host eukaryotic cells. Bioactive molecules of microbial origin are capable of inter-
action with the host cells through binding to the receptors of membranes, mitochon-
dria, ribosomes. These molecules can also be accumulated in intracellular exosomes
and regulate intercellular and intracellular channels, influencing the structure, syn-
thesis, transfer and function of regulatory and signaling molecules (Aguilar-Toaláa
et al. 2018; Bik et al. 2018; Engevik and Versalovic 2017; Gilbert et al. 2016; Lebeer
et al. 2018; Maguire and Maguire 2019; Shenderov 2011a; Shenderov 2011b; Singh
et al. 2018).
Unfortunately, in the last fifty years, numerous biotic and abiotic exogenous and
endogenous stress factors affecting the organism have disturbed the balance between
human symbiotic, eukaryotic and prokaryotic cells, have induced marked

https://doi.org/10.1007/978-3-030-34167-1_17
94 Conclusion
odifications in the composition and functions of its microbial symbiotic compo-

m
nent, which has led to the increased risk of many chronic metabolic disorders.
Initially, these damaging agents usually cause transitory negative changes in the
functioning of the “host-its microbiota” ecosystem, which are then followed by
metagenome, metaepigenome and information disorders. If the exposure is long-
lasting enough, so that it exceeds the system’s compensatory capabilities, various
pathological syndromes and conditions develop. Over the last 50 to 100 years,
anthropogenic negative impact on the environment, as well as the man-of-today’s
unbalanced diet came into collision with his ability to adapt. That is why these two
particular factors are now seen as two main causes of the progressively imbalanced
microecological profile in a good number of representatives of the human population.
Inadequate stress loads observed for several generations have induced deregula-
tion of those dietary and gut microbiome functions that formerly ensured human
dietary, microecological, metabolic, epigenetic, neurohormonal and immune
homeostasis, and have increased the risk of negative epigenome modifications
related to premature ageing and degenerative metabolic diseases (Shenderov 2008;
Shenderov 2011a; Shenderov 2011b). In order to prevent and restore these disor-
ders, various microecological techniques and technologies, as well as particular
probiotic therapeutics and food products, have been proposed and practically imple-
mented in the last decades. Unfortunately, long experience of their use has shown
that traditional probiotics have a wide range of drawbacks related, in the first place,
to their safety in case of long-term use.
Knowledge accumulated over many years about the nature of man as a peculiar
kind of superorganism consisting of a multitude of symbiotic eukaryotic and pro-
karyotic cells and viruses, about the role and molecular language of symbiotic
microorganisms in maintaining human health, as well as cellular and molecular
mechanisms of contemporary metabolic diseases, allowed to define a new biotech-
nical trend of “METABIOTICS.” It consists of using metabolomic potential of sym-
biotic (probiotic) microorganisms for commercial production of unique therapeutics,
dietary supplements and functional foods on the base of low microbial weight sub-
stances of a known chemical structure having diverse biological and/or pharmaco-
logical systemic or selective effects, as well as good human safety profile. These
microbial bioactive compounds can be used as biotechnological raw materials for
commercial production of microecological products free from viable microorgan-
isms, different in composition and in methods of administration, for prevention and
treatment of chronic conditions in humans, as well as for replenishment of various
nutritional deficiencies in sportsmen and old people. Presently, the world’s markets
of therapeutics, functional foods, dietary supplements can offer more than twenty
different metabiotics on the base of both microbial cell substances and various low
molecular metabolite, regulatory and signaling molecules (Table 15).
Various Bacillus species are among the most powerful producers of bioactive
secondary metabolites (up to 800 compounds), which allows to consider the strains
of these species as “the most perfect multifunctional probiotic bacteria.” The said
potential has been realized by Russian developers in Bactistatin produced by using
the Вacillus subtillis strain No.3. Bactistatin is a germfree filtrate of the В. subtilis
Conclusion 95
cultural fluid containing a complex of microbial low molecular weight compounds

immobilized on zeolite in admixture with soy flour hydrolysate. This product has
been proven in human clinical trials, normalizing microbial ecology of the digestive
tract, improving digestion, showing immune modulating and detoxicating effects,
normalizing motor and evacuation functions of the intestine. This product improve-
ment is attempted along the lines of creating combined therapeutics containing
added active target-focused ingredients.
One example of metabiotics on the base of cell components of probiotic lactoba-
cilli is Pylopass, made to control the Helicobacter pylori levels in the stomach and
to prevent the related disorders. The examination of over 700 lactobacilli conducted
in Germany allowed to isolate the Lactobacillus reuteri DSM 17648 strain which is
capable of binding to the Helicobacter pylori surface and cause the formation of
bacterial aggregates, which neutralized the activity of this pathogen and allowed for
eliminating it from the digestive tract of an infected individual (Hunt et al. 2011).
Moreover, no viable Pylopass cells are needed for the specific anti-H. pylori
action — lyophilized or spray dried cells are sufficient. The resulting complex of
Lactobacillus–Helicobacter is naturally evacuated from the stomach. It contributes
to considerable reduction in Helicobacter levels and offers an essentially new
approach to controlling bacterial load in the stomach after or in parallel with eradi-
cation therapy and as a prophylactic supplement. To date, it is a unique product with
a proven mechanism of action against Helicobacter pylori. The absence of live lac-
tobacilli considerably reduces the risk of potential side effects typical of probiotics
containing live strains; besides, it ensures good product stability for not less than
three years.
In the future, metabiotics will serve as a base for developing semi-synthetic or
synthetic metabiotic analogues. Antibiotics industry developed in much the same
way in its time (Shenderov et al. 2010; Shenderov 2011a). When practically imple-
menting the concept of “METABIOTICS,” it is critical to conduct detailed studies
of the metabolome of all known and potential probiotic strains in order to provide
an opportunity of using them in the future as starter cultures for commercial produc-
tion of microbial substances for different pharmacological or dietic applications.
Dozens and hundreds of microbial bioactive compounds – both known and new –
may be used as metabiotics in the years to come (Shenderov 2009; Shenderov 2017;
Shenderov et al. 2010; Cani 2018; Engevik and Versalovic 2017; Lebeer et al. 2018;
Maguire and Maguire 2019; Shenderov 2011a). As compared to traditional probiot-
ics on the base of live organisms, metabiotics have quite a few advantages: they are
characterized by known chemical structure, clear application targets; they are much
easier to dose, and to control as regards safety and pharmacological and biological
activity. They are more easily absorbed, metabolized and distributed to tissues and
organs, and more quickly removed from the organism. Finally, they have longer
storage periods (Aguilar-Toaláa et al. 2018; Osipov and Verkhovtseva 2011). Also,
bioactive compounds produced by the representatives of human symbiotic micro-
biota compare favorably to chemically alike substances of dietary or other origin.
96 Conclusion
For millions of years of evolution, human superorganism had been selecting pro-
karyotic and eukaryotic microorganisms from the environment, which were optimal
for its life and development from the functional and biological perspective. That is
why metabiotics constructed on the base of bio- and pharmacologically active low
molecular weight compounds of human symbiotic microorganisms will undoubt-
edly be most beneficial, effective and safe for health. Exact determination of the
quantitative content of microbial compounds with specific functional, metabolic or
signaling activity in human tissues and biological fluids will also help trace causal
relationship between the particular types of microecological imbalance and patho-
logical syndromes and diseases (Dorrestein et al. 2014; Gilbert et al. 2016;
Shenderov 2011a; Singh et al. 2018; Sonnenburg and Backhed 2016). The next gen-
eration of metabiotics (semi-synthetic, synthetic) will further promote the concept
of probiotics, improving efficiency, specificity and safety of classic probiotic effects;
they will also reduce the risks associated with currently used microecological tech-
niques of prevention and treatment of diseases related to the imbalance of mammal
symbiotic microbiota.
Speaking about metabiotics, it should be remembered that this applied scientific
biotechnical trend is now at the earliest stage of its development. To date, the range
of gut microorganisms capable of forming colonies on artificial culture media is
barely over 1500 species (Browne et al. 2016; Rajilic-Stojanovic and de Vos 2014).
Many hundreds and thousands of species of other germs colonizing human diges-
tive tract are yet to be isolated. Even such symbiotic microorganisms as lactobacilli
have not yet been fully characterized taxonomically (over 230 Lactobacillus species
have been identified; around 10 new species are being identified annually). Many
researchers, technologists and clinicians have insufficient knowledge of the quantity
and spectrum of newly found microbial low molecular weight compounds, their
detailed physical and chemical characteristics, targets and molecular mechanisms
of action, factors and conditions that influence their positive activity and side effects
on a microorganism, especially when using potential metabiotics in inadequate con-
centrations. Probably, when discussing metabiotics – these new forms of therapeu-
tics – functional foods or dietary supplements, it should be remembered that it is
necessary to create new adequate model systems highly sensitive to low concentra-
tions of these microbial compounds and their complexes. We suppose that for these
purposes could be used available banks of different eukaryotic cell lines obtained
from conventional and germfree animals, and also new model subcellular structures
may be created (membranes, mitochondria, ribosomes, exosomes, ion channels)
including those that will be created through synthetic biology techniques. This will
enable to determine, on molecular level, the effects of low metabiotic concentra-
tions on energy, protein, lipid and carbohydrate metabolism of cells, on the opera-
tion of their membranes, mitochondria, ion channels etc. The results obtained by
using these models will be even more informative if the newly created metabiotics
contain low molecular weight compounds carrying labeled isotopes in their
structure.
When constructing metabiotics, the primary requirement is for up-to-date ana-
lytical base that allows for expression and high-precision evaluation of raw products
Conclusion 97
for subsequent extraction of microbial ingredients, final metabolic products, the

content of the corresponding functional ingredients, their physical and chemical
characteristics that influence bio-accessibility and final specific effects (Table 1).
It is important to know the exact targets and functional ingredients of dietary and
endogenous (cellular and microbial) origin and the conditions that can bring about
the most pronounced favorable (or negative) effects. The use of various methods
from OMIC-technologies, germfree and gnotobiotic animals of different levels of
organization, as well as new and synthetic models of cell cultures and organelles
isolated from them will allow specialists that study and develop intended-use meta-
biotics to achieve even greater success in realizing this trend in practical medicine
and nutritiology. Russia is one of the first countries having taken stock of biotech-
nological prospects and importance of creation of various-purpose metabiotics that
can prevent, restore and regulate physiological functions, biochemical and behav-
ioral responses, signaling intra- and intercellular communication, epigenetic regula-
tion of gene expression and post-translational modification of their final products.
Unfortunately, metabiotics range and production volumes in our country are clearly
out of keeping with the importance of these microecological means of health sup-
port and preservation in humans, animals and plants. The difficulty of bulk develop-
ment and application of various-purpose metabiotics in the Russian Federation is
caused by a number of reasons:
–– the diversity of biological and pharmacological effects of many known low
molecular weight compounds of microbial origin;
Table 1 Factors influencing functional properties and bio-accessibility of physiologically active

ingredients of microbial origin included in the composition of metabiotics
Item
No. Factors
1 Physical and chemical characteristics of an ingredient:
– Molecular structure (L- or D-forms, α-, β-, γ- or other forms of a molecule; valency
and isotopic state of chemical elements included in the structure of the functional
ingredient etc.).
– Solubility, dispersity, bonds with ligands of macro- and micronutrients.
– Oxidation state.
– Interaction with other components (amplifiers and inhibitors of ingredient effect).
– Competition for specific transport proteins or absorption sites.
2 Physiological conditions related to the host organism:
– Gastric acidity.
– рН and redox potential in the gastric lumen.
– Metabolic activity of intestinal juices.
– Bowel motility.
– Digestive and physiological status (age, sex, pregnancy, lactation etc.).
– Different stress factors.
– Hereditary defects.
3 The state of symbiotic microbiota of the digestive tract.
4 The technologies of obtaining functionally active microbial connections.
5 Other factors.
98 Conclusion
–– the absence of economically sound techniques of industrial isolation and assess-

ment of physical and chemical structure of potential metabiotics, as well as the
interaction of their biologically and pharmacologically active microbial com-
pounds with other low molecular weight compounds of dietary or endogenous
origin;
–– insufficient use of OMIC-technologies and gnotobiotic models in our country for
the assessment of safety and efficiency of this new class of dietary supplements
and pharmaceuticals of microbial origin;
–– lack of biotechnological factories and skilled personnel educated in this sphere;
–– not enough museums of microorganisms and microbiocenoses of human origin
and microbial materials preserved therein.
Some of the above statements may be illustrated by the following examples. It is
known that in course of carbohydrates, poly- and oligosaccharides, proteins and
lipids fermentation, symbiotic microorganisms ever present in the digestive tract
form a considerable amount of SCFAs; participating in energy metabolism, these
fatty acids also display a multitude of other biological effects. SCFAs suppress the
growth of pathogenic and opportunistic bacteria, control gut barrier function, regu-
late cell differentiation, proliferation, apoptosis and mucous layer renewal, optimize
the growth and development of probiotic and symbiotic bacteria, take part in carbo-
hydrate and lipid metabolism, in water-salt exchange, in production and regulation
of hormone and neurotransmitter activity, cause immune effects, eliminate damaged
cells, prevent neoplastic transformation of normal cells, take part in LuxS protein
synthesis, in epigenome regulation of gene expression, quorum-sensing regulation
(Golovenko et al. 2011; Carding et al. 2015). Naturally, using them as metabiotics
will require that producers and doctors have full knowledge on particular targets
these microbial SCFAs are planned to be used against, concentrations, forms and
lengths of their administration etc.
Nowadays, there are over 600 different collections of microorganisms. But only
about 20 of them have the status of internationally recognized microorganism col-
lections, since only they are capable of performing the functions required from this
type of organizations. Their maintenance demands considerable investments from
the countries’ governments. To give an example, one could mention German
Collection of Microorganisms (DSMZ). Prokaryote, fungi and yeast section in
DSMZ supports 26 thousand cultures with 6 scientists and 18 employees on service.
In 2011, the cost of keeping this collection amounted to around EUR three million;
it distributes over 20,000 strains annually (40% domestically and 60% abroad). To
maintain the collection and conduct scientific research, about 1500 different culture
media are used. The first low-temperature bank in the Russian Federation for keep-
ing pure microorganism cultures was established at the Institute of Microorganism
Biochemistry and Physiology of Russian Academy of Sciences on the basis of All-
Russian Collection of Microorganisms. Unfortunately, though this Russian collec-
tion has been registered at the World Registry of Microorganism Collections, it is
not included among those complying with international requirements (Shenderov
et al. 2014).
Conclusion 99
Cryogenic banks of natural microbiocenoses of humans, animals, plants, surface

water bodies and soils deserve special mentioning. Cryogenic banks help preserve
intact (in viable state) symbiotic microbiocenoses of soils, water bodies, any cellu-
lar eukaryotic organisms having emerged in course of the evolution for extended
periods (practically for eternity) at low temperatures. The microorganisms kept in
cryobanks can subsequently be used for making personified foods, pharmaceuticals,
for maintaining and restoration of microbiocenoses in people of hazardous occupa-
tions, in the production of targeted microbial fertilizers designed for particular plant
species, in the preservation and restoration of rare or extinct animals and plants, in
the creation of specific pollutant destructors, in the remediation of soils and water
bodies, in the implementation of other cutting-edge biotechnological trends using
the genetic and biochemical potentials of the representatives of symbiotic microbi-
ota of different organisms. World’s first cryobank of natural microbiocenoses was
established in 1995–1996 on the basis of genetic resource cryopreservation labora-
tory at the Institute of Cell Biophysics, Russian Academy of Sciences (Pushchino-
on-Oka town, Russia). It was intended for long-term preservation of human colon
microbiocenoses. In concert with the researchers from Science and Innovation
Center of “Russian Yoghurt”, JSC, and later with the assistance from
G.N. Gabrychevsky Moscow Research Institute of Epidemiology and Microbiology,
the laboratory personnel had chosen optimal cryopreservation agents, worked out
viability assessment techniques for the preserved microorganism complexes, devel-
oped the cryobank structure, selected the necessary equipment, prepared a complete
feasibility report for establishing a cryoreservoir for 20,000 intestinal content sam-
ples. Biomaterial obtained from the children of cryopreservation laboratory person-
nel at the Institute of Cell Biophysics, Russian Academy of Sciences, was placed for
long-term storage. Multi-year research into the cryopreservation biomaterial had
shown that the invented technology for storage of natural human gut microbioceno-
ses is fully compliant with the requirements for live organism cryopreservation
(Shenderov et al. 1996; Shenderov et al. 2014; Shenderov et al. 2018a; Shenderov
et al. 2018b; Shenderov et al. 2018c).
Presently, a cryobank of natural human microbiocenoses containing dozens of
thousands of biomaterial samples was established and is functioning in France. The
groups of people whose symbiotic microbiocenoses are supposed to be sent for
long-term storage in cryobanks, should include, in the first place, all healthy chil-
dren aged 2 to 6, as well as young women (primarily, first-time mothers). Such
cryobanks should be created for people working for a long time under extreme
conditions (law enforcement personnel, pilots, submariners, astronauts, journalists,
public transport drivers, businessmen etc.), as well as for people living or working
in hazardous environments (chemical factory and nuclear power plant employees,
Arctic expedition participants etc.); practically all people willing to preserve their
natural microbiota for long periods of time and use it, if necessary, for microbial
autotransplantation would be able to send their natural microbiocenoses to such
cryobanks (Shenderov et al. 2018a; Shenderov et al. 2018b; Suvorov et al. 2018).
Considering the ever-increasing importance of symbiotic microbiota for preserv-
ing our planet’s biodiversity, for maintaining health and reducing the risks of
100 Conclusion
s o-called “lifestyle diseases,” one can easily understand why so much attention is
being paid worldwide to the construction of effective and economically sound
microecological strategies. Accumulated knowledge regarding the molecular lan-
guage of symbiotic microorganisms allows for constructing more effective and tar-
geted microecological products (therapeutics, dietary supplements, functional and
personal foods) for prevention and treatment of many metabolic diseases associated
with microecological disorders, changes in the epigenetic remodeling of chromatin,
DNA and proteins. In the future, microecological techniques of symbiotic micro-
biota correction will be developing and improving (provision is made for the cre-
ation of metabiotics on the base of microbial low molecular weight compounds
individually and in combination with various plant bioflavonoids; development of
national cryogenic banks of human microbiocenoses for long-term preservation of
individual microbiota, for constructing personal-use metabiotics and microecologi-
cal engineering; construction of probiotics and metabiotics by using the techniques
of synthetic biology; creation and implementation of new types of local-action anti-
microbial compounds.) Subsequent generations of simple, complex, semi-synthetic
and synthetic metabiotics (by analogy with antibiotics) will be analogues or
improved copies of natural low molecular weight compounds of probiotic and com-
mensal human microorganisms. It is beyond argument that before introducing the
specified groups of metabiotics into preventive and clinical medicine they should be
subjected to thorough pre-clinical trials, which must take into account the peculiari-
ties of their physical and chemical structure and mechanism of action on any given
targets of the host’s tissues and organs.
We suppose that in the years to come the following techniques for maintaining
and restoring human microbial ecology will be gaining momentum:
–– General-purpose, specific and personified metabiotics on the base of individual
and hybrid microbial low molecular weight compounds – individually and in
combination with different plant nutrients, regulatory and signaling molecules.
–– Cryogenic banks of human microbiocenoses for long-term preservation of indi-
vidual microbiota, for the construction of antibiotics for personal use and micro-
ecological engineering.
–– Probiotics made by using the techniques of synthetic biology.
–– New types of locally active antimicrobial compounds (phagobiotics, bacterio-
cins, other antimicrobial peptides, new versions of antibiotics etc.).
It is about time an international interdisciplinary program is created:
“METABIOTICS — NEW NUTRITIVE AND MICROECOLOGICAL
STRATEGY OF ACTIVE LONGEVITY AND PREVENTION OF CHRONIC
METABOLIC DISEASES,” whose implementation will allow for a dramatically
decreased risk and progression of the main “lifestyle diseases.” It could become a
useful, fruitful and consolidating program that would actively attract a wide range
of Russian scientists, biotechnologists and clinicians in diverse areas of expertise.
The transformation of probiotic strains into metabiotic therapeutics is an effec-
tive strategy intended to ensure high-quality active ingredients availability in a sta-
ble pharmaceutical formulation. Understanding the mechanism of action of probiotic
Conclusion 101
therapeutics responds to the needs of society and science, lays the foundation of the
development of new, safe and highly effective bioactive therapeutics. To better
understand human interaction with low molecular weight bioactive compounds of
microbial origin, experimental and clinical studies should be increasingly often
based on the leading-edge “multiOMIC technologies” (genomics and its variet-
ies — pathogenomics, probiogenomics; transcriptomics and its variety —
RNAomics; proteomics and its variety — interactomics; metabolomics and its
variety — fluxomics; epigenomics; phenomics). Simultaneous experimental use of
different germfree animals and gnotobiotes including those inoculated with symbi-
otic microorganisms taken from people of different ages, sex and races will help
obtain the most objective data on metabiotics application targets (Shenderov 2012;
Shenderov 2014a; Shenderov 2014b; Dorrestein et al. 2014; Karczewski and Snyder
2018; Martin et al. 2008).
When evaluating the interaction of humans with their symbiotic microbiota in ill
and in healthy people, it should always be remembered that every person is indi-
vidual both on genome and microbiome level. Preventive medicine for each particu-
lar person should be based on the knowledge about the peculiar features of his/her
genome and microbiome, on the understanding of molecular mechanisms of com-
munication between them and the role of environmental factors in developing,
maintaining and epigenetic modification of the host’s total metagenome functions.
Sudden and long-lasting defects of energetic metabolic processes in mitochondria
and bacterial membranes are often related to the deficiency or excessive ingestion of
substrates, co-factors or enzymes and disturbance of functioning of the microbiota–
mitochondria–epigenetics axis. It is accompanied by insufficient ATP generation,
increased content of high-reactive free radicals of oxygen, nitrogen and products of
peroxidation in cells, imbalance in antioxidant support network and compounds
involved in processes of gene expression regulation and in post-translational modi-
fication of final products of these genes. As a consequence, chronic inflammation is
induced, telomeres become shorter, processes of proteostasis are lost, cell ageing
occurs, stem cell pool diminishes, intercellular communication is disrupted – all this
results in quick ageing and risks of chronic pathological conditions (neurodegenera-
tive, metabolic, autoimmune, behavioral, mental, musculoskeletal, inflammatory
diseases, chronic infections, cancer) (Paul et al. 2015; Singh et al. 2018).
There are no food products, pharmaceuticals, physiological functional ingredi-
ents, which would have absolutely identical effect on everyone. Healthy and tai-
lored diet that takes into account a person’s individual needs helps restore the
expression of the polymorphic genes with adverse health effects, and, on the con-
trary, to optimize epigenomic control and realization of those allelic genes that
ensure better adaptation of the human “superorganism” and harmonize the work of
its symbiotic components in normal conditions, under stress and extreme condi-
tions. Understanding of this fact helps to further increase specificity, efficiency and
safety of microecological techniques aimed at prevention and treatment of diseases
related to mammal symbiotic microbiota imbalance (Shenderov 2017; Sharma and
Shukla 2016; Singh et al. 2018).
102 Conclusion
To date, we are only at the beginning of the new era of molecular personal bio-
therapy and nutrition. Nevertheless, it is beyond doubt that soon it will be possible
to intentionally and successfully manipulate both humans and their microbiota as a
result of interfering with their informational interaction, stability and epigenome
gene expression regulation by using different types of low molecular weight com-
pounds of eukaryotic, prokaryotic and dietary origin (Shenderov 2011a). Even pres-
ent-day knowledge on the structure and functions of human symbiotic microbiota in
all periods of life and development calls for revolutionary changes in the training of
future medical professionals with shifting the focus from this paradigm – “a human
being represents a community of eukaryotic cells” to another paradigm – “a human
being represents a superorganism, a community of eukaryotic, prokaryotic cells and
viruses”; given that, professional training programs should highlight that the state of
symbiotic microbiota is really the leading factor in human health.
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Index
A helicobacter and stomach health, 64

Acetate, 88 helinorm/pylopass strategy, 65
Antibiotics, 27 L. reuteri DSM17648, 63
fecal excretion, 20 metabiotic formulation, 68, 69
industry, 95 non-living probiotic cells, 63
Antimicrobial agents, 72 probiotics, helicobacter therapy,
Autoinducers, 33 64, 65
Autoprobiotics, 30, 90 pylopass (see Pylopass)
Co-aggregation activity, 68, 69
Complex dietary compounds, 13
B Cryogenic banks, 91, 99, 100
Bacillar antimicrobial peptides, 72
Вacillus subtillis, 71, 72
Bactistatin, 74, 94 D
Bactistatin dietary supplement, 73 Damaging agents, 94
Bifikol, 28 Dietary metabolism, 14
Bile acids, 17 Digestive tract microbiota, 18
Bioactive molecules, 15, 35, 50, 59, 74, DNA-methyltransferases (DNMT), 86
79, 93
Bioactive secondary metabolites, 94
Biofilm, 8, 19, 72, 79 E
Biotin, 17, 34, 72, 83, 86 Energy metabolism, 81
Epigenetic biochemical machinery, 40
Epigenomics, 55, 84–86, 101
C European Food Safety Authority (EFSA), 46
Cellular metabiotics Exohormones, 6
clinical evaluation, 69, 71
co-aggregation
activity, 68, 69 F
sites, 68 Fecal transplants, 91
growth phase, 68 Food & Drug Administration (FDA), 47
H. pylori, 63 Food products, 13, 41
H. pylori DSM 21031 vs. L. reuteri DSM Fortifiers, 90
1764, 66 Functionally dependent structures (FDS), 88

https://doi.org/10.1007/978-3-030-34167-1
120 Index
G endoecology, 6
Gastrointestinal Symptom Rating Scale fecal bacteria, 7
(GSRS), 65 Firmicutes and Bacteroides, 7
Gene transfer, 45 genome, 5
Gut bacteria, 14, 17 human chromosomes, 6
human physical and mental health, 5
human superorganism, 5, 6
H indigenous microbiota, 2
Health preservation, 27 large intestine, 6
Helicobacter pylori, 63, 64 long-term individual stability, 6
Histone-acetyltransferase (HAT), 86 metabolic activity, 6
Host-its microbiota system, 93, 94 metabolic changes, 8
Human gut microbiota metagenome, 5
antibiotics, 18, 20 microbial component, 5
bile acids, 17 microbial phyla, 7
biochemical metabolic pathways, 16 microbiocenosis, 5
biological effects, 15 microcolonies, 8
biological fluids, 21 multi-cellular organisms, 8
combiotics, 30 multiple low molecular weight
complex proteins, 17 compounds, 2
dietary, 14 regulatory substances, 6
digestive function, 13, 14 structure, 2
digestive tract, 15–18 superorganism, 5
endogenous substrates, 14 symbiosis-regulating systems, 8
energetic contribution, 16 therapeutics, 2
fecal excretion, nitrogen containing Human microbiota, 1, 7
compounds, 19 Human organism
feeding mice, 20 contemporary view, 1
free amino acids, 17 microbiome/microbiota, 1
functions, indigenous microbiota, 18 multiple and numerous intestinal
host organism, 18 microorganisms, 2
low molecular weight microbial Human superorganism, 5, 6
compounds, 21 Hybrid multicomponent microbial molecules
macro-and microelements, 16 acetate, 88
metabolic vs. signaling molecules, 15 bacterial cell development cycle, 88
metabolites, 21 commensal and symbiotic
metabolome analysis, 18 microorganisms, 87
metagenome, 16 FDS, 88, 89
microbial bioactive molecules, 15 hybrid metabolites, 89
microbial degradation, 18 hyperstructures, 88
microecological strategies, 27, 28, 30, 31 intercellular molecular exchange, 89
morphological changes, 19 polymodal effectors, 87
polyphenol degradation, 17 prokaryotic and eukaryotic cells, 88
principles and techniques, 28 SCFA, 88
probiotics, 29, 30 Hyperstructures, 88
propionic and butyric acids, 16
short peptides, 17
synbiotics, 30 I
vitamins, 17 International Probiotics Association (IPA), 12
Human gut symbiotic microbiota Intended-use metabiotics creation, 77–78
agents, 2
biofilm, 8
biological and abiotic factors, 2 L
composition, 6, 7 Lactobacillus reuteri DSM17648, 63, 66–70, 95
digestive tract, 8 Lactobacillus–Helicobacter, 95
Index 121
Lifestyle diseases, 100 market of therapeutics and functional

Low molecular microbial molecules foods, 63
antimicrobial compounds, 81 metabolite (see Metabolite metabiotics)
effector structural components, 81 microbial bioactive compounds
human microbial ecology, 100 targets, 51
approaches and techniques, 90 microbial products, 49
biotherapeutics, pharmaceutic microecological products, 49
activity, 90 modern techniques and technologies, 63
cryogenic banks, 91 molecular language, symbiotic
dietary supplements and functional microorganisms, 49, 50
foods, 90 potential forms, 58
epigenetic program, 90 primary requirement, 96
fecal transplants, 91 prokaryotic and eukaryotic cells, 50
microbiocenos, 91 SCDI, 59
microbiological cultures, 92 scientific biotechnical trend, 96
microecological techniques, 90 semi-synthetic/synthetic, 95
molecular genetic technologies, 91 signaling molecules, 3
pathogenicity factors, 91 starter cultures, 49
synthesis, toxins, 91 synthetic biology techniques, 96
synthetic biology techniques, 91 techniques and analytical technologies,
hybrid multicomponent microbial 53, 54
molecules, 87–89 therapeutics, 59, 96
microbial gas molecules, 87 traditional probiotic concept, 49
modulators traditional starters, 59
antioxidant status, 82 Metabolite metabiotics
energy metabolism, 81 amikacin А and B, 73
epigenome regulation, 84–86 antimicrobial agents, 72
immunity, 79 B. subtilis, 71, 72
intercellular information exchange, B. subtillis, 72, 73, 75
83, 85 bacillar antimicrobial peptides, 72
natural bioactive compounds, 84 bactistatin dietary supplement, 73
neuropsychic and social behavior, 85 bactistatin examination, 73
QS-regulation, 79, 80 characteristics, 72
neuromodulation effects and targets, 83 clinical assessment, 74
targets and approaches, 79, 80 germfree cultural fluids, 73
Low molecular peptides, 38 low molecular weight compounds,
74, 75
probiotics, 71
M Metabolome analysis, 18
Metabiotics Microbial bioactive compounds, 94
bio-accessibility, 97 Microbial degradation, 18
bioactive molecules, 50 Microbial gas molecules, 87
cellular (see Cellular metabiotics) Microbial low molecular weight cellular
chemical structure, 49 compounds
classification, 57 commensal and symbiotic
combined therapeutics, 61 microorganisms, 55
factors influencing functional modern techniques and methods,
properties, 97 53, 54
functional foods, 59 Microbial structural components, 82
international market, 59 Microecological disorders, 23
low molecular microbial molecules Microecological imbalance, 24
(see Low molecular microbial Microecological therapeutics, 57
molecules) Microorganisms, 1, 11
manufacturers, 59 Modern diet, 13
market of microecological products, 59 Molecular genetic technologies, 91
122 Index
Molecular language, symbiotic gut Probiotic potential, 29

microorganisms Probiotics
acetate and butyrate, 37 human population, 30
antimicrobial substances, 34 live organisms, 3
autoinducers, 33, 34 living microorganisms, 27
bacterial DNA, 38 market, 12
bioactive molecules synthesize, 35 market of therapeutics, 30
cell components, 39 metabiotics, 3
cell-free filtrate, 38 microorganisms, 28, 39
contemporary chromatography, 33 non-digestible carbon-containing
digestive tract, 34, 36 compounds, 28
DNA linear structure, 40 strains, 12, 30, 38, 100
energetically significant, 34 symbiotics, 27, 31
energy synthesis, 34, 40 traditional (see Traditional probiotics)
epigenetic biochemical machinery, 40, 42 Prokaryotic organisms, 89
eukaryotic human cells, 39 Proton pump inhibitor (PPI), 64
exogenous and endogenous factors, 41 Potential probiotic strains, 77
factors and agents, 40 Pylopass
food products, 41 characteristics, 67, 68
formation, human metabolome, 42 H. pylori, 64, 70
gastrointestinal tract, 36 helinorm, 63
live organism, 35 strain, 66, 67
low molecular peptides, 38
low molecular weight compounds, 39
mammal immune system, 36 Q
mass spectrometry, 33 Quorum sensing (QS), 79
metabolites and surface structures, 37
microbial metabolites, 35
microbial molecules, 35 R
microbial origin, 35 Regulatory substances, 6
microbial transformation, 35, 36 Riboflavin, 17
molecular mechanisms, 34
neuromediators, 40
pathophysiology, 36 S
physiology, 36 Semi-synthetic metabiotics, 57
probiotic lactobacilli, 39 Short-chain fatty acids (SCFA), 18, 20, 35–37,
probiotics (see Probiotics) 39, 50, 72, 81, 83, 87, 88, 98
SCFA, 37 Signaling molecules, 2, 55
skeleton P-CWS, 38 Simple and complex hybrid metabolites, 13
structure and function, 42 Skeleton P-CWS, 38
symbiont microbes vs. host cells, 35 Starter cultures for direct inoculation
TLR receptors, 38 (SCDI), 59
MultiOMIC technologies, 101 Superorganism, 5, 13, 94
Symbiosis-regulating systems, 8
Symbiotic microbiota
O antibiotics, 23
Over-feeding, 21 assessment, 25
Oxidant/antioxidant system, 82 cellular and molecular parameters, 24
Oxidative stress, 82 chronic diseases, 24
detection, violations, 25
diet, 23
P gastrointestinal diseases, 24
Probiotic lactobacilli, 95 host-microbiota system, 23
Probiotic microorganisms, 79 human microbiome variability, 23
Index 123
methods and technologies, 25 disease prevention, 43

microecological disorders, 23 DNA damage, eukaryotic cells, 46
microecological imbalance, 24 E. coli strains, 45
negative stress effects, 23 ecological and social consequences, 45
pharmaceuticals, 23 effectiveness and safety, 46
premature ageing, 24 EFSA, 46
Symbiotic microorganisms, 8 FDA, 47
glutamic acid, 11 gastrointestinal tract, 44
low molecular weight compounds, 93 gene transfer, 45
market, traditional probiotics, 12 indigenous microbiota, 43
microbial biotechnological products, 11 lactic acid bacteria, 46
intended-use metabiotics creation, 78 lactobacilli and bifidobacteria, 43
probiotic fermented milk products, 12 live organisms, 95
secondary metabolites, microbial origin, 11 market of pharmaceuticals, 43
workhorses, 11 metabiotics, 95
Symbiotics, 27 microbial transformation,
Synbiotics, 28 phosphatidylcholine, 44
Synthetic biology techniques, 91 recombination processes, 45
Synthetic metabiotics, 57 silent genes, 45
stains, 44
treatment, 43
T types, prebiotic substances, 47, 48
Traditional microecological therapeutics, 2 Traditional starters, 59
Traditional probiotics Triple therapy, 63
beneficial probiotic effects, 43 Tumor necrosis factor (TNF), 38
clinical observations, 45
clinical trials, 47
data, 44 W
dietary supplements, 43 Western pattern diet, 8
digestive tract, 44, 46 Workhorses, 11

Metabiotics: Boris A. Shenderov Alexander V. Sinitsa Mikhail M. Zakharchenko Christine Lang

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Metabiotics: Boris A. Shenderov Alexander V. Sinitsa Mikhail M. Zakharchenko Christine Lang

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Boris

ISBN 978-3-030-34166-4 ISBN 978-3-030-34167-1 (eBook)

© Springer Nature Switzerland AG 2020

related to understanding the interaction between metabiotics and host organism

Moscow, Russia Boris A. Shenderov

Cellular Metabiotics and Metabolite Metabiotics���������������������������������������� 63

Boris A. Shenderov is a physician, doctor of Medical Science (1976), professor of

Alexander V. Sinitsa is president of “Kraft” Group, Saint Petersburg, Russia, and

Mikhail M. Zakharchenko is a physician, candidate of Medical Science (2003),

Healthy Lifestyle (2007–2019). He leads the development of new products at

Christine Lang is an entrepreneur in biotechnology. She acts as a consultant for

All diseases start in the gastrointestinal tract.

© Springer Nature Switzerland AG 2020 1

© Springer Nature Switzerland AG 2020 5

it possible to consider symbiotic and commensal microbiota as a peculiar extracor-

and Firmicutes followed by Actinobacteria, Proteobacteria, Fusobacteria,

Microorganisms are essentially the main “workhorses” of contemporary biotech-

© Springer Nature Switzerland AG 2020 11

© Springer Nature Switzerland AG 2020 13

Symbiotic microorganisms ever-present in the organisms of adult people form over

© Springer Nature Switzerland AG 2020 15

o­ rigin often act as regulators of intra- and interpopulation interaction (nervous,

i­ntestine actively participate in the proteolysis of endogenous and exogenous pro-

Table 2 Influence of antibiotics on fecal excretion (Shenderov et al. 1990)

© Springer Nature Switzerland AG 2020 23

© Springer Nature Switzerland AG 2020 27

example of the Russian symbiotic is “Bifikol”, which includes E. coli, removing

Table 2 The list of certain probiotics commercially available in Russia

© Springer Nature Switzerland AG 2020 33

Among the metabolites of symbiotic bacteria having effect on immune composition

course of microbial transformation of L-histidine amino acid (Engevik and

Table 1 Some neuromediators formed by the representatives of human symbiotic microbiota

Physical climatic and Physical

of energetic and epigenetic biochemical machinery (Shenderov 2008; Shenderov

The growing market of pharmaceuticals, dietary supplements, and particularly

© Springer Nature Switzerland AG 2020 43

Fig. 1 The number of publications on clinical trials of probiotics according to PubMed.gov

The knowledge of molecular language of symbiotic (probiotic) microorganisms

© Springer Nature Switzerland AG 2020 49

scientific literature by the term “metabiotics” (Ardatskaya 2015; Ploskireva 2016;

© Springer Nature Switzerland AG 2020 53

Table 1 The techniques of removal of live starter microorganisms, destruction of microbial

Low molecular weight bioactive compounds formed by commensal and symbi-

Commercially available metabiotics can be divided according to their composition

© Springer Nature Switzerland AG 2020 57

Table 1 Potential forms of № Forms of metabiotics

© Springer Nature Switzerland AG 2020 59

Cellular metabiotics contains in its composition non-living probiotic cells. We will

© Springer Nature Switzerland AG 2020 63

Helicobacter and Stomach Health

Probiotics in Helicobacter Therapy

The use of probiotics, as monotherapy or synergistic therapy (in combination with

Mechanisms by which probiotics work in this application include suppressive

The Helinorm/Pylopass Strategy

Reducing the amount of H. pylori in the stomach by selective bacterial-bacterial cell

The Pylopass Strain

Pylopass specifically co-aggregates with H. pylori under in vitro conditions and

Expression of co-aggregation activity is dependent on the growth phase of L. reuteri

To understand if proteins are involved in binding, the susceptibility to protease

Moscow, Russia Boris A. Shenderov

Cellular Metabiotics and Metabolite Metabiotics�� 63

o rigin often act as regulators of intra- and interpopulation interaction (nervous,

intestine actively participate in the proteolysis of endogenous and exogenous pro-

Helicobacter and Stomach Health

Probiotics in Helicobacter Therapy

The Helinorm/Pylopass Strategy

The Pylopass Strain

Cell Walls Exhibit the Co-Aggregation Sites

The Power of Metabiotic Formulation

above- mentioned biological characteristics of B. subtilis are strain-specific

Modulators of Energy Metabolism

Modulators of Antioxidant Status

Modulators of Intercellular Information Exchange

Modulators of Neuropsychic and Social Behavior

Modulators of Epigenome Regulation

Metabiotics on the Base of Microbial Gas Molecules

ybrid Multicomponent Microbial Molecules as a Base

ther Contemporary Examples of the Techniques Needed

odifications in the composition and functions of its microbial symbiotic compo-