LWT - Food Science and Technology 125 (2020) 109246

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LWT - Food Science and Technology

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The potential of new bionic acids as prebiotics and antimicrobials T

a a b,∗
Aleksandra Wojciechowska , Robert Klewicki , Elżbieta Klewicka
a
Institute of Food Technology and Analysis, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Łódź, 90-924, Poland
b
Institute of Fermentation Technology and Microbiology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Łódź, 90-924, Poland

A R T I C LE I N FO A B S T R A C T

Keywords: A study was made to determine the effect of galactosyl derivatives of hydroxy acids on selected microorganisms
Hydroxy acid galactoside with probiotic features and on pathogenic bacteria associated with food, the digestive system and human skin. It
Lactobionic acid was shown that the highest tested concentration (0.05 M) of the gluconic acid and glucoheptonic acid derivatives
Lactic acid bacteria had a more favorable effect on the development of Lactobacillus bacteria than the corresponding concentration of
Pathogenic bacteria
lactobionic acid. The new bionic acids added to the culture medium at appropriate concentrations (0.05 M or
0.025 M gluconic acid derivative, 0.025 M or 0.0125 M glucoheptonic acid derivative) clearly stimulated the
growth of L. brevis strain MG451814.1. The derivatives also supported the formation of biofilm by selected
strains of L. acidophilus, L. brevis and L. casei. Antibacterial activity of gluconic and glucoheptonic acid deriva-
tives was observed against selected strains: S. Choleraesuis, S. epidermidis and Staphylococcus aureus. This pro-
vides a prospect of limiting the development of antibiotic-resistant strains. It is anticipated that the newly ac-
quired compounds have potential use as ingredients of dietary supplements, pharmaceutical preparations or
cosmetics, because of the properties demonstrated as well as their similarity to currently known bionic acids.

1. Introduction oligosaccharides or bionic acids.

Galactooligosaccharides have been classified as prebiotics. Such
Globalization and the pace of technological development in the substances are not digested in the human digestive tract, but they sti-
production of food favor the occurrence and spread of pathogenic mi- mulate the growth of beneficial intestinal microflora (Moreno, Corzo,
croorganisms (King et al., 2017; Soon, Brazier, & Wallace, 2020). In Montilla, Villamiel, & Olano, 2017). Cardelle-Cobas et al. (2011) tested
addition, the frequent use of antibiotics in the treatment of humans and in vitro the growth of bacteria of the genus Lactobacillus, Streptococcus
farm animals increases the number of antibiotic-resistant strains. In and Bifidobacterium on media containing as the sole carbon source se-
food production and mass catering operations, pathogenic bacteria lected trisaccharides (GOS) obtained from lactose (La) and lactulose
from the genera Staphylococcus, Enterococcus, Listeria and Salmonella (Lu); 6′-Gal-La and 6′-Gal-Lu allowed the growth of microorganisms at a
pose a significant health risk (European Food Safety Authority & similar or higher level as compared to lactose. In studies by Oh et al.
European Centre for Disease Prevention and Control, 2015). Probiotics (2017) there was an increase in almost all tested strains of the genus
are a factor that reduces the above-mentioned risk (by antagonistic Bifidobacterium in a medium containing GOS as the sole carbon source,
activity towards pathogenic microflora). Therefore, it is extremely im- while pathogens such as Listeria monocytogenes, Salmonella Typhi-
portant to look for new factors that stimulate the growth of probiotic murium and Enterococcus faecalis showed a minimal increase in analo-
microorganisms, while simultaneously limiting the increase in the gous conditions. Only Bacteroides fragilis, Clostridium ramosum and En-
number of antibiotic-resistant strains of pathogenic bacteria. terobacter cloacae had the ability to metabolize GOS.
Commercially available transgalactosylation products, as well as Ladirat et al. (2014) studied the influence of GOS on the re-
bionic acids produced by traditional methods, have found applications construction of intestinal microbiota after treatment with popular an-
in such fields as medicine and pharmacy, as well as in food and cos- tibiotics (amoxicillin, ciprofloxacin, doxycycline, clindamycin). GOS
metics (Alonso, Rendueles, & Diaz, 2013; Green, Yu, & Van Scott, 2009; supported the development of microbiota in a selective manner. After
Schaafsma, 2008). It may therefore be assumed that new compounds the decline in bifidobacteria under the influence of amoxicillin, re-
(with a structure similar to bionic acids) obtained through transga- generation of mainly the Bifidobacterium longum strain was observed. In
lactosylation of hydroxy acids will have bioactive properties similar to contrast, the presence of GOS did not affect the quantity of bacteria

∗
Corresponding author.
E-mail address: elzbieta.klewicka@p.lodz.pl (E. Klewicka).

https://doi.org/10.1016/j.lwt.2020.109246
Received 27 November 2019; Received in revised form 28 February 2020; Accepted 4 March 2020
Available online 05 March 2020
0023-6438/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
A. Wojciechowska, et al. LWT - Food Science and Technology 125 (2020) 109246

from the genera Bacteroides, Clostridium and Enterobacteriaceae, which monogastric animals (Klingeberg, Kozianowski, Kunz, Munir, & Vogel,
include pathogenic species for humans and animals. 2003) and as an active ingredient in antibacterial compositions, for
Wichienchot, Hemmaratchirakul, Jaturapiree, and Pruksasri (2016) example those added to food (Araki, Fuji, Ueno, Fuji, & Takahane,
compared the effect of a commercial preparation and GOS synthesized 2006).
by L. pentosus var. plantarum on fecal microorganisms obtained from The aim of the present study was to characterize the selected
adult healthy donors. Commercial GOS had a higher prebiotic index bioactive properties of galactosyl derivatives of gluconic and gluco-
and resulted in the growth of both lactic bacteria and bifidobacteria. heptonic acids: prebiotic activity against bacteria of the genus
The size of the Clostridium group decreased at the same time. GOS ob- Lactobacillus of intestinal origin and isolated from food, and anti-
tained using L. pentosus var. plantarum clearly increased the synthesis of bacterial activity against bacteria associated with the digestive system
acetic acid. and the human epidermis.
Cruz-Guerrero et al. (2014) showed that the cultivation of lacto-
bacilli in the presence of Oligomate 55® (55% w/w GOS) as a carbon 2. Materials and methods
source strongly stimulated the production of short-chain fatty acids,
H2O2 and bacteriocins. The resulting substances led to a significant (up 2.1. Materials
to 50%) slowing in the growth of pathogens (Escherichia coli, Listeria
innocua and Micrococcus luteus). According to Xin, Liang, and Wang Lactobionic acid (Carl Roth GmbH + Co. KG, Karlsruhe, Germany),
(2015), GOS stimulate B. longum and L. plantarum to produce exopo- lactic acid (Chempur, Piekary Śląskie, Poland), MRS Broth (Merck,
lysaccharides, which leads to antibacterial activity against E. coli. Darmstadt, Germany), Nutrient Broth (Merck, Darmstadt, Germany),
Sangwan, Tomar, Ali, Singh, and Singh (2015) tested a diet with a Bacto-Agar (DIFCO Laboratories, Detroit, Michigan, USA), DMSO
supplement of 6% GOS in mice infected with L. monocytogenes. Ga- (Sigma-Aldrich, Poznań, Poland), PBS (Sigma-Aldrich, Poznań, Poland),
lactooligosaccharides increased the concentration of IgA (small intes- crystal violet (Merck, Darmstadt, Germany), 96% ethanol (Stanlab,
tine) and IgG (blood) antibodies. This led to a decrease in the number of Lublin, Poland), pure disks for antibiogram (Oxoid Ltd, Basingstoke,
pathogens in the intestines, liver and spleen. UK), antibiotic disks (Oxoid Ltd, Basingstoke, UK), strains of
Kondepudi, Ambalam, Nilsson, Wadström, and Ljungh (2012) re- Lactobacillus bacteria: L. casei ŁOCK 0979, L. brevis ŁOCK 0944, L. brevis
ported the high antimicrobial activity of bifidobacteria against Clos- MG451814.1 (number in the NCBI database), L. brevis MG451813.1, L.
tridium difficile. The production of the NAP1/027 toxin was stopped. casei ŁOCK 0848, L. acidophilus ŁOCK 0937, L. acidophilus ŁOCK 0927,
This effect resulted from the production of appropriate organic acids in L. acidophilus ŁOCK 0926, L. acidophilus ŁOCK 0842 (ŁOCK – Łódzki
the presence of prebiotic oligosaccharides (including GOS, FOS, lactu- Ośrodek Czystych Kultur; currently: Collection of Pure Cultures of
lose) in the nutrient medium. Industrial Microbes), pathogenic bacteria: Salmonella Choleraesuis
From a chemical point of view, bionic acids are aldobionic. They are ATCC 1402, Salmonella Enteritidis 1307, Staphylococcus aureus ATCC
obtained from disaccharide (e.g. lactose, maltose, melibiose) by che- 27734, 25923 and 29737, Staphylococcus epidermidis ATCC 1533,
mical or enzymatic oxidation. Bionic acids have a number of interesting Escherichia coli ATCC 10536, Enterococcus faecalis 29212, Listeria
properties, which enable their use in various branches of science and monocytogenes ATCC 19111 and 19112.
industry. The most commonly used acid of this type is lactobionic acid.
Lactobionic acid has certain features of dietary fiber. It is not me- 2.2. Methods
tabolized in the small intestine, it is a medium for intestinal microbiota,
and it has a prebiotic effect (Schaafsma, 2008). Saarela, Hallamaa, 2.2.1. Isolation of galactosyl derivatives of bionic acids from post-reaction
Mattila-Sandholm, and Matto (2003) showed that this acid is used as a mixture
carbon source by Lactobacillus paracasei E-97949, Lactobacillus rham- A quantity of 10 ml of post-reaction mixture (Wojciechowska,
nosus E-97800 and Lactobacillus rhamnosus E-97948. The bacterial Klewicki, & Sójka, 2019; Wojciechowska, Klewicki, Sójka, & Klewicka,
growth was comparable to that obtained with other lactose derivatives 2017) containing a selected derivative of gluconic acid or glucohep-
with prebiotic properties (lactulose and lactitol). Excessive concentra- tonic acid was passed through a column (300 × 20 mm) filled with
tion of lactobionic acid in the environment, however, leads to a de- cation exchanger (Amberlite IR 120, hydrogen form, Sigma-Aldrich) to
crease in pH below 5 and may inhibit the growth of probiotic bacteria. remove metal ions. The collected fraction was concentrated to 30 °Bx
This effect was observed by Adebola, Corcoran, and Morgan (2014) for using an evaporator. Then 10 ml of the concentrated sample was loaded
L. reuteri NCIMB 11951 and L. acidophilus NCFM strains. When the onto a preparative chromatography column (1000 × 33 mm) con-
concentration of acid in the medium was 5%, the pH dropped below 4 taining cationite (Dowex 50WX4 hydrogen form, 100–200 mesh, Alfa
and growth of lactic bacteria was not observed. In a study by Kontula Aesar GmbH, Karlsruhe, Germany) to separate the acidic substrate.
et al. (2000) a number of strains of lactic acid bacteria were tested. L. Process conditions: mobile phase – water; flow rate 4.5 ml/min; tem-
salivarius most effectively used lactobionic acid (as compared to lactu- perature 40 °C; UV (210 nm) and RI detectors (KNAUER, Berlin, Ger-
lose, lactitol and arabinoxylo-oligosaccharides) as a carbon source. This many) connected in series. The separation was repeated many times;
acid also significantly increased the later growth of L. cellobiosus, which fractions (9 ml of each) with the highest product content were col-
was isolated from an environment enriched with arabinoxylo oligo- lected. The fractions were combined and concentrated to a volume of
saccharides. 5 ml. In the next purification step (separation of saccharide residues)
Bionic acid may also be a component used to support the anti- the sample was passed through a column (50 × 9 mm) filled with
bacterial activity of other substances. A study by Chen and Zhong anionite (Amberlite IRA-67 free base, Sigma-Aldrich) and eluted with
(2017) on a combination of nisin, thymol and lactobionic acid showed water (fraction discarded). The galactosyl derivative of acid retained on
that the minimum inhibitory concentrations (MIC) for L. monocytogenes the bed was released by rinsing the bed with 0.1% NaOH solution. Then
in the mixture were 31.25 IU/ml, 0.0625 mg/ml and 1.25 mg/ml re- the eluent was loaded onto a column (50 × 9 mm) containing cationite
spectively. For comparison, when each substance was used separately, (Amberlite IR120, hydrogen form, Sigma-Aldrich) to remove sodium.
the corresponding values were 125 IU/ml, 0.25 mg/ml and 10.0 mg/ The collected solution was concentrated with an evaporator.
ml.
Because of its properties, lactobionic acid can be used as a stimulant 2.2.2. Prebiotic activity of bionic acids towards bacteria of the genus
for the growth of bifidobacteria directly in culture and food products Lactobacillus
(Suguri, Yanagidaira, Kobayashi, & Shukke, 1995), as an additive to Solutions of 0.1 M gluconic acid and glucoheptonic acid derivatives
feed, supporting the treatment of bacterial intestinal infections in (isolated from the post-reaction mixture) and commercial lactobionic

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Table 1 Petri dishes (9 cm in diameter) were filled with 0.1 ml of 24-h liquid
Incubation conditions. cultivation of the selected pathogenic strains, and then Nutrient Agar
Strain Temperature [°C] Atmosphere 5% (v/v) CO2 was poured out. The density of the turf obtained was in the range
106–107 CFU/ml. After solidification of the substrate, clean disks were
L. acidophilus ŁOCK 0842 37 + applied to the antibiogram and soaked with 15 μl of the selected
L. acidophilus ŁOCK 0926 37 +
compound solution or 5% DMSO solution (negative test). For the po-
L. acidophilus ŁOCK 0927 37 +
L. acidophilus ŁOCK 0937 37 +
sitive sample, ready-made disks containing the appropriate antibiotic
L. brevis ŁOCK 0944 30 – were used (doxycyline for E. faecalis, vancomycin for other strains). The
L. brevis MG451813.1 30 – plates were incubated at 37 °C for at least 18 h until the growth of the
L. brevis MG451814.1 30 – tested bacteria in the form of turf. After this time, the diameter of the
L. casei ŁOCK 0848 37 +
zone of inhibition of microbial growth around the disks was de-
L. casei ŁOCK 0979 37 +
termined. The diameter of the disk (5 mm) was subtracted from the
read result. All tests were performed in triplicate and the results given
acid solution (0.1 M) were used. All solutions were filtered through as mean values (mm).
syringe filters (0.45 μm pore size) and diluted with sterile water 2, 4 or
8 times. 2.2.5. Statistical analysis
A quantity of 0.1 ml of MRS Broth (2 times concentrated) and 0.1 ml The results were examined statistically using one-way ANOVA
of solution of the selected acid at a defined concentration or 0.1 ml of (analysis of variance) and Duncan's post hoc test at a significance level
sterile distilled water were applied to a 96-well plate (Berthold 96). As a of p < 0.05 (Statistica 13 software, StatSoft Poland).
result, the concentration of compound in the final medium was 0.05,
0.025, 0.0125, 0.00625 or 0 M. Then 10 μl of liquid cultivation (24-h) 3. Results and discussion
of the selected strain of bacteria of the genus Lactobacillus
(2 × 109 CFU/ml) was added. After an incubation time of 0 and 48 h 3.1. Growth of Lactobacillus spp. bacteria in media containing polyhydroxy
(under the conditions described in Table 1), the absorbance at 520 nm acids
was measured using a TriStar2S LB942 Multimode Reader (Berthold
Technology). The increase in absorbance (optical density) after 48 h Due to the poor state of knowledge about derivatives of hydroxy
relative to the time 0 h was determined. All experiments were carried acids obtained through transgalactosylation, it is difficult to find reports
out in triplicate and the results given as mean values. describing their bioactive properties, including interactions with mi-
croorganisms. However, their similarity to substances with prebiotic
properties (such as lactobionic acid or gal-polyols) indicates the pre-
2.2.3. Effect of bionic acids on the ability of lactic acid bacteria to adhere to
biotic potential of these compounds. Therefore, attempts have been
abiotic surfaces
made to characterize the properties of galactosyl derivatives of gluconic
Tests were carried out using filtered solutions of lactobionic acid
and glucoheptonic acids, including prebiotic activity towards lactoba-
(0.1 M, 35.8 g/l), a galactosyl derivative of gluconic acid (0.1 M,
cilli bacteria of intestinal origin and isolated from the food environ-
35.8 g/l) and a galactosyl derivative of glucoheptonic acid (0.1 M,
ment, as well as antibacterial activity against bacteria associated with
37.9 g/l; a mixture of the acid derivative and its lactone).
the digestive system and human skin.
A quantity of 0.1 ml of MRS Broth (2 times concentrated) and 0.1 ml
Lactobacillus spp. bacteria are among the most frequently reported
of solution of the selected acid at a defined concentration or 0.1 ml of
probiotics. They occur on mucous membranes of the gastrointestinal
sterile distilled water were applied to a 96-well plate (Berthold 96). As a
tract, in the urogenital system of humans and animals, on plants, and in
result, the concentration of compound in the final medium was 0.05 or
spontaneously fermented food. These microorganisms have received
0 M. Then 10 μl of liquid cultivation (24-h) of the selected strain of
GRAS status, that is, they have been recognized as safe for humans and
bacteria of the genus Lactobacillus (2 × 109 CFU/ml) was added. No
animals (Cukrowska et al., 2009; Martinez, Prado Acosta, Candurra, &
culture was introduced into the wells marked as a control. The plates
Ruzal, 2012). One of the important physiological features of lactobacilli
were incubated for 24 h under the conditions described in Table 1. After
is their antagonistic activity towards both Gram-positive and Gram-
this time, free bacterial cells were removed by washing the wells three
negative bacteria (Fang, Shi, Huang, Chen, & Wang, 1996). These
times with 0.1 ml of PBS buffer, pH 7.4. After the plate was dried,
bacteria prefer a slightly acidic environment, and it can therefore be
0.05 ml of a 0.05% crystal violet solution was added, and the plate was
assumed that galactosyl derivatives of hydroxy acids may promote their
incubated for 45 min at room temperature on a shaker (80 rpm). The
growth. Consequently, the growth of selected strains of Lactobacillus
wells were again purged with PBS buffer (rinsing was repeated three
spp. bacteria on media containing 0.00625 M–0.05 M lactobionic acid,
times). The crystal violet was then extracted with 0.1 ml of 96%
a galactosyl derivative of gluconic acid or a galactosyl derivative of
ethanol. After 10 min absorbance was measured (λ = 490 nm, Multi-
glucoheptonic acid was examined.
mode Reader TriStar2S LB942 reader, Berthold Technology). The re-
For almost all of the tested Lactobacillus spp. bacteria (significant
sults were expressed as the coefficient of biofilm formation (Cbf) – the
statistical differences occurred in 7 out of 9 cases; Figs. 1–3) growth
ratio of the absorbance for the sample with the test strain to the ab-
inhibition was observed in a medium containing lactobionic acid with
sorbance for the medium not inoculated with the culture. All tests were
the highest tested concentration of 0.05 M (about 1.8% w/v). A similar
performed in triplicate and the results given as average values.
phenomenon associated with a lowering of the pH was recorded by
Adebola et al. (2014): L. reuteri NCIMB 11951 and L. acidophilus NCFM
2.2.4. Antimicrobial activity of bionic acids against selected pathogenic strains were incapable of growing in a medium with the addition of 5%
bacteria of this acid.
Tests were carried out using filtered solutions of lactobionic acid The presence of a galactosyl derivative of gluconic acid or gluco-
(0.1 M, 35.8 g/l), galactosyl derivative of gluconic acid (0.1 M, 35.8 g/ heptonic acid at a concentration of 0.05 M resulted in a reduction in the
l) and galactosyl derivative of glucoheptonic acid (0.1 M, about 37.9 g/ multiplication of 4 bacterial strains. This effect was less intensive than
l; a mixture of the acid derivative and its lactone). To determine the in the case of lactobionic acid. The strain most susceptible to the ad-
antimicrobial activity of the compounds, the diffusion-disk technique dition of bionic acids was L. brevis MG451814.1 (Fig. 2C). In this case,
recommended for the preparation of the antibiogram was used the results were also the most diverse. All bionic acids at a concentra-
(EUCAST, 2017). tion of 0.025 M clearly stimulated the development of this strain. In

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Fig. 2. Absorbance after 48 h incubation of strain A) L. brevis ŁOCK 0944, B) L.

brevis ŁOCK MG451813.1, C) L. brevis ŁOCK MG451814.1 depending on the
medium used. 0 - MRS broth without the addition of bionic acid. With the
addition of lactobionic acid: 1–0.05 M, 2–0.025 M, 3–0.0125 M, 4–0.00625 M;
with the addition of gluconic acid derivative: 5–0.05 M, 6–0.025 M,
7–0.0125 M, 8–0.00625 M; with the addition of glucoheptonic acid derivative:
9–0.05 M, 10–0.025 M, 11–0.0125 M, 12–0.00625 M. For a given strain, values
with the same letter (a, b, c, d, e, f, g, h) do not differ statistically significantly,
p < 0.05.

contrast, the derivative of glucoheptonic acid (in the entire con-

centration range tested) caused a weakening of bacterial growth. It can
Fig. 1. Absorbance after 48 h incubation of strain A) L. acidophilus ŁOCK 0842,
therefore be concluded that the concentration of the substance used is
B) L. acidophilus ŁOCK 0926, C) L. acidophilus ŁOCK 0927, D) L. acidophilus
an important factor. In addition, the bacterial response to the presence
ŁOCK 0937, depending on the medium used. 0 – MRS broth without the ad-
dition of bionic acid. With the addition of lactobionic acid: 1–0.05 M, of the compound is a strain trait.
2–0.025 M, 3–0.0125 M, 4–0.00625 M; with the addition of gluconic acid de- Lactobionic acid has already been used in several experiments re-
rivative: 5–0.05 M, 6–0.025 M, 7–0.0125 M, 8–0.00625 M; with the addition of lating to prebiotic properties. It was an effective source of carbon for
glucoheptonic acid derivative: 9–0.05 M, 10–0.025 M, 11–0.0125 M, bacteria of the genus Lactobacillus (L. paracasei E-97949, L. rhamnosus E-
12–0.00625 M. For a given strain, values with the same letter (a, b, c, d, e) do 97800 and L. rhamnosus E-97948), comparable to other lactose deri-
not differ statistically significantly, p < 0.05. vatives (lactulose and lactitol) (Saarela et al., 2003). In turn, in the
studies of Kneifel, Rajal, and Kulbe (2000), 2 out of 17 strains of Lac-
contrast, the presence of 0.05 M lactobionic acid or a glucoheptonic tobacillus (L. rhamnosus LC705 and L. rhamnosus L56) showed at least
acid derivative resulted in a lower optical density after 48 h of in- 50% intensity of growth compared with medium containing only glu-
cubation compared with the standard medium. In turn, the same con- cose as a carbon source.
centration of the gluconic acid derivative led to a significant increase in Galactooligosaccharides (GOS), being prebiotics already recognized
the growth of L. brevis. The positive effect of lactobionic acid and the on the market, have been thoroughly tested. In vitro tests using selected
glucoheptonic acid derivative was also found for a concentration of GOS as the sole carbon source confirmed growth of Lactobacillus spp. to
0.0125 M. In the case of L. casei ŁOCK 0848 (Fig. 3A), the growth in- an equal or greater degree than when using lactose (Cardelle-Cobas
hibition in the presence of the highest concentration of acids (0.05 M) et al., 2011). The cultivation of lactobacilli, for example L. casei Shirota,
was noted. Inhibition of bacterial growth was also observed in samples L. rhamnosus or L. plantarum, in a medium containing GOS leads to
containing 0.025 M lactobionic acid. In the case of L. casei ŁOCK 0979 stimulation of the production of antimicrobial substances, including
(Fig. 3B), no growth inhibition was found after the use of lactobionic exopolysaccharides (Cruz-Guerrero et al., 2014; Xin et al., 2015). In a
and gal-gluconic acid at 0.05 M and 0.00625 M concentrations. In study conducted by Hernandez-Hernandez et al. (2012)

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Fig. 4. Coefficients of biofilm formation (Cbf) for individual Lactobacillus

acidophilus strains depending on the medium used: MRS - MRS without the
addition of bionic acid; MRS LA - MRS with the addition of 0.05 M lactobionic
acid; MRS GAD - MRS with the addition of 0.05 M gluconic acid derivative;
MRS GhAD - MRS with the addition of 0.05 M glucoheptonic acid derivative.
For a given strain, the values with the same letter (a, b) do not differ statistically
significantly, p < 0.05.

Fig. 3. Absorbance after 48 h incubation of strain A) L. casei ŁOCK 0848, B) L.

casei ŁOCK 0979 depending on the medium used. 0 - MRS broth without the
addition of bionic acid. With the addition of lactobionic acid: 1–0.05 M,
2–0.025 M, 3–0.0125 M, 4–0.00625 M; with the addition of gluconic acid de-
rivative: 5–0.05 M, 6–0.025 M, 7–0.0125 M, 8–0.00625 M; with the addition of
glucoheptonic acid derivative: 9–0.05 M, 10–0.025 M, 11–0.0125 M,
12–0.00625 M. For a given strain, values with the same letter (a, b, c, d) do not
differ statistically significantly, p < 0.05.

galactooligosaccharides also had a favorable effect on the growth of L.

bulgaricus, L. casei, L. delbrueckii subsp. lactis, L. plantarum and L. sakei.
However, there was a slower synthesis of lactic acid compared with
cultivation in the presence of glucose or lactulose.

3.2. Adhesiveness of Lactobacillus spp. bacteria in the presence of

polyhydroxy acids
Fig. 5. Coefficients of biofilm formation (Cbf) for individual Lactobacillus brevis
One of the important characteristics of strains of the Lactobacillus strains depending on the medium used: MRS - MRS without the addition of
genus is their adhesiveness. Because of this property, these bacteria can bionic acid; MRS LA - MRS with the addition of 0.05 M lactobionic acid; MRS
compete with other microorganisms (including pathogens) for a place GAD - MRS with the addition of 0.05 M gluconic acid derivative; MRS GhAD -
MRS with the addition of 0.05 M glucoheptonic acid derivative. For a given
on the intestinal epithelium. Suppression of adhesion of pathogenic
strain, values with the same letter (a, b, c) do not differ statistically sig-
bacteria can limit the spread of these microorganisms, and conse-
nificantly, p < 0.05.
quently the occurrence of infections (Ma, Guo, Xu, You, & Ma, 2006;
Tuomola, Ouwehand, & Salminen, 1999). The introduction of bionic
acids into the growth environment of Lactobacillus strains may affect acidic transgalactosylation products on the adhesion of bacteria is ab-
their ability to adhere. Therefore, the coefficient of biofilm formation sent. Among the few data available are the results of in vitro studies by
(Cbf) was determined for selected strains of lactobacilli grown in a Kontula et al. (2000). They suggest that lactobionic acid can stimulate
medium with the addition of 0.05 M specific bionic acid (or without not only growth, but also the adhesion of the Lactobacillus cellobiosus
additive). strain to Caco-2 test cells, compared with other carbohydrate sources.
A comparison of Cbf values for 4 of the 9 strains showed significant
statistical differences between the media used (Figs. 4–6). It is therefore 3.3. Antipathogenic activity of Lactobacillus spp. bacteria in the presence of
possible to indicate bionic acids as a factor stimulating the adhesion of polyhydroxy acids
bacteria of the Lactobacillus genus. The addition of galactosyl derivative
of glucoheptonic acid to the medium resulted in an increase in Cbf for The study determined the antibacterial potential of bionic acids as
all strains, to varying degrees. Similarly, a beneficial effect on 7 and 8 direct factors inhibiting the development of selected bacteria associated
probiotic strains was demonstrated by galactosyl derivative of gluconic with the digestive system and human skin. The test strains included
acid and lactobionic acid respectively. Statistical differences were noted Salmonella Choleraesuis and Salmonella Enteritidis, Staphylococcus
in particular for L. brevis MG451814.1 (Fig. 5). The observed decrease aureus and Staphylococcus epidermidis, Escherichia coli, Enterococcus
in Cbf after culturing L. acidophilus strains ŁOCK 0842 and ŁOCK 0926 faecalis and Listeria monocytogenes. The results of the disk test are pre-
(Fig. 4) in the presence of gluconic acid derivative was statistically sented in Table 2. Among the analyzed bionic acids, lactobionic acid
insignificant. exhibited the strongest antibacterial activity, and at the same time had
The experiments described here can undoubtedly be considered an the ability to inhibit 6 out of 10 tested strains. The gluconic acid de-
important novel element of this work, because the available literature rivative was shown to inhibit the development of S. Choleraesuis ATCC
on this subject is very limited, and literature describing the impact of 1402, S. aureus ATCC 27734 and S. epidermidis ATCC 1533. The

5
A. Wojciechowska, et al. LWT - Food Science and Technology 125 (2020) 109246

(Shoaf, Mulvey, Armstrong, & Hutkins, 2006). Studies on mice infected

with L. monocytogenes showed that a diet containing GOS resulted in an
increase in the corresponding antibodies and, as a result, a decrease in
the number of pathogens (Sangwan et al., 2015).
Most of the available information, however, relates to the indirect
inhibition of pathogens, through the beneficial effects of specific com-
pounds on probiotic bacteria. GOS strongly stimulate strains of
Lactobacillus casei and Lactobacillus rhamnosus for the production of
antimicrobial compounds (Cruz-Guerrero et al., 2014), as well as Bifi-
dobacterium longum and Lactobacillus plantarum strains for the synthesis
of exopolysaccharides (Xin et al., 2015). In both cases, this resulted in
an antibacterial effect, among others in relation to E. coli. The presence
of GOS in a joint culture of Lactobacillus kefiri and Listeria monocytogenes
causes the growth of probiotic bacteria while inhibiting pathogen de-
velopment (Likotrafiti, Valavani, Argiriou, & Rhoades, 2015). Among
other transgalactosylation products, polyol derivatives (e.g. gal-xylitol)
deserve attention. Their addition to the culture medium has been
shown to broaden the antagonistic activity of Lactobacillus spp. against
Fig. 6. Coefficients of biofilm formation (Cbf) for individual Lactobacillus casei fungi (Alternaria alternata, A. brassicicola, Aspergillus niger, Fusarium la-
strains depending on the medium used: MRS - MRS without the addition of tenicum, Geotrichum candidum, Mucor hiemalis) and yeast (Candida vini)
bionic acid; MRS LA - MRS with the addition of 0.05 M lactobionic acid; MRS (Klewicka, 2007; Lipińska et al., 2016).
GAD - MRS with the addition of 0.05 M gluconic acid derivative; MRS GhAD -
MRS with the addition of 0.05 M glucoheptonic acid derivative. For a given 4. Conclusions
strain, values with the same letter (a, b) do not differ statistically significantly,
p < 0.05.
Derivatives of gluconic acid and glucoheptonic acid in a con-
centration of 0.05 M have a less inhibitory effect on the development of
glucoheptonic acid derivative was antibacterial for S. aureus ATCC bacteria of the Lactobacillus genus than the corresponding concentration
27734 and S. epidermidis ATCC 1533. of lactobionic acid.
The mechanisms of antagonistic activity of acidic compounds in- Bionic acids can be used as an agent to stimulate the adhesion of
volve increasing the permeability of the bacterial cell wall, as has been bacteria of the Lactobacillus genus; the intensity of changes in the
documented in the case of lactic acid (Alakomi et al., 2000). The sen- coefficient of biofilm formation depends on the substance used and the
sitivity of various groups of microorganisms to the pH of the environ- strain being tested.
ment is varied. The results of this study also indicate different reactions, Bionic acids exhibit antibacterial activity. Of the compounds ana-
for example between individual strains of S. aureus. It should also be lyzed, lactobionic acid has the strongest antibacterial activity, and at
remembered that the solution of the glucoheptonic acid derivative in the same time has the ability to inhibit the majority of tested patho-
fact contained a mixture of the derivative in the acid form and in the genic strains. The gluconic acid derivative has the ability to inhibit the
form of lactone, and therefore its effect may have been weakened. development of S. Choleraesuis ATCC 1402, S. aureus ATCC 27734 and
Among the literature data, only a few reports are available con- S. epidermidis ATCC 1533. The glucoheptonic acid derivative has an
cerning the direct antibacterial activity of lactobionic acid. For ex- antibacterial effect on the S. aureus ATCC 27734 and S. epidermidis
ample, a 4% content of this acid has been shown to contribute to a ATCC 1533 strains.
slowing of the growth of S. aureus isolates in liquid culture. As a result, In conclusion, the results of this study indicate the high potential of
it may be used as a component of preparations used for dry eye syn- new transgalactosylation products as non-antibiotic antibacterial sub-
drome (Olivieri et al., 2018). Chen and Zhong (2017) reported on the stances.
antibacterial activity of lactobionic acid against L. monocytogenes. Fur-
thermore, it is described as an active component of a patented anti- CRediT authorship contribution statement
bacterial composition for use in food (Araki et al., 2006). Other trans-
galactosylation products – galactooligosaccharides – have the ability to Aleksandra Wojciechowska: Formal analysis, Investigation,
reduce the adhesion of E. coli to the surface of intestinal epithelial cells, Resources, Writing - original draft, Visualization. Robert Klewicki:
which is also considered as a method of direct prevention of infections Conceptualization, Investigation, Writing - original draft, Writing -

Table 2
Zone of growth inhibition of pathogenic bacteria in the presence of bionic acids.
Strain 0.1 M lactobionic acid 0.1 M gluconic acid derivative 0.1 M glucoheptonic acid derivative antibiotica

diameter of zone [mm]

S. Choleraesuis ATCC 1402 6.33 ± 0.47 3.50 ni 3.33 ± 0.47
S. Enteritidis 1307 8.33 ± 0.94 ni ni 5.00 ± 0.00
S. aureus ATCC 27734 6.66 ± 1.70 4.50 6.00 14.33 ± 0.47
S. aureus ATCC 25923 6.66 ± 0.47 ni ni 9.00 ± 0.00
S. aureus ATCC 29737 ni ni ni 14.33 ± 0.47
S. epidermidis ATCC 1533 22.33 ± 1.25 13.66 ± 0.47 9.66 ± 0.94 36.00 ± 1.41
E. coli ATCC 10536 4.00 ± 0.82 ni ni 5.33 ± 0.47
E. faecalis 29212 ni ni ni 28.00 ± 0.82
L. monocytogenes ATCC 19111 ni ni ni 22.33 ± 3.30
L. monocytogenes ATCC 19112 ni ni ni 16.66 ± 1.70

ni – no inhibition.
a
In the case of E. faecalis doxycycline was used; for other strains – vancomycin.

6
A. Wojciechowska, et al. LWT - Food Science and Technology 125 (2020) 109246

review & editing. Elżbieta Klewicka: Methodology, Investigation, Kondepudi, K. K., Ambalam, P., Nilsson, I., Wadström, T., & Ljungh, Å. (2012). Prebiotic-
Resources, Writing - original draft, Writing - review & editing. non-digestible oligosaccharides preference of probiotic bifidobacteria and anti-
microbial activity against Clostridium difficile. Anaerobe, 18, 489–497. https://doi.
org/10.1016/j.anaerobe.2012.08.005.
Declaration of competing interest Kontula, P., Suihko, M.-L., Suortti, T., Tenkanen, M., Mattila-Sandholm, T., & von Wright,
A. (2000). The isolation of lactic acid bacteria from human colonic biopsies after
enrichment on lactose derivatives and rye arabinoxylo-oligosaccharides. Food
The authors declare that they have no known competing financial Microbiology, 17, 13–22. https://doi.org/10.1006/fmic.1999.0268.
interests or personal relationships that could have appeared to influ- Ladirat, S. E., Schuren, F. H. J., Schoterman, M. H. C., Nauta, A., Gruppen, H., & Schols, H.
ence the work reported in this paper. A. (2014). Impact of galacto-oligosaccharides on the gut microbiota composition and
metabolic activity upon antibiotic treatment during in vitro fermentation. FEMS
Microbiology Ecology, 87, 41–51. https://doi.org/10.1111/1574-6941.12187.
Acknowledgement Likotrafiti, E., Valavani, P., Argiriou, A., & Rhoades, J. (2015). In vitro evaluation of
potential antimicrobial synbiotics using Lactobacillus kefiri isolated from kefir grains.
International Dairy Journal, 45, 23–30. https://doi.org/10.1016/j.idairyj.2015.01.
The work was financed from statutory funds of the Institute of Food
013.
Technology and Analysis, Faculty of Biotechnology and Food Sciences, Lipińska, L., Klewicki, R., Klewicka, E., Kołodziejczyk, K., Sójka, M., & Nowak, A. (2016).
Lodz University of Technology, Poland. The antifungal activity of Lactobacillus sp. strains in the presence of xylitol and ga-
lactosyl xylitol. BioMed Research International, 5897486, 8. https://doi.org/10.1155/
2016/5897486.
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