Food Chemistry 430 (2024) 137006

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Bacterial, short-chain fatty acid and gas profiles of partially hydrolyzed

guar gum in vitro fermentation by human fecal microbiota
Xiong-E Pi a, *, Hao Fu a, Xiao-Xia Yang b, Zai-Chun Yu b, Wei-Lin Teng c, Yinjun Zhang b,
Xue-Wei Ye d, Hui Hui Quan a, Li-Zhi Lu a, *, Wei Liu a, *
a
State Key Laboratory for Managing Biotic and Chemical Treats to the Quality and Safety of Agro-Products, Zhejiang Academy of Agricultural Sciences, Hangzhou
310022, China
b
College of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China
c
Department of Infectious Disease Control and Prevention, HangZhou Center for Disease Control and Prevention, Hangzhou 310006, China
d
Shulan International Medical College, Department of Basic Medical Sciences, Zhejiang Shuren University, Hangzhou 310015, China

A R T I C L E I N F O A B S T R A C T

Keywords: Carbohydrates with different structures have metabolic differences in the human body, as well as individual
Partially hydrolyzed guar gum differences. The present study aimed to investigate the effects of bacterial, short-chain fatty acids (SCFAs) and
Gas production gas profiles of partially hydrolyzed guar gum (PHGG) on the fecal microbiota of 41 Chinese individuals by
SCFAs
simulated fermentation in vitro. Results showed that PHGG stimulated the growth of Bifidobacterium and Fae­
Gut microbiota
calibacterium, inhibited the growth of Escherichia-Shigella, Klebsiella, and Dorea, and induced the production of
fermentation gases (CO2, and H2) and SCFAs (acetic acid, butyric acid). Furthermore, Bifidobacterium was
significantly increased in the young female and the old male-originated samples, while Klebsiella was signifi­
cantly decreased in the old female ones after PHGG intervention, and there were also certain differences in gases
and SCFAs among different population samples. These findings indicate that PHGG can modulate gut microbiota
and metabolism well, whereas its use varies in different populations.

1. Introduction carob tree (Ceretonia siliqua) and is important sources of galactomannan

(von Freiesleben et al., 2019). Partially Hydrolyzed Guar Gum (PHGG),
Human health and disease are inextricably linked to gut microbiota. a guar gum hydrolysate, and is a novel functional dietary fiber raw
Prebiotics are beneficial to the host’s health because they cannot be material, which contains high amount of total dietary fiber (Mercier &
digested by the host but are preferentially metabolized by the gut Campargue, 2012). It is made up of β-1,4-D-mannose residue. Every 2
microbiota (Gibson et al., 2020). According to numerous scientific mannose contains a 1,6-α-D-galactose residue (Hu et al., 2019).The
studies, prebiotics are vital for human health. Dietary fibers or prebiotics composition of PHGG varies slightly due to different preparation pro­
are well-known factors modulating microbiota composition and func­ cesses. The total dietary fiber content of PHGG reached 90.6% in this
tion (Hoving et al., 2018). In the digestive tract, carbohydrate- experiment. Which is composed of mannose and galactose units in 2:1
containing fibers are commonly used as prebiotics because they pro­ ratio (Fig. S1 and Table S1). The PHGG is water-soluble and has a pre­
duce SCFAs during fermentation and beneficial metabolites for the body biotic effect by increasing the number of Bifidobacterium and Lactoba­
(Kim et al., 2018), However, the metabolism of prebiotics may vary cillus, as well as the amount of SCFAs (Yasukawa et al., 2019). PHGG has
among different populations due to differences in gut microbiota, and a variety of functions, among them, relieving abdominal distension, and
the functional differences of each prebiotic for different populations are other symptoms associated with functional gastrointestinal diseases,
also different. The metabolic differences of prebiotics among different including irritable bowel syndrome (IBS) (Niv et al., 2016). Further­
populations have been rarely studied. more, PHGG fermented by the gut microbiota increases the abundance
Guar gum, produced by the seeds of the guar plant (Cyamopsis tet­ of butyric acid. It is one of the essential energy sources of rectal
ragonolobus), and locust bean gum, which is enriched in the seeds of the epithelial cells. SCFAs have been linked to the regulation of gut health,

* Corresponding authors.
E-mail addresses: pixe@zaas.ac.cn (X.-E. Pi), zhangyj@zjut.edu.cn (Y. Zhang), 601215@zjsru.edu.cn (X.-W. Ye), quanhh@zaas.ac.cn (H.H. Quan), lulz@zaas.ac.cn
(L.-Z. Lu), liuw@zaas.ac.cn (W. Liu).

https://doi.org/10.1016/j.foodchem.2023.137006
Received 1 December 2022; Received in revised form 17 July 2023; Accepted 24 July 2023
Available online 27 July 2023
0308-8146/© 2023 Published by Elsevier Ltd.
X.-E. Pi et al. Food Chemistry 430 (2024) 137006

metabolism, and immunization (Koh, De Vadder, Kovatcheva-Datchary, 2.2. Growth conditions of human intestinal microbiota
& Bäckhed, 2016). However, there are significant physiological differ­
ences in the utilization of PHGG among different populations, which Preparation of control medium: 10 g tryptone, 2.5 g yeast extract, 1 g
may be closely related to differences in gut micro-biota among different L-cysteine, 2 mL heme solution, 0.9 g NaCl, 0.09 g CaCl2⋅6H2O, 0.45 g
populations. Currently, there is no research focusing on the differences KH2PO4, 0.45 g K2HPO4, and 0.09 g MgSO4⋅7H2O, 200 µL Vitamin I
in PHGG metabolism caused by gut microbiota among different solution, 1 mL resazurin solution, dissolved in 1 L of deionized water and
populations. stirred by magnetic stirrer. The PHGG medium is to add PHGG to control
The gastrointestinal tract is the body’s largest microecosystem. The medium at a ratio of 0.8 g/100 mL. After dissolving and boiling, nitro­
human gut microbiota is a dynamic system that begins after birth and is gen was filled to keep the liquid medium free of oxygen. Then, 4.5 mL
dominated by Bifidobacteria before being dominated by Bacteroidetes the medium was dispensed into 10 mL vials using a peristaltic pump
and Firmicutes in adult humans (Ottman, Smidt, De Vos, & Belzer, from Longer Co., Ltd. (Baoding, China) under anaerobic conditions.
2012). Studies have demonstrated that the composition of gut micro­ Then sealed the sample vials with lids, and autoclaved it for 15 min at
biota in the elderly population (78 ± 8 years) differs from that of the 115 ◦ C and 101 kPa by a heating pressure steam sterilizer from Shen An
human adult (36 ± 6 years) gut microbiota, resulting in a susceptible gut Co., Ltd. (Shanghai, China) (Chen et al., 2021).
system with impaired digestion and immunity (Claesson et al., 2012).
Similarly, the human gut microbiota differs significantly across genders
at phylum (ratio of Firmicutes/ Bacteroidetes), genus (Bacteroides), and 2.3. Human raw fecal sample collection
species levels (Haro et al., 2016), with females having a lower Bacter­
oidetes abundance than males. There were 41 healthy human volunteers from Hangzhou, China,
Fermentations of fecal gut microbiota were used to investigate the including 11 males and 10 females aged between 20 and 30 years, and
relationship between gut health and diet (both whole foods and bioac­ 10 males and females aged between 40 and 60 years (Table 1), respec­
tive compounds) (Wang et al., 2019). In vitro fermentation models are tively. All volunteers in this study were free of digestive disorders and
typically closed anaerobic environments used to study the ability of had not taken any medication, including antibiotics or prebiotics, for at
animal or human gut microbiota to metabolize various dietary fibers. least 4 weeks before sample collection. The Ethics Committee of the
Thus, in vitro fermentation using pooled or individual fecal samples is an Hangzhou Center for Disease Control and Prevention approved the
easy and efficient method for studying fermentation performance and collection of stool samples from all volunteers who provided written
short-term microbiota changes in response to a specific prebiotic sub­ informed consent (No. 202047). When the volunteers or their parents
strate (Li et al., 2022). signed the informed consent, consent was given. In a 30 mL 91 mm ×
With advances in sequencing technology, especially 16S rRNA gene Φ24mm sterile stool sample box from BioRise Co., Ltd. (Shanghai,
sequencing, there is increasing interest in the effects of the gut micro­ China), at least 3 g of intermediate stools with less food residue and less
biota and its metabolites on the host. PHGG has significant clinical and contact with air after defecation were quickly selected, and the volun­
nutritional effects due to its prebiotic effect (Kapoor, Sugita, Fukuzawa, teer’s information was indicated. The collected samples were stored at
& Okubo, 2017) and differences in the metabolism of PHGG by the gut 4 ◦ C and the experiment was completed within 4 h. In a fume hood, the
microbiota in different populations deserve to be studied. However, stool samples were diluted tenfold with phosphate-buffered saline (PBS)
owing to the lack of methods to study intestinal gases, the gases pro­ and then filtered to remove large particles using a 55 mm × 28 mm 300-
duced by fermentation of the intestinal microbiota have received little mesh stool screen processor from Huatuo Co., Ltd. (Shanghai, China) (Pi,
attention compared to the increasing interest in metabolites such as Yu, et al., 2022; Tao et al., 2022).
SCFAs. These fermentation gases are also closely related to the health of
the host. Patients with functional gut disorders, irritable bowel disease,
and related syndromes frequently attribute their symptoms to intestinal 2.4. Simulated fermentation in vitro
gas. Studies of intestinal gas dynamics have demonstrated subtle dys­
functions in intestinal motility (Azpiroz, 2005). And different carbohy­ The fecal supernatant (500 μL) was inoculated into a separate me­
drate structures may also produce different gas components, thereby dium, mixed, and incubated at 37 ◦ C for 24 h under anaerobic and kept
affecting host health (Gonlachanvit, Coleski, Owyang, & Hasler, 2004). under airtight conditions to determine gas composition and quantity.
Consequently, the purpose of this study was to investigate the reg­ The fermentation broth was centrifuged at 9000 r/min for 3 min
ulatory pattern of PHGG in the fecal microbiota composition of different (Eppendorf Centrifuge 5424, Germany), and the resultant pellet was
populations using in vitro fermentation including differences in micro­ used for DNA extraction (Pi, Hua, et al., 2022b).
biota regulation, SCFA regulation, and especially metabolic gas differ­
ences. This study fully demonstrates the metabolic differences of PHGG
in human gut microbiota. Hopefully, extension of these studies can thus 2.5. Effect of PHGG on gas production
provide a theoretical basis for the compatibility of PHGG as a prebiotic.
The fermentation vials were removed after 24 h and data on gas
2. Materials and methods production were recorded using a fermentation gas analyzer (APES-BC5-
B) with five sensitive gas transducers from Empaer Technology Co., Ltd
2.1. Materials (Shenzhen, China) while the vials cooled to room temperature (Ye et al.,
2022).
Tryptone, NaCl, yeast extract, L-cysteine, KH2PO4, K2HPO4, heme,
vitamin I, MgSO4⋅7H2O, CaCl2⋅6H2O, crotonic acid, resazurin, and gal­ Table 1
actomannan were purchased from Sigma Chemical Co., Ltd. (St. Louis, Baseline characteristics of the sampled population.
MO, USA). Sample diluent (NaCl, 9 g/L, Na2SO4, 1 g/L, phosphate, 0.1 Samples Number of Age Delivery Antibiotics
g/L, sodium benzoate, 0.1 g/L), NaOH, acetonitrile, PMP-methanol, samples mode usage
HCl, TFA were purchased from Sangon Biotech Co., Ltd (Shanghai, Young Males 11 20–30 Stool sample No
China). Young 10 20–30
Females
Old Males 10 40–60
Old Females 10 40–60

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X.-E. Pi et al. Food Chemistry 430 (2024) 137006

2.6. SCFAs quantification 2016) plugin in Qiime2 (version 2020.2) (Bolyen et al., 2019) produced
Amplicon sequence variants (ASVs). Based on the SILVA 16S rRNA
The samples after fermentation were centrifuged to obtain the su­ database (v138) and the Naive Bayes consensus taxonomy classifier
pernatant before being acidified with crotonic acid metaphosphoric acid implemented in Qiime2, ASVs were assigned a taxonomic classification.
mixed solution for 24 h. After acidification, the supernatant was
centrifuged and filtered with a 0.22 µm aqueous microporous membrane 2.9. Statistical analysis
(Millipore Express, Germany). Then, 150 µL of the filtered solution was
aspirated into a sample vial. The gas chromatograph (GC-2010 Plus, All the data were presented as mean ± standard error of the mean
Shimadzu, Japan) was prepared, loaded with the sample after which the (SEM). SPSS 23.0 (IBM Corp., USA) was used for data statistical signif­
aging procedure was performed. Column temperature heating condi­ icance analysis. GraphPad Prism 8.0.1 (GraphPad Software, USA) was
tions: column temperature at 80 ◦ C for 1 min, 10 ◦ C/min increased to used to plot gases, SCFAs, and five bacterial genera. The Shapiro-Wilk
190 ◦ C and maintained for 0.50 min; then increased to 240 ◦ C at a rate of test evaluated whether the data were normally distributed. The paired
40 ◦ C/min and maintained for 5 min; Flame ionization detector (FID) t-test was conducted if the data conformed to the normal distribution;
temperature at 240 ◦ C; gasification chamber temperature at 240 ◦ C; otherwise, the paired Wilcoxon rank-sum test was used. PCoA mapping
carrier gas: nitrogen flow rate 20 mL/min, hydrogen flow rate 40 mL/ is based on the bray-Curtis distance matrix. The correlation heatmap
min, air flow rate 400 mL/min. The obtained data were recorded. Trans- was measured using the Spearman correlation coefficient. All the above
2-butenoic acid was used as an internal standard. The composition of data analysis and mapping were carried out on the online Majorbio
SCFAs in culture filtrates, including acetic, propionic, butyric acids, Cloud Platform (https://www.majorbio.com).
isobutyric acid, valeric acid, and isovaleric acid, was determined using
GC fitted with a thin-film capillary column DB-FFAP 30 m × 0.32 mm × 3. Results
0.50 μm (Agilent Technologies, USA) (Pi, Hua, et al., 2022a).
3.1. Fecal microbiota analysis before and after fermentation
2.7. PHGG preparation
We used 16S rRNA gene sequencing technology to analyze the fecal
PHGG (Partially Hydrolyzed Guar Gum) was provided by Beijing microbiota composition of 41 healthy Chinese volunteers before and
Guarrun Technology Co., Ltd., and the preparation method was as after fermentation with incubating feces in the PHGG medium. To
described. PHGG is prepared by enzymatic hydrolysis of guar gum. Guar minimize the effects of sequencing depth on alpha and beta diversity,
gum is extracted from guar beans which is a plant grown in India or the number of sequences from each sample was rarefied to 58,756 the
Pakistan, and its main component is galactomannan. The mRmMan5A is minimum sequence of samples, which still yielded an average Good’s
an enzyme that hydrolyzes guar gum from Rhizomucor miehei CAU432 coverage of 97.90%. The raw fecal microbiota of the sampled population
by directed evolution. It was transformed into Pichia pastoris GS115 for was mainly composed of Firmicutes, Actinobacteria, Proteobacteria, and
heterologous expression, and its β-mannanase activity in the fermenta­ Bacteroidetes. Interestingly, the abundance of Bifidobacterium in the gut
tion broth reached 72,626 U/mL after high-density fermentation. PHGG microbiota composition of young females was higher than that of other
was hydrolyzed by β-mannanase from guar gum at pH 7.0 and 65 ℃ for samples, while the abundance of Escherichia-Shigella in the male-
6 h. The average molecular weight of PHGG is 2.5 × 104 Da, with 24.9% originated samples was higher than the other samples (Fig. S3). The
oligosaccharides (degree of polymerization <7), and up to 90.6% di­ alpha-diversity is an important index to quantity measure microbial
etary fiber. The hydrolysis rate was 88%, and the conversion rate of diversity within each sample. The alpha-diversity results showed that
galactomannan reached more than 95% (Katrolia et al., 2013; Li et al., PHGG did not alter the microbiota diversity compared to controls,
2017). The method of PHGG preparation was showed in the supple­ although there were significant differences in the phylogenetic diversity
mentary method. The structure of PHGG was measured by high per­ (pd) index of the raw fecal samples compared to controls (Fig. 1A and B,
formance liquid chromatography (HPLC) and the peak map was showed Fig. S4A). The microbiota community composition of the raw fecal
in supplementary Fig. 1 and supplementary Table 1. samples, the control samples, and the PHGG-treated samples was
investigated by PCoA analysis based on the Bray-Curtis distance. The
2.8. 16S rRNA gene sequencing results showed that there was a statistically significant difference (p =
0.001) between the PHGG-treated samples and the raw fecal samples
Bacterial genomic DNA was isolated from samples using a Stool DNA and the control samples in terms of community composition (Fig. 1C). In
Kit (200) according to the manufacturer’s instructions (Omega Bio-Tek, addition, Veen plot analysis indicated that 198 bacterial genera were
USA). The concentration of extracted DNA was determined using a present in all three groups of samples, and 14 bacterial genera were
NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific Bio­ unique to the PHGG-treated samples (Fig. S4B). At the genus level, the
tek, USA), and its integrity and size were confirmed by agarose gel abundance of Escherichia-Shigella in the PHGG-treated samples were
electrophoresis (1.0%) and stored at − 20 ◦ C. Using an ABI GeneAmp® lower than in the control samples, whereas the abundance of Bifido­
9700 PCR thermocycler (ABI, CA, USA), the hypervariable region V3-V4 bacterium and Streptococcus was higher than in the control samples
of the bacterial 16S rRNA gene was amplified with primer pairs 341F (5′- (Fig. 1D). After 24 h of fermentation, the relative abundances of Bifi­
FCCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATC­ dobacterium, Streptococcus and Faecalibacterium in the PHGG-treated
TAAT-3′). The 16S rRNA gene was amplified using PCR as follows: initial samples were significantly increased than in the control samples,
denaturation at 95 ◦ C for 3 min, followed by 23 cycles of denaturation at while the relative abundances of Escherichia-Shigella (P < 0.001), Kleb­
95 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, and extension at 72 ◦ C for 45 s, siella and Dorea in the PHGG-treated samples were significantly lower
and single extension at 72 ◦ C for 10 min, and storage at 4 ◦ C. Products than in the control samples (Fig. S2).
were detected using 2.0% agarose gel electrophoresis. The PE libraries In addition, the effects of gender and age on microbial community
were constructed using the NEXTFLEX® Rapid DNA-Seq kit (Bioo Sci­ composition were evaluated by the bacterial abundance at the genus
entific, USA).DNA was sequenced on an Illumina NovaSeq sequencing level of fecal samples obtained from various populations. The results
system platform operated by Shanghai Majorbio Biomedical Technology showed that the abundance of Escherichia-Shigella significantly
Co., Ltd. (Shanghai, China). All human consensus sequencing data were decreased in four different populations when compared with the control
submitted to the National Center for Biotechnology Information Short samples, including young male PHGG-treated samples, old male PHGG-
Read Archive under accession no. PRJNA774827. treated samples, young female PHGG-treated samples and old female
Denoising the optimized sequences using the DADA2 (Callahan et al., PHGG-treated samples (Fig. 2A). The abundance of Bifidobacterium was

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X.-E. Pi et al. Food Chemistry 430 (2024) 137006

Fig. 1. Microbiota structure analysis of primary fecal samples and fermentation samples at the genus level. A and B. Alpha-diversity analysis; C. Analysis of PCoA
based on bray-Curtis’s distance; D. The microbiota composition of the raw fecal samples and fermentation samples. RF: Raw fecal. Data are means ± SEM. *: 0.01 <
p ≤ 0.05; **: 0.001 < p ≤ 0.01; ***: p ≤ 0.001.

significantly increased in the old male PHGG-treated samples and young samples produced higher amount of H2 after fermentation. Meanwhile,
female PHGG-treated samples when compared with their control sam­ the female and young population samples showed better effect in pro­
ples (Fig. 2C-D). The abundance of Klebsiella was significantly decreased ducing H2 than that of male and old populations (Fig. 3C). The CO2
in the old female PHGG-treated samples when compared with old female content (p < 0.001) was significantly increased in different populations
control samples (Fig. 2E). (Fig. 3D). Compared with the control samples, the NH3 content of the
old-originated samples were significantly reduced (Fig. 3E). In our re­
sults we did not find significant differences in H2S production among the
3.2. Gases production after fermentation different populations (Fig. 3F). These findings suggest that the meta­
bolism and use of PHGG vary among populations.
The gas generated by gut microorganisms is an important component
in determining its production and effect on human health (Kalantar-
Zadeh et al., 2019). We measured five kinds of gases that produced in 3.3. SCFAs production after fermentation
different populations samples after fermentation. The results showed
that the total production of gas was significantly increased in the PHGG- SCFAs generated by gut microbiota are beneficial to human gut
treated samples (p < 0.001) compared to the control samples (Fig. 3A). health. Therefore, we investigated the effect of PHGG on SCFA pro­
The production of CO2 (p < 0.001) and H2 (p < 0.001) was significantly duction by gut microbiota. After 24 h of fermentation, the PHGG-treated
increased in the PHGG-treated samples compared to the control sam­ samples produced more SCFAs than the control samples, and the total
ples, while the production of NH3 and H2S was decreased (Fig. 3B). amount of SCFAs was significantly increased in the PHGG-treated
Compared to the control samples, both female and male fecal samples samples (p < 0.001) (Fig. 4A), particularly acetic acid and butyric
that fermented in the PHGG medium have produced higher amount of acid (Fig. 4B). We found no significant differences in acetic acid content
H2. As the same result about gender, both young and old population between populations (Fig. 4C). Male-originated samples produced

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X.-E. Pi et al. Food Chemistry 430 (2024) 137006

Fig. 2. Composition analysis of bacterial microbiota in different populations between the control samples and PHGG-treated samples. A. Comparative analysis of the
composition of microbiota in control samples and PHGG-treated samples of different populations at the genus level. B, C, D, E. The composition of five kinds of
bacteria in different populations. YM: Young male; OM: Old male; YF: Young female; OF: Old female. Data are means ± SEM. The Shapiro-Wilk test evaluated
whether the data were normally distributed. Every spot represents an individual sample. The unpaired t-test was conducted. *: 0.01 < p ≤ 0.05; **: 0.001 < p ≤ 0.01;
***: p ≤ 0.001.

significantly higher amount of propionic acid than that of the control SCFAs production and positively correlated with H2 and CO2 produc­
samples after fermentation, but the other three populations samples did tion, but neither is significant, and Faecalibacterium was positively
not show the significant effect. Moreover, old-originated samples pro­ correlated with propionic acid, valeric acid, acetic acid, butyric acid,
duced significantly higher amount of butyric acid than that of the con­ and CO2, H2S, Megasphaera was positively correlated with isobutyric
trol samples after fermentation, but the other three populations samples acid, isovaleric acid, valeric acid, and butyrate, while Escherichia-
also did not show the significant effect (Fig. 4D and E). Shigella was inversely correlated with propionic acid and butyric acid, as
well as CO2 and H2S (Fig. 5).
3.4. Correlation between SCFAs and gas production
4. Discussion
To investigate the association between fermentation microbiota and
At present, the models used to study intestinal microecological ef­
metabolites SCFAs and gases, this study conducted a correlation analysis
fects mainly include in vitro and in vivo models. The in vitro model is
between the top 15 different bacterial genera with the highest abun­
mainly the human intestinal simulated fermentation model. Compared
dance and all fecal metabolites after fermentation. The results demon­
with the in vivo experiment, the in vitro fermentation model does not
strated that different bacteria have different correlations with gases and
consider the factors such as intestinal absorption, gastrointestinal
SCFAs production, such as Bifidobacterium is negatively correlated with

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X.-E. Pi et al. Food Chemistry 430 (2024) 137006

Fig. 3. Effects of PHGG on fecal microbiota gas

production in different populations. A. Total gas
content; B. Contents of five gases produced by fecal
microbiota; H2 (C), CO2 (D), NH3 (E), and H2S (F)
produced. The production of H2S has reached the
maximum range 1000 of gas analyzer. Data are
means ± SEM. The Shapiro-Wilk test evaluated
whether the data were normally distributed. Every
spot represents an individual sample. The unpaired
t-test was conducted. *: 0.01 < p ≤ 0.05; **: 0.001
< p ≤ 0.01; ***: p ≤ 0.001.

secretion and defense system (Gibson & Fuller, 2000). However, the in system (Fan et al., 2016; Yin et al., 2017). By adjusting the culture
vivo experiments have some problems, such as moral and ethical safety, medium and fermentation conditions, Bacteroides and Prevotella have
high cost and long cycle, which make the implementation of research been successfully cultured in an in vitro intestinal simulated system.
more difficult. In our laboratory, we have established an in vitro simu­ Furthermore, by connecting a gas analyzer on top of the in vitro intes­
lated intestinal fermentation system and demonstrated through exten­ tinal simulated model, the production and composition of intestinal
sive experiments that 60–80% of bacterial species in the guts of Chinese bacterial gases can be studied under different fermentation conditions,
individuals can grow in a simulated in vitro intestinal fermentation especially under prebiotic supplementation conditions. This makes it

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X.-E. Pi et al. Food Chemistry 430 (2024) 137006

Fig. 4. Effects of PHGG on SCFAs production by fecal

microorganisms in different populations. A. Total
SCFAs content; B. Contents of five SCFAs produced by
various samples of fecal microbiotas, and Ace (C), Pro
(D), and But (E) contents produced by different
samples of fecal microbiota. Con, control; Ace, acetic
acid; Pro, propionic acid; Isob, isobutyric acid; But,
butyric acid; Isov, isovaleric acid; Pen, valeric acid.
Data are means ± SEM. The Shapiro-Wilk test evalu­
ated whether the data were normally distributed.
Every spot represents an individual sample. The un­
paired t-test was conducted. *: 0.01 < p ≤ 0.05; **:
0.001 < p ≤ 0.01; ***: p ≤ 0.001.

possible to design experiments to explore the relationship between gut populations than in young populations, particularly in old males. The
microbiota metabolism of different food components and digestive correlation between human gut microbiota and host health has recently
related diseases (Pan et al., 2023; Ye et al., 2023). Recently, the human received a lot of attention, and several studies have shed light on the
intestinal simulated fermentation model have been increasingly favored structure and function of human gut microbiota that impact host health
by researchers for studying the interaction between functional sub­ and play a role in disease occurrence. In previous studies, the relation­
stances and host gut microbiota (Feng et al., 2023; Wu et al., 2023). ship between ethnicity, gender, and age samples, as well as diet and gut
The present study investigated the effect of PHGG on the fecal microbiota has been evaluated (Rothschild et al., 2018).
microbiota of 41 healthy volunteers by simulated fermentation tech­ PHGG supplementation did not affect the alpha-diversity of micro­
nique in vitro and discovered that PHGG affected the structure of the gut biota before and after fermentation. It is worth noting that this study
microbiota altered to some extent, and the composition of gut micro­ found that there was a significant difference in the phylogenetic di­
biota differed between different age and gender populations. Previous versity index between the control samples and the raw fecal samples.
studies have found that the polysaccharides have different effects on gut The pd diversity index reflect that diversity of community lineages. But
microbiota in people or animals of different genders and ages (Yang PHGG did alter the microbiota community structure. The present results
et al., 2021). The differences of microbiota lead to differences in their found that the abundance of Escherichia-Shigella was significantly
metabolites. For example, the abundance of Bifidobacterium in young reduced in the PHGG-treated samples. Escherichia-Shigella is a gram-
populations was higher than in old populations, whereas the abundance negative bacillus and the most common pathogen of human bacillary
of Escherichia-Shigella, a harmful bacterium, was higher in old dysentery (Kotloff et al., 2017). Escherichia-Shigella may exacerbate

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X.-E. Pi et al. Food Chemistry 430 (2024) 137006

Fig. 5. Correlation analysis of fecal microbiota at the genus level with SCFAs (A) and gases (B) in the PHGG-treated samples. Ace, acetic acid; Pro, propionic acid;
Isob, isobutyric acid; But, butyrate; Isov, isovaleric acid; Pen, valeric acid. The correlation heatmap was measured using the Spearman correlation coefficient. *: 0.01
< p ≤ 0.05; **: 0.001 < p ≤ 0.01; ***: p ≤ 0.001.

colitis and increase intestinal permeability (Bian et al., 2020). Escher­ abundance of Klebsiella pneumonia, a bacterial pathogen causing organ
ichia-Shigella fimbriae can adhere to the surface of epithelial cells in the failure and death (Russo & Marr, 2019), was significantly reduced in the
terminal ileum and colonic mucosa and penetrate the epithelial cells via PHGG-treated samples.
invasive proteins, hence causing damage to the ileum and colon mucosa. Previous research has demonstrated a direct relationship between
In addition, Escherichia-Shigella could be regulated by prebiotic oligo­ Dorea and obesity-related metabolites (Ottosson et al., 2018). In agree­
saccharides, such as fructooligosaccharides and maltooligosaccharides. ment with these findings, daily supplementation with a mixture of inulin
Early studies have indicated these two prebiotics inhibited the adhesion and fructo-oligosaccharides (FOS) for three weeks (16 g/day) reduces
of pathogenic Escherichia coli to human epithelial cells (Cheon et al., Dorea in healthy individuals (Healey et al., 2018). Intriguingly, the
2023). Moreover, a diet of prebiotic fructooligosaccharides changed the present research also revealed that Dorea was significantly reduced in
abundance of Escherichia in the ileum of broiler chickens (Kumar, Shang, the PHGG-treated samples. These data suggested that PHGG is beneficial
& Kim, 2019). Hence, there is the strong possibility that PHGG will for intestinal health. Furthermore, the current study showed that the
regulate Escherichia-Shigella in the human gut. In addition, the abundance of Bifidobacterium was much higher in the PHGG-treated

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samples. Bifidobacterium, a beneficial bacterium that functions as a different correlations with the production of SCFAs and gases. Among
biological barrier in the body, has a significant effect on alleviating the top 15 bacterial genera in relative abundance, Faecalibacterium was
symptoms such as diarrhea and constipation (Aprile et al., 2021) and positively correlated with SCFAs and gases, after fermentation, the
preventing external biological invasion. An early study reported that relative abundance of Faecalibacterium increased, and Faecalibacterium is
probiotics including fructooligosaccharide could regulate the abun­ one of the butyric acid-producing gut microbiotas (Anand, Kaur, &
dance of Bifidobacterium lactis BL-99 (Zhang et al., 2022). PHGG might Mande, 2016). Therefore, it can be inferred that Faecalibacterium may
have potential in regulating the composition and structure of the in­ contribute to metabolic products. In this study, there have been no
testinal microbiota, especially Bifidobacterium. Some species of Strepto­ literature reports on the direct correlation between other significantly
coccus are used as probiotics, including Streptococcus thermophiles and positively correlated bacteria and SCFAs and gases production, which is
Streptococcus salivarius (Wei et al., 2020). Faecalibacterium is a potential worthy of further research. In addition, Escherichia-Shigella was
marker of intestinal health (De Filippis, Pasolli, & Ercolini, 2020). The inversely correlated with propionic acid and butyric acid, as well as CO2
abundances of Streptococcus and Faecalibacterium were significantly and H2S, while the proportion of Escherichia-Shigella significantly
increased in the PHGG-treated samples. These suggest a role for PHGG in decreased in the PHGG treated group, indicating that the presence of a
modulating the gut microbiota and maintaining human gut health. large amuont of Escherichia-Shigella may hinder the growth of other
Intestinal gas is produced predominantly in the colon, where unab­ SCFAs and gases producing bacteria.
sorbed meal residues are fermented by colonic bacteria (Flourié et al., We acknowledge that there are limitations with our study. First, the
1988; Levitt et al., 1987). People have discovered that the fermentation effects of gas on intestinal health are still poorly studied, especially the
of prebiotics by gut microbiota produces a large amount of gas, but the lack of tools and techniques to accurately measure the number of
composition and volume of gas produced are rarely known (Sahakian, different gases produced in the gut. Although we have measured the
Jee, & Pimentel, 2010). Previous studies have shown that the different production of a variety of gases through in vitro simulated fermentation
gases produced by intestinal fermentation are closely related to physi­ system, how these gases, such as H2S and NH3, regulate the gut micro­
ological functions. For example, some bacteria in the human gut can biota and influence the process of metabolism to promote human health
oxidize sulfur-containing substances to produce H2S, the end-product of still needs to be further studied and explored. Moreover, we didn’t find
sulfate-reducing bacteria metabolite. H2S plays important roles in the which bacterial species of gut had significant association with these
regulation of blood pressure, angiogenesis, smooth muscle cell prolif­ SCFAs and gases. It is hard to evaluate the regulating function to gut
eration and apoptosis, anti-oxidative stress, cardiac functions. Several microbiota from PHGG fermented metabolites. Furthermore, 16S rRNA
studies have demonstrated a significant increase in H2S in the stools of sequencing technology can’t provide information about functionality of
patients with ulcerative colitis, indicating that high H2S concentrations gut microbiota, thus we also can’t evaluate the metabolic pathway
may play an important role in causing mucosal inflammation and involved in these PHGG related metabolites. In addition, the metabolic
carcinogenesis (Carbonero, Benefiel, Alizadeh-Ghamsari, & Gaskins, mechanism of individual bacteria in gut microbiota is very complex,
2012; Ramasamy et al., 2006; Roediger, Duncan, Kapaniris, & Millard, which is worth further study in the future. Finally, more experimental
1993). CO2 is beneficial to gut microbiota homeostasis during colonos­ techniques and analytical methods need to be applied to explore the
copy in healthy subjects, which improves the growth of anaerobic pro­ regulatory effect of PHGG on intestinal health.
biotics such as Bacteroides caccae, Bacteroides finegoldii and Bacteroides
thetaiotaomicron (Sajid et al., 2015). NH3 is mainly produced in gastro­ 5. Conclusions
intestinal tract, then reaches the systemic circulation and improves
blood-NH3 concentration, which accounts for the hyperammonemia This research analyzed the effect of PHGG on the fecal microbiota of
observed in chronic liver disease (Levitt & Levitt, 2018), and gastroin­ 41 Chinese individuals by an in vitro simulated fermentation technique.
testinal diseases (Singh & Lin, 2015). Microbial molecular H2 cycling is A total of 41 volunteers who met the experimental criteria were selected
essential to metabolic homeostasis and microbial composition in the and divided into four groups of samples based on their age and gender.
human gastrointestinal tract (Wolf et al., 2016). The present study The abundance of Firmicutes and Proteobacteria was higher in raw fecal
revealed a lower H2S content in the PHGG-treated samples compared to samples of old females and males, respectively, while the abundance of
the control, demonstrating that PHGG could reduce H2S concentration. genera Bifidobacterium decreased with age. After 24 h of fermentation,
In addition, CO2 and H2 concentrations increased in the PHGG-treated the supplementation of PHGG increased the abundance of beneficial
samples, while NH3 concentrations decreased significantly. These find­ bacteria (such as Bifidobacterium, and Faecalibacterium) and reduced the
ings demonstrated that PHGG can reduce harmful gases in the intestine abundance of harmful bacteria (such as Escherichia-Shigella, Klebsiella,
and improve intestinal health. Dorea). As important metabolites of the fecal microbiota, the total pro­
In the present study, the overall production of SCFAs was signifi­ duction of SCFAs and gases were significantly increased in the PHGG-
cantly increased in the PHGG-treated samples, while the levels of acetic treated samples, among which the production of acetic acid, butyric
acid and butyric acid were increased. Short-chain fatty acids (SCFAs) are acid, CO2, and H2 was significantly increased, while the production of
crucial linkages between the host and the gut microbiota because they NH3 was significantly decreased. In addition, PHGG supplementation
are the primary degradation product of carbohydrates, including acetic differentially modulated the microbiota in each of the four groups of
acid, propionic acid, butyrate, and valeric acid by gut bacteria (Antunes samples. The abundance of Bifidobacterium was significantly increased in
et al., 2019). However, the acid production capacity of PHGG supple­ the young female-originated samples and the old male-originated sam­
mentation varied among different populations. PHGG significantly ples, and the abundance of Klebsiella in the old female-originated sam­
increased propionic acid content in the male-originated samples and ples was significantly decreased. Only in the old-originated samples did
butyric acid content in the old-originated samples. Additionally, propi­ PHGG significantly reduce the NH3 content. In addition, PHGG signifi­
onate and butyrate have been reported that could involve in supporting cantly increased the levels of propionic acid in the male-originated
the development of the intestinal immune system and maintain intesti­ samples and butyric acid content in the old-originated samples.
nal barrier function (Topping & Lockett, 2016). It was evident that there Finally, different bacteria displayed different correlations with the
were different metabolic mechanisms of SCFAs among populations due production of SCFAs and gases. PHGG is an excellent prebiotic that can
to differences in their gut microbiota, which played different physio­ improve microbiota, but its application must take into consideration the
logical roles, and this highlights the need for further in-depth research unique characteristics of the different populations. This study provides
on precision nutrition. accurate guidance and theoretical basis for the commercial application
Intestinal microbiota composition may affect the metabolic capacity of PHGG. Meanwhile, it provides a development concept for establishing
of intestinal bacteria This study indicated that different bacteria had an individualized nutrition system.

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Funding: This work was supported by the Hangzhou Agricultural Cheon, S., Kim, G., Bae, J. H., Lee, D. H., Seong, H., Kim, D. H., … Han, N. S. (2023).
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Institutional Review Board Statement: The study was conducted Claesson, M. J., Jeffery, I. B., Conde, S., Power, S. E., O’Connor, E. M., Cusack, S., …
according to the guidelines of the Declaration of Helsinki and approved O’Toole, P. W. (2012). Gut microbiota composition correlates with diet and health in
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Starch malabsorption and breath gas excretion in healthy humans consuming low-
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Xiong-E Pi: Funding acquisition, Data curation, Formal analysis, 0016-5085(88)90491-x
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Haro, C., Rangel-Zúñiga, O. A., Alcalá-Díaz, J. F., Gómez-Delgado, F., Pérez-Martínez, P.,
Declaration of Competing Interest Delgado-Lista, J., … Tena-Sempere, M. (2016). Intestinal microbiota is influenced by
gender and body mass index. PLoS One, 11(5), e0154090.
Healey, G., Murphy, R., Butts, C., Brough, L., Whelan, K., & Coad, J. (2018). Habitual
The authors declare that they have no known competing financial dietary fibre intake influences gut microbiota response to an inulin-type fructan
interests or personal relationships that could have appeared to influence prebiotic: A randomised, double-blind, placebo-controlled, cross-over, human
the work reported in this paper. intervention study. British Journal of Nutrition, 119(2), 176–189. https://doi.org/
10.1017/S0007114517003440
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Leiden.CETP mice. Scientific Reports, 8(1), 16515. https://doi.org/10.1038/s41598-
Data will be made available on request. 018-34970-y
Hu, H., Wang, H., Zhang, Y., Kan, B., Ding, Y., & Huang, J. (2019). Characterization of
Appendix A. Supplementary data genes in guar gum biosynthesis based on quantitative RNA-sequencing in guar bean
(Cyamopsis tetragonoloba). Scientific Reports, 9(1), 10991. https://doi.org/10.1038/
s41598-019-47518-5
Supplementary data to this article can be found online at https://doi. Kalantar-Zadeh, K., Berean, K. J., Burgell, R. E., Muir, J. G., & Gibson, P. R. (2019).
org/10.1016/j.foodchem.2023.137006. Intestinal gases: Influence on gut disorders and the role of dietary manipulations.
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