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Cooperative interactions between Veillonella ratti

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Cite this: DOI: 10.1039/d3fo03898j

and Lactobacillus acidophilus ameliorate
DSS-induced ulcerative colitis in mice†
Na Li,‡ Hejing Wang,‡ Huizhu Zhao, Mengyang Wang, Jin Cai, Yi Hao, Jia Yu,
Yun Jiang, Xin Lü and Bianfang Liu *

Received 13th September 2023,
Accepted 3rd October 2023
DOI: 10.1039/d3fo03898j
rsc.li/food-function
Veillonella and Lactobacillus species are key regulators of a healthy gut environment through metabolic
cross-feeding, influencing lactic acid and short-chain fatty acid (SCFA) levels, which are crucial for gut
health. This study aims to investigate how Veillonella ratti (V. ratti) and Lactobacillus acidophilus (LA) inter-
act with each other and alleviate dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in a mouse
model. We assess their metabolic interactions regarding carbon sources through co-culturing in a
modified medium. In the in vitro experiments, V. ratti and LA were inoculated in mono-cultures and co-
culture, and viable cell counts, OD600, pH, lactic acid, glucose and SCFAs were measured. For the in vivo
experiment, 60 C57BL/6 mice were randomly divided into five groups and administered V. ratti and LA
alone or in combination via oral gavage (1 × 109 CFU mL−1 per day per mouse) for 14 days. On the
seventh day, 2.5% DSS was added to the drinking water to induce colitis. The effects of these probiotics
on UC were evaluated by assessing intestinal barrier integrity and intestinal inflammation in the gut micro-
environment. In vitro results demonstrated that co-culturing V. ratti with LA significantly increased viable
cell numbers, lactic acid production, and SCFA production, while reducing pH and glucose levels in the
medium. In vivo findings revealed that intervention with V. ratti, particularly in combination with LA, alle-
viated symptoms, including weight loss, colon shortening, and tissue damage. These probiotics mitigated
intestinal inflammation by down-regulating pro-inflammatory molecules, such as IL-6, IL-1β, IL-γ, iNOS,
and IFN-γ, as well as oxidative stress markers, including MDA and MPO. Concurrently, they upregulated
the activity of anti-inflammatory enzymes, namely, SOD and GSH, and promoted the production of
SCFAs. The combined intervention of V. ratti and LA significantly increased acetic acid, propionic acid,
butyric acid, isobutyric acid, valeric acid, and total SCFAs in cecal contents. Furthermore, the intervention
of V. ratti and LA increased the abundance of beneficial bacteria, such as Akkermansia, while reducing the
abundance of harmful bacteria, such as Escherichia–Shigella and Desulfovibrio, thereby mitigating exces-
sive inflammation. These findings highlight the enhanced therapeutic effects resulting from the inter-
actions between V. ratti and LA, demonstrating the potential of this combined probiotic approach.

1. Introduction lished that UC is associated with dysbiosis of intestinal flora,

Ulcerative colitis (UC) is a chronic inflammatory bowel disease
primarily affecting the mucosa and submucosa of the colon.1
The DSS-induced UC mouse model exhibits oxidative damage
and local inflammation, resulting in clinical symptoms such
as diarrhea, weight loss, and colon lesions. It has been estab-
College of Food Science and Engineering, Northwest A&F University, Yangling
712100, Shaanxi, China. E-mail: bfliu9509@163.com; Fax: +86-13519116643;
Tel: +86-13519116643
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/
10.1039/d3fo03898j
‡ These authors contributed equally to this work.
disruption of mucosal barrier function, immune system altera-
tions, and a decrease in short-chain fatty acid (SCFA)-produ-
cing bacteria and SCFAs.2 Managing UC has become a challen-
ging task due to its high prevalence and complex pathogen-
esis, the tendency to recur, and the potential development of
colon cancer in some patients.3 Improving the gut mucosal
barrier integrity and the immune system, and adjusting the
gut microflora and their metabolites may contribute to redu-
cing colonic inflammation.4 Given that current treatments for
UC are prone to cause severe undesirable side effects after
long-term use,5 therefore, there is an urgent need for novel,
safe, and effective preventive and therapeutic strategies for UC,
such as probiotics.

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While the composition of the adult intestinal microbiota
exhibits interpersonal variation, certain core bacterial species
and their metabolites remain relatively stable.6 Among these
core species, Lactobacillus spp. and Veillonella spp. have been
identified in the human intestinal flora. Their absence or low
abundance has been associated with the manifestation of
symptoms.7
Probiotics such as Lactobacillus plantarum, Bifidobacterium,
Lactobacillus reuteri, and Lactobacillus acidophilus have been
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utilized for the treatment and prevention of colitis.8–10 These
lactic acid bacteria (LAB) produce substantial amounts of
lactic acid in the intestine. However, the accumulation of lactic
acid is limited by lactate-utilizing bacteria (LUB) that metab-
olize it. The metabolic pathways of LUB are crucial in prevent-
ing excessive lactate accumulation and promoting intestinal
ecosystem stability.11 Veillonella, an anaerobic commensal
LUB, plays a vital role in maintaining the stability and health
of the intestinal flora.6 It is essential for the establishment of
healthy and mature gut anaerobic microbiota and the recovery
of the intestinal microbiota after antibiotic use.12 Additionally,
Veillonella is also associated with the development of a healthy
immune system in infants and promotes immune system
development in early childhood.13 Veillonella can metabolize
lactic acid into acetic acid, propionic acid, and other SCFAs,
which are vital for gut health. Intestinal SCFAs influence UC
occurrence and development through multiple mechanisms,
including providing essential nutrients and energy to the host,
protecting the intestinal mucosal barrier, regulating intestinal
motility and host metabolism, and modulating the immune
system.2,14 Several studies have explored the physiological
functions of Veillonella in maintaining the dynamic balance
between lactic acid and SCFAs and its relevance to host health
and disease.15,16 A few studies have observed lactic acid
accumulation in the medium when Veillonella strains are co-
cultured with Enterococcus faecium17 or found that the medium
cultured with Lactobacillus and Veillonella isolated from
chicken cecum inhibits pathogenic intestinal bacteria.18
One of the crucial functions of Veillonella is the fermenta-
tion of organic acids to produce SCFAs; while some Veillonella
strains cannot ferment carbohydrates such as glucose and
lactose, they can ferment organic acids such as pyruvate and
lactic acid into acetic acid and propionic acid.19 On the other
hand, Lactobacillus acidophilus (LA) can ferment common
sugars and plant-derived carbohydrates such as cellobiose and
amygdalin.20 Some studies have demonstrated that cross-
feeding interactions in co-culture can promote the growth of
the two bacteria by converting intermediates such as succi-
nate, lactate, and acetyl CoA into acetate, propionate, butyrate,
and other SCFAs.21
Intestinal diseases, including UC and colorectal cancer, are
associated with a decrease in the number of LUB. Patients
with inflammatory bowel disease (IBD), unlike healthy individ-
uals, often exhibit high lactate accumulation in the colon,
because it cannot be efficiently metabolized by LUB into host-
friendly SCFAs.21 LUB plays a crucial role in maintaining the
stability of the human colonic microbial ecosystem. Chen et al.
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demonstrated that LUB significantly alleviated DSS-induced
colitis in mice, suggesting its potential as a probiotic for IBD
treatment.22
Our previous research demonstrated that a high abundance
of Veillonella spp. in the intestines of piglets inhibited entero-
hemorrhagic Escherichia coli infection.23 Therefore, the present
study aims to investigate the combined effects of Veillonella
ratti and Lactobacillus acidophilus on UC using an in vivo
mouse model. The findings from this study may serve as a
basis for the development of potential probiotic combinations
and have significant implications for future research on safe
and effective preventive and therapeutic strategies for UC.
2. Materials and methods
2.1 V. ratti and LA co-culture based on a modified
136 medium
2.1.1 Preparation of the co-culture medium. V. ratti was
isolated and identified in our laboratory and stored at the
China Center of Industrial Culture Collection (CICC25149,
GenBank accession no: OL597939.1). LA was obtained from
Chr. Hansen China (CGMCC 21802).
To prepare the co-culture medium, we used a modified
136 medium based on the Veillonella medium base, as rec-
ommended by DSMZ (German Centre for Microbial Strain
Conservation, DSMZ136 medium). The ingredients of the
medium per liter were as follows: trypticase (1 g), yeast extract
(0.6 g), Na-thioglycolate (0.75 g), Tween 80 (1 mL), glucose
(8 g), putrescine (3 mg), (NH4)2SO4 (8 g), L-cysteine (0.5 g),
K2HPO4 (2 g), MgSO4·7H2O (0.5 g), MnSO4 (0.04 g), and di-
ammonium hydrogen citrate (2 g). The pH of the medium was
adjusted to 7.5 using K2CO3.
2.1.2 Detection of the OD, pH, and viable counts in the co-
culture of V. ratti and LA. For the mono-cultures and co-
culture experiments, 1.5% and 0.5% of the inoculums (OD600
= 0.5) of V. ratti and LA were respectively added into 10 mL of a
modified 136 medium. The OD600 values and pH levels were
measured using a spectrophotometer (UV spectrophotometer
7200) and pH meter (PHSJ-3F), respectively. To determine the
viable counts, the spread plate method was employed, where
the samples were diluted and plated onto agar plates. The
plates were then incubated under suitable conditions, and the
number of viable bacteria was calculated using colony forming
units. Samples from the mono-cultures and co-culture were
preserved at −80 °C for subsequent analysis of lactic acid,
glucose, and short-chain fatty acids (SCFAs).
2.1.3 Lactic acid and glucose assessment. To measure
lactate production and glucose uptake in the medium, a
lactate assay kit and a glucose assay kit (Jiancheng
Bioengineering Institute, Nanjing, China) were used. A colori-
metric detection method was followed according to the manu-
facturer’s instructions.
2.1.4 Detection of SCFAs in the co-culture of V. ratti and
LA. For SCFA detection, the same method as described in
section 2.9 was employed, with slight modifications in the
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Food & Function
sample preparation. Briefly, a bacterial culture (6 mL) was cen-
trifuged at 5000 rpm (4 °C) for 10 min. Next, 5 mL of the fil-
trate and 1 mL of 50% H2SO4 (v/v) solution were added to a
centrifuge tube, followed by vortexing for 5 min. Subsequently,
5 mL of ether was added, and the mixture was allowed to
stand at 4 °C for 30 min, with intermittent shaking every
5 min. Afterward, the solution was centrifuged at 5000 rpm
(4 °C) for 20 min. Finally, the supernatant was extracted, con-
centrated using nitrogen blowing, and redissolved in 1 mL of
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ether.
2.2. Bacterial preparation for animal experiments
For the cultivation of V. ratti, BHI medium supplemented with
0.5% sodium lactate was used. The culture was incubated
under anaerobic conditions at 37 °C for 48 h. LA was cultured
in MRS broth at 37 °C for 12 h.
To collect the bacterial cells, the bacterial solution (10 mL)
was centrifuged at 8000 rpm (4 °C) for 10 minutes. The cells were
then washed twice with phosphate-buffered saline (PBS), resus-
pended in saline, and adjusted to a concentration of 109 CFU
mL−1 for subsequent administration via mouse gavage. It is
important to note that the V. ratti suspension should be used
immediately, and the gavage procedure should be completed
within 1 hour of preparation due to its strict anaerobic nature.
2.3. Animal experiment
Sixty male C57BL/6 mice (SPF grade) weighing 20 ± 2 g and
aged 6–8 weeks were obtained from Hunan SJA Laboratory Co.,
Ltd (Hunan, China). The mice were housed under controlled
conditions at a temperature of 25 °C with a 12-hour light/dark
cycle. They had access to standard chow and sterilized water
ad libitum. All animal experiments were conducted in compli-
ance with the guidelines and regulations approved by the
Animal Ethics and Welfare Committee of Northwest A&F
University, China (SP2022015).
The detailed experimental scheme is presented in Fig. 3A.
Following a week of acclimatization, the mice were randomly
assigned to five groups (n = 12): the control group with normal
drinking water (Ctrl), the DSS colitis group (DSS), the LA (109
CFU mL−1, 200 μL per day) intervention group (DSS + LA), the
V. ratti (109 CFU mL−1, 200 μL per day) intervention group (DSS +
V. ratti), and the V. ratti (109 CFU mL−1, 100 μL per day) + LA (109
CFU mL−1, 100 μL per day) intervention group (DSS + mixture).
During the first week of the acclimatization period, all
groups were fed standard chow and purified water. In the
intervention period, the Ctrl and DSS groups received 200 μL
of saline solution orally, while the probiotic intervention
groups received daily oral administration of 200 μL of bacterial
suspension (109 CFU mL−1).
After the seven-day intervention period, the UC mouse
model was induced by daily administration of 2.5% DSS (w/v,
MW: 36 000–50 000, American MP Biomedical Company) in the
drinking water, while the bacterial interventions continued.
Throughout the experiment, the body weight, food intake, and
water consumption of each mouse were recorded daily. On the
14th and 21st days, fecal samples were collected for microbial
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diversity analysis. On the 22nd day, all mice were anesthetized
and sacrificed. The entire colon tissues were harvested,
excised, and rinsed with cold PBS for macroscopic assessment
of changes.
2.4. Disease activity indexes
After oral administration, daily measurements were taken of
the body weight, stool consistency, and bleeding of the mice.
The severity of colitis was assessed using the disease activity
index (DAI) score, as described by Mennigen et al.24 The DAI
score takes into account weight loss, stool consistency, and
bleeding as indicators of colitis severity. The presence of fecal
occult blood was tested daily using a fecal occult blood kit
obtained from Nanjing Jiancheng Institute of Biological
Engineering. Specific DAI scores were calculated using a pre-
viously described method (Table S1†).25
2.5. Histopathological evaluation
Approximately 1 cm sections from the proximal and distal
colons of the mice were collected and fixed in a 4% (w/v) paraf-
ormaldehyde solution at 4 °C overnight. After fixation, the
colons were embedded in paraffin and sectioned into 5 μm
slices for hematoxylin and eosin (H&E) staining. The stained
sections were observed and their images were captured using a
microscope (DP27 digital microscope, Olympus, Japan). The
assessment of the colonic mucosa damage score was per-
formed following a previously described method (Table S2†).26
2.6. Real-time quantitative fluorescence polymerase chain
reaction (RT-qPCR)
Total RNA was extracted from 50 mg of colon tissue using a
TaKaRa RNAiso Plus kit (Beijing Haiding, China) following the
manufacturer’s protocol. The extracted RNA was then qualitat-
ively and quantitatively evaluated using a micro-spectrophoto-
meter Nano-200 (Hangzhou Allsheng Instruments, Korea).
Reverse transcription was performed using a first-strand cDNA
synthesis kit (Beijing Dining Biotechnology Co., Ltd), followed
by RT-qPCR amplification of 2 μL dilutions of cDNA using a
CFX96 Touch™ real-time PCR detection system (Bio-Rad,
Hercules, CA, USA) and PerfectStart® Green qPCR SuperMix
(TransGen Biotech, Beijing, China). The primer sequences for
the target genes can be found in Table S3.† GAPDH house-
keeping genes were used as an internal reference. The relative
expression of mRNA was calculated using the 2−ΔΔCt method.
2.7. Detection of oxidative factors in the colon
The levels of myeloperoxidase (MPO), malondialdehyde
(MDA), superoxide dismutase (SOD), and glutathione (GSH) in
mouse colonic tissues were measured using commercial kits
(Jiancheng Bioengineering Institute, Nanjing, China) following
the manufacturer’s instructions. To perform the measure-
ments, 100 mg of colon tissue was homogenized with 0.9 mL
of PBS buffer using a freezing grinder. The homogenate was
then centrifuged at 10 000 rpm for 15 minutes at 4 °C, and the
resulting supernatant was collected. The protein concentration
in the supernatant was determined using a bicinchoninic acid
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(BCA) protein assay kit (Jiancheng Bioengineering Institute,
Nanjing, China). The activities of GSH, MDA, and SOD in the
colon were expressed as nanomoles per milligram of protein
(nmol mg−1 protein) and units per milligram of protein (U
mg−1 protein).
2.8. Fecal microbiota analysis
Fecal samples from the mice were collected and immediately
frozen at −80 °C. The frozen samples were then placed on dry
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ice and sent to LC Biotechnologies Co., Ltd (Hangzhou, China)
for microbiota diversity analysis. Total DNA was extracted from
the fecal samples using an OMEGA stool DNA kit. The bac-
terial 16S rRNA genes in the V3–V4 region were amplified
using the forward primer 341F (5′-
CCTACGGGNGGCWGCAG-3′) and the reverse primer 805R (5′-
GACTACHVGGGTATCTAATCC-3′). The resulting amplicons
were pooled in equal amounts, and paired-end sequencing
with a read length of 2 × 250 bp was performed using the
Illumina platform. After sequencing, all sequences were classi-
fied into operational taxonomic units (OTUs) with a similarity
level of 97%. To investigate the bacterial members responsible
for the observed differences the between groups, LEfSe ana-
lysis was conducted.
2.9. Short-chain fatty acid (SCFA) and lactic acid detection
Short-chain fatty acids (SCFAs) in the feces of the mice were
measured using a gas chromatograph (GC-2014C, Shimadzu
Corporation, Japan) equipped with a DB-FFAP capillary
column (30 m × 0.25 µm × 0.25 µm, Agilent Technologies, Palo
Alto, CA, USA) and a flame ionization detector. Fecal samples
weighing 200 mg were dissolved, homogenized, and then cen-
trifuged at 10 000 rpm (4 °C) for 10 minutes. The resulting
supernatant was collected and filtered through a 0.22 µm
organic membrane into gas-phase vials for GC-MS analysis.
The gas chromatography-mass spectrometry (GC-MS) con-
ditions were as follows: nitrogen (N2) was used as the carrier
gas with a split ratio of 10 : 1. Temperature programmed gas
chromatography (TPGC) involved an initial temperature of
50 °C for 1 minute, followed by an increase to 120 °C at a rate
of 15 °C min−1. The temperature was then raised to 170 °C at a
rate of 5 °C min−1, and finally raised to 240 °C at 15 °C min−1
and held for 3 minutes. The injector and detector tempera-
tures were set to 250 °C and 270 °C, respectively. Standard
SCFA curves were prepared using acetic acid, propionic acid,
isobutyric acid, butyric acid, isovaleric acid, and valeric acid
(Sigma-Aldrich, St Louis, MO, USA).
Lactic acid in the feces of the mice was measured using a
lactate assay kit (Jiancheng Bioengineering Institute, Nanjing,
China) through colorimetric detection following the manufac-
turer’s instructions. The supernatant solution of the feces of
the mice was standardized based on the sample weight.
2.10. Statistical analysis
The trial data were analyzed and graphed for significance
using GraphPad Prism 8.0.1 software (GraphPad Software, Inc.,
San Diego, CA, USA). The data obtained from five independent
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experiments were presented as mean ± standard deviation
(SD). Statistical analysis of differences between multiple
groups was conducted using two-way ANOVA, followed by
Tukey’s multiple comparison test, with a significance level set
at P < 0.05.
3. Results
3.1. Cross-feeding between V. ratti and LA in a modified
136 medium
In our previous experimental study, both V. ratti and LA
showed normal growth in the 136 medium (to be published).
Considering the dynamic metabolic responses to changes in
carbon sources during the co-culture of these two strains, we
adjusted the carbon source amount in the co-culture medium
accordingly. As depicted in Fig. 1, compared to mono-cultures,
V. ratti co-cultured with LA exhibited an accelerated entry into
the logarithmic growth phase, with a higher OD600 observed
from 6 hours and a significantly increased number of viable
bacteria from 9 hours (P < 0.05, Fig. 1A and C). Additionally,
the co-culture led to a noticeable decrease in the medium’s pH
compared to the mono-cultures (Fig. 1B). Measurements of
lactic acid and glucose in the medium demonstrated that the
co-culture of V. ratti with LA consumed more glucose and
exhibited enhanced lactic acid production compared to mono-
cultures (P < 0.05, Fig. 1D and E).
Gas chromatography analysis was performed to detect the
short-chain fatty acids (SCFAs) produced in the co-culture and
mono-cultures of V. ratti and LA. The results showed that the
two bacteria significantly produced higher amounts of acetic
acid (378.29 ± 4.34 mmol L−1), propionic acid (253.32 ±
0.9 mmol L−1), isobutyric acid (162.35 ± 0.42 mmol L−1) and
butyric acid (149.24 ± 0.49 mmol L−1), and total SCFAs
(1218.76 ± 3.92 mmol L−1) after 48 hours of co-culture (Fig. 2).
There was no significant difference in the production of valeric
acid and isovaleric acid between the mono-cultures and the co-
culture (data not shown). These findings indicate that the co-
culture of V. ratti and LA promotes bacterial growth and pro-
liferation through cross-feeding interactions, where LA con-
sumes glucose and converts it into lactic acid, which in turn
serves as a carbon source for V. ratti to produce SCFAs.
3.2. V. ratti and LA restore body weight and relieve colitis
symptoms in DSS-induced colitis mice
The effects of V. ratti and LA on colitis symptoms induced by DSS
in mice were investigated (Fig. 3). The probiotic intervention was
assessed by monitoring the body weight, food intake, and general
conditions of the animals (Table S4†). Throughout the interven-
tion period, mice treated with V. ratti and LA for one week
showed no signs of toxicity compared to the Ctrl group. All
groups of mice experienced weight loss during the UC model
induction period (Fig. 3B). Notably, the DSS + mixture group
exhibited significantly less weight loss (P < 0.05).
Compared to the Ctrl group, the DAI score of the DSS group
began to increase on day three (P < 0.01, Fig. 3C). In contrast,
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Fig. 1 V. ratti and LA mono-cultures and co-culture in vitro. (a) The growth curves of the mono-culture and co-culture of V. ratti and LA; (b)
changes of pH in the mono-cultures and co-culture of V. ratti and LA; (c) changes in the number of viable cells in the mono-cultures and co-culture
of V. ratti and LA; (d) changes in the content of glucose; and (e) changes in the content of lactic acid; the analysis was performed in triplicate (n = 3).
All significant differences were analyzed using two-way ANOVA and Tukey’s post hoc test. LA compared with co-culture, *p < 0.05; **p < 0.01; ***p
< 0.001; ****p < 0.0001. V. ratti compared with co-culture, #p < 0.05; ##p < 0.01; ##p < 0.001; ####p < 0.0001.

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Fig. 2 The SCFAs produced in the co-culture and mono-cultures of the V. ratti and LA. (a)–(e) show acetic acid, propionic acid, butyric acid, isobu-
tyric acid, and the total short-chain fatty acids, respectively. The analysis was performed in triplicate (n = 3). All significant differences were analyzed
using two-way ANOVA and Tukey’s post hoc test. LA compared with co-culture, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. V. ratti compared
with co-culture, #p < 0.05; ##p < 0.01; ##p < 0.001; ####p < 0.0001.

the DSS + V. ratti, DSS + LA, and DSS + mixture groups dis-
played significantly lower DAI scores than the DSS group on
day seven (P < 0.01, P < 0.05, or P < 0.001). Fecal occult blood
yielded showed negative results in the Ctrl group, with virtually
no discoloration within 2 minutes, and positive results were
observed in the DSS group, with an immediate shift to dark
blue-black. However, the levels of occult blood were minimal
in the DSS + V. ratti and DSS + mixture groups, indicating that
V. ratti intake improved hematochezia in the mice (Fig. 3D).
Enlarged spleens and temporary shrinkage of the thymus
were observed in mice.
The weighing of the spleens showed that the thymus index
exhibited a significant decrease, while the spleen index
showed a significant increase in the DSS group compared to
the Ctrl group. Furthermore, the thymus index exhibited a sig-
nificant decrease, while the spleen index showed a significant
increase in the DSS group compared to the Ctrl group.
Importantly, the thymus index for the DSS + mixture group

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Fig. 3 V. ratti and LA ameliorate colitis clinical symptoms. (a) Experimental design and grouping schematic diagram. Colitis was induced by the
administration of 2.5% DSS in drinking water for 14 days. LA, V. ratti, or LA + V. ratti were supplemented in the saline solution for 7 days. Ctrl: healthy
control group fed with regular diet. (b) Rate of weight loss in mice during the UC model induction period; (c) schematic diagram of the trend of the
DAI score; (d) occult blood condition in the modeling period; (e) spleen index (%); and (f ) thymus index (%). All significant differences were analyzed
using one-way ANOVA and Tukey’s post hoc test. Compared with the Ctrl group, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Compared with
the DSS group, #p < 0.05; ##p < 0.01; ##p < 0.001; ####p < 0.0001.

and the Ctrl group showed no significant difference, F). These findings suggest that interventions of V. ratti and LA
suggesting that the intervention of V. ratti and LA led to partial promote body weight recovery and alleviate colitis symptoms
recovery of the health of the spleen and thymus (Fig. 3E and in DSS-induced colitis mice.

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3.3. V. ratti and LA reduce damage to the colon tissues
The colon length of mice (5.414 ± 0.2424 cm) was significantly
shortened due to the DSS treatment compared to the Ctrl
group (8.786 ± 0.2807 cm) (Fig. 4A and B). However, the DSS +
V. ratti, DSS + LA, and DSS + mixture intervention groups effec-
tively prevented colon shortening compared to the DSS group
(6.786 ± 0.4562 cm, 6.443 ± 0.2010 cm, or 7.257 ± 0.2553 cm,
respectively).
In the Ctrl group, the mucosal epithelium appeared intact
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and continuous, the mucosal villous glands were neatly
arranged, the presence of lymphocytes in the lamina propria
was minimal, and there was no infiltration of inflammatory
cells. Conversely, the DSS group exhibited severe damage to
the colon tissues, including deformed crypt structures,
destruction or even absence of goblet cells, extensive necrosis
of the colonic mucosal layer, damage to the mucosal epi-
thelium, and infiltration of inflammatory cells. In contrast,
both the DSS + V. ratti and DSS + mixture intervention groups
effectively alleviated colitis. The mucosal tissues exhibited stra-
tification, the arrangement of mucosal villous glands was less
disordered, and the infiltration of inflammatory cells was sig-
nificantly reduced. Additionally, the DSS + mixture group
showed significantly lower histological scores and more intact
colonic tissue compared to the DSS + V. ratti group (P < 0.01;
Fig. 4C and D).
3.4. V. ratti and LA regulate the expression of related genes
in the colon
To investigate whether V. ratti and LA regulated the expression
of related genes in the colon, we conducted an assessment of
the mRNA expressions of TJ proteins occludin, mucins (MUC2
and MUC3) and cytokines (IL-6, IL-1β, IL-17a, IL-γ, IFN-γ, TNF-
α, and iNOS) in the colon tissue using RT-qPCR. The levels of
inflammatory cytokines were significantly elevated in the DSS
group compared to the Ctrl group (P < 0.05; Fig. 5). However,
these inflammatory cytokines decreased significantly in the
DSS + V. ratti and DSS + mixture groups compared to the DSS
group (P < 0.05). Notably, the DSS + mixture group exhibited a
pronounced effect in suppressing these inflammatory cyto-
kines (P < 0.05). The level of TNF-α did not differ significantly
between the groups (Fig. 5E). Additionally, the expression of
PPAR-γ was reduced in the DSS group (0.019 ± 0.0045; Fig. 5F)
compared to the Ctrl group (1 ± 0.196). However, adminis-
tration of V. ratti and LA restored PPAR-γ expression levels in
the colon of the mice in the mixture group (0.322 ± 0.039).
To further assess the preventative effect of V. ratti and LA
on the epithelial mucosa in the mice with DSS-induced colitis,
we examined the expression of MUC2 and MUC3 genes, which
are crucial for mucosal integrity The expression of both genes
was significantly down-regulated in the DSS group compared
to the Ctrl group (P < 0.0001, P < 0.0001; Fig. 5I). However, the
expression of these genes was prominently up-regulated in the
DSS + mixture group (P < 0.001, P < 0.1). Furthermore, the
expression of the tight junction protein, occludin, was sup-
pressed in the DSS group compared to the Ctrl group (P <
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0.001), indicating damage to the tight junction structure. In
contrast, the expression of occludin was significantly increased
in the DSS + V. ratti group (P < 0.0001). These results indicate
that the combination of V. ratti and LA may prevent DSS-
induced disruption of intestinal integrity by effectively regulat-
ing the expression of related genes in the colon, including the
enhanced mucin mRNA and tight junction protein expressions
(Fig. 5I).
3.5. V. ratti and LA alleviate oxidative stress in the colons of
the UC mice
We assessed the levels of superoxide dismutase (SOD), malon-
dialdehyde (MDA), myeloperoxidase (MPO), and reduced gluta-
thione (GSH) in the colonic homogenate supernatant of mice
with colitis using a commercial kit. As shown in Fig. 6C and D,
the levels of MDA and MPO were significantly elevated by
approximately three-fold (P < 0.001) in the DSS group com-
pared to the Ctrl group. In contrast, the activity of SOD and the
levels of GSH were significantly decreased (P < 0.001; Fig. 6A
and B) in the DSS group compared to the Ctrl group. However,
in the DSS + mixture group, the contents of SOD and GSH
were significantly increased (P < 0.01), while the levels of MDA
and MPO were decreased compared to the DSS and individual
treatment groups (Fig. 6). These results suggest that the combi-
nation treatment of V. ratti and LA is more effective in alleviat-
ing oxidative stress of UC than the single treatment.
3.6. V. ratti and LA contribute to restoring the intestinal flora
homeostasis of UC mice
We collected fecal samples from mice to analyze the compo-
sition of the gut microbiota. In total, we identified 31 genera
in all samples during the probiotic intervention period.
Importantly, the relative abundance of Ligilactobacillus and
total lactic acid bacteria (LAB) was significantly higher in the
mixture group compared to the other groups (Fig. 7A–C, P <
0.05), indicating that the interactions between V. ratti and LA
promotes the growth of LAB, which is consistent with the
in vitro results of this study.
During the UC model induction period, DSS treatment sig-
nificantly reduced the diversity of the gut microbiota (P < 0.05,
Fig. 8A–E). Fig. 8F illustrates the 15 major phyla, with
Bacteroidetes and Firmicutes being the dominant ones. DSS
treatment increased the relative abundance of Firmicutes,
while decreasing the relative abundance of Bacteroidetes com-
pared to the Ctrl group (P < 0.05, P < 0.01). This shift resulted
in an elevated Firmicutes to Bacteroidetes ratio in the DSS
group compared to the Ctrl group (P < 0.001, Fig. 8J). In con-
trast, the F/B ratio was decreased in the DSS + mixture group
compared to the DSS group (P < 0.01). It is worth noting that
the ratio of F/B has been reported to increase in the animal
models of UC, which is consistent with our findings, However,
a combined intervention of V. ratti and LA significantly miti-
gated this increase, indicating a positive impact on the gut
microbiota composition.
Furthermore, we identified 30 genera in all samples
(Fig. 8G). The relative abundance of
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Fig. 4 V. ratti and LA interventions and the histopathological evaluations of their effects in the colon. (a) Macroscopic pictures of the colons; (b)
colon length of the mice; (c) histopathological score of the colon; and (d) H&E-stained sections of the mouse colon tissue (×10 and ×40). All signifi-
cant differences were analyzed using one-way ANOVA and Tukey’s post hoc test. Compared with the Ctrl group, *p < 0.05; **p < 0.01; ***p < 0.001;
****p < 0.0001. Compared with the DSS group, #p < 0.05; ##p < 0.01; ##p < 0.001; ####p < 0.0001.

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Fig. 5 V. ratti and LA regulated the expression of related genes in the colon. (a) Gene expression of IL-6 in the colonic epithelial tissue; (b) gene
expression of IL-1β in the colonic epithelial tissue; (c) gene expression of IL-17a in the colonic epithelial tissue; (d) gene expression of IL-γ in the
colonic epithelial tissue; (e) gene expression of TNF-α in the colonic epithelial tissue; (f ) gene expression of PPAR-γ in the colonic epithelial tissue;
(g) gene expression of IFN-γ in the colonic epithelial tissue; (h) gene expression of iNOS in the colonic epithelial tissue; and (i) gene expression of
tight junction proteins and mucins in the colonic epithelial tissue. All significant difference analyses were performed using one-way ANOVA and
Tukey’s post hoc test. Compared with the Ctrl group, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Compared with the DSS group, #p < 0.05;
##
p < 0.01; ###p < 0.001.

Desulfovibrionaceae_unclassified, Escherichia–Shigella, and
Parasutterella was significantly increased, whereas the relative
abundance of Muribaculaceae_unclassified, Alloprevotella,
Ruminococcus, Roseburia, and Ligilactobacillus was significantly
decreased in the DSS group compared to the Ctrl group (P <
0.05). However, the relative abundances of Akkermansia and
Bacteroides were significantly increased in the DSS + mixture
group compared to the DSS group, which indicates that the
intervention of V. ratti and LA enriched the two genera in the
intestines of mice.
To further analyze the gut microbiota diversity among the
five groups, we employed the LDA effect size (LEfSe) approach.

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Fig. 6 V. ratti and LA regulated oxidative stress in the colons of UC mice. (a) GSH; (b) SOD; (c) MDA; and (d) MPO. All significant difference analyses
were performed using one-way ANOVA and Tukey’s post hoc test. Compared with the Ctrl group, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Compared with the DSS group, #p < 0.05; ##p < 0.01; ##p < 0.001; ####p < 0.0001.
The LDA score histogram was generated to identify statistically
significant biomarkers and the dominant microorganisms in
each group (Fig. 8H and I). Dominant communities were
identified in the Ctrl, DSS, DSS + V. ratti, DSS + LA, and DSS +
mixture groups, respectively. The Ctrl group showed higher
relative abundances of the bacterial family Ruminococcaceae
and the genera Alloprevotella, Roseburia, and Muribaculum. In
contrast, the DSS group exhibited a significant enrichment of
potentially pathogenic bacteria, such as Proteobacteria,
Desulfobacterota, Enterobacteriaceae, and Escherichia–Shigella.
Notably, the DSS + mixture group showed an increase in ben-
eficial bacteria, including Verrucomicrobiota, Akkermansia and
Bacteroides (Fig. 8J).
3.7. V. ratti and LA modulate the production of SCFAs, SCFA-
producing bacteria and lactate
We conducted an analysis of short-chain fatty acids (SCFAs)
and lactate content in feces using gas chromatography (GC)
and a lactate assay kit, respectively. The DSS group showed sig-
nificantly reduced levels of acetic acid (P < 0.001), isobutyric
acid (P < 0.01), valeric acid (P < 0.05), isovaleric acid (P < 0.05),
and total SCFAs, while lactate content was increased (P <
0.0001) compared to the Ctrl group. In contrast, the DSS +
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mixture group exhibited significantly increased levels of acetic
acid and isobutyric acid (P < 0.001), and reduced lactate
content (P < 0.0001) compared to the DSS, DSS + LA and DSS +
V. ratti groups. Importantly, the DSS + mixture group demon-
strated a higher total SCFA content and a lower lactate content
than the other groups (Fig. 9, P < 0.0001). These results indi-
cate that when the UC mice were administered with a combi-
nation of V. ratti and LA, the lactate produced by LA was effec-
tively converted into SCFAs by V. ratti, thus maintaining intesti-
nal health and alleviating colitis in mice.
4. Discussion
Gut microbiota dysbiosis is a significant factor in the occurrence
and development of UC.27 Furthermore, patients with severe UC
often exhibit elevated levels of fecal lactate.28 Previous studies
have illuminated the beneficial impact of introducing feces-
enriched LUB flora in ameliorating colitis in murine models.22
Notably, L. acidophilus (LA) has shown promising potential in alle-
viating UC in mice by modulating immune responses, influen-
cing miRNA expression, restoring gut microbiota balance, and
producing essential metabolites.10
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Fig. 7 Effect of V. ratti and LA pretreatment on the intestinal microbiota in healthy mice. During the intervention period: (a) the relative abundances
of the gut microbiota at the genus level; (b) the relative abundance of Ligilactobacillus; and (c) the total relative abundance of LAB. Compared with
the Ctrl group, **p < 0.01; compared with the DSS group, ##p < 0.01; DSS + V. ratti vs. DSS + mixture, *p < 0.05.

Veillonella, a key member of the LUB group found in the use of lactic acid bacteria as intestinal probiotics for
the intestines, actively engages in cross-feeding interactions UC, there remains a paucity of reports on the combined
with lactic acid bacteria to regulate the intestinal environ- probiotic effect of Veillonella and LA in relieving colitis in
ment of the host. While numerous studies have examined mice. Thus, in this study, we aimed to assess the probiotic

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Fig. 8 V. ratti and LA contributed to restore the intestinal flora homeostasis of UC mice. During the UC model induction period: (a) rarefaction
curves comparing the number of sequences with the number of OTUs found in the 16S rRNA gene libraries from the microbiota in the feces of the
mice in the five groups; (b) alpha diversity (Pielou’s e) illustrating the diversity of each group; (c) Venn diagrams of OTUs demonstrating overlap
among the groups; (d and e) beta diversity (PCA and NMDS) illustrating the diversity of each group; (f ) the top 16 phylum-level stacked-bar; (g) the
top 30 genus-level stacked-bar; (h) histogram of LDA scores >2 for p_phylum, c_class, o_order, f_family, g_genus and s_species; and (i) taxonomic
cladogram. Branching the evolutionary tree diagram, the circles radiating from inside to outside in the diagram represent the taxonomic levels from
phylum to genus (the innermost one with yellow circles is the boundary). Each small circle at a different taxonomic level represents a taxon at that
level, and the diameter size of the small circles represents the relative abundance size. ( j) The relative abundances of Firmicutes, Bacteroidota, the
ratio of Firmicutes to Bacteroidota, Verrucomicrobiota, Bacteroides, Escherichia–Shigella and Desulfovibrionaceae in the Ctrl, DSS, DSS + LA and
DSS + V. ratti, DSS + mixture groups. Compared with the Ctrl group, **p < 0.01; compared with the DSS group, ##p < 0.01; DSS + V. ratti vs. DSS +
mixture, *p < 0.05.

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Fig. 9 V. ratti and LA regulated the production of lactate, SCFAs, and SCFA-producing bacteria. (a) The levels of acetic acid, propionic acid, isobuty-
ric acid, butyric acid, isovaleric acid and valeric acid; (b) the levels of total SCFAS; (c) the production of lactate in the feces; (d) the relative abundance
of acetate-producing bacteria; and (e) the relative abundance of butyrate-producing bacteria. Data are presented as mean ± SD, n = 5/group.
Significant differences were calculated by one-way ANOVA, followed by Tukey’s test for multiple comparisons,*p < 0.05; **p < 0.01; ***p < 0.001;
****p < 0.0001, compared to the Ctrl group; #p < 0.05; ##p < 0.01; ##p < 0.001; ####p < 0.0001, compared to the DSS group.

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combination of V. ratti and LA in a DSS-induced colitis
mouse model.
As expected, the co-administration of V. ratti and LA posi-
tively influenced the intestinal microenvironment, leading to
an improved colitis status in mice. The intervention with
V. ratti and LA yielded multiple beneficial outcomes, including
an increase in colon length, a reduction in disease activity,
lower disease activity index (DAI) scores, elevated levels of anti-
oxidant factors, reduced expression of pro-inflammatory
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factors, and relief from colitis symptoms. Notably, the combi-
nation of V. ratti and LA demonstrated enhanced efficacy com-
pared to administering mice with them separately. These find-
ings suggest that this novel probiotic blend holds promise as a
potential treatment for alleviating inflammatory bowel disease.
Oxidative stress and immune response are key factors in
tissue damage associated with UC.29,30 The excessive pro-
duction of reactive oxygen species (ROS) disrupts the balance
between oxidants and antioxidants, resulting in oxidative
damage to the host.31 In our experimental study, we observed
a significant increase in myeloperoxidase (MPO) accumulation
in the colon tissue of mice in the DSS group, indicating heigh-
tened levels of inflammation. However, the co-administration
of V. ratti and LA significantly reduced MDA and MPO pro-
duction, improved SOD activity, and decreased GSH levels in
DSS-challenged mice (Fig. 6). These findings underscore the
potential of the V. ratti and LA combination to mitigate the
inflammatory responses in the colon triggered by DSS, enhan-
cing antioxidant activity and decreasing oxidative damage.
UC is often characterized by uncontrolled and highly acti-
vated inflammation within the intestinal mucosa.32 Previous
studies have reported elevated iNOS expression in the colonic
mucosal epithelium and crypts of individuals suffering from
ulcerative colitis (UC), which has been confirmed through
in vitro cellular experiments.33,34 Moreover, pro-inflammatory
cytokines such as IL-1β, IL-γ, IL-6, IL-17a, and IFN-γ are com-
monly involved in initiating intestinal inflammation in UC.35
In our study, DSS treatment increased the expression of these
pro-inflammatory cytokines, while the co-administration of
V. ratti and LA significantly regulated their levels (Fig. 5). We
speculate that this combination may alleviate colitis by inhibit-
ing the signaling pathway and activating the NRF-2 signaling
pathway, both of which play roles in regulating inflammatory
responses.36,37 Disruption of the intestinal epithelial barrier
function is also implicated in UC.38 We analyzed the
expression of proteins that maintain the intestinal barrier
function and found that V. ratti combined with LA positively
modulated the DSS-induced decrease in the expression of
barrier-maintaining proteins such as MUC2, MUC3 and occlu-
din in mice. The results indicate that V. ratti combined with
LA is able to restore epithelial barrier integrity and relieve UC
symptoms.
Bacteria engage in both competition for nutrients and
cooperative processes such as metabolic cross-feeding, where
one strain’s metabolites serve as nutrition for another.39
Previous studies conducted in the rumen and the pig GI tract
have established that Veillonella are capable of utilizing lactate
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and converting it primarily into acetate and propionate.40 In
our previous study, we conducted experiments adjusting the
glucose and sodium lactate concentrations to 4.0 g L−1 and
4.5 g L−1, respectively, based on medium 136, to assess the
yield of SCFAs in the medium after 24 hours of co-culture. The
results demonstrated a significant increase, approximately
tenfold, in both acetic and propionic acid production during
co-culture (data unpublished). To further explore the dynamics
of cross-feeding between V. ratti and LA under conditions with
limited carbon sources, we reduced the amount of trypsin and
yeast dip powder by five-fold, removed sodium lactate, and
increased glucose to 8.0 g L−1. Our experimental data showed
that V. ratti and LA in co-culture primarily produced acetic
acid, propionic acid, and butyric acid, all of which were at sig-
nificantly higher levels compared to mono-cultures. These
findings suggest the mutual promotion of proliferation by
V. ratti and LA through cross-feeding during co-culture,
wherein LA rapidly converts glucose into lactate, which, as an
available carbon source for V. ratti, is further utilized to
produce SCFAs.
Short-chain fatty acids (SCFAs), including acetate, propio-
nate, and butyrate, play a crucial role in maintaining intestinal
homeostasis and serve as an important fuel for intestinal epi-
thelial cells, strengthening the integrity of the gut barrier.2,14
These SCFAs have exhibited benefits in preventing intestinal
diseases, including IBD.41–43 Moreover, previous research has
highlighted their capacity to inhibit the proliferation of patho-
gens within the host’s intestinal microenvironment.44
In order to gain deeper insights into how this probiotic
combination influences the intestinal microflora and their
metabolites, we conducted analyses of the gut microbiota and
metabolites (including SCFAs and lactic acid) in the UC mice
subjected to treatment with V. ratti and LA.
In our study, we observed a significant enrichment of
Ligilactobacillus in the mixture group during the intervention
period. Ligilactobacillus salivarius is known for its anti-
microbial and immune-regulatory properties, which contribute
to the regulation of intestinal microbiota.45,46 We speculate
that the one-week intervention with V. ratti and LA in mice
effectively increased the relative abundance of the beneficial
bacterium Ligilactobacillus, thereby preventing the develop-
ment of colitis. Furthermore, during the UC model induction
period, the DSS group displayed a significant increase in the
relative abundance of pro-inflammatory bacteria such as
Desulfovibrio, Escherichia–Shigella, and Helicobacter, contribut-
ing to greater intestinal inflammation. In contrast, the DSS +
mixture group exhibited an increase in the relative abundance
of beneficial bacteria, including Akkermansia, Ruminococcus,
and Eubacterium, which are known for their capacity to
produce SCFAs (Fig. 9D and E). And their increased abundance
in the DSS + mixture group corresponded to higher levels of
SCFAs.47,48 The above findings suggest that the intervention
with V. ratti and LA reversed the microbiota alterations and
alleviated the inflammatory responses in mice.
The microbial community of the human colon contains
numerous lactic acid-producing bacteria. However, lactate is
Food Funct.

Paper
normally detected at low concentrations (<5 mM) in feces from
healthy individuals.22,49 In our study, the DSS group exhibited
a significantly higher lactic acid content than the Ctrl group.
Interestingly, the DSS + mixture group exhibited a lower lactic
acid content compared to both the DSS and DSS + V. ratti
groups. This finding indicates that LA promotes the growth of
V. ratti, facilitating efficient consumption of lactic acid and its
conversion into SCFAs. As anticipated, the DSS + mixture
group demonstrated higher levels of total SCFAs in the feces.
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Collectively, the colonization with V. ratti and LA in mice
resulted in increased SCFA levels. This modulation of the gut
microbiota and metabolites contributed to the maintenance of
intestinal barrier integrity and control of intestinal inflam-
mation through enhanced immune functions. Therefore, the
administration of combined V. ratti and LA promoted the pro-
duction of SCFAs, which played a vital role in regulating intes-
tinal health.
This study has several limitations that should be acknowl-
edged. First, we are yet to delve deeply into the workings of the
mechanisms of inflammatory and antioxidant factors that
underlie the regulatory effects of V. ratti and LA in chronic UC.
Second, while our results are promising, the application of
V. ratti and LA as a probiotic combination in UC patients
requires rigorous safety evaluation and clinical intervention
trials to confirm the results observed in our animal model.
Third, our focus on lactic acid and short-chain fatty acids rep-
resents only a small fraction of the potential intestinal metab-
olites influenced by V. ratti and LA. We recognize the need for
more comprehensive investigations on the broader spectrum
of intestinal metabolites in future studies.
5. Conclusion
The co-administration of V. ratti and LA positively impacted
the intestinal microenvironment, resulting in notable improve-
ments in the colitis status of mice. This was evident through
several key observations: an increase in colon length, a
reduction in disease activity, and enhanced antioxidant
activity. These findings underscore promising therapeutic
potential of this novel probiotic combination for mitigating
inflammatory bowel disease.
Author contributions
Na Li: conceptualization, methodology, and writing – original
draft. Hejing Wang: investigation, data curation and software.
Huizhu Zhao: methodology, software and validation.
Mengyang Wang: visualization, investigation and formal ana-
lysis. Yi Hao: data curation. Jia Yu: conceptualization and
resources. Jin Cai: methodology and software. Yun Jiang:
writing – review and editing. Xin Lü: supervision. Bianfang
Liu: supervision and funding acquisition. All authors have
read and agreed to the published version of the manuscript.
Food Funct.
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Ethical approval
The animals were allowed free access to standard chow and
sterilized water. All experiments involving animals were
approved by the Animal Ethics and Welfare Committee of the
Northwest A&F University, China (SP2022015).
Data availability
The datasets analyzed during the current study are available
from the corresponding author on reasonable request.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by Grant No. 31972043 from the
National Natural Science Foundation of China and Grant
2023-YBNY-146 from the General project – agricultural field.
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