Journal of Functional Foods 80 (2021) 104424

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Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Probiotic-fermented Chinese dwarf cherry [Cerasus humilis (Bge.) Sok.]

juice modulates the intestinal mucosal barrier and increases the abundance
of Akkermansia in the gut in association with polyphenols
Chang-e Guo , Qingyu Cui , Jinghe Cheng , Jiaji Chen , Zihan Zhao , Ran Guo , Xi Dai ,
Zhijiang Wei , Weidong Li *
a
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, PR China
b
Engineering Research Center of Good Agricultural Practice for Chinese Crude Drugs, Ministry of Education, 102488 Beijing, PR China

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

Keywords: Polyphenols have long been used as functional compounds and are beneficial for host immunity and gut health.
Chinese dwarf cherry This three-phased study investigated the function of Ouli fermented juice (OF) in improving the immunity of
Polyphenol cyclophosphamide (CTX)-immunosuppressed mice and regulating their intestinal microbiota and mucosal bar­
Fermentation
rier. Lactobacillus plantarum fermentation increased the phenolic content in the OF. OF effectively improved host
Intestinal mucosal barrier
immunity and the intestinal mucosal barrier, including increasing serum immunoglobulin (Ig)A, IgG, and IgM
Akkermansia
levels and secreted IgA (sIgA) levels in the colon, and reduced CTX-induced pathological damage to the spleen
and jejunum. Importantly, OF effectively increased the abundance of Lachnospiracea, Roseburia, Akkermansia, and
butyric acid-producing microbes, subsequently increasing the levels of short chain fatty acids in the colon. OF
with polyphenols exhibited better efficacy at altering the intestinal flora, which might affect the host immunity
by changing the host-mucosal barrier-microbiota interaction.

1. Introduction
Immune suppression is an unbalanced, abnormal state of the immune
system that is caused by damage to immune cells, tissues and organs.
Imbalanced host immunity leads to a variety of diseases, such as cancer,
asthma, urticaria, and vitiligo. The intestinal mucosa barrier plays a role
in host immunity; this barrier is composed of epithelial cells that are
protected by an overlying host-secreted mucosal layer (Martens, Neu­
mann, & Desai, 2018). The intestinal epithelium consists of differenti­
ated cell types with defined functions: enterocytes, enteroendocrine
cells, microfold cells, etc. Different types of cells secrete a wide array of
cytokines that contribute to host immunity and the immunomodulatory
signals produced by them. Secreted immunoglobulin A (sIgA) is stored
in mucus and is the most abundant antibody isotype in humans. Mi­
crobes and their metabolites, short chain fatty acids (SCFAs), also play
key roles in innate and adaptive immunity (Johnson & Kudsk, 2000).
Microbes may modulate the growth of lymphoid cells and the ultra­
structure of the mucosa. SCFAs, which are produced by microbes in the
colon (Mileo, Nistico, & Miccadei, 2019), modulate the mucosal barrier
and the peripheral immune system by regulating the activities of
GPR41/43 (Vieira et al., 2015) in intestinal epithelial cells. Intestinal
mucosal immune disorders potentially lead to ulcerative colitis, Crohn’s
disease, colorectal cancer and other systemic diseases, and thus must be
resolved immediately.
Traditional Chinese medicine and natural plants have attracted
increasing attention due to their bioactive substances and functions in
regulating host immunity. Common immunopotentiators, such as poly­
saccharides (Tang et al., 2018), phenolic compounds (A. R. Williams
et al., 2017), crude extracts (Lin et al., 2019), and fermented juices of
plants (Lu, Toshima, Wu, Zhang, & Cai, 2007), may adjust host immu­
nity and influence the mucosal barrier. Purple potato polysaccharides
and Momordica charantia L. juice increase the abundance of beneficial

Abbreviations: Ouli, Chinese dwarf cherry; OF, fermented Ouli juice; CTX, cyclophosphamide; sIgA, secreted immunoglobulin A; SCFAs, short chain fatty acids; IL,
interleukin; OJ, Ouli juice; PP, prevention phase; MP, model phase; CP, continuation phase; Z, normal group; M, model group; OFL, OFM and OFH, low, middle, and
high concentration OF groups, respectively; P, positive group; SI, spleen indices; TI, thymus indices; CAC, cecal contents; COC, colon contents; OTUs, operational
taxonomic units.
* Corresponding author at: School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, PR China.
E-mail address: liweidong2005@126.com (W. Li).

https://doi.org/10.1016/j.jff.2021.104424
Received 26 October 2020; Received in revised form 24 February 2021; Accepted 26 February 2021
Available online 23 March 2021
1756-4646/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
C.-e. Guo et al. Journal of Functional Foods 80 (2021) 104424

microbes and levels of butyric acid in the intestines (Tang et al., 2018).
The polyphenols in red wine significantly increase the release of inter­
leukin (IL)-21 and reduce IL-1β and IL-6 levels (Nash et al., 2018). Along
with their many other functions, an important issue is how to take
advantage of these active ingredients of fruits and plants.
Fermentation technology has been used to prolong food storage time
and alter the nutritional and sensory qualities of fruits and vegetables.
During fermentation, secondary metabolites such as polyphenol, poly­
saccharides, and saponins are digested into small molecules. Different
types of fruits, including blueberry (Vuong, Martineau, Rarnassarny,
Matar, & Haddad, 2007), pomegranate (Tomas-Barberan & Espin, 2019) Fig. 1. Different treatments and phases of study in the animal experiments.
and mulberry (Kwaw et al., 2018), have been fermented and used as
functional foods. Fermented cashew apple (Kaprasob et al., 2019)
products regulate type 2 diabetes. Fermented papaya juice (Somanah Table 1
et al., 2016) regulates oxidative damage, reduces inflammation and Treatments received by the experimental mice in different phases and groups.
improves immune function by regulating nitric oxide synthesis and TNF- Groups Explanation
α secretion from RAW 267.7 macrophages. In other words, fermentation
Z Z1 (Z-PP) Water for 14 days
is a good method for the microbial transformation of fruits and vege­ Z2 (Z-MP) Z-PP followed by water + i.p. saline for 3 days
tables used as functional foods. Z3 (Z-CP) Z-MP followed by water for 14 days
Chinese dwarf cherry (Ouli) (Cerasus humilis (Bge.) Sok.) belongs to M M1 (M-MP) Z-PP followed by water + i.p. CTX (80 mg/kg) for 3 days
M2 (M-CP) M-MP followed by water for 14 days
the Rosaceae family and the Cerasus genus (Ye et al., 2017). Ouli has
OFL OFL-PP OF juice low dose (0.625 g/kg bw) for 14 days
been described as the “calcium fruit” because its calcium content is OFL-MP OFL-PP followed by OF juice + i.p. CTX (80 mg/kg) for
significantly higher than kiwi, strawberry, and apple (Li et al., 2014). 3 days
Ouli is abundant with polyphenol, the concentration of which ranges OFL-CP OFL-MP followed by OF juice (0.625 g/kg bw) for 14 days
from 217.13 to 458.20 mg/100 g in different districts, and possesses a OFM OFM-PP OF juice medium dose (1.25 g/kg bw) for 14 days
OFM-MP OFM-PP followed by OF juice + i.p. CTX (80 mg/kg) for
good antioxidant capacity (Li et al., 2014). However, because Ouli is a
3 days
soft and friable fruit, the market value of this fruit is limited. Conse­ OFM-CP OFM-MP followed by OF juice (1.25 g/kg bw) for 14 days
quently, a reasonable approach is to prepare Ouli as a fermented func­ OFH OFH1 (OFH- OF juice high dose (2.5 g/kg bw) for 14 days
tional product, which would extend its marketability and increase its PP)
economic value. Dietary polyphenols have many functions, such as anti- OFH2 (OFH- OFH-PP followed by OF juice + i.p. CTX (80 mg/kg) for
MP) 3 days
allergic (Kou, Han, Li, Xue, & Zhou, 2016), intestinal mucosal barrier OFH3 (OFH- OFH-MP followed by OF juice (2.5 g/kg bw) for 14 days
-regulating (A. R. Williams et al., 2017) and tumor cell growth- CP)
inhibiting activities (Ding, Jiang, & Fang, 2018). Polyphenol in plums OJ OJ1 (OJ-PP) OJ juice dose (2.5 g/kg bw) for 14 days
(Septembre-Malaterre, Remize, & Poucheret, 2018) modulates the ac­ OJ2 (OJ-MP) OJ-PP followed by OJ juice + i.p. CTX (80 mg/kg) for
3 days
tivities of the Akt/mTOR pathway and microRNAs, both of which are
OJ3 (OJ-CP) OJ-MP followed by OJ juice (2.5 g/kg bw) for 14 days
potential factors contributing to the incidence of colon cancers. P P-PP Levamisole dose (1.25 mg/kg bw) for 14 days
Polyphenol-enriched cranberry (Anhê et al., 2015) extract prevents P-MP P-PP followed by levamisole + i.p. CTX (80 mg/kg) for
obesity, intestinal inflammation and other diseases and improves the 3 days
distribution of Akkermansia bacteria in high-fat/high-sucrose models. A P-CP P-MP followed by levamisole (1.25 mg/kg bw) for
14 days
polyphenol-enriched diet and Ascaris suum infection modulate mucosal
immune responses by stimulating the growth of intestinal epithelial
cells, modulating the gut microbiota composition and increasing SCFA The fruits were cut, and the seeds were removed. The non-edible parts
levels in pigs (A. R. Williams et al., 2017). However, only a small amount were removed, and the remaining portion was juiced without adding
of the polyphenols is absorbed in the small intestine; most of the poly­ any water. The juice was first sterilized at 90 ◦ C for 8 min. The sterilized
phenols accumulate in the colon and are then metabolized by flora juice was inoculated with a solution of Lactobacillus plantarum strain
before absorption, which results in a low oral bioavailability (Mileo BNCC194165 (5%, m/V) (this strain was purchased from the BeNa
et al., 2019). Although much is known regarding the effects of Culture Collection, Suzhou, China) and then fermented at 37 ◦ C for 72 h
polyphenol-rich foods on the mucosal barrier, no related reports have to prepare the OF. Non-fermented Ouli juice (OJ) was prepared as a
examined the function of Ouli fermented juice (OF) in regulating im­ control under the same conditions without inoculation and fermenta­
munity and the intestinal mucosal barrier. tion. The OF and OJ were sterilized at 90 ◦ C for 10 min, and these juices
This study investigated the function of OF on improving the immune were stored at − 80 ◦ C.
function of cyclophosphamide (CTX)-immunosuppressed mice and
regulating the intestinal mucosal barrier. The study was divided into
three phases to obtain complete data for analysis. This study provides 2.2. Determination of the contents of the main components
guidance for the use of Chinese dwarf cherry as a functional food for
humans and animals. The total phenolic content was determined using the Folin-Ciocalteu
method (Zuhaili, Sujatha, Noor, & Jamilah, 2018). The standard curve
2. Materials and methods was y = 0.0013x + 0.0271 (60–600 μg/mL), r2 = 0.9993. The total
flavonoid content was determined using the Al(NO3)3-NaOH-NaNO2
2.1. Ouli fruit treatment and preparation of Ouli fermented juice method (Zuhaili et al., 2018). The standard curve was y = 00016x-
0.0174 (50–600 μg/mL), r2 = 0.999. The DMAC method was used to
The ‘Jingou 2′ variety of Ouli was used in this experiment. Plants determine the total procyanidin content (Prior et al., 2010). The stan­
were grown in the Xulun Hoh Banner of Inner Mongolia (No 2. Sub­ dard curve was y = 0.0025x + 0.0147 (10–200 μg/mL), r2 = 0.9994. The
sidiary of Wuyi Ranch, located at N45◦ 52′ E119◦ 43′ ), China. The fresh differential pH method was used to determine the total anthocyanin
fruits were harvested, transported at a low temperature to the labora­ content (Zuhaili et al., 2018). The SCFAs in OF and OJ were detected by
tory, washed, divided into different containers, and stored at − 80 ◦ C. gas chromatography (GC) (Yu et al., 2020).

2
C.-e. Guo et al.
2.3. Animals and experimental design
Male ICR mice (22.0 ± 2.0 g, 4 weeks old) were purchased from
Beijing Vital River Laboratory Animal Technology Co., Ltd. (SYXK
(Beijing) 2016–0006, China). All mice were placed in clean poly­
propylene cages and housed in animal shelters. The mice were housed at
a constant temperature of 25 ◦ C on a 12-h light–dark cycle and provided
free access to water and food. All animal experiments were performed in
accordance with the National Institutes of Health guide for the care and
use of Laboratory animals (NIH Publications No. 8023, revised 1978)
and approved by the Ethics Committee of Experimental Animal Care at
Beijing University of Chinese Medicine. After one week of adaptation, all
200 mice were randomly divided into three phases and twenty groups
(ten mice in each group) as described in Fig. 1 and Table 1. The
experiment was divided into three phases: the prevention phase (PP);
the model phase (MP); and the continuation phase (CP). The PP included
six groups: a normal group (Z); the low, middle, and high concentration
OF groups (OFL, OFM and OFH groups, respectively); the OJ group (OJ);
and the positive group (P). The MP and CP both included an additional
model group (M), in which mice received OF and OJ orally in the
morning and then received CTX (80 mg/kg) or saline through intra­
peritoneal injection 12 h later. During these phases, mouse body weight
and food intake were recorded daily. The mice were sacrificed at 24 h
after the last observation time.
2.4. Determination of spleen and thymus indices
Three mice in each group were euthanized, and the spleen and
thymus were dissected and weighed. The organ indices were calculated
as follows: spleen index (SI) (g/100 g) = spleen weight × 100 / body
weight; thymus index (TI) (g/100 g) = thymus weight × 100 / body
weight.
2.5. Hematological analysis
The blood was collected into tubes containing the anticoagulant
EDTA to analyze the hematological parameters, such as the erythrocyte
(RBC) count, leukocyte (WBC) count, monocyte (MO) count, hemoglo­
bin (HGB) levels, and platelet (PLT) count, using an auto-analyzer (MEK-
6410C Auto-hematology analyzer, Nihon Kohden Corp., Japan).
2.6. Determination of the levels of cytokines, immunoglobulins in serum
and sIgA levels in the colon
Blood samples were collected and centrifuged at 3500g for 15 min at
4 ◦ C after incubation for at least 1 h. The serum was stored and divided
into sterile centrifuge tubes for subsequent experiments. The levels of IL-
2, IL-6, IFN-γ and TNF-α were determined using a Multifactor Detection
Kit (Millipore cat# MCYTMAG-70K-PX32, mouse, Germany). The IgA,
IgM and IgG levels in serum and the sIgA levels in the colon were
analyzed using an ELISA kit (Shanghai Enzyme-linked Biotechnology
Co., Ltd, China). We used the Multifactor Detection Kit to detect IL-2, IL-
6, IFN-γ and TNF-α content in serum, whose minimum detection limits
were 1, 0.98, 0.49, and 0.49 pg/mL, respectively. ELISA methods were
used to detected IgA, IgM and IgG levels in serum and the sIgA levels in
the colon, whose minimum detection limits were 20 μg/mL, 200 μg/mL,
1.25 mg/mL, and 2 μg/mL, respectively.
2.7. Histopathological analysis of the spleen, thymus, jejunum and colon
The spleen and thymus were fixed with 10% formalin for the histo­
pathological analysis. The lower jejunum and the anterior colon were
flushed with PBS buffer and then fixed with 10% formalin. Thin sections
of these tissue samples were prepared using semi-automatic paraffin
slicers (RM2245, Leica, Germany), then stained with hematoxylin and
eosin and analyzed under a Nikon Ts2/Ts2-FL microscope (Japan) for
3
Journal of Functional Foods 80 (2021) 104424
histological changes.
2.8. Determination of SCFAs content
After the mice were sacrificed, the cecum and colon were removed.
The cecal contents (CAC) and colon contents (COC) were squeezed into
tubes, freeze-dried, and stored at − 80 ◦ C. The internal standard (IS) was
cyclone. Then, 50 mg of CAC and COC freeze-dried powders were
weighed, 0.5 mL of an aqueous solution (containing 0.4 mM IS) was
added, and the pH was adjusted to 2–3 with sulfuric acid. The mixture
was vortexed for 1 min, incubated for 15 min, and then centrifuged at
10000 rpm for 10 min. The supernatant was then filtered with a 0.22-μm
membrane and stored until further analysis.
The levels of SCFAs, including acetic acid, propionic acid, iso-butyric
acid, butyric acid, iso-valeric acid, and valeric acid, were determined
using gas chromatograph (GC) (Tang et al., 2018). The GC analysis of the
extracted samples was performed with an Agilent 7890A gas chro­
matograph equipped with a flame ionization detector (FID), automatic
sampler, and DB-Wax capillary column (30 m × 0.25 μm × 0.25 mm).
The ratio of hydrogen, air, and nitrogen was 1:10:1. The carrier gas flow
rate was 1.0 mL/min. The column temperature was maintained at
100 ◦ C for 1 min, then increased to 130 ◦ C at a rate of 5 ◦ C/min and
maintained for 1 min, and finally increased to 200 ◦ C at a rate of 20 ◦ C/
min and maintained for 1 min. The split ratio was 1:40. The injection
volume was 2 μL.
2.9. Gut microbe 16S rRNA sequencing and bioinformatics analysis
Microbial DNA was extracted from fecal samples using an E.Z.N.A.®
Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the
manufacturer’s protocols. The final DNA concentration was determined
using a NanoDrop 2000 UV–vis spectrophotometer (Thermo Scientific,
Wilmington, DE, USA), and DNA quality was assessed using 1% agarose
gel electrophoresis. The V3-V4 hypervariable regions of the bacterial
16S rRNA gene were amplified with primers 338F (5′ - ACTCCTACGG­
GAGGCAGCAG-3′ ) and 806R (5′ -GGACTACHVGGGTWTCTAAT-3′ )
using a PCR thermocycler system (GeneAmp 9700, ABI, USA). PCR was
conducted using the following program: 3 min of denaturation at 95 ◦ C;
27 cycles of 30 s at 95 ◦ C, 30 s of annealing at 55 ◦ C, and 45 s of elon­
gation at 72 ◦ C; and a final extension step at 72 ◦ C for 10 min. PCR was
performed in triplicate with a 20-μL mixture containing 4 μL of
5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM),
0.4 μL of FastPfu Polymerase and 10 ng of the template DNA. The
resulting PCR products were extracted from a 2% agarose gel and further
purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences,
Union City, CA, USA) and quantified using QuantiFluor™ -ST (Promega,
USA) according to the manufacturer’s protocols. Purified amplicons
were pooled in equimolar concentrations and paired-end sequenced
(2 × 300) using an Illumina MiSeq platform (Illumina, San Diego, CA,
USA) according to standard protocols by Majorbio Bio-Pharm Technol­
ogy Co. Ltd. (Shanghai, China). Raw fastq files were filtered for quality
by Trimmomatic and merged with FLASH. Operational taxonomic units
(OTUs) were clustered with a 97% similarity cutoff using UPARSE
(version 7.1 http://drive5.com/uparse/) with a novel ‘greedy’ algo­
rithm that simultaneously performs chimaera filtering and OTU clus­
tering. The taxonomy of each 16S rRNA gene sequence was analyzed
using the RDP Classifier algorithm (http://rdp.cme.msu.edu/) and
compared with the Silva (SSU132) 16 s_bacteria database using a con­
fidence threshold of 70%.
2.10. Statistical analysis
All data are presented as the mean ± standard error of the mean
(SEM). The data were analyzed using SPSS 20.0 software (IBM Co., NY,
USA). Differences among various groups were analyzed by one-way
analysis of variance (ANOVA) and Turkey’s test for multiple
C.-e. Guo et al.
Fig. 2. Contents of the main components of the OF and OJ (x ± SEM, n = 3).
Fig. 3. Effcts of OF and OJ on body weight and total food intake in mice during three phases (x ± SEM, n = 10).
comparisons. Differences were considered significant at P < 0.05. The
correlation analysis was conducted using R software. The images were
created using GraphPad Prism 8.0 software (GraphPad Software, San
Diego, CA, USA).
3. Results
3.1. Contents of the main components and SCFAs in the OF and OJ
As shown in Fig. 2, total phenolic and flavonoid content in the OF
were almost double the amount in the OJ. Total procyanidin content in
the OJ was 395.2 ± 8.165 mg/100 mL, which was nine times greater
than that in the OF. Total anthocyanin content in the OJ was
13.75 ± 0.06303 mg/100 mL, which was two times greater than that in
the OF. After fermentation, the acetic acid, iso-butyric acid, and butyric
acid contents were higher in the OF than that in the OJ. However, the
contents of propionic acid, valeric acid, and iso-valeric acid did not reach
limit of quantification.
3.2. Effects of OF on immunity in the three phases
3.2.1. Effects of the OF on body weight and food intake
During the PP, the body weight maintained a stable increase, and
significant differences in food intake were not observed (Fig. 3). During
the MP and after injection with CTX, body weight and food intake
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Journal of Functional Foods 80 (2021) 104424
showed a decreasing trend, except in the normal group, indicating that
CTX altered mouse growth. Moreover, during the CP, supplementation
with OF and OJ did not produce significant differences compared with Z.
Overall, the intake of OF and OJ did not affect the growth and devel­
opment of mice.
3.2.2. Effects of OF on the hematological parameters of mice
During the PP (Table 2), compared with normal mice, OF-
supplemented mice showed significantly increased PLT counts
(P < 0.001). A significant decrease in the hematological parameters of
the CTX model group, except for MO, was observed during the MP
(P < 0.05), while HGB levels and RBC counts increased in both the OFM
and OFH groups. During the CP, each hematological parameter slowly
increased after the CTX injection and significantly decreased in the HGB
level and RBC count compared to normal mice. In all three stages, no
differences were observed among the Z, OFM, OFH and OJ groups.
3.2.3. Effects of the OF on the spleen and thymus indices in mice
The effects of OF on the SI and TI in immunosuppressed mice are
shown in Fig. 4A. During the PP, OFL, OFM and OFH significantly
increased the SI (P < 0.05), while only OFH increased the TI in normal
mice (P < 0.05). During the MP, CTX exerted substantial effects on the SI
and TI in model mice (P < 0.001). Although the SI and TI decreased,
their changes were slightly less than the changes observed in the model
mice. After 14 days, the SI of the M group was significantly lower than
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Table 2
Effects of OF and OJ on hematological parameters during three phases (‾x ± SEM, n = 10).
PP

Group HGB (g/L) MO (109/L) PLT (109/L) RBC (1012/L) WBC (109/L)

Z 147.2 ± 2.950 0.4667 ± 0.1155 796.2 ± 80.44 7.808 ± 0.1797 5.300 ± 1.337
OFL 147.2 ± 4.324 0.7667 ± 0.2082* 1094 ± 127.1*** 7.986 ± 0.3917 5.080 ± 1.252
OFM 151.0 ± 7.937 0.5600 ± 0.1517 1092 ± 151.0*** 7.785 ± 0.4904 5.367 ± 1.079
OFH 150.7 ± 4.716 0.7800 ± 0.2280* 995.7 ± 104.9*** 8.180 ± 0.5339 5.540 ± 1.489
OJ 148.2 ± 4.025 0.6000 ± 0.1732 929.4 ± 135.2 7.910 ± 0.3908 4.800 ± 0.424
P 149.6 ± 5.224 0.5200 ± 0.0837 1058 ± 154.2*** 8.234 ± 0.4021 3.517 ± 0.731*
MP
Z 202.3 ± 27.10 0.5667 ± 0.2422 1049 ± 77.54 11.17 ± 1.656 5.460 ± 1.212
M 152.0 ± 8.093* 0.3125 ± 0.0571 815.5 ± 86.09* 8.049 ± 0.3319* 1.367 ± 0.2422*
OFL 176.0 ± 19.21 0.4333 ± 0.0577 952.7 ± 129.8 9.498 ± 1.573 2.083 ± 0.5193*
OFM 184.3 ± 11.81## 0.3000 ± 0.1414 982.1 ± 128.0 10.31 ± 0.9944# 1.875 ± 0.0500*,#
OFH 185.1 ± 18.59# 0.5000 ± 0.0828 1135 ± 174.9## 10.19 ± 1.372# 2.550 ± 0.3536
OJ 169.7 ± 21.91 0.3000 ± 0.0660 988.6 ± 190.0 9.428 ± 1.672 2.200 ± 0.6831*
P 186.5 ± 27.72 0.5000 ± 0.0814 937.3 ± 153.0 10.44 ± 1.835 2.080 ± 0.3564*
CP
Z 197.5 ± 24.17 0.8000 ± 0.0914 867.6 ± 87.10 9.645 ± 1.631 3.200 ± 0.8679
M 148.0 ± 9.827* 0.6800 ± 0.0583 756.4 ± 132.8 8.292 ± 0.8035* 3.100 ± 0.1826
OFL 174.5 ± 16.90 0.8000 ± 0.1097 843.3 ± 83.03 8.791 ± 0.9597 4.056 ± 0.779
OFM 169.3 ± 8.077## 0.8429 ± 0.0902 884.0 ± 118.8# 9.133 ± 0.5911 4.500 ± 0.754*,#
OFH 160.4 ± 3.259 0.8667 ± 0.0704 914.8 ± 195.6# 8 483 ± 0.5005 3.900 ± 0.712
OJ 173.3 ± 16.26 # 0.8571 ± 0.1512 917.9 ± 84.93## 9.356 ± 0.9689## 5.411 ± 0.712*,#
P 187.0 ± 21.94# 1.1800 ± 0.1271 840.6 ± 124.6 9.346 ± 1.115 4.233 ± 0.734*,#

Note: Z, normal group; M, model group; OFL, OFM and OFH, low, middle, and high concentration OF groups, respectively; P, positive group. *, #, P < 0.05 means the
difference is significant; **, ##, P < 0.01 means the difference is very significant; ***, P < 0.001 means the difference is extremely significant. * means comparing with Z
group. # means comparing with M group.

that of the OFL, OFM and OFH groups (P < 0.01). In addition, the TI did
not increase in any group. However, the effects of OJ on the SI and TI
were not obvious.
3.2.4. Effects of OF on serum IL-2, IL-6, IFN-γ, and TNF-α levels
In the three phases, serum IL-2, IL-6, IFN-γ, and TNF-α levels
(Fig. 4B) ranged from 0.8920 to 5.808 pg/mL, 0.5360 to 3.005 pg/mL,
0.2812 to 12.41 pg/mL, and 5.095 to 12.43 pg/mL, respectively. A
remarkable increase in IL-6 and IFN-γ levels was observed in model mice
(P < 0.001), while a decrease in TNF-α levels (P < 0.001) was observed
during the MP, indicating that CTX suppressed immune activities. OFM
and OFH increased the TNF-α concentration in mice (P < 0.05), and OFH
also significantly decreased IL-6 levels (P < 0.05). The IL-2, IL-6, and
IFN-γ levels in M mice did not recover normally during the CP, and IFN-γ
levels showed compensatory increases (P < 0.001). Following OJ sup­
plementation, every abovementioned index of OFM and OFH completely
returned to the values of the normal group, and the IL-6 and IFN-γ levels
decreased significantly (P < 0.05). Therefore, the OF reversed CTX-
induced immunosuppression and improved immunity by modulating
cytokine production.
3.2.5. Effects of the OF on serum IgA, IgM and IgG levels
Immunoglobulins, including IgA, IgG and IgM, directly reflect host
immunity. The serum concentrations of IgA, IgM and IgG in mice during
the PP ranged from 497.4 to 538.9 μg/mL, 14.33 to 16.65 μg/mL, and
2548 to 3488 μg/mL, respectively (Fig. 4C). Compared with normal
mice, OFH and OJ obviously increased the IgM content. The secretion of
IgA, IgM and IgG in CTX-treated mice was significantly inhibited
(P < 0.001) during the MP. Different concentrations of the OF and OJ
increased IgA secretion (P < 0.05); OFM and OFH increased IgG secre­
tion (P < 0.05), and IgM levels were obviously increased by OFH.
Finally, during the CP, we observed significant increases in IgG and IgM
levels of up to 16.10 μg/mL and 2797 μg/mL, respectively (P < 0.05 and
P < 0.01, respectively).
3.2.6. Effects of the OF on spleen and thymus histopathology
Based on the results described above, OFH displayed the best efficacy
at improving immunity in normal and CTX-immunosuppressed mice;
therefore, the animals in the OFH group were selected for HE staining of
the spleen and thymus. As shown in Fig. 4D, the structure of the spleen in
group Z was complete, the white and red myelin structures were clear,
and the splenic corpuscle was complete, clear and well arranged. The
white myelin and splenic corpuscle in the spleen exhibited atrophy and
the number of lymphocytes decreased in the model mice. In the OFH
group, white myelin atrophy was reduced. Moreover, red myelin hy­
perplasia in the spleen was observed, and the germinal center was
obvious, indicating that after a period of treatment, the spleen showed
signs of regenerative repair. The thymus slice results are not presented,
because there were no significant changes among these groups.
3.3. Effects of the OF on intestinal mucosal barrier during the three phases
3.3.1. Effects of the OF on sIgA level in the colon
The sIgA level was detected in the colon to evaluate the mucosal
barrier. As shown in Fig. 5A, during the PP, a significant increase in sIgA
levels was observed in the OFH group (P < 0.001). Compared with
normal mice, significant decreases in sIgA levels were observed in CTX-
suppressed mice during both the MP and CP. SIgA levels recovered to
normal following the administration of OFM and OFH supplements
during the MP (P < 0.05), and OJ significantly increased the secretion of
sIgA in the colon during the CP (P < 0.05).
3.3.2. Effects of the OF on the SCFA contents in the CAC and COC
The contents of six SCFAs in the CAC and COC were determined to
further examine the effects of OF on SCFAs (Fig. 5B and S1). The butyric
acid contents are shown in Fig. 5B. Significant increases in SCFA con­
tents were observed following the OF treatment during the three phases
(P < 0.05), and the OJ produced smaller increases.
3.3.3. Effects of the OF on histopathological changes in the jejunum and
colon
OFH displayed the best efficacy at improving the mucosal barrier in
normal and CTX-immunosuppressed mice, and thus, this group was
selected for jejunum and colon staining (Fig. 5C). HE stain could give an
image on intestine structure rather than function, which could help us
understand structure changes of intestine with immunosuppression. As

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Fig. 4. Effects of OF and OJ on spleen and thymus indices (A), serum IL-2, IL-6, IFN-γ, and TNF-α levels (B) and serum IgA, IgG and IgM levels (C) in mice during the
three phases (x ± SEM, n = 10). (D) Effects of OFH and P on histopathological changes in the mouse spleen during the three phases (×10). Notes: Z, normal group; M,
model group; OFL, OFM and OFH, low, middle, and high concentration OF groups, respectively; P, positive group. Black arrows indicate corpuscles in the spleen
tissue; red arrows indicate extramedullary hematopoiesis in the spleen tissue. Compared with the model group, *, P < 0.05 indicates a significant difference; **,
P < 0.01 indicates a very significant difference; ***, P < 0.001 indicates an extremely significant difference.

shown in Fig. 5C, the jejunum structure and mucous layer were com­
plete. Significant changes in the submucosal muscular layer or adven­
titia were not observed. However, the lacteal structure in the cilium
disappeared in the model mice. The lactea is a capillary lymphocyte in
the intestine surrounding by some lymphocytes and is related to the host
immune response and absorption of macromolecules. In summary, these
results indicated that supplementation with OF could improve intestinal
mucosal barrier and indirectly affect host immunity.
3.3.4. Effects of the OF on gut microbes during the three phases
3.3.4.1. The gut microbiome data. Six hundred eighty OTUs in the PP,
810 OTUs in the MP and 863 OTUs in the CP were identified from
24700, 30655 and 23835 valid sequences, respectively. The α-diversity
indices are shown in Fig. S2 of the Online Resource. Based on the un­
weighted UniFrac analysis, β-diversities reflect the microbial structure
and evaluate differences in the whole microbiota among different

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Fig. 5. Effects of the OF and OJ on sIgA levels in the colon (A) and butyric acid contents in the CAC and COC (B) from mice during the three phases (x ± SEM,
n = 10). (C) Effects of OFH and P on histopathological changes in the mouse jejunum during the three phases (×10). Circles indicate that the lacteal part of the
jejunum disappeared, and the red arrows indicate lactea. Note: PP, prevention phase; MP, model phase; CP, continuation phase; Z, normal group; M, model group;
OFL, OFM and OFH, low, middle, and high concentration OF groups, respectively; P, positive group; CAC, cecal contents; COC, colon contents. Compared with
normal group or model group, *, P < 0.05 indicates significant differences; **, P < 0.01 indicates very significant differences; ***, P < 0.001 indicates extremely
significant differences. Different letters indicate significant differences (P < 0.05).

groups. During the three phases, samples were pooled, which reflected
similarities in the community composition within Z, M, OFH and OJ
mice. As shown in Fig. 6A, different groups were separated in PCoA
plots, and differences were observed among the different groups.
3.3.4.2. Dynamic alterations in gut taxa after supplementation with OF and
OJ. At the phylum level, the remaining sequences belonged to 11, 13
and 11 phyla (Fig. 6B), among which Firmicutes and Bacteroidetes were
highly abundant in all groups and phases. Firmicutes and Bacteroidetes
accounted for approximately 95.0% of the phyla. OF and OJ both
regulated the structure at the phylum level. During the PP, compared
with the Z group, the abundance of Proteobacteria, Tenericutes, unclassi­
fied_k_norank_d_Bacteria, and Cyanobacteria increased significantly, and
the abundance of Firmicutes decreased (P < 0.05) after supplementation
with OFH. During the MP, compared with Z2, the abundance of Firmi­
cutes and unclassified_k_norank_d_Bacteria decreased significantly, and
the abundance of Bacteroidetes increased significantly (P < 0.05) in
model mice. Compared with M1, we observed a decrease in the abun­
dance of Firmicutes (25.5% vs. 40.23%, P < 0.05) and a significant in­
crease in the abundance of Proteobacteria (0.93% vs. 0.1381%) and
Verrucomicrobia (0.073% vs. 0%, P < 0.01), of which Verrucomicrobia
was only detected after supplementation with OFH. Similar changes
were observed in M2 during the CP compared to Z3. Epsilonbacteraeota
(2.30% vs. 0.257%, P < 0.05) showed a significant increase in the OFH3
group. Both the OF and OJ regulated the abundance of bacterial phyla
and increased the number of beneficial microbes. Genus alteration of gut
flora is shown in Fig. S3-S4. Alistipes, unclassified_o_Bacteroidales, Para­
sutterella, and Akkermansia were enriched in the OFH2 group (LDA
score > 2.0 with P < 0.05).
Below, we focus on changes at the genus level in the M1 and OFH2
groups during the MP, illustrating that OFH regulated the microbiota in
CTX model mice. The results of the LEfSe analysis and LDA scores are
shown in Fig. 6-C/D. Ruminococcus_1, norank_f_Clostridiales_
vadinBB60_group, Roseburia, Ruminiclostridium_6, Kurthia, and Rumino­
coccaceae_UCG_013 were significantly enriched (LDA score > 2.0 with
P < 0.05) in the M1 group. Meanwhile, in the OFH2 group, Para­
bacteroides, Escherichia-Shigella, unclassified_f_Muribaculaceae, and
Akkermansia were enriched (LDA score > 2.0 with P < 0.05).
3.3.5. Comparison of the regulatory effects of OFH and OJ on the gut
microbiota
Significant differences were observed between the effects of OFH and
OJ on regulating gut microbiota, particularly in the MP. The LEfSe
analysis and LDA scores of MP showed that Parabacteroides, Escherichia-
Shigella, Ruminococcaceae_NK4A214_group, Faecalibaculum, Butyr­
icimonas, and Akkermansia were enriched after treatment with OFH
(Fig. 7A-B, LDA score > 2.0 with P < 0.05). As shown in Fig. 7C-D,
Lachnospiraceae_NK4A136_group, unclassified_f_Lachnospiraceae, Anaero­
truncus, Roseburia, and Biophila in the OFH1 group and Helicobacter and
Ruminococcus_1 in the OFH3 group were significantly enriched in the PP
and CP, respectively. The administration of OJ in the PP and CP regu­
lated the abundance of some genera, such as Odoribacter, nor­
ank_o_Mollicutes, norank_o_Chloroplast, Marvinbryantia, norank_f_
Clostridiales_vadinBB60_group, and Lachnospiraceae_UCG-006.

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Fig. 6. Effects of the OF and OJ on β-diversities (A) and gut taxa at the phylum level (B) in mice during the three phases. (C-D) LEfSe and LDA analyses of the M1 and
OFH2 groups. Taxa displaying significant and biologically consistent differences are shown. Only the taxa with a significant logarithmic LDA threshold score of > 2
and P < 0.05 are shown. Notes: PP, prevention phase; MP, model phase; CP, continuation phase; Z1/2/3, normal group in PP, MP and CP; M1/2, model group in MP
and CP; OFH1/2/3, high concentration OF groups in PP, MP and CP; OJ1/2/3, OJ groups in PP, MP and CP.

3.3.6. Correlations among the gut microbiota, host immunity and intestinal
mucosal barrier
The correlations among host immunity, the mucosal barrier, and
microbiota were determined by calculating Spearman’s correlation co­
efficients (Fig. 8 and S5). Differences in the results were observed among
three periods. Some genera related to host immunity that produce SCFAs
were enriched in both the MP and CP. A negative correlation was
observed between body weight and most microbial genera in the PP and
MP, while the correlation was positive in the CP.
The SI and TI in the PP were positively correlated with GCA-
900066225, Roseburia, unclassified_f_Lachnospiraceae, Blautia, and Lach­
nospiraceae_NK4A136_group. Meanwhile, in the CP, a positive correla­
tion was observed between SI and unclassified_o_Bacteroidetes and
Lachnospiraceae_UCG-006, and a negative correlation was observed be­
tween TI and Roseburia, Parabacteroides, Helicobacter, Bacteroides. Host
immunity was in the normal or activated state, and serum IL-2 and IFN-γ
levels positively correlated with unclassified_o_Bacteroidetes, Ruminococ
caceae_UCG-004, Muribaculum, Parabacteroides, Odoribacter, Alistipes,
Lachnospiraceae_NK4A136_group, norank_f_Muribaculaceae, and Para­
sutterella. The association between TNF-α levels and the genera was
modest, as positive correlations with Parasutterella, Dubosiella and nor­
ank_f_Muribaculaceae was only observed in the CP. Few correlations were
observed between serum IgA, IgG and IgM levels and microbes, and
Butyricicoccus, GCA-900066225, Cetobacterium, ASF356, and Lachno­
spiraceae_NK4A136_group showed associations with immunoglobulin
secretion.
During the MP, the intestinal microbiome was altered drastically in a
remarkably short period after the CTX injection. Negative correlations
were observed among weight, SI, TI and microbial genera, such as
Parasutterella, Escherichia-Shigella, unclassified_o_Bacteroidates, Para­
bacteroides, Alloprevotella, Alistipes, norank_f_Muribaculaceae. Serum IL-6
levels positively correlated with unclassified_o_Bacteroidates,
Prevotellaceae_UCG-001, Alloprevotella, Alistipes, and norank_f_Mur­
ibaculaceae, while negative correlations were observed between IFN-γ

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Fig. 7. Differences in the bacterial communities determined using the LEfSe analysis of the 16S rRNA sequences. (A-B) LEfSe and LDA analyses of the OFH2 and OJ2
groups during the MP. Taxa with significant and biologically consistent differences are shown. Only the taxa with a significant logarithmic LDA threshold score of > 2
and P < 0.05 are shown. (C) Comparison between the OFH1 and OJ1 groups in the PP using the Wilcoxon rank-sum test. (D) Comparison between the OFH3 and OJ3
groups in the CP using the Wilcoxon rank-sum test. Notes: OFH1/2/3, high concentration of Ouli fermentation juice in PP, MP and CP; OJ1/2/3, Ouli juice in PP, MP
and CP. Compared with the OJ group, *, P < 0.05 indicates significant differences; **, P < 0.01 indicates very significant differences.

levels and these genera. Similar positive correlations were observed
between serum IgA, IgG and IgM levels and Lactobacillus.
The sIgA level generally correlated with most genera in different
periods. Strong positive correlations were observed between sIgA levels
and Cetobacterium, Anaerotruncus, Mucispirillum, and unclassified_­
f_Lachnospiraceae, whereas Odoribacter and Ruminococcaceae_UCG-014
negatively correlated with sIgA levels. Positive correlations with Parvi­
bacter, norank_f_Erysipelotrichaceae, Parasutterella, Escherichia-Shigella,
unclassified_o_Bacteroidales, Ruminococcus_2, Parabacteroides, Alloprevo­
tella, Bacteroides, and norank_f_Muribaculaceae were observed in the MP
and CP.
3.3.7. Correlations among the gut microbiota, immunity and chemical
compounds in OF during the MP
In summary, the most dramatic alterations in various genera
occurred in the MP. We chose the MP to perform a deeper analysis of the
correlations among the gut microbiota, host parameters and chemical
compounds in OF, expecting to obtain more theoretical support for the
ability of phenolics to improve immunity. As intuitively shown in
Fig. 9A, the Firmicutes and Bacteroidetes phyla were closely related to the
SI, TI, IgA levels, and IgG levels. Meanwhile, Bacteroidetes positively
correlated with sIgA and butyric acid levels in the colon, including
Alloprevotella, unclassified_o_Bacteroidates, Alistipes, Bacteroides,

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Fig. 8. Correlations between the gut microbiota, host immunity and intestinal mucosal barrier during the PP (A), MP (B), and CP (C). Associations of the changes in
the gut microbial community with other host parameters were determined using R software. Spearman’s correlation analysis was performed after screening the
variance inflation factor (VIF). If the VIF was greater than 10, the index was removed, and another index was used for the correlation analysis. Notes: PP, prevention
phase; MP, model phase; CP, continuation phase; SI, spleen indices; TI, thymus indices; COC, colon contents. × indicates that the correlation is significant, P < 0.05;
£ indicates that the correlation is very significant, P < 0.01.
norank_f_Muribaculaceae, and Parabacteroides. Ruminococcaceae_UCG-
014 negatively correlated with IL-2 levels in serum. Serum IL-6 levels
positively correlated with Prevotellaceae, Odoribacter, and nor­
ank_o_Gastranaerophilales. Serum IFN-γ levels were mainly negatively
correlated with the Bacteroidetes phylum. The relationship between the
microbiome and body weight was negative.
Fig. 9B showed the correlations between different chemical compo­
nents in OF and the gut flora. Parasutterella, unclassified_o_Bacteroidales,
and Parabacteroides were significantly positively correlated with total
phenolic, flavonoid, procyanidin and anthocyanin contents. Helicobacter
was significantly negatively correlated with the aforementioned pa­
rameters. Unclassfied_f_Prevotellaceae, Escherichia-Shigella, and nor­
ank_f_Muribaculaceae displayed significant positive correlations with
total phenolic and flavonoid contents. Ruminococcu_2, norank_o_Gas­
tranaerophilales, and Alloprevotella positively correlated with total pro­
cyanidin and anthocyanin content, while Ruminiclostridium_5 displayed
a significant negative correlation with the aforementioned parameters.
As shown in Fig. 9C, these four substances exhibited significant
positive correlations with sIgA and butyrate levels in the colon, and
significant negative correlations with body weight. Serum IFN-γ levels
exhibited strong negative correlations with these four substances.
4. Discussion
In our study, 200 mice were divided into three phases and 20 groups
(Table 1). OF at 0.625, 1.25, and 2.5 g/kg was orally administered to
mice, and 80 mg/kg CTX was administered by intraperitoneal injection
to mimic immune suppression. During the prevention phase and model
phase, normal and CTX-treated mice were used as control groups,
respectively. Also, the effects of OF and OJ on host immunity and the
intestine mucosal barrier were compared. Studies showed that OF
resulted in positive physiological changes on CTX-treated immunosup­
pressed mice, including the efficient prevention of immune damage by
CTX and marked changes in the serum IL-2, IL-6, IFN-γ, IgA, IgM, and
IgG levels. The changes in these biochemical parameters were further
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Journal of Functional Foods 80 (2021) 104424
supported by evidence obtained from pathological sections, as OF
ameliorated the disrupted morphology and function of the spleen and
jejunum (Figs. 4 and 5). The increase in the sIgA and butyric acid levels
in colon attracted our interest in exploring the interactions among
chemical components in OF, mucosal barrier, especially gut microbiota,
as emerging evidence has linked alterations in the gut microbiota to food
with polyphenols (Wankhade et al., 2019; A. Williams, Andersen-Civil,
Zhu, & Blanchard, 2020).
CTX is an important agent for cancer treatment and other autoim­
mune diseases. Recently, some studies have focused on the effects of CTX
on host immunity and gut microbiota. In CTX-depressed mice, the
number of opportunistic pathogenic microorganisms increased, such as
Enterbacteraceae and Escherichia coli, which will lead to gut dysbacter­
iosis. Also, according to Kong et al. (Kong, Gao, Yan, Huang, & Qin,
2018), probiotics increase the abundance of pro-inflammatory microbes
in mice fed a high-fat and high-sugar diet, such as Alistipes, Acinetobacter,
Anaerotruncus, Bacteroides, Blautia, and Escherichia-Shigella. In our study,
the abundance of these genera was also increased, indicating that CTX
disrupts the balance in the intestinal environment and activates some
related microbes to combat inflammatory reactions during the MP and
CP. It has been demonstrated that changes in gut microbiota caused by
CTX could induce the translocation of several species of Gram-positive
bacteria in secondary lymphoid organs and further affect immune re­
sponses (Viaud et al., 2013). In our study, model mice induced by CTX
showed lower weight, atrophy of spleen and thymus, and disorders of
cytokine in serum, which might be caused by a microbiota disorder.
Chinese dwarf cherry fruits are rich in polyphenol and have the
potential to be a functional food. According to previous studies of Chi­
nese hawthorn (Zheng et al., 2018) and pomegranate (Mosele, Macià,
Romero, Motilva, & Rubió, 2015), proanthocyanins and flavonoid
rutinosides modulate the gut microbiota by exerting a classical
prebiotic-like effect, and are metabolized into caffeic acid, dihydrox­
yphenyl propionic acid, and epicatechin (González-Quilen et al., 2020).
These molecules are absorbed in the colon. Together with the results for
host immunity and intestinal mucosal barrier, we postulate that OF may
C.-e. Guo et al. Journal of Functional Foods 80 (2021) 104424

Fig. 9. Correlations among the gut microbiota, immunity-related parameters and chemical compounds in OF during the MP. (A) Analysis of networks between the
gut microbiome and immunity-related parameters. (B) Analysis of networks between the gut microbiome and chemical compounds in OF. (C) Spearman’s rank
correlation analysis of immunity-related parameters and chemical compounds in OF. The species correlation network diagram mainly calculates the correlation
coefficients, such as Spearman’s rank correlation coefficients, between species to reflect the correlation between species; species with P < 0.05 are displayed by
default in the figure. Notes: SI, spleen indices; TI, thymus indices; COC, colon contents; TPC, total phenolic content; TFC, total flavonoid content; TPAC, total
procyanidin content; TAC, total anthocyanin content. The size of the nodes in the figure represents the abundance of species. Different colors indicate different
species. The line colors indicate positive and negative correlations and the thickness of line indicates the size of the correlation coefficient, where a thicker line
represents a greater correlation between species. In Fig. 9C, *P < 0.05 and **P < 0.01 indicates that the correlation is significant or very significant, respectively.

have a potential immunity-promoting function.
Numerous studies have investigated the ability of various
polyphenol-rich functional foods to restore balanced immune function
in mice and rats with acute and chronic diseases (Fernando F. Anhê
et al., 2015). When colitis occurs, the T-helper (Th)1 and Th17 immune
responses might be restrained. Grape seed procyanidin reduce diarrhea
in piglets after weaning, which is associated with increased levels of
cytokines and antibodies in serum (Chen, Wang, Li, Huang, & Ma,
2018). On the other hand, after supplementation with a polyphenol-rich
diet, antibody secretion and the cellular immune response in the gut are
also enhanced (Denis et al., 2015). Therefore, functional foods with
more polyphenols might enhance host immune responses and regulate
the intestinal mucosal barrier.
However, the mechanism by which diets with more polyphenols
regulate intestinal mucosal barrier remains unclear. The mechanisms
that have been identified are described below. Firstly, some studies have
confirmed that procyanidin or other substances with probiotic-like
functions selectively promote the growth of certain beneficial bacteria,
which is consistent with the results of our study. Polyphenol-rich foods,
such as pomegranates, tea, and nuts, promote the growth of Bifido­
bacterium and lactic acid-producing bacteria (Tomas-Barberan & Espin,
2019). Polyphenol in blueberries (Jiao et al., 2019) alter the gut taxa
and the abundance of Proteobacteria, Deferribacteres, Actinobacteria,
Bifidobacterium, Adlercreutzia, Helicobacter, Flexispira and Prevotella. The
gut taxa detected during different phases were complex and varied. After
supplementation with OFH and OJ, the abundance of Lachnospiracea
was increased and correlated with propionic acid production. These
results were consistent with a previous study showing that Lachnospir­
acea was beneficial to the host and metabolized plant fibers into SCFAs.
Roseburia, Faecalibaculum, and particularly Akkermansia were
confirmed to be beneficial microbes in humans. Akkermansia belongs to
the phylum Verrucomicrobiaceae, which was enriched in OFH2 (Brahe
et al., 2015). Akkermansia is the most abundant mucus-soluble bacteria
in the healthy gut. Lower Akkermansia numbers in the intestines results
in a thinner mucous membrane layer, which may lead to a decrease in
the intestinal barrier function and allow toxins in the intestines to more

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Fig. 10. Effects of OF and OJ on intestinal mucosal barrier and host immunity in normal and CTX-treated immunosuppressed mice. Intestinal mucosal barrier is an
important component of host immunity and is divided into four parts. The biological barrier includes commensal bacteria, such as Roseburia, Faecalibaculum, and
Akkermansia. Chemical barriers include SCFAs, other microbial metabolites and sIgA. The mechanical barrier consists of epithelial cells, lymphocytes and receptors.
The immunological barrier includes different types of immune cells, cytokines and immunoglobulins. Roseburia, Faecalibaculum, and Akkermansia produce SCFAs.
SCFAs are important energy sources for intestinal cells that potentially modulate the activity of GPR41/GPR43 and FFAR2 to enhance mucosal barrier function or
directly affect peripheral immune cells. Intestinal epithelial cells produce sIgA, and its concentrations reflect the intestinal immune status. Peripheral immune cells,
such as Tregs and macrophages, secrete cytokines to balance host immunity.

easily invade the host. Patients with inflammatory bowel disease,
obesity and type II diabetes have lower levels of Akkermansia. Cran­
berries exert a probiotic-like effect on Akkermansia because procyanidin
promote the differentiation of goblet cells and mucus production to
provide a better environment for the growth of Akkermansia (Fernando
F. Anhê et al., 2015). Additionally, caffeic acid increases the abundance
of Akkermansia and promote its anti-inflammatory abilities (Zhang et al.,
2016). PP-rich grape, pomegranate, and rhubarb extracts, among others,
promote Akkermansia muciniphila, Faecalibacterium prausnitzii, or Rose­
buria spp. growth (Tomas-Barberan & Espin, 2019). Roseburia was only
detected in the OFH and OJ groups. According to a previous study,
supplementation with PP-rich whole grain increases the abundance of
Roseburia in human feces (Zhong, Nyman, & Fak, 2015). The results
from these studies are consistent with our study, providing supporting
evidence that the PP-rich OF regulates the abundance of gut taxa and
increases the levels of beneficial bacteria.
Secondly, diets with more polyphenols may increase the production
of SCFAs (Solano-Aguilar et al., 2018). SCFAs are important energy
sources for intestinal cells and might modulate the expression of GPR41/
GPR43 on intestinal epithelial cells to enhance mucosal barrier function
or directly affect immune cells (Smith et al., 2013). For example, acetic
acid promotes Treg cell differentiation by regulating the differentiation
of goblet cells, and acetic acid or butyric acid induce the production of
Th1/Th2 cells to alter cytokine levels in the host (Allaire et al., 2018).
Butyric acid regulates gene expression by inhibiting histone deacetylase,
which appears to be the mechanism by which the gut microbiota
regulates peripheral Treg cell differentiation. The manure of pigs fed a
grape pomace rich in polyphenols contains a higher concentration of
propionate. SCFAs affect host defenses by modulating the activity of
group 3 lymphoid cells through FFAR2 and macrophages via metabolic
reprogramming (Garrett, 2019). In our study, Muribaculaceae, Allopre­
votella, Parabacteroides, and Ruminococcus_2 were closely related to
butyric acid production, and the abundance of all of these genera was
increased in the OFH group. Parabacteroides was closely related to
butyric acid production during the MP, and its abundance was increased
during the CP. Recently, Parabacteroides was reported to exhibit a po­
tential immune regulating function in mice with DSS-induced ulcerative
colitis (Kverka et al., 2011). The abundance of Ruminococcus was
increased during the MP and CP and was related to butyric acid pro­
duction. Liang et al. (Liang et al., 2019) reported that Ruminococcus is
associated with increases in host malnutrition and promoted body
weight recovery.
Finally, SCFAs produced by intestinal flora may inhibit the produc­
tion of inflammatory factors, which instead promote the production of
sIgA (Seifert & Watzl, 2007). The administration of proanthocyanins
alone to mice promotes the production of sIgA. Intestinal epithelial cells
produce sIgA, and its levels reflect the intestinal immunity (Honda &
Littman, 2016). During the different phases, both OFH and OJ increased
the sIgA contents in the colon. Based on the histopathological changes in
the jejunum, lacteal structures in the cilium disappeared only in model
mice. The lactea is a lymph capillary in the intestine surrounded by some
lymphocytes and is related to the host immune response and absorption

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of macromolecules. In summary, supplementation with OF improves
mucosal barrier and indirectly affects host immunity.
The relationships among chemical compounds, immunity-related
and microbiota in this study were determined using a correlation anal­
ysis. After administering the OF to immunosuppressed mice, the levels of
the four effective substances were positively correlated with the sIgA
and butyric acid concentrations in the colon. Together with the results
described above, polyphenol-rich functional foods modulate the intes­
tinal immune response. Alloprevotella, unclassified_o_Bacteroidates, Alis­
tipes, Bacteroides, norank_f_Muribaculaceae, Parabacteroides and other
beneficial bacteria also significantly increase the sIgA and butyric acid
levels in the intestine. Procyanidin regulates the activity of dendritic
cells and Th cells in the intestine, thereby reducing the production of
IFN-γ, which is consistent with the results of our experiments. Based on
these results, OF regulates innate and acquired immunity and maintains
intestinal homeostasis (Fig. 10), which has the potential to activate the
immune system. The effects of OF on the colon and the mechanism by
which polyphenol reacted with microbes require further analyses of
their chemical compounds, which will be the next step in our long-term
study of the mechanism of regulating immunity.
5. Conclusions
Chinese dwarf cherry fruits exert better pharmacological effects after
fermentation by an Lactobacillus plantarum strain than OJ. OF improved
host immunity, but exerted more substantial effects on intestinal
mucosal barrier and the gut microbiome, which were significantly
different from mice treated with OJ. The regulation of the immune
response might be associated with host-intestinal-microbiota in­
teractions, mostly due to the alteration of the microbiota, production of
SCFAs, and production of sIgA in the colon, which eventually alter in­
testinal mucosal barrier to modulate the host immune response. After
supplementation with OF, more beneficial microbiota were produced
and the abundance of Roseburia, Faecalibaculum, and Akkermansia, and
butyric acid-producing microbes increased, which provides a valuable
reference for guiding the generalization and application of functional
foods with polyphenols.
6. Ethics statement
All animal experiments were carried out in accordance with the
National Institutes of Health guide for the care and use of Laboratory
animals (NIH Publications No. 8023, revised 1978) and approved by the
Ethics Committee of Experimental Animal Care at Beijing University of
Chinese Medicine.
CRediT authorship contribution statement
Chang-e Guo: Data curation, Formal analysis, Methodology. Qingyu
Cui: Project administration. Jinghe Cheng: Project administration.
Jiaji Chen: Project administration. Zihan Zhao: Project administration.
Ran Guo: Project administration. Xi Dai: Project administration. Zhi­
jiang Wei: Project administration. Weidong Li: Conceptualization,
Funding acquisition, Resources, Supervision, Writing - review & editing.
Acknowledgements
Sequencing service was provided by Majorbio Bio-Pharm Technol­
ogy Co. Ltd. (Shanghai, China). We also thank American Journal Experts
for providing the comprehensive language guidance.
Funding
This work was supported financially by Key Research and Develop­
ment Programs in the Ningxia Hui Autonomous Region, China (No.
2020BBF02027), Independent Research Project of Beijing University of
13
Journal of Functional Foods 80 (2021) 104424
Chinese Medicine (No. 2019-JYB-XS-090), Beijing Municipal Natural
Science Foundation (5212014).
Declaration of interests
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jff.2021.104424.
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