RESEARCH ARTICLE

Probio-X Relieves Symptoms of Hyperlipidemia by Regulating

Patients’ Gut Microbiome, Blood Lipid Metabolism, and Lifestyle
Habits
Huan Wang,a,b,c,d,e Cuicui Ma,a,b Yan Li,a,b Lei Zhang,c,d,e lima A,a,b Chengcong Yang,c,d,e Feiyan Zhao,c,d,e Haifeng Han,a,b
Dongyang Shang,a,b Fan Yang,a,b Yuying Zhang,a,b Heping Zhang,c,d,e Zhihong Sun,c,d,e Ruifang Guoa,b

a Department of Clinical Nutrition, Inner Mongolia People’s Hospital, Hohhot, Inner Mongolia, China
b Inner Mongolia Key Laboratory of Nutrition and Health, Inner Mongolia People’s Hospital, Hohhot, Inner Mongolia, China
c Inner Mongolia Key Laboratory of Dairy Biotechnology and Engineering, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China
d Key Laboratory of Dairy Products Processing, Ministry of Agriculture and Rural Affairs, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China
e Key Laboratory of Dairy Biotechnology and Engineering, Ministry of Education, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China

Huan Wang and Cuicui Ma contributed equally to this work. Author order was determined by type of their contributions.

ABSTRACT Hyperlipidemia is a key risk factor for cardiovascular disease, and it is asso-
ciated with lipid metabolic disorders and gut microbiota dysbiosis. Here, we aimed to
investigate the beneficial effects of 3-month intake of a mixed probiotic formulation in
hyperlipidemic patients (n = 27 and 29 in placebo and probiotic groups, respectively).
The blood lipid indexes, lipid metabolome, and fecal microbiome before and after the
intervention were monitored. Our results showed that probiotic intervention could sig-
nificantly decrease the serum levels of total cholesterol, triglyceride, and low-density
lipoprotein cholesterol (P , 0.05), while increasing the levels of high-density lipoprotein
cholesterol (P , 0.05) in patients with hyperlipidemia. Probiotic recipients showing
improved blood lipid profile also exhibited significant differences in their lifestyle habits

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after the 3-month intervention, with an increase in daily intake of vegetable and dairy
products, as well as weekly exercise time (P , 0.05). Moreover, two blood lipid metab-
olites (namely, acetyl-carnitine and free carnitine) significantly increased after probiotic
supplementation cholesterol (P , 0.05). In addition, probiotic-driven mitigation of hyperlip-
idemic symptoms were accompanied by increases in beneficial bacteria like Bifidobacterium
animalis subsp. lactis and Lactiplantibacillus plantarum in patients’ fecal microbiota. These
results supported that mixed probiotic application could regulate host gut microbiota
balance, lipid metabolism, and lifestyle habits, through which hyperlipidemic symptoms
could be alleviated. The findings of this study urge further research and development of
probiotics into nutraceuticals for managing hyperlipidemia.
IMPORTANCE The human gut microbiota have a potential effect on the lipid metab-
olism and are closely related to the disease hyperlipidemia. Our trial has demon-
strated that 3-month intake of a mixed probiotic formulation alleviates hyperlipid-
emic symptoms, possibly by modulation of gut microbes and host lipid metabolism. Editor Yuan Pin Hung, Tainan Hospital,
The findings of the present study provide new insights into the treatment of hyper- Ministry of Health and Welfare

lipidemia, mechanisms of novel therapeutic strategies, and application of probiotics- Copyright © 2023 Wang et al. This is an open-
access article distributed under the terms of
based therapy. the Creative Commons Attribution 4.0
International license.
KEYWORDS hyperlipidemia, probiotics, gut microbiota, carnitine, lipid metabolism
Address correspondence to Ruifang Guo,
[email protected].
The authors declare no conflict of interest.

T he rapid development of the world economy has dramatically changed people's living
standards and diet structure, and the fast-food diet structure has also led to an obvious
increase in the proportion of high-fat foods in the daily diet (1). A long-term high-fat diet will
Received 2 November 2022
Accepted 20 March 2023
Published 6 April 2023
not only disrupt the balance of lipid metabolism in the body and cause metabolic disorders

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(2), but also lead to chronic diseases, such as hyperlipidemia (3), type 2 diabetes (4), hyper-
tension (5), and obesity (6). Apart from being one of the most common metabolic disorders
caused by a high-calorie diet, hyperlipidemia is also an important contributing factor in car-
diovascular disease (7). The main therapeutic drugs for hyperlipidemia are statins, such as
fluvastatin, atorvastatin, and rosuvastatin (8). However, it is worth noting that long-term use
of statins can cause certain side effects to the body, and the drugs are relatively costly.
Therefore, it is of interest to find alternative strategies for hyperlipidemia.
Several studies have found a link between hyperlipidemia and human gut microbiota,
for example, significant differences in the gut microbiota profile between hyperlipidemic
and healthy individuals (including animals) (9). It is also thought that alterations in the gut
microbiome are an important cause promoting the onset and progression of hyperlipid-
emia (10). Long-term intake of a high-fat diet not only disrupts gut homeostasis, leading
to gut microbial dysbiosis (11), but potentially damages the gastrointestinal barrier and
permeability (12). Moreover, a long-term high-fat diet may lead to the reduction in both
the ratio of gut Firmicutes to Bacteroidetes and the abundance of fatty acid-producing
bacteria, such as Bacteroides, Lactiplantibacillus, and Blautia (13). In fact, the host gut micro-
biota are closely associated with some liver diseases, implicating the existence of an enter-
ohepatic axis, which influences hepatic (fat) metabolism (14, 15). The liver is an important
site for fat synthesis; however, liver cells can synthesize but not store the fat. Therefore, the
synthesized fat is transported into the bloodstream after combining with apolipoproteins
and cholesterols in the form of very low-density lipoproteins (16). The small intestine is
another important site of lipid synthesis, and it is anticipated that the resident microbiota
could play a critical role in the synthesis and transport of body fat.
Wang et al. found that feeding animals with high-fat diets disrupted both the host’s
antioxidant capacity and gut microbiota balance, ultimately leading to the exacerbation of
hyperlipidemic symptoms (17). Sun et al. found that a high-sucrose diet drastically shifted
the gut microbiota in rats, characterized by an increase in the ratio of Bacteroidetes/
Firmicutes, decrease in alpha diversity, and changes in diurnal oscillations, suggesting that
a high-sucrose diet could reduce the level of blood fat via modulating the gut microbiota
community (18). Ke et al. discovered that symbiotic intervention can reverse high fat diet
(HFD)-induced changes in body weight gain, cholesterol, triglycerides, and microbial pop-

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ulations in mice through metabolic pathways involved in carbohydrate, amino acid, and
energy metabolism (19). Yan et al. found that probiotics reversed the intestinal imbalance
caused by hyperlipidemia by increasing the abundance of short-chain fatty acids-produc-
ing bacteria and thus the total amount of colonic short-chain fatty acids (13). In addition,
supplementing probiotics-based products significantly reduced the weight gain and
visceral obesity in hyperlipidemic mice and attenuated dyslipidemia via improving oxi-
dative stress and chronic inflammation (20). Some Lactiplantibacillus spp. can regulate
bile acid metabolism by downregulating farnesoid X receptor (FXR) genes and reduce
exogenous cholesterol absorption by regulating Niemann-Pick C1-Like 1 (NPC1L1) (21).
Provided the close association between the gut microbiota and hyperlipidemia, they can
be regarded as a potential target for hyperlipidemia treatment. A growing body of litera-
ture shows that probiotics can modulate and even restore a healthier gut microbiota from
disease-associated microbial dysbiosis states; thus, it is worth further investigating the clinical
effects of probiotic application in managing hyperlipidemia.
This study hypothesized that probiotics could regulate the gut microbiota balance
in hyperlipidemic patients and improve hyperlipidemic symptoms via modulating the
host lipid metabolism. In this 3-month intervention trial, a total of 56 hyperlipidemic
patients were recruited and randomized into the placebo and probiotic (receiving a
mixed probiotic formulation) groups. The fecal microbiome and serum lipids of these
patients were analyzed using metagenomics and liquid chromatography–mass spec-
trometry (LC-MS), respectively. Meanwhile, changes in patients’ physiological and bio-
chemical parameters were recorded. This study confirmed the beneficial effect of Probio-X
in managing hyperlipidemia.

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TABLE 1 Information of patients

Groups Chi-square test

Patients’ information Placebo (n = 27) Probiotic (n = 29) X2/t P

Gender (male/female) 14/13 12/17 0.617 0.432
Ethnicity (Han/other) 20/7 21/8 0.020 0.889
Age (mean 6 SD, yrs) 48.67 6 11.79 51.17 6 10.14 0.855 0.397
Ht (mean 6 SD, m) 1.68 6 0.85 1.63 6 0.65 1.137 0.261
Wt (mean 6 SD, kg) 76.30 6 18.02 70.91 6 13.76 0.917 0.363
BMI (mean 6 SD, kg/m2) 27.09 6 5.79 26.44 6 4.16 0.507 0.614
Working status (full-time employment/other) 19/8 20/9 0.013 0.909
Marital status (married/other) 25/2 27/2 0.006 0.941
Education level (bachelor's degree or above/other) 20/7 20/9 0.179 0.672

RESULTS
Probio-X improved blood lipid indexes in a hyperlipidemia population. Body
weight, height, and work employment were collected and analyzed (Table 1). No significant
difference was observed in the gender, ethnicity, age, height, weight, BMI, work status, mari-
tal status, and education level between probiotic and placebo groups (P . 0.05 in all cases).
There were no significant differences in all blood biochemistry between the placebo and
probiotic groups at baseline. The blood analysis of hyperlipidemic patients showed that the
total cholesterol (TC) and low-density lipoprotein (LDL-C) levels decreased significantly after
probiotic consumption (paired t test, P , 0.05; Fig. 1A), while the high-density lipoprotein
(HDL-C) levels increased significantly (paired t test, P , 0.05). In contrast, in the placebo
group, only blood HDL-C levels (paired t test, P . 0.05), but not TC, triglyceride (TG), and
LDL-C levels (paired t test, P , 0.05), significantly decreased after the 3-month intervention
period (P , 0.05). However, both groups showed a significant decrease in fasting glucose
(paired t test, P , 0.05). The consumption of Probio-X not only significantly reduced the
body weight, visceral fat, and blood fat of patients, but also effectively prevented loss in their
bone mineral density (paired t test, P . 0.05; Fig. 1B). The analysis of gut mucosa indicators

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FIG 1 Probio-X improved blood lipid indexes in hyperlipidemia patients. (A) Comparative analysis of (A) blood indicators; (B) body weight and composition; (C) gut
mucosa indicators. (D) Number of patients that were responsive toward probiotic treatment, evaluated by changes in four key blood lipid indicators. Chi-square test
was performed to evaluate the overall difference between two groups. LDL-C, low-density lipoprotein; TC, total cholesterol; HDL-C, high-density lipoprotein;
TG, triglyceride; *, P , 0.05; **, P , 0.01; ***, P , 0.001 (t test). Pla_0M and Pla_3M indicate before and 3 months after the placebo intervention, respectively,
and Pro_0M and Pro_3M before and 3 months after the probiotic intervention, respectively.

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(Fig. 1C) revealed no significant change in diamine oxidase, D-lactate, and endotoxin
in both groups of patients (paired t test, P . 0.05), though it is worth noting that the pro-
biotic group had significantly lower values in the aforementioned indicators (unpaired t test,
P , 0.05).
We further analyzed changes in the four main blood lipid indicators in the placebo
and probiotic groups (Fig. 1D), and our results revealed that significantly more patients
in the probiotic group showed improvements in TC, LDL-C, and HDL-C than the placebo
group (chi-square test, P , 0.05), while no significant difference was found in the number
of patients showing improvement in TG (chi-square test, P . 0.05). These results demon-
strated that the consumption of Probio-X could significantly improve some major clinical
indicators related to hyperlipidemia.
Probio-X improved dietary and exercise habits in hyperlipidemic patients. A
self-administered questionnaire was applied to record the dietary and exercise habits
of the hyperlipidemic patients during the trial intervention. We thus analyzed changes
in dietary and exercise habits in patients showing improvements in TC, LDL-C, and HDL-C.
Interestingly, patients in the probiotic group that showed a significant decrease in TC
(Fig. 2A) also increased their daily vegetable and dairy product consumption, as well as
time of weekly exercise, compared to the preintervention period (chi-square test, P , 0.05),
while those in the placebo group did not show any significant changes in these aspects
after the intervention (chi-square test, P . 0.05). Similarly, patients in the probiotic group
that had lowered LDL-C levels (Fig. 2B) increased daily vegetable and dairy product con-
sumption (chi-square test, P , 0.05), though no significant change was observed in their
weekly exercise time compared to preintervention (chi-square test, P . 0.05). Such changes
were not observed in the placebo group (chi-square test, P . 0.05). In contrast, patients
from both the probiotic and placebo groups showing improvement in HDL-C (Fig. 2C)
did not exhibit obvious changes in their dietary and exercise habits (chi-square test, P . 0.05).
Meanwhile, the patients’ feeling of gastrointestinal wellness was analyzed (Fig. 2D).
Significantly less diarrhea frequency was observed in subjects given Probio-X compared
with those receiving placebo material (chi-square test, P , 0.05), while there was no
significant difference in the occurrences of abdominal pain, bloating, and constipation
between the two groups (chi-square test, P . 0.05). These results together showed that
the improvement in major blood lipid indexes was accompanied by healthier lifestyle

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changes.
Changes in patients’ fecal microbiota after Probio-X intervention. Although numer-
ous studies in recent years have observed a strong association between the gut microbiota
and onset/progression of hyperlipidemia, our microbial alpha diversity analysis (Shannon
and J indexes) of subjects’ fecal microbiota revealed no significant intra-/intergroup differen-
ces (paired Wilcoxon test, P . 0.05; unpaired Wilcoxon test, P . 0.05, respectively; Fig. 3A
and B), suggesting that probiotic application did not significantly change the alpha diversity
of subjects. Then, a principal coordinate analysis (Bray-Curtis distance, Fig. 3C) was performed
to evaluate differences between groups/subgroups. On the principal coordinate score plot,
symbols of different groups/subgroups showed a high degree of overlap, indicating no signif-
icant differences in the overall intra-/intergroup microbiota structure. The fecal microbiota
composition was similar to those reported in previous studies (22), and the major species
were mostly common microbes residing in the human intestine (Fig. 3D). Differential
microbes between probiotic and placebo groups after the 3-month intervention were
detected using linear discriminant analysis effect size (LEfSe), and a total of 22 differential
species were found (Fig. 3E). Notably, both Bifidobacterium animalis and Lactiplantibacillus
plantarum were significantly enriched after probiotic intervention.
This study also analyzed the profile of metagenome-assembly genomes (MAGs) in
the fecal microbiota (Fig. 3F). Some differential MAGs were detected, including B14_32
(corresponding to Bifidobacterium animalis subsp. lactis), that were significantly more
enriched in the probiotic group than the placebo group after the 3-month intervention
(Fig. 3G). Finally, by mapping the reads to the genomes of probiotics, the relative abundance
of all five applied strains of probiotics could be identified from patients’ gut microbiome.
For the placebo group, the relative abundances of all five strains did not show obvious

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FIG 2 Probio-X improved dietary and exercise habits and gastrointestinal wellness in hyperlipidemia patients. Comparative analysis of habits
of vegetables/dairy product consumption and weekly exercise in (A) patients with normal total cholesterol (TC); (B) low-density lipoprotein (LDL-C)
populations; and (C) high-density lipoprotein (HDL-C) remission populations. (D) Comparative analysis of patients’ feelings of gastrointestinal wellness.
Pla_0M and Pla_3M indicate before and 3 months after the placebo intervention, respectively, and Pro_0M and Pro_3M before and 3 months after
the probiotic intervention, respectively.

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FIG 3 Changes in fecal microbiota after Probio-X intervention. (A) Shannon index. (B) J index. (C) Relative abundance of dominant species. (D) Principal coordinate
analysis (Bray-Curtis distance). (E) Linear discriminant analysis effect size (LEfSe) analysis. Distribution of (F) dominant metagenome-assembled genomes (MAGs) in
each subject; (G and H) the MAG, B14_32, and the applied probiotic strains (Probio-M8, Bifidobacterium lactis Probio-M8; V9, Bifidobacterium lactis V9; Probio-M9,
Lactiplantibacillus rhamnosus Probio-M9; Zhang, Lactiplantibacillus casei Zhang; P-8, Lactiplantibacillus plantarum P-8) in different sample groups. Pla_0M and Pla_3M
indicate before and 3 months after the placebo intervention, respectively, and Pro_0M and Pro_3M before and 3 months after the probiotic intervention, respectively.
A1 represents Pla_0M, A2 represents Pla_3M, B1 represents Pro_0M, and B2 represents Pro_3M.

changes after the 3-month treatment (paired Wilcoxon test, P . 0.05; Fig. 3H), while significant
increases were observed in three of the strains (Bifidobacterium animalis Probio-M8,
Bifidobacterium animalis V9, and Lactiplantibacillus plantarum P-8) after 3 months of pro-
biotic mix intervention (paired Wilcoxon test, P , 0.05; Fig. 3H).
Changes in patients’ serum lipid profile after Probio-X intervention. It is known
that gut microbiota interacts with the host metabolism and influences the blood metabo-
lome. Therefore, patients’ lipid blood metabolic profile was analyzed by principal-compo-
nent analysis (Fig. 4). However, symbols representing the four subgroups (placebo and
probiotic groups at baseline and after 3-month intervention) largely overlapped on the
principal-component analysis score plot, indicating that the probiotic treatment did not
cause significant changes in the overall blood lipid metabolome structure (Fig. 4A and B).
Then, volcano plot analysis was performed to look for differential metabolites between
placebo and probiotics groups after the intervention, which identified only two significantly
increased differential metabolites, acetyl-carnitine and free carnitine after probiotic inter-
vention (fold change [FC] . 2, P , 0.05; Fig. 4C and D). There was, however, a general
decrease in other differential metabolites identified after postprobiotic intervention (identi-
fied only based on paired Wilcoxon test, P , 0.05; Fig. 4E). A finer analysis of the blood lipid

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FIG 4 Changes in serum lipid profile after Probio-X intervention. (A and B) Principal-component analysis of serum lipid metabolomes of subjects. Each dot
represents the serum lipid metabolome of each subject. (C) Volcano map showing detected serum lipid metabolites. Non-SIG represented metabolites
without significant increase after probiotic intervention, UP represented metabolites with significant increase after probiotic intervention. Significantly
increased metabolites are shown in red. (D) Relative abundance heatmap of differential metabolites. The color scale represents the relative abundance of
metabolites. (E) Box plots showing the normalized amounts of acetyl-carnitine and free carnitine after probiotic intervention. (F) Violin plots showing
differences in various lipid metabolites after probiotic intervention. G, monoglyceride; CAR, acyl carnitine; CE, cholesterol lipids; FFA, free fatty acids; LPI,
lysophosphatidylinositol; PI, phosphatidylinositol; PS, phosphatidyl serine. Pla_0M and Pla_3M indicate before and 3 months after the placebo intervention,
respectively, and Pro_0M and Pro_3M before and 3 months after the probiotic intervention, respectively.

metabolites based on class grouping (into eight categories: MG, monoglyceride; CAR, acyl
carnitine; CE, cholesterol lipids; FFA, free fatty acids; LPI, lysophosphatidylinositol; PI,
phosphatidylinositol; PS, phosphatidyl serine, TG, triglyceride; Fig. 4F) revealed no signifi-
cant difference in all metabolite categories between probiotic and placebo groups after
the intervention (unpaired Wilcoxon test, P . 0.05).
Changes in predicted gut metabolites after Probio-X intervention. The probiotic
treatment did not seem to drastically modulated patients’ fecal microbiota structure and
composition; thus, we further investigated the mechanisms of regulation of hyperlipidemia
by analyzing probiotics-driven changes in predicted gut metabolites from the MAGs. A total
of 41 gut metabolites were predicted across 337 MAGs (selection criteria: integrity greater
than 80, contamination less than 10, and relative abundance greater than 1%; data not
shown). The gut metabolite, MGB043, was common to the majority of MAGs, while MGB034,
MGB055, MGB056, and MF0059 were predicted to be present exclusively in only one MAG.

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FIG 5 Predicted gut metabolites. (A and B) Relative abundance of predicted gut metabolites. (C) Violin
plots showing differential gut metabolites. Correlation heatmap of (D) metagenome-assembly genome
(MAGs) and gut metabolites; (E) MAGs and blood indicators. Red indicates positive correlation, blue indicates
negative correlation, and the color intensity is the strength of correlation. *, P , 0.05; **, P , 0.01;
***, P , 0.001.

A total of 20 gut metabolites were predicted from the MGB database, including the major
metabolites MGB011 and MGB043 (Fig. 5A), while a total of 21 gut metabolites were pre-
dicted from the MF database (MF0103 and MF0067 as the major metabolites; Fig. 5B).
Forty-one significantly differential metabolites between the probiotic and placebo groups
were detected after the intervention (Fig. 5C); nine were significantly enriched (e.g., cortisol
degradation, glycolysis, and pentose phosphate pathway; unpaired Wilcoxon test, P , 0.05
in each case) or deprived (e.g., tryptophan synthesis, quinolinic acid degradation, and galac-
turonate degradation I et al.; unpaired Wilcoxon test, P , 0.05 in each case) after the probi-
otic treatment.

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Correlation analysis revealed significant positive correlations between three predicted

differential gut metabolites (cortisol degradation, glycolysis, and pentose phosphate
pathway) and almost all differential MAGs (Spearman's test, P , 0.05; Fig. 5D), while
other differential gut metabolites showed significantly negative correlations with almost
all differential MAGs (Spearman 's test, P , 0.05; Fig. 5D), except Paraprevotella clara, which
showed an opposite trend. Moreover, acetyl-carnitine and free carnitine correlated signifi-
cantly and positively with two MAGs, Bifidobacterium animalis and Blautia obeum (Spearman's
test, P , 0.05; Fig. 5E); HDL-C was significantly and negatively correlated with Paraprevotella
clara, but was positively correlated with Bifidobacterium animalis, Veillonella parvula, and
Erysipelatoclostridium ramosum (Spearman's test, P , 0.05 in all cases; Fig. 5E). Serum uric
acid showed a significant positive correlation with Paraprevotella clara, but negatively corre-
lated with Bifidobacterium animalis (Spearman's test, P , 0.05; Fig. 5E).

DISCUSSION
Gut microbiota are associated with the host metabolism and may influence the onset
and development of hyperlipidemia (23). Probiotics have been shown to lower blood TC,
LDL-C, and TG, alleviating metabolic syndrome in hyperlipidemic rats (21). This study
analyzed the effects of a mixed probiotic formulation on hyperlipidemia, with focus on
changes in patients’ gut microbiota and their metabolic potential.
Our data supported that the intake of the probiotic mix could effectively reduce the
serum levels of TC and LDL-C, while increasing serum HDL-C levels, in patients with hyperlipid-
emia. Such results supported that probiotic application could regulate hosts’ lipid metabolism.
There are different subtypes of dyslipidemia (e.g., high cholesterol and/or high triglycerides),
which would require different treatment approaches. For example, Yan et al. found that
oryzanol could reduce the TC of serum in rats (24), while Liu et al. found that Tartary
buckwheat protein could significantly reduce LDL-C levels and increase HDL-C levels in
the sera of hyperlipidemic rats (25). Although these functional products could reduce
some of the blood lipid indices in hyperlipidemic rats, only the Probio-X applied to this
could effectively modulate all three important blood lipid indexes, TC, LDL-C, and HDL-C,
in hyperlipidemic subjects, suggesting that probiotics are potential functional products
for alleviating hyperlipidemia.

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The results of the self-administered questionnaire revealed interesting and desirable life-
style habit changes (such as increased daily vegetable consumption, regular dairy consump-
tion, and increased weekly exercise time) in hyperlipidemic patients that showed significant
improvement in blood lipid indexes (TC, LDL-C, and HDL-C) after probiotic consumption.
Indeed, there was a strong association between the desirable changes in patients’ lifestyle
habits and lowering of these indexes. Wastyk et al. believe that diet and gut microbiota
are interacting bidirectionally. In other words, diet regulates the gut microbiota, and vice
versa (26). A previous study also demonstrated an influential role of intake of vegetables/
dairy products and exercise in regulating serum levels of TC, LDL-C, and HDL-C (27). Moreover,
apart from lower hyperlipidemia, the results of this study also found that taking Probio-X
could significantly alleviate diarrhea. Similarly, McFarland et al. confirmed the effectiveness
of probiotics in treating diarrhea (28). The meta-analysis of Lu et al. observed a significantly
reduced total diarrhea rate in cancer patients when probiotics were used in conjunction
with the conventional treatment (29). Numerous studies consistently showed that probiotic
intake could alleviate diarrhea, which was likely achieved by regulating the host's gut micro-
bial balance (30). Thus, the Probio-X applied in this study could alleviate hyperlipidemia and
diarrhea.
Although a large body of study has supported that the gut microbiota serve as an
important mediator of lipid metabolism, our study did not observe drastic probiotic-driven
changes in the fecal microbiota structure and composition, which could be related to the
high complexity of the human gut microbiota. Similar observation was made by Liu et al.,
where the authors investigated effects of probiotics on the gut microbiome of asthmatic
patients (31). Differential species were only identified between probiotic and placebo
groups at a fine taxonomic level. For example, we noted significant increases in the species

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Bifidobacterium animalis subsp. lactis and Lactiplantibacillus plantarum postprobiotic inter-

vention. The increases in some of the applied strains in the mixed probiotic preparation
(including Probio-M8, V9, and P-8) in the fecal microbiome of the probiotic group were
confirmed by MAG analysis. A previous investigation showed that intestinal Bifidobacterium
animalis subsp. lactis could improve hyperlipidemia by regulating bile acid metabolism
through downregulating FXR genes while reducing exogenous cholesterol absorption
through modulating the NPC1L1 gene (21). Chen found that Lactiplantibacillus planta-
rum could reduce serum levels of TG, TC, and LDL-C, which in turn inhibited excessive
accumulation of liver fat (32). Our results supported that the Probio-X-driven hyperlip-
idemic symptom improvement was accompanied by modulating a specific portion of
the intestinal microbiota.
The blood lipid metabolome of subjects did not change significantly regardless of
probiotic intervention, but further analysis of lipid metabolites identified two differen-
tial metabolites, i.e., acetyl-carnitine and free carnitine, that were significantly increased
after the mixed probiotic intervention. The human body, as an extremely complex microeco-
system, has a strong capacity to maintain organismal stability by self-regulation, which may
be one of the reasons for the nonsignificant change in the overall blood metabolome and
low number of differential metabolites. However, it is worth noting that acetyl-carnitine
plays an integral role in lipid metabolism by participating in the b -oxidation of long-chain
fatty acids (33). Seccombe demonstrated that acetyl-carnitine could alleviate hepatic steato-
sis caused by a high-fat diet and reduce plasma levels of TC and TG (34). Additionally, free
carnitine is beneficial in lowering blood lipids (35).
Our further analysis found that Bifidobacterium animalis subsp. lactis correlated positively
with acetyl-carnitine and free carnitine, as well as the predicted gut metabolic pathway,
representing g -aminobutyric acid (GABA) synthesis. Since acetyl-carnitine and free carnitine
are known to have high affinity with certain GABA uptake sites (36, 37), we speculate that
Bifidobacterium animalis subsp. lactis may promote the transport of acetyl-carnitine and
free carnitine through GABA synthesis, then elevate their serum levels and thus improve
the blood lipid profile. In conclusion, our data support that administering Probio-X could
improve hyperlipidemia by modulating gut microbes and their metabolites.
Conclusions. We performed a 3-month intervention trial and confirmed the beneficial
effects of application of a probiotic mix in alleviating hyperlipidemia, at least in part, by regu-

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lating the blood lipid indexes (lowering serum TC, LDL-C, and HDL-C), lipid metabolism, and
gut microbiota. Although insignificant changes were observed in the lipid metabolome
and gut microbiota structure, some interesting fecal bacteria (e.g., Bifidobacterium animalis
and Lactiplantibacillus plantarum) and blood metabolites (e.g., acetyl-carnitine and free car-
nitine) increased significantly after Probio-X intervention, suggesting the probiotic-driven
treatment improvement of hyperlipidemic effects could be through modulation of these
gut microbes and host lipid metabolism. Collectively, our study serves as an additional study
supporting that probiotic administration is a promising approach in managing hyperlipid-
emia and improving public health.

MATERIALS AND METHODS

Probiotic administration. Probio-X (3  1010 CFU/g) contains equal proportions of Lactiplantibacillus
casei Zhang (L. casei Zhang), Bifidobacterium lactis V9 (B. lactis V9), Bifidobacterium lactis Probio-M8 (B. lactis
Probio-M8), Lactiplantibacillus rhamnosus Probio-M9 (L. rhamnosus Probio-M9), and Lactiplantibacillus planta-
rum P-8 (L. plantarum P-8). Lactiplantibacillus casei Zhang was isolated from a sour horse milk product in Inner
Mongolia; Bifidobacterium lactis V9 was isolated from the feces of healthy children; Bifidobacterium lactis
Probio-M8 and Lactiplantibacillus rhamnosus Probio-M9 were isolated from breast milk samples of healthy
mothers; and Lactiplantibacillus plantarum P-8 was isolated from a traditionally fermented dairy product in
Inner Mongolia. L. casei Zhang, B. lactis V9, B. lactis Probio-M8, L. rhamnosus Probio-M9, and L. plantarum P-
8 showed good tolerance to conditions simulating the gastrointestinal tract (such as in environments con-
taining gastric acid, intestinal fluid, and bile) and abilities to improve the host gut microbiota structure and
health.
Trial design and subject recruitment. This study was a 3-month randomized controlled double-
blind trial. A total of 112 patients with hyperlipidemia were recruited. All patients were recruited by the Inner
Mongolia People's Hospital and Inner Mongolia Agricultural University. The inclusion criteria were as follows: (i)
male or female aged between 18 and 65 with diagnostic criteria for hyperlipidemia; and (ii) willingness to com-
mit throughout the trial. In contrast, the exclusion criteria were as follows: (i) a history of major diseases, such

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Probiotic Effects in Hyperlipidemia Microbiology Spectrum

as cancer, kidney disease, gastrointestinal diseases, mental illness, or type I diabetes; (ii) takin antibiotics, pro-
biotics, prebiotics, postbiotics, and immunosuppressive agents within 1 month before the intervention or
during the intervention; (iii) irregular eating habits during the intervention and follow-up period; and (iv) fail-
ure to collect stool and blood samples twice. Forty-three subjects were excluded after the first round of
screening; sixty-nine patients were randomly assigned to probiotic (n = 35) and placebo (n = 34) groups.
After being informed of the specific experimental guidelines and details, three patients withdrew from the
trial; seven patients discontinued intervention for their consumption of antibiotics or yogurt; and three
patients lacked stool samples. Thus, 29 and 27 subjects subsequently remained in the probiotic and placebo
groups. Both participants and researchers were unaware of the group allocation throughout the trial. The
two groups of patients received a daily dose of two grams of probiotic or placebo preparation, respectively.
The placebo preparation comprised maltodextrin, which was identical to the probiotic preparation in pack-
aging and in sensory aspects (e.g., taste and smell). The probiotic preparation contained the placebo material
(excipient) plus probiotics (a total of 3  1010 CFU/g). No patients took any probiotics, antibiotics, or yogurt
during the trial. Patients’ diet and lifestyle information were collected by a self-administered questionnaire
for subsequent correlation analyses. Written, informed consent from legal guardians was obtained from
each participant.
Collection and processing of stool and blood samples. The study was conducted for 3 months,
and feces and blood were collected from all participants at days 0 and 90 after the start of the study.
Stool samples were self-collected by each subject at the specified time point at home using a uniformly
provided sterile stool sampler with DNA protection solution (Guangdong Longhai Biomedical Co., Ltd.,
Guangzhou, China). All patients were given prior training in the fecal collection procedure to prevent
fecal contamination during the collection process. Collected samples were transported to the hospital at low
temperature and were stored in a 280°C freezer before the next steps of sample processing for deep metage-
nomic sequencing.
All patients were asked to visit the hospital after stool sample collection, when blood samples (after a
12-h fast) were taken. All collected blood samples were stored in sterile centrifuge tubes, before centrifug-
ing at 4°C for 15 min at 3,000 g for serum collection. Collected serum samples were stored at 280°C pending
further analysis.
The study was approved by the Ethics Committee of the Inner Mongolia People's Hospital (no. 201811002)
and was registered on the Chinese Clinical Trial Registry (http://www.chictr.org.cn/; registration number
ChiCTR1900026469). Informed consent was obtained from all recruited subjects prior to the study.
Analysis of biochemical indicators. Blood samples were used for monitoring physiological and bio-
chemical changes in the subjects. The analyzed physiological/biochemical parameters mainly focused
on lipid metabolism, namely, LDL-C, TC, HDL-C, high-sensitivity C-reactive protein [hsCRP], fasting serum
glucose, TG, serum uric acid, diamine oxidase, D-lactate, and endotoxin. All biochemical analysis was performed
by the Inner Mongolia People's Hospital.
Fecal metagenomics analysis. (i) DNA extraction. After thawing the fecal samples, two grams of
each fecal sample was weighed and used for metagenomic DNA extraction. The metagenomic DNA was
extracted with the QIAamp DNA Stool minikit (Qiagen, Germany) according to the manufacturer's
instructions. The purity and quality of the extracted DNA were ensured by a NanoDrop spectrophotome-

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ter (Thermo Scientific, USA) and agarose gel electrophoresis. All samples were stored in a 220°C freezer
prior to further processing (38).
(ii) Shotgun metagenomic sequencing. One microgram DNA per sample was used for sequencing
library preparation. Sequencing libraries were generated using NEBNext Ultra DNA Library Prep kit for Illumina
(NEB, USA) following the manufacturer’s recommendations. Index codes were applied to attribute sequences
of each sample. Shotgun sequencing was performed on the HiSeq 2500 platform (Illumina, CA, USA). Paired-
end reads (151 bp in length) were generated.
(iii) Bioinformatic analysis. The reads were trimmed using FastQC (v 0.11.9) (https://github.com/s
-andrews/FastQC) and were subsequently aligned to the human genome to remove host DNA sequences.
The remaining high-quality reads were further analyzed. After removing the human and low-quality sequences,
a total of 1258.36 Gb of high-quality sequence data was generated from all samples (average of 11.24 Gb per
sample). J-Index and Shannon index were used to characterize the abundance of community microorganisms of
subjects’ fecal microbiota (14).
The fecal microbiota composition was obtained by comparing high-quality reads with standard databases
using Metaphlan 2, ver. 2.0 (39). High-quality reads were assembled into contigs using MegaHit, ver. 1.0 (40),
and the obtained contigs were binned into high-quality scaffolds by MetaBAT2, ver. 2.12.1 (41), VAMB, ver. 1.0
(42), and Maxbin2, ver. 2.0 (43). Meanwhile, the obtained high-quality scaffolds were screened for metage-
nome-assembly genomes (MAGs) using the Das tool, v 1.1.2 (44). The quality of the obtained MAGs was
assessed by CheckM, ver. 1.0.18 (45) to screen for high-quality MAGs and gene set construction
(completeness . 80% and contamination , 5%). High-quality MAGs were clustered by dRep, ver.
2.2.4 (46) to define species-level genome bins (SGBs). The taxonomy of each MAG was annotated against
the National Center for Biotechnology Information (NCBI) nonredundant Nucleotide Sequence Database
(NT) using BLASTn. The Kyoto Encyclopedia of Genes and Genomes (KEGG) was annotated using eggnog-
mapper. Metabolic profiles of the fecal microbes were predicted using MelonnPan (https://huttenhower
.sph.harvard.edu/melonnpan/).
(iv) Nucleotide sequence accession numbers. The sequence dataset generate in this study has
been deposited in the NCBI Sequence Read Archive (SRA) database (PRJNA946655).
Serum metabolomics analysis. (i) Sample preparation and extraction. Serum samples were thawed,
vortexed for 10 s, and centrifuged for 5 min (4°C and 3,000 rpm). Then, 50 m L of each sample was accurately
pipetted into a prelabeled centrifuge tube containing 1 mL of lipid extract (including the standard mixture).

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Probiotic Effects in Hyperlipidemia Microbiology Spectrum

The mixture was vortexed for 2 min, sonicated for 5 min, before adding 500 m L of distilled water, vortexed for
1 min, and centrifuged for 10 min (4°C and 1,2000 rpm). Then, 500 m L of the supernatant was accurately pipet-
ted into a new prelabeled centrifuge tube, which was then concentrated and reconstituted into a final volume
of 100 m L with acetonitrile containing 0.04% acetic acid (i.e., the mobile phase B for LC- tandem mass spec-
trometry, MS/MS, analysis).
(ii) Ultrahigh performance liquid chromatography-quadrupole time-of-flight mass spectrome-
try (UPLC-Q-TOF MS). Serum lipid profile was mainly performed by UPLC Shim-pack UFLC SHIMADZU
CBM30A, SHIMADZU, Kyot liquid phase were: the column (Water ACQUITY UPLC HSS T3 C18 1.8 m m,
2.1 mm*100mm); mobile phases, ultrapure water (0.04% acetic acid) for phase A and acetonitrile (0.04%
acetic acid) for phase B; flow rate of 0.4 mL/min; column temperature of 40°C; injection volume of 5 m L.
The MS conditions were as follows: electrospray ionization (ESI) temperature of 550°C; MS voltages of
5,500 V (positive) and 24,500 V (negative); ion source gas I of 55 lb/in2, gas II of 60 lb/in2, curtain gas of
25 lb/in2; and collision-activated dissociation parameters set to high (47). Qualitative analysis was per-
formed using a self-built metware database, MWDB, identifying metabolites based on retention time,
ion pair information, and secondary spectrum data of the detected substances.
Statistical analysis and data visualization. All statistical analyses were performed using the R soft-
ware (v 4.0.3), and data were expressed as mean 6 SD. Wilcoxon test and t test were used to evaluate differen-
ces in blood indicators, fecal microbiome, and lipid metabolites between groups/subgroups. The chi-square
test was used to calculate the difference between counts. The R package vegan was used for alpha and beta
diversity analysis. The R package Psych was used for Spearman’s correlation analysis. Principal coordinate analy-
sis (Bray-Curtis distance) and principal-component analysis were performed with R. LEfSe analysis was per-
formed with R. Data were visualized with R software.

ACKNOWLEDGMENTS
This research was supported by the Inner Mongolia Science and Technology Major
Projects (2021ZD0014), the National Natural Science Foundation of China (31720103911),
the Health Science and Technology Planning Project of Inner Mongolia (202202026), and
the earmarked fund for CARS36.
We declare no conflicts of interest.

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