Effects of grape pomace and seed polyphenol extracts on the recovery of gut microbiota after antibiotic treatment in high‐fat diet‐fed mice

Abstract The widespread use of antibiotics all over the world increases the risk of many metabolic diseases by altering the gut microbiota. Grape by‐products are of particular interest in the prevention of metabolic diseases, while only minimum amounts of these wastes are up‐graded or recycled at present. The study investigated the effect of grape pomace (GPE) and seed (GSE) polyphenol extracts on the recovery of gut microbiota after antibiotic cocktail treatment in high‐fat diet‐fed (HFD) mice. C57BL/6J mice were fed HFD together with antibiotic treatment by drinking water for 3 weeks. Then, GPE and GSE were supplemented for 7 days after withdrawal of antibiotics. The gut microbiota was characterized by a significant loss of diversity and certain important taxon after a 3‐week antibiotic cocktail treatment. The GPE and GSE supplementation during the restore period of gut microbiota had some positive effects. The relative abundance of gut microbiota was improved by GPE and GSE compared to the spontaneous recovery group. And gut microbiota diversity was also greatly changed by GPE and GSE, being indicated by the changes of Verrucomicrobia and Akkermansia in feces. These findings suggested that grape polyphenol extracts have a great influence on the recovery of gut microbiota after antibiotics and high‐fat diet treatment.

Thus, it seems to be very important to find out that how to partly reverse the negative effects of antibiotics on gut microbiota (Dethlefsen & Relman, 2011;Suárez-Zamorano et al., 2015).
Growing evidence supported that grape polyphenol extracts may play beneficial roles on the physiological health of human due to its interaction with microbiota (Milenkovic, Jude, & Morand, 2013;Del Rio et al., 2013). Dietary polyphenols as the parent compounds or their metabolites pass to the colon where they are degraded by the action of the local microbiota, giving rise principally to small phenolic acid and aromatic catabolites that are absorbed into the circulatory system (Cardona et al., 2013;Choy et al., 2014;Lee, Jenner, Low, & Lee, 2006;Monagas et al., 2010;Del Rio et al., 2013;Tenore, Campiglia, Ritieni, & Novellino, 2013). Meanwhile, polyphenols and their metabolites may also modify the composition, metabolism, or activity of gut microbiota, further affect intestinal ecology (Kim et al., 2015;Laparra & Sanz, 2010 Proanthocyanidin-rich extract from grape seeds given to healthy adults for 2 weeks was able to significantly increase the number of Bifidobacterium (Dolara et al., 2005;Kim et al., 2015;Queipo-Ortuño et al., 2012;Rastmanesh, 2011;Tzounis et al., 2008). However, the concentration on the recovery effect of polyphenol on gut microbiota, after antibiotics damages, remains quite limited.
Thus, the main goal of this work was to investigate the effects of grape pomace polyphenol extracts (GPE) and grape seed polyphenol extracts (GSE) on the recovery of gut microbiota after antibiotic cocktails treatment in HFD mice so that elaborate the potential beneficial effects of polyphenols on gut microbiota.

| Extracts of polyphenols from grape pomaces (GPE)
Extracts of polyphenols used in the investigation were extracted from the Kyoho Grape (Vitis vinifera"Kyoho") pomace according to the methods of Ghafoor, Choi, Jeon, and Jo (2009) were provided by Tianjin Jianfeng Natural (Tianjin Jianfeng Natural R&D Co., Ltd.).
The UV was used to determine total polyphenol content and total proanthocyanidin content of GPE and GSE. Total polyphenol content of GPE and GSE, expressed as of equivalent gallic acid (mg of gallic acid equivalents (GAE)/g extract), was 80.60 g GAE/100 g and 86.33 g GAE/100 g, respectively. Proanthocyanidins, the main polyphenolic components in grape pomace, was expressed as equivalent catechin, namely, mg of catechin equivalents (CAT)/g extract. The contents were 63.47 g CAT/100 g and 91.00 g CAT/100 g in GPE and GSE, respectively. The anthocyanin and phenolic profile determined by UPLC-MS is shown in Table 1 and Figure 1, where the specific types of proanthocyanidins of GPE were showed as proanthocyanidins dimer, galloyl proanthocyanidin dimer, and proanthocyanidins trimer (Table 1). Among total proanthocyanidins, oligomeric proanthocyanidin accounts for 63.14% and proanthocyanidin B2 accounts for 1.84%.

| Animals and experimental design
All animal experimental procedures were performed and approved by the Ethical Committee of Peking University, Health Science Center (Beijing, China) with mice housed in specific pathogen-free (SPF) conditions. Male C57BL/6J mice at the age of 6 weeks were purchased from Vital River Laboratories and were housed under the standard laboratory conditions (22 ± 2°C, 55 ± 5% relative humidity, a 12-hr light/dark cycle) with free access to food and drinking water. Mice acclimatized on water and standard chow diet ad libitum for 1 week prior to the initiation of the experiment. All efforts were made to minimize animal suffering.
Acclimatized animals were weighed and randomly divided into five groups of 10 animals each and kept five per cage. One group of animals were normally raised with normal chow (NC) diet containing 10% kcal from fat (D12450B, Research Diets Inc.), without antibiotics as healthy controls (namely, NC + Abx − group). Four experiment groups were given a high-fat diet containing 60% Kcal from fat (D12492, Research Diets Inc.) during the experiment process and were initially given a 3-week treatment of the following antibiotics in their drinking water (0.5 g/L of vancomycin, 1 g/L of ampicillin, 1 g/L of neomycin sulfate, 1 g/L of metronidazole), as previously described (Suez et al., 2014) with some modifications.
And the diet composition is exhibited in Table 2 according to the information supplied by manufacturer. Antibiotics were supplied every 2 days. One of the experiment groups was only received antibiotics for 3 weeks, without recovery (namely, HFD + Abx + group).
While other three groups, after 3-week antibiotic treatment, have been immediately repopulated with microbiota for 7 days after withdrawal of antibiotics, meanwhile these three groups additionally being received by daily gavage either normal saline (namely, HFD + Abx +/− group) or a solution of 200 mg/kg·bw of GPE in normal saline (namely, HFD + Abx +/− + GPE group) and GSE (namely, F I G U R E 1 UPLC/MS profile of grape pomace extracts detected at (a) 280 nm for phenolic acid, (b) 520 nm for anthocyanins HFD + Abx +/− + GSE group) in normal saline. Body weight of each mouse was recorded weekly, and the volume of solutions to force feed was adjusted according to the weight of mice. Feces were collected from all mice of five groups in the last two consecutive days.
Stool samples were snap-frozen in liquid nitrogen before storage at −80°C until using.

| Biochemical analysis of serum lipid
For analysis of serum lipid, a 3,100 automatic biochemistry analyzer (Hitachi Ltd.) was used to determine triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) content in serum.

| PCR amplification and sequencing analysis
Fecal DNA samples were used as the template for PCR amplification of the V4 hypervariable regions of 16S rRNA genes in a PCR system (Bio-Rad). The V4 region is one of the commonly used regions for microbiota sequencing (Claesson et al., 2011;David et al., 2014;Lozupone et al., 2013;Lukens et al., 2014;Yatsunenko et al., 2012). Some studies have found that the results of sequencing in the V4 region and sequencing in the V4-5 region are very close (Walters et al., 2016). That is to say, the V4 region can represent the V4-5 region and can circumvent the problem of shorter sequence sequencing reducing quality of detection. The ampli- And chimeric sequences were identified and removed using UCHIME.

| Statistical analysis
All data were analyzed using one-way analysis of variance (ANOVA) and expressed as mean ± SEM (standard error of mean). Significant differences between the means were further analyzed using the Tukey test (p < .05).
TA B L E 2 The composition of normal and high-fat diet

| Effects of GPE and GSE on the recovery of serum lipid parameter
As shown in Table 3, HFD + Abx + group and HFD + Abx +/− group mice exhibited dyslipidemia as evidenced by significant increased levels of TC, TG, and LDL and decreased level of HDL. GSE supplementation presented a significantly decreasing effect on plasma TC in mice (p < .05).

| Sequencing depth and community diversity
Changing community composition was assessed in five groups (ND + Abx − group, HFD + Abx + group, HFD + Abx +/− group, HFD + Abx +/− + GPE group, HFD + Abx +/− + GSE group). Good's coverage (estimated probability that the next read will belong to a refOTU that has already been found) was up to 99.6%-99.8% for individual samples (Table 4), showing that the sequencing can be representative and that the sequencing depth covered rare new phylotypes and most of the diversity.
The 3-week treatment of antibiotics did significantly reduce the richness and diversity of fecal microbiota. The refOTUs decreased in relative abundance significantly after Abx-treatment and present an increasing tendency after withdrawal of antibiotics, as well as the same change with the abundance and diversity indexes of ACE, Chao, Shannon, and Simpson. In spite there was no significant difference among HFD + Abx +/− group, HFD + Abx +/− + GPE group and HFD + Abx +/− + GSE group on the fecal microbiota according to UniFrac-based principal analysis (PCA) revealed a distinct clustering of microbiota composition of ND + Abx − group versus HFD + Abx + , HFD + Abx +/− HFD + Abx +/− + GPE and HFD + Abx +/− + GSE group ( Figure 2).

| Effects on microbiota composition
Two bacterial phyla dominate the gut of normal mice: Firmicutes return to their initial state, but the return was incomplete, as described in (Figure 3a,b). However, GPE and GSE supplementation during the resurrecting process of microbiota increased its composition and improved its complexity (Figure 3a,b). At phylum level, relative abundance of Verrucomicrobia tended to be significantly greater while relative abundance of Actinobacteria was markedly lowered by GPE and GSE compared to HFD + Abx +/− group (Figure 3a).
Further analysis of the bacterial phyla at a genus level showed post-antibiotic a significant loss on certain important genuses, such as Bacteroides, Blautia, Alloprevotella, and Akkermansia (Figure 3b). In HFD + Abx + group, after 1-week recovery, the relative abundance F I G U R E 2 PCA analysis of some genuses has increased, for example, Escherichia-Shigella and Acinetobacter (Figure 3b), where the return of the whole gut microbiota community was limited. While GPE supplementation and GSE supplementation during the resurrecting process of microbiota contribute to the recovery of gut microbiota, for increasing its composition and improving its complexity on the genus level (Figure 3b).
According to Figure 3b and Table 5, the decreased relative abundance of Akkermansia in feces was markedly recovered by GPE and GSE compared to the HFD + Abx +/− group (0.01%), changing from 0.0496% after treatment of antibiotic to 13.35% and 9.61%, respectively, even higher than the initial state (5.01%).  TA B L E 5 GPE and GSE administration modulated the recovery of gut microbiota of antibiotic-treated mice in genus level (%) treatment of antibiotic) to 0.1731%. Taking GPE and GSE not only increased microbiota abundance, but also decreased some taxon.

GPE and GSE also increased
Relative abundance of Streptococcus was significantly lowered by GPE and GSE intake (Table 5). Although the relative abundance of these microbiota was less than 1%, the restorative effect of 1-week GPE and GSE supplementation on them could still reflect the trend of recovery. Figure 3c shows the heatmap of microbiota 16S rDNA analysis. It can be seen that GPE and GSE had a modest effect on gut microbiota restore. After treatment of antibiotic and restore, Gram-negative bacterium generated greater changes compared to Gram-positive bacterium. Unclassified Enterobacteriaceae (gram-negative bacterium) had the highest proportion after recovery, with relative abundance of Akkermansia (gram-negative bacterium) close behind. In contrast, GPE and GSE had no good for the restore of Bifidobacterium

| D ISCUSS I ON
The human distal gut is one of the most complex ecosystems on the planet. However, it may be a tractable and powerful system for the study of both basic ecological principles and health-related community interactions through the exploitation of disturbance (Dethlefsen & Relman, 2011). The association between the health benefits of grape polyphenols, such as amelioration of cardiovascular and obesity risk factors, and changes in intestinal microbiota composition had profound implication for the relationship between diet and chronic disease (Kim et al., 2015). Interactions of gut microbiota with functional food components and nutraceuticals, like polyphenols, and the impact of gut microbiota on human health have already been studied (Laparra & Sanz, 2010). Therapeutic potential of gut microbiota has caused wide attention (Smits, Bouter, De Vos, Borody, & Nieuwdorp, 2013). Therefore, the present study provided further evidence for the potential role of grape polyphenols in the regulation of gut microbiota thus may suggest the role of grape polyphenol for the regulation of host health.
Antibiotic treatment had tremendous impact on the overall taxonomic composition of gut microbiota. Even with short-term administration of antibiotics may shift the microbiota to a long-term state of dysbiosis (Lange et al., 2016). According to the study conducted by Vrieze et al. (2014), antibiotic reduced the absolute number of  Clostridium, and Enterobacteraciae (Rakoff-Nahoum, Paglino, Eslami-Varzaneh, Edberg, & Medzhitov, 2004;Wang et al., 2012) and can be recolonized with normal gut microbiota after withdrawal of antibiotics (Suárez-Zamorano et al., 2015). In the current study, the gut microbiota, which was composed of diverse populations of commensal bacterial species, were mostly removed by a 3-week antibiotic cocktail treatment with a significant loss of diversity and certain important taxa, such as Akkermansia, Alloprevptella, and Prevotella and then uncompletely recovered after withdrawal of antibiotics for 7d. This consisted of previous investigations.
The effect of ciprofloxacin on the gut microbiota was profound and rapid, with a loss of diversity and a shift in community composition occurring within 3-4 days of drug initiation, and communities began to return to their initial state by 1 week after the end of each course, but the return was often incomplete (Dethlefsen & Relman, 2011). After withdrawal of antibiotics, Abx-treated mice were repopulated with microbiota immediately from their conventional former littermates by co-housing them for 7 days (Suárez-Zamorano et al., 2015). In the present study, gut microbiota was mainly composed of the phyla Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria, and the Verrucomicrobia phylum was occasionally observed. While the post-antibiotic dysbiosis was characterized by a significantly reduced diversity of the phyla Firmicutes, Bacteroidetes, and Actinobacteria together, with a markedly increase of the family Enterobacteriacea of Proteobacteria, in accordance with previous study (Lange et al., 2016).
It has been reported that grape polyphenols have physiological effect on human health and some of which is closely linked to modulation of gut microbiota (Kim et al., 2015). The bioavailability and effects of polyphenols also greatly depended on their transformation by specific components of the gut microbiota; meanwhile, polyphenols and their metabolites may also inhibit or stimulate the growth of specific bacteria, exert prebiotic-like effects, modify the composition or activity of the gut microbiota, thus affect the intestinal ecology (Kim et al., 2015;Laparra & Sanz, 2010).
In our work, supplementation of GPE and GSE after the withdrawing of antibiotics did had some positive effects on the recovery of gut microbiota. The richness and diversity of microbiota in feces of GPE and GSE administration animals were increased compared to the HFD + Abx +/− group. GPE and GSE supplementation during the resurrecting process of gut microbiota improved the composition and total number of gut microbiota. Relative abundance of Verrucomicrobia was significantly increased by GPE and GSE administration. A study conducted by Zhang et al. (2018) consisted with our result. The Verrucomicrobia phylum had a phylogenetically close relationship with Chlamydiae and Planctomycetes and was mainly made up by environmental microorganisms. In this phylum, Akkermansia was an attractive bacterium that was first isolated from human feces (Dubourg et al., 2013). While relative abundance of Actinobacteria was markedly lowered by GPE and GSE administration compared to HFD + Abx +/− group, consisting with a previous research (Jiao et al., 2018).
After further analysis, we found that relative abundance of Akkermansia in feces was greatly recovered by GPE and GSE supplementation compared to the HFD + Abx +/− group. This was in accordance with a previous study (Anhê et al., 2017). It has been reported that Akkermansia, a Gram-negative mucin degrading bacterium, which accounts for 1%-5% of total gut microbiota in health mammals. Hubert et al. have observed that the abundance of Akkermansia decreased during obesity and diabetes compared with healthy people, and that higher baseline abundance was significantly associated with the improvement of cardiometabolic parameters in individuals with obesity undergoing caloric restriction (Plovier et al., 2016). And feeding of Akkermansia enhanced mucus thickness, intestinal endocannabinoid production, and gut barrier function in mice on HF diets, which resulted in reduced fat mass, endotoxemia, adipose tissue inflammation, and insulin resistance (Everard et al., 2013;Kim et al., 2015;Shin et al., 2014), suggesting that GPE and GSE may ameliorate obesity and diabetes partly by increasing the relative abundance of Akkermansia in the gut. Moreover, since Akkemansia belongs to Verrucomicrobia, the improved Akkemansia was the major contributor to the observed increase of the relative abundance of Verrucomicrobia in present investigation.

| CON CLUS ION
Our findings suggested a light modulation of gut microbiota by grape pomace polyphenols on the restore of gut microbiota. The gut microbiota was characterized by a significant loss of diversity and certain important taxa after a 3-week antibiotic cocktail treatment.
Compared to the HFD + Abx +/− group, supplementation of GPE and GSE during the recovery period of gut microbiota had some positive effects on relative abundance and diversity of microbiota in feces.
Especially relative abundance of Akkermansia in feces was greatly elevated by GPE and GSE intake. This study also suggested that pharmacological or nutritional modulation of gut microbiota was an effective therapeutic method for the intestinal disorders making by antibiotics. Foundation.

CO N FLI C T O F I NTE R E S T
The authors notify that there are no conflicts of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.