Wenjing Liao, Wenjiao Li, Suyu Liu, Dong Tang, Yunxi Chen, Yijun Wang,Zhongwen Xie, Jinbao Huang*

State Key Laboratory of Tea Plant Biology and Utilization, Key Laboratory of Food Nutrition and Safety,School of Tea & Food Science and Technology, Anhui Agricultural University, Hefei 230036, China

Keywords:

Black tea

Intestinal microbiota

Anaerobic fermentation

Short-chain fatty acid

A B S T R A C T

Black tea is a healthy and popular tea beverage.However, its main bioactive compounds (theaflavins and thearubigins) are not easily absorbed.The aim of this study was to investigate the modulation of intestinal microbiota by the nonabsorptive components of Keemun black tea (KBT) and Dianhong black tea (DBT),and fructooligosaccharide (FOS) was selected for use in the control group.KBT, DBT, and FOS significantly increased total short-chain acid production.Specifically, FOS treatment predominantly increased the production of acetic acids and black tea treatments increased the production of acetic, propionic, and butyric acids at similar rates.Moreover, FOS exerted a strong bifidogenic effect after 24 h of fermentation; KBT and DBT increased the abundance of the beneficial genus Bacteroides and Roseburia.In summary, the nonabsorptive components of KBT and DBT could serve as novel prebiotics, the underlying mechanisms of which are quite different from those of FOS.

1.Introduction

The human intestine harbors a complex bacterial community,which is a potential source of novel therapeutics [1].Gut microflora reportedly play a pivotal role in the host’s nutrient absorption, metabolism, and immunity [2].As the major metabolites of microorganisms, short-chain fatty acids (SCFAs)are vital in the health-promoting functions of gut flora [3].A structurally disrupted gut microbiota is associated with various chronic diseases [4], including obesity, type 2 diabetes, chronic kidney diseases, and even Parkinson’s disease [5-7].Microbial alterations may be caused by either endogenous or environmental factors, and dietary intervention is a prominent method for modulating the gut microbiota [8].

Phenolic compounds are the most prevalent secondary metabolites in the plant kingdom and are abundant in plant-derived foods such as vegetables, fruits, and tea [9].Dietary polyphenols reportedly evade digestion in the small intestine and then reach the large intestine intact [10].Studies have indicated that dietary polyphenols could modulate gut microbiota by promoting the growth of potentially beneficial bacteria and inhibiting that of pathogenic bacteria [11].Zhou et al.[12]indicated thatin vitroanaerobic fermentation with grape polyphenols promotes the growth ofBifidobacteriumandLactobacillusbut inhibits that of other groups such asClostridiumspp.A similar affinity ofBifidobacteriumto red wine was reported by Queipo-Ortuno et al.[13].In addition, the modulation of the gut microbiota was observed in the fermentation of polyphenol-rich extract from chokeberry [14].

Tea, the most consumed beverage worldwide, is primarily made from the shoots and young leaves of the tea plantCamellia sinensisvar.assamicaorC.sinensisvar.sinensis[15].The health benefits of consuming tea, a rich source of polyphenols, have been extensively reported and include reduction in body weight; prevention of metabolic abnormality, diabetes, and cancer; alleviation of metabolic syndrome; and regulation of the gut microbiome [16,17].According to the manufacturing process, tea can be classified into green, white,yellow, oolong, black, or dark tea [18].Black tea is fully fermented and accounts for approximately 75% of global tea consumption.Most of the phenolic compounds of black tea are oxidized, dimerized to form theaflavins, and polymerized to form thearubigins, which do not easily penetrate the intestinal barrier to reach systematic sites.Many studies have already confirmed the low bioavailability of epigallocatechin gallate, the most prominent phenolic compound in tea [19].The oxidized and polymerized forms of tea polyphenols theoretically yield much lower serum concentrations after consumption.However, similar health benefits were obtained from green and black tea in obese mice fed a high-fat diet in another study we conducted [20].Intestinal metabolism and microorganisms potentially play vital roles in regulating processes.Few studies have focused on the prebiotic effects of black tea’s nonabsorptive components and how they regulate intestinal microbiota.

In this study, two typical categories of Chinese black tea were selected: Keemun black tea (KBT) and Dianhong black tea (DBT).KBT, which is grown mainly in Qimen county, Anhui province,China, is processed from the tea plant varietyC.sinensisvar.sinensisand possesses unique floral and honey aromas [21]; DBT, which is grown mainly in Yunnan province, China, is processed fromC.sinensisvar.assamicaand possesses a higher phenolic content and strong, mellow aromas [22,23].Black tea powder was successively subjected toin vitrosimulated digestion and dialysis.The black tea components obtained were then fermented in an anaerobic tank for 24 h with gut flora from healthy volunteers, and fructooligosaccharide(FOS) was selected as a positive control.The aim was to clarify the effects of the nonabsorptive components on the production levels of several SCFAs and the gut flora after fermentation.

2.Materials and methods

2.1 Samples and chemicals

KBT and DBT with the same maturity (one bud and two young leaves) were analyzed in this study.The sample of KBT was purchased from local tea factory (Qimen, Anhui, China), and DBT was supplied by the tea planting base of Yunnan Dianhong Group Co., Ltd.(Fengqing, Yunnan, China).All samples were sealed stored at -20 °C before analysis.

FOS (> 90%) and Folin–Ciocalteu reagent were provided by Beijing Solarbio Science & Technology Co., Ltd.(Beijing, China).Salivaryα-amylase, pepsin, gastric lipase and pancreatin were purchased from Sigma Chemical Co.(St.Louis, MO, USA).Bile salt was obtained from Shanghai Ryon Biological Technology Co., Ltd.(Shanghai, China).Standards of acetic, propionic,n-butyric, isobutyric,n-valeric, isovaleric, and 2-ethylbutyric acids of high-performance liquid chromatography (HPLC) grade were purchased from Aladdin Co.(Shanghai, China).Standards for gallic acid (GA, > 98%), theobromine (THB, > 98%), caffeine(CAF, > 98%), (–)-epigallocatechin gallate (EGCG, > 98%),(–)-epicatechin gallate (ECG, > 98%), (–)-epicatechin (EC, >98%), (+)-catechin (C, > 98%), (–)-epigallocatechin (EGC, > 98%),theanine (> 99% purity), thea flavin (TF, ＞ 95%), thea flavin-3-gallate(TF-3-G, ＞ 95%), theaflavin-3’-gallate (TF-3’-G, ＞ 95%), and thea flavin-3,3’-digallate (TF-3,3’-diG, ＞ 95%) were purchased from Yuanye Biotechnology Company (Shanghai, China).HPLC-grade acetonitrile, methanol, and water were purchased from Thermo Fisher Scientific Co.(Fair Lawn, NJ, USA).All other reagents used in the study were of analytical grade.

2.2 Chemical composition of black tea

2.2.1 Determination of total phenolic content

The total polyphenol content of the black tea was measured using the Folin-Ciocalteu method with a few modifications [24].In brief,0.2 g of black tea powder was extracted, combined with 10 mL of 70% methanol, and maintained at 70 °C for 20 min.The extract was then centrifuged at 3 500 r/min for 10 min, and the supernatants were diluted with water.Thorough mixing of 1.0 mL of the sample,5.0 mL of 10% Folin-Ciocalteu reagent, and 4.0 mL of 7.5% sodium carbonate was performed.After standing at 30 °C in darkness for 1 h, the absorbance measurements were obtained at 765 nm.The total polyphenol content was standardized against GA.

2.2.2 HPLC analysis

Black tea samples were prepared according to a reported method,with some modifications [25].The contents of tea catechins, caffeine,theanine, and theaflavin in the black tea were determined using an Agilent 1260 series HPLC (Agilent Technologies, Palo Alto, CA,USA).Separation was performed in an Agilent ZORBAX SB-Aq C18column (250 mm × 4.6 mm, 5 μm).The chromatographic conditions were as described by Zhang et al.[26].

2.3 Simulated digestion of black tea in vitro

In vitrosimulated salivary, gastric, and small intestinal digestion of black tea was performed as reported by Hu et al.[27], Smith et al.[28],and Chen et al.[29].In brief, the method involved a three-step digestion procedure.

To simulate oral digestion, simulated salivary fluid was prepared by dissolving KCl, KH2PO4, NaHCO3, MgCl2·6H2O, and (NH4)2CO3in distilled water.Next, 10 g of black tea (KBT and DBT) was mixed with 8 mL of simulated salivary fluid; 1 mL of salivaryα-amylase solution was added, followed by 50 μL of 0.3 mol/L CaCl2and 950 μL of water at pH 7.0.Subsequently, incubation was performed at 37 °C for 2 min.

For the resulting saliva phase, the pH was then adjusted to 3.0 with a 0.1 mol/L HCl solution.To prepare the simulated gastric fluid,KCl, KH2PO4, NaHCO3, NaCl, MgCl2·6H2O, and (NH4)2CO3were dissolved in distilled water.Subsequently, 1 mL of gastric pepsin solution and gastric lipase solution were added to the simulated gastric fluid, and the mixture was maintained at 37 °C for 2 h.

After gastric digestion, the solution pH was adjusted to 7.0.To prepare simulated intestinal fluid, KCl, KH2PO4, NaHCO3, and MgCl2·6H2O were dissolved.To simulate small intestinal digestion,10.0 mL of pancreatin solution and 5.0 mL of bile salt were added to the simulated intestinal fluid.The mixture was then maintained at 37 °C for 2 h.Once small intestinal digestion was completed, the solution was dialyzed and freeze dried for 48 h.

2.4 Anaerobic fermentation in vitro

Afterin vitrodigestion, Thein vitromodel of fermentation was used to investigate the effects of black tea on human intestinal microbiota according to the method described by Chen et al.[30],with slight modification.The growth medium was prepared with 2 g/L peptone, 2 g/L yeast extract, 0.1 g/L NaCl, 0.04 g/L K2HPO4,0.04 g/L KH2PO4, 0.01 g/L MgSO4·7H2O, 0.01 g/L CaCl2·6H2O,2 g/L NaHCO3, 0.02 g/L hemin, 0.5 g/L cysteine HCl, 0.5 g/L bile salts, 1 mg/L resazurin, 2 mL/L Tween 80, and 10 μL/L vitamin K1.The medium was adjusted to pH 7.0 and then sterilized at 121 °C for 20 min.

Fresh fecal samples were obtained from two healthy volunteers(one male and one female, age 22–28 years) who had never had gastrointestinal disorders and had not ingested antibiotics in the preceding 3 months.The fecal samples were mixed with autoclaved physiological saline solution (8.5 g/L NaCl) to obtain a 10% (m/V)suspension and subsequently centrifuged at 4 °C (800 ×g, 5 min).The resulting fecal slurry was used for microbial fermentation, which was initiated through the addition of 3.0 mL of fecal slurry to 27.0 mL of culture medium.The systems were as follows: a positive control containing FOS and the KBT, DBT (10.0 g/L), and no-additive (BLK)groups.Each experiment was performed in triplicate.Batch cultures were performed in an MGC Anaero Pack system (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan).All samples were incubated at 37 °C for 24 h, and gentle shaking was performed every 4 h.After fermentation, samples (3 mL) were collected for bacterial DNA extraction and SCFA analysis.

2.5 Determination of SCFAs

The fermentation products were centrifuged at 12 000 r/min for 10 min.Supernatants were used for determining the contents of SCFAs according to the method described by Tian et al.[31]with some modifications.Chromatographic analysis was performed on a GC instrument (7 890 A, Agilent Technologies) equipped with an HP-INNOWAX column (30 m × 0.25 mm × 0.25 μm, Agilent)and a flame ionization detector (FID).The column temperature was regulated as follows: 100 °C maintained for 1 min, an increase at 5 °C/min for 16 min to achieve a temperature of 180 °C, which was then maintained for 4 min.

2.6 Analysis of gut microbiota

After fermentation for 24 h, the culture media were centrifuged,and the DNA of the total bacteria was immediately extracted with a TIANamp Stool DNA Kit (TIANGEN Biotech, Beijing, China)according to the protocols of manufacturers.The total bacterial DNA was sent to Novogene Co., Ltd.(Beijing, China) in dry-ice conditions.For high-throughput sequencing, the hypervariable region V3-V4 of the 16S rRNA gene was selected for amplification.Operational taxonomic units (OTUs) were clustered with the similarity of 97% by UPARSE, and the OTUs were subsampled randomly [32].α-Diversity analysis was performed to evaluate the complexity of species diversity.β-Diversity analysis was used to evaluate differences among samples in terms of species complexity.Theα- andβ-diversities of both weighted and UniFrac results were performed by using R software (Version 2.15.3 http://www.r-project.org/) [33].

2.7 Statistical analysis

All experiments were performed at least in triplicate, and the results were analyzed with SPSS version 22.0 (Chicago, IL, USA) and expressed as the mean ± standard error of the mean (SEM).Multiple samples were compared through one-way analysis of variance(ANOVA) by using the Tukey test.Values ofP≤ 0.05 were defined as statistically significant.

3.Results

3.1 Measurement of the active ingredients in black tea

The total phenolic contents of KBT and DBT were determined to be 7.94% and 12.70% (the results are expressed as g gallic acid equivalents per 100 g of sample), respectively, by using the Folin–Ciocalteu method.HPLC analysis was conducted to determine the contents of catechins, alkaloids, theanine and theaflavins in the KBT and DBT samples (Table 1).Catechins and caffeine were present at higher concentrations in DBT compared with in KBT, and the levels of EGC and ECG were higher in DBT.The content of theanine in KBT was threefold higher than that in DBT.Four monomers of theaflavins were identified in the black tea samples: TF, TF-3-G, TF-3’-G,and TF-3,3’-diG.TF-3,3’-diG was the most abundant, followed by TF-3-G, TF-3’-G, and TF.The contents of TF (1.34 mg/g) and TF-3’-G (3.23 mg/g) were higher in DBT than in KBT (1.19 mg/g,2.48 mg/g), but the TF-3-G (6.35 mg/g) and TF-3,3’-diG (19.02 mg/g)contents were lower in DBT than in KBT (7.78 mg/g and 24.58 mg/g,respectively).

Table 1Comparison of compound contents of KBT and DBT detected by HPLC (mg/g).

3.2 Effects of black tea on SCFAs production

To investigate the effects of black tea or FOS supplementation on the gut microbiotain vitro, the contents of SCFAs before and after fermentation were analyzed (Table 2).KBT, DBT, and FOS significantly increased total SCFA production by 44%, 46%, and 64%, respectively.Among the SCFAs identified, acetic and propionic acids were the most abundant organic acids, followed by butyric acid.After fermentation for 24 h, the FOS treatment significantly increased the production of acetic and propionic acids by 123% and 13%,respectively; no effects onbutyric acid were observed.KBT treatment promoted the production of acetic, propionic, andn-butyric acids by 58.90%, 61.73%, and 40.14%, respectively, compared with BLK treatment.The rate of increase in the DBT group was comparable to that in the KBT group.

Table 2Concentrations of SCFAs and total acids.

3.3 Overall difference in microbial structure

A total of 12 samples (three samples per group) were used for 16S rRNA sequencing analysis, and 754 216 effective reads and 648 OTUs were obtained.As shown in Fig.S1, with the increase in sequencing depth, the refraction curves continued to increase.However, the Shannon index values of all 12 samples exhibited a plateau, indicating that most species had been identified, including new phylotypes.Theα-diversity indexes are shown in Table S1.The mean values for the Chao1, abundance-based coverage estimator (ACE), and Shannon indexes were not significantly different among the groups,the Simpson index was slightly increased by KBT (P＜ 0.05,KBT compared with FOS).

The overall differences among the samples tested were evaluated at OTU levels.PCoA indicated that the samples were clearly divided into three groups: the control group (BLK), FOS, and black tea treatments (Fig.1A), and two black tea treatments were clustered into one group.The cluster analysis also yielded similar results(Fig.1B).A significant separation among BLK, FOS, and black tea groups was observed, with BLK and FOS constituting one subgroup and two black tea treatments constituting another subgroup.

Fig.1 Effects of fermentation products from black tea on microbial community structure.(A) Principal coordinates analysis (PCoA); (B) cluster analysis on the basis of weighted_unifrac distance.

The linear discriminant analysis effect size (LEfSe; LDA = 4)was used to determine the bacteria most likely to explain differences among groups.As shown in Fig.2, the number of discriminative features identified in the microbiota was 16 in the FOS group, 8 in the KBT group, and 10 in the DBT group.At the phylum level,Actinobacteria exhibited greater abundance in the FOS group, whereas Fusobacteria and Bacteroidetes were prevalent in samples collected from the KBT and DBT groups.At the class level, the relative abundances of Fusobacterila and Bacteroidia increased and those of Gammaproteobacteria, Negativicutes, and Clostridia decreased in the KBT and DBT groups.At the order level, KBT and DBT increased the abundances of Fusobacteriales and Bacteroidales and reduced those of Clostridiales, Selenomonadales, and Enterobacteriales.At the family level, KBT and DBT enhanced the abundances of Fusobacteriaceae, Bacteroidaceae, and Burkholderiaceae and inhibited those of Lachnospiraceae, Tannerellaceae, Acidaminococcaceae,and Enterobacteriaceae.

Fig.2 LDA effect size of 16S rRNA sequencing results of the BLK, FOS,KBT, and DBT groups.

3.4 Changes in microbiota composition at the phylum and genus levels

Changes to the microbial community were also analyzed at the phylum and genus levels.At the phylum level, the microorganisms identified comprised mainly Bacteroidetes,Firmicutes, Proteobacteria,Actinobacteria, and Fusobacteria(Fig.3A).Black tea supplementation significantly reduced the relative abundance of Firmicutes (P< 0.000 1,Fig.3B) and increased the level of Bacteroidetes (P< 0.000 1,Fig.3C).The ratio of FirmicutestoBacteroidetes(F/B) exhibited reductions of 68.21%, 75.50%, and 45.03%, respectively, after KBT,DBT, and FOS treatments compared with in the BLK group(Fig.3D).Moreover, the relative abundance of Actinobacteria was increased only by FOS treatments compared with in the BLK group (P< 0.000 1,Fig.3E).

Fig.3 Black tea modulation of microbial community composition at the phylum level.(A) stack column; (B) relative abundance of Firmicutes; (C) relative abundance of Bacteroidetes; (D) The ratio of F/B; (E) relative abundance of Actinobacteria.Data are presented as the mean ± standard error of the mean (n = 3).Different letters indicate significant difference values (analysis of variance, P ＜ 0.05).

At the genus level, the microbial compositions and relative abundances in the black tea groups were also substantially different from those for the FOS treatment (Fig.4).In this study, the predominant bacterial genera identified wereBacteroides,Collinsella,Tyzzerella,Fusobacterium,Lachnoclostridium,Faecalibacterium,Sutterella,Phascolarctobacterium,Bifidobacterium, andRoseburia(Fig.4A).We focused mainly on the top 30 genera with relative abundances greater than 1%, and the relative abundances of gut bacteria are shown in Fig.S2.We noticed that the relative abundances ofCollinsella,Faecalibacterium,Bifidobacterium,andSubdoligranulumincreased in the FOS group (Figs.S2A, B);notably, the abundances of the beneficial bacteriaCollinsella,Faecalibacterium, andBifidobacteriumwere significantly higher than those in other groups (P< 0.000 1, Figs.4B–D).In the KBT and DBT groups, the abundances ofBacteroides,Fusobacterium,Sutterella, andRoseburiaincreased (Figs.S2A, B), whereas those ofEnterobacteriaceae,Phascolarctobacterium,Tyzzerella, andRuminococcaceaedecreased compared with those in the BLK group(Figs.S2A, B).Our data showed that KBT and DBT significantly increased the relative abundance ofBacteroidesby 107% and 114%,respectively (P< 0.000 1 Fig.4E), and increased the abundance ofFusobacteriumby 167% and 134%, respectively (P< 0.000 1, Fig.4F).We also observed that the relative abundance ofRoseburiaincreased by 415% and 453% after KBT and DBT treatment, respectively(P< 0.000 1, Fig.4G), compared with the BLK group.The results suggest that the consumption of black tea might benefit certain gut microbiota,and the data were consistent with results at the phylum level.

Fig.4 Microbial community composition at the genus level.(A) stack column; relative abundance of (B) Collinsella, (C) Faecalibacterium, (D) Bifidobacterium,(E) Bacteroides, (F) Fusobacterium, and (G) Roseburia.Results are presented as the mean ± standard error of the mean (n = 3).Different letters indicate significant difference values (ANOVA, P ＜ 0.05).

Fig.4 (Continued)

3.5 Relationship between gut microbiota composition and SCFAs production

Pearson correlation analysis revealed a relationship between the relative abundances of bacteria and SCFA concentration (Fig.5).The heat map illustrates significant positive correlations between the production of acetic acid and the abundances ofCollinsella,Faecalibacterium,Bifidobacterium,Subdoligranulum,Blautia,Butyricicoccus,Agathobacter,Anaerostipes, andDialister.However, the abundances ofTyzzerella,Lachnoclostridium,Phascolarctobacterium,Allisonella,Parabacteroides,Desulfovibrio,andAkkermansiawere negatively correlated with acetic acid production.Propionic acid production exhibited a positive correlation with the abundances ofBacteroides,Fusobacterium,Sutterella,Roseburia, andLachnospiraand a negative correlation with the abundances ofTyzzerella,Blautia,Alistipes,Desulfovibrio,Akkermansia, andVeillonella.In addition, butyric acid production exhibited a positive correlation with the abundances ofBacteroides,Fusobacterium,Sutterella, andRoseburiaand a negative correlation with the abundances ofSubdoligranulum,Blautia,Alistipes, andVeillonella.We found that the bacterial genera whose abundances were positively associated with acetic acid production were enhanced by FOS treatment and that those whose abundances were positively correlated with propionic acid and butyric acid production were enhanced by black tea treatment.Notably, an increase in the abundance of the genusBifidobacterium(a genus that is positively associated with acetate) was observed mainly after FOS treatment.The generaBacteroides(a genus whose abundance is positively associated with propionate) andRoseburia(a genus whose abundance is positively associated with propionate and butyrate) exhibited the greatest enhancement in response to black tea.

Fig.5 Heat map of Pearson correlation between gut microbiota and SCFAs.Solid circles indicate a greater abundance in the black tea and FOS groups compared with in the BLK group; hollow circles indicate a lower abundance in the black tea groups and FOS group compared with in the BLK group.Colors of squares represent P values (from 1.00 to ?1.00).Significant correlations are indicated by *P ＜ 0.05, **P < 0.01, and ***P < 0.001.

3.6 Microbial function prediction

The abundances for functional categories of the microbial community profiles were predicted with Tax4Fun (Fig.S3).At Kyoto Encyclopedia of Genes and Genomes (KEGG) level 2, the metabolic function of microbiota involved in metabolic pathways—including carbohydrate metabolism, energy metabolism and the metabolism of other amino acids, and genetic information processing(folding, sorting, and degradation)—were predicted to be enhanced by both black tea and FOS treatments.Significant differences in the abundances of 10 functional genes were observed between the black tea and FOS groups (P＜ 0.05, Figs.S3A-C), compared with in the BLK group, including genes involved in membrane transport, translation, replication and repair, glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, lipid metabolism,enzyme families, transcription, metabolism, and cellular processes and signaling.Specifically, compared with the FOS group, in which the functional pathways were enriched, in the black tea group,metabolic functions (glycan biosynthesis and metabolism, metabolism of cofactors and vitamins, enzyme families, and lipid metabolism)and cellular processes and signaling were significantly improved.The pathways related to genetic information processing (replication and repair, transcription, and translation) and environmental information processing (membrane transport) were significantly reduced by black tea treatments.At KEGG level 3, the FOS treatment significantly increased the level of pyruvate metabolism, which was related to acetate metabolism, compared with in the BLK group (P= 0.002,Fig.S3D).Propanoate metabolism, which is related to propionate metabolism, was increased by black tea treatment compared with the BLK group (P＜ 0.01, Figs.S3E, F).Overall, the microbial communities present in the black tea and FOS groups could be distinguished according to their functions.

4.Discussion

The gut microbiota plays a major role in human health [34], and it is closely associated with nutrient acquisition and energy regulation in hosts [35].Black tea is the most consumed tea beverage worldwide.The major phenolic compounds of black tea are oxidized and dimerized to form theaflavins, which are of large molecular weight and not easily absorbed by the small intestine.Using anin vitroanaerobic fermentation system, alterations of human gut microbiota by black tea and FOS treatments were investigated in this study, and changes in SCFA production were also determined.

We first analyzed the caffeine, theanine, catechins, and thea flavins contents in the two black tea samples used.The major components of polyphenol in black tea are thea flavins, which are a mixture of TF,TF-3-G, TF-3’-G, and TF-3,3’-diG [36].Greater quantities of TF(1.34 mg/g) and TF-3’-G (3.23 mg/g) were found in DBT; TF-3-G(7.78 mg/g) and TF-3,3’-diG (24.58 mg/g) were more abundant in KBT.According to Henning et al.[20], black tea polyphenols are utilized by gut microbiota and offer health benefits.Thus, the effects of the non-absorptive components of KBT and DBT on the gut microbiota were further analyzed.

To investigate changes to intestinal flora caused by experimental treatments, the production of SCFAs in the fermentation fluid was analyzed.SCFAs are the major metabolites of the gut microbiota, and they play a prominent role in the regulation of host health [37].SCFAs can act as major sources of epithelial energy or can protect against pathogenic bacteria and protect the intestinal mucosal barrier.Acetic acid is converted into propionic and butyric acid by the intestinal flora, and these help to regulate the secretion of intestinal mucin [38].Propionate is involved in stimulating the secretion of peptide tyrosine-tyrosine and glucagon-like peptide-1, increasing insulin sensitivity [39].Furthermore, propionate contributes to reducing serum cholesterol and regulating inflammation [40].Moreover,propionic acid may play a crucial role in appetite regulation [41].As a preferred energy source for colon epithelial cells, butyrate is critical for regulating host gene expression and also inhibits colorectal cancer [42].In our study, the total SCFA production in all groups was increased after fermentation for 24 h, and FOS incubation resulted in the greatest total quantity of SCFAs, which reached (42.88 ± 0.22) mmol/L.FOS treatment significantly increased the production of acetic and propionic acid by 123% and 13%, respectively, but had no effect on butyric acid.Black tea treatments enhanced the contents of these three fatty acids in similar proportions (40%–60%).Therefore, a difference in the effects of FOS and black tea treatments on SCFAs indicated that differences may also exist in the function of flora.

We profiled gut microbiota compositions after anaerobic fermentation by using a 16S rRNA sequencing technique.PCoA of our data indicated distinct differences in the gut microbiota compositions for the BLK, FOS, and black tea groups.Similar results were observed in the unweighted pair group method of average clustering.The results indicated that the intestinal microbial compositions in black tea groups were quite different from those in the FOS group, whereas KBT and DBT treatments produced similar microorganism profiles.Similarly, distinct LEfSe discriminative features were defined for FOS and black tea.LEfSe revealed differentially abundant taxa between the black tea groups and the FOS group.The majority of the bacterial taxa in FOS comprised the Coriobacteriaceae, Ruminococcaceae, and Bifidobacteriaceae families.The abundances of the Fusobacteriaceae, Bacteroidaceae,and Burkholderiaceaefamilies were identified as discriminative features in black tea groups.Thus, different dominant microbial community structures were exhibited for FOS and black tea treatment.

Severalin vitroandin vivostudies have suggested that polyphenols can modulate gut microbiota, and they have been considered for use as prebiotics [43].In the present study, KBT and DBT significantly reduced the F/B at the phylum level in a manner similar to Fuzhuan brick tea polysaccharide fermentation by human intestinal microbiota [44].Notably, the F/B in the KBT and DBT groups were significantly lower than that in the FOS group.The F/B is closely associated with the energy harvesting of the gut microbiota [45], which is closely related to a decreased risk of obesity in humans [46].Thus, KBT and DBT are believed to reduce the risk of obesity.As well as using LEfSe, we evaluated the abundances ofFusobacterium,Collinsella,Faecalibacterium,Bifidobacterium,Subdoligranulum,Bacteroides,Sutterella,Roseburia,Tyzzerella,Lachnoclostridium, andPhascolarctobacterium.At the genus level, significant increases inCollinsella,Faecalibacterium,Bifidobacterium, andSubdoligranulumwere observed in the FOS group.Perez-Burillo et al.[47]reported that polyphenol-rich tart cherries exert potential health benefits by promoting the relative abundances ofBacteroidesandCollinsella.In addition,Faecalibacteriumis a butyrate-producing bacterium, playing a vital role in protecting against gut inflammation [48].Bifidobacteriumenrichment can be viewed as a marker of intestinal health, and numerous studies have demonstrated the beneficial effects of FOS,including the inhibition of pathogens and cardiovascular disease prevention [49].Subdoligranulumcan produce butyrate and may exert anti-inflammatory effects [50].Notably, in our study, KBT and DBT did not affect the abundance ofBifidobacterium, but they increased the abundances ofBacteroidesandRoseburiacompared with well-established prebiotics; this supports the results of anin vitrofermentation experiment in which the fermentation ofβ-glucan was found to have no effect onBifidobacteriumand to result in increasedRoseburiaabundance [51].In addition, the abundances ofFusobacteriumandSutterellawere significantly increased in the black tea groups.Although studies have shown that colon cancer is related to increasedFusobacteriumlevels, the genus may contain potentially beneficial bacteria.According to the reports of Gillespie et al.[52],Sutterellais a potentially beneficial bacteria that improves the feed conversion rate in chickens.After black tea supplementation,Lachnoclostridium,Phascolarctobacterium, andTyzzerellalevels decrease significantly.Lachnoclostridiumis associated with digestive diseases [53].Phascolarctobacteriumhas been reported to produce butyrate with anti-inflammatory effects [54].Tyzzerellabelongs to the Lachnospiraceae family and was reported to be associated with increased cardiovascular disease risk [55].

The concept of prebiotics have changed, and they now selectively increase species other thanBifidobacterium[56].In the present study,community compositions were comparable for the KBT and DBT groups, with a significantly increased abundance ofBacteroidesandRoseburia(members of Firmicutes) compared with that in the FOS and BLK groups.Microalgae can reportedly be used as a functional food, increasing propionate concentrations and the abundance ofBacteroides[57].Moreover,β-glucan was reported to constitute a potential novel prebiotic [58].An increase in the relative abundance ofBacteroideswas also observed in mice administered EGCG3”Me [59].Bacteroidesis associated with anti-inflammatory properties and plays a valuable role in modulating the gut microbiota [60].Members of the genusRoseburiaare SCFA-producing bacteria that produce both butyrate and propionate [61].An increase inRoseburiawas observed in human fecal fermentation with propionylated starch [62].Furthermore,Roseburiaincreased in the fecal microbiota of human volunteers on almond-based diets [63].IncreasedRoseburiacould stimulate the differentiation of Tregcells, ameliorating intestinal inflammation [64].Bacteroidesis a genus that produces propionate [65].Numerous studies have demonstrated that bothBacteroidesandRoseburiaare beneficial gut microbiota.Moreover, intestinal bacteria were reported to participate in the decomposition and metabolism of polyphenols [12].Therefore,BacteroidesandRoseburiamight be key microorganisms for the utilization of KBT and DBT.

Our correlation analyses identified 23 key variables that exhibited negative or positive correlations with the production of acetate, propionate, and butyrate after black tea or FOS treatments.Treatment with FOS significantly enhanced the relative abundances ofCollinsella,Bifidobacterium, andAnaerostipes, which are beneficial for gut flora.These genera were positively associated with the production of acetate; additionally, they have been implicated in gut health, and the bacteria of the generaAnaerostipeswere described as acetate-producing bacteria [66].Treatment with black tea significantly increased the proportions ofBacteroidesandRoseburia, the abundances of which were positively correlated with the production of propionate and butyrate.van Hul et al.[67]reported that the abundance ofRoseburiaincreased significantly in C57BL/6J mice fed with grape pomace.Moreover,RoseburiaandBacteroidesare considered to benefit gut health through the improvement of metabolic disorders associated with obesity [68].The results of correlation analyses further confirmed the observed effects exerted by black tea and FOS treatments on SCFAs and gut flora.The microorganisms whose abundances were significantly correlated with the production of the three SCFAs were all significantly affected by black tea or FOS treatments.

The bacterial compositions differed between the black tea and FOS groups; the functional KEGG analysis with Tax4Fun also supported this result, revealing that many of the KEGG pathways of gut microorganisms were altered by FOS or black tea treatments.At KEGG level 2, carbohydrate metabolism, energy metabolism,metabolism of other amino acids, and folding, sorting, and degradation were all increased in the black tea and FOS groups.Notably, the level 3 KEGG pathways of propanoate metabolism (SCFA metabolism pathway) were more abundant in the black tea groups compared with in the BLK group, and pyruvate metabolism was more abundant in the FOS group compared with in the BLK group, which corresponds with the production levels of acetate and propionate.Pyruvate,propanoate, and butanoate metabolism are closely related to acetate,propionate, and butyrate metabolism.In addition, 10 pathways (level 2 KEGG pathways) differed between the black tea and FOS groups.Five differential pathways were more abundant in the black tea groups than in the FOS group.These elevated expression pathways were related mainly to metabolism (glycan biosynthesis and metabolism,metabolism of cofactors and vitamins, enzyme family, and lipid metabolism).The results of function prediction not only revealed that black tea and FOS treatments promote beneficial functions in intestinal microorganisms but also further confirmed the results regarding how these treatments affect SCFAs and intestinal flora.According to the above experimental results, it could be reasonably inferred that the possible mechanism of black tea improving gut health was shown in Fig.6.

Fig.6 Possible mechanism of black tea improving gut health.

Conditions forin vitrofermentation are similar to those for colonic fermentation.However,in vitrofermentation nonetheless has some limitations [51], a main one being uncontrolled pH.SCFAs formedin vivoare rapidly absorbed in the colon, whereas inin vitromodels, SCFAs are not absorbed, which can alter medium pH and lead to a reduction in pH; the pH of the media used in this study was uncontrolled throughout the experiment.Another limitation of this study was the short fermentation time.The present study was conducted only for 24 h and may not have been able to fully illustrate the long-term effects of black tea on the gut microbiota.Thus, we will conduct a long-term animal experiment in the future to further investigate the effects of KBT and DBT on the microbiota and metabolite production.Besides, thearubigins and tea polysaccharides are important ingredients of the nonabsorptive components of black tea used, and whether they play vital roles in promoting the gut health warrants further research.

5.Conclusion

In conclusion, the microbiota composition and SCFA concentrations after treatment with KBT or DBT differed compared with those after treatment with FOS (a well-established prebiotic).The present results demonstrate that KBT and DBT modulate the gut microbiota by stimulating the growth ofBacteroidesandRoseburiabut exert no effects onBifidobacterium.Therefore, KBT and DBT are potential novel prebiotics for improving gut health.

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by the Key Research and Development Program of Anhui Province (201904b11020038, 1804b06020367),a Key Joint Grant for Regional Innovation and Development from National Sciences Foundation of China (U19A2034), the National Natural Science Foundation (31972459), and an Earmarked fund for China Agriculture Research System (CARS-19).

Appendix A.Supplementary data

Supplementary data associated with this article can be found in the online version, at http://doi.org/10.1016/j.fshw.2021.12.022.