Guijie Chen, Ziqi Zeng, Minho Xie, Yuji Peng, Wngting Zhou, Weiqi Xu,Yi Sun, Xioxiong Zeng,*, Zhonghu Liu*

a College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

b Collaborative Innovation Center for Modern Grain Circulation and Safety, College of Food Science and Engineering,Nanjing University of Finance and Economics, Nanjing 210023, China

c Key Laboratory of Ministry of Education for Tea Science, Hunan Agricultural University, Changsha 410128, China

d National Research Center of Engineering Technology for Utilization of Botanical Functional Ingredients, Changsha 410128, China

Keywords:

Fuzhuan brick tea

Polysaccharide

Fermentation characteristics

Gut microbiota

A B S T R A C T

Polysaccharides from Fuzhuan brick tea (FBTPS), one of most important bioactive components in tea, showed various health-promoting functions.Our previous work demonstrated that the crude FBTPS (CFBTPS) could modulate the gut microbiota.However, which purified fraction in CFBTPS contributing to the modulation of gut microbiota remains unclear.Thus, the fermentation characteristics and probiotic activity of a purified fraction (FBTPS-2-1) of CFBTPS were evaluated in this work.The results showed that gut microbiota could utilize FBTPS-2-1 to produce short-chain fatty acids including acetic, propionic, n-butyric and n-valeric acids.FBTPS-2-1 could modulate the structure and metabolic pathways of gut microbiota.FBTPS-2-1 could increase the health-promoting gut microbiota such as Prevotellaceae and Bifidobacteriaceae, and decreased the harmful bacteria such as Enterobacteriaceae and Fusobacteriaceae.The results of metagenomics showed that Prevotella copri and Megamonas funiformis were the dominant bacteria after fermentation of FBTPS-2-1.Furthermore, FBTPS-2-1 could regulate the biosynthesis and metabolism pathways of gut microbiota.Thus,the enrichment of food with FBTPS-2-1 is expected as a potential strategy for promoting human health due to modulation of gut microbiota.

1.Introduction

There are approximately 100 trillion microorganisms existing in the human gastrointestinal tract, and most of them are bacteria,which can encode over three million genes and produce thousands of metabolites [1].The microbiome has breadth of functions, such as promotion of immune maturation, hydrolysis of indigestible dietary fibers, modulation of metabolic homeostasis, biosynthesis of vitamins,defense of pathogen and metabolism of xenobiotic [2].Given the diverse functional repertoire of gut microbiota, thus, it is not surprising that gut microbiota plays a critical role in the human health [3].As a result, gut microbiota has been regarded as a virtual organ of the body.However, the dysbiosis of gut microbiota may also lead to a broad range of chronic diseases, such as inflammatory bowel disease(IBD), brain disorders, cardiovascular diseases, obesity, nonalcoholic fatty liver disease, type 2 diabetes mellitus and cancer [4-8].Thus, it is important to improve gut microbiota for human health.

The composition and function of gut microbiota are influenced by many factors, including genetics of the host, lifestyle, medication,dietary patterns and so on [9,10].Thereinto, dietary intervention is a promising and effective way to modulate gut microbiota [11,12].Furthermore, increasing evidences have showed that gut microbiota could be regarded as an important modulator of the crosstalk between diet and host health [13,14].In recent decades, a potentially promising strategy, based on modulation of gut microbiota by dietary intervention, has been presented to improve the human health.Especially, dietary fiber, as important resource of prebiotics, shows a superior modulated effect on the gut microbiota [15].Moreover,dietary fibers with different sources or structures have different probiotic activities on gut microbiota [16,17].In recent years, some dietary fibers, such as inulin, have been widely used as probiotics in pharmaceutical and food industries [18].However, not all the polysaccharides could positively modulate the gut microbiota.For example, carboxymethylcellulose, one commonly used emulsifier,could impact the mice gut microbiota promoting colitis and metabolic syndrome [19,20].Furthermore, a recent work showed that pectin could ameliorate the colitis, while inulin exacerbated the severity of the disease, which is related to the different modulated effects on the gut microbiota [21].Thus, development of microbiota-directed dietary fibers, which can selectively stimulate the proliferations of beneficial gut microbes and inhibit the harmful pathogen, is still highly needed for improve human health.

Fuzhuan brick tea (FBT), a traditional Chinese dark tea mainly produced in Hunan province of China, is made from the coarse leaves and branches ofCamellia sinensiswith the help of fungal fermentation [22-24].It is widely consumed as a popular beverage by people in the frontier regions of China as an ethno-botanical remedy [25,26].In recent decades, a growing amount of evidence suggests that FBT has various health-promoting functions, such as antioxidant [27],anti-inflammatory [28], hepatoprotective [29]and anti-obesity [22]activities.Thus, FBT has been gradually accepted by other tea lovers due to its uniquely flavor and potential beneficial bioactivities.Recently, it has been reported that the crude polysaccharides from FBT (CFBTPS), one of the important bioactive components in FBT,showed probiotic effect on gut microbiotain vitroandin vivo[23,24].The CFBTPS were further separated by chromatography of DEAE Sepharose Fast Flow to afford two main purified fractions (FBTPS-2 and FBTPS-3).Thereinto, FBTPS-3 also showed modulated effects on the composition and metabolism of gut microbiota [30].Moreover,FBTPS-2 was further purified by a column of Sephadex G-50 to afford homogeneous fraction of FBTPS-2-1, and its structural characterization and immunostimulatory activity were evaluated in our previous work [31].FBTPS-2-1 is a typical heteropolysaccharide with the molecular weight of 748 kDa and monosaccharide composition of galactose (Gal), arachidonic acid (Ara), glucose (Glc), mannose(Man), rhamnose (Rha), galacturonic acid (GalA) and glucuronic acid (GlcA) in molar ratio of 46.59:22.13:13.57:8.20:6.02:2.12:1.38,and its hypothetical structure of is shown in Fig.S1.However, the fermentation characteristics and probiotic activity of FBTPS-2-1 is still unknown.Thus, the fermentation characteristics of FBTPS-2-1 were investigated using an anaerobic fermentation modelin vitro.The probiotic activity of FBTPS-2-1 was evaluated by sequencing of the bacterial 16S rDNA V4 region and metagenomics.It is expected that this work could further enhance our understanding of potential probiotic activity of FBTPS-2-1.

2.Materials and methods

2.1 Preparation of FBTPS-2-1

The FBT samples were obtained from Hunan Yiyang Tea Processing Factory Co., Ltd.(Yiyang, China).The preparation of FBTPS-2-1 was carried out according to our previous work [31].Briefly, the dried FBT samples were treated with ethanol solution(85%,V/V) to remove the polyphenols and pigments.Then, CFBTPS was obtained through water extraction (1:10,m/V) at 70 °C,precipitation by adding triple volumes of ethanol, removal of protein by Sevag method, and dialysis using dialysis bag (8-14 kDa).The CFBTPS was separated and purified by a DEAE fast flow column and a Sephadex G-50 column to afford the purified fraction FBTPS-2-1.

2.2 Fermentation in vitro

The fermentation characteristics and probiotic activity of FBTPS-2-1 was evaluated by a fermentation model with gut microbiota according to our previous work [23].Brie fly, the samples of gut microbiota were kindly provided by 4 healthy volunteers who had no gastrointestinal diseases and did not take probiotics, prebiotics or antibiotics.The fecal samples were immediately mixed with 9-fold of autoclaved modified physiological saline solution (NaCl 8.5 g/L, cysteine-HCl 0.5 g/L), and the 10% (m/V) of fecal slurry was obtained after centrifugation at 500 g for 5 min at 4 °C.The solution for fermentation experiment was composed of 1.0 mL fecal slurry, 100 mg of FBTPS-2-1, and 9.0 mL of basal nutrient medium(containing 2.0 g/L peptone, 4.0 g/L yeast extract, 0.1 g/L NaCl,0.04 g/L K2HPO4, 0.04 g/L KH2PO4, 0.01 g/L MgSO4, 0.01 g/L CaCl2,2.0 g/L NaHCO3, 0.02 g/L hemin, 0.46 g/L cysteine-HCl, 0.50 g/L bile salt, 1.0 g/L resazurin, 2.0 mL/L Tween 80 and 10 mL/L vitamin K1).Then the fermentation experiment was carried out in an Anaero Pack System (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan).Under the same conditions, the BLK (without any carbon source), INL (with inulin as carbon source) and GLU (with glucose as carbon source)were designed as the control groups.The gut microbiota before fermentation was named as the ORI group.The fermentation samples of FBTPS-2-1 were collected at 6, 12 and 24 h for evaluation of fermentation characteristics.After fermentation for 24 h, all samples were collected and stored at –80 °C for further analysis.

2.3 Evaluation of fermentation characteristics

The contents of reducing sugar and residual carbohydrates during the fermentation were evaluated by dinitrosalicylic acid (DNS) and phenol-sulphuric acid methods, respectively.The changes of molecular weight during the fermentation were measured by HPLC instrument(Shimadzu Corp., Tokyo, Japan) equipped with a TSK G4000PWXL column (7.8 mm × 300 mm, Tosoh Crop., Tokyo, Japan) and an evaporative light-scattering detector (ELSD) as described in the previous work [32].After centrifugation and filtered through a 0.45 mm membrane, 20 mL fermentation solution was loaded onto column of HPLC.The deionized water was used as mobile phase,the flow rate was set at 0.5 mL/min, and the temperature of column was 35 °C.The pH value of solution was measured by a pH meter(Mettler-Toledo instruments Ltd., Shanghai, China).

2.4 Determinations of short-chain fatty acids (SCFAs) and lactic acid

After fermentation, the levels of SCFAs in fermentation solution, including acetic, propionic,n-butyric, isobutyric,nvaleric and isovaleric acids, were measured using a GC (7890A,Agilent) equipped with a flame ionization detector (FID) and an HPINNOWAX column (30 m × 0.25 mm × 0.25 mm, Agilent)according to the previous work [7].2-Ethylbutyric acid was dissolved in 0.2 mol/L HCl solution to afford internal standard solution(0.3 mg/mL).After centrifugation, the fermentation solution was mixed with same volume of internal standard solution.After filtered through a 0.45 mm membrane, 1 mL sample was loaded onto GC.The initial oven temperature of GC was 100 °C for 1 min, then gradually increased to 180 °C at a speed of 5 °C/min, and further maintained at 180 °C for 4 min.The concentration of lactic acid in fermentation solution was determined by commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.5 Gut microbiota analysis

After centrifugation, the precipitate of fermentation samples was used to analyze the structure of gut microbiota.Brie fly, the DNA of gut microbiota in fermentation samples was obtained by QiAamp DNA stool Mini Kit (NO.51504, Qiagen, Germany).Then, the DNA was sent to DeepBiome Co., Ltd.(Jinan, China) for sequencing of the bacterial 16S rDNA V3-V4 region by Illumina MiSeq platform.The raw data were quality- filtered using Trimmomatic (version 0.36)and merged using USEARCH fastq_mergepairs command (version 11.2.64), and further clustered into operational taxonomic units(OTUs) using USEARCH with 97% similarity cutoff.The principal component analysis (PCA), principal co-ordinates analysis (PCoA)and hierarchical cluster analysis according to the abundances of OTUs were analyzed by Vegan package in R (V3.5.2).Based on the results of sequencing of the bacterial 16S rDNA V4 region, the gut microbiota of the FBTPS-2-1 and GLU groups were further analyzed by metagenomics.In brief, the quality of the raw reads extracted from the results of sequencing was evaluated by FastQC (Version 0.11.9).Trimmomatic (Version 0.38) was used to trim the adaptor sequences in the raw reads.The taxonomy profiling was carried out according to Braken (version 2.0.8b), Kaiju (version 1.6.3) and Kracken (version 2.5.0).The assembly was carried out using metaSPAdes (version 3.13.2) and MEGAHIT (version 1.2.9).The gene prediction of the assembled metagenomic contigs was analyzed using MetaProdigal(version 2.6.3).

2.6 Statistical analysis

All values are presented as means ± standard deviation (SD).The statistical analysis was carried out using one-way (ANOVA)procedure followed by Tukey test by SPSS 22 software (IBM).Comparison of microbiota and KEGG categories among different groups was analyzed by linear discriminant analysis (LDA) effect size(LEfSe, http://huttenhower.sph.harvard.edu/galaxy/).

3.Results and discussion

3.1 Fermentation characteristics of FBTPS-2-1

Our previous work has shown that the CFBTPS could reach the large intestine without being hydrolyzed in the simulated saliva,gastric and small intestinal conditions, but they could be broken down in large intestine by gut microbiota [23].Thus, in the present work, the fermentation characteristics of FBTPS-2-1 were further investigated.The distribution of molecular weight during the fermentation is shown in Fig.1.It was obvious that the response of FBTPS-2-1 in HPLC gradually decreased, and some fragments with low molecular weight were observed after fermentation.Furthermore,both the residual carbohydrates and reducing sugars were significantly decreased during the fermentation process (Table S1).Thus, the results showed that FBTPS-2-1 could be utilized by gut microbiota.After fermentation of 24 h, the content of residual carbohydrates was 46.6%, suggesting that more than 50% of FBTPS-2-1 could be metabolized into non-carbohydrate products (Table S1).In fact, a lot of works have showed that the polysaccharides remaining intact without digestion by digestive enzymes in the upper gut could be metabolized by the gut microbiota in the cecum and colon [33].For example, theβ-glucans from yeast and polysaccharides from the seeds ofPlantago asiaticaL.could be broken down by gut microbiota [34,35], which is consistent with result in this work.

Fig.1 HPGPC chromatograms of FBTPS-2-1 during the fermentation by gut microbiota.

3.2 pH and SCFAs

After fermentation, the polysaccharide could be used to produce a serious of metabolites mainly including SCFAs by gut microbiota [36,37],which will decrease the pH value of fermentation solution.Thus, the pH values of different groups during the fermentation were compared(Fig.S2).The results showed that fermentation without carbon source (BLK group) could increase the pH compared with the ORI group, whereas, the additions of glucose, inulin and FBTPS-2-1 all decreased the pH value, which might be due to the production of SCFAs [32,34,38].Thus, the concentrations of SCFAs, including acetic, propionic,n-butyric, isobutyric, nvaleric, and isovaleric acids,and lactic acid were measured to find the evidence of utilization of FBTPS-2-1 by gut microbiota.As shown in Table 1, the SCFAs in fermentation solution were mainly acetic and propionic acids, which were significantly enhanced due to the addition of carbon source compared with those in the BLK group.Specially, FBTPS-2-1 group showed the highest levels of both acetic and propionic acids.Furthermore, FBTPS-2-1 exhibited considerable modulation on the level ofn-butyric andn-valeric acids.As a result, the content of total acids for the FBTPS-2-1 group ((42.41 ± 1.71) mmol/L) was much higher than that for the BLK ((20.51 ± 3.58) mmol/L), GLU((34.64 ± 4.59) mmol/L) or INL ((32.39 ± 1.61) mmol/L) group.SCFAs, as the main metabolites produced by bacterial fermentation of polysaccharides in the cecum and colon, play a key role in intestinal homeostasis, human health and disease [39].In recent decades, it has been widely reported that the polysaccharide-derived SCFAs might affect the psychological function [38], IBD [2], control the body weight and insulin sensitivity [40], etc.In this work, FBTPS-2-1 could promote the productions of acetic, propionic,n-butyric andn-valeric acids compared with the other groups, which may thereby contribute to prevent disease and improve host health.

Table 1The concentrations of SCFAs (mmol/L) in fermentation solutions.

3.3 Overall structural changes of gut microbiota

Evidence is accumulating that the gut microbiome is involved in the human health, such as protecting against overgrowth of pathogen,providing a source of energy biogenesis, regulating bone density,neurologic signaling and intestinal endocrine functions, maturation and continued education of the host immune response, biosynthesizing neurotransmitters and vitamins, whereas, the dysbiosis of gut microbiota (imbalances in the composition and function of gut microbiota) could lead to various diseases, including behaviour and brain disorders, metabolic diseases, cardiovascular disease, IBD,chronic kidney diseases, cancers etc.[3].Dietary nutrient is not only essential for human health, but also provides energies for the survival of the trillions of gut microbiota, thus, diet play a critical role in modulating the structure and function of gut microbiota [13].Recently, a potential strategy, based on modulation of gut microbiota by diet polysaccharides, was proposed to improve human health and prevent diseases.Thus, the probiotic activity of FBTPS-2-1 was evaluated using the high throughput sequencing analysis.As shown in Figs.2a–2c, theβ-diversity analysis including PCA, PCoA and hierarchical cluster analysis at the OTUs level was used to evaluate the effects of different samples on the structure and composition of gut microbiota.Similar to the previous findings [41,42],it was obvious that the structure of gut microbiota exhibited significantly different responses to different carbon sources during the fermentation.The FBTPS-2-1 group showed a clear separation with other groups, suggesting that FBTPS-2-1 showed a different probiotic activity compared with glucose and inulin.

Fig.2 The β-diversity and composition of gut microbiota at phylum level.(a) PCA (b) PCoA, (c) hierarchical cluster analysis of gut microbiota at the OTU level,(d) the bacterial taxonomic profiling at phylum level, the relative abundances of (e) Firmicutes and (f) Bacteroidetes, (g) the ratio of Firmicutes/Bacteroidetes, the relative abundances of (h) Proteobacteria and (i) Actinobacteria.The different letters mean significance differences between different groups (P ＜ 0.05) analyzed using One-way ANOVA procedure followed by Tukey test.

Fig.2 (Continued)

As shown in Fig.2d, the compositions of gut microbiota at the phylum level in all groups were mainly composed of Firmicutes, Bacteroidetes,Proteobacteria and Actinobacteria, which is similar with other report [43].Differently, GLU could significantly increase the relative abundance of Firmicutes and decrease the relative abundance of Bacteroidetes, which led to the higher level of Firmicutes/Bacteroidetes compared with other groups.An increasing number of findings showed that high level of Firmicutes/Bacteroidetes might increase the energy harvest, which may contribute to the metabolic diseases [44,45].Thus, the maintenance of Firmicutes/Bacteroidetes ratio by inulin and FBTPS-2-1 might be beneficial to metabolic health.The relative abundance of Proteobacteria in the BLK and INL groups was significantly higher than that in the ORI, GLU or FBTPS-2-1 groups.Proteobacteria,considered as microbial signature of dysbiosis of gut microbiota,shows positive correction with many diseases [34].It is noted that the FBTPS-2-1 and GLU groups showed higher level of Actinobacteria compared with the ORI, BLK and INL groups.It is well known that Actinobacteria, containing various health-promoting gut microbiota such as Bifidobacteria, is considered as probiotics.Thus, FBTPS-2-1 could promote the growth of health-promoting probiotics and inhibit the proliferation of detrimental gut microbiota, which may contribute to improvement of human health.

The effects of FTPS-2-1 on gut microbiota at the family level were analyzed, and the results are shown in Fig.3.Compared with the ORI group, the BLK group showed higher levels of Enterobacteriaceae,Veillonellaceae, Bacteroidaceae, Acidaminococcaceae, Fusobacteriaceae,Porphyromonadaceae, Clostridiaceae_1 and lower levels of Prevotellaceae, Lachnospiraceae, Ruminococcaceae after fermentation.In contrast, the addition of carbon source could prevent these changes.Both INL and FBTPS-2-1 could increase the relative abundance of Prevotellaceae.GLU, INL and FBTPS-2-1 could decrease the relative abundances of Enterobacteriaceae, Fusobacteriaceae,Porphyromonadaceae, and Clostridiaceae_1.Both GLU and FBTPS-2-1 could reduce the relative abundance of Bacteroidaceae.Besides, FBTPS-2-1 could reduce the relative abundance of Acidaminococcaceae.More importantly, GLU and FBTPS-2-1 could significantly increase the relative abundance of Bifidobacteriaceae.Thus, FBTPS-2-1 showed superior modulation on the gut microbiota at the family level.The heatmap of gut microbiota composition at genus level is shown in Fig.S3, and the comparative analysis was also carried out.The results showed thatPrevotellain the ORI group was the most dominated microbiota (Table 2), but it was significantly decreased after fermentation, whereasMegamonasdramatically increased after fermentation in groups with addition of carbon sources.Furthermore, it is worth noting that FBTPS-2-1 could significantly increase the relative abundances ofBifidobacteriumandCollinsella,and decrease the relative abundance ofEscherichia/Shigella.The comparison of microbiota among different groups was further evaluated by LEfSe.Compared with the ORI group, FBTPS-2-1 could significantly increase some beneficial bacteria such asBifidobacterium, and reduce the harmful microbiota such as Clostridiales and Erysipelotrichales after fermentation (Fig.4a).Compared with the BLK, INL and GLU groups, FBTPS-2-1 could significantly enhance the relative abundances ofPrevotella copriandCollinsella aerofaciens.The different OTUs of gut microbiota in the same species may show different responses to addition of different carbon source, thus, it is necessary to evaluate the difference at the OTUs level.As shown in Fig.5, it was observed that 10 OTUs were significantly different (P＜ 0.05), and 2, 2, 2 and 4 OTUs in the INL,GLU, FBTPS-2-1 and BLK groups were higher than other three groups according to the LDA scores, respectively.In the BLK group,the proliferation of Enterobacteriaceae (OTU5, OTU3 and OTU85)was observed, which was significantly reduced in other groups.P.copri(OTU2) andC.aerofaciens(OTU6) could be promoted by the addition of FBTPS-2-1.Prevotellastrains are usually related to plant-rich or dietary fiber-rich diets [46], which are more prevalent in populations with non-Westernized lifestyles [47].It has been reported that a lot of polysaccharides could stimulate the growth ofPrevotellastrains [48,49].On the other hand, thePrevotellastrain could encodes specific polysaccharide utilization loci (PULs) enabling the utilization of polysaccharides [50,51].Recently,Prevotellastrains showed correlation with host health, such as improvement of glucose homeostasis and protection against food allergy [52,53], which is expected as a potential eubiotic versus dysbiotic biomarker of diet [54].C.aerofaciens, a butyric acid-producingCollinsellabacterium, could fermentD-lactose,D-fructose andD-maltose to biosynthesize butyric acid [55], which may contribute to the high level ofn-butyric acid in the FBTPS-2-1 group.

Table 2The comparative analysis of gut microbiota at the genus level.

Fig.3 Bacterial taxonomic profiling of gut microbiota at the family level, including relative abundances of (a) Enterobacteriaceae, (b) Prevotellaceae,(c) Veillonellaceae, (d) Lachnospiraceae, (e) Bacteroidaceae, (f) Acidaminococcaceae, (g) Coriobacteriaceae, (h) Ruminococcaceae, (i) Bifidobacteriaceae,(j) Fusobacteriaceae, (k) Porphyromonadaceae, and (l) Clostridiaceae_1.The different letters mean significance differences between different groups (P ＜ 0.05)analyzed using One-way ANOVA procedure followed by Tukey test.

Fig.4 Comparison of microbiota (a) between the ORI and FBTPS-2-1 groups, and (b) among Blank, FBTPS-2-1, GLU and INL groups evaluated by LEfSe analysis.

Fig.4 (Continued)

Fig.5 Comparison of gut microbiota among Blank, FBTPS-2-1, GLU and INL groups at OTUs level using LEfSe analysis.(a) Histogram of LDA scores with more than 4 in different groups, (b) Heatmaps showing significant different gut bacteria (log 2 transformation) based on result of LEfSe, and (c) the information of gut microbiota including phylum, family, genus and species.

As shown in Fig.5b, both GLU and FBTPS-2-1 could increase the relative abundance ofBifidobacterium(OTU8).However,the species of OTU8 could not be identified by sequencing of the bacterial 16S rDNA V3-V4 region.Thus, the metagenomics was used to further evaluate the gut microbiota in the FBTPS-2-1 and GLU groups.As shown in Fig.S5, the structures of gut microbiota in the GLU and FBTPS-2-1 groups were significantly different in results of PCoA and hierarchical cluster analysis based on the data of Metagenomics.As shown in Figs.6a–6c, the composition of gut microbiota at the phylum, family and genus levels are similar with the results of sequencing of the bacterial 16S rDNA V3-V4 region.In Fig.6d, it was found thatM.funiformiswas dominant bacterium in the GLU group with relative abundance of more than 50%.Whereas,P.copriandM.funiformisbecame the main gut microbiota with more than 70%, suggesting thatP.copriandM.funiformismaybe key gut microbiota for hydrolysis and utilization of FBTPS-2-1.Furthermore,two species in the genus ofBifidobacteriumincludingB.longumandB.adolescentiswere observed in both the GLU and FBTPS-2-1 groups.B.longumandB.adolescentishave showed potential healthpromoting functions, which may be used as potential probiotics [56-59].The significant difference of KEGG categories between the GLU and FBTPS-2-1 groups was analyzed by LEfSe (Fig.S6), it was found that FBTPS-2-1 could modulate the metabolic pathways of gut microbiota, mainly promoting the biosynthesis of secondary metabolites (map01110), amino acids (map01230), polyketide sugar unit (map00523), peptidoglycan (map00550), and metabolisms of pyrimidine (map00240), purine (map00230), amino sugar and nucleotide sugar (map00520), alanine, aspartate and glutamate(map00250), riboflavin (map00740), sphingolipid (map00600),thiamine (map00730), glycerophospholipid (map00564).It has been reported that the modulation of metabolic pathways of gut microbiota,such as amino acid metabolism, may contribute to the host health [60].Thus, FBTPS-2-1 could potentially improve the host health by modulating the metabolic pathways of gut microbiota.However, the potential molecular mechanisms of FBTPS-2-1 regulating the biosynthesis and metabolism pathways of gut microbiota is still unknown in this work.

Fig.6 The bacterial taxonomic profiling at (a) phylum, (b) family, (c) genus and (d) species level obtained from metagenomics.

Recently, the microbiota-directed food intervention for prevention of diseases and improvement of human health has been presented [61].Furthermore, a new clinical trial showed a microbiota-directed complementary food prototype could be served as a dietary supplement for young children with moderate acute malnutrition,which targeted manipulation of microbiota components linked to growth including bone growth and neurodevelopment [62].Based on the above encouraging findings in this work, FBTPS-2-1 is expected to be a microbiota-directed food to improve our health targeting promotion of healthy changes of gut microbiota.However, the results of this work were obtained fromin vitroexperiments, which restricted its further application.Thus, the animal experiments and clinical trials are still highly needed, which will be our next work.

4.Conclusion

In this work, the fermentation characteristics and probiotic activity of FBTPS-2-1 were evaluated.The results showed that FBTPS-2-1 could be broken down and metabolized into SCFAs by gut microbiota.FBTPS-2-1 could significantly stimulate the productions of acetic, propionic,n-butyric andn-valeric acids.On the other hand,FBTPS-2-1 could modulate the gut microbiota.At the phylum level,FBTPS-2-1 could maintain the ratio of Firmicutes/Bacteroidetes,decrease the relative abundance of Proteobacteria and increase the relative abundance of Actinobacteria.At family level, FBTPS-2-1 decreased the relative abundances of Enterobacteriaceae,Fusobacteriaceae, Porphyromonadaceae, Bacteroidaceae,Acidaminococcaceae and Clostridiaceae_1 and increased the relative abundances of Prevotellaceae and Bifidobacteriaceae.At species level,P.copriandM.funiformismight be key gut microbiota with more than 70% for hydrolysis and utilization of FBTPS-2-1.Two species in the genus ofBifidobacteriumincludingB.longumandB.adolescentiswere also enriched by FBTPS-2-1.Furthermore, FBTPS-2-1 could modulate the biosynthesis and metabolism pathways of gut microbiota.Thus,FBTPS-2-1 is expected to be a potential functional food for promoting healthy changes in the human gut microbiota.

Declaration of competing interest

The authors declare no competing financial interests.

Acknowledgments

The study was supported by the National Natural Science Foundation of China (No.32001645 and No.31972025), the National Key Research and Development Program of China(2018YFC1604404), and the Fundamental Research Funds for the Central Universities (KJQN202154).

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.2021.12.030.