Optimization And Application Of In Vitro Prebiotic Evaluation Based On Batch Fermentation Models

Prebiotics,which typically refer to selectively fermented nondigestible food ingredients that specifically support the growth and/or activity of health-promoting bacteria colonized in the gastrointestinal tract,commonly assessed by specific stimulation effects on the bifidobacteria and lactobacilli.Recently,a revised prebiotic concept— “a nondigestible compound that,through its metabolization by microorganisms in the gut,modulates composition and/or activity of the gut microbiota,thus conferring a beneficial physiological effect on the host” has been defined.The effect of a prebiotic was commonly evaluated through in vitro models,animal models and clinical trials.Because in vitro batch fermentation model is economical and convenient to be established,it has been used most widely in preliminary prebiotics screening for subsequent animal and clinical trials among numerous food ingredients.However,the same prebiotics have been reported to exert different microbial modulatory effects in different in vitro studies.In the present work,we aim to reveal the resources underlying the discrepancies,such as trophic status of batch model,the use of different controls,and the microbial analysis methods,to optimize the method for in vitro prebiotic evaluation.Firstly,we observed the changes of microbial compositions and activities in the oligotrophic and eutrophic batch fermentation models,including 5 treatment groups: oligotrophic phosphate buffered saline medium(low-protein,low-bile salts)inoculated with 5%(P5),10%(P10),16%(P16)fecal microbiota;and eutrophic culture medium(high protein,high-bile salts)inoculated with 1%(B1)and 5%(B5)fecal slurry.We used initial 0-hour culture as the baseline control and the culture without prebiotics as the negative control.The microbial compositions were mapped by using denaturing gradient gel electrophoresis fingerprints and 454 pyrosequencing.Metabolic activities were detected through the measurement of pH,short-chain fatty acids(SCFAs)and bile salts.Moderate and consistent shifts of microbiota were shown among the three oligotrophic systems,but drastic and inconsistent microbial changes were demonstrated between the two eutrophic systems.In the eutrophic models,we found a bloom of proteolytic and bile resistant bacteria but decrease or even depletion of bile sensitive bacteria,a feature similar to human gut under a high protein diet.The pre-existing nutrients in the fermentation models thus have a significant impact on the microbiota.Use of a baseline control accounts for the way bacterial communities change over time in the culture,and use of a negative control allows determination of which changes are due to the prebiotics.Therefore,inclusion of negative controls is essential for evaluating the modulatory effects of prebiotics on microbiota in vitro.We suggest that P5 and B1 modesl should be used to demonstrate whether the trophic status influence the prebiotic evaluations.To further understand how the trophic status and methods for gut microbiota analysis may impact the evaluations,the P5 and B1 systems were established to evaluate modulatory effects of the one prebiotic formula on the same original fecal microbiota with 454 pyrosequencing.The prebiotic formula significantly modulated the overall microbiota in both systems,but with quite different patterns.There were only 8(7 positive and 1 negative responders)consistently modulated OTUs in both systems out of the 50 key prebiotic responders.In P5,the prebiotic stimulated the bacteria distributed across Faecalibacterium and Prevotella.In B1,the prebiotic stimulated the bacteria distributed across Faecalibacterium,Bacteroides,Lactobacillus and Bifidobacterium,and inhibited the bacteria distributed across Streptococcus,Escherichia/Shigella and Bilophila.Furthermore,the prebiotic formula modulated a large number of bacteria,which were predominant but less well characterized in the intestinal tract,for example,Oscillibacter,Dialister and Alistipes.We also observed that the prebiotic increased some OTUs but decreased others in the same genera in the same condition.Therefore,the overall modulatory effects of putative prebiotics based on the targeted microbial analysis methods should be re-analized by using new technology such as pyrosequencing on the species level.Based on these findings,we proposed that to reveal the modulatory effects of a prebiotic on the microbiota,using both oligotrophic and eutrophic models,comparing to both baseline and the negative controls,and analyzing the microbial changes down to the OTU level with next generation sequencing,should be the optimized method for in vitro prebiotic evaluation.Finally,two widely used prebiotics,fructooligosaccharide(FOS)and resistant starch,were re-evaluated by using this proposed method to reveal their impacts on the microbial structures and metabolic functions.Both prebiotics significantly shifted the overall microbiota,with similar inhibitory effects on the opportunistic pathogenic bacteria,but different preference on promoting the potentially beneficial bacteria.FOS stimulated bacteria belonging to Faecalibacterium in the oligotrophic system,whereas stimulated Blautia,Lactobacillus,un_Lachnospiraceae,Faecalibacterium,Megamonas and Bifidobacterium in the eutrophic system.Resistant starch mainly stimulated the bacteria of un_Lachnospiraceae in both systems.The inhibitory effects on the proteolytic specific iso-SCFAs were similar between the both prebiotics,but the SCFAs types promoted were different.FOS produced the consistently large acetate pool in both models,whereas a large butyrate pool depending on the oligotrophic environment.Fibosol-2 promoted the stablely large propionate and butyrate pool.The modulatory effects of the two prebiotics on the metabolic functions were evaluated based on the promoting effects of fermentation supernatants on the Caco-2 intestinal cells.Only a promotion was detected after fermentation with FOS in the oligotrophic system.Taken together,the two different prebiotics stimulated the predominant intestinal bacteria differentially,and the better modulatory effects on the beneficial bacteria and the larger stimulation of the metabolites that promoting growth of intestinal cells were displayed with FOS,whereas its beneficial effects was affected in fermentation trophic environments.Overall,we proposed an optimized method for in vitro prebiotic evaluation,and demonstrated its validity and sensitivity by evaluating the modulatory effects on microbial compositions and metabolic functions of two widely used prebiotics.This study may serve as a foundation for future standardization of the in vitro prebiotic evaluation models.