Effects Of Daidzein On The Gastrointestinal Microbial Community Of Piglets And Isolation And Study Characterisation Of Equol-producing Bacteria

Daidzein is one of the major metabolites from isoflavones,and it can be further metabolized into equol with more estrogenic activity by intestinal microorganisms. However,little information is available on the daidzein effect on the intestinal microflora of animals,and only a few intestinal bacteria have so far been identified to metabolize daidzein.In the present work,a PCR/DGGE approach was used to investigate the effect of daidzein on the gastrointestinal bacterial community of piglets.For the first time this study investigated the possibility of conversion of daidzein to equol by pig fecal microflora. Three selective media used for enrichment of three large groups of bacteria were then compared for equol production and subsequently for the isolation of equol-producing bacteria from pig faecal materials.This dissertation was described in the following five sections.1 Effect of daidzein on the intestinal microflora of pigletsSix litters of piglets were randomly allocated into two groups:the control and the treatment.Each group had three litters.The piglets of the treatment group were fed with daidzein orally on Day 7,9 and 11.They were all weaned on Day 21.One piglet of each group was slaughtered on Day 14,21,24 and 35,respectively.The digesta of duodenum, jejunum,ileum,caecum,colon and rectum were collected and used for DNA extraction and followed by PCR/DGGE analysis.The diversity of intestinal microbial populations of the piglets was determined based on the shannon's index.The 16S rRNA sequences of intestinal bacterial species were analysed.Piglets of the treatment group showed a higher bacterial diversity and more DGGE bands compared with that of the control on Days 24,35. Three DGGE bands existing in the control disappeared in the treatment group.The three clones corresponding to the three bands had their sequences most closely related to species of Clostridium thermocellum,Lactobacillus pontis and Streptococcus sp.,respectively.The other three DGGE bands were present only in the large intestine of piglets in the treatment group,and their corresponding clones had their 16S rRNA gene sequences most closely related to species of Butyrate-producing bacterium SL7/1,Clostridium butyricum, Ruminococcus obeum,respectively.The results suggested that daidzein could increase the diversity of the piglets intestinal bacterial community,and stimulate some specific bacteria to grow.2 In vitro fermentation reveals the conversion of daidzein to equol by fecal microflora of Erhualian pigsThe possibility of conversation of daidzein to equol by fecal microflora of Erhualian pigs was investigated by in vitro culture with fecal sample as inoculum.Faecal samples of three litters(A,B,C) of Erhualian pigs were collected.Each litter includes the sow and three piglets.Faecal samples were used to inoculate culture bottles with and without daidzein.After 48 h incubation,the cultures were taken to measure equol and daidzein concentrations by HPLC,and to analyze the microflora by PCR/DGGE.Results showed that microflora of eight pigs from the total 12 Erhualian pigs could transform daidzein into equol.For litter A,microflora of two piglets could metabolize daidzein to produce equol. For litter B,microflora of sow and one piglet could metabolize daidzein to produce equol. For litter C,microflora of sow and all the piglets could metabolize daidzein to produce equol.DGGE profiles revealed that in each litter,the pigs that could transform daidzein to equol showed higher similarities,while they had lower similarities to those that did not produce equol.The results showed that faecal microflora of Erhualian pigs had the ability to transform daidzein to equol.3 Medium selection for the isolation of equol-produciug bacterial strains from cultures of pig faecesThree selective media were compared for equol production using faeces from the Erhualian piglets which could transform daidzein into equol during in vitro fermentation as inoculum.Subsequently,three single bacterial strains capable of transforming daidzein into equol were isolated by using the selected medium.The bacterial strains were then identified using morphorlogical and biochemical parameters and by 16S rRNA gene sequence analysis.Results showed that among the three selective media,equol was detected only in the medium M1,which was selective medium for butyrate-producing bacteria,suggesting that butyrate-producing bacteria may transform daidzein to equol.Three single bacterial strains(D1,D2,D3) capable of transforming daidzein into equol in their pure cultures were then isolated using medium M1.At the genus level,D1 and D2 were both identified as Eubacterium sp.,while D3 as Enterococcus sp.based on morphological and physiological characteristics.The 16S rRNA gene sequence analysis showed that strains D1 and D2 were most closely related to previous daidzein-metabolisming bacteria isolated from humen and ruman faecal sample,respectively.Strain D3 was most closely related to Enterococcus faecium with sequence similarity as 97%.Strain D1 was further investigated in its characteristics of growth and equol production in vitro fermentation system.The results showed that the basal media supplemented with glucose,maltose,FOS,inulin,could stimulate the growth of strain D1,and media supplemented with FOS and inulin better supported it to grow.FOS and inulin could stimulate equol production.Media supplemented with acetate,butyrate and lactate failed to stimulate the growth of strain D1. But media supplemented with acetate and butyrate could stimulate equol production, whereas it was found that adding lactate was inhibitory for equol production.The results demonstrated that the present approach using a simple medium enabled to isolate bacterial strains capable of transforming daidzein into equol.The stains D1,D2 and D3 isolated could directly transforme daidzein into equol and oligosaccharide supplemented into the basal medium could stimulate the productiong of equol.4 The variation of faecal microorganisms of Erhualian pigs after being subcultured forty times in vitroMicroflora community change in the Erhualian pig facece and its equol production during subcultures was investigated.The results showed that the faecal bacteria were still able to transform daidzein into equol after being subcultured forty times.PCR/DGGE analysis showed that the number of DGGE bands decreased with the culture times increasing.Some DGGE bands in the culture disappeared,their 16S rRNA sequences most close to species of Megasphaera elsdenii strain YJ-4(91%),Megasphaera elsdenii strain 5T(99%),Clostridium sp.cTPY-17(87%),Succinivibrio dextrinosolvens(96%), Bacteroides sp.MANG(94%),respectively.Other bands enriched after 40 subcultures.The dominant bands remained in the culture after 40 subculturing had their 16S rRNA sequences most close to species of Coporococcus comes strain ATCC 27758(92%), Clostridium bacterium JN18_V41_S(93%),Eubacterium callanderi(99%), Bifidobacterium pseudolongum subsp,globosum strain 7#-3-7(94%),respectively.The results indicated that though some bacterial strains could not directly transform daidzein into equol,they play an important role during the time of daidzein being metabolized into equol.