Microbial Mechanism Of Resistant Starch Degradation In The Hindgut Of Piglets

Diarrhoea in piglets is a big promblem in livestock production, since it can cause serious economic losses. Weaning and changes of environment cause microbiota disorder in the gut of piglets. As antibiotics gradually fade into history, researchers hold the hope that they can modify the flora in intestinal tract by dietary component. Rsistant starch (RS) is defined as the sum of starch and products of starch degradation which are not absorbed in the small intestine of healthy individuals. Studies in vitro or in animals and human subjects suggest that RS increase short-chain fatty acids (SCFA) production in hindgut. Production of SCFA, mainly of butyate, has been deemed a favorable trait of colon-targeted functional foods due to their beneficial physiological effects on the growth and differentitation of colonic epithelial cells. The microbiota involved in degradation of RS and production of butyrate is complex, and the process is complicated. So far, the mechanism is unclear and the evidence from studies is equivocal. Is there difference between different types of RS? How metabolic intermediate affect the production of butyrate? What bacterial are able to degradate starch? To understand such questions, in vitro fermentation, Hungate tube technique, DGGE and Real-time PCR were employed to study the process of starch degradation by pig large intestinal bacteria. This thesis includes four sections.In the first section, to compare the fermentation character of amylopectin and resistant starch by hindgut microbiota of piglets, two type of resistant starch (RS2and RS4) and amylopectin were used as substances, colonic and cecal digesta of piglets as inocula, in an in vitro fermentation test. The difference in the fermentation character of RS2between colonic inoculum and cecal inoculum was also conducted. Results showed that as compared with resistant starch, amylopectin could be degraded easily by colonic microbiota of piglets, cumulative gas production and concentrations of volatile fatty acid were significantly higher, pH value was significantly lower (P<0.05). Similar changes in the concentration of lactic acid were observed when resistant starch RS2and amylopectin were used as substances. The concentration increased significantly in6h after inoculation, then decreased gradually. Results also showed that resistant starch RS4could not be degraded by microbiota in the hindgut of piglets. By comparing the difference in the fermentation character of RS2between colonic and cecal inocula, lower producing rate of lactic acid was observed when cecal digesta was used as inoculum. However, there was no significant difference in the concentration of volatile fatty acid.The second section includes two parts. Part1:To investigate the difference between the fermentation of amylopectin and resistant starch by colonic and cecal digesta of piglets,16S rRNA gene based denaturing gradient gel electrophoresis (DGGE) and sequencing techniques and real-time PCR were used for microbial analysis. Results showed that the number of bands in DGGE profiles from amylopectin group was higher than that in RS group. Samples at6h time point in RS group and all samples in amylopectin group gathered in the same cluster with the similarity higher than80%. DGGE profiles from RS fermentation samples by colonic and cecal inocula shared most of dominant bands. One of the most dominant bands, band A was identified to related to Subdoligranulum variablie (99%). Bands B and C appared in profiles from samples after6h time point belonged to Blautia producta (99%). Real-time PCR analysis showed that the numbers of16S rRNA gene copys of total bacteria and Bacteroides in amylopectin fermentation samples were significantly higher than in RS samples (P<0.01). The numbers of Bifidobacterium increased gradully during the fermentation in both groups. Part2:To investigate the diversity of starch-degrading bacteria in the intestinal tract of piglets, Hungate tube technique and plate streaking in anaerobic workstation were used to isolate these strains. DNA of these strains were extracted and used for sequencing their16S rRNA gene. More than forty strains were isolated, of all these strains, thirteen strains were identified as follow: strain1, Clostridium perfringens, strain2, Escherichia coli, strain4, Streptococcus suis, strain6, Anaerovibrio lipolyticus, strain8, Bacillus licheniformis, strain10, Streptococcus caballi, strain12, Streptococcus lutetiensis, strain64, Clostridium butyricum, strain65, Prevotella buccae, strain70, Enterococcus canintestini, strain74, Clostridium glycolicum, strain75, Ruminococcus gnavus and strain33, Streptococcus bovis. It was demonstarted that strain1,4,12,65and33may could efficiently utilize resistant starch through release starch-degrading enzymes in the medium, since clear zones were produced around the strains. In addition, strain2,8,64,74and75might utilize resistant starch via adhere to starch granules. Strain6and10could utilize resistant starch via adhere to starch granules. In the third section, the concentration of L-lactic acid and D-lactic acid were determinated in Landrace sows, Landrace piglets and Meishan piglets. Inter-individual variation was clearly showed in the result. According to this result, in vitro fermtation with L-lactic acid and D-lactic acid as substrate, feces of piglets as inocula was conducted to investigate the utilizing character of L-lactic acid and D-lactic acid by microbiota in the gut of piglets. Results showed that there was no significant difference in utilizing rate among L-, D-and L, D-lactic acid when5mmol/L lactic acid was used in fermentation system. A higher utilizing rate of L, D-lactic acid and a slower utilizing rate of D-lactic acid were observed if25mmol/L lactic acid was used as substates. Acetate and propionate were the main endpoint fermentation products in all groups. The mixed of two types lactic acid produced more gas than the other singal lactic acid in the same concentrion.