Construction Of Acid-tolerant Selenomonas Ruminantium And Effect On The Fermentation Of Ruminal Microorganisms

Based on the isolation program of lactobacillus and using the SL culture mediums which were specific for isolating acid-tolerant lactobacillus from ruminal fluids.Eight acid-tolerant lactobacilli(named 1,2,3,4,5,6,7,8) were isolated from ruminal fulid and the morphological and biochemical characteristics were observe and analyzed.The consensus primers were designed according to the conservative region of bacterial 16S rRNA and the 16S rRNA genes were cloned from their genome.The 16S rRNA genes of the eight strains were amplified by polymerase chain reaction (PCR) and sequenced. The sequence obtained was 98% identical to those of Lactobacillus casei, Lactobacillus curvatus, Lactobacillus coryniformis, Lactobacillus brevis, Lactobacillus mucosae in GenBank.The strains 1,2,3,4,5,6,7,8 were identified through morphology and biochemical characteristics and 16S rRNA gene sequence as Lactobacillus casei, Lactobacillus curvatus, Lactobacillus coryniformis, Lactobacillus casei,Lactobacillus curvatus, Lactobacillus brevis, Lactobacillus mucosae, Lactobacillus mucosae,respectively.The growth curve of Selenomonas ruminantium K6 and Lactobacillus mucosae lm4208 were drawn and the logarithmic phase of them are definited. The lm4208's logarithmic phase is 4h to 12h and the K6's is 8h-16h.To establish the optimum conditions of the protoplast preparation and regeneration of the lactobacillus mucosae lm4208 and Selenomonas ruminantium K6,the L9(34)orthogonal experiments were used.The results showd that the optimized conditions of the strain of lm4208 as follows: 10h incubation was for protoplast release,100μg/mL lysing enzyme for protoplast preparation, 37℃and 60min. for the release of protoplast.And the ratios of protoplast preparation and regeneration were 98.3% and 24%,respectively.The optimum conditions of K6 were as follows: 30min of treating time, 600μg/mL lysozyme and 12h incubation time.Under the optimum conditions,the ratios of formation and regeneration protoplast were 94% and 17.2%,resperctively.The thermal inactivating conditions of strains lm4208 were determined by survival rate.In 56℃,the strain lm4208 treated for 60min were optimal,and the lowest pH of K6 to grow is 5.4.Following all conditions were optimized, preparation,fusion and regeneration of protoplast were done and four recombinant strains were selected according to the rate of survival strains at the pH5.4 in the regenerating medium.After the future generation,the fusant F-K6-lm4208 was gained at last.The fusant F-K6-lm4208 can't make gelatin liquefy,but can hydrogenize the nitrate,and ferment maltose, xylose, glucose, fructose, cellobiose, lactose, glucopyranoside, sorbose and mannose,but can't ferment rhamnose,aesculin and salicin.The biochemical and physiological characteristics of the fusants F-K6-lm4208 are similar to Selenomonas ruminantium K6. The molecular weight of the fusants was larger than their parents. According to the morphological,the biochemical and physiological characteristics,the molecular weight and the acid resistant experiment, the fusants were identified as acid-tolerant fusants and can survive at pH5.1.The influence of the liminary strain K6 and the engineering bacteria F-K6-lm4208 on in vitro rumen fermentation was studied using co-incubating the strains with mixed rumen micro-organisms of dairy cows as incoculums. Culture fluid and ruminal fluid was sampled for analysis of pH and Volatile Fatty Acid (VFA) at 0,2,4,6,8,10,12,14,16,18h.The results showed that the pH in engineering bacteria group was the lowest after 4h incubation then stepped up significantly.The lactic acid was not accumulated and at the 18h ,its concentration was 0.53 mmol/L.Compared to control group at 18h,the concentration of VFAs in the liminary bacteria group and the engineering bacteria group increased by 24% and 105%,the ratio of acetic acid decreased by 12% and 16%, the ratio of propanoic acid increased by 22% and 35%,respectly.And the ratio of acetic acid to propanoic acid decreased 0.42and 0.57,respectly.This demonstrate that the liminary strains K6 and its engineering bacteria both can have effect on the fermentation and the ratio of acetic acid to propanoic acid,and can make the fermentation prone to propionic acid production but the effects of the engineering bacteria were more marked higher than the liminary strains K6.