| Pig industry is an animal husbandry with the biggest grains consumption in China. Research on biological characteristics of crude feed tolerance in some good Chinese local pig breeds is of great significance in cultivating feed-saving new pig breeds and developing feed-saving pig industry.Tibetan pig is a breed growing on Qinghai-Tibet Plateau with local characteristics, adapting to graze on all-year-round pasture and mainly feed on grass. It has attracted a lot of attention from all around the world for its good characteristics, such as herbivorous characteristics, good resistance to disease and cold, aromaticity, environmental protection and so on. Herbivorous characteristic in Tibetan pigs is closely related to its special gut microbial community. To reveal the grazing characteristic of Tibetan pigs, we did the systematic study on the diversity and components of the gut microbial community of Tibetan pigs, isolation and screening of the cellulolytic bacterium from Tibetan pigs, characterization of the cellulase, cellulase production, cloning and expressing of cellulase genes and probiotic properties of the cellulolytic bacterium. The results were as belows:1. We studied the diversity and composition of bacteria communities from Tibetan pigs’ gastrointestinal tract with DGGE. The bacterial species were the most abundant in the cecum, then stomach, ileum, jejunum and duodenum. All bacteria in the gastrointestinal tract were divided into five phyla(Bacteroidetes, Proteobacteria, Firmicutes, Actinobacteria and Spirochaetes), and 61% of them are the new bacterium, Among them, the dominant bacteria is different in the different regions of the gastrointestinal tract. We studied that the diversity and composition of bacterial communities in the cecum from Tibetan pigs and lean-pigs with DGGE. The results showed that the cecal bacteria in Tibetan pigs were more diverser,and the similarity of the bacterial community composition in the cecum is more than 80%, but lean-pigs is that of 65%. Both numbers of specific bands and DNA abundance in the DGGE map about the Tibetan pigs were higher than those in lean-pigs. Bacteroidetes and Firmicutes were dominant in both of two pig breeds, but the richness of Bacteroidetes was much higher than that of Firmicutes. Furthermore, various fiber degradation bacteria were discovered in both of the two pig breeds, such as Prevotella, Ruminiclostridium, Anaerovibrio, Sphaerochaeta, Bacteroides and Parabacteroides. However, the abundant of cellulolytic bacterium from Tibetan pigs were higher 11.2% than that of lean-pigs.2. We used Illumina Miseq high-throughput sequencing to study the diversity and composition of the bacterial community from cecum samples of Tibetan pigs and lean-pigs. The results showed that 75,069 and 74,759 valid sequences of 16 S rRNA gene V4-V5 region came from five Tibetan pigs and lean-pigs, respectively, and the average length was 393.8 bp. Clustering analysis at the 97% similarity level obtained 660 OTU and 668 OTU from Tibetan pigs and lean-pigs, respectively. Composition of the cecal bacteria community was similar in the two pig breeds. The cecal bacteria community in Tibetan pigs could be divided into 15 phyla and 70 genus, and its could be divided into 15 phyla and 74 genus in lean-pigs. However, the bacteria community in Tibetan pigs were stable. 256 OTU were shared in different individuals, and the core microbiota took up 88.10% in total intestine bacteria sequence which could be divided into 7 phyla. Yet, lean-pigs took up 81.29% which could be divided into 6 phyla. Bacteroidetes and Firmicutes were the most abundant bacterium in the cecum of two pig breeds. Besides, the abundance of Bacteroidetes was higher than Firmicutes. However, compared with lean-pigs, Tibetan pigs had a high abundance in Bacteroidetes and a low abundance in Firmicutes, and 42.4% of them in cecal bacterium belonged to unclassified and uncultured. In addition, Proteobacteria which is closely related to intestine diseases was lower than lean-pigs at the very significant level(P<0.01). We found some cellulolytic bacterium from the cecum of two pig breeds, such as Ruminococcus、Bacteroides、Prevotella、Clostridium、Butyricicoccus、Fibrobacte、Lachnospira、Anaerovibrio、Parabacteroides et al. Therefore, long-term herbivorous characteristic of Tibetan pigs was related to stability and complexity of gut microflora and a large number of uncultured and unclassified bacterium, as well as the special digestive physiology formed in their long-term intake of green crude feed.3. We isolated a cellulolytic bacterium from Tibetan pig’s cecum with Carboxymethylcellulose agar plates and shake-flask cultures. It was identified based on morphological, physiological and biochemical characteristics, as well as 16 S rRNA partial sequence analysis, and named it as Bacillus subtilis BY-2. Bacillus subtilis BY-2 grew fast, its growth curve entered the logarithmic phase after 8-12 h and the stable growth phasebetween 20 and 36 h, reproduction went into recession gradually after 36 h. Optimal reaction conditions of carboxymethylcellulase from Bacillus subtilis BY-2 was at pH 5.5 and 65°C with a reaction time of 5 min, and the carboxymethylcellulase has strong acid and alkali resistance and thermal stability. In addition, the best carbon source of Bacillus subtilis BY-2 for fermentation was 1% corn flour, while 2% peptone and yeast powder mixture(1:1)were the best nitrogen sources. The initial pH during fermentation was 5.5, with 4% inoculum, resulting in a high and stable amount of enzyme in 24-36 h. Compared with the original carboxymethylcellulase activity of Bacillus subtilis BY-2(0.40 U/mL), the carboxymethylcellulase activity increased nearly 8.6-fold after the reaction and production conditions of carboxymethylcellulase were optimized when fermentation reached 24 h(3.43 U/mL).4. Carboxymethylcellulase gene egls-BY was cloned from the Bacillus subtilis BY-2 genomic DNA. The length was 1500 bp, and was able to continuously encoding 499 amino acids. The sequence analysis showed that the carboxymethylcellulase which is coded by egls-BYgene was belonged to glycoside hydrolase family 5, and it contain a CMB-3 domain. The gene was expressed in the E.coli, and its protein molecular weight was at about 55 kDa, Most of expressing products reside in the form of inclusion bodies inside cells.5. Experiments in vitro showed that Bacillus subtilis BY-2 had strong resistance. The survival rate of Bacillus subtilis BY-2 was between 89.83% and 91.07% when it was kept at 65-85 °C for 20 min. Cultured in artificial gastric juice in pH1-4 for 4 h, Bacillus subtilis BY-2 survival rate was between 73.78% and 83.61%. Treated with artificial gastric juice for 2 h, and with artificial intestinal juice and intestinal juice with different bile salts for 4 h, the survival rate of Bacillus subtilis BY-2 was still above 75%. The antimicrobial substances from Bacillus subtilis BY-2 could inhibit E. coli, Salmonella and Staphylococcus aureus, and the cytotoxicity is low for ZYM-SIEC02. In addition, the adhesion ability of Bacillus subtilis BY-2 was poor for ZYM-SIECO2, but has particular adhesion inhibition for E. coli, Salmonella and Staphylococcus aureus. Therefore, the cellulolytic bacterium Bacillus subtilis BY-2 with the fast growth, producing carboxymethylcellulase early and stability, and strong resiliencenot only have the great significance to reveal the grazing mechanism of Tibetan pigs, but also provide the necessary experimental basis for industrial development. |
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