| Lactic acid bacterias were used as material which isolated from 115 alfalfa samples by collectting from five province of heilongjiang, jilin, shanxi, henan, and hebei. In order to obtain the excellent lactic acid bacterias for improving quality of alfalfa silage, we used three indexs of antimicrobial activity, fermentation characte ristics for alfalfa meal, utilization capacity of structural sugars to screen. And study of its influence on microbial diversity, fermentation quality and nutritional value in process of fermentation dynamic. Finally breeding the lactic acid bacterias by methods of domestication and ion beam mutagenesis.Research has shown that the screenned lactic acid bacteria could effectively improve quality of alfalfa silage by the strong ability for using or fermentated alfalfa meal and promoting polysaccharide or cellulosein used in alfalfa silage. Methods of breeding in this research has also provided a new mentality for alfalfa silage additives ulteriorly be able to development. The test results are as follows:Bacteriostatic results has shown that there are 18 strains in number of 264 lactic acid bacteria presente strong antimicrobial activity for Escherichia coli, Salmonella and Listeria monocytogenes by Oxford cup of double plate methods. There are 18 strains produce much acid in fermentation results of Alfalfa meals (0.15mg/ml) and 5 strains antibacterial better, especially strains of ZZU A340, ZZU A345 and ZZU A38 0 shows a relatively low pH value in above the 5 strains of Alfalfa meal (0.4 g/ml) fermentation among number of 72 strains isolated from Shanxi Province. Strains of ZZU A345 presente the minimum value after fermentation on 24 hours in dextran (MW:3000) pure solutions with concentration of 1 gram per hundred milliliter, it was was identified as Lactobacillus plantarum by its physiological and biochemical characteristics and 16S rRNA sequence analysis.In order to attest utilization ability of lactic acid bacteria, this paper has studied its properties of fermentation by dextran or xylan, decomposition by CMC-Na and utilization carbon source, which contains different structural sugar material on the cell wall. Results show that the pH value on 24 hours by vaccination ZZU A313 and ZZU A344 all were low in dextran MRS and dextran pure solutions with different molecular weight and pH value of vaccination ZZU A341 were low both in xylan MRS and xylan pure solutions when compared without the control of bacterias. All of the above 3 strains show clear transparent circles on CMC-Na plate after the Congo red stained. Fermentation of carbons show that strains of ZZU A313 and ZZU A341 could use of L-arabinose, D-xylose, glucose, fructose and mannose, but strains of ZZU A344 and ZZU A345 could use of L-arabinose, glucose, fructose and galactose when fermentated in sugars which made of cellulose and hemicellulose on cell walls.The silage results indicated that both vaccination strains of ZZU A313 and ZZU A345 could obviously reduce value of the pH when compared with natural silage of no bacterias. When compared with results of commercial strains, there has a low pH and low amount of propionic or butyric, but has a large number of lactic acid when ensiling on 60 and 100 days by inoculated strains of ZZU A313. While, there also has a low pH and less Escherichia Coli for ensiling on 100 days by inoculated strains of ZZU A345, but it present high amount of acetic and protein.After domested the strains of highly utilization capacity for structural sugar by ZZU A313, ZZU A341 and ZZU A344, the study obtained that strain of 313XH-7-9 has a power ability to use dextran than that of 344XH-9-7 and as well has a good ability to use xylan for strain of 341XH-7-7 when compared with the original strain. According to these results, it shows that the utilizing ability of offspring strains after domesticated were superior to its original strain. On circumstances of simulated silage to fermentation alfalfa meals(0.4 g/ml), all pH of alfalfa meals fermented liquids were much lower when on 12,18,72 hours and all hemicellulose, cellulose, soluble ash were slightly difference by inoculated strain of 313XH-7-9 (p<0.05). But the pH value at fermentation on 12 hours was obviousily lower than fermentation on 18 and 72 hours of alfalfa meals, and merely cellulose decreased significantly when fermentation on 72 hours (p<0.05). This result shows that the excellent strains after domesticated has a good value in used for alfalfa and can be applied to the alfalfa silage so as to further improvement the quality of alfalfa silage.Ion beam mutate these screened strains of ZZU A340, ZZU A345 and ZZU A380, which have strong power of antibacterial activity and fermentation for alfalfa meals, and we discovered that all the highest rate of positive mutation was the ZZU A345 by fermentated in alfalfa meals (0.4 g/ml), dextran and xylan pure solutions (0.01 g/ml). Results of CMC-Na plate test showed that mutant strains which transparent circle diameter exceed 12 mm was occupied at 21.55%of all forward mutation strains(transparent circle diameter of origina strain was 4.5 mm). The genetic stability for decomposition of CMC-Na showed that both strains 345-25 and 345-177 could biggish appear to 11 generations transparent circle of all 13 generati ons for mutant strains of ZZU A345. These indicated that ion beam mutagenesis could improve the fermentation ability for alfalfa meals, glucan and xylan pure solutions. |
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