Breeding Of Cellulolytic Lactic Acid Bacteria

In this study,acid production capability in different carbon sources of 8 lactic acid bacteria?LAB?strains isolating from fresh alfalfa samples with good antibacterial capability were tested.LAB strain with good characteristics as an alfalfa ensiling additive was screened out,and applied in alfalfa ensiling experiment.Ion beam implantation was performed on the strain,and mutant strains with enhanced cellulolytic capability were screened out.The screening system was also improved.Effects of the strains before and after the implantation on fermentation quality during the ensiling process was studied.Changes on 3 cellulolytic related gene expression quantities of the strains after implantation was also tested.8 lactic acid bacteria?LAB?strains isolating from fresh alfalfa samples of Shanxi province with good antibacterial capability were applied in alfalfa meal fermentation test.After 48 h fermentation,samples treated with A340,A344,A345 and A357showed the lowest pH?pH<5.20?.The 8 LAB strains were inoculated in 1%glucose,glucan?MW:3000?,and microcrystalline cellulose?MCC?solution separately and fermented for 24 h.Among the strains,A345 showed the lowest pH in all the four carbon source solutions.A345 was screened out and identified as Lactobacillus plantarum by 16s rRNA sequencing analysis.morphological,physiological and biochemical characteristics of A345 were also determined.A345 was applied in alfalfa ensiling test.After 65 d ensiling,A345 showed a better performance than commercial strain FG1.Silage treated with A345 performed the lowest pH?pH of 4.67?,and the highest content of lactic acid and acetic acid,6.25g/L%FM and 2.66 g/L%FM,respectively.The ammonia-N content and Escherichia coli count of silage treated with A345 was also significantly lower than silage treated with FG1.Principle component analysis?PCA?demonstrated that A345 showed a better performance on alfalfa Jinneng,than on alfalfa Sandeli.J x A345 performed lower ammonia-N and acetic acid content,and lower count of LAB,Escherichia coli,and Bacillus than S x A345.While Sandeli showed better ensiling quality than Jinneng when no additive was treated?P<0.05?.This indicated that A345 could significantly improve alfalfa ensiling quality,especially for those alfalfa species showed difficulty in natural ensiling.Results of qPCR indicated that expression quantities of the 3 cellulolytic related genes of A345 in CMC medium were significantly higher than that of commercial strain FG1,and cellulose degradation capability of A345 might be induced by CMC.Ion beam implantation was performed on A345.At the dose of 1×10¹55 N⁺·cm^-2,the radiation lethality of the strain was 70.44%.There was no significant difference in radiation lethality between the doses of 5×10¹55 N⁺·cm^-22 and 1×10¹66 N⁺·cm^-2.Both reached more than 90%.The Congo red staining method was used to determine the fiber degradation performance of the strains.After the first round of implantation,mutant strain Ei-177 with increased enzymatic hydrolysis was screened out and used as a starting strain for the second round of implantation.After the second round of implantation,mutant strains zw1-33,zw2-8,zw2-21,zw2-23,zw3-57 with increased enzymatic hydrolysis were screened out.The five strains were applied in alfalfa meal fermentation test and CMCase activity test.The results showed that zw2-21 showed the lowest pH after 48 h of alfalfa meal fermentation and the highest CMC activity in value?P>0.05?.A plurality of treatment groups?A345 added,zw2-21 added,commercial strain FG1 added,commercial strain YX added?were subjected to alfalfa ensiling experiments.Analysis by the general linear model showed that the pH of the treated group with zw2-21 was the lowest,which decreased to 5.15?P<0.05?.The addition of A345 and its mutant strain zw2-21 can effectively inhibit the growth of E.coli.At the same time,the content of lactic acid and acetic acid in the treatment group with zw2-21 was significantly higher than that in the treatment group with the starting strain A345?P<0.05?.Ion beam implantation was performed on A345,at the doses of 1×10¹44 N⁺·cm^-2,5×10¹44 N⁺·cm^-2,1×10¹55 N⁺·cm^-2,5×10¹55 N⁺·cm^-22 and 1×10¹66 N⁺·cm^-2.At doses of 1×10¹⁴N⁺·cm^-22 and 5×10¹44 N⁺·cm^-2,there was no significant difference in the lethality of the strains,which were both less than 40%.The lethality at the other three doses was consistent with previous experiments.The Congo red staining method was optimized to improve the screening efficiency.Among 1000 strains,60 mutant strains were screened out with increased enzymatic hydrolysis.3 mutant strains with enhanced cellulolytic capability and genetic stability were finally screened out,through glucan?MW:3000?fermentation test,alfalfa meal fermentation test,and genetic stability test.Among the strains,F-54 performed the lowest pH in glucan?MW:3000?fermentation test?P<0.05?and alfalfa meal fermentation test?P>0.05?.F-54 also showed the highest lactic acid content in alfalfa meal fermentation test?P<0.05?.Results of qPCR indicated that expression quantities of the 3 cellulolytic related genes of F-54reached 5.87,4.62,and 5.49 folds than that of A345,respectively.A345 and F-54showed no significant difference on inhibition capability of E.coli,Salmonella and Listeria monocytogenes.In conclusion,we established a high-efficiency screening system for cellulolytic LAB strains,and obtained 3 mutant strains with good characteristics and genetic stability.Results of the alfalfa ensiling experiment indicated that enhancing cellulolytic activity of the LAB strains might be beneficial to improving the fermentation quality of alfalfa ensiling.Besides,analysis of the cellulolytic related gene expression quantities proved that cellulolytic capability of the mutant strain was enhanced after implantation on transcriptional scale,which explained the mutation mechanism of mutant strains at the molecular level preliminarily.