Isolation Of Lactic Acid Bacteria And Their Effects On Aerobic Stability In Silage

Silage is a hibernation feed for ruminants that is formed by storing green crops in a low-oxygen environment and fermented by lactic acid bacteria.The hetero-fermented lactic acid bacteria can produce organic acids such as lactic acid,acetic acid and other substances which lower the pH value,inhibit the growth of harmful bacteria,and play an important role in improving the nutritional quality and aerobic stability of the silage.Therefore,the screening of lactic acid bacteria with fast acid production,rapid growth and bacteriostatic effect as a silage inoculant has attracted much attention.Silage fermentation is a dynamic process in which various microorganisms participate in the interaction and evolution of different microbial communities.The microorganisms involved in different stages of silage are different,such as the most common lactic acid bacteria,aerobic spores,and molds.The first understanding of the dynamic distribution of micro-organisms was the use of culture-depended methods.The presence of uncultured micro-organisms limits its overall understanding of complex micro-organisms in habitats.Molecular identification techniques such as PCR-DGGE as a well-developed molecular biology method are widely used to analyze the genetic diversity of complex microflora and to monitor population dynamics.In this study,the inhibitory effects of lactic acid bacteria to each strain on the silage spoilage bacteria which were isolated from silage were analyzed.The preferred hetero-fermentative strains were applied to the 2 L silage tank for corn stalk fermentation,and the fermentation indicators in the silage process were assayed to determine the effect of added starter on feed quality and aerobic stability.At the same time,in order to screen the excellent type of fermented lactic acid bacteria,and lay a foundation for the development of silage fermentation agent,we used PCR-DGGE technology to analyze the dynamic changes of lactic acid bacteria during silage fermentation.The results are as follows:1.Screening of Lactic Acid Bacteria with Antimicrobial Ability in SilageWe have isolated and identified 17 strains of lactic acid bacteria in silage from Lanzhou,Jinan,Yucheng:Lb.parafarraginis 1021,Lb,parafarraginis 1022,Lb.parafarraginis 1024,Lb.parafarraginis 1025;Lb.buchneri 1031,Lb,buchneri 1032,Lb.buchneri 1034,Lb.buchneri 1036,Lb.buchneri 1037;Lb.farcinimis 1101,Lb.farcinimis 1102;Lb.plantarum 4001;Lb,plantarum 4002;Lb,fermentum 5001,Lb.acidophilus 6001;Lb.oris 1091;Lb.brevis 0991.Besides measuring the growth,pH,and organic acids content(lactic acid,acetic acid,and propionic acid)of these 17 strains of lactic acid bacteria,we used Bacillus sp.YY2,Escherichia coli DH5a,Candida boidinii 121,Issatchenkia orientalis 111,Candida ethanolica 131,Aspergillus niger 141 and Penicillium citrinum 151 as indicator bacteria to screen for lactic acid bacteria that can inhibit the growth of indicator bacteria by using the Oxford cup method and the double plate method.The results showed that Lb.oris 1091,Lb.brevis 0991,Lb,parafarraginis 1021 and Lb.buchneri 1036 grew fast,produced organic acid quickly,and could produce abundant lactic acid,acetic acid and propionic acid;All four strains of lactic acid bacteria were capable of inhibiting Bacillus sp.YY2,Escherichia coli DH5a,Candida boidinii 121 and Candida ethanolica 131,as well as Aspergillus niger 141 and Penicillium citrinum 151.Among them,the inhibitory effect of Lb.oris 1091 on bacteria and yeast was best,Lb.parafarraginis 1021,Lb.buchneri 1036 had the best inhibitory effect on Aspergillus niger 141.2.Effect of Hetero-fermentative Lactic Acid Bacteria on Fermentation Quality and Aerobic Stability of SilageThe 4 strains of lactic acid bacteria which were above-mentioned and had a bacteriostatic effect were inserted into the corn stalk at an inoculation amount of 1010 CFU/g.After mixing,they were put into a 2 L silage tank and stored naturally.And then,the silage fermentation indicators and aerobic stability on the 1st,3rd,5th,7th,15th,30th,and 45th day were measured,and silage without lactic acid bacteria was treated as Control.The experimental results showed that the number of lactic acid bacteria was higher in the experimental group than in the control group at the 3rd day of fermentation.The number of lactic acid bacteria in the experimental group was 2.24×108 CFU/g,and the number of lactic acid bacteria in the control group was 1.48×107 CFU/g.At the 7th day of fermentation,the number of undesired bacteria was the lowest in the experimental group and the control group.The number of undesired bacteria in the experimental group was 2.05×1O7 CFU/g,while the number of undesired bacteria in the control group was 3.15×108 CFU/g.The pH value of the experimental group decreased faster than that of the control group.The initial pH value of the silage was 5.5,the lowest pH value of the experimental group was 3.8,and the lowest pH value of the control group was 3.93.The content of lactic acid and acetic acid in silage with inoculant was higher than that of silage without lactic acid bacteria.At the 7th day of fermentation,the highest lactic acid content of experimental group was 22.62 mM,at this time the lactic acid of control group was 17.59 mM.At 15th day of fermentation,the maximum values of acetic acid were reached in the experimental group and the control group,and the acetate content was 13.67 mM and 11.63 mM,respectively.From the ratio of dry weight to wet weight of the experimental group and the control group,it is a critical period for silage fermentation when it is fermented for 3th day to 15th day,during these days,the highest proportion of nutrient preservation in the control group was 38.19%,while the highest proportion of nutrient preservation in the experimental group was 47.84%.In addition,during the 7th day of fermentation,the lowest content of reducing sugar in the control group was 18 mg/g,and the lowest was 13 mg/g in the experimental group.The free reducing sugar content in the control group was higher than that in the experimental group.The experimental results showed that the lactic acid bacteria inoculants could better improve the fermentation quality of silage.When the aerobic spoilage stability of silage was measured,the shelf life of silage in the experimental group was 216 hours,while that of the silage in the control group was 264 hours,this was not the desired result.The stability of the experimental group was slightly poor.So,we speculated that the aerobic stability of the feed cannot be judged by simply relying on the increase in temperature because the hetero-fermented lactic acid fermentation strain has a good inhibition on mold,and the inhibitory activity against bacteria needs to be improved because the breeding of spoilage bacteria can also lead to an increase in feed temperature.3.Analysis of Lactic Acid Bacteria in Silage by PCR-DGGESilage can be well preserved because of various lactic acid bacteria which play different roles in fermentation stages of silage.Therefore,we used PCR-DGGE technology to analyze the lactic acid bacteria in the fermentation process of silage mentioned above,it was investigated whether lactic acid bacteria inoculants could play a role,and which lactic acid bacteria were involved in the fermentation of silage.First,the DGGE map model was established using standard lactic acid bacteria to facilitate the rapid identification of lactic acid bacteria in silage.Experimental results demonstrated,in the total fermentation period of silage,the main strains in the silage were Lb.pentosus,Lb.plantarum and Lb.brevis.The two strains of lactic acid bacteria could grow rapidly and initiate the fermentation.The main strains present during the late fermentation of silage were Lb.parafarraginis and Lb.buchneri.Lb,parafarraginis grew well and Lb.buchneri could produce acetic acid and other substances to improve the aerobic stability of silage.From the DGGE graph,we could see that Lb.parafarraginis and Lb.buchneri appeared earlier in the experimental group,illustrating that the silage starter played a role.Using PCR-DGGE technology,we successfully analyzed the lactic acid bacteria flora in silage and made a DGGE model,which helped to quickly identify lactic acid bacteria in complex samples.4.Inhibition of silage spoilage organisms by Short-chain volatile organic acidsWhen silage is opened,silage comes into contact with oxygen,and molds,yeasts and bacteria on the surface of the feed begin to grow and secondary fermentation occurs,resulting in increased pH value.In order to improve the aerobic stability of the feed,we screened lactic acid bacteria producing large amounts of lactic acid,acetic acid,and propionic acid as the silage inoculants.In order to analyze their inhibitory mechanisms against spoilage bacteria,we used Bacillus sp.YY2,Escherichia coli DH5a,and Candida boidinii 121,Pichia kudriavzevii 111,Candida ethanolica 131,Aspergillus niger 141 and Penicillium citrinum 151 which were isolated from rotten silage as indicator bacteria to determine the inhibitory effect of lactic acid,acetic acid and propionic acid.The experimental results showed that propionic acid had the best inhibitory effect on spoilage microorganisms,and the lactic acid inhibition effect was the worst.In addition,the combination of lactic acid,acetic acid,and propionic acid had the best inhibitory effect on spoilage microorganisms,and the amounts of lactic acid,acetic acid,and propionic acid were the lowest.Bacillus sp.YY2 and E.coli DH5a were most easily inhibited,and yeasts and molds were more difficult to inhibit.Candida ethandica 131 was more difficult to suppress than Pichia kudriavzevii 111.By comparing the content of organic acids in silage in Chapter 3,it was known that the production of organic acids in the fermentation of hetero-type lactic acid bacteria was low,which could not effectively inhibit the growth of spoilage microorganisms in silage,and the lactic acid content of silage added with hetero-fermentative lactic acid bacteria was high.High-concentration lactic acid bacteria were used by acid-tolerant ·yeast in silage to induce aerobic spoilge of silage.In summary,inhibiting the growth of yeast in silage plays a key role in improving the aerobic stability of silage.We need to increase the concentration of propionic acid or acetic acid in silage to inhibit the growth of spoilage microorganisms.