Investigation On The Variation Of Flora In The Fermentation Process Of Corn Stalk Biological Feed

The change of microorganisms in the fermentation process of bio-feed is very important.The application of fluorescence quantitative PCR in the detection of intestinal microflora has been reported,but the application of fluorescence quantitative PCR in the fermentation process of corn stalk bio-feed has not been reported.In this paper,the microbial fermentation of corn stalk was used as a route,combined with fluorescence quantitative PCR technology to explore the changes of microbial flora in the process of corn stalk fermentation,in order to provide a reference for the high-quality production of corn stalks and other biological feeds,and greatly improving the economic and social benefits of breeding industry.Firstly,the specific primers of Bacillus subtilis,Bacillus licheniformis,Lactobacillus plantarum,Lactobacillus acidophilus,Saccharomyces cerevisiae,Candida tropicalis,Bifidobacterium longum,Lactobacillus casei and Lactobacillus rhamnosus were established,and the fluorescence quantitative PCR method for the detection of the change of microflora in the fermentation process of biological feed was preliminarily established.Then,the traditional plate method and the fluorescence quantitative PCR method was used to determine the number of bacteria in the fermentation process of corn stalk biological feed respectively.The results showed that the results of fluorescence quantitative PCR were generally greater than those measured by the plate method,but the changes of flora in the fermentation process were basically the same.After analysis,the fluorescence quantitative PCR method was more time-saving,more accurate and more specific than the traditional plate method.It can be used for quantitative analysis of flora in the fermentation process of biological feed.Secondly,adding soybean dregs or bran as an auxiliary material in corn stalk to determine the change of flora during the fermentation process of corn stalk biological feed.From the peak value of fermentation,the number of Bacillus subtilis in thesamples supplemented with bran was 4.27 times as much as that of Bacillus subtilis in the samples without bran,1.73 times as much as that of Lactobacillus plantarum and1.48 times as much as that of Saccharomyces cerevisiae,the results showed that adding bran as supplementary material was more helpful to the growth of microflora in fermented feed.Then,the results of repeated sampling and non-repeated sampling during fermentation were compared.From the peak value of fermentation,the number of Bacillus subtilis and Lactobacillus plantarum in non-repeated samples was 1.51 times and 10.94 times of those in repeated samples.This indicated that non-repeated sampling reduced the entry of bacteria and air in the process of sampling and was more conducive to the fermentation of anaerobic probiotics.As for the data without repeated sampling,the maximum value of aerobic bacteria mostly appeared on the third or fifth day of fermentation,and the fermentation strains used in the experiment did not show any inhibition phenomena such as non-growth or large-scale decrease of bacterial quantity in the fermentation process,indicating that there was no antagonism effect between the fermentation strains added in the experiment,which could be used for co-fermentation.Then,under the condition of sterilization of raw materials,the change of microflora during anaerobic fermentation were analyzed after adding Bacillus subtilis,Lactobacillus plantarum and Saccharomyces cerevisiae simultaneously for one day,two days and three days respectively.From the peak value of fermentation,the growth of Lactobacillus plantarum was basically the same,the number of Saccharomyces cerevisiae and Bacillus subtilis was the largest in the process of anaerobic fermentation after three days of aerobic fermentation,but the results were similar to those in aerobic two days.Therefore,in order to save production time and optimize fermentation process,it is advisable to carry out anaerobic fermentation after two days of aerobic fermentation.Five kinds of anaerobic probiotics of Lactobacillus plantarum,Lactobacillus acidophilus,Bifidobacterium longum,Lactobacillus casei and Lactobacillus rhamnosus were selected for single and mixed bacterial fermentation.The change of microbial flora of anaerobic probiotics during the fermentation of corn stalk bio-feed were analyzed.It was found that the bacterial quantity of corn stalk bio-feedfermented by anaerobic probiotic fermentation alone and mixed anaerobic probiotic fermentation was slightly larger than that measured by raw material sterilization,but the difference was not significant.Therefore,in the large-scale production practice of bio-feed,it can be considered that the raw material are directly fermented without sterilization.At the same time,compared with the aerobic and anaerobic probiotic bacteria were added step by step to ferment and anaerobic probiotic bacteria and aerobic bacteria were added at the same time,from the samples mixed with five kinds of bacteria,the number of Lactobacillus plantarum was 2.45 times of the step fermentation,the number of Lactobacillus acidophilus was 5.62 times of the step fermentation,the number of Bifidobacterium longum was 2.24 times of the step fermentation,the number of Lactobacillus casei and Lactobacillus rhamnosus is 1.91 and 2.82 times of the step fermentation,so in order to simplify the fermentation process,if only the growth of anaerobic probiotics is considered,aerobic and anaerobic bacteria can be added simultaneously for one-step fermentation.Finally,the results show that the maximum value of anaerobic probiotics occurs on the seventh day of fermentation,and the total bacterial content can be significantly increased by adding five kinds of anaerobic probiotics at the same time.The number of bacteria in the mixed fermentation process is close to that measured by single fermentation,which indicates that the five kinds of anaerobic probiotics have more application value in synergistic fermentation.This study provides a rapid,accurate and specific detection method for the detection of bacterial flora in the fermentation process of corn stalk bio-feed,which lays an experimental foundation for the determination of different kinds of bacteria in feed in the future,and also provides guidance for large-scale mixed bacterial fermentation of bio-feed.