| During pre-and postweaning periods,diarrhea often occurs in piglets due to weaning stress,dietary anti-nutritional factors,environmental pathogens and other factors,which causing huge losses to the pig industry.Feed antibiotics are generally used to solve this problem in swine production.Due to the prohibition of feed antibiotics,antibiotics alternatives like probiotics,oligosaccharides,acidifiers,and fermented feeds are currently used.Based on previous researches,in the present study,lactic acid bacteria from piglet manure were isolated as fermented starter,milk powder and oligosaccharides as fermentation substrates to develop functional fermented yogurt for weaning piglets,which contains functional ingredients of fructooligosaccharides,galacto-oligosaccharides,lactic acid bacteria and acidulants.The weaning stress of weaned piglets treated with fermented yoghurt was alleviated and the nutrition was supplemented.The thesis consists of the following three parts:1.Isolation of swine lactic lcid bacteria and screening of strains for yoghurt fermentationThis chapter aims to isolate lactic acid bacteria from pig intestines,and to screen strains with strong acid-producing capacity and excellent anti-stress ability as starter strains for fermented yogurt.In the experiment,21 strains of lactic acid bacteria were isolated from fresh piglet manure,and 4 different strains were identified through 16S r RNA gene sequencing:Enterococcus faecium,Lactobacillus animalis,Lactobacillus johnsonii and Enterococcus hirae.The results of in vitro culture showed that the acid-producing performance of the four lactic acid bacteria from high to low was:Lactobacillus johnsonii>Enterococcus hirae>Enterococcus faecium>Lactobacillus animalis.These lactic acid bacteria have a certain tolerance to the environment of p H 3,and the tolerance to the environment of p H 2.5 is relatively poor.Enterococcus hirae had the best acid resistance under the conditions of p H 3 and 2.5.In the 0.1%bile salt treatment group,Enterococcus faecium was the most tolerant,followed by Enterococcus hirae and Lactobacillus johnsonii,and Lactobacillus animalis was the weakest to 0.1%bile salt;in the 0.5%bile salt treatment group,Lactobacillus animalis is not tolerant to it,and Enterococcus hirae is the strongest.Based on the above test results,all strains of Lactobacillus animalis have the weakest stress resistance.Enterococcus faecium L8,Lactobacillus johnsonii L19 and Enterococcus hirae L20 were selected as starter strains for fermented yogurt.2.Optimization of fermentation conditions of porcine lactic acid bacteria yoghurtThis chapter aims to optimize the combination of strains,substrates and fermentation conditions for fermented yogurt.By measuring the influence of single strain,double strain and three strain fermentation on the result of fermented yogurt,the combination with the largest number of viable lactic acid bacteria,the highest lactic acid production and the lowest p H value was selected as the best starter combination.Fermentation was carried out with this starter combination selected in chapter 1,and the influence of different milk powder addition,sucrose addition,oligosaccharide addition and addition ratio on the fermentation results were compared,and then the orthogonal experiment design was carried out to determine the optimal addition amount.Then determine the effects of inoculum,fermentation temperature and fermentation time on the number of viable lactic acid bacteria,lactic acid production and p H value of fermented yogurt after 24 hours of fermentation.The results show that the effect of three-strain compound fermentation is better than that of two-strain fermentation and single-strain fermentation.Among them,the combination of starter with the ratio of Lactobacillus johnsonii:Enterococcus faecalis:Enterococcus hirae is3:2:3.When the fructose:galactose ratio is 3:1,the fermentation effect is the best.The optimal addition range is determined by single factor experiment as 15-20%milk powder,6-10%sucrose,1.5-2.5%oligosaccharide.The orthogonal test results show that the best raw material is milk powder.The addition amount is 15%,the addition amount of sucrose is8%,and the addition amount of oligosaccharide is 1.5%.The best fermentation effect is under the conditions of 3%strain inoculation,37℃fermentation temperature,and 24hours fermentation time.In summary,the best combination of compound starter is Lactobacillus johnsonii:Enterococcus faecalis:Enterococcus hirae=3:2:3,and the best fermentation process is 15%milk powder,8%sucrose,the addition amount of oligosaccharide is 1.5%,fructose:galactose=3:1,the inoculum amount is 3%,the fermentation temperature is 37℃,and the fermentation time is 24 h.3.Screening of compound acidifiers and determination of shelf life of yogurtThis chapter aims to screen the compound acidifier with the best antibacterial effect and determine the shelf life of the fermented yogurt.In the experiment,lactic acid,citric acid,caprylic acid and calcium formate were selected as the ingredient s of compound acidifiers,and the combination of compound acidifiers with the strongest antibacterial effect on Escherichia coli and Salmonella was screened through a uniform test design,and the effect of the added amount of acidifier on the number of viable lactic acid bacteria in yogurt was determined.Analyzing the number of viable lactic acid bacteria and changes in p H value of yogurt under different temperature conditions to determine the shelf life.The results showed that when the compound acidifier consisted of 40%lactic acid,10%citric acid,8%caprylic acid,14%calcium formate,and 28%emulsifier,the antibacterial effect was the best.The addition of 0.5%,0.75%,and 1%acidifier test group had a strong inhibitory effect on the number of viable cells in yogurt.Addition of 0.25%acidifier had the least effect on the number of viable cells in yogurt.Therefore,the optimal dosage was0.25%.In addition,when the yogurt was stored at 4℃for 21 d,the number of viable bacteria was still higher than 10~8CFU/m L,while on the 28th day,the number of viable lactic acid bacteria in the yogurt was reduced to 10~7CFU/m L,and the tissue status had been stratified.,flocculent precipitation appears.Under 15℃storage conditions,the number of viable lactic acid bacteria in the yogurt decreased to 10~7CFU/m L on the third day,and the number of viable bacteria decreased to 10~6CFU/m L on the 7th day,and there was obvious stratification on the 14th day.And the yogurt produced flocculent precipitation.The number of viable lactic acid bacteria was reduced to 10~4CFU/m L.Under the storage condition of25℃,it has been stratified at 7 days,and flocculent precipitation has occurred.During this period,all groups of molds,yeasts,E.coli and Salmonella were not detected.In summary,the compound acidulant combination with the best antibacterial effect was 40%lactic acid,10%citric acid,8%caprylic acid,14%calcium formate,and 28%emulsifier,and the optimal concentration was 0.25%.The best storage condition for yogurt was 4℃,and the shelf life was 21 d.In summary,this study screened out 3 strains of porcine intestinal-derived lactic acid bacteria with strong acid-producing and stress-resistance properties,including Enterococcus faecalis L8,Lactobacillus johnsonii L19 and Enterococcus hirae L20 as starter strains for fermented yogurt.The best compound starter combination for fermented yogurt is Lactobacillus johnsonii:Enterococcus faecalis:Enterococcus hirae=3:2:3.The best fermentation process was 15%milk powder,8%sucrose,and oligosaccharides.The addition amount was 1.5%,fructose:galactose=3:1,the inoculum amount was 3%,the fermentation temperature was 37℃,and the fermentation time was 24 h.The compound acidulant combination with the best antibacterial effect was 40%lactic acid,10%citric acid,8%caprylic acid,14%calcium formate,and 28%emulsifier,and the optimal dosage was 0.25%.The best storage condition for yogurt was 4℃,and the shelf life was 21d. |
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