Isolation And Screening Of High Quality Lactic Acid Bacteria From Kefir And Development Of Yogurt Compound Strain Starter

In recent years,yoghurt has been deeply loved by consumers due to its unique flavor,rich nutrition and certain health effects.However,the homogenization of yoghurt products on the market is serious now,and it can no longer meet people's diversified demands for yoghurt products.The flavor and quality of yoghurt products depend largely on the composition and proportion of the bacteria in the yoghurt starter used.The research on yogurt starters in China started late,and there are few commercial starters with independent intellectual property rights.The market for yogurt starters has basically been monopolized by foreign companies.Therefore,it is of great significance to carry out applied basic research,such as screening lactic acid bacteria(LAB)with excellent fermentation performance and developing novel yogurt composite starters with independent intellectual property rights.In this study,the traditional culture method was used to isolate the LAB in kefir grains from different sources,and the 16S rDNA sequence analysis method was used for identification.Secondly,the excellent LAB were selected through the measurement of fermentation performance.Thirdly,the selected strains were combined and fermented according to their functional characteristics to obtain the best yogurt complex strain starter formula.Finally,an orthogonal test was carried out on the basis of a single factor test to obtain the optimal fermentation conditions for the composite strain starter.And the performance of this yogurt starter was tested and evaluated by comparing with the commercially available foreign starter.The main research contents and results achieved are as follows:(1)Taking 8 kinds of kefir grains from different regions as the isolation source,a total of 43 strains of LAB were isolated and identified,and their phylogenetic tree was constructed.Eight kinds of kefir grains were collected from Xinjiang,Tibet,Inner Mongolia and Northeast China,and they were used as the isolation source of LAB.Through morphology,catalase and Gram stain observation and analysis,43 strains of LAB were obtained.The obtained strains were subjected to specific amplification,determination and homology comparison analysis of 16S r DNA sequences to identify their species,and the phylogenetic tree was constructed.The identification results of 43 strains of LAB are as follows:12 strains of Lactobacillus kefiri,7 strains of Lactobacillus paracasei,5 strains of Lactococcus lactis,5 strains of Streptococcus thermophilus,4 strains of Lactobacillus acidophilus,3 strains of Lactobacillus plantarum,2 strains of Lactobacillus delbruecki subsp.bulgaricus,2 strains of Leuconostoc pseudomesenteroides,1 strain of Leuconostoc mesenteroides,1 strain of Enterococcus durans,and 1 strain of Lactobacillus rhamnosus.(2)The 43 strains of LAB were tested and screened according to the fermentation performance,and 3 excellent strains were obtained.First,43 strains of LAB were screened for genetic stability,curd time,and acid production capacity.12 strains of LAB with better genetic stability,moderate curd time,and better acid production capacity were obtained.They were 4 strains of Lactobacillus kefiri,4strains of Lactococcus lactis,2 strains of Streptococcus thermophilus,1 strain of Lactobacillus rhamnosus,and 1 strain of Enterococcus durans.Secondly,the 12strains were tested for water holding capacity,viscosity production capacity,aroma production capacity,proteolysis capacity,bacteria vitality,and sensory evaluation of their single-bacteria fermented milk.Based on the comprehensive test results,three strains of LAB with the best performance,which can be used for the development of the composite strain starter,were selected:Streptococcus thermophilus YKHH2,Lactococcus lactis SFCE1 and Lactobacillus kefir YKGX5.The water-holding capacity and initial apparent viscosity of Streptococcus thermophilus YKHH2 were64.62%and 9.21 Pa,respectively,which was the most outstanding in terms of water holding capacity and viscosity production capacity.Lactococcus lactis SFCE1 had good acetaldehyde production capacity(14.34?g/mL)and diacetyl production capacity(6.03?g/mL),and a suitable ratio between this two substances(2.38:1),which was the most outstanding in terms of aroma production capacity.Lactobacillus kefir YKGX5 had a strong pre-acid production capacity(87.49°T)and a weak post-acidification capacity(6.35°T).In addition,its amino nitrogen content was353.75?g/mL,and the viable count was as high as 1.31×10~9 CFU/mL,which was also the most outstanding in terms of proteolysis and bacteria vitality.The sensory scores of YKHH2,SFCE1 and YKGX5 single-bacteria fermented milk samples were79.50,76.00 and 65.80 points,respectively,and the evaluation was good in terms of color,flavor and texture.The three strains screened met the requirements of industrial fermentation and had excellent commercial application value and prospects.(3)Three excellent strains of LAB screened were optimized for the compound fermentation,and the best formula of yogurt composite starter was obtained,which was Lactococcus lactis SFCE1:Streptococcus thermophilus YKHH2:Lactobacillus kefir YKGX5=30:300:1.Taking the three strains of excellent LAB as the research object,the antagonistic test between strains and the growth curve test in milk were first carried out.The results showed that there was no antagonism among the three strains and they grew well in milk.Secondly,the best combination ratio test between cocci was conducted,and the results showed that the best ratio was SFCE1:YKHH2=1:10.Yogurt samples at this ratio had better acidity,water holding capacity,hardness and adhesiveness,and the sensory evaluation was the best,with a score of 83.20 points.Finally,on the basis of the best combination ratio between the cocci obtained,the best compounding ratio test between cocci and bacilli was carried out,and the results showed that the best compounding ratio was 30:1.The yoghurt samples at this ratio performed better in acidity,water holding capacity,hardness and adhesiveness,and had the best sensory evaluation,with 84.20 points.The best formula of the yogurt composite strain starter was finally determined to be SFCE1:YKHH2:YKGX5=30:300:1.The fermented yogurt product at this ratio has suitable acidity,good water holding capacity and organizational structure,and the highest sensory score.(4)The fermentation conditions of the compound starter were optimized by orthogonal test.The optimized fermentation test verified that the property of the yogurt composite strain starter was excellent,and the overall performance was better than that of commercially available foreign starters.Taking inoculum amount,fermentation temperature and sucrose addition as influencing factors,orthogonal optimization test was carried out on the basis of single factor test results.The optimal fermentation conditions were obtained as follows:fermentation temperature 33?,inoculation amount 3×10~7 CFU/mL,and sucrose addition amount6%.The sensory evaluation of fermented yogurt under this condition was the best,with a score of 88.10 points,which was higher than the highest score in the orthogonal test group(87.20 points),indicating that the orthogonal test was optimized successfully.Under the optimal fermentation conditions,the yogurt composite starter was evaluated from the aspects of conventional fermentation performance,TPA and rheological characteristics,and volatile aroma components.The results showed that,compared with the commercially available foreign starters,the compound starter had a moderate curd time and was excellent in acid production capacity,water holding capacity,and bacterial vitality.Yogurt fermented by the composite starter had better curd state and texture.When subjected to external forces,the change of its tissue state was relatively small,and the recovery speed was relatively fast.Meanwhile,it has richer volatile aroma components and better sensory evaluation.In summary,the yogurt composite starter obtained in this study had excellent properties,and its overall performance was better than that of commercially available foreign starters.It has excellent industrial application prospects and economic value.