Isolation,Mutation And Screening Of Antioxidant Strains In Gannan Traditional Yak Yogurt

The yak milk is rich in plateau pastoral areas.For generations,nomads use traditional and ancient natural fermentation methods to make yogurt.The fermented milk of yak contains lactic acid bacteria with good genetic stability,strong resistance and excellent fermentation performance.Although domestic scholars have done a lot of research on the screening of lactic acid bacteria,there are still few lactic acid bacteria that can be put into commercial application.Gannan’s unique ecological environment breeds rich and diverse microbial resources.Lactic acid bacteria with strong fermentation and antioxidant capacity are the key to improving the quality of food.In this study,the traditional natural fermented yak yoghurt in Gannan pastoral area was used as the test material.Based on the determination of the fermentation performance of the selected dominant strains.A comparative analysis of its antioxidant properties was conducted,and heavy ion accelerated mutagenesis was carried out on dominant strains.The aim is to select lactic acid bacteria with excellent fermentation performance and antioxidant performance from traditional fermented dairy products in China.The research results are as follows:1.Isolation,identification and characteristics of lactic acid bacteria.A total of 46 strains were isolated from Gannan traditional yak yoghurt samples.Six dominant strains were screened out through the determination of hydrogen peroxide resistance,acid resistance,bile salt resistance and curd resistance.Screening strains were determined by measuring antioxidant activity.Strain M5 was identified as L paracasei by sugar fermentation test and16S rDNA.The DPPH,?OH,and O₂^-·clearance rate of M5 were 48.4%,4.76%,and 49.9%under the cell-free extract with a cell concentration of 1×10⁹ CFU/mL.The yeast survival cell model verified that the cell-free extracts,fermentation supernatant,and intact cells of M5strain increased the survival rate of S.cerevisiae by 95.3%,130%,and 28.5%,respectively.2.Screening of M5 strain by heavy ion beam mutagenesis.The M5 bacterial suspension was irradiated with a heavy ion beam of 0-360 Gy.The results showed that the lethal rate of360 Gy was 90.13%;the positive mutation rate of 100-140 Gy was 26.9%-21.3%,which was significantly higher than the negative mutation rate of 14.3%-11.8%,and it can help the strain to mutate at a dose of 100Gy.After screening with calcium carbonate plates,24-well deep-well plates and shake flask fermentation rescreening,the mutant N1 with strong fermentation and anti-oxidation at a dose of 20 Gy was selected.The acid production capacity of N1 liquid culture fermentation at 26h is 14.4g/L,which was an increase of 42.15%compared with the original strain M5.The total anti-oxidation of cell-free extract,hydroxyl radical inhibitory power and metal Fe²⁺chelating power are 0.166mM FeSO₄ and49.19U/mL,3.06%respectively,increased by 23.8%、34%and 402%compared with M5.The9th generation slant was subcultured and the mutant N1 had good genetic stability.3.Mutant strain characteristics.The growth curves N1 and M5 strains were not significantly different.The absorbance of OD₆₀₀00 in the 24h culture was 1.8-1.9.The survival rate of strain N1 at pH3.0 and 0.3%bovine bile salt was 58.9%and 45.7%,which was 10.5%and 4.58%higher than M5,and the self-cohesion increased by 20.1%.Stomach fluid tolerance and surface hydrophobicity of strains N1 and M5 had no significant difference,and their basic morphology had changed.N1’s ability to tolerate intestinal fluid decreased by4.29%compared to M5.4.Characteristics of fermented milk.Under the same fermentation conditions,N1 and M5 fermented milk had no significant sensory differences at 7 days,but in the evaluation of nutritional components,N1 fermented milk had a higher first limiting amino acid score.The DPPH·scavenging activity of fermented milk was 92.31%and 89.96%,the chelating power of ferrous ions at 60 min was 76.15%and 67.11%,and the H₂O₂ scavenging activity was25.01%and 11.85%,respectively.The antioxidant activity was better than M5 strain.The above research shows that the L.paracasei M5 was screened by Gannan traditional yoghurt,the dominant strain N1 that was obtained by heavy ion beam mutagenesis.has improved acid production capacity and antioxidant capacity.The fermented milk has good probiotic properties.It has the potential to produce fermentation fungus.