| Glucose repression is a ubiquitous phenomenon in the microbial community.In the long evolutionary process,yeast has obtained a preferential mechanism to utilize efficient carbon sources,giving them a competitive advantage to adapt to changes in environmental conditions.From the perspective of the fermentation industry,the presence of this glucose inhibits the use of other carbon sources by yeast cells,which is often one of the main obstacles affecting the utilization of raw materials and reducing production efficiency.In this study,a natural anti-glucose repression K.marxianus 246 was screened from three samples of Koumiss collected from Inner Mongolia.The effect of glucose on KM246 and control strain DSMZ5422 gene expression was significantly studied by RNA-Seq technique.The set of metabolic pathways,the molecular mechanism of its anti-glucose repression.The anti-glucose repression effect of KM246 strain was further verified by the difference in lactase activity and gene expression.The process conditions for the preparation of the direct injection type starter of KM246 strain were optimized.The results obtained are as follows:(1)Screening and identification of anti-glucose repressor yeast strainA strain of anti-glucose repression yeast was screened from 3 traditional Koumiss collected from Inner Mongolia.According to the cell and colony morphology,physiological and biochemical characteristics,and molecular biology method,it was identified as K.marxianus,named it KM246.(2)Transcriptome analysis of anti-glucose repressor strain KM246By culturing KM246 and K.marxianus standard strain DSMZ5422 on separate carbon source medium(glucose or lactose)and mixed sugar medium(glucose and lactose),according to the residual sugar amount curve,cells with different carbon sources and different growth stages Transcriptome sequencing was performed to analyze the expression levels of related genes and their regulatory mechanisms.It is speculated that due to the decrease in the expression of MIG1 gene in KM246 strain,the transcription of structural genes in lactose permease,lactase and galactose metabolism networks regulated by them is derepressed,so the related genes of lactobacillus yeast KM246 are adaptively evolved.Thereby having an anti-glucose repression effect.(3)Study on lactase activity and gene expression of anti-glucose repressor strain KM246In this study,DSMZ5422 strain was used as a control to analyze the differences in the trends of lactase activity and gene expression of KM246 and DSMZ5422 strains.The results showed that the carbon source utilization,lactase activity and lactase gene expression in KM246 and DSMZ5422 were basically consistent with the trend of lactase activity and expression,and the anti-glucose repression of KM246 was also verified from the aspects of lactase activity and expression.(4)Preparation of KM246 strain direct-injection starterIn this study,the number of viable bacteria after lyophilization was used as an indicator to optimize the freeze-drying process of KM246 by single factor experiment and orthogonal experiment.The results showed that the conditions of the proliferation medium were glucose 7%,potassium nitrate 3%,yeast powder 2%,initial pH 8.0,inoculum amount 2%,and the fermentation was terminated after incubation at 30°C,225 r/min for 20 h.3.04×10~8 CFU/mL high density fermentation broth.The best combination of protective agent is skim milk powder 10%,sucrose 7.5%,sodium glutamate 2.5%,mannitol 5%.After centrifugation at 6000 rpm for 15 min,mix thoroughly with the protective agent in a ratio of 5:1.Freeze-drying at-80°C for 1 h,when the lyophilization time is 40 h,the water content drops to 6.9%.Under these conditions,a dry powder preparation having a viable cell count of 4.4×10~8 CFU/g was obtained,which satisfies the production requirements. |
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