Study On Screening And Identification Of Zygosaccharomyces Mellis Strains In Stored Honey And Their Metabolic Characteristics

Yeast was often restricted by the conditions of high sugar in the process of natural reproduction or industrial training and its physical properties was affected.Osmotic regulation of yeast cells is the very important biological reactions.Sugar tolerance of regular yeast is limit,which affect the understanding of the sugar tolerance mechanism.The sugar tolerance yeast in environment with high concentration of sugar,fermentation to grow,produce ethanol and carbon dioxide,which has important application value on the entanol fermentation under high concentrations of sugar.Most of having sugar tolerance yeast root in the conditions of high concentrations of sugar,but its resistance mechanism is not yet clear.The ability of the resistance of sugar tolerance yeast had different features.This study intends to screening of sugar tolerance yeast isolated from honey,identification of their species and resistance.Provides the foundation about thick mash fermentation and yeast for alcohol resistant strains of high sugar mechanism research,and the high sugar stress stress mechanism were studied.The results are as follows:Sugar tolerance yeast is a fungus,which in environment with high concentration of sugar,fermentation to grow,produce ethanol and carbon dioxide.The charcteristics:reproducing fast,conversion ability,can fermentation in the harsh environment of high sugar.It was often affected by the conditions of high sugar in the process of natural reproduction or industrial trainingand.Thus,cell osmotic regulation about yeast is the very important biological reactions.Currently,HST yeasts have been widely investigated.However,all the reported yeast strains had HST below 400 g/L at the sugar content ranging from 300 g/L to 650 g/L.Since the yeast strains with HST of above 650 g/L have not been well documented,this study is aiming at screening out HST yeast and identifying their strains.Furthermore,the selected yeast stress tolerance,including A,sugar tolerance,B,ethanol tolerance,C,acid tolerance,D,SO₂ tolerance performerce were evaluated to provide important information for ethanol HGF and HST mechanism.This study intends to screening of sugar tolerance yeast isolated from honey,identification of their species and resistance.Provides the foundation about thick mash fermentation and yeast for alcohol resistant strains of high sugar mechanism research,and the high sugar stress stress mechanism were studied.The results are as follows:（1）The morphological features of this experiment is mainly by physiological and biochemical characteristics:molecular biology characteristics,tolerance,assimilation.Screening to sugar tolerance yeast to conduct a comprehensive system of resistance identification.Finally,6 strains of sugar tolerance yeast isolated from 17 out dated honey were identified as:The strain 7-7142,7-7352,6-7431,7-7551,7-7651,7-7751were Zygo Saccharomyces.mellis.6 strains of yeast have good sugar tolerance,alcohol tolerance,acid tolerance,SO₂ tolerance and assimilation performance is also well.The tolerance can be best and the most comprehensive nutrient utilization ability about the yeast strain 6-7431.The lag phase is 22 h,tolerance of highest sugar concentration of 700 g/L,tolerance of high alcohol content is 12%,the lowest p H of tolerance is 2.5;Among the six strains tested,the 6-7431 yeast strain utilized the widest range of nutrients;it effectively used carbon sources such as glucose,fructose,maltose,sucrose,galactose and glycerol.This strain also utilized urea,ammonium sulfate,peptone,yeast extract powder and other nitrogen sources.Interestingly,the 6-7431 yeast strain survived in vitamin-free culture media.（2）Choosing the yaest strain 6-7431 haved the best the physiological and biochemical properties optimizated alcoholic fermentation parameters and visual analysis with orthogonal assistant software under the condition of sugar concentration of 300 g/L,400 g/L,500 g/L,600 g/L.The results that the ethanol fermentation was affected by yeast inoculation quantity,fermentation temperature,initial p H of the significant effect（P<0.05）about the yeast strain6-7431.The effect factors of the size about fermentation is yeast inoculation quantity>fermentation temperature>initial p H under the condition of sugar concentration of 300 g/L,400 g/L,500 g/L,600 g/L.Namely,the yeast inoculation quantity is the main factors influencing the yeast fermentation,followed by the fermentation temperature and the initial p H.The best yeast 6-7431 fermentation conditions:the Inoculation amount is 10%,the culture temperature is 30℃,the initial p H is 6.0.（3）The yeast strain 6-7431 metabolic network flux in a series of changes have taken place under the condition of high-glucose stress.The carbon flow of the yeast stain 6-7431towards the metabolism branch of synthesis of trehalose,glycerol,ethanol and TCA.Compared with the conventional cultivation,the content of trehalose,glycerol and TCA was increased by 50.00%,17.73%and 4.79%under high-glucose stress,respectively.The change of the output flux of trehalose and glycerol of the yeast strain under the condition of high-glucose stress reflects it,s important protection fuction.The flow of Carbon of TCA of high-glucose stress higher than conventional culture’s.The change of the output flux of TCA of the yeast strain under the condition of high-glucose stress reflects it,s important protection fuction.（4）Specifically,the effects of high-glucose conditions on the morphology and reproduction pattern of the 6-7431 yeast strain were studied by scanning electron microscopy,and the characteristics of stress-induced metabolites were studied by high performance liquid chromatography（HPLC）.The results show that under high-glucose conditions of 300～550g/L,the 6-7431 yeast strain exhibited normal bud reproduction,and the concentrations of alcohol,glycerol,trehalose,organic acids（malic acid,α-ketoglutarate,lactic acid and lemon acid）and other substances were much higher under high-glucose conditions than under low-glucose conditions of 100 g/L sugar.Under high-glucose conditions of 550–700 g/L,the 6-7431 yeast strain gradually underwent spore reproduction from bud reproduction,the concentration of alcohol was lower under high-glucose conditions than under low-glucose conditions,whereas the concentrations of malic acid andα-ketoglutarate increased.Under high-glucose conditions of 750 g/L,the 6-7431 yeast strain was severely dehydrated and the morphology was dry,which halted fermentation activity.