Preliminary Study On The Optimization Of Fermentation Technology And Immune Activity Of Eurotium Cristatum

Eurotium cristatum EC,a probiotic,is a natural probiotic cell grown by the“flowering” process under the specific temperature and humidity conditions,commonly known as “golden flower”.And it has good anti-oxidation,anti-bacterial and anti-tumor functions,but the research of these functions is mainly based on solid fermentation and pure culture fermentation.In order to study the optimization and activity of liquid fermentation conditions of Eurotium cristatum,the paper explores the paper.The carbon source of the medium and the influence of nitrogen source factors on the fermentation were determined,and the optimal fermentation conditions of the sclerotium were determined by single factor experiment,orthogonal experiment and response surface experiment.Subsequent evaluation of the preliminary cytotoxic activity of the fermentation product provides a theoretical basis for screening natural anti-tumor drugs.1 Determination of the best carbon source and nitrogen source in the fermentation conditions of Eurotium cristatumLactose,galactose,sucrose,maltose,glucose and soluble starch are used as the carbon source in the liquid fermentation of the sclerotium.The nitrogen source is selected from peptone LP0037,LP0021,ammonium sulfate,corn flour,yeast powder,and betaine.Fermented by strains,the liquid volume is 100 mL,the inoculum is 5%,the fermentation temperature is 28 °C,the rotation speed is 200 r/min,and the shaker is cultured for 6 days,8 days,10 days,12 days,and the fermentation broth is determined.The immunological activity was a single indicator,and the optimal fermentation medium carbon source was determined to be soluble starch,and the nitrogen source was corn flour,and the immune activity was best on the 8th day of fermentation.2 Optimization of fermentation conditions by single factor experiment and orthogonal experimentSoluble starch is the best carbon source,corn flour is the best nitrogen source,potassium dihydrogen phosphate,magnesium sulfate heptahydrate is used as inorganic salt,and the fermentation activity and the dry weight of mycelium are used as indicators to determine the best fermentation raw material.formula.Fermentation temperature(22?,25 °C,28 °C,30 °C),pH(4.5,5,5.5,6),inoculum(4%,6%,8%,10%),liquid volume(50 mL),70 mL,100 mL,120 mL),fermentation time(6 days,8days,10 days,12 days)and other five factors determine the best conditions,through shake flask fermentation,250 mL shake flask volume of 100 mL,The inoculation amount was 5%,the rotation speed was 200 r/min,and the shaker was cultured for 8days.The immunological activity of the fermentation broth and the dry weight of the mycelium were determined.The results of single factor experiments showed that the temperature was 25?,the pH was 6,and the liquid volume was 100 mL,the inoculum size was 8%,and the mycelium had the highest dry weight when the fermentation time was 10 days.On the basis of single factor experiment,the orthogonal experimental design of five factors and four levels of L16(45)was used to further optimize the fermentation conditions of Eurotium cristatum.The results showed that: Within the experimental range,various factors were The factors affecting the fermentation conditions are temperature > liquid loading > inoculum >time > pH.3 Application of response surface methodology to optimize the fermentation conditions of Eurotium cristatumIn order to further optimize the fermentation conditions of the sclerotium sclerotium,the dry weight of the mycelium and the immunological activity of the fermentation broth were used as the evaluation indicators,and the inoculation amount,the liquid volume,and the fermentation of the bacterium in the fermentation process were measured by the response surface method.Temperature,fermentation time,four main conditions for optimization experiments.The results showed that the stability of the fermentation process optimized by the response surface method was good.Finally,the fermentation conditions of the process were as follows: fermentation temperature was 24.04,fermentation days were 9.27 days,liquid volume was 106.54 mL,and inoculum volume was 8.93%.It was analyzed that the optimum mycelium weight was0.923 g.As a result of the verification of the process,the weights of the three bottles of mycelium were 0.875 g,0.911 g,and 0.862 g,respectively.The average value is0.883 g,the variance is 0.0254,and the absolute deviation from the predicted value is<1%,which indicates that the experimental results are in good agreement with the model prediction.4 Evaluation of Cytotoxic Activity of Eurotium cristatumFour kinds of cells,such as A549,B16,SW480,U251 were used as experimental subjects to inhibit tumor cells by C.The role was screened.The results showed that the crude extract of S.cerevisiae had certain inhibitory effects on the four tumor cells,and the inhibition effect on SW480 cells was the best.5 Preliminary study on the immunological activity of crude extracts from the fermentation of Eurotium cristatumThe immunological activity of crude extracts from the fermentation broth of Eurotium cristatum was studied by u RAW264.7 as a model.The crude extract of the fermentation broth was applied to the immune cell RAW264.7 for activity tracking,and found to have a certain proliferation effect on the cells.After endotoxin testing using an endotoxin kit,it was found that the drug did not contain endotoxin.After the endotoxin interference was excluded,the next immunological activity study was carried out,and the immunological activity of the crude extract of the fermentation broth of Eurotium cristatum was evaluated from the level of gene regulation and expression of immune cytokines.The results of RT-QPCR showed that the expressions of INOS,TNF-?,and IL-6 genes were up-regulated compared with the normal group,and There is a dose-effect relationship between drug concentrations.The release amount of cytokines IL-6,IL-1? and TNF-? released from immune cells was detected by using an ELISA test kit.The release of cytokines IL-6 and IL-1? was more than that of the normal group,but the release amount was not significant,and there was no significant quantitative effect relationship with the drug concentration.When the drug concentration is between 6.25 ?g/ml and 50 ?g/ml,the amount of TNF-? released by the cells decreases with increasing concentration.When the drug concentration is 100 ?g/ml,the cells promote the release of TNF-?.It also showed an upward trend,and the amount of TNF-? released by cells was significantly higher than that of the blank control group.There was no significant dose-effect relationship between the amount of TNF-? released by the cells and the drug concentration.