Study on Glutathione from Yeast Extract

A large number of studies have shown that the physiological activity of glutathione is of great value to people's production and life, and it has played an important role in various fields such as food, medicine and health care products. At present, the most commonly used method for the industrial production of glutathione is the pure strain fermentation method, and the strains used are mainly Saccharomyces cerevisiae and Escherichia coli. The purebred fermentation strains have relatively harsh requirements on environmental conditions, and the fermentation time is relatively long. In the process of optimizing the fermentation to increase the glutathione content, the increase in glutathione yield is based on the conditions of the strains themselves, so in order to obtain a higher yield of glutathione, it is necessary to work on the selection of strains with high yield, and to improve the ability of the strains to synthesize glutathione through certain technological means (mutation breeding and crossbreeding of genetic engineering, etc.). Through certain technical means (mutation breeding, genetic engineering hybridization, etc.), the ability of the strain to synthesize glutathione can be improved. This method has been widely used in industrial production.

Brewer's yeast is very rich in nutrients, and it has been determined that it contains a large amount of proteins, essential amino acids, and a small amount of RNA, and the cell wall of the yeast cells contains 25%-35% of yeast polysaccharides, mainly glucans and mannans[87] . In recent years, with the rapid development of China's brewing industry, the production of beer has increased year by year. From the latest data, China's beer production will be around 30-40 million tons from 2016 to 2021, and the annual beer production is far ahead of other countries, and every 200 t of beer can produce 3.0 t of brewer's yeast sludge with a water content of about 80%.

The purpose of this experiment is to biosynthesize glutathione directly from fresh beer yeast after beer production from the perspective of economy and environmental protection. The advantage of this experiment is to utilize the waste cells of beer yeast after fermentation, and add a large amount of physiologically active fresh yeast directly into the glutathione production process without the need of pre-cultivation of yeast, which not only shortens the production cycle and saves energy, but also makes the beer yeast cheaper and more abundant. This method does not require strict cultivation conditions, and the synthesis of glutathione can be carried out with sufficient raw materials under the conditions of less time-consuming and low cost, which can not only create more economic benefits for beer manufacturers and realize the effective use of resources, but also solve the problems of waste of resources and environmental pollution, which is of important economic and social effects.

1. Introduction

The production of glutathione by yeast fermentation has been studied at home and abroad, and the fermentation method has obvious advantages, and the strains used for the production of glutathione are mainly bacteria or yeast cells. These cells are easy to cultivate, simple to handle, and the reaction conditions are mild. Therefore, fermentation has become the most commonly used method for glutathione production.

The optimization of the brewer's yeast culture process focuses on the nutritional conditions and environmental factors of glutathione fermentation, including the influence of nitrogen and carbon sources, metal ion concentrations and precursor amino acids on the synthesis of glutathione by the cells.

There are not many reports on the direct synthesis of glutathione from fresh brewer's yeast after tankering. In this chapter, the effects of culture components, culture conditions and precursor amino acid addition strategies on glutathione synthesis by brewer's yeast were investigated to find out the optimal biosynthesis conditions and amino acid addition strategies through one-way optimization experiments and response surface analysis experiments in order to improve the glutathione production.

2.Experiment

2.1 Experimental Materials

Brewer's yeast was provided by Yantai Muping Brewery (fresh yeast from the brewery after the beer production). Since the fresh brewer's yeast has a limited life span and is prone to lose its physiological activity if left for a long time, long-term experiments need to use different batches of brewer's yeast, which inevitably have differences in physiological activity and thus in their ability to synthesize glutathione, so in order to minimize the experimental errors and ensure the accuracy of the data, we tried to ensure that the same batch of beer yeast was used under the same factors and levels. In order to minimize the experimental errors and ensure the accuracy of the data, we try our best to use the same batch of brewer's yeast for the experiments under the same factors and levels.

2.2 Materials and Reagents

Table 2. 1 Materials and Reagents

2.3 Main Instruments and Equipment

Table 2.2 Instruments and Equipment

2.3 Basic Media

Shake flask medium: 25 g/L glucose, 10 g/L peptone, 1.0 g/L K2HPO4, 1.0 g/L KH2PO4, 0.3 g/L CaCl2, 2.5 g/L MgSO4, 0.05 g/L FeSO4.

The pre-medium composition is as above, and the post-medium will have some adjustments in the optimization process.

2.4 Cultivation Conditions

A certain amount of pre-treated brewer's yeast sludge was added into a conical flask, and mixed thoroughly according to the ratio of 20% of the volume of yeast sludge to the volume of medium to dissolve the yeast sludge. The fermentation time was 24 h, the fermentation temperature was 28℃, the shaking speed was 160 rpm, and the volume of liquid was 24 mL/300 mL. The samples were taken at intervals of 3.0 h. The cell biomass, total glutathione and intracellular content were determined in three parallel sets of experiments.

2.5 Measurement Methods

2.5.1 Brewer's Yeast Pretreatment

Pre-treatment of fresh beer yeast slurry: In the fresh beer yeast slurry from Muping Brewery, add twice the volume of deionized water and stir and rinse 4~5 times, when the yeast slurry settles, discard the supernatant and floating matter, rinse repeatedly until the upper layer of the liquid becomes colorless and clear from yellow, and then discard the supernatant after sufficiently static and get the yeast slurry into 4.0℃ refrigerator to be used.

Brewer's yeast cell pretreatment for glutathione determination: Take 5.0 mL of fermentation broth of brewer's yeast cells and put it in a centrifuge tube, centrifuge at 4500 rpm, discard the supernatant, add 40% ethanol solution, extract for 2.0 h at 40℃, centrifuge at 5000 rpm, and take the supernatant and dilute it for a certain number of times, which will be used as the samples for the determination of glutathione.

2.5.2 Determination of Cellular Biomass of Brewer's Yeasts

Brewer's yeast organisms obtained by centrifugation and the centrifuge tube were dried in an oven at 80°C to a constant weight and weighed accurately, and the total weight of the brewer's yeast organisms and the centrifuge tube was weighed as (w1). Then the dried brewer's yeast organisms in the centrifuge tube were washed out, and the centrifuge tube was dried to a constant weight, and the weight of the centrifuge tube was weighed as (w0), and the total weight of the centrifuge tube was weighed as (w0). w(g)=w1-w0.

2.5.3 Methods for the Determination of Glutathione

Tetroxidine method [64]: Take 1.0 mL of the sample solution to be tested, add 0.5 mL of 0.1 mol/L glycolic acid, 3.5 mL of 0.24 mol/L phosphate buffer solution, and 1.0 mL of 1.0 g/L tetroxidine solution in order to fully react for 20 min, and then the absorbance value of A305 was measured by ultraviolet spectrophotometer at 305 nm.

2.6 Experimental Methods

2.6.1 Determination of Beer Yeast Fermentation Time

The pre-treated brewer's yeast sludge was mixed with the medium at 20% by volume and added to shake flasks at 8% volume. The fermentation was incubated continuously for 30 h at a temperature of 28℃ and a rotational speed of 160 rpm. The total glutathione, intracellular content and cellular biomass of the yeast were measured by taking 5.0 mL of the yeast fermentation broth every 3 hours. Three groups of parallel experiments were set up.

2.6.2 Main Culture Conditions and the Effect of Nutrient Composition

2.6.2.1 Effect of Loading Volume on Synthesized Glutathione

In this experiment, 300 mL shake flasks were filled with 6%, 8%, 10%, 12% and 14% of brewer's yeast fermentation broth and incubated for 24 h at a temperature of 28 ℃ and a rotational speed of 160 rpm. The optimal loading was determined by calculating the total amount of glutathione, the intracellular glutathione and the biomass of the brewer's yeast cells, and three groups were set up for the parallel experiments.

2.6.2.2 Effect of Temperature on the Synthesis of Glutathione

In this experiment, the optimum temperatures were determined by calculating the total glutathione, intracellular glutathione and cellular biomass of brewer's yeast cells at 24℃, 26℃, 28℃, 30℃ and 32℃, and the optimum loading volume was determined in Experiment 2.6.2.1, and the incubation was carried out for 24 hours at a rotational speed of 160 rpm. Three parallel experiments were set up.

2.6.2.3 Effect of Glucose Concentration on Synthesized Glutathione

In this experiment, four levels of glucose concentration, namely 15 g/L, 20 g/L, 25 g/L, 30 g/L and 35 g/L, were set up and incubated for 24 h at 160 rpm under the optimal loading volume and temperature as determined in the previous experiments 2.6.2.1 and 2.6.2.2. The optimal glucose concentration was determined by calculating the total glutathione, intracellular glutathione content and cellular biomass of the brewer's yeast. Three parallel experiments were set up.

2.6.2.4 Effect of Peptone Concentration on Synthesized Glutathione

Four levels of peptone concentration, 15 g/L, 20 g/L, 25 g/L, 30 g/L, and 35 g/L, were used in this experiment. The incubation was carried out at 160 rpm for 24 h under the optimum loading, temperature and glucose concentration determined in the previous experiments. The optimum peptone concentration was determined by calculating the total glutathione, intracellular glutathione and cellular biomass of brewer's yeast. Three parallel groups were set up.

2.6.2.5 Effect of Magnesium Sulfate Concentration on Synthesized Glutathione

In this experiment, four levels of magnesium sulfate were added at 1.5 g/L, 2.0 g/L, 2.5 g/L, 3.0 g/L, 3.5 g/L, respectively. Under the optimal loading volume, optimal temperature and optimal glucose concentration determined in the previous experiments, the incubation was carried out at 160 rpm for 24 hours. The optimal concentration of magnesium sulfate was determined by calculating the total amount of glutathione, the intracellular glutathione content and the biomass of brewer's yeast cells. Three parallel experiments were set up.

2.6.3 Response Surface Methodology to Optimize Culture Conditions

Based on the results of the one-way experiment, the three factors of glucose concentration (A), magnesium sulfate concentration (B) and temperature (C) were selected, and then the experimental design was optimized by Box-Behnken, and the factors and levels of the experiment are shown in Table 2.3.

Table 2.3 Experimental factors and levels

Factor Symbol Level 1 Level 2 Level 3

2.6.4 Effect of Precursor Amino Acid Concentration and Time of Addition

In this experiment, we optimized the addition strategies of precursor amino acids in the biosynthesis process of brewer's yeast cells, including the concentration of glycine (Gly), glutamic acid (Glu) and cysteine (Cys), and the time of addition of the three precursor amino acids, and finally, we determined the optimal addition concentration and time by the total amount of synthesized glutathione. The initial addition concentrations of the three precursor amino acids were 10 mmol/L for Gly, 6 mmol/L for Glu, and 4 mmol/L for Cys, and the initial addition time was 21 h for brewer's yeast cells, and 24 h for brewer's yeast cells.

2.6.4.1 Effect of Gly Concentration on Glutathione Synthesis

The three precursor amino acids were added together, and the initial concentrations of Glu and Cys were kept unchanged at 6 mmol/L and 10 mmol/L, respectively, while the concentration of Gly was changed to 3.0 mmol/L, 6.0 mmol/L, 9.0 mmol/L, 12 mmol/L and 15 mmol/L. The optimal concentration of Gly was determined by calculating the total glutathione, intracellular glutathione content and cellular biomass indexes. The total glutathione, intracellular glutathione content and cellular biomass of brewer's yeast were calculated to determine the optimal concentration of Gly and to investigate its effect on glutathione synthesis. Three parallel experiments were set up.

2.6.4.2 Effect of Glu Concentration on Glutathione Synthesis

The optimal concentration of Glu was set at 3.0 mmol/L, 6.0 mmol/L, 9.0 mmol/L, 12 mmol/L, and 15 mmol/L, and the optimal concentration of Glu was determined by calculating the total amount of glutathione, the intracellular glutathione content and the biomass indexes of brewer's yeast cells. The total amount of glutathione, intracellular glutathione content and cellular biomass of brewer's yeast were calculated to determine the optimal concentration of Glu, and to investigate the effect of Glu on glutathione synthesis. Three groups of parallel experiments were set up.

2.6.4.3 Effect of Cys Concentration on Glutathione Synthesis

The optimal concentrations of Gly and Glu in the above experiments were kept unchanged, and the concentrations of Cys were changed to 2.0 mmol/L, 4.0 mmol/L, 6.0 mmol/L, and 8.0 mmol/L. The total amount of glutathione and the intracellular glutathione content and the biomass indexes of brewer's yeast cells were calculated to determine the optimal concentration of Cys to investigate its effect on glutathione synthesis. The optimal concentration of Cys was determined by calculating the total amount of glutathione, the intracellular glutathione content and the biomass indexes of brewer's yeast cells to investigate the effect of Cys on glutathione synthesis. Three parallel experimental groups were set up.

2.6.5 Response Surface Methodology to Optimize Precursor Amino Acid Concentrations

Based on the results of the one-way experiments, three factors, namely, glycine concentration (A), glutamic acid concentration (B) and cysteine concentration (C), were selected, and then Box-Behnken optimized the design of experiments, and the factors and levels of the experiments are shown in Table 2.4.

Table 2.4 Experimental factors and levels

Note: Glycine, glutamic acid, and cysteine concentrations are in mmo/L.

2.6.6 Effect of Timing of Precursor Amino Acid Addition on Glutathione Synthesis

After determining the optimal concentrations of the three precursor amino acids, the effects of the addition time of the precursor amino acids on the synthesis of glutathione by brewer's yeast were investigated, and the addition times were determined to be 13 h, 16 h, 19 h, 21 h, and 24 h, respectively. The brewer's yeast cells were cultured for 24 h. The optimal addition time of precursor amino acids was determined by calculating the total amount of glutathione, the intracellular glutathione content and the biomass of the brewer's yeast cells. Three parallel experiments were set up.

2.6.7 Determination of the Shelf Life of Brewer's Yeasts

In this experiment, we measured the changes in cell growth and the total amount of glutathione produced by brewer's yeast over a period of 25 d. The results were obtained from the first day of fresh brewer's yeast collection to the 25th day. The cellular biomass, total glutathione and intracellular glutathione content of the brewer's yeast were measured every day from the first day of collection to the 25th day of incubation of fresh brewer's yeast stored in a refrigerator at 4℃ before incubation, and after incubation of brewer's yeast optimized for 24 h with the addition of precursor amino acids, and the incremental increase in cellular biomass, total glutathione and intracellular glutathione content of brewer's yeast before and after incubation were calculated. The incremental changes in the total glutathione and intracellular glutathione content were calculated before and after the incubation of the yeast.

2.7 Results and Analysis

2.7.1 Determination of Fermentation Time of Beer Yeast Cells

According to the results of total glutathione, intracellular glutathione content and biomass of yeast cells, which were measured and calculated after 30 h of brewer's yeast cell culture, the results were plotted in Fig. 2.1, from which it can be seen that brewer's yeast cells were synthesizing intracellular glutathione during the period from 0 h to 13 h as the cells continued to grow, and that the brewer's yeast cells basically ceased to grow and were still synthesizing glutathione from 13 to 26 h. The total glutathione content was measured and calculated after 30 h of culture, and the total glutathione content and biomass of yeast cells were plotted in Fig. 2.1. At 13 h to 26 h, the growth of brewer's yeast cells basically stopped while glutathione was still being synthesized. Studies have shown [62] that the addition of precursor amino acids at these two stages can increase the production of glutathione. The total amount of glutathione, the intracellular glutathione content and the biomass of the yeast cells were 102.42 mg/L, 4.66 mg/g and 21.46 g/L in the fresh yeast cells from the brewery just after the production of beer. From the experimental data, it can be seen that if glutathione is extracted directly, the glutathione yield is low and the economic benefit is low, so it is considered to utilize the fresh yeast just out of the tank for targeted recultivation, making full use of the glutathione synthetizing enzyme system in the fresh yeast cells, providing suitable nutrition and culture conditions for further biosynthesis of glutathione. As shown in Figure 2.1, the total glutathione, intracellular glutathione and yeast cell biomass reached the maximum values of 26.82 g/L, 7.79 mg/g and 208 mg/L, respectively, after 24 h of incubation. Considering the time factor, the optimal fermentation time of 24 h was determined, which saved a lot of time and resources compared with the pure fermentation.

Figure 2. 1 Brewer's yeast growth and glutathione synthesis curves.

2.7.2 Effects of Major Culture Conditions and Nutrients

2.7.2.1 Effect of Loading Volume on Glutathione Synthesis

The amount of liquid loaded in the experiment is mainly considered to be the level of dissolved oxygen in the brewer's yeast cell culture medium, which affects the growth of the brewer's yeast cells as well as the synthesis of intracellular glutathione to a certain extent.

Figure 2.2 Effect of loading volume on glutathione synthesis

Fig.2.2 Effect of liquid loading on glutathione synthesis

Figure 2.2 shows that the total amount of glutathione, the intracellular glutathione content and the biomass of the yeast cells increased and then decreased with the increase of the loading volume. The main reason for this may be that when the loading volume was small, the oxygen supply was sufficient and the growth of brewer's yeast was vigorous, and the increase in the biomass of brewer's yeast cells led to the increase in the total amount of glutathione, and the biomass of brewer's yeast cells reached the maximum value of 27.12 g/L at a loading volume of 8%, and the biomass of the brewer's yeast cells was gradually reduced with the increase of the loading volume, so the growth of brewer's yeast cells would be inhibited at a larger loading volume, and the growth of brewer's yeast cells would be inhibited at a larger loading volume. The total glutathione and intracellular glutathione reached the maximum value of 212.07 mg/L and 7.82 mg/g, respectively, at 8%.

2.7.2.2 Effect of Temperature on Glutathione Synthesis

Temperature is one of the most important factors affecting the growth of yeast cells. Temperature affects glutathione synthetase activity, which in turn affects the ability of brewer's yeast to synthesize glutathione, leading to differences in glutathione production. The effect of temperature on enzyme activity is that, in general, too low or too high a temperature will result in a decrease in enzyme activity, and only at the optimum temperature will enzyme activity be maximized.

As shown in Figure 2.3, the biomass of yeast cells and the ability to synthesize glutathione increased with increasing temperature, and the maximum values of total glutathione, intracellular glutathione and yeast cell biomass were reached at 28℃, which were 216.51 mg/L, 7.85 mg/g and 27.62 g/L, respectively.

Figure 2.3 Effect of temperature on glutathione synthesis

2.7.2.3 Effect of Glucose Concentration on Glutathione Synthesis

Glucose is essential for the growth and reproduction of yeast cells and the biosynthesis of glutathione. Glucose can provide yeast cells with the energy they need for growth, and on the other hand, it also provides ATP for the synthesis of glutathione, which in turn promotes the synthesis of glutathione. The right concentration of glucose not only facilitates the growth of brewer's yeast cells but also the synthesis of glutathione by the yeast cells.

Figure 2.4 Effect of glucose concentration on glutathione synthesis

Fig.2.4 Effect of glucose concentration on glutathione synthesis

As shown in Figure 2.4, the biosynthesis of glutathione increased with the increase of glucose concentration, and the total amount of glutathione, the intracellular glutathione content and the biomass of yeast cells reached the maximum values at 25 g/L glucose, which were 211.52 mg/L, 7.82 mg/g and 27.82 g/L, respectively. The total amount of glutathione, intracellular glutathione and yeast cell biomass reached the maximum values at 211.52 mg/L, 7.82 mg/g and 27.82 g/L, respectively.

2.7.2.4 Effect of Peptone Concentration on Glutathione Synthesis

Nitrogen source is one of the important conditions for the growth of yeast cells, and different organic nitrogen sources have a certain effect on the growth and synthesis of intracellular substances in the yeast. The experiment mainly investigated the effect of different peptone concentrations on the synthesis of glutathione by brewer's yeast. As can be seen from Fig. 2.5, the effect of peptone concentration on the total amount of glutathione is not significant, and the effect on the biomass of brewer's yeast cells is also not significant. Overall, the effect of peptone concentration on the production of glutathione is not obvious, and from the perspective of cost saving, it can be considered that no peptone should be added in the culture medium in the later stage of the study.

Fig. 2.5 Effect of peptone concentration on glutathione synthesis

2.7.2.5 Effect of Magnesium Sulfate Concentration on Glutathione Synthesis

Magnesium ion is essential for the synthesis of glutathione, which can increase the activity of glutamine-cysteine synthetase (glutathione 1) and glutathione synthetase (glutathione 2), reduce the feedback inhibition of glutathione 1, and improve the enzyme activity[54] . Therefore, the addition of appropriate magnesium sulfate during the incubation process is beneficial to the synthesis of glutathione and improves the total production of glutathione.

From Fig. 2.6, it can be concluded that the concentration of MgSO4 has a relatively large effect on the synthesis of glutathione by brewer's yeast, and also has a large effect on the growth of brewer's yeast cells. With the increase of MgSO4 concentration, the total amount of glutathione and cell biomass increased, and the total amount and intracellular content and cell biomass of brewer's yeast cells reached the maximum when the concentration of MgSO4 was 2.5 g/L, which were 212.88 mg/L, 7.81 mg/g, 27.25 g/L, so the optimal concentration of MgSO4 can be determined as 2.5 g/L. 212.88 mg/L, 7.81 mg/g, and 27.25 g/L, so the optimal concentration of MgSO4 can be determined as 2.5 g/L. The concentration of MgSO4 was 2.5 g/L.

Figure 2.6 Effect of MgSO4 concentration on glutathione synthesis

2.7.3 Response Surface Design Experiments to Optimize Culture Conditions

Based on the results of the above one-way experiments on the optimization of the culture conditions for the biosynthesis of glutathione in brewer's yeast, the results of the

A response surface design (RSD) experiment was conducted using Design Expert 10.0 to investigate the effects of glucose concentration (A), magnesium sulfate concentration (B) and temperature (C) on the total amount of glutathione (Y), which was analyzed by response surface analysis (RSA).

2.7.3.1 Box-Behnken regression analysis

Table 2.5 Box-Behnken design and results

A three-factor, three-level response surface design experiment on glutathione synthesis in brewer's yeast was carried out using Design Expert 10.0 software, and the results were analyzed by regression analysis. The quadratic multinomial regression equation of the selectivity coefficients of the total glutathione biosynthesized by brewer's yeast with respect to the concentrations of glucose (A), magnesium sulphate (B), and temperature (C) was obtained as follows: Y= 194.56+ 5.04*A - 5.61*B - 7.53*C + 0.14*AB - 0.20*AC + 5.07*BC + 7.99*A - 2 5.04*A-12.61*B-7.53*C+0. 14*AB-0.20*AC+5.07*BC+7.99*A2-

37. 13*B2-46.98*C2 2.7.3.2 Analysis of Variance (ANOVA)

Table 2.6 Analysis of Variance for Regression Equations

Note: R2=0.9969; R2Adj=0.9930; "*" denotes significant difference (P<0.05); "**" denotes highly significant difference (P<0.01).

As can be seen from Table 2.6, the F value of the model was 252.64, and the P value was <0.0001, which indicated that the difference of the regression model was highly significant, R2=0.9969, which indicated that 99.69% of the variation could be explained by the model, R2Adj=0.9930, which indicated that the model was significant, and the values of R2and R2adj were relatively close to each other, which indicated that the regression model was more reasonable, and the equation was well fitted. The R2 and R2adj values are close to each other, which means that the regression model is reasonable, the fit of the equation is high and the experimental error is small, and the model can be used to analyze and predict the culture conditions of glutathione synthesis in brewer's yeast. The P-value of the misfit term was 0.9820＞0.01, which indicated that the difference of the misfit term was not significant, i.e., the model did not have misfit phenomenon, and the model could describe the experiment well, and the model was used to analyze the effects of glucose, magnesium sulfate and temperature on the production of glutathione, so as to obtain the optimal response molecule level.

In the model, except for AB, AC and BC, all the items are significant, of which A is significant, B, C, A2, B2 and C2 are highly significant, indicating that glucose has a great influence on glutathione production, magnesium sulfate and temperature have a great influence on glutathione production, and the relationship between the influence of the three factors is B (magnesium sulfate) > C (temperature) > A (glucose).

2.7.3.3 Response Surface Analysis

Combining the results of the first-order and second-order terms of the model equation, it can be concluded that the effects of glucose (A), magnesium sulfate (B) and temperature (C) on the synthesis of glutathione by Brewer's yeast are complex, and are not simple linear relationships, and the response surface effect is significant. The response surface diagram is shown in Fig. 2.7.

Figure 2.7 Response surface plot of factors interaction on glutathione synthesis by brewer's yeast

(a) magnesium sulfate - glucose (b) glucose - temperature (c) temperature - magnesium sulfate

Fig.2.7 Response surface diagram of the interaction of various factors to the synthesis of glutathione by beer yeast

From Figure 2.7, it can be seen that the results of the above one-way experiments are consistent with the results of the response surface design (RSD) experiment, in which the total amount of glutathione increases and then decreases with the increase in the levels of the influencing factors. The response surface design experiment can clearly reflect the important influence of the interaction of various factors on the experimental results. The slope of the response surface represents the strength of the effect of the factor on the total amount of glutathione.

The closer the contour lines at the level of the response surface are to an ellipse, the more significant the effect of the interaction between the two factors is on the results of the experiment. When the contour lines of the response surface are closer to circles, the interaction between the two factors has a less significant effect on the experimental results. From the figure, it can be seen that the interactions between the factors were significant except for the interaction between temperature and magnesium sulfate, which was not significant on the total amount of glutathione.

By analyzing the regression model, the optimal conditions for glutathione synthesis by brewer's yeast were glucose concentration of 24.66 g/L, magnesium sulfate concentration of 2.41 g/L, and temperature of 28.10 ℃, and the total amount of glutathione was 195.64 mg/L under these conditions. In the validation experiment, the above conditions were repeated, and the actual total glutathione amount was 198.62 mg/L. The difference between the total glutathione amount obtained from the repeated experiments and the predicted value was not big, which indicated that this model could predict the culture conditions of the brewer's yeast for the synthesis of glutathione better, and the total glutathione amount obtained from the response surface analysis was a little bit lower than that obtained from the one-way optimization experiment, which was mainly due to the fact that the response surface analysis experiment and the one-way optimization experiment were different. The total amount of glutathione obtained after response surface analysis was a little bit lower than that of the one-way optimization, mainly because the response surface analysis experiment and the one-way optimization experiment were conducted with different batches of brewer's yeast, so the results were slightly different, as mentioned in 2.2.1 Experimental materials.

2.7.4 Effect of Precursor Amino Acid Concentration and Addition Time

The synthesis of glutathione in brewer's yeast cells requires a two-step reaction, in which precursor amino acids are involved in the synthesis of glutathione as intermediate substances in the reaction, so for the reaction system, the concentration of the reactants has a certain influence on the synthesis of the products, and the addition of the required substances at the right time will have a certain effect on the synthesis of the products, so we need to add the precursor amino acid and optimize the concentration and the addition time at the optimal time of the biosynthesis stage of brewer's yeast. Therefore, it is necessary to add the precursor amino acids at the optimal time during the biosynthesis stage of brewer's yeast, and optimize the concentration of precursor amino acids and the addition time.

2.7.4.1 Effect of Gly Concentration on Synthesized Glutathione

Gly, as a reactant in the second step of glutathione synthesis, is synthesized from the intermediate γ-glutamylcysteine, together with ATP and Gly, and catalyzed by glutathione 2. Therefore, the concentration of Gly has a certain effect on the synthesis of glutathione.

Figure 2.8 Effect of Gly concentration on synthesized glutathione

Figure 2.8 shows that the total glutathione and intracellular glutathione content as well as cellular biomass increased with the increase of Gly concentration, which indicates that increasing the Gly concentration not only promotes the growth of brewer's yeast cells, but also facilitates the synthesis of glutathione by brewer's yeast. When the concentration of Gly was 12 mmol/L, the total glutathione and intracellular content reached the maximum value, and with the increase of the concentration, the total glutathione and intracellular content began to decrease, and at this time, the total glutathione was 302.54 mg/L, the intracellular content was 8.98 mg/g, and the cellular biomass was 33.62 g/L. Therefore, the optimal concentration of Gly was 12 mmol/L at the time of the maximum value of the total glutathione. Therefore, the optimal concentration of Gly was 12 mmol/L at the maximum value of total glutathione.

2.7.4.2 Effect of Glu Concentration on Glutathione

Glu is the primary reactant for the synthesis of glutathione, and glutathione1 catalyzes the synthesis of γ-glutamylcysteine under the energy supply of ATP, so the concentration of Glu has a certain effect on the synthesis of glutathione. Figure 2.9 shows that the total amount of glutathione gradually increased with the increase of Glu concentration, and the total amount of glutathione, intracellular content and cellular biomass reached the maximum value when the Glu concentration was 6.0 mmol/L, which were 309.85 mg/L, 9.12 mg/g and 33.86 g/L, respectively, and with the increase of the Glu concentration, the total amount and intracellular content began to decrease, but the effect on the cellular biomass began to decrease. As the concentration of Glu increased further, the total amount of glutathione and the intracellular content of glutathione began to decrease, but the overall effect on cell biomass was not significant. Therefore, the optimal concentration of Glu for glutathione synthesis was determined to be 6.0 mmol/L. The concentration of Glu in the synthesis phase of glutathione was determined to be 6.0 mmol/L.

Figure 2.9 Effect of Glu concentration on glutathione

2.7.4.3 Effect of Cys Concentration on Glutathione

Both Cys and Glu are the initial substrates for the synthesis of glutathione, and the synthesis of the glutathione intermediate (γ-glutamylcysteine) is catalyzed by ATP and glutathione 1, so the concentration of Cys has an effect on the synthesis of glutathione.

Figure 2. 10 Effect of Cys concentration on glutathione

As shown in Figure 2.10, with the increase of Cys concentration, the total amount of glutathione and cell biomass firstly increased and then gradually decreased, so it can be seen that Cys had an inhibitory effect on the growth of brewer's yeast, and the higher the concentration of Cys was, the more obvious inhibition on the growth of brewer's yeast cells. When the concentration of Cys was 4.0 mmol/L, the total glutathione and intracellular content reached the maximum value, and the maximum value of total glutathione was 310.42 mg/L, which was 51.33% higher than that of the control group.

2.7.5 Response Surface Methodology to Optimize Precursor Amino Acid Concentrations

Based on the results of the above one-way experiments on the synthesis of glutathione in brewer's yeast optimizing the concentration of precursor amino acids.

Response surface experiments were conducted using Design Expert 10.0 to investigate the effects of glycine concentration (A), glutamate concentration (B) and cysteine concentration (C) on the total amount of glutathione (Y), and the response surface analysis was performed, and the results of the experiments are shown in Table 2.7 and the analysis of variance (ANOVA) is shown in Table 2.8.

2.7.5.1 Box-Behnken Regression Analysis

Table 2.7 Box-Behnken design and results

Using Design Expert 10.0 software, a 3-factor, 3-level response surface design of experiments was conducted on glutathione synthesis conditions in brewer's yeast, and the results were analyzed by regression analysis to obtain the total amount of glutathione biosynthesized by brewer's yeast.

Quadratic multinomial regression equation for the selectivity coefficients of glycine concentration (A), glutamate concentration (B) and cysteine (C): Y=313.77+10.08*A+2.62*B-14.92*C+0. 17*AB+0.018*AC-0. 15*BC- 23.88*A2-13.29*B2-53.22*C2

2.7.5.2 Analysis of Variance (ANOVA)

As can be seen from Table 2.8, the F value of the model was 275.81, and the P value was <0.0001, which indicated that the difference of the regression model was highly significant. R2=0.9972 indicated that 99.72% of the variation could be explained by the model, and R2Adj=0.9960 indicated that the significance of the model was good, and the values of R2 and R2adj were relatively close to each other, which indicated that the regression model was more reasonable, and the equation fit was good. The values of R2 and R2adj were close to each other, which indicated that the regression model was reasonable, the fit of the equation was high, and the experimental error was small.

Table 2.8 Analysis of Variance for Regression Equations

Note: R2=0.9972; R2Adj=0.9936; "*" denotes significant difference (P<0.05); "**" denotes highly significant difference (P<0.01).

The P-value of the out-of-fit term was 0.1092＞0.01, which indicated that the difference of the out-of-fit term was not significant, i.e., there was no out-of-fit phenomenon in the model, and the model was able to describe the experiments well. The model was used to analyze the effect of glutamate, glutamate, and cysteine on glutathione production and to find out the optimal response molecule level. In the model, all items were significant except AB, AC and BC, of which B was significant, and A, C, A2, B2 and C2 were highly significant, indicating that glutamate concentration had a strong effect on glutathione yield, and glycine and cysteine concentrations had a strong effect on glutathione yield, and the relationship between the three factors was C (cysteine)>A (glycine)>B (glutamate). C (cysteine) > A (glycine) > B (glutamic acid).

2.7.5.3Response Surface Analysis

Combining the results of the first-order and second-order terms of the model equations, it can be seen that the effects of glycine concentration (A), glutamic acid concentration (B) and cysteine concentration (C) on the synthesis of glutathione by Brewer's yeast are complex, not simple linear relationships, and the response surface effect is significant. The response plots are shown in Figure 2.11.

Fig. 2. 11 Response surface plots of precursor amino acids to the synthesis of glutathione by brewer's yeast by interaction of factors

(a) Glutamate-glycine (b) Glycine-cysteine (c) Cysteine-glutamate

Figure.2. 11 Response surface diagram of interaction of precursor amino acids with beer yeast for synthesis of glutathione

From Figure 2.11, it can be seen that the results of the above one-way experiments are consistent with the results of the response surface design (RSD) experiment, in which the total amount of glutathione increases and then decreases with the increase in the levels of the influencing factors. The response surface design experiment can clearly reflect the important influence of the interaction of various factors on the experimental results. The slope of the response surface reveals the strength of the effect of each factor on the total amount of glutathione. The closer the contour of the horizontal plane of the response surface is to an ellipse, the more significant is the effect of the interaction between the two factors on the experimental results.

The closer the contour of the horizontal plane of the response surface is to a circle, the less significant is the effect of the interaction between the two factors on the experimental results. As can be seen from the figure, the interactions between the factors were significant, except for the interaction between glycine and cysteine, which was not significant for the total glutathione.

By analyzing the regression model, the optimal conditions for glutathione synthesis in brewer's yeast were 12.64 mmol/L for glycine, 6.30 mmol/L for glutamate, and 3.72 mmol/L for cysteine, and the total amount of glutathione was 316.01 mg/L under these conditions. In the validation experiment, the above conditions were repeated, and the actual glutathione amount was 315.65 mg/L. The total amount of glutathione obtained from the repeated experiments was not much different from the predicted value, which indicated that this model could well predict the culture conditions for glutathione synthesis in brewer's yeast.

2.7.6 Effect of Precursor Amino Acid Addition Time on Glutathione

Brewer's yeast cell growth is divided into two phases: the first phase is the growth of the yeast cell itself, in which the yeast cell itself grows and increases in number, with a small amount of synthesis of the yeast glutathione; the second phase is the synthesis of intracellular glutathione, due to the depletion of glucose and other nutrients in the culture, the cell itself reaches a peak in number, and then the synthesis of cellular contents, including a large amount of glutathione synthesis, Liang Bin et al. The second stage is the intracellular glutathione synthesis stage, in which the number of cells peaks due to the depletion of glucose and other nutrients in the culture components, followed by the synthesis of cellular contents, including the large amount of glutathione synthesis. After 30 h of incubation, the intracellular content was 10.41 g/L, which was 10.5% higher than the control, and the glutathione production was 278 mg/L, which was 116% higher than the control.

Relevant studies have shown that the addition of precursor amino acids at the stage of glutathione synthesis will further increase the total amount of glutathione, therefore, according to the synthesis of glutathione in the brewer's yeast cells in this experiment, we set the time of addition of precursor amino acids to the brewer's yeast cells at the 13th, 16th, 19th, and 24th h of cell culture.

Fig. 2. 12 Effect of time of addition of mixed precursor amino acids on glutathione

Fig.2. 12 Effect of precursor amino acid addition time on glutathione

As shown in Figure 2.12, with the increase of addition time, the glutathione content and intracellular content reached the maximum value at 21 h, and the cell biomass also reached the maximum value, but there was not much difference in the overall level, and the total amount of glutathione, the intracellular glutathione content, and the biomass of the yeast cells decreased significantly at 24 h, which was close to the maximum production of glutathione in yeast cells under optimized cultivation conditions but without the addition of precursor amino acids. The total glutathione amount, intracellular glutathione content and yeast cell biomass decreased significantly at 24 h, which was close to the maximum production of glutathione in yeast cells under optimized culture conditions without the addition of precursor amino acids. The reason for this may be that the addition of precursor amino acids at 24 h was the end of the fermentation of brewer's yeast cells and the yeast cells were unable to synthesize their own glutathione from the precursor amino acids in a short period of time. When the addition time was 21 h, the maximum glutathione content was 315.87 mg/L, which was 52.51% higher than that of the control group, so the optimal addition time was determined to be 21 h. The results showed that the glutathione production of the yeast was higher than that of the control group.

2.7.7 Determination of Cell Retention Time of Brewer's Yeasts

During the experimental investigation of brewer's yeast, it was found that the ability of the yeast cells to synthesize glutathione under the same conditions decreased with the extension of the storage time after the fresh brewer's yeast was retrieved, and the ability of the yeast to synthesize glutathione weakened with the extension of the storage time. Therefore, in order to ensure the stability of the experimental data, the storage time of brewer's yeast cells was measured to ensure that the experiments were conducted as soon as possible before the decline in the ability of brewer's yeast cells to synthesize glutathione, so as to accurately grasp the accuracy of the results of the experiments.

Fig. 2. 13 Changes in cell biomass and incremental glutathione production in brewer's yeast before and after optimization over 25 d.

From Fig. 2.13, it can be seen that with the increase of storage time, the biomass enhancement rate of the optimized cultured brewer's yeast cells gradually decreased, which indicated that the growth of brewer's yeast cells with longer storage time did not increase significantly after optimization, and this may be mainly due to the fact that the activity of brewer's yeast cells was lost a lot, and the increase of the total amount of glutathione was small, which was not conducive to the subsequent isolation experimental operation.

The physiological activity of brewer's yeast decreased with the increase of the storage time, and its growth and glutathione production capacity decreased with the increase of the storage time at low temperature. The total amount of glutathione, the intracellular glutathione content and the increase rate of yeast cell biomass decreased by 5%, 7% and 9%, respectively, when the storage time was longer than 5 d. Therefore, it is better to cultivate the yeast within the first 5 d after the fresh yeast was dropped from the tanks in order to increase the yield of the biologically synthesized glutathione in brewer's yeast. Therefore, it is better to incubate the yeast for glutathione production within the first 5 d after taking fresh brewer's yeast out of the tank in order to increase the yield of glutathione biosynthesis by brewer's yeast.

3. Summary

In this experiment, we used brewer's yeast cells as strains, and investigated the culture conditions and precursor amino acid addition strategy to enhance the ability of brewer's yeast to synthesize glutathione by itself. The following conclusions were obtained:

（1）Through the one-way optimization of the culture conditions, it was finally determined that the main components of the culture medium were glucose and MgSO4, with the concentrations of 22 g/L~28 g/L and 2.0 g/L~3.0 g/L, respectively, and the external conditions of 26℃~28℃, with a liquid volume of 8%. On the basis of the response surface design method, the three main factors of glucose concentration, magnesium sulfate concentration and temperature were further optimized to obtain the optimal process conditions: glucose concentration of 24.66 g/L, magnesium sulfate concentration of 2.41 g/L, and the temperature of 28.10 ℃. Under these conditions, the total amount of glutathione was 198.62 mg/L, which was 93.93% higher than the initial amount of glutathione. The optimal incubation time of the brewer's yeast cells was 24 h, which was determined by measuring the growth changes of the brewer's yeast.

（2）Through the one-way optimization of glycine, glutamic acid and cysteine, the three precursor amino acids were finally determined to be added at a concentration of 9.0 mmol/L to 15 mmol/L for Gly, 3.0 mmol/L to 9.0 mmol/L for Glu and 2.0 mmol/L to 6.0 mmol/L for Cys, and based on this, the optimum process conditions were obtained by using the Response Surface Design method to further optimize the three main factors Gly concentration, Glu concentration and Cys concentration. On this basis, the three main factors Gly concentration, Glu concentration and Cys concentration were further optimized using response surface design to obtain the optimal process conditions: glycine concentration of 12.64 mmol/L, glutamic acid concentration of 6.30 mmol/L, cysteine concentration of 3.72 mmol/L, and the total amount of glutathione obtained under these conditions was 315.65 mg/L. Compared with the optimal process conditions, the total amount of glutathione obtained under the optimal process conditions was 315.65 mg/L. The total amount of glutathione was increased by 58.92% compared with the optimal conditions and 208.19% compared with the initial amount of glutathione. By optimizing the addition time of precursor amino acids, it was determined that the addition time was the 21st hour after the incubation of brewer's yeast.

(3) Finally, the preservation time of brewer's yeast cells was investigated. After 25 days, the study showed that the glutathione production, intracellular content and cellular biomass of brewer's yeast decreased with the prolongation of the preservation time, and the percentage of the increase in the glutathione production, intracellular content and cellular biomass of brewer's yeast decreased with the addition of precursor amino acids, so the experiment should be carried out within 5 days after fresh brewer's yeast was taken out of the tank to increase its production. Therefore, the experiment should be carried out within 5 d after the fresh brewer's yeast was taken out of the tank in order to increase the yield and ensure the stability of the experimental data.