A yeast freeze-drying protective agent screening method, preparation method and application thereof

Abstract

The application discloses a yeast freeze-drying protective agent screening method and application, and belongs to the field of yeast freeze-drying protective agents.A yeast freeze-drying protective agent comprises skimmed milk powder, trehalose, a phosphate buffer and sorbitol; wherein, the concentration of the skimmed milk powder is 12.84%, the concentration of the trehalose is 16.90%, the concentration of the phosphate buffer is 2.05%, and the concentration of the sorbitol is 14.58% in terms of the total mass percentage concentration of the protective agent.

Description

Technical Field

[0001] This invention relates to the field of yeast freeze-drying protectants, specifically to a method for screening, preparing, and applying a yeast freeze-drying protectant. Background Technology

[0002] L-lactic acid has wide applications in food, pharmaceuticals, cosmetics, and environmentally friendly materials. Currently, the industry mainly produces L-lactic acid through calcium salt fermentation, but this method suffers from high production costs, a long process, and generates a large amount of calcium sulfate solid waste, causing significant environmental pollution. Against this technological backdrop, more researchers are focusing on solid waste-free methods for producing lactic acid, eliminating the need for neutralizing agents during fermentation.

[0003] It has been reported that various yeast strains can grow in harsh environments with pH < 3.0. During fermentation, no alkaline neutralizing agent is needed to maintain the fermentation pH, and no calcium sulfate solid waste is produced. This aligns with the national concept of sustainable development and has certain social and economic benefits.

[0004] Vacuum freeze-drying is the most commonly used method for industrial microbial strain preservation. However, freeze-drying inevitably causes certain damage or structural changes to the cells, resulting in a decrease in the survival rate of freeze-dried bacterial powder and a reduction in the activity of the strain after rehydration. Therefore, screening out suitable freeze-drying protectants and their addition ratios is a necessary and urgent technical problem to be solved. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a method for screening, preparing, and applying yeast freeze-drying protectants.

[0006] The objective of this invention can be achieved through the following technical solutions: A first aspect of the present invention relates to a yeast freeze-drying protectant comprising: skim milk powder, trehalose, phosphate buffer, and sorbitol; Of which, based on the total mass percentage concentration of the protective agents, the concentration of the skim milk powder is 12.84%, the concentration of the trehalose is 16.90%, the concentration of the phosphate buffer is 2.05%, and the concentration of the sorbitol is 14.58%.

[0007] Optionally, the freeze-drying protectant is optimized using response surface methodology, including the following steps: First, the effects of different types of protective media on the survival rate of the strain are screened through single-factor optimization experiments to determine the optimal value of each single factor; then, the Plackett-Burman test is used to screen out the main factors that significantly affect the survival rate of the strain; next, the steepest ascent test is used to determine the center point of the factors in the response surface optimization experiment; further, a central composite design is adopted, with the survival rate as the response value, to establish a regression equation between the response value and each significant factor, thereby screening out a set of optimal composite freeze-drying protectant formulations.

[0008] Optionally, the survival rate of the strain is determined by: rehydrating the freeze-dried bacterial powder with YPD medium; taking 1 mL of bacterial solution before and after freeze-drying and performing serial dilutions; then spreading the diluted bacterial solution evenly on the solid culture medium with a spreading stick; and incubating in a 30℃ incubator for 24 h before counting.

[0009] Optionally, the formula for calculating the freeze-dried survival rate of the strain is as follows: Optionally, the specific design of the single-factor experiment is as follows: using skim milk powder as the basic protective medium, setting different concentrations and mixing it thoroughly with sterile water, as a single-component protective agent.

[0010] Optionally, the concentrations of skim milk powder are set at 1%, 5%, 10%, 15%, and 20%.

[0011] Optionally, the specific design of the single-factor experiment is as follows: glucose, trehalose and maltodextrin are used as sugar protection media, and each is thoroughly mixed with sterile water at the same mixing concentration of 10% as a single-component protectant.

[0012] Optionally, the specific design of the single-factor experiment is as follows: using citrate, phosphate buffer and histidine as pH-adjusting protective media, each mixed with sterile water at the same concentration of 1%, as single-component protective agents.

[0013] Optionally, the specific design of the single-factor experiment is as follows: using sorbitol, sucrose, and glycerol as osmotic pressure regulating protective media, each at the same mixed concentration of 10%, they are thoroughly mixed with sterile water to serve as single-component protective agents.

[0014] Optionally, the specific design of the single-factor experiment is as follows: vitamin C, cysteine ​​and monosodium glutamate are used as antioxidant protective media, and are mixed thoroughly with sterile water at the same concentration of 1% to serve as single-component protective agents.

[0015] A second aspect of the present invention relates to a method for preparing freeze-dried yeast powder, comprising the following steps: The yeast sludge was mixed with the freeze-drying protectant mentioned above to obtain a mixed bacterial solution; The mixed bacterial solution is pre-frozen, and the pre-freezing includes a first pre-freezing step and a second pre-freezing step, wherein the first pre-freezing step is carried out at -20°C and the second pre-freezing step is carried out at -80°C; The pre-frozen mixed bacterial solution was then subjected to vacuum freeze-drying.

[0016] Optionally, after the protective agent is prepared, it is mixed with yeast sludge at a ratio of (2-3):1. 1-2 mL of the mixture is then dispensed into vials, sealed with rubber stoppers, and pre-frozen at -20°C for 2-3 hours. After pre-freezing, the sample is transferred to -80°C and frozen for another 5-6 hours. The sample is then removed from -80°C and transferred to a vacuum freeze dryer for 24-30 hours to obtain the freeze-dried sample. The freezing temperature of the vacuum freeze dryer is -65°C, and the vacuum degree is <10 Pa.

[0017] A third aspect of the present invention relates to the use of the above-mentioned yeast freeze-drying protectant in the preparation of freeze-dried yeast powder for maintaining the L-lactic acid fermentation activity of Pichia pastoris.

[0018] Optionally, the yeast is Pichia pastoris, with the preservation number CCTCC NO: M 20251051.

[0019] The beneficial effects of this invention are: Based on single-factor experiments, this invention screened a group of composite freeze-drying protectants with excellent effects on the freeze-drying preservation of yeast through Plackett-Burman experiments and response surface methodology, thereby improving the survival rate of yeast after rehydration. Furthermore, fermentation experiments on rehydrated freeze-dried yeast powder verified that it still possessed good fermentation activity. Attached Figure Description

[0020] The invention will now be further described with reference to the accompanying drawings.

[0021] Figure 1 This is a comparison chart of the results of single-factor tests on different protective media in Experiment Example 1 of this invention; Figures 2-4 The images show the three-dimensional surface plot and contour plot of the effect of the interaction between skim milk powder, trehalose, phosphate buffer and sorbitol on the survival rate in Example 1 of this invention. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: Screening, preparation method, and application of a yeast freeze-drying protectant 1. Strains Pichia pastoris strain preservation number CCTCC NO: M 20251051 2. Reagents and materials YPD medium: 1% yeast extract, 2% peptone, 2% glucose; YPD solid medium: Add 2% agar powder to YPD medium.

[0024] 3. Test methods Strain activation: Remove the glycerol tubes stored at -80℃ and thaw at 4℃. Use an inoculation loop to take a small amount of bacterial suspension and streak it onto YPD solid medium, incubate at 30℃ for 18-20 hours. Pick a single colony and inoculate it onto YPD liquid medium, incubate at 30℃ and 220 rpm for 15-20 hours.

[0025] Preparation of mycelial sludge: Centrifuge the activated seed liquid obtained above at 4℃ and 5000 rpm / min for 10 min, discard the supernatant, wash the precipitate with sterile water to obtain mycelial sludge.

[0026] Preparation of freeze-dried powder: Part of the obtained yeast sludge was counted before freeze-drying, and the other part was mixed with the protectant at a ratio of (2-3):1. 1-2 mL of the mixture was dispensed into vials, sealed with rubber stoppers, and pre-frozen at -20℃ for 2-3 hours. After pre-freezing, the sample was transferred to -80℃ and frozen for another 5-6 hours. The sample was then removed from -80℃ and transferred to a vacuum freeze dryer for vacuum freeze-drying for 24-30 hours to obtain the freeze-dried sample. The freezing temperature of the vacuum freeze dryer was -65℃, and the vacuum degree was <10 Pa.

[0027] Calculation of strain survival rate after freeze-drying: The freeze-dried bacterial powder was rehydrated using YPD medium; bacterial counting was performed using the gradient plate dilution method, with 1 mL of bacterial solution taken before and after freeze-drying for counting; the formula for calculating the strain survival rate is as follows: 4. Shake-flask fermentation experiment The fermentation medium formula is as follows: 4% glucose, 1% yeast extract, 0.1-1% dipotassium hydrogen phosphate, 0.005-0.05% magnesium sulfate, 0.002-0.015% manganese chloride, 0.005-0.02% sodium chloride, and the balance being water. Incubate at 30-35℃ and 200-220 rpm for 48 hours.

[0028] Example 1: Screening and preparation method of a yeast freeze-drying protectant, including: This experimental example uses response surface methodology to optimize the formulation of a yeast freeze-drying protectant. First, single-factor experiments were used to determine the optimal composition affecting strain survival. Then, Plackett-Burman assays identified trehalose, sorbitol, skim milk powder, and phosphate buffer as key factors influencing strain survival. Next, steepest ascent experiments were used to determine the centroid of the response surface optimization factors. Finally, a central composite design was employed, using survival rate as the response value, to establish regression equations between the response value and each significant factor, thus obtaining the optimal compound protectant formulation for yeast. The specific implementation steps are as follows: 1. Activation of the strain Remove the yeast glycerol tubes stored at -80℃ and thaw at 4℃. Use an inoculation loop to take a small amount of bacterial culture and streak it onto YPD solid medium, incubating at 30℃ for 18-20 hours. Pick a single colony and inoculate it onto YPD liquid medium, incubating at 30℃ and 220 rpm for 15-20 hours to obtain activated seed culture. Centrifuge at 4℃ and 5000 rpm for 10 minutes, discard the supernatant, and wash the precipitate with sterile water to obtain bacterial sludge. The obtained yeast sludge was divided into two parts. One part was used for pre-freeze-drying microbial counting, and the other part was mixed with the preservative at a ratio of (2-3):1. 1-2 mL of the mixture was dispensed into vials, sealed with rubber stoppers, and pre-frozen at -20℃ for 2-3 hours. After pre-freezing, the samples were transferred to -80℃ for 5-6 hours. The samples were then removed from -80℃ and transferred to a vacuum freeze dryer for 24-30 hours to obtain the freeze-dried samples. The freeze dryer temperature was -65℃, and the vacuum degree was <10 Pa. The viable cell count was determined, and the survival rate was calculated.

[0029] 2. Single-factor optimization experiment Using lyophilized survival rate as the response value, the effects of basic protective media (different concentrations of skim milk powder), sugar-based protective media, pH-adjusting protective media (pH adjusted to 5.5-6.0), osmotic pressure-adjusting protective media, and antioxidant protective media on the lyophilized survival rate of yeast were evaluated. The results of the single-factor experiments are as follows: Figure 1 As shown.

[0030] 3. Plackett-Burman test Based on the single-factor experiments, the Plackett-Burman experimental design was used to screen the above-mentioned influencing factors. Each factor was tested at two levels, low (-1) and high (+1), for a total of 15 experimental combinations. The experimental design is shown in Table 1. Each treatment was repeated three times, and the average value was taken as the experimental result.

[0031] Table 1. Factor Levels in the Plackett-Burman Experimental Design The results of determining the response values ​​using the Plackett-Burman experimental design are shown in Table 2. The results of the analysis of variance using Mintab 19.1 are shown in Table 3.

[0032] As shown in Table 3, the model P value was 0.038 (P < 0.05), indicating that the Plackett-Burman experimental design factors had a significant impact on the survival rate of the strains within the selected level range.

[0033] Table 2. Results of Plackett-Burman Experimental Design and Response Values Table 3. Results of ANOVA for the Plackett-Burman experimental design 4. Steepest Climb Test Based on the Plackett-Burman test results, significant factors affecting strain survival were selected: trehalose, sorbitol, skim milk powder, and phosphate buffer. A steepest climb experiment was then conducted, with the climb direction and step size determined by the coefficients of each factor obtained from the Plackett-Burman test: factors with positive estimated values ​​were selected at higher levels; factors with negative estimated values ​​were selected at lower levels. The peak value was the center point of the Box-Behnken test analysis.

[0034] The design and results of the steepest ascent experiment are shown in Table 4. As can be seen from the table, the highest survival rate of the strain (80.15%) was achieved when the amounts of skim milk powder, trehalose, phosphate buffer, and sorbitol added were 12%, 16%, 2%, and 14%, respectively.

[0035] Table 4. Experimental Design and Results for the Steepest Climb 5. Response surface optimization Using the central composite design principle of Box-Behnken, three levels were assigned to each of the four significant influencing factors—skim milk powder, trehalose, phosphate buffer, and sorbitol—based on the center point obtained from the steepest climb experiment, as shown in Table 5.

[0036] Table 5 Factor Levels in the Box-Behnken Experiment A response surface methodology was designed with 29 experimental sites across 4 factors and 3 levels. The results are shown in Table 6.

[0037] Table 6 Box-Behnken Test Protocol and Results Each experiment was repeated three times, and the average value was taken. Regression analysis was performed on the experimental data using Design-Expert software to obtain the regression equation with survival rate (Y) as the response value: Y=78.06+1.65A+2.36B-0.0367C+1.28D-0.2550AB+0.7525AC+1.36AD-3.33BC-2.93BD+0.0050CD-4.57A 2 -5.84B 2 -4.79C 2 -4.40D 2 .

[0038] Regression analysis was performed on the regression model, and the results are shown in Table 7. The model's p-value < 0.05 (P = 0.0139) indicates that the quadratic equation of the model is significant, while the lack-of-fit term is not significant (P = 0.1135). The coefficient of determination R0 of the regression equation is also shown. 2 The value of 0.9743 indicates that the regression equation accurately describes the true relationship between each factor and the response value. The order of influence of each factor on survival rate is: trehalose > sorbitol > skim milk powder > phosphate buffer.

[0039] Table 7 Analysis of Variance of the Box-Behnken Trial Note: P < 0.05, the difference is significant; P < 0.01, the difference is highly significant.

[0040] Based on the above regression equation and the analysis of variance of the experimental results, the Design-Expert V11.0.1 software was used to plot the three-dimensional surface graphs and corresponding contour plots of the response surface to yeast survival rate by trehalose, sorbitol, skim milk powder, and phosphate buffer, as shown below. Figure 2 As shown in the diagram, each response surface plot visually represents the impact of the other two factors on survival rate when one factor is at a zero level. The closer the contour lines are to a circle, the less significant the interaction between the two factors; the closer the contour lines are to an ellipse, the more significant the interaction between the two factors. All six response surfaces have extreme points and are convex spheres opening downwards.

[0041] Based on the response surface methodology, the optimal compound freeze-drying protectant formulation is as follows: 12.84% skim milk powder, 16.90% trehalose, 2.05% phosphate buffer, 14.58% sorbitol, with the remainder being water. The theoretically predicted maximum survival rate of the strain is 79.45%.

[0042] 6. Verification Test The optimal compound ratio of yeast strains was verified in a test, with an average survival rate of 81.03% after three replicates. Simultaneously, shake-flask fermentation experiments were conducted on the lyophilized yeast powder and the subcultured culture broth under this compound protectant formulation. The lactic acid content of the fermentation broth was measured, and the differences between the two were small, at 32.52 g / L and 32.15 g / L respectively. This indicates that the lyophilized powder treated with this compound protectant not only has a high strain survival rate but also maintains good fermentation activity.

[0043] Comparative Example 1: The freeze-drying protectants in this comparative example included skim milk powder at a concentration of 12.84%, glucose at a concentration of 10%, phosphate buffer at a concentration of 2.05%, and sucrose at a concentration of 10%. The freeze-dried yeast survival rate was measured to be 63.79%.

[0044] Comparative Example 2: The freeze-drying protectant in this comparative example included skim milk powder at a concentration of 12.84%, glucose at a concentration of 10%, phosphate buffer at a concentration of 2.05%, and sucrose at a concentration of 5%. The freeze-dried yeast survival rate was measured to be 61.80%.

[0045] Comparative Example 3: The freeze-drying protectants in this comparative example included skim milk powder at a concentration of 12.84%, glucose at a concentration of 10%, phosphate buffer at a concentration of 2.05%, and trehalose at a concentration of 13%. The freeze-dried yeast survival rate was measured to be 65.15%.

[0046] Comparative Example 4: The freeze-drying protectants in this comparative example included skim milk powder at a concentration of 12.84%, glucose at a concentration of 10%, phosphate buffer at a concentration of 2.05%, and glycerol at a concentration of 5%. The freeze-dried yeast survival rate was measured to be 57.80%.

[0047] Comparative Example 5: The freeze-drying protectants in this comparative example included 12.84% skim milk powder, 10% glucose, 2.05% phosphate buffer, and 7% glycerol. The freeze-dried yeast survival rate was measured to be 58.35%.

[0048] Comparative Example 6: The freeze-drying protectants in this comparative example included 12.84% skim milk powder, 16.90% trehalose, 2.05% phosphate buffer, and 10% sorbitol. The freeze-dried yeast survival rate was measured to be 59.21%.

[0049] Comparative Example 7: The freeze-drying protectants in this comparative example included skim milk powder at a concentration of 12.84%, trehalose at a concentration of 16.90%, phosphate buffer at a concentration of 2.05%, and sorbitol at a concentration of 18%. The freeze-dried yeast survival rate was measured to be 55.42%.

[0050] Comparative Example 8: The freeze-drying protectants in this comparative example included 12.84% skim milk powder, 20% trehalose, 2.05% phosphate buffer, and 14.58% sorbitol. The freeze-dried yeast survival rate was measured to be 67.30%.

[0051] Comparative Example 9: The freeze-drying protectants in this comparative example included 12.84% skim milk powder, 10% trehalose, 2.05% phosphate buffer, and 14.58% sorbitol. The freeze-dried yeast survival rate was measured to be 56.32%.

[0052] Comparative Example 10: The freeze-drying protectants in this comparative example included 5% skim milk powder, 16.90% trehalose, 2.05% phosphate buffer, and 14.58% sorbitol. The freeze-dried yeast survival rate was measured to be 50.26%.

[0053] Comparative Example 11: The freeze-drying protectants in this comparative example included skim milk powder at a concentration of 9%, trehalose at a concentration of 16.90%, phosphate buffer at a concentration of 2.05%, and sorbitol at a concentration of 14.58%. The freeze-dried yeast survival rate was measured to be 56.52%.

[0054] Comparative Example 12: The freeze-drying protectant in this comparative example included skim milk powder at a concentration of 12.84%, trehalose at a concentration of 16.90%, histidine at a concentration of 1%, and sorbitol at a concentration of 14.58%. The freeze-dried yeast survival rate was measured to be 66.57%.

[0055] The results of the above comparative studies show that different substances and concentrations have significant differences in their freeze-drying protection effects on yeast strains, indicating that there are requirements for the composition and content of substances for freeze-drying protection of Pichia pastoris.

[0056] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0057] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A yeast freeze-drying protectant, characterized in that, include: Skim milk powder, trehalose, phosphate buffer, and sorbitol; Of which, based on the total mass percentage concentration of the protective agents, the concentration of the skim milk powder is 12.84%, the concentration of the trehalose is 16.90%, the concentration of the phosphate buffer is 2.05%, and the concentration of the sorbitol is 14.58%.

2. A method of screening for a freeze-drying protectant formulation for yeast cells, characterized by, The steps are as follows: a) Preliminary screening of protective media components and their concentrations was conducted through single-factor experiments; b) Using the Plackett-Burman assay, identify the key factors that have a significant impact on yeast survival from the components screened in step a); c) Apply the steepest ramp test to determine the center point region for concentration optimization based on the key factors mentioned above; d) Apply response surface methodology to design and optimize experiments within the central region, establish a regression model between the concentration of the key factor and the yeast survival rate, and determine the concentration ratio that makes the predicted yeast survival rate reach its peak based on the regression model.

3. The method of claim 2, wherein, The key factors that significantly affect yeast survival rate as described in step b) are skim milk powder, trehalose, phosphate buffer, and sorbitol.

4. The method according to claim 2 or 3, characterized in that, The response surface methodology described in step d) employs a Box-Behnken design.

5. A method for preparing a freeze-dried yeast powder of a yeast strain, characterized in that, Includes the following steps: The yeast sludge is mixed with the freeze-drying protectant described in claim 1 to obtain a mixed bacterial solution; The mixed bacterial solution is pre-frozen, and the pre-freezing includes a first pre-freezing step and a second pre-freezing step, wherein the first pre-freezing step is carried out at -20°C and the second pre-freezing step is carried out at -80°C; The pre-frozen mixed bacterial solution was then subjected to vacuum freeze-drying.

6. The preparation method according to claim 5, characterized in that, The first pre-freezing step lasts for 2-3 hours, and the second pre-freezing step lasts for 5-6 hours.

7. The preparation method according to claim 6, characterized in that, The volume ratio of the yeast sludge to the freeze-drying protectant is 1:(2-3).

8. The preparation method according to claim 7, characterized in that, The vacuum freeze-drying step is carried out under conditions of a cold trap temperature of -65°C and a vacuum degree of less than 10 Pa for 24-30 hours.

9. The use of the yeast freeze-drying protectant according to claim 1 in the preparation of freeze-dried yeast powder for maintaining the L-lactic acid fermentation activity of Pichia pastoris.

10. The application of the yeast freeze-drying protectant according to claim 1 as described in claim 9 in the preparation of freeze-dried yeast powder for maintaining the L-lactic acid fermentation activity of yeast, characterized in that, The yeast strain is Pichia pastoris, with the preservation number CCTCC NO: M 20251051.

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