Preparation of probiotic freeze-drying protective agent and application of probiotic freeze-drying protective agent in improving survival rate of strains

Abstract

The invention discloses preparation of a probiotic freeze-drying protective agent and application of the probiotic freeze-drying protective agent in improving the survival rate of strains, and belongs to the technical field of microbial protection. The preparation method has a relatively good protection effect on the lactobacillus plantarum TD017, and the survival rate of the lactobacillus plantarum TD017 can be effectively improved; and compared with a blank group, the survival rate of the strain is improved by 81.19%. Vacuum freeze-drying is one of effective methods for protecting the survival rate of lactic acid bacteria, but strains are inevitably damaged in the freeze-drying process; therefore, the preparation of the excellent probiotic freeze-drying protective agent optimizes the formula and perfects the freeze-drying process at the same time, so that the damage of the strain in the freeze-drying process is reduced, and the survival rate of the strain is increased.

Description

Technical Field

[0001] The invention relates to the preparation of a freeze-drying protective agent and the application of the freeze-drying protective agent in improving the survival rate of bacterial strains, belonging to the technical field of microbial protection. Background Art

[0002] Lactic acid bacteria (LAB) are a type of Gram-positive bacteria that are non-spore-forming, anaerobic or facultative anaerobic, and can ferment a series of sugars with lactic acid as the main end product. They have the functions of regulating intestinal flora, maintaining intestinal flora balance, enhancing human immunity, and improving the gastrointestinal tract. Lactobacillus plantarum is a type of facultative lactic acid bacteria with curved rods, short rods with round ends, and chains, and does not produce spores. It is the most widespread lactic acid bacteria in nature and the most closely related to human life. In order to improve the survival rate of the strains, extend the storage time of the strains, and produce highly active and stable bacterial powder, we use vacuum freeze-drying technology, but the proteins and enzymes in the cells of the strains will be severely damaged during the vacuum freezing process, resulting in a decrease in the survival rate.

[0003] In order to improve the survival rate of strains, prolong the storage time of strains, and produce highly active and stable bacterial powder, it is necessary to add appropriate freeze-drying protective agents. These protective agents act as supports and receptors during the rehydration process, provide a certain skeleton structure for dry matter, and protect strain cells during the freeze-drying process, thereby minimizing or effectively avoiding the damage caused by vacuum freeze-drying to strains, adjusting the environment of probiotics during freeze-drying, reducing damage to cells during freeze-drying, and maintaining the original physiological and biochemical characteristics and biological activity of microorganisms as much as possible. Screening suitable strain protective agents from a large number of freeze-drying protective agents and determining their optimal concentrations can significantly improve the survival rate of strains. However, a single protective agent may not achieve the expected protective effect, cannot fully guarantee the survival of strains, and there are significant differences in the protective effects of protective agents of different concentrations. Therefore, we usually adopt a mixed strategy of multiple protective agents to optimize their protective efficacy for strains by accurately adjusting the composition and proportion of the protective agents.

[0004] Vacuum freeze drying is one of the effective methods to protect the survival rate of lactic acid bacteria, but the bacteria will inevitably be damaged during the freeze drying process. Therefore, it is very important to choose a suitable protective agent. Summary of the invention

[0005] The invention aims to reduce the damage of bacteria in the freeze-drying process by preparing a freeze-drying protective agent, and provides a theoretical reference for the freeze-drying research of plant lactobacillus and the preparation of high-efficiency bacterial powder.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] A preparation of a freeze-dried protective agent and its application in improving the survival rate of bacterial strains, comprising the following steps:

[0008] Step 1: Activation and pure culture of strains. (1) Prepare skimmed milk powder into a skimmed emulsion with a volume concentration of 100g / L, and place it at room temperature for more than 0.5h until the milk protein is completely rehydrated. (2) Then use a fermentation tank for high-temperature automatic sterilization. Sterilize at 110℃ in an autoclave for 10 minutes, then cool and return to room temperature; (3) Take out the bacteria preservation tube from the low-temperature refrigerator, thaw it at room temperature, take a loop of the strain and use an inoculation loop to inoculate it into the skimmed emulsion, and place it in a constant temperature incubator at 37±1℃ for 24h of activation culture.

[0009] Step 2: Liquid culture and bacterial sludge collection. Prepare 100 mL of MRS broth in a triangular flask, use an inoculation loop to take a single colony from the plate and inoculate it into the MRS broth and culture it in a constant temperature incubator at 37°C for 18 hours. Then transfer it to a 250 mL sterile centrifuge cup with a lid, centrifuge at 4000 r / min, 4°C, high-speed refrigerated centrifugation for 20 minutes, discard the supernatant, wash with sterile saline, shake, repeat centrifugation twice under the same conditions, and discard the supernatant to obtain bacterial sludge.

[0010] Step 3: Optimization of freeze-drying protective agent conditions. The freeze-drying survival rate of the freeze-drying protective agent is affected by skim milk powder, trehalose and glycerol. Determine the optimal concentration of skim milk powder, trehalose and glycerol.

[0011] (1) Single-factor test of lyophilized protective agent. Different masses of lyophilized protective agents were added to 100 mL of sterile water to prepare a single protective agent, with a total of 15 groups of protective agents of different concentrations. Sterilize at 121°C for 15 minutes, mix the bacterial sludge with skim milk powder solutions of different concentrations in a 1:1 ratio to prepare a bacterial suspension, pre-freeze at -40°C for 2 hours, and immediately transfer to a vacuum freeze dryer and dry at -60°C, 25 Pa for 24 hours to obtain freeze-dried bacterial powder. After taking out the bacterial powder, rehydrate and count, and compare the survival rates of the strains before and after freeze-drying to obtain the optimal concentrations of skim milk powder, trehalose and glycerol. The amount of skim milk powder added was 6, 8, 10, 12, and 14 g; the amount of trehalose added was 4, 5, 6, 7, and 8 g; and the amount of glycerol added was 2, 3, 4, 5, and 6 g. (2) Based on the results obtained from single factors, the formula of the lyoprotectant for Lactobacillus plantarum TD017 was optimized through response surface experiment, and the compound ratio of the lyoprotectant was determined to be 12.3 g / 100 mL of skim milk powder, 7.3 g / 100 mL of trehalose, and 4.2 g / 100 mL of glycerol.

[0012] Step 4: Preparation of lyoprotectant. Skim milk powder, trehalose and glycerol were used as protectants, and the lyoprotectant formula of Lactobacillus plantarum TD017 was optimized by response surface test. The lyoprotectant was prepared according to the lyoprotectant conditions obtained in step 3.

[0013] The freeze-drying protective agent prepared by the invention has the function of improving the survival rate of bacterial strains.

[0014] The beneficial effects achieved by the present invention are:

[0015] 1. In order to reduce the damage of the strains during the freeze-drying process, suitable protective agents can be added to protect the strains, thereby improving the phenomenon of low survival rate of the strains and increasing the survival rate of the strains.

[0016] 2. The average survival rate of Lactobacillus plantarum TD017 before and after freeze-drying is basically consistent with the theoretical value of 92.65%. Compared with the blank group, the survival rate of the strain was significantly improved, indicating that the freeze-drying protection effect of the freeze-drying protectant of Lactobacillus plantarum TD017 optimized by response surface methodology is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Determination of the optimal concentration of skim milk powder;

[0018] Figure 2 To determine the optimal concentration of trehalose;

[0019] Figure 3 To determine the optimal concentration of glycerol;

[0020] Figure 4 The contour map and three-dimensional surface map are the effects of the interaction between skim milk powder and trehalose on the survival rate of bacteria.

[0021] Figure 5 The contour map and three-dimensional surface map are the effects of the interaction between skim milk powder and glycerol on the survival rate of bacteria.

[0022] Figure 6 Contour map and three-dimensional surface map of the effect of the interaction between trehalose and glycerol on the survival rate of bacteria. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present invention.

[0024] A preparation of a freeze-drying protective agent and its application in improving the survival rate of bacterial strains, comprising the following steps:

[0025] Step 1: Activation and pure culture of strains. (1) Prepare skimmed milk powder into a skimmed emulsion with a volume concentration of 100g / L, and place it at room temperature for more than 0.5h until the milk protein is completely rehydrated. (2) Then use a fermentation tank for high-temperature automatic sterilization. Sterilize at 110℃ in an autoclave for 10 minutes, then cool and return to room temperature; (3) Take out the bacteria preservation tube from the low-temperature refrigerator, thaw it at room temperature, take a loop of the strain and use an inoculation loop to inoculate it into the skimmed emulsion, and place it in a constant temperature incubator at 37±1℃ for 24h of activation culture.

[0026] Step 2: Liquid culture and bacterial sludge collection. Prepare 100 mL of MRS broth in a triangular flask, use an inoculation loop to take a single colony from the plate and inoculate it into the MRS broth and culture it in a constant temperature incubator at 37°C for 18 hours. Then transfer it to a 250 mL sterile centrifuge cup with a lid, centrifuge at 4000 r / min, 4°C, high-speed refrigerated centrifugation for 20 minutes, discard the supernatant, wash with sterile saline, shake, repeat centrifugation twice under the same conditions, and discard the supernatant to obtain bacterial sludge.

[0027] Step 3: Optimization of freeze-drying protective agent conditions. The freeze-drying survival rate of the freeze-drying protective agent is affected by skim milk powder, trehalose and glycerol. Determine the optimal concentration of skim milk powder, trehalose and glycerol.

[0028] (1) Single factor test of lyoprotectant. Different masses of lyoprotectants were added to 100 mL of sterile water to prepare a single protectant, with a total of 15 groups of protectants of different concentrations. Sterilize at 121°C for 15 minutes, mix the bacterial sludge with skim milk powder solutions of different concentrations in a 1:1 ratio to prepare a bacterial suspension, pre-freeze at -40°C for 2 hours, and immediately transfer to a vacuum freeze dryer and dry at -60°C, 25 Pa for 24 hours to obtain freeze-dried bacterial powder. After taking out the bacterial powder, rehydrate and count it, and compare the survival rate of the strain before and after freeze-drying to obtain the optimal concentrations of skim milk powder, trehalose and glycerol, among which the amount of skim milk powder added was 6, 8, 10, 12, 14 g; the amount of trehalose added was 4, 5, 6, 7, 8 g; and the amount of glycerol added was 2, 3, 4, 5, 6 g. (2) Based on the results obtained from the single factor, the formula of the lyoprotectant for Lactobacillus plantarum TD017 was optimized through response surface experiment.

[0029] Determination of the optimal concentration of skimmed milk powder:

[0030] The results of the effect of skim milk powder concentration on the survival rate of Lactobacillus plantarum TD017 are as follows Figure 1As shown in the figure, with the increase of skim milk powder concentration, the survival rate of the strain showed a trend of first increasing and then decreasing, and reached a peak of 59.6% at 12g / 100ml. Therefore, the skim milk powder concentration in the best protective agent formula for preparing Lactobacillus plantarum TD017 was determined to be 12g / 100mL. Studies have shown that the freeze-drying protective effect of skim milk powder comes from its structure. Skim milk powder is a macromolecular substance with good freeze-drying effect. During freeze-drying, the whey protein in the milk powder can form a protein membrane protective layer outside the bacteria to protect the cells and fix freeze-dried enzymes

[16] . However, when the concentration of skim milk powder reaches a certain concentration, the survival rate of the strain decreases. The reason is that the high concentration of protective agent accelerates the protein polymerization in the cells, forming a strong glass structure, which is not conducive to the low-temperature preservation of bacterial cells and the rehydration effect is poor.

[0031] Determination of the optimal concentration of trehalose:

[0032] The results of the effect of trehalose concentration on the survival rate of Lactobacillus plantarum TD017 are as follows Figure 2 As shown in the figure, with the increase of trehalose concentration, the survival rate of the strain showed a trend of increasing first and then decreasing, and reached a peak of 82.3% at 7g / 100ml, so it was more appropriate to determine the trehalose concentration in the best protective agent formula for preparing Lactobacillus plantarum TD017 as 7g / 100mL. Studies have shown that the protective effect of trehalose comes from hydroxyl groups. Trehalose has multiple hydroxyl groups and is a non-reducing disaccharide with extremely strong stability. It can combine with free radicals on the surface of bacteria to prevent bacteria from being exposed to the medium. At the same time, it can form hydrogen bonds with proteins to replace water and stabilize cell membranes and protein structures. However, when the concentration of trehalose is too high, it will cause an imbalance in the osmotic pressure inside and outside the bacterial cells, leading to the death of the bacteria and reducing the survival rate of the bacteria.

[0033] Determination of the optimal concentration of glycerol:

[0034] The results of the effect of glycerol concentration on the survival rate of Lactobacillus plantarum TD017 are as follows Figure 3 As shown in the figure, with the increase of glycerol concentration, the survival rate of the strain showed a trend of increasing first and then decreasing, and reached a peak of 38.7% at 4g / 100ml. Therefore, it is more appropriate to determine the glycerol concentration in the best protective agent formula for preparing Lactobacillus plantarum TD017 as 4g / 100mL. The study found that the protective effect of glycerol comes from its properties. Glycerol can penetrate into the bacteria, enhance the ability of the bacteria to bind water, reduce the free water content, and increase the viscosity of the solution, thereby slowing down the growth of the crystal nucleus and making the formed ice crystals smaller, so as to achieve the purpose of protecting the bacteria. However, since glycerol has a strong water absorption capacity, when the glycerol concentration is too high, it may cause the bacterial cells to dehydrate, resulting in a decrease in the survival rate of the bacteria.

[0035] Response surface optimization test results of protective agent formula:

[0036] The results obtained from the single-factor experiments were optimized by response surface design using Design-Expert 8.0.6.1 software. Taking the skim milk powder concentration A (g / 100 mL), trehalose concentration B (g / 100 mL), and glycerol concentration C (g / 100 mL) as independent variables, and the survival rate (%) of Lactobacillus plantarum TD017 as the response value, a three-factor and three-level response surface experiment was conducted according to the Box-Behnken experimental design principle. There were a total of 17 experimental points, with 5 being zero points, used to evaluate the experimental error.

[0037] Table 1 Box-Behnken experimental scheme

[0038]

[0039]

[0040] Using Design-Expert 8.0.6.1 software for multiple regression fitting, the regression equation for the strain survival rate was obtained as: Y = 91.28 + 3.26A + 7.82B + 1.14C + 2.81AB - 2.52AC + 5.71BC - 12.93A2 - 16.58B2 - 8.49C2

[0041] As can be seen from Table 2, the P value of the model < 0.01, that is, the regression model is extremely significant; the P value of the lack-of-fit term > 0.05, indicating that the model fitting effect is good and the error is small; from the effects of each factor on the survival rate of Lactobacillus plantarum TD017 obtained by equation fitting, it can be concluded that the P value of glycerol > 0.05; the P value of skim milk powder > 0.01 and < 0.05; the P value of trehalose < 0.01, that is, trehalose has the greatest impact on the survival rate of Lactobacillus plantarum, followed by skim milk powder on the strain survival rate, and glycerol has the least impact on the strain survival rate.

[0042] Table 2 Analysis of variance of the survival rate of Lactobacillus plantarum TD017

[0043]

[0044]

[0045] Note: ** indicates extremely significant (P < 0.01), * indicates significant (0.01 < P < 0.05), and no * indicates not significant (P > 0.05)

[0046] Analyze the response surface and two-dimensional contour plots:

[0047] Figure 4 , 5, 6 are the contour plots and response surface plots made by Design-Expert.V8.0.6.1 software, which can reflect the interaction effects between factors and the response value.

[0048] With the increase of skim milk powder concentration and trehalose concentration, the survival rate of Lactobacillus plantarum TD017 strain showed a trend of first increasing and then decreasing. The slope of skim milk powder and trehalose was steeper, the contour lines were denser, and the elliptical curvature was larger. This shows that the interaction between skim milk powder and trehalose was significant. Moreover, the contour lines of trehalose concentration were denser, indicating that the main effect of trehalose concentration was greater than that of skim milk powder concentration.

[0049] The survival rate of Lactobacillus plantarum TD017 showed a trend of increasing first and then decreasing with the increase of skim milk powder concentration and glycerol concentration. The slope of skim milk powder and glycerol was gentle, the contour lines were sparse, and the ellipse curvature was small, indicating that the interaction between skim milk powder and glycerol was not significant.

[0050] The survival rate of Lactobacillus plantarum TD017 showed a trend of increasing first and then decreasing with the increase of trehalose concentration and glycerol concentration. The slopes of trehalose and glycerol were steeper, the contour lines were denser, and the elliptical curvature was larger, indicating that the interaction between trehalose and glycerol was significant. Moreover, the contour lines of trehalose concentration were denser, indicating that the main effect of trehalose concentration was greater than that of glycerol concentration.

[0051] Step 4: Preparation of lyoprotectant. Skim milk powder, trehalose and glycerol were used as protectants, and the lyoprotectant formula of Lactobacillus plantarum TD017 was optimized by response surface test. The lyoprotectant was prepared according to the lyoprotectant conditions obtained in step 3. The obtained lyoprotectant was used for subsequent Lactobacillus plantarum TD017 survival rate (%) experiment.

[0052] Embodiment 1: The results obtained from the single factor experiment were optimized by response surface design using Design-Expert 8.0.6.1 software. With skim milk powder concentration A (g / 100 mL), trehalose concentration B (g / 100 mL), and glycerol concentration C (g / 100 mL) as independent variables and Lactobacillus plantarum TD017 survival rate (%) as response value, a 3-factor 3-level response surface experiment was performed according to the Box-Behnken experimental design principle, with a total of 17 test points, 5 of which were zero points, to evaluate the experimental error.

[0053] The survival rates of the above 17 groups of Lactobacillus plantarum TD017 were measured.

[0054] In summary, the optimal freeze-dried protective agent formula has a good protective effect on Lactobacillus plantarum TD017 and can effectively improve the survival rate of Lactobacillus plantarum TD017.

Claims

1. A preparation of a freeze-dried protective agent and its application in improving the survival rate of bacterial strains, characterized in that: Skim milk powder 12.28g / 100mL, trehalose 7.27g / 100mL, glycerol 4.14g / 100mL.

2. The preparation of a freeze-dried protective agent according to claim 1 and its use in improving the survival rate of bacterial strains, characterized in that: Freeze-dried protective agents have the function of improving the survival rate of bacteria.

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