CN117402739A - Method for preparing high-expression cold shock protein lactobacillus bulgaricus agent - Google Patents

Method for preparing high-expression cold shock protein lactobacillus bulgaricus agent Download PDF

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CN117402739A
CN117402739A CN202311417584.8A CN202311417584A CN117402739A CN 117402739 A CN117402739 A CN 117402739A CN 202311417584 A CN202311417584 A CN 202311417584A CN 117402739 A CN117402739 A CN 117402739A
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freeze
lactobacillus bulgaricus
soybean lecithin
drying
agent
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关海滨
贾鑫
麻剑南
李晶
张英
梁宇光
董至恒
田欣圆
刘梦呵
岳艳君
赵璟
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Inner Mongolia Medical University
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    • C12R2001/225—Lactobacillus

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Abstract

The invention relates to a method for preparing a high-expression cold shock protein lactobacillus bulgaricus agent, which comprises the following steps: adding soybean lecithin into lactobacillus bulgaricus fermentation broth, mixing uniformly, placing in a constant-temperature water bath for treatment, centrifuging, discarding supernatant, collecting thalli, adding a freeze-drying protective agent, mixing uniformly, and vacuum drying to obtain freeze-drying bacterial powder. The lyoprotectant comprises skimmed milk powder, lactose, ascorbic acid and soybean lecithin. Soybean lecithin is added in the processes of inducing CspA expression by lactobacillus bulgaricus and freeze-drying, so that the stability of a bacterial liquid system can be improved, the stress resistance of cells is enhanced, the integrity of cell membranes is protected, and the lactobacillus is helped to better cope with challenges in the freeze-drying process, so that the survival rate of lactobacillus bulgaricus after freeze-drying is obviously improved.

Description

Method for preparing high-expression cold shock protein lactobacillus bulgaricus agent
Technical Field
The invention relates to the technical field of freeze-drying protection of thalli, in particular to a method for preparing a Lactobacillus bulgaricus agent with high expression of cold shock proteins.
Background
Lactobacillus bulgaricus (Lactobacillus bulgaricus) belongs to the subspecies bulgaricus of lactobacillus delbrueckii, and is a lactic acid bacterium (lactic acid bacterium, LAB) which is gram-positive in staining, facultative anaerobic, not moving and not producing spores, and is widely applied to fermentation of yoghourt at present because of being capable of endowing the yoghourt with unique taste; in addition, lactobacillus bulgaricus has physiological effects of regulating intestinal flora balance, enhancing immunity, resisting invasion of pathogenic microorganisms and the like, so that the lactobacillus bulgaricus is widely applied to main components of health-care products and medicines; the lactobacillus bulgaricus can be obtained in large quantity by a liquid fermentation method, and bacterial powder is prepared by a freeze-drying method, and is the optimal preservation mode of bacterial, so that the lactobacillus bulgaricus can be used as a direct-vat starter of yoghourt, and can be used in the fields of medicines and health products.
In the industrial production process, the freeze-drying process can cause serious damage to the lactobacillus bulgaricus, and the factors of mechanical damage to cell membranes, denaturation and inactivation of proteins, change of cell membrane permeability, DNA damage and the like are mainly represented by the formation of ice crystals; however, in order to adapt to the low-temperature environment, the thalli also generate cold stress reaction, and a series of low-temperature resistant proteins, namely cold shock proteins (cold shock proteins, CSPs) are generated to resist the damage of the low-temperature environment to the thalli.
The applicant is dedicated to the anti-freezing research of lactobacillus bulgaricus, as in research results of the applicant, namely, research on the influence of low temperature on the expression of the CspA gene of lactobacillus bulgaricus, and the expression of cold shock protein A, the freeze thawing survival rate and the freeze drying survival rate of thalli are examined by carrying out low-temperature induction treatment on the lactobacillus bulgaricus, and the results show that: the low-temperature treatment can obviously improve the expression quantity of the CspA, and the mRNA expression quantity of the CspA is highest after the treatment for 4 hours at 20 ℃, and the freeze-thawing survival rate and the freeze-drying survival rate of thalli are obviously improved, and the CspA can improve the freeze-thawing survival rate and the freeze-drying survival rate of lactobacillus bulgaricus.
The optimization of the lactobacillus bulgaricus freeze-drying protective agent takes lactobacillus bulgaricus as an initial strain, takes the viable count of the freeze-dried bacterial powder of the lactobacillus bulgaricus as an index, optimizes the addition amount of 4 common freeze-drying protective agents, and the optimal freeze-drying protective agent ratio of the lactobacillus bulgaricus is lactose, sodium glutamate, ascorbic acid and skimmed milk powder=3:1:2:7.
Other researches, such as CN115478017A, use cryoprotectants containing glycerol, L-linoleic acid and alpha-linolenic acid to realize the great improvement of the viable count and the viable stability of the Lactobacillus bulgaricus; CN103923835B uses mixed polysaccharide extracts of coix seed and Chinese yam as freeze-drying protective agent, so that the bacterial powder has higher activity.
On the basis of the research of the prior art, the applicant tries to further induce the generation of cold shock proteins and improve the survival rate of the thalli after freezing; lecithin can be classified into animal lecithin and vegetable lecithin according to its origin; lecithin is a fat mixture containing nitrogen and phosphorus which is separated from egg yolk for the first time in 1844; soybean lecithin was also found in byproducts of soybean oil processing in the 30s of the 20 th century; soybean lecithin mainly consists of Phosphatidylcholine (PC) (lecithin), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and soybean oil. Natural lecithins are a mixture of several lecithins containing different fatty acids. Common among the fatty acids of lecithin molecules are palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, and the like. The molecular structure can make lecithin possess emulsifying property, dispersivity, stability and freeze protection property.
In view of the problems of a precipitation state of bacterial liquid which is left for a long time, insufficient stability and freezing resistance of the existing freeze-drying protective agent, and the like, the soybean lecithin is tried to be used for preparing the Lactobacillus bulgaricus agent.
Disclosure of Invention
The present invention has been made in view of the above-mentioned and conventional problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide a method for preparing the lactobacillus bulgaricus agent with high expression cold shock and high survival rate after freeze-drying of thalli.
The specific scheme is as follows:
uniformly mixing lactobacillus bulgaricus fermentation liquor and soybean lecithin liquid, placing in a constant-temperature water bath for treatment, centrifuging, discarding supernatant, collecting thalli, adding a freeze-drying protective agent, uniformly mixing, and vacuum drying to obtain freeze-drying bacterial powder.
Further, the concentration of the soybean lecithin liquid is 0.1% -1%.
Further, the mixing ratio of the fermentation liquor and the soybean lecithin liquor is 1:0.5-2 (V/V).
Further, the temperature of the constant temperature water bath is 10-25 ℃.
Further, the temperature of the constant temperature water bath is 1-6h.
Further, the lyoprotectant comprises skimmed milk powder, lactose, ascorbic acid and soybean lecithin.
Further, the lactobacillus bulgaricus fermentation broth is prepared by the following method: the liquid fermentation culture adopts a four-stage culture method, wherein the first, second and third stages are seed culture mediums, the fourth stage is a fermentation culture medium, the first-stage seed culture medium is inoculated with freeze-dried strains, a constant-temperature anaerobic incubator is placed for culture, the second-stage seed culture medium is inoculated with the cultured first-stage seed liquid, and the culture is carried out for 8 hours; and so on, passage to a fourth fermentation stage.
Technical effects
The soybean lecithin is added in the processes of inducing the CspA expression and freezing the lactobacillus bulgaricus, so that the stability of a bacterial liquid system can be improved, the CspA expression is induced, the stress resistance of thalli is enhanced, the integrity of cell membranes is protected, the lactobacillus bulgaricus is helped to better cope with challenges in the freezing process, and the survival rate of the lactobacillus bulgaricus after freezing is obviously improved.
Drawings
FIG. 1 shows the relative expression levels of CspA mRNA induced at low temperature by adding different concentrations of soybean lecithin;
FIG. 2 shows the freeze-drying survival rate of the thalli after low-temperature induction by adding different concentrations of soybean lecithin;
FIG. 3 shows the freeze-drying viability of cells using different concentrations of soybean lecithin as an anti-freeze agent;
FIG. 4 shows the freeze-drying survival rate of thalli using different lyoprotectants;
Detailed Description
Other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein, and it is apparent that the described embodiments are only some of the embodiments of the present application, not all of the embodiments. The present application may be embodied or carried out in other specific embodiments, and the details of the present application may be modified or changed from various points of view and applications without departing from the spirit of the present application. The features of the following embodiments and examples may be combined with each other without any conflict.
In the following description, specific details are provided in order to provide a thorough understanding of the examples. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details.
The specific scheme is as follows: uniformly mixing lactobacillus bulgaricus fermentation liquor and soybean lecithin liquid, placing in a constant-temperature water bath for treatment, centrifuging, discarding supernatant, collecting thalli, adding a freeze-drying protective agent, uniformly mixing, and vacuum drying to obtain freeze-drying bacterial powder.
Further, the concentration of the soybean lecithin liquid is preferably 0.2% to 0.9%, preferably 0.2% to 0.8%, preferably 0.2% to 0.7%, preferably 0.2% to 0.6%, preferably 0.2% to 0.5%.
Further, the mixing ratio of the fermentation broth to the soybean lecithin broth is preferably 1:0.8-1.2 (V/V), most preferably 1:1.
Further, the temperature of the thermostatic water bath is preferably 12-25 ℃, preferably 13-25 ℃, preferably 14-25 ℃, preferably 15-25 ℃, preferably 16-25 ℃, preferably 17-24 ℃, preferably 18-23 ℃, preferably 19-22 ℃.
Further, the time of the constant temperature water bath is preferably 2 to 5 hours, preferably 3 to 5 hours, preferably 3.5 to 4.5 hours.
Further, the lyoprotectant comprises 10-40% of skimmed milk powder, 5-15% of lactose, 5-20% of ascorbic acid and 0.05-0.5% of soybean lecithin, and the balance of water or starch. Wherein the soybean lecithin content is further preferably 0.05-0.4%, preferably 0.05-0.35%.
The freeze-drying protective agent is prepared by adopting a common conventional method, for example, a certain amount of each component is taken, added with sterile water, stirred and mixed, and then sterilized.
The addition of the soybean lecithin in the freeze-drying protective agent can reduce interfacial tension between oil and water phases, has good emulsifying dispersion stability, enables bacterial liquid to form dispersion liquid with certain stability in the induction process of the lactobacillus bulgaricus low-temperature induction CspA test, enhances the stability of a system, enables the temperature balance of the system in the induction process of the fermentation liquid, and further induces the expression of the CspA; in addition, the soybean lecithin is one of the main components of the cell membrane, and can protect the cell membrane of the lactobacillus bulgaricus and prevent the cell membrane from being damaged in the freezing process, thereby reducing the death rate of the cell and improving the survival rate of the cell; the soybean lecithin can also maintain the normal metabolic activity of lactobacillus cells, so that the cells can maintain a better physiological state in the freezing process, thereby better resisting adverse factors in the freezing process; the further soybean lecithin can enhance the stress resistance of the lactobacillus bulgaricus, so that the lactobacillus bulgaricus has stronger cold resistance and oxidation resistance in the freezing process, thereby being better suitable for the freezing environment. Furthermore, soybean lecithin contains fatty acids such as unsaturated fatty acids and linoleic acid, which can assist in transporting fatty acids in lactobacillus bulgaricus at low temperatures. Fatty acids are important substances for cellular energy metabolism and signal transduction, so that the recovery and growth of lactobacillus bulgaricus after freezing can be promoted.
Specifically, the Lactobacillus bulgaricus freeze-drying protective agent contains 10-40% of skimmed milk powder, 5-15% of lactose, 5-20% of ascorbic acid, 0.05-0.35% of soybean lecithin and the balance of water or starch.
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The specific techniques or conditions are not noted in the examples, and are carried out according to the techniques or conditions described in the literature in the field, or based on the previous studies by the inventors of the present application (see "study of the influence of low temperature on the expression of the CspA gene of Lactobacillus bulgaricus"), or according to the product specifications; the reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Examples
Four-stage fermentation culture of lactobacillus bulgaricus
The liquid fermentation culture adopts a four-stage culture method, wherein the first, second and third stages are seed culture mediums, the fourth stage is a fermentation culture medium, the first stage seed culture medium is inoculated with freeze-dried strain, and the freeze-dried strain is placed in a constant-temperature anaerobic incubator for static culture at 37 ℃ for 12 hours. The secondary seed culture medium is inoculated with the cultured primary seed liquid according to the inoculation amount of 5 percent, and is cultured for 8 hours; and so on, passage to a fourth fermentation stage.
(1) Lactobacillus bulgaricus low-temperature induction CspA test
Uniformly mixing the lactobacillus bulgaricus four-stage fermentation liquor with soybean lecithin liquid 1:1 (V/V) with concentration of 0%, 0.25%, 0.5%, 0.75% and 1.0%, placing the mixture in a constant-temperature water bath at 20 ℃ for treatment for 4 hours, setting 3 repetitions for each group of tests, and detecting the expression quantity of CspA by using an RT-PCR method.
Cloning the Lactobacillus bulgaricus CspA gene, detecting the expression quantity of mRNA by using an RT-PCR method, designing a primer for detecting the CspA gene by using Prime5.0 software, wherein the primer sequence (F is '5-TTGCCTTGTTCAACATCGTA', R is '5-GGGCTTTGGGTTTATCAC-3'), the product length is 123bp, and the primer is synthesized by Invitrogen company; extracting total RNA of lactobacillus bulgaricus, operating according to the method of RNAiso Plus (D9108A Takara) kit instruction, detecting the ratio of OD260 to OD280 by using an enzyme-labeled instrument, wherein the ratio is 1.8-2.0, and the RNA is the RNA meeting the requirements.
RNA loading was 2.0. Mu.L (about 500 ng) following the protocol described for the PrimeScript RT reagent Kit (DRR 037A Takara) kit, reaction conditions: 37 ℃ for 15min; the reaction was completed at 85℃for 5 seconds to obtain cDNA.
Fluorescent quantitative PCR was performed according to the protocol of SYBR Premix Ex Taq (DRR 041A Takara), using cDNA as a template, and CspA primers were added, and the CspA gene was amplified by a Real-time fluorescent quantitative PCR (RT PCR) method.
Reaction conditions: pre-denaturation at 95℃for 30s; denaturation at 95℃for 5s, annealing at 60℃for 34s and reading for 40 cycles; making a melting curve from 65 ℃ to 95 ℃; the relative expression quantity of the CspA is determined by taking 16srDNA as an internal reference gene (length 210 bp); and (5) carrying out electrophoresis detection analysis and sequencing analysis on the product.
Analysis of the expression level of CspA:
as shown in the figure 1, the addition of soybean lecithin at the induction temperature of 20 ℃ for 4 hours can significantly affect the expression level of CspA mRNA, which indicates that the addition of soybean lecithin can further induce the bacterial cells to produce CspA. Under the condition of mixing with 0.25-0.5% concentration soybean lecithin, the expression quantity of the bacterial strain CspA reaches the highest.
(2) Lactobacillus bulgaricus freeze-drying verification test
Uniformly mixing lactobacillus bulgaricus fermentation liquor with soybean lecithin liquid 1:1 (V/V) with concentration of 0%, 0.25%, 0.5%, 0.75% and 1.0%, placing in a constant-temperature water bath at 20 ℃ for 4 hours, centrifuging for 15 minutes at 10000r/min, discarding supernatant, collecting thalli, pre-cooling for 30 minutes at-40 ℃, and vacuum drying for 24 hours to obtain freeze-dried bacterial powder.
Precisely weighing 1.00g of bacterial powder, suspending and dissolving in 10mL of PBS, uniformly mixing, and measuring the viable count of the suspension by a plate bacterial mixing counting method, wherein each group of tests are repeated for 3 times; the freeze-drying survival rate (total viable count of freeze-dried bacteria powder/total viable count of fermentation broth) of each group of samples was calculated.
As shown in figure 2, the freeze-drying survival rate of the thalli is 28.36% only through low-temperature induction, and the freeze-drying survival rate of the soybean lecithin added into the fermentation liquor is remarkably improved, and the survival rate of the thalli reaches a maximum value 47.86% under the condition of 0.25% concentration of the soybean lecithin, which indicates that the soybean lecithin can further protect the thalli and reduce the damage of the freeze-drying to the thalli.
(3) Influence of different lyoprotectants on cell freezing resistance
Soybean lecithin as lyoprotectant
Uniformly mixing the lactobacillus bulgaricus four-stage fermentation liquor with 0.25% soybean lecithin solution in a ratio of 1:1 (V/V), placing in a constant-temperature water bath at 20 ℃ for 4 hours, centrifuging at 10000r/min for 15 minutes, discarding supernatant, collecting thalli, respectively adding soybean lecithin solutions with the concentration of 0.05%, 0.1%, 0.2%, 0.3% and 0.5% as a freeze-drying protective agent, uniformly mixing the freeze-drying protective agent with bacterial mud according to a mass ratio of 3:1, pre-cooling at-40 ℃ for 30 minutes, and vacuum drying for 24 hours to obtain freeze-dried bacterial powder. Precisely weighing 1.00g of bacterial powder, suspending and dissolving in 10mL of PBS, uniformly mixing, and measuring the viable count of the suspension by a plate bacterial mixing counting method, wherein each group of tests are repeated for 3 times; the freeze-drying survival rate (total viable count of freeze-dried bacteria powder/total viable count of fermentation broth) of each group of samples was calculated.
As shown in figure 3, the freeze-drying survival rate of thalli can be obviously improved by taking the soybean lecithin solution with different concentrations as a freeze-drying protective agent; the 0.2 percent soybean lecithin solution is used as a freeze-drying protective agent, and the maximum freeze-drying survival rate reaches 63.43 percent.
(4) The soybean lecithin is matched with other freeze-drying protective agents for use
Uniformly mixing lactobacillus bulgaricus fermentation liquor with soybean lecithin solution with the concentration of 0.25% at a ratio of 1:1 (V/V), placing in a constant-temperature water bath at 20 ℃ for 4 hours, centrifuging at 10000r/min for 15 minutes, discarding supernatant, collecting thalli, adding different lyoprotectant groups 1-6, uniformly mixing the lyoprotectant and bacterial mud according to the mass ratio of 3:1, pre-cooling at-40 ℃ for 30 minutes, and vacuum drying for 24 hours to obtain the lyoprotectant powder.
Precisely weighing 1.00g of bacterial powder, suspending and dissolving in 10mL of PBS, uniformly mixing, and measuring the viable count of the suspension by a plate bacterial mixing counting method, wherein each group of tests are repeated for 3 times; the freeze-drying survival rate (total viable count of freeze-dried bacteria powder/total viable count of fermentation broth) of each group of samples was calculated.
Lyoprotectant 1:25% nonfat dry milk +10% lactose +10% ascorbic acid;
the preparation method comprises the following steps: a certain amount of skimmed milk powder, lactose and ascorbic acid were taken, mixed with sterile water with stirring, and then sterilized (the same applies hereinafter).
Lyoprotectant 2: adding 0.05% soybean lecithin based on the freeze-drying protective agent 1;
lyoprotectant 3: adding 0.1% soybean lecithin based on the freeze-drying protective agent 1;
lyoprotectant 4: adding 0.2% soybean lecithin based on the freeze-drying protective agent 1;
lyoprotectant 5: adding 0.3% soybean lecithin based on the freeze-drying protective agent 1;
lyoprotectant 6: adding 0.5% soybean lecithin based on the freeze-drying protective agent 1;
as shown in figure 4, on the basis of the lyoprotectant 1 (25% of skimmed milk powder, 10% of lactose and 10% of ascorbic acid), the addition of soybean lecithin can greatly improve the freeze-drying survival rate of thalli, and the addition amount of 0.1% -0.2% of soybean lecithin can greatly improve the freeze-drying survival rate by more than 89% compared with 71.82% of the lyoprotectant 1.

Claims (10)

1. The lactobacillus bulgaricus lyoprotectant is characterized by comprising 10-40% of skimmed milk powder, 5-15% of lactose, 5-20% of ascorbic acid and 0.05-0.35% of soybean lecithin.
2. A method for preparing a cold shock protein-highly expressed lactobacillus bulgaricus agent, comprising the steps of: 1) Uniformly mixing lactobacillus bulgaricus fermentation liquor and 0.2% -0.75% of soybean lecithin liquor, wherein the mixing ratio of the fermentation liquor to the soybean lecithin liquor is 1:0.5-1.5 (V/V), placing the mixture in a constant-temperature water bath at 12-25 ℃ for 2-5h, centrifuging, removing the supernatant, and collecting thalli;
2) Adding a freeze-drying protective agent into the thalli, uniformly mixing, and vacuum drying to obtain freeze-drying bacterial powder; the freeze-drying protective agent contains 10-40% of skimmed milk powder, 5-15% of lactose, 5-20% of ascorbic acid and 0.05-0.35% of soybean lecithin.
3. The method according to claim 2, wherein the concentration of soybean lecithin in step 1) is 0.2% -0.6%.
4. The method according to claim 2, wherein the concentration of soybean lecithin in step 1) is 0.2% -0.5%.
5. The method according to claim 2, characterized in that the temperature of the thermostatic water bath is 15-25 ℃.
6. The method according to claim 2, characterized in that the thermostatic water bath takes 3-5 hours.
7. The method according to claim 2, wherein the mixing ratio of the fermentation broth to the soybean lecithin broth is 1:0.8-1.2 (V/V).
8. The method of claim 2, wherein the lyoprotectant comprises 0.08-0.25% soy lecithin.
9. The method of claim 2, wherein the lyoprotectant balance is water.
10. A lactobacillus bulgaricus agent prepared by the method for preparing a lactobacillus bulgaricus agent with high expression of cold shock protein according to claim 2.
CN202311417584.8A 2023-10-30 2023-10-30 Method for preparing high-expression cold shock protein lactobacillus bulgaricus agent Pending CN117402739A (en)

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