CN116218676A - Preparation for improving bacillus survival bacterial storage amount, composite protective agent and application thereof - Google Patents

Preparation for improving bacillus survival bacterial storage amount, composite protective agent and application thereof Download PDF

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CN116218676A
CN116218676A CN202310220634.7A CN202310220634A CN116218676A CN 116218676 A CN116218676 A CN 116218676A CN 202310220634 A CN202310220634 A CN 202310220634A CN 116218676 A CN116218676 A CN 116218676A
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bacillus
protective agent
parts
preparation
composite protective
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安健
曹海鹏
顾颖
王会聪
李家胜
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Lianyungang Marine And Fisheries Development Promotion Center
Lyuao Environmental Protection Technology Shanghai Co ltd
Shanghai Ocean University
Jiangsu Polytechnic College of Agriculture and Forestry
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Lianyungang Marine And Fisheries Development Promotion Center
Lyuao Environmental Protection Technology Shanghai Co ltd
Shanghai Ocean University
Jiangsu Polytechnic College of Agriculture and Forestry
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    • C12N1/04—Preserving or maintaining viable microorganisms
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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Abstract

The invention relates to a preparation for improving the survival bacterial load of bacillus, a composite protective agent adopting the preparation and application of the composite protective agent; the preparation for improving the storage and survival of bacillus comprises the following components in parts by mass: 4-6 parts of skim milk powder; 3-5 parts of sorbitol; sodium glutamate, 2-4 parts. The preparation adopts the combination of skimmed milk powder, sorbitol and sodium glutamate to synergistically increase the effective viable bacteria survival rate of the probiotic bacillus liquid preparation in the storage process. The preparation and the compound protective agent are easy to operate, do not need special equipment and do not take a long time.

Description

Preparation for improving bacillus survival bacterial storage amount, composite protective agent and application thereof
Technical Field
The invention relates to a preparation for improving the storage and survival of bacillus, a composite protective agent and application thereof, and belongs to the technical field of storage of probiotics.
Background
The protective agent is the most important external factor influencing the activity of probiotics in the microecological preparation, has the characteristics of no immunogenicity, no toxic or side effect, easy dissolution, full maintenance of biological activity and the like, and can fully exert the good protective effect in the storage process of the microecological preparation. For example, in order to prevent denaturation of effective live bacteria during lyophilization (lyophilization after 2h for 10-20 h) of probiotics, many researchers have added a protective agent to the probiotics to effectively reduce or prevent damage to bacterial cells by lyophilization, so that the probiotics maintain original physicochemical properties and biological activities, and thus the protective agent has been effectively utilized as a key technology for improving the survival rate of the bacterial species (Carvalho A S, silva J, ho P, et al, survivin of freeze-dried Lactobacillus plantarum and Lactobacillus ramnosus during storage in the presence of protections, biotechnology letters, 2002, 24:1587-1591.). At present, related scholars have optimized saccharomycetes (Li Zhen, ji Shengxin, liang Jingjing, etc. A yeast lyoprotectant mainly comprising oat beta-glucan, modern food technology, 2022,7:. Li Zhen, etc. A composite yeast lyoprotectant, patent application number CN 202110920034.2), lactobacillus (Yuan Yahong, etc. research on a protective agent of freeze-dried high-activity lactobacillus powder, university of northwest agriculture and forestry science and technology, nature science edition, 2003,31 (10): 82-84), paramycola Diels (Jixiao, etc. A method for improving the freeze-drying survival rate of Paramycola Diels and application of the protective agent are carried out, but the protective agent is added in the freeze-drying process of the probiotics, and a large amount of live bacteria still exist after freeze-dryingLoss of bacteria. For example, tang Erming studies have found that Saccharomyces cerevisiae has significantly changed in viable cell count before and after lyophilization, from 28X 10 before lyophilization 5 CFU/ml was reduced to 28X 10 after lyophilization 3 CFU/ml (Tang Er, survival of microorganisms in freeze-dried preservation, food and fermentation industries, 1993, 3:80-80.) thus demonstrating that the development of optimal cryoprotectants for a single species of probiotic is important. In general, a single protecting agent cannot meet the condition that the bacteria resist the external severe environment, and each protecting agent in the composite protecting agent plays a respective role in freeze drying and has a synergistic effect. Therefore, the development of the compound protective agent is the main research direction for improving the survival rate of strains (Yuan Yahong, et al, research on freeze-dried high-activity lactobacillus powder protective agent, university of northwest agriculture and forestry science and technology, nature science edition, 2003,31 (10): 82-84).
The probiotics bacillus such as bacillus subtilis (Bacillus subtilis), bacillus licheniformis (Bacillus licheniformis) are microecological preparation strains which are allowed to be used by the agricultural rural department of China, the liquid preparation of the probiotics is a common commercial microecological preparation in China, and the probiotics have great economic, social and ecological benefits, have wide application prospects and play an important role in green high-quality development of agriculture, aquaculture industry, animal husbandry and feed industry. However, the existing probiotic bacillus liquid preparation is easy to generate bacterial death in the storage process, so that the effective viable bacteria survival rate is low, the shelf life is short, and the product quality is seriously influenced. How to increase the stored live bacteria amount of the probiotic bacillus liquid preparation is a bottleneck problem faced in prolonging the shelf life and guaranteeing the product quality. At present, bacillus is generally subjected to freeze drying by taking skimmed milk as a cold-resistant protective agent to improve the storage live bacteria amount (Wang Pingzhu, et al; bacillus is subjected to freeze vacuum drying and change of survival rate in sand tube preservation processes; industrial microorganisms, 1995, 25 (3)), but the method requires special equipment, is complicated in operation process and is large in operation difficulty (Li Xiaoping, et al; strain preservation method and comparison of advantages and disadvantages thereof; health professional education, 2009, 27 (20): 134-135).
Therefore, the development of the protective agent for improving the survival bacterial amount of the probiotics bacillus is of great significance to agriculture, aquaculture industry, animal husbandry and feed industry.
Disclosure of Invention
In view of the above-mentioned problems and/or other problems of the related art, the present invention provides a method for enhancing the viable bacteria storage amount of a probiotic bacillus preparation by a freezing method based on a composite protective agent, which is simple and convenient to operate, does not require special equipment, does not take a long time, and does not easily cause death of viable bacteria during storage.
In order to achieve the aim of the invention, the invention firstly provides a preparation for improving the stored viable bacteria amount of bacillus, which comprises the following components in parts by mass: 4-6 parts of skim milk powder; 3-5 parts of sorbitol; sodium glutamate, 2-4 parts.
The preparation comprises the following preferable components in parts by mass: skim milk powder, 5 parts; sorbitol, 5 parts; sodium glutamate, 2 parts.
The preparation comprises the following preferable components in parts by mass: skim milk powder, 5 parts; 4 parts of sorbitol; sodium glutamate, 4 parts.
The preparation comprises the following preferable components in parts by mass: skim milk powder, 5 parts; 4 parts of sorbitol; sodium glutamate, 4 parts.
Secondly, the invention provides a composite protective agent for improving the storage and survival bacterial amount of bacillus, which is an aqueous solution of the preparation for improving the storage and survival bacterial amount of bacillus, and the mass volume concentration of the aqueous solution is 10-20%.
Preferably, the mass volume concentration of the composite protective agent is 20%.
Finally, the invention provides application of a composite protective agent for improving the storage and survival bacterial load of bacillus: the composite protective agent is added into bacillus liquid for storage, and the storage temperature is-20 ℃; the volume ratio of the composite protective agent to the bacterial liquid is 1:3-3:1; the bacterial liquid contains 3×10 10 CFU/mL bacillus.
Preferably, the volume ratio of the composite protective agent to the bacterial liquid is 1:2-2:1.
Further preferably, the volume ratio of the composite protective agent to the bacterial liquid is 2:1.
The bacillus is bacillus subtilis A4, bacillus licheniformis C1, bacillus subtilis JK-2 or bacillus licheniformis AQ19-11.
The invention provides a preparation for improving the survival bacterial storage amount of a probiotic bacillus preparation by a freezing method based on a composite protective agent, the composite protective agent and application thereof, wherein the combination of skimmed milk powder, sorbitol and sodium glutamate is adopted to synergistically improve the effective survival rate of live bacteria in the storage process of a probiotic bacillus liquid preparation. The preparation and the compound protective agent are easy to operate, do not need special equipment and do not take a long time.
Detailed Description
The present invention will be further described with reference to the following specific embodiments, but the present invention is not limited to these specific embodiments.
Materials, reagents and the like used in the following specific embodiments are commercially available unless otherwise specified. Wherein: bacillus subtilis A4 (China center for type culture collection (cctccc) No. m 20221167), isolated from pond bottom mud [ An Jian 2022] by the present laboratory; bacillus licheniformis C1 (China center for type culture collection (CCTCC) No. M2020680), bacillus subtilis JK-2 (China center for type culture collection (CCTCC) No. M20221568), bacillus licheniformis AQ19-11 (China center for type culture collection (CCTCC) No. M2022012), providencia stuartii KM4 as positive control strains provided by Shanghai marine university aquatic animal pathogen library; glucose, fructose, sucrose, trehalose, mannitol, sorbitol, sodium glutamate, glycine, glycerol, gelatin, maltodextrin, polyvinylpyrrolidone, nutrient broth, nutrient agar, purchased from national pharmaceutical community chemical reagent company; skim milk powder, available from Heng Natural commerce Co.
The preparation method of the bacillus subtilis A4 bacterial liquid in the following specific embodiment comprises the following steps: aseptic technique inoculating Bacillus subtilis A4 into aseptic nutrient broth, culturing at 30deg.C and 200r/min for 48 hr, and making into final concentrationIs 3X 10 10 CFU/mL bacterial liquid.
The specific techniques or conditions are not identified below, either as described in the literature in this field or as a matter of product specifications.
Examples of preparations for increasing the amount of viable bacteria stored in Bacillus
Example 1
The preparation for improving the storage viable bacteria amount of bacillus in the example 1 comprises the following components in parts by mass:
4 parts of skim milk powder; sorbitol, 3 parts; sodium glutamate, 2 parts.
The testing method comprises the following steps:
the components of the preparation according to the embodiment are respectively weighed skim milk powder, sorbitol and sodium glutamate to make the total mass of the skim milk powder and the sorbitol to be 20g, distilled water is added to a volume of 100mL to prepare a compound protective agent with the effective content of 20 percent, the compound protective agent is sterilized at the temperature of 121 ℃ for 20 minutes and cooled to the room temperature, and the compound protective agent prepared by different compound proportions and the bacterial concentration of 3 multiplied by 10 are prepared 10 The bacillus subtilis A4 bacterial liquid with CFU/mL is uniformly mixed in a ratio of 1:1, an equal amount of sterile water bacterial liquid is added as a control, and then the mixture is placed at the temperature of minus 20 ℃ for 60 days, and a plate counting method is adopted to detect the viable count of the bacillus subtilis A4 bacterial liquid treated by the composite protective agent prepared in the embodiment 1.
The effective content is 20%, namely the mass volume concentration is 20%, namely the solution concentration is 0.2g/mL. The effective contents appearing below are all mass volume concentrations.
Example 2
The preparation for improving the storage viable bacteria amount of bacillus in example 2 comprises the following components in parts by mass: 4 parts of skim milk powder; 4 parts of sorbitol; sodium glutamate, 3 parts.
The test method is the same as that of embodiment 1, and detailed description thereof will be omitted.
Example 3
The preparation for improving the storage viable bacteria amount of bacillus in example 3 comprises the following components in parts by mass: 4 parts of skim milk powder; sorbitol, 5 parts; sodium glutamate, 4 parts.
The test method is the same as that of embodiment 1, and detailed description thereof will be omitted.
Example 4
The preparation for improving the storage viable bacteria amount of bacillus in example 4 comprises the following components in parts by mass: skim milk powder, 5 parts; sorbitol, 3 parts; sodium glutamate, 3 parts.
The test method is the same as that of embodiment 1, and detailed description thereof will be omitted.
Example 5
The preparation for improving the storage viable bacteria amount of bacillus in example 5 comprises the following components in parts by mass: skim milk powder, 5 parts; 4 parts of sorbitol; sodium glutamate, 4 parts.
The test method is the same as that of embodiment 1, and detailed description thereof will be omitted.
Example 6
The preparation for improving the storage viable bacteria amount of bacillus in example 6 comprises the following components in parts by mass: skim milk powder, 5 parts; sorbitol, 5 parts; sodium glutamate, 2 parts.
The test method is the same as that of embodiment 1, and detailed description thereof will be omitted.
Example 7
The preparation for improving the storage viable bacteria amount of bacillus in example 7 comprises the following components in parts by mass: 6 parts of skim milk powder; sorbitol, 3 parts; sodium glutamate, 4 parts.
The test method is the same as that of embodiment 1, and detailed description thereof will be omitted.
Example 8
The preparation for improving the storage viable bacteria amount of bacillus in example 8 comprises the following components in parts by mass: 6 parts of skim milk powder; 4 parts of sorbitol; sodium glutamate, 2 parts.
The test method is the same as that of embodiment 1, and detailed description thereof will be omitted.
Example 9
The preparation for improving the storage viable bacteria amount of bacillus in the example 9 comprises the following components in parts by mass: 6 parts of skim milk powder; sorbitol, 5 parts; sodium glutamate, 3 parts.
The test method is the same as that of embodiment 1, and detailed description thereof will be omitted.
Examples of composite protectants employing the formulation obtained in example 6 above
Example 10
10g of the preparation prepared in example 6 is weighed, distilled water is added to a volume of 100mL, and the composite protective agent with the effective content of 10% is prepared.
The testing method comprises the following steps:
the concentration of the composite protective agent and bacteria prepared in the application example is 3 multiplied by 10 10 And (3) mixing the bacillus subtilis A4 bacterial liquid with the concentration of CFU/mL at a ratio of 1:1, freezing and storing for 60 days at the temperature of minus 20 ℃, and detecting the viable count of the bacillus subtilis A4 bacterial liquid treated by the composite protective agents with different effective contents by adopting a plate counting method. The bacterial liquid added with the same amount of sterile water is used as a control.
Example 11
Embodiment 11 differs from embodiment 10 described above only in that: the effective content of the composite protective agent of the example 11 is 20%; the other conditions such as the composition of the components in parts by mass of the formulation used, the test method, etc. are the same as those of example 10, and detailed descriptions thereof are omitted.
Example 12
Embodiment 12 differs from embodiment 10 described above only in that: the effective content of the composite protective agent in the example 12 is 30%; the other conditions such as the composition of the components in parts by mass of the formulation used, the test method, etc. are the same as those of example 10, and detailed descriptions thereof are omitted.
Example 13
Embodiment 13 differs from embodiment 10 described above only in that: the effective content of the composite protective agent of the example 13 is 40%; the other conditions such as the composition of the components in parts by mass of the formulation used, the test method, etc. are the same as those of example 10, and detailed descriptions thereof are omitted.
Application example of composite protectant prepared by the above example 11
Application example 1
Sterile procedure the composite protectant prepared in example 11 above was used at a concentration of 10 10 CFU/mL of bacillus subtilis A4 bacterial liquid is mixed according to the volume ratio of 1:3, and frozen at the temperature of minus 20 ℃.
The testing method comprises the following steps: after 60d of freezing storage, detecting the viable count of the bacillus subtilis A4 bacterial liquid of the application example by adopting a plate counting method, and calculating the survival rate according to a formula (1).
Formula 1 survival rate = viable count before freezing/viable count after freezing x 100%
Application example 2
Application example 2 differs from application example 1 described above only in that: the volume ratio of the composite protective agent to the bacillus subtilis A4 bacterial liquid in the application example 2 is 1:2. The components such as the components in parts by mass of the preparation used, the testing method and the like are the same as those of application example 1, and detailed description thereof is omitted.
Application example 3
Application example 3 differs from application example 1 described above only in that: the volume ratio of the composite protective agent to the bacillus subtilis A4 bacterial liquid in the application example 3 is 1:1. The components such as the components in parts by mass of the preparation used, the testing method and the like are the same as those of application example 1, and detailed description thereof is omitted.
Application example 4
Application example 4 differs from application example 1 described above only in that: the volume ratio of the composite protective agent to the bacillus subtilis A4 bacterial liquid in the application example 4 is 2:1. The components such as the components in parts by mass of the preparation used, the testing method and the like are the same as those of application example 1, and detailed description thereof is omitted.
Application example 5
Application example 5 differs from application example 1 described above only in that: the volume ratio of the composite protective agent to the bacillus subtilis A4 bacterial liquid in the application example 5 is 3:1. The components such as the components in parts by mass of the preparation used, the testing method and the like are the same as those of application example 1, and detailed description thereof is omitted.
Each of the above examples 1 to 13 and application examples 1 to 5 was repeated 3 times.
Single protectant control experiments
Respectively weighing 20g glucose, fructose, sucrose, trehalose, mannitol, sorbitol, sodium glutamate, glycine, glycerol, gelatin, maltodextrin, skim milk powder and polyvinylpyrrolidone, adding distilled water to constant volume to 100mL, respectively preparing into protective agent with effective content of 20%, sterilizing at 121deg.C under high pressure for 20min, cooling to room temperature, and sterilizing to obtain powder with bacterial concentration of 3×10 10 CFU/mL of bacillus subtilis A4The liquid is uniformly mixed in a ratio of 1:1, bacterial liquid added with equal amount of sterile water is used as a control, and after the liquid is placed at 25 ℃,4 ℃ and minus 20 ℃ for 60 days, the bacterial liquid of the bacillus subtilis A4 treated by different protective agents is respectively subjected to viable count detection by adopting a plate counting method. Each treatment was repeated 3 times.
Experiment on cryopreservation protection effect of bacillus composite protective agent on different bacillus strains
Sterile inoculating Bacillus subtilis JK-2, bacillus licheniformis C1, bacillus licheniformis AQ19-11 and providencia stuartii KM4 into sterile nutrient broth, and culturing at 30deg.C and 200r/min for 48 hr to obtain bacterial solutions of Bacillus subtilis JK-2, bacillus licheniformis C1, bacillus licheniformis AQ19-11 and providencia stuartii KM4 with bacterial solution concentrations of 6.7X10 9 CFU/mL、9.4×10 9 CFU/mL、7.7×10 9 CFU/mL and 1.7X10 7 CFU/mL。
The composite protectant prepared in example 11 is mixed with bacterial solutions of bacillus subtilis JK-2, bacillus licheniformis C1 and bacillus licheniformis AQ19-11 respectively in a ratio of 2:1, frozen at-20 ℃ for 60d, viable count detection is carried out on bacterial solutions of all strains treated by the composite protectant by adopting a plate counting method, and the survival rate is calculated according to a formula (1). The providencia rette KM4 bacterial liquid treated by the composite protective agent under the same condition is used as a positive control, and each bacterial liquid added with the same amount of sterile water is used as a negative control. Each treatment was repeated 3 times.
Effect data
1. The preparation and the composite protective agent have the application effect of improving the survival bacterial load of bacillus:
tables 1-3 show that the formulations of examples 1-9 with different compounding ratios all have higher viable counts than the bacillus subtilis A4 treated without the addition of the protective agent under the same conditions.
Example 6 is an optimal compounding ratio, the composition ratio of skim milk powder, sorbitol and sodium glutamate is 5:5:2, and the viable count of the bacillus subtilis A4 treated by the compound protectant prepared in the compounding ratio at-20 ℃ is 1.19X10 10 CFU/mL, compared with the dried bud treated without the protective agent under the same conditionViable count of Bacillus sp.A4 (1.21×10) 9 CFU/mL) is 882.07% (P)<0.05). Meanwhile, the viable count of the bacillus subtilis A4 treated by the preparation of the example 6 is obviously higher than the effect of single skim milk powder, sorbitol or sodium glutamate; this demonstrates that: in example 6, the combination of skim milk powder, sorbitol and sodium glutamate was synergistic.
In addition, the main and secondary factors of the influence of 3 excellent protective agents on the viable count of the bacillus subtilis A4 at the temperature of minus 20 ℃ are sorbitol > skim milk powder > sodium glutamate, namely the influence of sorbitol on the viable count of the bacillus subtilis A4 at the temperature of minus 20 ℃ is the largest, and then the main and secondary factors are skim milk powder and sodium glutamate.
Table 4 shows that the compound protective agents with the effective contents of 10%, 20%, 30% and 40% are prepared by compounding the compounds of the examples 10-13, and the viable count of the treated bacillus subtilis A4 at the temperature of minus 20 ℃ is 1.08X10 respectively 10 CFU/mL、1.19×10 10 CFU/mL、5.73×10 9 CFU/mL and 4.82×10 9 CFU/mL, compared with the viable count of Bacillus subtilis A4 treated without the addition of the protective agent under the same conditions (1.21X10) 9 CFU/mL) is 788.43% (P)<0.05)、882.07％(P<0.05)、373.80％(P<0.05 And 298.10% (P)<0.05). Therefore, the composite protective agent provided by the application has a remarkable effect.
In example 11, skim milk powder, sorbitol and sodium glutamate are compounded in a ratio of 5:5:2 to prepare a composite protective agent with an effective content of 20%, and the composite protective agent has the best protective effect on bacillus subtilis A4 at-20 ℃.
TABLE 1 results of orthogonal experiments examples 1-9
Figure BDA0004116452430000091
Figure BDA0004116452430000101
Note that: different letters indicate significant difference (P < 0.05), the same letters indicate insignificant difference (P > 0.05) table 2 orthogonal experimental analysis of variance
Figure BDA0004116452430000102
Note that: * Represents P <0.01
TABLE 3 frozen viable count of Bacillus subtilis A4 treated with each Single protectant
Figure BDA0004116452430000103
Figure BDA0004116452430000111
Note that: different letters indicate significant differences (P < 0.05), the same letters indicate insignificant differences (P > 0.05)
TABLE 4 influence of the effective content of the composite protectant on the number of viable bacteria of Bacillus subtilis A4 frozen
Figure BDA0004116452430000112
Note that: different letters indicate significant differences (P < 0.05), the same letters indicate insignificant differences (P > 0.05)
2. The preparation, the composite protective agent and the bacterial liquid have the effects of different mixing ratios:
the results of the experiments (Table 5) showed that the survival rates of Bacillus subtilis A4 after the respective experiments of application examples 1 to 5 were 53.11% (application example 5), 89.50% (application example 4), 79.22% (application example 3), 66.67% (application example 2) and 33.85% (application example 1), respectively. Therefore, the volume ratio of 2:1 in application example 4 is preferable as the mixing ratio of the composite protectant and the bacterial liquid.
TABLE 5 effects of the composite protectant on the cryopreservation of Bacillus subtilis A4 at different mixing ratios
Figure BDA0004116452430000113
Note that: the same column with different letters indicates that the difference is significant (P < 0.05), and the same column with the same letters indicates that the difference is not significant (P > 0.05)
3. Analysis of cryopreservation protection effect of composite protective agent on different bacillus strains
The experimental results (Table 6) show that the composite protective agent has good protective effects on bacillus licheniformis C1, bacillus subtilis JK-2 and bacillus licheniformis AQ19-11 preserved at-20 ℃ besides the bacillus subtilis A4. The specific expression is as follows: the viable count of the bacillus licheniformis C1, the bacillus subtilis JK-2 and the bacillus licheniformis AQ19-11 treated by the composite protective agent still reaches 5.4x10 after the bacillus licheniformis C1, the bacillus subtilis JK-2 and the bacillus licheniformis AQ19-11 are frozen at the temperature of minus 20 ℃ for 60 days 9 Above CFU/mL, survival rates were 90.73%, 81.50% and 88.15%, respectively. In addition, the viable count of the control strain-Provemuraden Lei KM4 treated by the composite protective agent after being frozen at-20 ℃ for 60 days is obviously lower than that of bacillus and is only 8.0 multiplied by 10 6 CFU/mL, survival rate is only 46.64%, which indicates that the composite protective agent is suitable for the cryopreservation protection of bacillus.
TABLE 6 cryopreservation protective Effect of the composite protective Agents on different strains
Figure BDA0004116452430000121
Note that: the same column with different letters indicates that the difference is significant (P < 0.05), and the same column with the same letters indicates that the difference is not significant (P > 0.05)
In summary, the invention provides a preparation for improving the survival bacterial storage amount of a probiotic bacillus preparation by a freezing method based on a composite protective agent, the composite protective agent and application thereof, and the preparation adopts the combination of skimmed milk powder, sorbitol and sodium glutamate to synergistically improve the effective survival rate of the probiotic bacillus liquid preparation in the storage process. The preparation and the compound protective agent are easy to operate, do not need special equipment and do not take a long time.
It should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is for clarity only, and that the skilled artisan should recognize that the embodiments may be combined as appropriate to form other embodiments that will be understood by those skilled in the art.
The above list of detailed descriptions is only specific to practical embodiments of the present invention, and they are not intended to limit the scope of the present invention, and all equivalent embodiments or modifications that do not depart from the spirit of the present invention should be included in the scope of the present invention.

Claims (8)

1. The preparation for improving the storage and survival of bacillus is characterized by comprising the following components in parts by mass: 4-6 parts of skim milk powder; 3-5 parts of sorbitol; sodium glutamate, 2-4 parts.
2. The preparation according to claim 1, wherein the preparation comprises the following components in parts by mass: skim milk powder, 5 parts; sorbitol, 5 parts; sodium glutamate, 2 parts.
3. A composite protective agent for improving the storage and survival of bacillus is characterized in that: the composite protective agent is an aqueous solution of the preparation for improving the storage viable bacteria amount of bacillus according to claim 1 or 2, and the mass volume concentration of the aqueous solution is 10% -20%.
4. The composite protective agent for improving the survival bacterial load of bacillus according to claim 3, wherein the composite protective agent is characterized by: the mass volume concentration of the composite protective agent is 20%.
5. The use of a composite protectant for increasing the viable count of bacillus according to claim 4, wherein: adding the composite protective agent as claimed in claim 4 into bacillus liquid for storage, wherein the storage temperature is-20 ℃; the volume ratio of the composite protective agent to the bacterial liquid is 1:3-3:1; the bacterial liquid contains 3×10 10 CFU/mL bacillus.
6. The use of a composite protectant for increasing the viable count of bacillus according to claim 5, wherein: the volume ratio of the composite protective agent to the bacterial liquid is 1:2-2:1.
7. The use of a composite protectant for increasing the viable count of bacillus according to claim 6, wherein: the method is characterized in that: the volume ratio of the composite protective agent to the bacterial liquid is 2:1.
8. The use of a composite protectant for increasing the viable count of bacillus according to claim 7, wherein: the bacillus is bacillus subtilis A4, bacillus licheniformis C1, bacillus subtilis JK-2 or bacillus licheniformis AQ19-11.
CN202310220634.7A 2023-03-09 2023-03-09 Preparation for improving bacillus survival bacterial storage amount, composite protective agent and application thereof Pending CN116218676A (en)

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