Lactobacillus mucilaginosus freeze-dried powder with shelf life stability and preparation method of lactobacillus mucilaginosus freeze-dried powder

Abstract

The invention provides freeze-dried lactobacillus mucus powder with shelf life stability and a preparation method of the freeze-dried lactobacillus mucus powder, and belongs to the technical field of microbial agents. The freeze-drying protective agent comprises the following components in parts by weight: 2-15 parts of protein, 5-25 parts of sugar alcohol, 0-1 part of sodium ascorbate and 0-10 parts of dextrin. The freeze-drying protective agent provided by the invention is suitable for preparing the fermented lactobacillus mucus freeze-dried powder, so that the fermented lactobacillus mucus freeze-dried powder has the effect of high stability, and the survival rate still reaches 90% or above after the fermented lactobacillus mucus freeze-dried powder is stored at 4 DEG C for 90 days. Meanwhile, the raw materials of the freeze-drying protective agent are food-grade, so that the prepared freeze-dried bacterial powder is good in edible safety, and the freeze-drying protective agent is widely applied to industrial production or preparation of foods and medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial agents, and particularly relates to a freeze-dried fermented Lactobacillus mucilaginosus powder with shelf-life stability and a preparation method thereof. Background Art

[0002] Fermented mucus lactobacillus belongs to the genus Lactobacillus and is a natural probiotic extracted by humans. It can survive in artificial gastrointestinal fluid with low pH, produce lactic acid and other beneficial substances, and has multiple physiological functions such as promoting intestinal health, improving immunity, anti-oxidation, improving digestive function, preventing constipation and lowering cholesterol. It is often used in various production fields such as food and medicine. In order to promote the utilization and development of fermented mucus lactobacillus in the production field, it is particularly important to develop a preparation method for a highly stable freeze-dried bacterial powder of fermented mucus lactobacillus.

[0003] At present, in industrial production, the preparation of fermented mucus lactobacillus freeze-dried powder is often subject to the constraints of various conditions, such as high production cost, low survival rate of viable bacteria, short shelf life cycle number, fast degradation rate, etc. In particular, fermented mucus lactobacillus has high requirements for external living conditions, and it is easy to be subjected to low temperature stress, freezing stress damage, etc. during freeze drying. However, the preparation of freeze-dried powder often uses the freeze drying method, so the addition of freeze-drying protective agent reduces the mechanical damage and solute damage to the bacterial body during vacuum freeze drying, so as to improve the freeze-drying survival rate and stability during preservation. Different strains have significant differences in the proportion and composition of protective agent, so research and development of freeze-drying protective agent suitable for fermented mucus lactobacillus is an extremely important step. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a freeze-drying protective agent suitable for fermenting Lactobacillus mucilaginosus, which can improve the survival rate during the shelf life.

[0005] The invention provides a freeze-drying protective agent suitable for fermenting mucus lactobacillus, comprising the following components in parts by weight: 2-15 parts of protein, 5-25 parts of sugar alcohol, 0-1 part of ascorbic acid sodium salt and 0-10 parts of dextrin.

[0006] Preferably, the lyoprotectant comprises the following components in parts by weight: 4 to 12 parts of protein, 10 to 25 parts of sugar alcohol, 0.5 to 1 part of sodium ascorbic acid, and 5 to 10 parts of dextrin.

[0007] Preferably, the protein comprises skim milk; and the sugar alcohol comprises at least one of the following: lactose, trehalose and sorbitol.

[0008] Preferably, the lyoprotectant further comprises water;

[0009] The freeze-drying protective agent comprises an aqueous solution containing the following mass percentage concentrations: 6% skim milk, 1% lactose, 12% trehalose, 12% sorbitol, 10% dextrin and 1% sodium ascorbic acid.

[0010] The invention provides a freeze-dried bacterial powder of fermented Lactobacillus mucilaginosus prepared by the freeze-drying protective agent.

[0011] The invention provides a preparation method of freeze-dried bacterial powder of fermented Lactobacillus mucoides. The method comprises the following steps: inoculating seed liquid of fermented Lactobacillus mucoides into a fermentation medium for fermentation culture, during which a carbon and nitrogen source is added, and controlling the pH value of the system to be 5.5-6; collecting bacterial bodies from the fermentation liquid, mixing the cells with a freeze-drying protective agent, and freeze-drying the cells to obtain freeze-dried bacterial powder of fermented Lactobacillus mucoides.

[0012] Preferably, the fermentation medium is an aqueous solution containing the following components: 20-30 g / L sucrose, 20-40 g / L peptone, 10-20 g / L yeast extract, 1-2 g / L sodium acetate, 0.2-0.3 g / L magnesium sulfate, 0.1-0.2 g / L manganese sulfate and 1-2 g / L Tween 80.

[0013] Preferably, the inoculation amount of the fermented Lactobacillus mucilaginosus seed solution is 3% to 6%;

[0014] The solvent used for controlling the pH value of the system is a food-grade sodium hydroxide solution with a mass percent concentration of 30% or an ammonia solution with a volume percent concentration of 30%.

[0015] Preferably, the fermentation culture cycle is 14 to 18 hours;

[0016] The fermentation culture is aerated; the aeration rate during the aeration culture is 1 L / min.

[0017] Preferably, the carbon source added is sucrose, and the nitrogen source added is peptone and yeast extract powder; the mass ratio of the carbon source to the nitrogen source is 2-5:3-6; the mass ratio of the peptone to the yeast extract powder is (1-3):(1-2); the volume of the carbon and nitrogen sources added is 80% of the volume of the initial fermentation medium;

[0018] The time for adding the carbon and nitrogen source is 4 to 12 hours for fermentation; the adding of the carbon and nitrogen source includes uniform flow addition or step-by-step differential flow addition; during the uniform flow addition, the flow acceleration of the carbon and nitrogen source is 4.5 ml / min; during the step-by-step differential flow addition, the flow acceleration of the carbon and nitrogen source is 4 ml / min for 4 to 7 hours of fermentation; after 7 hours to 12 hours of fermentation, the flow acceleration of the carbon and nitrogen source is 8 ml / min.

[0019] The present invention provides a freeze-dried protective agent suitable for fermenting mucus lactobacillus, comprising the following components in parts by weight: 2 to 15 parts of protein, 5 to 25 parts of sugar alcohol, 0 to 1 part of sodium ascorbate and 3 to 10 parts of dextrin. The freeze-dried bacterial powder of fermented mucus lactobacillus is prepared by using the freeze-dried protective agent provided by the present invention. After storage for 90 days at 4°C, the survival rate is still as high as more than 40%, while the freeze-dried bacterial powder of fermented mucus lactobacillus prepared by the freeze-dried agent composed of protein and trehalose in the control group has a survival rate of only about 1%. This shows that the freeze-dried protective agent provided by the present invention is suitable for the preparation of freeze-dried bacterial powder of fermented mucus lactobacillus, which can greatly improve the activity of fermented mucus lactobacillus in a long-term low temperature environment and greatly improve the shelf life stability. At the same time, the components of the present invention are all food-grade raw materials, with high safety, and the production operation steps are simple. The obtained freeze-dried powder can be used for industrial production, and can also be mixed with other substances to make solid beverages, tablet candies or medicines, and is widely used. DETAILED DESCRIPTION

[0020] The present invention provides a freeze-drying protective agent suitable for fermenting mucus lactobacillus, comprising the following components in parts by weight: 2 to 15 parts of protein, 5 to 25 parts of sugar alcohol, 0 to 1 part of sodium ascorbate and 3 to 10 parts of dextrin. The protein can form a protein film on the surface of the bacteria during the freeze-drying process to prevent the bacteria from being exposed to the outside world, causing cell rupture and damage; the sugar alcohol has multiple hydroxyl groups, which can replace water molecules to form hydrogen bonds with phosphate groups in the phospholipids of the bacterial cell membrane during the freeze-drying process to avoid the integrity of the cell membrane and protein structure and function from being damaged; the dextrin can reduce the loss of cell bound water, prevent the cells from excessive water loss leading to cell death, and improve cell stability; the sodium ascorbate can reduce the redox potential of the protective agent, consume part of the oxygen during the test process, and reduce the oxidation and deterioration of the freeze-dried powder.

[0021] In the present invention, the lyophilization protective agent preferably includes the following components in parts by weight: 4-13 parts of protein, 8-25 parts of sugar alcohol, 0-1 part of sodium ascorbate and 4-10 parts of dextrin, or 4-12 parts of skim milk, 10-25 parts of sugar alcohol, 0.5-1 part of sodium ascorbate and 5-10 parts of dextrin. The protein preferably includes skim milk. The sugar alcohol preferably includes at least one of the following: lactose, trehalose and sorbitol. The lyophilization protective agent preferably includes 6 parts of skim milk, 12 parts of trehalose, 12 parts of sorbitol and 10 parts of dextrin or 12 parts of skim milk, 12 parts of trehalose, 12 parts of sorbitol and 1 part of sodium ascorbate. The lyophilization protective agent preferably also includes water; the lyophilization protective agent preferably includes an aqueous solution containing the following mass percentage concentrations: 6% skim milk, 1% lactose, 12% trehalose, 12% sorbitol, 10% dextrin and 1% sodium ascorbate. The preparation method of the freeze-dried protective agent comprises dissolving skimmed milk, lactose, trehalose, sorbitol, dextrin and sodium ascorbic acid in water, heating the solution to 105° C. and performing a mild Maillard reaction for 10 minutes to obtain the freeze-dried protective agent. The freeze-dried protective agent is cooled to 4° C. for storage.

[0022] The invention provides application of the freeze-drying protective agent in preparing freeze-dried bacterial powder of fermented Lactobacillus mucilaginosus.

[0023] In the present invention, the lyophilization protectant makes the fermented mucus lactobacillus thalline reduce along with water loss and temperature in the freeze drying process, protects the thalline from losing activity and regulating osmotic pressure, and provides the necessary nutrients for maintaining survival in long-term low temperature storage. The present invention does not make special restrictions on the strain type of the fermented mucus lactobacillus, adopts the fermented mucus lactobacillus known in the art, and in embodiments of the present invention, in order to prove the effect of the lyophilization protectant, the fermented mucus lactobacillus strain A21252 is illustrated as an example. The fermented mucus lactobacillus strain A21252 is a known strain disclosed in the prior art, referring to the fermented mucus lactobacillus strain A21252 recorded in the patent of publication number CN117165488A.

[0024] The invention provides a preparation method of freeze-dried bacterial powder of fermented Lactobacillus mucoides. The method comprises the following steps: inoculating seed liquid of fermented Lactobacillus mucoides into a fermentation medium for fermentation culture, during which a carbon and nitrogen source is added, and controlling the pH value of the system to be 5.5-6; collecting bacterial bodies from the fermentation liquid, mixing the cells with a freeze-drying protective agent, and freeze-drying the cells to obtain freeze-dried bacterial powder of fermented Lactobacillus mucoides.

[0025] In the present invention, the fermentation medium is preferably an aqueous solution containing the following components: 20-30 g / L sucrose, 20-40 g / L peptone, 10-20 g / L yeast extract, 1-2 g / L sodium acetate, 0.2-0.3 g / L magnesium sulfate, 0.1-0.2 g / L manganese sulfate and 1-2 g / L Tween 80, which can be 20 g / L sucrose, 40 g / L peptone, 20 g / L yeast extract, 1 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 1 g / L Tween 80, and the remainder is water. The fermentation medium is sterilized at 121 ° C for 15 min and cooled to 37 ° C for standby use. The fermentation medium is used for fermentation and culture of Lactobacillus mucosa, and high-density fermentation can be achieved. The results of the embodiment of the present invention show that the fermentation culture medium used for fermentation and culture of Lactobacillus mucosa can greatly increase the number of bacteria in the fermentation broth compared with the ordinary culture medium.

[0026] In the present invention, the inoculation amount of the fermented Lactobacillus mucilaginosus seed solution is preferably 3% to 6%, and can be 4% or 5%. The fermentation culture cycle is preferably 14 to 18 hours, and can be 15 hours, 16 hours or 17 hours. By increasing the inoculation amount of the fermented Lactobacillus mucilaginosus seed solution and extending the fermentation cycle, it is beneficial to increase the initial viable count.

[0027] In the present invention, the pH value of the control system is controlled by a solvent of 30% by mass percent concentration of food-grade sodium hydroxide solution or 30% by volume percent concentration of ammonia solution, preferably 30% by mass percent concentration of food-grade sodium hydroxide solution. The results of the embodiment show that the type of the pH value of the control system can affect the storage stability of the prepared freeze-dried bacterial powder, and the food-grade sodium hydroxide solution of 30% by mass percent concentration is more conducive to improving the shelf life survival rate of the freeze-dried bacterial powder for 90 days than the ammonia solution of 30% by volume percent concentration.

[0028] In the present invention, the carbon source added is preferably sucrose, and the nitrogen source added is preferably peptone and yeast extract powder. The mass ratio of the carbon source and the nitrogen source is preferably 2-5:3-6, which can be 1:2, 2:5, 1:3, 1:1, 3:4, 3:5, 4:3, 4:5, 4:5, 2:3 or 5:6; the mass ratio of peptone and yeast extract powder is preferably (1-3): (1-2), which can be 1:1, 1:2, 2:1, 2:3, 3:1 and 3:2; the volume of the carbon and nitrogen sources added is preferably 80% of the volume of the initial fermentation medium. The time for adding carbon and nitrogen sources is preferably 4 to 12 hours of fermentation; the addition of carbon and nitrogen sources preferably includes uniform flow addition or step-by-step differential flow addition; during the uniform flow addition, the flow acceleration of the carbon and nitrogen source is preferably 4.5 ml / min; during the step-by-step differential flow addition, the flow acceleration of the carbon and nitrogen source is preferably 4 ml / min for 4 to 7 hours of fermentation; after 7 hours to 12 hours of fermentation, the flow acceleration of the carbon and nitrogen source is preferably 8 ml / min. The method of adding carbon and nitrogen sources is also a factor that affects the number of viable bacteria in the freeze-dried powder. Although the step-by-step differential flow addition of carbon and nitrogen sources can increase the initial number of viable bacteria, after 90 days of storage, the freeze-dried powder prepared by the uniform flow addition method has a higher survival rate of more than 90%.

[0029] The viable count of the fermented mucus Lactobacillus freeze-dried powder prepared by the above preparation method was 2×10 11 CFU / g, compared with the initial number of live bacteria, the survival rate of live bacteria was above 60%, with the highest reaching 93.9%.

[0030] The following is a detailed description of a freeze-dried bacterial powder of fermented Lactobacillus mucilaginosus with shelf-life stability and a preparation method thereof provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1

[0032] A method for preparing a high-stability fermented Lactobacillus mucilaginosus strain A21252 freeze-dried powder comprises the following steps:

[0033] (1) Resuscitation and activation of strains: The fermented Lactobacillus mucilaginosus stored in a glycerol tube was thawed at room temperature, streaked for the first time on MRS solid medium, and incubated at 37°C for 48 hours. A single colony was picked from the first streaked plate, streaked again on MRS solid medium, and incubated at 37°C for 48 hours.

[0034] (2) Seed solution preparation: Pick a single colony of medium size after activation, inoculate it into a culture medium containing MRS liquid, and culture it at a constant temperature of 37°C for 12 hours to obtain a seed solution. Inoculate the activated seed solution into MRS liquid culture medium at a 3% inoculum amount, and culture it at 37°C for 12 hours to obtain a working seed solution.

[0035] (3) High-density culture: The basic culture medium for fermenting mucus lactobacillus is formulated as 20 g / L sucrose, 40 g / L peptone, 20 g / L yeast extract, 1 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 1 g / L Tween 80, and the remainder is water. The basic culture medium for fermenting mucus lactobacillus is sterilized at 121°C for 15 min and cooled to 37°C for use. The basic culture medium is placed in a fermenter, and a working seed solution of 3% of the fermentation volume is added at a temperature of 37°C. The stirring speed is adjusted to 55 rpm. During the fermentation process, a carbon and nitrogen source is added, and the flow addition volume is 80%. The flow addition is uniformly added for 4 to 12 hours, and the flow acceleration rate is 4.5 ml / min. The pH is controlled to 5.5 with a food-grade sodium hydroxide solution of 30%. The aeration rate during the fermentation process is 1 L / min, and the fermentation is terminated after 14 hours of fermentation.

[0036] (4) Bacterial cell collection: centrifuge at 10,000 g for 5 min under low temperature conditions, and wash the resulting bacterial sludge with sterile saline to remove metabolites and residual culture medium from the fermentation process.

[0037] (5) The freeze-drying protective agent formula of the high-stability fermentation mucus lactobacillus is an aqueous solution containing the following mass percentage concentrations: 6% skim milk, 1% lactose, 12% trehalose, 12% sorbitol, 10% dextrin and 1% sodium ascorbate. After being dissolved in water, it is heated to 105°C for a mild Maillard reaction for 10 minutes and then cooled to 4°C for use.

[0038] (6) Addition of freeze-drying protective agent: The bacterial sludge and the stable freeze-drying protective agent were mixed in a mass ratio of 1:2 and emulsified for 45 min to obtain an emulsion.

[0039] (7) Freeze drying: Place the emulsion on a freeze drying tray for vacuum freeze drying. Freeze drying parameter settings: -40°C pre-freezing for 4.5 hours, cooling rate of 1°C / min, vacuum degree of 5-30Pa, and freeze drying time of 25 hours.

[0040] (8) Collection of bacterial powder: The freeze-dried material is crushed and packaged as required.

[0041] Example 2

[0042] A method for preparing a freeze-dried powder of fermented Lactobacillus mucilaginosus strain A21252 comprises the following steps:

[0043] (1) Resuscitation and activation of strains: The fermented Lactobacillus mucilaginosus stored in a glycerol tube was thawed at room temperature, streaked for the first time on MRS solid medium, and incubated at 37°C for 48 hours. A single colony was picked from the first streaked plate, streaked again on MRS solid medium, and incubated at 37°C for 48 hours.

[0044] (2) Seed solution preparation: Pick a single colony of medium size after activation, inoculate it into a culture medium containing MRS liquid, and culture it at a constant temperature of 37°C for 12 hours to obtain a seed solution. Inoculate the activated seed solution into MRS liquid culture medium at a 3% inoculum amount, and culture it at 37°C for 12 hours to obtain a working seed solution.

[0045] (3) High-density culture: The basal culture medium was placed in a fermenter, and a working seed solution of 3% of the fermentation volume was added at a temperature of 37°C. The stirring speed was adjusted to 55 rpm. During the fermentation process, a carbon and nitrogen source was added in a flow rate of 80%. The flow rate was divided into two sections: ① 4 to 7 h of flow addition, with a flow rate of 4 ml / min; ② 7 to 10 h of flow addition, with a flow rate of twice that of the previous section. The pH was controlled at 6 with a 30% food-grade sodium hydroxide solution. The ventilation rate during the fermentation process was 1 L / min, and the fermentation was terminated after 14 days of fermentation.

[0046] (4) Bacterial cell collection: centrifuge at 10,000 g for 5 min under low temperature conditions, and wash the resulting bacterial sludge with sterile saline to remove metabolites and residual culture medium from the fermentation process.

[0047] (5) The freeze-drying protective agent formula of fermented mucus lactobacillus is the same as that of Example 1.

[0048] (6) Addition of freeze-drying protective agent: The bacterial sludge and the stable freeze-drying protective agent were mixed in a mass ratio of 1:2 and emulsified for 45 min to obtain an emulsion.

[0049] (7) Freeze drying: Place the emulsion on a freeze drying tray for vacuum freeze drying. Freeze drying parameter settings: -40°C pre-freezing for 4.5 hours, cooling rate of 1°C / min, vacuum degree of 5-30Pa, and freeze drying time of 25 hours.

[0050] (8) Collection of bacterial powder: The freeze-dried material is crushed and packaged as required.

[0051] Example 3

[0052] A method for preparing a freeze-dried powder of fermented Lactobacillus mucilaginosus strain A21252 comprises the following steps:

[0053] (1) Resuscitation and activation of strains: The fermented Lactobacillus mucilaginosus stored in a glycerol tube was thawed at room temperature, streaked for the first time on MRS solid medium, and incubated at 37°C for 48 hours. A single colony was picked from the first streaked plate, streaked again on MRS solid medium, and incubated at 37°C for 48 hours.

[0054] (2) Seed solution preparation: Pick a single colony of medium size after activation, inoculate it into a culture medium containing MRS liquid, and culture it at a constant temperature of 37°C for 12 hours to obtain a seed solution. Inoculate the activated seed solution into MRS liquid culture medium at a 3% inoculum amount, and culture it at 37°C for 12 hours to obtain a working seed solution.

[0055] (3) High-density culture: The basal culture medium was placed in a fermenter, and a working seed solution of 6% of the fermentation volume was added at a temperature of 37°C. The stirring speed was adjusted to 55 rpm. During the fermentation process, a carbon and nitrogen source was added at a flow rate of 80% of the flow volume. The flow rate was uniformly added for 4 to 12 hours, and the flow rate was 4.5 ml / min. The pH was controlled to 5.5 with a 30% ammonia solution. The ventilation rate during the fermentation process was 1 L / min, and the fermentation was terminated after 18 hours.

[0056] (4) Bacterial cell collection: centrifuge at 10,000 g for 5 min under low temperature conditions, and wash the resulting bacterial sludge with sterile saline to remove metabolites and residual culture medium from the fermentation process.

[0057] (5) The freeze-drying protective agent formula of fermented mucus lactobacillus is the same as that of Example 1.

[0058] (6) Addition of freeze-drying protective agent: The bacterial sludge and the stable freeze-drying protective agent were mixed in a mass ratio of 1:2 and emulsified for 45 min to obtain an emulsion.

[0059] (7) Freeze drying: Place the emulsion on a freeze drying tray for vacuum freeze drying. Freeze drying parameter settings: -40°C pre-freezing for 4.5 hours, cooling rate of 1°C / min, vacuum degree of 5-30Pa, and freeze drying time of 25 hours.

[0060] (8) Collection of bacterial powder: The freeze-dried material is crushed and packaged as required.

[0061] Example 4

[0062] A method for preparing a freeze-dried powder of fermented Lactobacillus mucilaginosus strain A21252, specifically comprising the following steps:

[0063] (1) Bacterial recovery and activation: Same as Example 1.

[0064] (2) Seed solution preparation: same as Example 1.

[0065] (3) High-density culture: Same as Example 1.

[0066] (4) Bacteria collection: Same as Example 1.

[0067] (5) The freeze-dried protective agent formula of fermented mucus lactobacillus is composed of common components: 6% skim milk, 12% trehalose, 12% sorbitol, 10% dextrin, and the balance is water.

[0068] (6) Addition of freeze-drying protective agent: same as in Example 1.

[0069] (7) Freeze drying: same as in Example 1.

[0070] (8) Collection of bacterial powder: Same as in Example 1.

[0071] Example 5

[0072] A method for preparing a freeze-dried powder of fermented Lactobacillus mucilaginosus strain A21252, specifically comprising the following steps:

[0073] (1) Bacterial recovery and activation: Same as Example 1.

[0074] (2) Seed solution preparation: same as Example 1.

[0075] (3) High-density culture: Same as Example 1.

[0076] (4) Bacteria collection: Same as Example 1.

[0077] (5) The freeze-drying protective agent formula of fermented mucus lactobacillus is composed of common components: 12% skim milk, 12% trehalose, 12% sorbitol, 1% sodium ascorbate, and the balance is water.

[0078] (6) Addition of freeze-drying protective agent: same as in Example 1.

[0079] (7) Freeze drying: same as in Example 1.

[0080] (8) Collection of bacterial powder: Same as in Example 1.

[0081] Comparative Example 1

[0082] A method for preparing a freeze-dried powder of fermented Lactobacillus mucilaginosus strain A21252, specifically comprising the following steps:

[0083] (1) Bacterial recovery and activation: Same as Example 1.

[0084] (2) Seed solution preparation: same as Example 1.

[0085] (3) High-density culture: Same as Example 1.

[0086] (4) Bacteria collection: Same as Example 1.

[0087] (5) The freeze-dried protective agent formula of fermented Lactobacillus mucilaginosus is composed of common components: 12% skim milk, 12% trehalose, and the balance is water.

[0088] (6) Addition of freeze-drying protective agent: same as in Example 1.

[0089] (7) Freeze drying: same as in Example 1.

[0090] (8) Collection of bacterial powder: Same as in Example 1.

[0091] Example 6

[0092] The freeze-dried bacterial powder of fermented mucus lactobacillus obtained in the above Examples 1 to 4 and Comparative Examples 1 to 2 was stored at 4° C. in the same manner, and samples were taken out at 30 days and 90 days, respectively. The number of viable bacteria of fermented mucus lactobacillus was detected and the shelf life survival rate was calculated according to the method of the international "GB 4789.35-2016 National Food Safety Standard Food Microbiology Detection Lactic Acid Bacteria Detection". The calculation formula of the shelf life survival rate is shown in Formula I:

[0093] Shelf life survival rate (%) = shelf life viable bacteria count ÷ initial viable bacteria count × 100% Formula I.

[0094] Table 1 Survival rate of freeze-dried powder of fermented Lactobacillus mucilaginosus stored at 4℃ during shelf life

[0095]

[0096]

[0097] (1) As can be seen from Table 1, the viable count and shelf life survival rate of the freeze-dried bacterial powder of fermented mucus Lactobacillus prepared by the methods of Examples 1 to 5 are significantly higher than those of Comparative Example 1. The survival rate of Comparative Example 1 after 90 days is only 1%, while that of Examples 1 to 5 is above 40%, and that of Example 1 reaches 93.9%, which is much higher than that of Comparative Example 1.

[0098] (2) Compared with Example 2, Example 1 changed the method of high-density fermentation culture. Although the initial viable bacteria count and the 30-day shelf life viable bacteria count of Example 2 were higher than those of Example 1, the viable bacteria count at 90 days was lower than that of Example 1, and the survival rate was 30% lower than that of Example 1.

[0099] (3) Compared with Example 3, Example 1 increased the inoculation amount, changed the solution for adjusting pH, and prolonged the fermentation time. There was also a significant difference in the storage stability between the two.

[0100] (4) Compared with Examples 4 and 5, the high stability lyophilization protective agent used in Example 1 is more than 37% higher than the ordinary lyophilization protective agent. Although the initial viable bacterial count in Example 5 reached 4.42×10 11 CFU / g, but under shelf storage, the 90-day survival rate is much lower than that of Example 1.

[0101] In summary, the high-density culture process of fermentation and the freeze-drying protective agent formula need to be combined to obtain the highly stable freeze-dried bacterial powder of fermented Lactobacillus mucilaginosus A21252, so that the bacterial powder can be protected from more mechanical damage and solute damage in the storage environment and maintain a high shelf life stability.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A freeze-drying protective agent suitable for fermenting mucus lactobacillus, characterized in that: The invention comprises the following components in parts by weight: 2 to 15 parts of protein, 5 to 25 parts of sugar alcohol, 0 to 1 part of ascorbic acid sodium salt and 0 to 10 parts of dextrin.

2. The lyophilized protective agent according to claim 1, characterized in that The freeze-drying protective agent comprises the following components in parts by weight: 4 to 12 parts of protein, 10 to 25 parts of sugar alcohol, 0.5 to 1 part of ascorbic acid sodium salt and 5 to 10 parts of dextrin.

3. The lyoprotectant according to claim 2, characterized in that: The protein includes skim milk; The sugar alcohol includes at least one of the following: lactose, trehalose and sorbitol.

4. The lyophilization protective agent according to claim 3, characterized in that The lyoprotectant also includes water; The freeze-drying protective agent comprises an aqueous solution containing the following mass percentage concentrations: 6% skim milk, 1% lactose, 12% trehalose, 12% sorbitol, 10% dextrin and 1% sodium ascorbic acid.

5. A freeze-dried bacterial powder of fermented Lactobacillus mucilaginosus prepared by using the freeze-drying protective agent according to any one of claims 1 to 4.

6. A method for preparing freeze-dried powder of fermented mucus lactobacillus, characterized in that: The seed liquid of fermented mucus lactobacillus is inoculated into a fermentation medium for fermentation culture, during which a carbon and nitrogen source is added, and the pH value of the system is controlled to be 5.5-6; the bacteria are collected from the fermentation liquid, mixed with the freeze-drying protective agent described in any one of claims 1 to 4, and freeze-dried to obtain freeze-dried bacterial powder of fermented mucus lactobacillus.

7. The preparation method according to claim 6, characterized in that: The fermentation medium is an aqueous solution containing the following components: 20-30 g / L sucrose, 20-40 g / L peptone, 10-20 g / L yeast extract, 1-2 g / L sodium acetate, 0.2-0.3 g / L magnesium sulfate, 0.1-0.2 g / L manganese sulfate and 1-2 g / L Tween 80.

8. The preparation method according to claim 6, characterized in that: The inoculation amount of the fermented mucus lactobacillus seed liquid is 3% to 6%; The solvent used for controlling the pH value of the system is a food-grade sodium hydroxide solution with a mass percent concentration of 30% or an ammonia solution with a volume percent concentration of 30%.

9. The preparation method according to claim 6, characterized in that: The fermentation culture cycle is 14 to 18 hours; The fermentation culture is aerated; the aeration rate during the aeration culture is 1 L / min.

10. The preparation method according to any one of claims 6 to 9, characterized in that: The carbon source added is sucrose, and the nitrogen source added is peptone and yeast extract powder; the mass ratio of the carbon source to the nitrogen source is 2-5:3-6; the mass ratio of the peptone to the yeast extract powder is (1-3):(1-2); the volume of the carbon and nitrogen sources added is 80% of the volume of the initial fermentation medium; The time for adding the carbon and nitrogen source is 4 to 12 hours for fermentation; the adding of the carbon and nitrogen source includes uniform flow addition or step-by-step differential flow addition; during the uniform flow addition, the flow acceleration of the carbon and nitrogen source is 4.5 ml / min; during the step-by-step differential flow addition, the flow acceleration of the carbon and nitrogen source is 4 ml / min for 4 to 7 hours of fermentation; after 7 hours to 12 hours of fermentation, the flow acceleration of the carbon and nitrogen source is 8 ml / min.

Images