Freeze-drying protection composition and application thereof in preparation of lactobacillus fermentum TY-F13 freeze-drying bacteria

Abstract

The invention belongs to the technical field of microbial agents, and particularly relates to a freeze-drying protection composition and application thereof in preparation of lactobacillus fermentum TY-F13 freeze-dried bacteria, and the freeze-drying protection composition comprises skimmed milk powder, trehalose and yeast dietary fibers. The lactobacillus fermentum TY-F13 freeze-dried bacteria prepared on the basis of the freeze-drying protection composition are high in strain survival rate, and the freeze-drying survival rate can reach 93% or above; and after the freeze-dried bacteria are stored at the low temperature of-18 DEG C for 2 years, the number of viable bacteria is not obviously changed, and the freeze-dried bacteria are excellent in stability. Moreover, the original characteristic of inhibiting mould can still be kept, the shelf life of the fermented milk can be effectively prolonged, and meanwhile, the original quality of the fermented milk is ensured not to be affected by any adverse effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial inoculant, and particularly relates to a freeze-drying protective composition and application thereof in preparation of freeze-dried Lactobacillus fermentum TY-F13 bacteria. BACKGROUND

[0002] Yogurt, as a globally popular fermented dairy product, is favored by consumers for its unique flavor and nutritional value. However, the shelf life of yogurt is significantly affected by mold and yeast contamination, which has become a key bottleneck restricting the industrial scale development. Mold and yeast can still reproduce in low temperature environments, leading to stratification, rancidity and flavor deterioration in yogurt, not only reducing product quality, but also potentially causing food safety risks and economic losses to production enterprises.

[0003] To solve the above problems, the industry traditionally uses chemical preservatives (such as potassium sorbate), strict environmental control (such as sterile filling), or packaging material upgrading to extend the shelf life. However, chemical additives are controversial for health, and physical control measures face problems such as high cost, complex operation, or limited antibacterial effect. In recent years, lactic acid bacteria have become a research hotspot due to their natural antibacterial properties and probiotic functions. Among them, direct-fed lactic acid bacteria powder gradually replaces traditional starter cultures to become the core raw material for yogurt production due to its advantages such as no need for activation and convenient use. However, in the existing preparation process of direct-fed lactic acid bacteria powder, the freeze-drying process easily leads to damage of lactic acid bacteria cell membranes and loss of active ingredients, and the freeze-dried bacteria powder has a rapid decrease in activity during storage and transportation, with insufficient stability of antibacterial efficacy. At the same time, the production cycle is long and the efficiency is low, which is difficult to meet the needs of high activity, long shelf life and low cost of bacteria powder for industrial production.

[0004] In view of the technical bottlenecks such as low activity, poor stability, fluctuation of antibacterial efficacy, and insufficient production efficiency of lactic acid bacteria powder in the prior art, the present application aims to provide an optimized preparation method for direct-fed lactic acid bacteria powder. By innovating the formula and preparation process of freeze-drying protective agents, the freeze-drying survival rate and storage stability of lactic acid bacteria are improved, and their inhibition ability against mold and yeast is enhanced, thereby effectively extending the shelf life of yogurt and maintaining the original quality of the product. At the same time, the production process is simplified and the cost is reduced, providing an efficient and safe technical solution for the fermented dairy product industry. SUMMARY

[0005] Based on this, the present application provides a freeze-drying protective composition suitable for Lactobacillus fermentum TY-F13 and a freeze-drying protective agent. The freeze-dried Lactobacillus fermentum TY-F13 bacteria prepared based on the freeze-drying protective composition and the freeze-drying protective agent have high freeze-drying survival rate, good stability, and effectively maintain their original characteristics of inhibiting mold.

[0006] To achieve the above-mentioned purposes, the present application can adopt the following technical solutions:

[0007] The present application provides a freeze-drying protective composition in one aspect, the freeze-drying protective agent comprising skimmed milk powder, trehalose and yeast dietary fiber.

[0008] Preferably, in the freeze-drying protective composition, the mass ratio of skimmed milk powder, trehalose and yeast dietary fiber is 15:(7-8):(7-8).

[0009] More preferably, in the freeze-drying protective composition, the mass ratio of skimmed milk powder, trehalose and yeast dietary fiber is 2:1:1.

[0010] The present application provides a freeze-drying protective agent in another aspect, the freeze-drying protective agent comprising the freeze-drying protective composition and an aqueous medium.

[0011] The present application provides a freeze-dried Lactobacillus fermentum TY-F13 bacteria in still another aspect, the freeze-dried Lactobacillus fermentum TY-F13 bacteria comprising the freeze-drying protective composition and Lactobacillus fermentum TY-F13.

[0012] The present application provides a preparation method of the freeze-dried Lactobacillus fermentum TY-F13 bacteria in still another aspect, the preparation method comprising:

[0013] (1) fermenting Lactobacillus fermentum TY-F13 to obtain a fermentation broth, and centrifuging the fermentation broth to obtain a bacterial slurry;

[0014] (2) mixing the freeze-drying protective agent and the bacterial slurry to obtain an emulsion;

[0015] (3) freeze-drying the emulsion to obtain the freeze-dried Lactobacillus fermentum TY-F13 bacteria.

[0016] Preferably, the preparation method satisfies one or more of the following conditions:

[0017] Condition (i):

[0018] In step (1), the fermentation process uses a culture medium for culture; the culture medium comprises yeast extract, peptone, glucose, sodium acetate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and manganese sulfate; wherein the yeast extract is selected from one or more combinations of yeast extract, yeast extract powder FM803, yeast extract powder FM802 or yeast extract powder FM503; and / or the peptone is selected from one or more combinations of bovine bone peptone, yeast peptone FP102, cottonseed peptone or soybean peptone;

[0019] Condition (ii):

[0020] In step (2), the mass ratio of the freeze-drying protective agent and the bacterial slurry is (10-20):1; and / or the emulsification comprises: emulsifying at 0°C-4°C, 550r / min-650r / min for 10min-20min;

[0021] Condition (iii):

[0022] In step (3), the freeze-dried Lactobacillus TY-F13 bacteria are crushed to obtain freeze-dried Lactobacillus TY-F13 bacteria powder.

[0023] More preferably, in condition (i) of the above preparation method,

[0024] The culture medium comprises, by weight: 13-17 parts of yeast extract, 8-12 parts of proteose peptone, 28-32 parts of glucose, 4-6 parts of sodium acetate, 1-3 parts of potassium phosphate dibasic, 1-3 parts of potassium phosphate monobasic, 0.05-0.15 parts of magnesium sulfate, and 0.04-0.06 parts of manganese sulfate, and 1000 parts of medium water; wherein,

[0025] The yeast extract is selected from one or more of yeast extract FM803 or yeast extract FM802; and / or

[0026] The proteose peptone is selected from one or more of yeast peptone FP102 or cottonseed peptone.

[0027] In another aspect of the present application, the freeze-dried Lactobacillus TY-F13 bacteria described above are used in the preparation of fermented milk.

[0028] In another aspect of the present application, the freeze-dried Lactobacillus TY-F13 bacteria described above are used to inhibit spoilage molds in fermented milk, including one or more of Penicillium, Trichoderma longibrachiatum, Aspergillus aculeatinus, Aspergillus fumigatus, Cladosporium halotolerans, Aspergillus Sydowii, and Aspergillus japonicus.

[0029] The beneficial effects of the present application include:

[0030] (1) The strain survival rate of the freeze-dried Lactobacillus TY-F13 bacteria prepared based on the freeze-drying protective composition provided by the present application is high, and the freeze-drying survival rate can reach more than 93%; and after storage at -18℃ for 2 years, the number of live bacteria does not change significantly, and the freeze-dried bacteria have excellent stability.

[0031] (2) The freeze-dried Lactobacillus TY-F13 bacteria prepared based on the freeze-drying protective composition provided by the present application still maintain their original mold inhibiting properties, can effectively prolong the shelf life of fermented milk, and at the same time ensure that the original quality of the fermented milk is not adversely affected.

[0032] (3) The freeze-dried Lactobacillus fermentum TY-F13 prepared based on the freeze-drying protective composition provided by the application is easy to operate, short in production time, easy to industrialize, and has a direct injection design, which further simplifies the use process and provides convenience for application. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 A comparison result diagram of yogurts prepared for the experimental group carrying Penicillium CICC2515 and the control group stored at 10℃;

[0034] Figure 2 A comparison result diagram of yogurts prepared for the experimental group carrying Penicillium CICC2515 and the control group stored at 28℃;

[0035] Figure 3 A comparison result diagram of yogurts prepared for the experimental group carrying factory mixed mold and the control group stored at 28℃. DETAILED DESCRIPTION

[0036] The embodiments are used to better illustrate the application, but are not the only embodiments of the application, and are limited to the embodiments. Therefore, those skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above description, which still belong to the protection scope of the application.

[0037] The terms used herein are used only to describe specific embodiments and are not intended to limit the present disclosure. Unless there is a clear different meaning in the context, the singular form includes the plural form. As used herein, it should be understood that terms such as "include", "have", "contain", "comprise", etc. are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present application are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations can exist or can be added. As used herein, " / " can be interpreted as "and" or "or" depending on the circumstances.

[0038] In a first aspect, the embodiments of the present application provide a freeze-drying protective composition, and the freeze-drying protective agent comprises skimmed milk powder, trehalose and yeast dietary fiber.

[0039] It should be noted that the present application compares the effects of trehalose and skimmed milk powder and single glucose and skimmed milk powder prepared lyophilized protectant, it is found that the lyophilized survival rate of the lyophilized bacteria powder prepared by trehalose is improved from 70% to 85%, and the number of live bacteria is still higher after 2 years of storage, which shows that trehalose has good freeze protection effect, it can form a glassy structure around the cells, reduce the damage of ice crystal formation to the cell membrane, thereby improving the survival rate and storage stability of probiotics in the freeze-drying process; in addition, the lyophilized bacteria powder prepared by the lyophilized protectant based on single yeast dietary fiber and skimmed milk powder shows very stable live bacteria maintenance ability (2.0x10 11 and 1.7x10 11 CFU / g) during 1 year and 2 years of storage, which shows that the yeast dietary fiber may effectively slow down the damage of the bacteria during storage through physical barrier or water adsorption mechanism, and has good long-term protection potential. In addition, trehalose and yeast dietary fiber are combined in an equal proportion, and the performance is the most outstanding: the freeze-drying survival rate is 93%, the number of live bacteria after freeze-drying is 4.0x10 11 CFU / g, which is significantly higher than that of other groups; after 1 year and 2 years of storage, the number of live bacteria is 4.3x10 11 and 3.9x10 11 CFU / g, which is much higher than that of single component formula. This shows that the combination of trehalose and dietary fiber can play a synergistic effect, which not only improves the freeze-drying efficiency, but also enhances the low-temperature storage stability.

[0040] In some specific examples, the mass ratio of skimmed milk powder, trehalose and yeast dietary fiber in the above lyophilized protection composition is 15:(7-8):(7-8).

[0041] It should be noted that the mass ratio of skimmed milk powder, trehalose and yeast dietary fiber in the present application can be 15:(7-8):(7-8), for example 15:7:8, 15:8:7, 15:7:7 or 15:8:8, etc.

[0042] In some specific examples, the mass ratio of skimmed milk powder, trehalose and yeast dietary fiber in the above lyophilized protection composition is 2:1:1.

[0043] It should be noted that the mass ratio of skimmed milk powder, trehalose and yeast dietary fiber in the present application can be preferably the above ratio, and more preferably 2:1:1 (i.e. 15:7.5:7.5), and the performance of the freeze-dried bacteria of Lactobacillus fermentum TY-F13 prepared under this ratio is better.

[0044] In the second aspect, the present application provides a lyophilized protectant, which comprises the above lyophilized protection composition and a water medium.

[0045] It should be noted that the freeze-dried protective composition in the present application can be mixed with water medium uniformly, and then sterilized at 110 DEG C for 10 minutes to obtain the freeze-dried protective agent.

[0046] In a third aspect, the present application provides a freeze-dried Lactobacillus fermentum TY-F13 bacteria, which comprises the freeze-dried protective composition and the Lactobacillus fermentum TY-F13.

[0047] It should be noted that the freeze-dried protective composition and the freeze-dried protective agent in the present application are suitable for the Lactobacillus fermentum TY-F13. The preservation number of the Lactobacillus fermentum TY-F13 is CGMCC No. 25740. In addition, it should be understood that the Lactobacillus fermentum is now also called Limosilactobacillus fermentum.

[0048] In a fourth aspect, the present application provides a preparation method of the freeze-dried Lactobacillus fermentum TY-F13 bacteria, which comprises the following steps:

[0049] (1) fermenting the Lactobacillus fermentum TY-F13 to obtain a fermentation broth, and centrifuging the fermentation broth to obtain a bacterial slurry;

[0050] (2) mixing the freeze-dried protective agent of claim 4 and the bacterial slurry to obtain an emulsion;

[0051] (3) freeze-drying the emulsion to obtain the freeze-dried Lactobacillus fermentum TY-F13 bacteria.

[0052] In some specific examples, in step (1) of the preparation method, the fermentation process uses a culture medium for culture; the culture medium comprises yeast extract, peptone, glucose, sodium acetate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and manganese sulfate; wherein the yeast extract is selected from one or more combinations of yeast extract, yeast extract powder FM803, yeast extract powder FM802 or yeast extract powder FM503; and / or the peptone is selected from one or more combinations of bovine bone peptone, yeast peptone FP102, cottonseed peptone or soybean peptone.

[0053] It should be noted that the culture medium in the present application is well known to those skilled in the art, and the culture medium containing the above components is more suitable for the culture of the Lactobacillus fermentum TY-F13. In addition, the "yeast extract" in the present application refers to a kind of yeast extract products such as yeast extract and yeast extract powder, and the "yeast extract" only refers to the substance extracted from yeast by traditional methods, and does not include yeast extract powder.

[0054] In some specific examples, in step (1) of the preparation method,

[0055] The culture medium comprises, by weight: 13-17 parts of yeast extract, 8-12 parts of proteose peptone, 28-32 parts of glucose, 4-6 parts of sodium acetate, 1-3 parts of dipotassium hydrogen phosphate, 1-3 parts of potassium dihydrogen phosphate, 0.05-0.15 parts of magnesium sulfate, and 0.04-0.06 parts of manganese sulfate, and 1000 parts of medium water; wherein,

[0056] The yeast extract is selected from one or more of yeast extract powder FM803 or yeast extract powder FM802; and / or

[0057] The proteose peptone is selected from one or more of yeast proteose peptone FP102 or cottonseed proteose peptone.

[0058] It should be noted that in the above culture medium, the component quantities are preferably the above-mentioned quantities. In addition, the yeast extract and the proteose peptone in the culture can be further preferably the above-mentioned yeast extract powder FM803 or yeast extract powder FM802 and yeast proteose peptone FP102 or cottonseed proteose peptone, which are more suitable for the proliferation of Lactobacillus fermentum TY-F13.

[0059] In some specific examples, in step (2) of the above preparation method,

[0060] The mass ratio of the freeze-drying protective agent to the bacterial slurry is (10-20):1, such as 1:3:1, 15:1, or 17:1, etc.; and / or

[0061] The emulsification comprises: emulsifying at 0-4℃ and 550-650 r / min (such as 570 r / min, 600 r / min, or 630 r / min, etc.) for 10-20 min (such as 13 min, 15 min, or 17 min, etc.).

[0062] In some specific examples, in step (3) of the above preparation method, the freeze-dried Lactobacillus TY-F13 bacteria are crushed to obtain freeze-dried Lactobacillus TY-F13 bacterial powder.

[0063] It should be noted that the present application can further prepare the freeze-dried Lactobacillus TY-F13 bacteria into different forms, such as powder, and the preparation method is known in the art.

[0064] In a fifth aspect, the present application provides a use of the above-mentioned freeze-dried Lactobacillus fermentum TY-F13 in the preparation of fermented milk.

[0065] In a sixth aspect, the application provides a use of the freeze-dried Lactobacillus fermentum TY-F13 bacteria in inhibiting spoilage molds in fermented milk, wherein the spoilage molds include one or more of Penicillium, Trichoderma longibrachiatum, Aspergillus aculeatinus, Aspergillus fumigatus, Cladosporium halotolerans, Aspergillus Sydowii, and Aspergillus japonicus.

[0066] It should be noted that the freeze-dried Lactobacillus fermentum TY-F13 bacteria prepared based on the freeze-drying protective composition provided by the application still has the original characteristics of inhibiting molds, and can effectively prolong the shelf life of fermented milk while ensuring that the original quality of the fermented milk is not adversely affected.

[0067] In order to better understand the application, the application will be further illustrated below in conjunction with specific examples, but the application is not limited to the following examples.

[0068] In the following examples, the Lactobacillus fermentum TY-F13 used has the preservation number CGMCC No. 25740.

[0069] In the following examples, the raw materials of the culture medium used are all commercially available raw materials.

[0070] In the following examples, the yeast extract, yeast extract powder FM 803, yeast extract powder FM 802, and yeast extract powder FM 503 are all purchased from Angel Yeast Co., Ltd.

[0071] Example 1

[0072] The application provides a preparation method of the Lactobacillus fermentum TY-F13 bacteria powder, which is specifically as follows.

[0073] (1) Preparation of fermentation bacteria: under sterile conditions, a freeze tube of the Lactobacillus fermentum TY-F13 is taken out, the bacteria liquid stored at -80°C is restored to room temperature, and then streaked on a solid culture medium and cultured at 37°C for 32 hours to obtain a purified bacteria strain; after the culture, a single colony is picked on the plate and inoculated in 5 mL of liquid culture medium, and cultured at 37°C for 16 hours for activation to obtain a first-stage seed liquid; the first-stage seed liquid is transferred to 100 mL of liquid culture medium, and cultured at 37°C for 8 hours for activation to obtain a second-stage seed liquid.

[0074] (2) fermentation culture: the secondary seed liquid is transferred to 6L liquid medium, and cultured at 37℃ for 8h for expansion culture. After the expansion culture is completed, it is transferred to 70L liquid medium, and cultured at 37℃ for 12h. During the fermentation, the pH of the fermentation broth is maintained at 5.6 by adding 20% sodium hydroxide solution dropwise. When the OD value of the fermentation broth no longer rises, the fermentation is terminated, and the temperature of the fermentation broth is reduced to 20℃;

[0075] (3) centrifugation and emulsification: the above cooled fermentation broth is centrifuged at 12000rpm / min using a centrifuge to collect the bacterial slurry. After centrifugation, the bacterial slurry is mixed with the freeze-drying protectant at a mass ratio of 1:10. The freeze-drying protectant is controlled at about 4℃, and emulsified at 600r / min for 15min to obtain an emulsion;

[0076] (4) freeze-dried and crushed: the freeze-drier plate temperature is reduced to 5℃ in advance, the sterilized tray is placed, and the emulsion is poured into the tray for freeze-drying (-40℃ for 48h). After freeze-drying is completed, the semi-finished product in the freeze-drying tray is collected into a designated container that has been sterilized, processed into small pieces, slowly poured into a powder sifter, and siftered to obtain freeze-dried powder;

[0077] In the above preparation method, the preparation method of the liquid medium is as follows: yeast extract FM 80315g, yeast peptone FP10210g, glucose 30g, sodium acetate 5g, potassium hydrogen phosphate 2g, potassium dihydrogen phosphate 2g, magnesium sulfate 0.1g, and manganese sulfate 0.05g are uniformly mixed with 1000mL distilled water, and then sterilized at 121℃ for 15min;

[0078] In the above preparation method, the preparation method of the solid medium is as follows: 1.5% agar is added to the liquid medium to prepare it;

[0079] In the above preparation method, the preparation method of the freeze-drying protectant is as follows: 15g skimmed milk powder, 7.5g trehalose, and 7.5g yeast dietary fiber are uniformly mixed with 100mL distilled water, and then sterilized at 110℃ for 10min.

[0080] Examples 2 to 5

[0081] Examples 2 to 5 are substantially the same as Example 1, except that the raw material components for preparing the freeze-drying protectant are different, and the rest is the same as Example 1 to prepare freeze-dried powder. In Examples 2 to 5, the raw material components for preparing the freeze-drying protectant are as follows:

[0082] In Example 2, the raw material components of the freeze-drying protectant are: 15g skimmed milk powder, 7g trehalose, 7.5g yeast dietary fiber, and 100mL distilled water;

[0083] The raw material components of the freeze-drying protective agent in Example 3 are: skimmed milk powder 15 g, trehalose 8 g, yeast dietary fiber 7.5 g, and 100 mL of distilled water;

[0084] The raw material components of the freeze-drying protective agent in Example 4 are: skimmed milk powder 15 g, trehalose 7.5 g, yeast dietary fiber 7 g, and 100 mL of distilled water;

[0085] The raw material components of the freeze-drying protective agent in Example 5 are: skimmed milk powder 15 g, trehalose 7.5 g, yeast dietary fiber 8 g, and 100 mL of distilled water.

[0086] Examples 6 to 11

[0087] Examples 6 to 11 provide liquid culture media suitable for culturing Lactobacillus fermentum TY-F13, the liquid culture medium components of Examples 6 to 11 are substantially the same as those in Example 1, the difference is that the components in the liquid culture medium are different, as shown below:

[0088] The liquid culture medium of Example 6 uses yeast extract instead of yeast extract FM803 in the liquid culture medium of Example 1, and the others are the same as Example 1;

[0089] The liquid culture medium of Example 7 uses yeast extract FM802 instead of yeast extract FM803 in the liquid culture medium of Example 1, and the others are the same as Example 1;

[0090] The liquid culture medium of Example 8 uses yeast extract FM503 instead of yeast extract FM803 in the liquid culture medium of Example 1, and the others are the same as Example 1;

[0091] The liquid culture medium of Example 9 uses bovine bone peptone instead of yeast peptone FP102 in the liquid culture medium of Example 1, and the others are the same as Example 1;

[0092] The liquid culture medium of Example 10 uses cottonseed peptone instead of yeast peptone FP102 in the liquid culture medium of Example 1, and the others are the same as Example 1;

[0093] The liquid culture medium of Example 11 uses soybean peptone instead of yeast peptone FP102 in the liquid culture medium of Example 1, and the others are the same as Example 1.

[0094] Comparative Examples 1 to 4

[0095] Comparative Examples 1 to 4 are substantially the same as Example 1, the difference is that the raw material components for preparing the freeze-drying protective agent are different, and the others are the same as Example 1, and the freeze-dried powder is prepared; wherein the raw material components for preparing the freeze-drying protective agent in Comparative Examples 1 to 4 are as follows:

[0096] The raw material components of the freeze-drying protectant in Comparative Example 1 were: skim milk powder 15 g, glucose 15 g, and 100 mL of distilled water;

[0097] The raw material components of the freeze-drying protectant in Comparative Example 2 were: skim milk powder 15 g, trehalose 15 g, and 100 mL of distilled water;

[0098] The raw material components of the freeze-drying protectant in Comparative Example 3 were: skim milk powder 15 g, yeast dietary fiber 15 g, and 100 mL of distilled water;

[0099] The raw material components of the freeze-drying protectant in Comparative Example 4 were: skim milk powder 15 g, glucose 7.5 g, yeast dietary fiber 7.5 g, and 100 mL of distilled water.

[0100] Related tests

[0101] (I) Strain survival rate and stability test in freeze-dried powder

[0102] In the following tests, the viable cell count and freeze-drying survival rate were determined as follows: 1 g of freeze-dried powder sample was weighed into a sterilized test tube, 10-fold diluted with sterile physiological saline, and then the sample was fully dissolved and mixed using a hand shaker and a shaker. The sample was diluted to the appropriate gradient, and after incubation in 37℃ medium for 36h, the survival rate was calculated; freeze-drying survival rate (%) =(N1 / N0)*100%; Note: N0 is the viable cell count before freeze-drying, and N1 is the viable cell count after freeze-drying.

[0103] The viable cell count test after storage at -18℃ for 1 or 2 years was as follows: the freeze-dried powder after freeze-drying was stored in a sealed aluminum foil composite bag at -18℃ under atmospheric pressure; 1 g of freeze-dried powder was weighed at -18℃ every year, rehydrated in sterile physiological saline, and the rehydrated bacterial cells were diluted by gradient dilution, then plated and incubated at 37℃ anaerobically for 36h, and the viable cell count was determined; storage survival rate (%) =(N1 / N0)*100%; Note: N0 is the viable cell count at 0 days of storage, and N1 is the viable cell count after N days of storage.

[0104] The freeze-drying survival rate and viable cell count after freeze-drying of the freeze-dried powders prepared in Examples 1 to 5 and Comparative Examples 1 to 4 were tested, and the viable cell count after storage at -18℃ for 1 year and 2 years was tested, and the results are shown in Table 1 below.

[0105] Table 1 Effect of different freeze-drying protectants on strain freeze-drying survival rate and low-temperature storage stability

[0106]

[0107]

[0108] As can be seen from Table 1 above,

[0109] The freeze-dried survival rate and the number of viable bacteria after freeze-drying of the freeze-dried bacterial powder prepared in Examples 1 to 5 were significantly higher than those of the Comparative Examples, and the reduction rate of the number of viable bacteria after storage at -18°C for 1 year and 2 years was also significantly lower than that of the Comparative Examples;

[0110] Among them, by comparing Example 1 with Comparative Example 2 and Comparative Example 3, it can be seen that the effective components of the freeze-drying protective agent in Comparative Example 2 are skim milk powder 15 g and trehalose 15 g, the effective components of the freeze-drying protective agent in Comparative Example 3 are skim milk powder 15 g and yeast dietary fiber 15 g, and the effective components of the freeze-drying protective agent in Example 1 are skim milk powder 15 g, trehalose 7.5 g and yeast dietary fiber 7.5 g; through analysis of various test data in Table 1, it can be seen that the freeze-dried bacterial powder prepared by the freeze-drying protective agent prepared by combining trehalose and yeast dietary fiber has various performance indicators far higher than those of single trehalose and single yeast dietary fiber, and not a simple additive effect, indicating that trehalose and yeast dietary fiber have a synergistic effect in preparing freeze-dried bacterial powder suitable for Lactobacillus fermentum TY-F13.

[0111] In addition, by comparing Comparative Example 1, Comparative Example 3 and Comparative Example 4, it can be seen that the effective components of the freeze-drying protective agent in Comparative Example 2 are skim milk powder 15 g and trehalose 15 g, the effective components of the freeze-drying protective agent in Comparative Example 3 are skim milk powder 15 g and yeast dietary fiber 15 g, and the effective components of the freeze-drying protective agent in Comparative Example 4 are skim milk powder 15 g, glucose 7.5 g and yeast dietary fiber 7.5 g; through analysis of various test data in Table 1, it can be seen that the freeze-dried bacterial powder prepared by the freeze-drying protective agent prepared by combining glucose and yeast dietary fiber has various performance indicators higher than those of single trehalose, but far lower than those of single yeast dietary fiber, further indicating that not all combinations of sugars and yeast dietary fiber have a synergistic effect.

[0112] (II) Bacteriostatic test of freeze-dried powder in yogurt

[0113] (1) Fungal standard strain Penicillium CICC2515 bacteriostatic test

[0114] 1) Penicillium CICC2515 was cultured on PDA plates at 28°C for 4 days until spores were formed; the surface of the plate was washed with sterile water containing 0.05% Tween 80, filtered with sterile gauze to obtain a spore suspension, and the concentration of Penicillium spores was adjusted to an appropriate concentration using a hemocytometer to obtain a Penicillium liquid, which was stored in a 4°C refrigerator for standby.

[0115] 2) Defatted reconstituted milk (defatted milk powder added amount 120 g / L, sugar added amount 60 g / L) was heated to 95°C in a water bath for sterilization for 15 minutes to obtain an emulsion, and the emulsion was cooled to about 42°C and inoculated with a starter culture (commercial starter culture with product number S4.01T from Zelenye Linii LLC, Russia, same below); in addition, in order to make the results of the antibacterial experiment more obvious, Penicillium sp. liquid was added to the above emulsion according to a final concentration of 100 spores / cup (30 mL);

[0116] 3) The emulsion to which the Penicillium sp. liquid was added was divided into a control group, a positive group, a low-dose experimental group, and a high-dose experimental group; wherein the control group was not treated; the positive group was added with commercial preservative bacteria powder Y (commercial starter culture with product number S3.02 from Zelenye Linii LLC, Russia, same below) according to a final concentration of 10 5 CFU / mL of emulsion; the low-dose experimental group was added with TY-F13 freeze-dried bacteria powder (prepared in Example 1) according to a final concentration of 10 5 CFU / mL of emulsion; the high-dose experimental group was added with TY-F13 freeze-dried bacteria powder (prepared in Example 1) according to a final concentration of 10 6 CFU / mL of emulsion; different groups of emulsions were respectively treated according to the above, and then stirred uniformly and filled into 30 mL / cup;

[0117] 4) The different samples after the above treatment were all fermented in a 42°C constant temperature incubator for 5h to a pH of 4.6, and then ripened at 4°C for 12h to obtain different yogurt samples;

[0118] 5) Different yogurt samples were respectively stored at 10°C and 28°C, and the state of the yogurt samples was observed regularly.

[0119] The state change of different groups of yogurt samples at 10°C and 28°C is shown in Figure 1 and Figure 2 The results show that for Penicillium CICC2515, the growth and reproduction speed is significantly accelerated under the storage condition of 28°C than under the storage condition of 10°C; although temperature has a significant effect on the growth of mold, the freeze-dried bacteria powder shows a consistent trend in preservation effect under different temperature storage conditions, and the specific mold prevention effect ranking is: TY-F13 bacteria powder (10 6 cfu / mL) > TY-F13 bacteria powder (10 5 cfu / mL) > commercial preservative bacteria powder Y (10 5 cfu / mL) > blank control. This shows that the TY-F13 bacteria powder has the best mold prevention effect at a concentration of 10 6 cfu / mL, and even at a concentration of 10 5 cfu / mL is better than the commercial preservative bacteria powder Y (105 TY-F13 powder showed significant mold inhibition effect at different storage temperatures, and the optimal mold inhibition effect was at 10 6 CFU / mL, which was significantly better than the commercial preservative powder Y.

[0120] (2) Mixed mold inhibition test in production workshop

[0121] 1) Fresh yogurt was exposed in the open air for 7 days in the production workshop of Chongqing Tianyou Dairy Co., Ltd., and then the spoilage molds were obtained by isolation and purification identification. The spoilage molds included Trichoderma longibrachiatum JD1, T. longibrachiatum JD2, Aspergillus pseudotuberculus JB, A. fumigatus DHZ926-1, Cladosporium resinae JD926-2, Aspergillus sydowii CS926-2, and A. japonicus DHZ1.

[0122] 2) The mold indicator bacteria of the above spoilage molds were cultured on PDA plates at 28°C for 4 days until spores were generated. The plate surface was washed with sterile water containing 0.05% Tween 80, filtered with sterile gauze to obtain a spore suspension, and the concentration of mold spores was adjusted to an appropriate concentration using a hemocytometer. The mixed mold liquid was obtained by mixing the seven mold indicator bacteria at an appropriate concentration and storing it in a 4°C refrigerator.

[0123] 3) The skimmed reconstituted milk (skimmed milk powder addition amount 120 g / L, sugar addition amount 60 g / L) was heated to 95°C in a water bath for sterilization for 15 minutes to obtain a milk emulsion, which was cooled to about 42°C for inoculation of the starter culture. In order to make the results of the inhibition experiment more obvious, the mixed mold liquid was added to the above milk emulsion according to a final concentration of 100 spores / cup (30 mL).

[0124] 4) The milk emulsion with the added mixed mold liquid was divided into a control group, a positive group, a low-dose experimental group, a medium-dose experimental group, and a high-dose experimental group. The control group was not treated. The positive group was added with the commercial preservative powder Y according to a final concentration of 10 8 CFU / mL of the milk emulsion (the optimal addition amount recommended by the manufacturer). The low-dose experimental group was added with the TY-F13 freeze-dried powder (prepared in Example 1) according to a final concentration of 10 4 CFU / mL of the milk emulsion. The medium-dose experimental group was added with the TY-F13 freeze-dried powder (prepared in Example 1) according to a final concentration of 10 5 CFU / mL of the milk emulsion. The high-dose experimental group was added with the TY-F13 freeze-dried powder (prepared in Example 1) according to a final concentration of 10 6 CFU / mL of the milk emulsion. The different groups of milk emulsions were stirred evenly after the above treatments and then filled into 30 mL / cup.

[0125] 5) The different group samples after the above treatment are fermented in a constant temperature incubator at 42°C for 5h to end fermentation at pH 4.6, and are post-ripened at 4°C for 12h to obtain different yogurt samples;

[0126] 6) The different yogurt samples are stored at 28°C, and the state of the yogurt samples is observed regularly.

[0127] The state change of the different group yogurt samples at 28°C is shown in Table 3, and the results show that:

[0128] For the 7 mixed molds separated from the production workshop of Chongqing Tianyou Dairy Co., Ltd., the control group yogurt without any preservative strains began to have a small amount of colonies on the 5th day, and a large amount of mold appeared as time went on; the control group yogurt added with commercial preservative strain powder Y showed strong antibacterial effect in the early stage, but its antibacterial effect gradually weakened as the culture time was prolonged, but the coverage area of the mold was still lower than that of the control group; for the experimental group, the yogurt added with different concentrations (10 4 CFU / mL, 10 5 CFU / mL and 10 6 CFU / mL) of TY-F13 preservative strain powder showed obvious antibacterial effect, and the antibacterial effect was enhanced as the concentration increased. Even at a lower concentration (10 4 CFU / mL), the growth of the mold was significantly inhibited, and the coverage area of the mold was still much less than that of the control group and the commercial preservative strain Y group on the 16th day. In summary, the TY-F13 preservative strain powder has a significant inhibitory effect on the growth of mold in yogurt, and the antibacterial effect is enhanced as the added concentration increases, showing a long-term antibacterial performance better than that of the commercial preservative strain powder Y.

[0129] (Three) Different medium culture test

[0130] The effects of different yeast extracts in the liquid medium in Test Example 1 and Examples 6 to 8 on the growth performance of Lactobacillus fermentum TY-F13 were tested as follows: under sterile conditions, a frozen tube of Lactobacillus fermentum TY-F13 was taken out, and the bacteria liquid stored at -80°C was restored to room temperature, then streaked on a solid medium and cultured at 37°C for 32h to obtain a purified strain; after culture, a single colony was picked on the plate and inoculated in 5mL of liquid medium, and cultured at 37°C for 16h for activation to obtain a first-stage seed liquid; the first-stage seed liquid was transferred to 100mL of liquid medium and cultured at 37°C, and the viable cell count was determined at 5h, 8h, 12h and 24h, respectively; wherein, the solid medium was prepared by adding 1.5% agar by mass fraction to the liquid medium.

[0131] The viable counts of Lactobacillus fermentum TY-F13 after culturing in culture media containing different yeast extracts for different periods of time are shown in Table 2 below.

[0132] Table 2 Effects of different yeast extracts on the growth performance of TY-F13

[0133]

[0134] From Table 2 above, we can see that:

[0135] The culture medium prepared with yeast extract powder FM803 in Example 1 and yeast extract powder FM802 in Example 7 reached a concentration of nearly 9.0×10 8 The number of viable bacteria CFU / g was significantly higher than that of the yeast extract in Example 6 and the yeast extract powder FM503 in Example 8, indicating that yeast extract powder FM803 and yeast extract powder FM802 can quickly provide the nutrients required by Lactobacillus fermentum TY-F13 (such as amino acids, nucleotides, and vitamins), promoting it to enter the logarithmic growth phase. In contrast, the ordinary yeast extract (Example 6) and FM503 (Example 8) only reached 10 in 5 hours. 8 level, indicating that its nutrient release rate or ingredient adaptability is weak;

[0136] At 12 hours, the viable counts of both Example 1 and Example 7 exceeded 1.7×10 9 CFU / g, significantly better than Example 1 (4.1×10 8 ) and Example 8 (4.1×10 8 ), showing stronger nutritional support ability and bacterial expansion potential; especially Example 7 (FM802), which reached 2.0×10 9 CFU / g, which was the highest value among all groups, indicating that it had a stronger growth-promoting effect at a specific stage;

[0137] By 24 hours, Example 1 and Example 7 still maintained at 10 7 CFU / g, while Example 6 and Example 8 have dropped to 10 6 CFU / g, indicating that yeast extracts FM803 and FM802 not only promote rapid growth but also extend the stable period of the strain and reduce mortality. This may be related to their rich content of natural buffer substances, antioxidants, or cytoprotective factors, which help alleviate the toxic pressure caused by the accumulation of metabolic byproducts.

[0138] It can be seen from the above that, compared with traditional yeast extract and other yeast extracts (such as FM503), the yeast extract FM803 and FM802 can accelerate the growth start of the strain, shorten the lag phase, increase the maximum viable cell density, enhance the fermentation efficiency, prolong the stable phase of the strain, and improve the survival stability, and have good industrial application prospects.

[0139] In addition, the influence of different protein peptones in the liquid medium in Test Example 9 and Examples 6 to 8 on the growth performance of Lactobacillus fermentum TY-F13 was tested as follows: under sterile conditions, a frozen tube of Lactobacillus fermentum TY-F13 was taken out, and the bacteria liquid stored at -80°C was restored to room temperature, then streaked on a solid culture medium and cultured at 37°C for 32h to obtain a purified strain; after culturing, a single colony was picked on the plate and inoculated in 5mL of liquid medium, and cultured at 37°C for 16h for activation to obtain a first-stage seed liquid; the first-stage seed liquid was transferred to 100mL of liquid medium and cultured at 37°C, and the viable cell count was determined at 5h, 8h, 12h and 24h, respectively; wherein, the solid culture medium was prepared by adding 1.5% agar by mass fraction to the liquid culture medium.

[0140] The viable cell count of Lactobacillus fermentum TY-F13 after being cultured in the medium containing different protein peptones for different time is shown in Table 3.

[0141] Table 3 Influence of different protein peptones on the growth performance of TY-F13

[0142] Group 5h viable count 8h viable count 12h viable count 24h viable count Example 1 1.9 x 10 9 ]]> 3.1 x 10 9 ]]> 3.2 x 10 9 ]]> 4.0 x 10 7 ]] Example 9 7.0 x 10 8 ]]> 1.8 x 10 9 ]]> 1.5 x 10 9 ]]> 1.5 x 10 7 ]]> Example 10 1.7 x 10 9 ]]> 3.4 x 10 9 ]]> 3.3 x 10 9 ]]> 5.5 x 10 7 <!-- 10 -->]]> Example 11 7.8 x 10 8 ]] 1.0 x 10 9 ]]> 1.2 x 10 9 ]]> 1.6 x 10 7 ]]>

[0143] It can be seen from the above Table 3 that, when yeast peptone FP 102 (Example 1) and cottonseed peptone (Example 10) are selected as the medium components, the viable cell count of Lactobacillus fermentum TY-F13 in each growth stage is significantly higher than that when soybean peptone (Example 11) and bovine bone peptone (Example 1) are used. This indicates that the nutritional factors in yeast peptone FP 102 and cottonseed peptone are more in line with the metabolic requirements of Lactobacillus fermentum TY-F13, which is beneficial to the proliferation thereof.

[0144] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application but not limit the same, and although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions of the present application, and all of them should be covered in the scope of the claims of the present application.

Claims

1. A freeze-dried protective composition, characterized in that The freeze-dried protective composition comprises skim milk powder, trehalose and yeast dietary fiber.

2. The freeze-dried protective composition according to claim 1, characterized in that The mass ratio of skim milk powder, trehalose and yeast dietary fiber is 15:(7-8):(7-8).

3. The freeze-dried protective composition according to claim 2, characterized in that The mass ratio of skim milk powder, trehalose and yeast dietary fiber is 2:1:

1.

4. A freeze-drying protective agent, characterized in that The invention comprises the freeze-dried protection composition according to any one of claims 1 to 3 and an aqueous medium.

5. Lactobacillus fermentum TY-F13 freeze-dried bacteria, characterized in that The invention comprises the freeze-dried protection composition according to any one of claims 1 to 3 and Lactobacillus fermentum TY-F13.

6. The method for preparing the freeze-dried Lactobacillus fermentum TY-F13 according to claim 5, characterized in that: The preparation method comprises: (1) fermenting Lactobacillus fermentum TY-F13 to obtain a fermentation broth, and centrifuging the fermentation broth to obtain bacterial sludge; (2) mixing the freeze-dried protective agent according to claim 4 with bacterial sludge and emulsifying the mixture to obtain an emulsion; (3) The emulsion is freeze-dried to obtain freeze-dried Lactobacillus fermentum TY-F13.

7. The preparation method according to claim 6, characterized in that The preparation method meets one or more of the following conditions: Condition (i): In step (1), the fermentation process is cultured using a culture medium; the culture medium comprises yeast extract, peptone, glucose, sodium acetate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and manganese sulfate; wherein the yeast extract is selected from one or more combinations of yeast extract, yeast extract powder FM803, yeast extract powder FM802 or yeast extract powder FM503; and / or the peptone is selected from one or more combinations of bovine bone peptone, yeast peptone FP102, cottonseed peptone or soy peptone; Condition (ii): In step (2), the mass ratio of the freeze-dried protective agent to the bacterial sludge is (10-20):1; and / or the emulsification comprises: emulsification at 0°C to 4°C, 550r / min to 650r / min for 10min to 20min; Condition (iii): In step (3), the freeze-dried Lactobacillus TY-F13 bacteria are crushed to obtain freeze-dried Lactobacillus TY-F13 bacteria powder.

8. The preparation method according to claim 7, characterized in that In condition (i), The culture medium comprises, by weight, 13 to 17 parts of yeast extract, 8 to 12 parts of peptone, 28 to 32 parts of glucose, 4 to 6 parts of sodium acetate, 1 to 3 parts of dipotassium hydrogen phosphate, 1 to 3 parts of potassium dihydrogen phosphate, 0.05 to 0.15 parts of magnesium sulfate, 0.04 to 0.06 parts of manganese sulfate, and 1000 parts of medium water; wherein, The yeast extract is selected from one or more of yeast extract powder FM803 or yeast extract powder FM802; and / or The peptone is selected from one or more of yeast peptone FP102 and cottonseed peptone.

9. Use of the freeze-dried Lactobacillus fermentum TY-F13 according to claim 5 in the preparation of fermented milk.

10. Use of the freeze-dried Lactobacillus fermentum TY-F13 according to claim 5 in inhibiting spoilage molds in fermented milk, wherein the spoilage molds include one or more of Penicillium, Trichoderma longibrachiatum, Aspergillus aculeatinus, Aspergillus fumigatus, Cladosporium halotolerans, Aspergillus Sydowii and Aspergillus japonicus.

Images