A postbiotic preparation for improving firmness and hydration and its production process and application based on liquid-solid fermentation combination

Abstract

The present invention belongs to the technical field of microbial preparations, and in particular to a postbiotic preparation for improving firmness and moisture and a production process and application thereof based on the combination of liquid-solid fermentation. The present invention screens and obtains a strain of Lactobacillus johnsonii Nice-LJ, and successfully develops a postbiotic product based on the strain. It is verified by experiments that it has the effect of promoting eln1 and eln2 expression to improve elastin synthesis, and improves skin elasticity. And the situation of excessive expression of aqp3a caused by hypertonicity can be reduced, so as to play the effect of moisturizing, and finally realize the effect of improving the firmness and moisture of skin (mucous membrane). At the same time, by optimizing the preparation process condition parameters of the postbiotic preparation, Lactobacillus johnsonii is optimized by combining liquid-solid fermentation with process, so as to be more suitable for industrial production, and therefore has the value of good practical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial preparations, and specifically relates to a postbiotic preparation for improving firmness and hydration, and a production process and application thereof based on a combination of liquid-solid fermentation. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Elastin is an important extracellular matrix protein, second only to collagen in the content of organisms. It plays an irreplaceable role in tissue expansion and contraction, resistance to external force deformation, and can give tissue elasticity. Elastin plays an important role in regulating a variety of cell functions, including promoting cell adhesion, proliferation, differentiation, chemotaxis and migration. With age, the surface of elastic fibers becomes rough, the elastin network degenerates, and separates from the microfibrils, making elastin more easily degraded by metalloproteinases in cells, accelerating the loss of elastin. Studies have shown that elastin peptides can activate fibroblasts in human skin and significantly promote fibroblasts to produce collagen and elastin, which has a good effect on improving skin elasticity, flexibility and wrinkles. Aquaporin, also known as aquaporin, is a protein (intrinsic membrane protein) located on the cell membrane. It can form "pores" on the cell membrane to control the entry and exit of water in the cell, just like a "cell water pump". Aquaporin 3 (AQP3A) is a member of the aquaglycerol channel protein subfamily, expressed in the epidermis and upper basal layer, and plays a role in regulating the entry of water and glycerol into cells. The inventors found that there are almost no oral products that target both elastin and aquaporin. Therefore, it is of great significance to research and develop a product with significant effects.

[0004] At the same time, non-viable microorganisms, as well as their cellular components and metabolites, can also affect health. In 2013, Katerina from Spain formally proposed the concept of "postbiotics". In 2021, the International Scientific Association of Probiotics and Prebiotics (ISAPP) proposed a definition of postbiotics, pointing out that postbiotics are "preparations of inanimate microorganisms and / or their components that are beneficial to the health of the host". Probiotics and their metabolites are also mainly absorbed by intestinal epithelial cells to further exert their effects. The water content in the intestines of adults is 4-5L, and the water content of healthy stools is 25-75%. Ordinary probiotics and their metabolites can only be cultured in liquid form. After passing through gastric acid and bile salt digestive enzymes, the number of live bacteria that actually reach the intestine is difficult to maintain dominance, and it is difficult to successfully colonize and exert their effects. Therefore, the fermentation method of postbiotics has a very large impact on their effects. Summary of the invention

[0005] Based on the above-mentioned deficiencies in the prior art, the present invention provides a postbiotic preparation for improving firmness and hydration, and a production process and application thereof based on the combination of liquid-solid fermentation. Specifically, the present invention screens and obtains a strain of Lactobacillus johnsonii, and obtains the corresponding postbiotic preparation product through a postbiotic production process combined with liquid-solid fermentation. It has been verified by experiments that it has the effects of improving elastin synthesis and moisturizing, thereby exerting the effect of firming and moisturizing the skin and mucous membranes, thereby effectively improving the firmness and hydration of the skin (mucous membrane). Based on the above research results, the present invention is completed.

[0006] In order to achieve the above technical objectives, the present invention relates to the following technical solutions:

[0007] In one aspect of the present invention, a strain of Lactobacillus johnsonii Nice-LJ is provided, which is deposited in the China Center for Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), with a preservation date of July 8, 2024, and its biological preservation number is CCTCC NO: M 20241500.

[0008] The second aspect of the present invention provides a postbiotic preparation, which at least comprises the inactivated Lactobacillus johnsonii and / or its culture.

[0009] In the present invention, the culture of Lactobacillus johnsonii refers to the material obtained during the fermentation and culture of Lactobacillus johnsonii (all materials in the culture container).

[0010] The third aspect of the present invention provides a method for preparing the above-mentioned postbiotic preparation, which specifically adopts a liquid-solid combined production process, that is, firstly, liquid fermentation is used to enrich and grow Lactobacillus johnsonii, and then solid-state fermentation is used to simulate the fermentation environment and metabolic process of healthy human intestinal flora, so as to efficiently obtain the active substances of Lactobacillus johnsonii and its metabolites.

[0011] More specifically, the preparation method comprises:

[0012] (1) strain activation: streaking the Lactobacillus johnsonii Nice-LJ into an activation medium to obtain a pure strain;

[0013] (2) Preparation of primary seed solution: a single colony of Lactobacillus johnsonii Nice-LJ obtained in step (1) is selected and placed in a primary culture solution at 35-37° C. for 12-14 hours to obtain a primary seed solution;

[0014] (3) Preparation of secondary seed solution: inoculate the primary seed solution into the secondary culture solution, incubate at 35-37° C. for 12-14 hours to obtain the secondary seed solution, with the inoculation amount being 1-5% (preferably 3%, v / v);

[0015] (4) Lactobacillus johnsonii primary liquid fermentation: the secondary seed liquid is inoculated into a fermentation tank containing sterilized culture medium, the inoculation amount is 1-5% (preferably 3%), 35-38° C. (preferably 37° C.), 80-120 r / min (preferably 100 r / min), the air pressure is maintained at 0.02-0.08 MPa (preferably 0.05 MPa), the pH is adjusted to weak acidity (preferably pH 6.5), and the culture is carried out for 16-18 hours;

[0016] (5) Post-treatment of the primary liquid fermentation of Lactobacillus johnsonii: After the liquid fermentation is completed, 50% of the fermentation solution is spray-dried to obtain powder A, and the remaining 50% of the fermentation solution is freeze-dried to obtain powder B. The above powders A and B are used for further solid fermentation respectively;

[0017] (6) Lactobacillus johnsonii secondary solid fermentation: powder A and powder B are mixed in a mass ratio of 1-2:2-4, and the solid fermentation humidity is set to 30-45%, 28-32° C. (preferably 30° C.), the stirring speed is 10-30 r / min, the nitrogen pressure is maintained at 0.02-0.08 MPa (preferably 0.05 MPa), the pH is natural, and the fermentation is performed for 15-18 hours (preferably 16 hours); the temperature is raised for sterilization, and after cooling to room temperature (such as 30° C.), freeze-drying is performed.

[0018] A fourth aspect of the present invention provides the use of the above-mentioned postbiotics in the preparation of products for improving firmness and hydration.

[0019] Furthermore, the product for improving firmness and hydration has the effects of increasing elastin synthesis and replenishing water and moisturizing, thereby exerting the effect of firming and moisturizing the skin and mucous membranes. In particular, experiments have shown that the postbiotic preparation product of the present invention can not only relieve dryness, lack of water and wrinkles on the skin (such as facial skin), but also has the effects of replenishing water, moisturizing and firming the skin. In addition, female users indicate that the product also has the effect of promoting firmness, plumpness and hydration of the vagina.

[0020] Beneficial technical effects of one or more of the above technical solutions:

[0021] The above technical solution screened and obtained a strain of Lactobacillus johnsonii, and successfully developed a postbiotic product based on the strain. It has been verified by experiments that it has the effect of promoting the expression of eln1 and eln2 to increase elastin synthesis and improve skin elasticity. It can also reduce the excessive expression of aqp3a caused by hypertonicity, thereby playing a role in moisturizing and ultimately achieving the effect of improving the firmness and hydration of the skin (mucous membrane). At the same time, by optimizing the preparation process parameters of the postbiotic preparation, the liquid-solid fermentation combined process of Lactobacillus johnsonii is optimized.

[0022] Specifically, the above technical solution adopts the method of first liquid fermentation and then solid fermentation to cultivate Lactobacillus johnsonii, and uses the simulated intestinal environment as the background, which can maximize the application value and health benefits of Lactobacillus johnsonii. This specific fermentation strategy not only simulates the natural conditions in the intestine, but also optimizes the growth environment of Lactobacillus johnsonii and enhances its positive impact on host health. In the liquid fermentation stage, Lactobacillus johnsonii can proliferate rapidly and accumulate primary metabolites that are beneficial to intestinal health. The subsequent solid fermentation stage not only further stimulates the metabolic activity of Lactobacillus johnsonii, but also produces more secondary metabolites that are beneficial to health, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 This is the result of eln1 gene expression in zebrafish after postbiotic action;

[0025] Figure 2 This is the result diagram of eln2 gene expression in zebrafish after postbiotic action;

[0026] Figure 3 Analysis of zebrafish tail fin area after incubation for metabiotic samples;

[0027] Figure 4 The effect of postbiotic samples on the expression of zebrafish water channel protein 3 gene aqp3a;

[0028] Figure 5 The effects of postbiotics on crow's feet before and after taking them. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0030] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0031] In a typical embodiment of the present invention, a strain of Lactobacillus johnsonii Nice-LJ is provided, which is deposited in the China Type Culture Collection (address: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province), the preservation date is July 8, 2023, and its biological preservation number is CCTCC NO: M 20241500.

[0032] The second aspect of the present invention provides a postbiotic preparation, which at least comprises the inactivated Lactobacillus johnsonii and / or its culture.

[0033] In the present invention, the culture of Lactobacillus johnsonii refers to the material obtained during the fermentation and culture of Lactobacillus johnsonii (all materials in the culture container).

[0034] The third aspect of the present invention provides a method for preparing the above-mentioned postbiotic preparation, which specifically adopts a liquid-solid combined production process, that is, firstly, liquid fermentation is used to enrich and grow Lactobacillus johnsonii, and then solid-state fermentation is used to simulate the fermentation environment and metabolic process of healthy human intestinal flora, so as to efficiently obtain the active substances of Lactobacillus johnsonii and its metabolites.

[0035] More specifically, the preparation method comprises:

[0036] (1) strain activation: streaking the Lactobacillus johnsonii Nice-LJ into an activation medium to obtain a pure strain;

[0037] The composition of the activation culture medium is as follows: each 1000 mL contains 10-15 g of peptone, 5-10 g of beef powder, 2-5 g of yeast powder, 1-4 g of glucose, 1-2 mL of Tween 80, 1-4 g of dipotassium hydrogen phosphate, 3-6 g of sodium acetate, 1-3 g of triammonium citrate, 0.2-0.4 g of magnesium sulfate, and 0.05-0.1 g of manganese sulfate, and the pH is adjusted to 6.5.

[0038] More specifically, the composition of the activation culture medium is as follows: per 1000 mL, it contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and the pH is adjusted to 6.5.

[0039] (2) Preparation of primary seed solution: a single colony of Lactobacillus johnsonii Nice-LJ obtained in step (1) is selected and placed in a primary culture solution at 35-37° C. for 12-14 hours to obtain a primary seed solution;

[0040] The composition of the primary culture solution is as follows: per 1000 mL, it contains 10-15 g of peptone, 5-10 g of beef powder, 2-5 g of yeast powder, 1-4 g of glucose, 1-2 mL of Tween 80, 1-4 g of dipotassium hydrogen phosphate, 3-6 g of sodium acetate, 1-3 g of triammonium citrate, 0.2-0.4 g of magnesium sulfate, 0.05-0.1 g of manganese sulfate, 0.2-0.5 g of vitamin C, and 0.2-0.5 g of collagen tripeptide, and the pH is adjusted to 6.5.

[0041] More specifically, the composition of the primary culture medium is as follows: per 1000 mL, it contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 15 g agar powder, 0.2 g vitamin C, 0.2 g collagen tripeptide, and the pH is adjusted to 6.5.

[0042] (3) Preparation of secondary seed solution: inoculate the primary seed solution into the secondary culture solution, incubate at 35-37° C. for 12-14 hours to obtain the secondary seed solution, with the inoculation amount being 1-5% (preferably 3%, v / v);

[0043] More specifically, the composition of the secondary culture medium is as follows:

[0044] Each 1000mL contains 10-15g peptone, 5-10g beef powder, 2-5g yeast powder, 1-4g oligofructose, 1-2mL Tween 80, 1-4g dipotassium hydrogen phosphate, 3-6g sodium acetate, 1-3g triammonium citrate, 0.2-0.4g magnesium sulfate, 0.05-0.1g manganese sulfate, 0.2-0.5g vitamin C, 0.2-0.5g collagen tripeptide, and the pH is adjusted to 6.5.

[0045] More specifically, the composition of the secondary culture medium is as follows: every 1000 mL contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 4 g oligofructose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 0.3 g vitamin C, 0.3 g collagen tripeptide, and the pH is adjusted to 6.5.

[0046] (4) Lactobacillus johnsonii primary liquid fermentation: the secondary seed liquid is inoculated into a fermentation tank containing sterilized culture medium, the inoculation amount is 1-5% (preferably 3%), 35-38° C. (preferably 37° C.), 80-120 r / min (preferably 100 r / min), the air pressure is maintained at 0.02-0.08 MPa (preferably 0.05 MPa), the pH is adjusted to weak acidity (preferably pH 6.5), and the culture is carried out for 16-18 hours;

[0047] The composition of the culture solution is as follows: per 1000 mL, it contains 10-20 g soybean peptide, 15-25 g wheat peptide, 20-45 g oligofructose, 20-25 g tomato, 10-15 g water-soluble starch, 20-35 g collagen tripeptide, 5-10 g vitamin C, 5-10 g vitamin E, 0.5-2 g polyether defoamer for real-time defoaming, and adjusts pH 6.5.

[0048] Specifically, the composition of the culture solution is as follows: per 1000 mL, it contains 20 g soybean peptide, 25 g wheat peptide, 45 g oligofructose, 25 g tomato, 15 g water-soluble starch, 20 g collagen tripeptide, 5 g vitamin C, 5 g vitamin E, 0.5 g polyether defoamer for real-time defoaming, and adjusts pH to 6.5.

[0049] (5) Post-treatment of the primary liquid fermentation of Lactobacillus johnsonii: After the liquid fermentation is completed, 50% of the fermentation solution is spray-dried to obtain powder A, and the remaining 50% of the fermentation solution is freeze-dried to obtain powder B. The above powders A and B are used for further solid fermentation respectively;

[0050] (6) Lactobacillus johnsonii secondary solid fermentation: powder A and powder B are mixed in a mass ratio of 1-2:2-4, and the solid fermentation humidity is set to 30-45%, 28-32° C. (preferably 30° C.), the stirring speed is 10-30 r / min, the nitrogen pressure is maintained at 0.02-0.08 MPa (preferably 0.05 MPa), the pH is natural, and the fermentation is performed for 15-18 hours (preferably 16 hours); the temperature is raised for sterilization, and after cooling to room temperature (such as 30° C.), freeze-drying is performed.

[0051] The composition of the culture medium for solid fermentation is as follows in parts by mass: 100-200 parts of powder A, 200-400 parts of powder B, 100-200 parts of soybean peptide, 50-150 parts of wheat peptide, 50-100 parts of oligofructose, 80-150 parts of tomato powder, 30-80 parts of casein, 10-15 parts of water-soluble starch, 30-65 parts of collagen tripeptide, 5-10 parts of vitamin C, 5-10 parts of vitamin E, 5-10 parts of vitamin B, 10-15 parts of glycine, 30-45 parts of water, and natural pH.

[0052] Specifically, the composition of the culture medium is as follows: per 1000 g, it contains: 150 g powder A, 300 g powder B, 100 g soybean peptide, 100 g wheat peptide, 50 g oligofructose, 120 g tomato powder, 35 g casein, 15 g water-soluble starch, 55 g collagen tripeptide, 10 g vitamin C, 10 g vitamin E, 10 g vitamin B, 10 g glycine, 35 g water, and natural pH.

[0053] A fourth aspect of the present invention provides the use of the above-mentioned postbiotics in the preparation of products for improving firmness and hydration.

[0054] Furthermore, the product has the effects of improving elastin synthesis and replenishing water and moisturizing, thereby exerting the effect of firming and moisturizing the skin and mucous membranes. In particular, experiments have shown that the postbiotic preparation product of the present invention can not only relieve dryness, lack of water and wrinkles on the skin (such as facial skin), but also play the role of replenishing water, moisturizing and tightening the skin. In addition, female users said that the product also has the effect of promoting vaginal firmness, plumpness and hydration.

[0055] The product can be food or medicine, which is not specifically limited here.

[0056] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0057] Example 1

[0058] Under a sterile operating table, Lactobacillus johnsoniiNice-LJ stored in a -80°C low-temperature refrigerator was streaked onto a culture medium plate (each 1000 mL contained 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and the pH was adjusted to 6.5), single colonies were screened, and the single colonies were activated 3 times to obtain a pure strain;

[0059] Pick a single colony and put it in 100 mL of liquid culture medium (each 1000 mL contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 15 g agar powder, 0.2 g vitamin C, 0.2 g collagen tripeptide, and adjust the pH to 6.5), and culture at 37 ° C for 14 hours to obtain the first-level seed solution; the first-level seed solution was inoculated with Plant in a triangular flask culture medium (each 1000mL contains 10g peptone, 5g beef powder, 4g yeast powder, 4g oligofructose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 0.3g vitamin C, 0.3g collagen tripeptide, and adjust pH to 6.5), with an inoculation amount of 3%, and then place in a constant temperature box and culture at 37°C for 14h, which is called secondary seed liquid;

[0060] The secondary seed liquid was inoculated into a fermenter containing 100L sterilized culture solution (each 1000mL contained, 20g soybean peptide, 25g wheat peptide, 45g oligofructose, 25g tomato, 15g water-soluble starch, 20g collagen tripeptide, 5g vitamin C, 5g vitamin E, 0.5g polyether defoamer for real-time defoaming, pH 6.5 was adjusted), the inoculation amount was 3%, 37°C, 100r / min, air pressure was 0.05MPa, 20% NaOH was used to adjust pH 6.5, and the culture was carried out for 18 hours; after the liquid fermentation was completed, half of the fermentation solution was spray-dried to obtain powder A, and the remaining half of the fermentation liquid was freeze-dried to obtain powder B, and powder A and powder B were mixed in a ratio of 1:2 to prepare a solid culture medium (each 1000g contained, powder A 150 g, powder B 300 g, 100g soybean peptide, 100g wheat peptide, 50g oligofructose, 120g tomato powder, 35g casein, 15g water-soluble starch, 55g collagen tripeptide, 10g vitamin C, 10g vitamin E, 10g vitamin B, 10g glycine, 35g water, pH natural), set the solid fermentation humidity to 35%, 30°C, stirring speed 20r / min, nitrogen pressure to 0.05MPa, pH natural, fermentation for 16 hours; after 16 hours, the fermentation tank was heated to 90°C, maintained for 40min, then cooled to 30°C, and freeze-dried to obtain the sample.

[0061] Example 2

[0062] Under a sterile operating table, Lactobacillus johnsoniiNice-LJ stored in a -80°C low-temperature refrigerator was streaked onto a culture medium (each 1000mL contained 10g peptone, 5g beef powder, 4g yeast powder, 2g glucose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, and the pH was adjusted to 6.5) plate, single colonies were screened, and the single colonies were activated 3 times to obtain a pure strain;

[0063] Pick a single colony and put it in 100 mL of liquid culture medium (each 1000 mL contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 15 g agar powder, 0.2 g vitamin C, 0.2 g collagen tripeptide, and adjust the pH to 6.5), and culture at 37 ° C for 14 hours to obtain the first-level seed solution; the first-level seed solution was inoculated with Plant in a triangular flask culture medium (each 1000mL contains 10g peptone, 5g beef powder, 4g yeast powder, 4g oligofructose, 1mL Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 0.3g vitamin C, 0.3g collagen tripeptide, and adjust pH to 6.5), with an inoculation amount of 3%, and then place in a constant temperature box and culture at 37°C for 14h, which is called secondary seed liquid;

[0064] The secondary seed liquid was inoculated into a fermentation tank containing 100L of sterilized culture medium (each 1000mL contained, 20g soybean peptide, 25g wheat peptide, 45g oligofructose, 25g tomato, 15g water-soluble starch, 20g collagen tripeptide, 5g vitamin C, 5g vitamin E, 0.5g polyether defoamer for real-time defoaming, and pH adjusted to 6.5), with an inoculation amount of 3%, 37°C, 100r / min, air pressure of 0.05MPa, 20% NaOH to adjust pH to 6.5, and culture for 18 hours; after the liquid fermentation was completed, the sample was freeze-dried.

[0065] Example 3

[0066] A single colony of Lactobacillus johnsonii Nice-LJ was picked and incubated in 100 mL of liquid culture medium (each 1000 mL contained 10 g of peptone, 5 g of beef powder, 4 g of yeast powder, 2 g of glucose, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 5 g of sodium acetate, 2 g of triammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 15 g of agar powder, 0.2 g of vitamin C, and 0.2 g of collagen tripeptide, and the pH was adjusted to 6.5), and cultured at 37°C for 14 hours to obtain a primary seed solution; The first-level seed solution was inoculated into a triangular flask culture medium (each 1000 mL contained 10 g peptone, 5 g beef powder, 4 g yeast powder, 4 g oligofructose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 0.3 g vitamin C, 0.3 g collagen tripeptide, and the pH was adjusted to 6.5) with an inoculation amount of 3%, and then placed in a constant temperature box and cultured at 37°C for 14 h. This is called the second-level seed solution;

[0067] The secondary seed liquid was inoculated into a fermenter containing 100L sterilized culture solution (each 1000mL contained, 20g soybean peptide, 25g wheat peptide, 45g oligofructose, 25g tomato, 15g water-soluble starch, 20g collagen tripeptide, 5g vitamin C, 5g vitamin E, 0.5g polyether defoamer for real-time defoaming, pH 6.5 was adjusted), the inoculation amount was 3%, 37°C, 100r / min, air pressure was 0.05MPa, 20% NaOH was used to adjust pH 6.5, and the culture was carried out for 18 hours; after the liquid fermentation was completed, the powder was obtained after spray drying, and then the solid fermentation was inoculated according to the 3% inoculation amount, the solid fermentation humidity was set to 35%, 30°C, the stirring speed was 20r / min, the nitrogen pressure was maintained at 0.05MPa, the pH was natural, and the fermentation was carried out for 16 hours; after 16 hours, the fermenter was heated to 90°C, maintained for 40min, then cooled to 30°C, and the sample was obtained after freeze drying.

[0068] Effect verification

[0069] 1. Postbiotics that promote the expression of eln1 and eln2 and increase elastin synthesis, the verification method is:

[0070] (1) Wild AB zebrafish of 4 dpf (day post fertilization) were used as experimental animals. Normal zebrafish were selected under a stereomicroscope and randomly divided into a blank control group and a metazoan group, with 10 fish in each group, and 3 replicate wells were set. Zebrafish culture water was added to the blank control group, and sample solutions with final concentrations of 0.5%, 1.0%, and 1.5% were added to the metazoan group, respectively. The zebrafish in the above groups were placed in a 28°C constant temperature incubator and cultured for 24 hours;

[0071] (2) The cultured zebrafish were washed with fish water, and the fish bodies were homogenized under low temperature conditions. The total RNA of each group of zebrafish was extracted using an RNA extraction kit and then reverse transcribed into cDNA. Using cDNA as a template and β-actin as an internal reference gene, the relative expression levels of elastin genes (eln1 and eln2) were detected using RT-qPCR technology, and statistical analysis was performed using GraphPad Prism software. P<0.05 indicated that the difference was statistically significant.

[0072] After the postbiotic effect of combined fermentation, the expression levels of eln1 and eln2 genes in zebrafish were significantly increased.

[0073] like Figure 1 and Figure 2 As shown, zebrafish experiments have proved that the product of the present invention has the effect of increasing the expression of eln1 and eln2 genes and increasing the synthesis of elastin.

[0074] 2. Reduce the overexpression of aqp3a caused by hypertonicity, thereby achieving the effect of hydrating and moisturizing. The verification method is:

[0075] (1) Wild AB zebrafish of 2 dpf with normal development were used as experimental animals. They were placed in a 1 mg / mL pronase solution for about 3 minutes to remove the outer egg membrane of the zebrafish embryo. The zebrafish with demembraned zebrafish were randomly divided into a blank control group, a NaCl modeling group, and a postbiotic group, with 10 fish in each group, and 3 replicates were set. The blank control group was added with zebrafish breeding water. Except for the blank control group, the remaining groups were added with a final concentration of 1% NaCl solution. At the same time, the postbiotic sample groups with different concentrations were added with sample solutions with final concentrations of 0.5%, 1%, and 1.5%, respectively. The above groups of zebrafish were placed in a 28°C constant temperature incubator and cultured for 20 hours.

[0076] (2) 8-10 zebrafish were randomly selected from each group and observed and photographed under a microscope. The tail fin area of ​​zebrafish was calculated using Image-Pro Plus software, and statistical analysis was performed using GraphPad Prism software. The statistical results were expressed as mean±SEM, and the results were used to analyze and evaluate the hydrating and moisturizing effects of the samples. P<0.05 indicated that the difference was statistically significant.

[0077] The tail fin area of ​​zebrafish in the NaCl modeling group was significantly reduced. Compared with the NaCl modeling group, the samples in the metazoan group could increase the tail fin area of ​​zebrafish to varying degrees after incubation, showing significant hydrating and moisturizing effects.

[0078] (3) The zebrafish were washed with fish water, and the fish bodies were homogenized under low temperature conditions. The total RNA of each group of zebrafish was extracted using an RNA extraction kit and then reverse transcribed into cDNA. Using cDNA as a template and β-actin as an internal reference gene, the relative expression level of the aquaporin 3 (aqp3a) gene was detected by RT-qPCR technology, and statistical analysis was performed using GraphPadPrism software. P<0.05 indicated that the difference was statistically significant.

[0079] Since hypertonic culture in 1% NaCl solution will cause dehydration in zebrafish skin, the expression of aqp3a gene will increase significantly, and water loss will be serious. Postbiotics can significantly downregulate the increased expression of aqp3a gene caused by hypertonic culture in NaCl solution, and play a regulatory role in water balance.

[0080] 3. Small-scale population experiment:

[0081] 30 women aged 20-50 years old who were willing to participate in the experiment and were able to complete the questionnaire and record were included in the experimental group. 600 mg of postbiotics (the product prepared in Example 1) were taken orally on an empty stomach every morning for 4 weeks. The original eating habits were not changed during the drinking period, and the diet was normal. After the taking period, 100% of the users said that the dry and dehydrated skin conditions before taking the product were significantly improved, and the crow's feet around the eyes were also well improved (see Figure 5 ), and a skin tightening effect was seen. And 80% of users said that the vagina felt tighter, fuller and more hydrated than before taking it.

[0082] Table 1 Statistics of postbiotic population trials

[0083]

[0084]

[0085] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus johnsonii ( Lactobacillus johnsonii )Nice-LJ, deposited in China Center for Type Culture Collection, the deposit date is July 8, 2023, and its biological deposit number is CCTCC NO: M 20241500.

2. A postbiotic preparation, characterized in that The invention at least comprises the inactivated Lactobacillus johnsonii according to claim 1 and a culture thereof, wherein the culture is a substance obtained during the fermentation and cultivation process of Lactobacillus johnsonii.

3. The method for preparing the postbiotic preparation according to claim 2, characterized in that: The preparation method specifically adopts a liquid-solid combined production process, including: (1) Activation of bacteria: The Lactobacillus johnsonii described in claim 1 Lactobacillus johnsonii Nice-LJ was streaked into activation medium to obtain pure strains; (2) Preparation of primary seed solution: a single colony of Lactobacillus johnsonii Nice-LJ obtained in step (1) is selected and placed in primary culture solution at 35-37° C. for 12-14 hours to obtain a primary seed solution; (3) Preparation of secondary seed solution: inoculate the primary seed solution into the secondary culture solution, incubate at 35-37°C for 12-14 hours to obtain the secondary seed solution. The inoculation amount is 1-5%; (4) Lactobacillus johnsonii primary liquid fermentation: inoculate the secondary seed solution into a fermentation tank containing sterilized culture medium at an inoculation rate of 1-5%, 35-38°C, 80-120 r / min, air pressure of 0.02-0.08 MPa, adjust the pH to weak acidity, and culture for 16-18 hours; (5) Post-treatment of the primary liquid fermentation of Lactobacillus johnsonii: After the liquid fermentation is completed, 50% of the fermentation solution is spray-dried to obtain powder A, and the remaining 50% of the liquid is freeze-dried to obtain powder B. The above powders A and B are used for further solid fermentation respectively; (6) Secondary solid fermentation of Lactobacillus johnsonii: Mix powder A and powder B in a mass ratio of 1-2:2-4, set the solid fermentation humidity to 30-45%, 28-32 °C, stirring speed to 10-30 r / min, nitrogen pressure to maintain 0.02-0.08 MPa, pH to natural, ferment for 15-18 hours; heat sterilize, cool to room temperature, and freeze-dry.

4. The preparation method according to claim 3, characterized in that: In step (3), the inoculation amount is 3%; In step (4), the secondary seed solution is inoculated into a fermenter containing sterilized culture medium at an inoculation rate of 3%, 37°C, 100 r / min, air pressure of 0.05 MPa, pH adjusted to 6.5, and cultured for 16-18 hours.

5. The preparation method according to claim 3, characterized in that: The composition of the activation culture medium is as follows: per 1000 mL, it contains 10-15 g of peptone, 5-10 g of beef powder, 2-5 g of yeast powder, 1-4 g of glucose, 1-2 mL of Tween 80, 1-4 g of dipotassium hydrogen phosphate, 3-6 g of sodium acetate, 1-3 g of triammonium citrate, 0.2-0.4 g of magnesium sulfate, and 0.05-0.1 g of manganese sulfate, and the pH is adjusted to 6.

5.

6. The preparation method according to claim 5, characterized in that: The composition of the activation culture medium is as follows: per 1000 mL, it contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and the pH is adjusted to 6.

5.

7. The preparation method according to claim 3, characterized in that: The composition of the primary culture medium is as follows: per 1000 mL, it contains 10-15 g peptone, 5-10 g beef powder, 2-5 g yeast powder, 1-4 g glucose, 1-2 mL Tween 80, 1-4 g dipotassium hydrogen phosphate, 3-6 g sodium acetate, 1-3 g triammonium citrate, 0.2-0.4 g magnesium sulfate, 0.05-0.1 g manganese sulfate, 0.2-0.5 g vitamin C, 0.2-0.5 g collagen tripeptide, and the pH is adjusted to 6.

5.

8. The preparation method according to claim 7, characterized in that: The composition of the primary culture medium is as follows: per 1000 mL, it contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 15 g agar powder, 0.2 g vitamin C, 0.2 g collagen tripeptide, and the pH is adjusted to 6.

5.

9. The preparation method according to claim 3, characterized in that: The composition of the secondary culture solution is as follows: Each 1000 mL contains 10-15 g peptone, 5-10 g beef powder, 2-5 g yeast powder, 1-4 g oligofructose, 1-2 mL Tween 80, 1-4 g dipotassium hydrogen phosphate, 3-6 g sodium acetate, 1-3 g triammonium citrate, 0.2-0.4 g magnesium sulfate, 0.05-0.1 g manganese sulfate, 0.2-0.5 g vitamin C, 0.2-0.5 g collagen tripeptide, and the pH is adjusted to 6.

5.

10. The preparation method according to claim 9, characterized in that: The composition of the secondary culture medium is as follows: per 1000 mL, it contains 10 g peptone, 5 g beef powder, 4 g yeast powder, 4 g oligofructose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 0.3 g vitamin C, 0.3 g collagen tripeptide, and the pH is adjusted to 6.

5.

11. The preparation method according to claim 3, characterized in that: In the primary liquid fermentation of Lactobacillus johnsonii, the composition of the culture solution is as follows: per 1000 mL, it contains 10-20 g soybean peptide, 15-25 g wheat peptide, 20-45 g oligofructose, 20-25 g tomato, 10-15 g water-soluble starch, 20-35 g collagen tripeptide, 5-10 g vitamin C, 5-10 g vitamin E, 0.5-2 g polyether defoamer, and the pH is adjusted to 6.

5.

12. The preparation method according to claim 11, characterized in that: The composition of the culture solution is as follows: per 1000 mL, it contains 20 g soybean peptide, 25 g wheat peptide, 45 g oligofructose, 25 g tomato, 15 g water-soluble starch, 20 g collagen tripeptide, 5 g vitamin C, 5 g vitamin E, 0.5 g polyether defoamer, and the pH is adjusted to 6.

5.

13. The preparation method according to claim 3, characterized in that: The composition of the solid fermentation culture medium is as follows in parts by mass: 100-200 parts of powder A, 200-400 parts of powder B, 100-200 parts of soybean peptides, 50-150 parts of wheat peptides, 50-100 parts of oligofructose, 80-150 parts of tomato powder, 30-80 parts of casein, 10-15 parts of water-soluble starch, 30-65 parts of collagen tripeptide, 5-10 parts of vitamin C, 5-10 parts of vitamin E, 5-10 parts of vitamin B, 10-15 parts of glycine, 30-45 parts of water, and natural pH.

14. The preparation method according to claim 13, characterized in that: The composition of the solid fermentation culture medium is as follows: per 1000 g, it contains: 150 g powder A, 300 g powder B, 100 g soybean peptide, 100 g wheat peptide, 50 g oligofructose, 120 g tomato powder, 35 g casein, 15 g water-soluble starch, 55 g collagen tripeptide, 10 g vitamin C, 10 g vitamin E, 10 g vitamin B, 10 g glycine, 35 g water, pH natural.

15. Use of the postbiotic preparation according to claim 2 or the postbiotic preparation obtained by the preparation method according to any one of claims 3 to 14 in the preparation of a product that improves firmness and hydration, wherein the product is a food; the product has the effects of increasing elastin synthesis and replenishing water and moisture, thereby exerting a firming and hydrating effect on the skin and mucous membranes.

16. The use according to claim 15, characterized in that The product not only relieves dry skin, dehydration and wrinkles, but also has the effect of moisturizing and tightening the skin, and at the same time has the effect of promoting vaginal firmness, plumpness and moisture.

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