A Bifida ferment lysate, its preparation method and application

Abstract

This invention proposes a Bifida ferment lysate, its preparation method, and its application, belonging to the field of cosmetic technology. The first fermentation product is obtained by enzymatic fermentation of Ganoderma lucidum, Poria cocos, Nostoc commune, oats, lactoferrin, and ginseng. Konjac gum hydrolysate and collagen and glycosaminoglycans extracted from tilapia skin are added, and fermentation continues to obtain a second fermentation product. Modified lysozyme is added for enzymatic hydrolysis, followed by centrifugation. The supernatant is collected, and copper and calcium salts are added and stirred to obtain a Cu-Ca chelated fermentation lysate. The product is then encapsulated to obtain the Bifida ferment lysate, which has excellent whitening, moisturizing, antibacterial, soothing, skin-repairing, and pH-regulating effects on the skin. It is safe, non-toxic, non-irritating to the skin, highly absorbable by the skin, and has a long shelf life, showing broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to a Bifida ferment lysate, its preparation method, and its application. Background Technology

[0002] In recent years, microbiome skincare has become a hot trend, utilizing probiotics on human skin to maintain its microecology and reduce oxidative damage to skin cells from free radicals. This has made probiotic fermentation products a popular product development approach in the cosmetics industry. Bifidobacteria are among the most important probiotics in the intestines of humans and animals, playing a crucial role in maintaining intestinal microecological balance. Research reports indicate that the number of Bifidobacteria in the human body declines with age. However, Bifidobacteria can often be detected in the intestines of healthy, long-lived elderly individuals. Therefore, Bifidobacteria have significant effects on maintaining human health and anti-aging.

[0003] Bifida ferment lysate is a metabolite obtained from the fermentation of Bifidobacterium. Its main components include organic acids (lactic acid, citric acid, and succinic acid, etc.), vitamins (B1, B2, B6, B12, pantothenic acid, folic acid, etc.), bacteriocins (mostly water-soluble polypeptides), polysaccharides, proteins, and amino acids. Lactic acid in Bifida ferment lysate is a natural moisturizing factor present in human skin, effectively removing fine lines and wrinkles and accelerating keratin renewal. Folic acid can activate epidermal cells, promote moisture retention and nutrient absorption, and reduce the rate of keratinization, thus achieving the purpose of beautifying and softening the skin. In addition, active ingredients such as amino acids, lipids, polysaccharides, adenosine, vitamins, trace minerals, and B vitamins moisturize and nourish the skin, helping it resist damage caused by external environmental factors and stress. It also promotes the expression of various protein genes related to epidermal differentiation and skin barrier function, exerting effects such as wrinkle reduction, repair, moisturizing, and enhanced skin elasticity.

[0004] Therefore, Bifida ferment lysate has great market application potential. However, the current preparation process of Bifida ferment lysate filtrate used as a cosmetic raw material is complex and often requires the continuous supplementation of exogenous nutrients to promote the fermentation and growth of Bifidobacteria. This results in some exogenous additives remaining in the metabolites and also increases the production cost of fermentation.

[0005] Chinese patent CN114854643B relates to a culture medium that promotes the synergistic proliferation of Lactobacillus and Bifidobacterium and its application. This patent only optimizes the ratio between the components of the prebiotic combination culture medium and does not involve a systematic study on simplifying the composition of fermentation culture medium components and cell lysis and separation techniques. Summary of the Invention

[0006] The purpose of this invention is to provide a Bifida ferment lysate, its preparation method, and its application. This invention has excellent whitening, moisturizing, antibacterial, soothing, skin repair, and pH regulation effects. It is safe and non-toxic, does not irritate the skin, has high skin absorption and utilization, and has a long shelf life. It has broad application prospects.

[0007] The technical solution of this invention is implemented as follows:

[0008] This invention provides a method for preparing Bifida ferment lysate. The method involves enzymatically fermenting Ganoderma lucidum, Poria cocos, Nostoc commune, oats, lactoferrin, and ginseng to obtain a first fermentation product. Then, konjac gum hydrolysate and collagen and glycosaminoglycans extracted from tilapia skin are added, and fermentation continues to obtain a second fermentation product. Modified lysozyme is added for enzymatic hydrolysis, followed by centrifugation. The supernatant is collected, and copper and calcium salts are added. The mixture is stirred to obtain a Cu-Ca chelated fermentation lysate. The product is then encapsulated to obtain the Bifida ferment lysate.

[0009] As a further improvement to the present invention, the following steps are included:

[0010] S1. Enzymatic hydrolysis of konjac gum: Add konjac gum to water, add β-mannanase for enzymatic hydrolysis, centrifuge, filter, add ethanol to the supernatant for precipitation, dry, and obtain konjac gum enzymatic hydrolysis product;

[0011] S2. Processing of tilapia skin: Wash the tilapia skin, remove the scales and fish meat, add water, add lipase to deesterify, filter, soak the filter residue in alkaline solution, take it out and wash it, add water to homogenize, add enzyme to hydrolyze, inactivate the enzyme, add salt to precipitate, collect solid protein and supernatant, dissolve the solid protein in acetic acid solution and dialyze, freeze dry to obtain collagen, dialyze the supernatant and dry to obtain glycosaminoglycan, mix the obtained collagen and glycosaminoglycan evenly to obtain the tilapia skin processed product;

[0012] S3. Enzyme-assisted fermentation: Ganoderma lucidum, Poria cocos, Ge Xianmi, oats, lactoferrin and ginseng are washed, dried and crushed, mixed to obtain a mixed powder, added to water, added compound enzyme, pre-enzymatic hydrolysis, sterilized, inoculated with fermentation bacteria, and anaerobic fermentation culture to obtain the first fermentation product.

[0013] S4. Re-fermentation: Mix the konjac gum enzymatic hydrolysate obtained in step S1 and the tilapia skin treatment product obtained in step S2 evenly, add it to the first fermentation product in step S3, and continue anaerobic fermentation culture to obtain the second fermentation product.

[0014] S5. Synergistic treatment with modified lysozyme: Add modified lysozyme to the second fermentation product obtained in step S4, heat for enzymatic hydrolysis, inactivate the enzyme, centrifuge, collect the supernatant, and obtain the fermentation lysate product.

[0015] S6. Chelation of metal ions: Add copper and calcium salts to the fermentation lysate obtained in step S5, stir the reaction, filter, freeze dry, and obtain Cu-Ca chelated fermentation lysate.

[0016] S7. Encapsulation: Add the Cu-Ca chelated fermentation lysate obtained in step S6 to water, add sodium alginate and emulsifier, stir and mix evenly, add to fish oil, emulsify, add calcium ion solution dropwise, solidify at room temperature, centrifuge, wash, dry, and obtain Bifida ferment lysate.

[0017] As a further improvement of the present invention, in step S1, the mass ratio of konjac gum to β-mannanase is 100:0.5-1, the enzymatic hydrolysis temperature is 40-45℃, the time is 1-3 hours, ethanol is added until the ethanol content in the system is 80-85 wt%, and the precipitation time is 3-5 hours; in step S2, the mass ratio of tilapia skin to lipase is 100:0.2-0.4, the enzymatic hydrolysis temperature is 37-40℃, the time is 0.5-1 hours, and the alkaline solution is... The process involves using a 0.1-0.15 mol / L NaOH or KOH solution. In the enzymatic hydrolysis, the enzyme is a mixture of neutral protease and papain in a mass ratio of 3-5:2. The amount of enzyme added is 0.5-1 wt% of the total system mass. The hydrolysis time is 2-4 hours at a temperature of 35-45°C. In the salting-out process, the salt is sodium chloride or ammonium sulfate, added until the salt content in the system is 0.9-1 mol / L. The salting-out time is 5-7 hours at a temperature of 3-5°C.

[0018] As a further improvement of the present invention, in step S3, the mass ratio of Ganoderma lucidum, Poria cocos, Ge Xianmi (a type of herb), oats, lactoferrin, and ginseng is 3-5:2-4:5-7:3-5:0.5-1:1-2; the mass ratio of the mixed powder, water, and compound enzyme is 15-20:100-150:0.5-1; the compound enzyme is selected from at least two of cellulase, pectinase, hemicellulase, papain, fig protease, bromelain, and neutral protease; the pre-enzymatic hydrolysis temperature is 40-45℃, and the time is 1-3 hours; the fermentation bacteria are selected from Bifidobacterium adolescentis, Bifidobacterium angularis, and... The anaerobic fermentation culture contains at least two of the following: *Bifidobacterium animalis*, *Bifidobacterium bifidum*, *Bifidobacterium breve*, *Bifidobacterium longum*, *Bifidobacterium infantis*, *Bifidobacterium pseudostreptorum*, and *Bifidobacterium thermophilum*. The anaerobic fermentation culture is carried out under hypoxic conditions at 37-39°C, 100-120 r / min, and fermentation culture for 36-48 h. In step S4, the mass ratio of the konjac gum enzymatic hydrolysate, the tilapia skin treatment product, and the first fermentation product is 3-5:5-7:200-220. The continued anaerobic fermentation culture is carried out under hypoxic conditions at 37-39°C, 100-120 r / min, and fermentation culture for 24-36 h.

[0019] As a further improvement of the present invention, the composite enzyme is hemicellulase and papain in a mass ratio of 3-5:7; the fermentation bacteria are a seed culture of *Bifidobacterium adolescentis*, *Bifidobacterium infantis*, and *Bifidobacterium longum*, and the bacterial count of the seed culture is 10. 8 -10 9 The inoculation amounts of the seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis, and Bifidobacterium longum were 1-3 v / v%, 2-4 v / v%, and 1-2 v / v, respectively.

[0020] As a further improvement of the present invention, in step S5, the mass ratio of the second fermentation product to the modified lysozyme is 1000:3-5, the temperature of the enzymatic hydrolysis is 40-45℃, and the time is 1-3h; in step S6, the mass ratio of the fermentation lysate, copper salt, and calcium salt is 1000:2-4:1-3, the copper salt is copper chloride or copper nitrate, the calcium salt is calcium chloride or calcium nitrate, and the stirring reaction time is 15-20min; in step S7, the mass ratio of the Cu-Ca chelated fermentation lysate, sodium alginate, and emulsifier is 15-20:17-22:0.5-1, the emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, and Tween-80, and the solidification time at room temperature is 20-30min.

[0021] As a further improvement of the present invention, the preparation method of the modified lysozyme is as follows:

[0022] T1. Heat treatment of lysozyme: Add lysozyme to water, adjust the pH of the solution to 7.5-8, heat for 10-15 minutes to obtain a heat-treated lysozyme system;

[0023] T2. Modification treatment: Dithiothreitol and hexadecyltrimethylammonium bromide are added to the lysozyme system that was heat-treated in step T1, and the mixture is stirred at 30-35℃ for 1-3 hours. Small molecules are removed by dialysis, and the mixture is freeze-dried to obtain modified lysozyme.

[0024] As a further improvement of the present invention, the temperature of the heat treatment in step T1 is 75-80°C, and the mass ratio of the lysozyme to water is 3-5:100; the mass ratio of the lysozyme system, dithiothreitol and hexadecyltrimethylammonium bromide in the heat treatment in step T2 is 150-200:0.5-1:0.2-0.4.

[0025] The present invention further protects a Bifida ferment lysate prepared by the above-described preparation method.

[0026] This invention further protects the application of the above-mentioned Bifida ferment lysate in the preparation of cosmetics with anti-aging, whitening, moisturizing, anti-inflammatory, anti-allergic, and skin barrier repair properties.

[0027] The present invention has the following beneficial effects:

[0028] Poria cocos contains abundant active substances such as poria polysaccharides, poria acid, and sterols. Its fermentation products are effective prebiotics that can promote the proliferation and growth of bifidobacteria and lactobacilli. At the same time, these prebiotics can also serve as nutrients for the facial skin microbiome, maintaining the diversity, richness, and overall health of the microbiome, promoting the normal growth and reproduction of beneficial bacteria on the face, thereby improving skin texture.

[0029] Nostoc commune is a freshwater microalgae rich in polysaccharides and proteins. It is rich in phycobiliproteins, and the products after fermentation include C-phycocyanin. Experiments have shown that C-phycocyanin not only has good antioxidant properties, but can also inhibit melanin synthesis through a dual mechanism.

[0030] After fermentation, Ganoderma lucidum contains abundant polysaccharides, flavonoids, polypeptides, and other substances, which have excellent antioxidant, whitening, and anti-aging activities. It significantly improves the scavenging rate of DPPH free radicals and hydroxyl free radicals, effectively promotes fibroblast proliferation, and has a high inhibition rate of tyrosinase activity, thus having better anti-aging and whitening effects.

[0031] Oats contain beta-glucan. After fermentation, the oat cell walls rupture, releasing a large amount of beta-glucan, which greatly improves the product's moisturizing effect.

[0032] Ginseng contains abundant active substances such as ginsenosides and ginseng polysaccharides. After fermentation by fermentation bacteria, a large number of common saponins are transformed into rare saponins, such as Rk, Rh1, Rh3, Rg3, and Rh4, which exert better antioxidant, anti-aging, and whitening activities.

[0033] This invention utilizes a combination of raw materials that undergo fermentation. During the synergistic fermentation of *Bifidobacterium adolescentis*, *Bifidobacterium infantis*, and *Bifidobacterium longum*, various metabolites are produced, including polysaccharides, organic acids, amino acids, polypeptides, proteins, nucleotides, and vitamins. These active components provide a variety of nutrients to the human skin and have beneficial biological effects. The products can effectively prevent skin aging, increase skin radiance, activate fibroblasts, inhibit melanin production, and effectively prevent skin aging and whiten the skin. Containing a large amount of protease and lipase, they can effectively cleanse and decompose excessive sebum secreted by sebaceous glands, break down keratinized cells in hair follicles, prevent sebaceous gland blockage, eliminate the environment conducive to the growth of acne-related pathogens, and effectively prevent acne breakouts.

[0034] Konjac gum, after being hydrolyzed by β-mannanase, produces oligoglucomannan, which significantly promotes the growth of Bifidobacteria and provides a carbon source for fermentation. Tilapia skin is rich in collagen and glycosaminoglycans, providing a nitrogen source for fermentation. Simultaneously, after fermentation, glycosaminoglycans degrade and exhibit wound-healing, anti-inflammatory, and antiviral activities. Collagen peptides and small-molecule amino acids obtained from collagen egg white fermentation promote skin growth and improve skin elasticity. When these two are combined and added to the system, they work synergistically to enhance the stress resistance of the fermentation bacteria, prolong their stable phase, and enable the production of more beneficial products, including trace elements, vitamins, enzymes, proteins, amino acids, and other trace elements and bioactive substances. These can promote metabolism, blood circulation, enhance cell vitality, improve the regenerative and self-healing abilities of immune cells, and prevent skin damage caused by sunlight and ultraviolet radiation.

[0035] The lysozyme of this invention is first thermally modified to retain most of its activity while simultaneously increasing the exposure of its hydrophobic groups, thus enhancing its ability to destroy both Gram-negative and Gram-positive bacteria. Further modification is achieved using dithiothreitol and hexadecyltrimethylammonium bromide. Dithiothreitol reduces the disulfide bonds in the lysozyme, resulting in greater molecular flexibility, exposing more hydrophobic surface regions, and altering its circular dihedral properties, thereby enhancing its affinity for cell membranes. The amino group in hexadecyltrimethylammonium bromide reacts with the lysozyme, and its long hydrophobic alkyl chain increases the hydrophobicity of the lysozyme surface, significantly improving its ability to destroy and disinfect bacteria. This allows fermentation bacteria to rupture, promoting the dissolution of their cellular contents and greatly increasing the abundance of active substances in the product. Furthermore, it enhances the product's bactericidal ability, thereby extending its shelf life.

[0036] The second fermentation product is treated with modified lysozyme, which causes the fermentation bacteria to rupture and a large number of active substances to dissolve. The intracellular active products have a good ability to inhibit tyrosinase activity, affect melanin metabolism, reduce melanin to colorless, and peel off melanin. They help strengthen the skin capillary walls, promote blood circulation, allow nutrients to penetrate the skin tissue more quickly, improve the skin microecology, and make the skin appear rosy and healthy like that of a young person. They provide deep moisturization to the skin and thoroughly prevent problems such as dryness and tightness. They can also break down the oil on the skin to achieve the effect of tightening pores and making the skin firm and smooth.

[0037] Furthermore, by chelating the fermentation lysate with copper and calcium ions, amino acid-copper and amino acid-calcium complexes are obtained. These significantly improve the skin's absorption of trace elements and amino acids, and are safe, non-toxic, and stable. On the other hand, the formed amino acid-copper complex effectively inhibits tyrosinase activity, while the amino acid-calcium complex maintains skin calcium ion homeostasis, promotes laminar secretion and keratinocyte membrane formation, reduces capillary permeability, and has anti-inflammatory and anti-allergic effects, contributing to skin barrier repair. The synergistic effect of these two compounds can prevent the release of the immunosuppressant IL-10 while simultaneously preventing the inhibition of IL-12, maintaining the IL-10 / IL-12 balance to combat photoaging and chronic photodamage, and repairing UV-induced DNA damage.

[0038] Encapsulating Cu-Ca chelated fermentation lysate can prevent the oxidation and damage of amino acids and active antioxidants by oxygen and moisture, thus avoiding a reduction in skin care effects. After encapsulation, the microcapsule shell will rupture under simple friction during actual use, releasing the contents and thus achieving better skin care effects.

[0039] The composition of this invention, under the fermentation action of Bifidobacterium microorganisms, can biosynthesize small molecules such as amino acids, organic acids, flavonoids, and polyphenols that are easily absorbed by the skin. It has excellent moisturizing, repairing, whitening and pH-regulating effects on the skin. The active substances such as amino acids, antibiotics and vitamins produced in the fermentation cells also have whitening, moisturizing, antibacterial and soothing and skin-repairing effects.

[0040] The bifida ferment lysate prepared by this invention has excellent whitening, moisturizing, antibacterial, soothing, skin repair, and pH regulation effects on the skin. It is safe and non-toxic, does not irritate the skin, has high skin absorption and utilization, and has a long shelf life, showing broad application prospects. Detailed Implementation

[0041] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0042] Konjac gum, content >99%, purchased from Chengdu Wanxiang Hongrun Biotechnology Co., Ltd.; lysozyme, 2 million U / g, lipase, 100,000 U / g, neutral protease, 50,000 U / g, papain, 100,000 U / g, hemicellulase, 50,000 U / g, purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd.; β-mannanase, 10,000 U / g, purchased from Wuhan Kemike Biomedical Technology Co., Ltd.; Bifidobacterium adolescentis, 10 billion CFU / g, Bifidobacterium infantis, 10 billion CFU / g, Bifidobacterium longum, 10 billion CFU / g, all purchased from Zhongke Jiayi Bioengineering Technology Co., Ltd.

[0043] The preparation method of spore liquids of *Bifidobacterium adolescentis*, *Bifidobacterium infantis*, and *Bifidobacterium longum* is as follows: *Bifidobacterium adolescentis*, *Bifidobacterium infantis*, and *Bifidobacterium longum* were inoculated into Gao's medium and fermented at 38℃, 100 r / min, and under anaerobic conditions for 18 h to obtain spores with a bacterial count of 10... 8 -10 9 CFU / mL bacterial seed solution.

[0044] For any undisclosed operating methods, follow the conventional methods in this field.

[0045] Preparation Example 1: Preparation of Modified Lysozyme

[0046] The method is as follows:

[0047] T1. Heat treatment of lysozyme: Add 3 parts by weight of lysozyme to 100 parts by weight of water, adjust the pH of the solution to 7.5, heat to 75°C, and treat for 10 min to obtain the heat-treated lysozyme system.

[0048] T2. Modification treatment: Add 0.5 parts by weight of dithiothreitol and 0.2 parts by weight of hexadecyltrimethylammonium bromide to 150 parts by weight of the lysozyme system subjected to heat treatment in step T1, stir and react at 30°C for 1 hour, remove small molecules by dialysis, freeze dry, and obtain modified lysozyme.

[0049] Preparation Example 2: Preparation of Modified Lysozyme

[0050] The method is as follows:

[0051] T1. Heat treatment of lysozyme: Add 5 parts by weight of lysozyme to 100 parts by weight of water, adjust the pH of the solution to 8, heat to 80°C, and treat for 15 min to obtain the heat-treated lysozyme system.

[0052] T2. Modification treatment: Add 1 part by weight of dithiothreitol and 0.4 parts by weight of hexadecyltrimethylammonium bromide to 200 parts by weight of the lysozyme system subjected to heat treatment in step T1, stir and react at 35°C for 3 hours, remove small molecules by dialysis, freeze dry, and obtain modified lysozyme.

[0053] Preparation Example 3: Preparation of Modified Lysozyme

[0054] The method is as follows:

[0055] T1. Heat treatment of lysozyme: Add 4 parts by weight of lysozyme to 100 parts by weight of water, adjust the pH of the solution to 7.7, heat to 77°C, and treat for 12 min to obtain the heat-treated lysozyme system.

[0056] T2. Modification treatment: Add 0.7 parts by weight of dithiothreitol and 0.3 parts by weight of hexadecyltrimethylammonium bromide to 170 parts by weight of the lysozyme system subjected to heat treatment in step T1, stir and react at 32°C for 2 hours, remove small molecules by dialysis, freeze dry, and obtain modified lysozyme.

[0057] Comparative Preparation Example 1

[0058] The difference compared to Preparation Example 3 is that step T1 was not performed.

[0059] Comparative Preparation Example 2

[0060] The difference from Preparation Example 3 is that dithiothreitol was not added in step T2.

[0061] Comparative preparation example 3

[0062] The difference from Preparation Example 3 is that hexadecyltrimethylammonium bromide was not added in step T2.

[0063] Comparative preparation example 4

[0064] The difference compared to preparation example 3 is that step T2 was not performed.

[0065] Example 1

[0066] This embodiment provides a method for preparing Bifida ferment lysate, including the following steps:

[0067] S1. Enzymatic hydrolysis of konjac gum: 100 parts by weight of konjac gum were added to 1000 parts by weight of water, 0.5 parts by weight of β-mannanase were added, and the mixture was enzymatically hydrolyzed at 40°C for 1 hour. After centrifugation and filtration, ethanol was added to the supernatant until the ethanol content in the system was 80 wt%. After precipitation for 3 hours, the mixture was dried to obtain the konjac gum enzymatic hydrolysis product.

[0068] S2. Processing of tilapia skin: 100 parts by weight of tilapia skin were washed, scales and flesh were removed, and the skin was added to 1000 parts by weight of water. 0.2 parts by weight of lipase were added, and the mixture was enzymatically hydrolyzed at 37°C for 0.5 h. The mixture was filtered, and the residue was soaked in 0.1 mol / L NaOH solution for 1 h. The residue was then washed, and 500 parts by weight of water were added to homogenize the mixture. Enzyme was added at 35°C for 2 h to inactivate the enzyme. Sodium chloride was added until the sodium chloride content in the system was 0.9 mol / L. Salting out was carried out at 3°C ​​for 5 h. Solid protein and supernatant were collected. Solid protein was dissolved in 1 wt% acetic acid solution and dialyzed for 5 h. Then it was dialyzed in water for 5 h and freeze-dried to obtain collagen. The supernatant was dialyzed for 2 h and dried to obtain glycosaminoglycan. The collagen and glycosaminoglycan were mixed evenly to obtain the tilapia skin processed product.

[0069] The enzyme is a mixture of neutral protease and papain in a mass ratio of 3:2;

[0070] S3. Enzyme-assisted fermentation: 3 parts by weight of Ganoderma lucidum, 2 parts by weight of Poria cocos, 5 parts by weight of Ge Xian Mi, 3 parts by weight of oats, 0.5 parts by weight of lactoferrin and 1 part by weight of ginseng were washed, dried and pulverized, and mixed to obtain a mixed powder. 200 parts by weight of water and 0.5 parts by weight of compound enzyme were added. The mixture was pre-enzymatically hydrolyzed at 40°C for 1 hour, sterilized, and inoculated with seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum. The inoculation amounts of the seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum were 1v / v%, 2v / v%, and 1v / v, respectively. Under anaerobic conditions, fermentation was carried out at 37°C and 100 r / min for 36 hours to obtain the first fermentation product.

[0071] The complex enzyme is hemicellulase and papain in a mass ratio of 3:7.

[0072] S4. Re-fermentation: Mix 3 parts by weight of the konjac gum enzymatic hydrolysate obtained in step S1 and 5 parts by weight of the tilapia skin treatment product obtained in step S2 evenly, add it to 200 parts by weight of the first fermentation product in step S3, and ferment under anaerobic conditions at 37°C and 100 r / min for 24 h to obtain the second fermentation product.

[0073] S5. Co-treatment with modified lysozyme: Add 3 parts by weight of the modified lysozyme prepared in Preparation Example 1 to 1000 parts by weight of the second fermentation product obtained in step S4, heat to 40°C, enzymatically hydrolyze for 1 h, inactivate the enzyme, centrifuge, collect the supernatant, and obtain the fermentation lysate product.

[0074] S6. Chelation of metal ions: Add 2 parts by weight of copper chloride and 1 part by weight of calcium chloride to 1000 parts by weight of the fermentation lysate obtained in step S5, stir and react for 15 min, filter, freeze dry to obtain Cu-Ca chelated fermentation lysate.

[0075] S7. Encapsulation: 15 parts by weight of the Cu-Ca chelated fermentation lysate obtained in step S6 were added to 200 parts by weight of water, 17 parts by weight of sodium alginate and 0.5 parts by weight of Tween-20 were added, and the mixture was stirred for 10 min. The mixture was then added to 500 parts by weight of fish oil and emulsified at 10000 r / min for 15 min. 20 parts by weight of 3 wt% calcium ion solution were added dropwise, and the mixture was solidified at room temperature for 20 min. The mixture was then centrifuged, washed, and dried to obtain the Bifida ferment lysate.

[0076] Example 2

[0077] This embodiment provides a method for preparing Bifida ferment lysate, including the following steps:

[0078] S1. Enzymatic hydrolysis of konjac gum: 100 parts by weight of konjac gum were added to 1000 parts by weight of water, 1 part by weight of β-mannanase was added, and the mixture was enzymatically hydrolyzed at 45°C for 3 hours. After centrifugation and filtration, ethanol was added to the supernatant until the ethanol content in the system was 85 wt%. After precipitation for 5 hours, the mixture was dried to obtain the konjac gum enzymatic hydrolysis product.

[0079] S2. Processing of tilapia skin: 100 parts by weight of tilapia skin were washed, scales and flesh were removed, and the skin was added to 1000 parts by weight of water. 0.4 parts by weight of lipase were added, and the mixture was enzymatically hydrolyzed at 40°C for 1 hour. The mixture was filtered, and the residue was soaked in 0.15 mol / L KOH solution for 1 hour. The residue was then washed, and 500 parts by weight of water were added to homogenize the mixture. Enzyme was added at 45°C for 4 hours to inactivate the enzyme. Ammonium sulfate was added until the ammonium sulfate content in the system was 1 mol / L. The mixture was salted out at 5°C for 7 hours. Solid protein and supernatant were collected. Solid protein was dissolved in 1 wt% acetic acid solution and dialyzed for 5 hours. Then it was dialyzed in water for 5 hours and freeze-dried to obtain collagen. The supernatant was dialyzed for 2 hours and dried to obtain glycosaminoglycans. The obtained collagen and glycosaminoglycans were mixed evenly to obtain the tilapia skin processed product.

[0080] The enzyme is a mixture of neutral protease and papain in a mass ratio of 5:2;

[0081] S3. Enzyme-assisted fermentation: 5 parts by weight of Ganoderma lucidum, 4 parts by weight of Poria cocos, 7 parts by weight of Ge Xianmi, 5 parts by weight of oats, 1 part by weight of lactoferrin and 2 parts by weight of ginseng were washed, dried and pulverized, and mixed to obtain a mixed powder. The mixed powder was added to 200 parts by weight of water, 1 part by weight of compound enzyme was added, and pre-enzymatic hydrolysis was carried out at 45℃ for 3 hours. After sterilization, the seed culture of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum was inoculated. The inoculation amount of the seed culture of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum were 3v / v%, 4v / v%, and 2v / v, respectively. Under anaerobic conditions, fermentation was carried out at 39℃ and 120r / min for 48 hours to obtain the first fermentation product.

[0082] The complex enzyme is hemicellulase and papain in a mass ratio of 5:7.

[0083] S4. Re-fermentation: Mix 5 parts by weight of the konjac gum enzymatic hydrolysate obtained in step S1 and 7 parts by weight of the tilapia skin treatment product obtained in step S2 evenly, add it to 220 parts by weight of the first fermentation product in step S3, and ferment under anaerobic conditions at 39℃ and 120r / min for 36h to obtain the second fermentation product.

[0084] S5. Co-treatment with modified lysozyme: Add 5 parts by weight of the modified lysozyme prepared in Preparation Example 2 to 1000 parts by weight of the second fermentation product obtained in step S4, heat to 45°C, enzymatically hydrolyze for 3 hours, inactivate the enzyme, centrifuge, collect the supernatant, and obtain the fermentation lysate product.

[0085] S6. Chelation of metal ions: Add 4 parts by weight of copper nitrate and 3 parts by weight of calcium nitrate to 1000 parts by weight of the fermentation lysate obtained in step S5, stir for 20 min, filter, freeze dry to obtain Cu-Ca chelated fermentation lysate.

[0086] S7. Encapsulation: 20 parts by weight of the Cu-Ca chelated fermentation lysate obtained in step S6 were added to 200 parts by weight of water, 22 parts by weight of sodium alginate and 1 part by weight of Tween-40 were added, and the mixture was stirred for 10 min. The mixture was then added to 500 parts by weight of fish oil and emulsified at 10000 r / min for 15 min. 20 parts by weight of 3 wt% calcium ion solution were added dropwise, and the mixture was solidified at room temperature for 30 min. The mixture was then centrifuged, washed, and dried to obtain the Bifida ferment lysate.

[0087] Example 3

[0088] This embodiment provides a method for preparing Bifida ferment lysate, including the following steps:

[0089] S1. Enzymatic hydrolysis of konjac gum: 100 parts by weight of konjac gum were added to 1000 parts by weight of water, 0.7 parts by weight of β-mannanase were added, and the mixture was enzymatically hydrolyzed at 42°C for 2 hours. After centrifugation and filtration, ethanol was added to the supernatant until the ethanol content in the system was 82 wt%. After precipitation for 4 hours, the mixture was dried to obtain the konjac gum enzymatic hydrolysis product.

[0090] S2. Processing of tilapia skin: 100 parts by weight of tilapia skin were washed, scales and flesh were removed, and the skin was added to 1000 parts by weight of water. 0.3 parts by weight of lipase were added, and the mixture was enzymatically hydrolyzed at 38°C for 1 hour. The mixture was filtered, and the residue was soaked in 0.12 mol / L NaOH solution for 1 hour. The residue was then washed, and 500 parts by weight of water were added to homogenize the mixture. Enzyme was added at 0.7 wt% of the total mass of the system. The mixture was enzymatically hydrolyzed at 40°C for 3 hours to inactivate the enzyme. Sodium chloride was added until the sodium chloride content in the system was 0.95 mol / L. Salting out was carried out at 4°C for 6 hours. Solid protein and supernatant were collected. Solid protein was dissolved in 1 wt% acetic acid solution and dialyzed for 5 hours. Then it was dialyzed in water for 5 hours and freeze-dried to obtain collagen. The supernatant was dialyzed for 2 hours and dried to obtain glycosaminoglycans. The collagen and glycosaminoglycans were mixed evenly to obtain the tilapia skin processed product.

[0091] The enzyme is a mixture of neutral protease and papain in a mass ratio of 4:2;

[0092] S3. Enzyme-assisted fermentation: 4 parts by weight of Ganoderma lucidum, 3 parts by weight of Poria cocos, 6 parts by weight of Ge Xian Mi, 4 parts by weight of oats, 0.7 parts by weight of lactoferrin and 1.5 parts by weight of ginseng were washed, dried and pulverized, and mixed to obtain a mixed powder. The mixed powder was added to 200 parts by weight of water, and 0.7 parts by weight of compound enzyme was added. The mixture was pre-enzymatically hydrolyzed at 42°C for 2 hours, sterilized, and inoculated with seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum. The inoculation amounts of the seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum were 2v / v%, 3v / v%, and 1.5v / v, respectively. Under anaerobic conditions, the mixture was fermented at 38°C and 110 r / min for 42 hours to obtain the first fermentation product.

[0093] The complex enzyme is hemicellulase and papain in a mass ratio of 4:7.

[0094] S4. Re-fermentation: Mix 4 parts by weight of the konjac gum enzymatic hydrolysate obtained in step S1 and 6 parts by weight of the tilapia skin treatment product obtained in step S2 evenly, add them to 210 parts by weight of the first fermentation product in step S3, and ferment under anaerobic conditions at 38°C and 110 r / min for 30 h to obtain the second fermentation product.

[0095] S5. Co-treatment with modified lysozyme: Add 4 parts by weight of the modified lysozyme prepared in Preparation Example 3 to 1000 parts by weight of the second fermentation product obtained in step S4, heat to 42°C, enzymatically hydrolyze for 2 hours, inactivate the enzyme, centrifuge, collect the supernatant, and obtain the fermentation lysate product.

[0096] S6. Chelation of metal ions: Add 3 parts by weight of copper nitrate and 2 parts by weight of calcium nitrate to 1000 parts by weight of the fermentation lysate obtained in step S5, stir for 17 min, filter, freeze dry to obtain Cu-Ca chelated fermentation lysate.

[0097] S7. Encapsulation: 17 parts by weight of the Cu-Ca chelated fermentation lysate obtained in step S6 were added to 200 parts by weight of water, 20 parts by weight of sodium alginate and 0.7 parts by weight of Tween-80 were added, and the mixture was stirred for 10 min. The mixture was then added to 500 parts by weight of fish oil and emulsified at 10000 r / min for 15 min. 20 parts by weight of 3 wt% calcium ion solution were added dropwise, and the mixture was solidified at room temperature for 25 min. The mixture was then centrifuged, washed, and dried to obtain the Bifida ferment lysate.

[0098] Example 4

[0099] The difference from Example 3 is that the complex enzyme is a single hemicellulase.

[0100] Example 5

[0101] The difference from Example 3 is that the complex enzyme is a single papain.

[0102] Comparative Examples 1-4

[0103] The difference from Example 3 is that the modified lysozyme was prepared from Comparative Preparation Examples 1-4.

[0104] Comparative Example 5

[0105] The difference from Example 3 is that no compound enzyme was added in step S3.

[0106] Specifically as follows:

[0107] S3. Fermentation: 4 parts by weight of Ganoderma lucidum, 3 parts by weight of Poria cocos, 6 parts by weight of Ge Xian Mi, 4 parts by weight of oats, 0.7 parts by weight of lactoferrin and 1.5 parts by weight of ginseng were washed, dried and pulverized, and mixed to obtain a mixed powder. The mixed powder was added to 200 parts by weight of water, sterilized, and inoculated with seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum. The inoculation amounts of the seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum were 2v / v%, 3v / v%, and 1.5v / v, respectively. Under anaerobic conditions, fermentation was carried out at 38°C and 110 r / min for 42 h to obtain the first fermentation product.

[0108] Comparative Example 6

[0109] The difference from Example 3 is that no seed culture of Bifidobacterium adolescentis was inoculated in step S3.

[0110] Specifically as follows:

[0111] S3. Enzyme-assisted fermentation: 4 parts by weight of Ganoderma lucidum, 3 parts by weight of Poria cocos, 6 parts by weight of Ge Xian Mi, 4 parts by weight of oats, 0.7 parts by weight of lactoferrin and 1.5 parts by weight of ginseng were washed, dried and pulverized, and mixed to obtain a mixed powder. The mixed powder was added to 200 parts by weight of water, and 0.7 parts by weight of compound enzyme were added. The mixture was pre-enzymatically hydrolyzed at 42°C for 2 hours, sterilized, and inoculated with seed cultures of Bifidobacterium infantis and Bifidobacterium longum. The inoculation amounts of the seed cultures of Bifidobacterium infantis and Bifidobacterium longum were 3v / v% and 1.5v / v%, respectively. Under anaerobic conditions, the mixture was fermented at 38°C and 110 r / min for 42 hours to obtain the first fermentation product.

[0112] The complex enzyme is a combination of hemicellulase and papain, with a mass ratio of 4:7.

[0113] Comparative Example 7

[0114] The difference from Example 3 is that no inoculum of Bifidobacterium infantis seed solution was inoculated in step S3.

[0115] Specifically as follows:

[0116] S3. Enzyme-assisted fermentation: 4 parts by weight of Ganoderma lucidum, 3 parts by weight of Poria cocos, 6 parts by weight of Ge Xian Mi, 4 parts by weight of oats, 0.7 parts by weight of lactoferrin and 1.5 parts by weight of ginseng were washed, dried and pulverized, and mixed to obtain a mixed powder. The mixed powder was added to 200 parts by weight of water, and 0.7 parts by weight of compound enzyme was added. The mixture was pre-enzymatically hydrolyzed at 42°C for 2 hours, sterilized, and inoculated with seed cultures of Bifidobacterium adolescentis and Bifidobacterium longum. The inoculation amounts of the seed cultures of Bifidobacterium adolescentis and Bifidobacterium longum were 2v / v% and 1.5v / v%, respectively. Under anaerobic conditions, the mixture was fermented at 38°C and 110 r / min for 42 hours to obtain the first fermentation product.

[0117] The complex enzyme is a combination of hemicellulase and papain, with a mass ratio of 4:7.

[0118] Comparative Example 8

[0119] The difference from Example 3 is that no spore liquid of Bifidobacterium longum was inoculated in step S3.

[0120] Specifically as follows:

[0121] S3. Enzyme-assisted fermentation: 4 parts by weight of Ganoderma lucidum, 3 parts by weight of Poria cocos, 6 parts by weight of Ge Xian Mi, 4 parts by weight of oats, 0.7 parts by weight of lactoferrin and 1.5 parts by weight of ginseng were washed, dried and pulverized, and mixed to obtain a mixed powder. The mixed powder was added to 200 parts by weight of water, and 0.7 parts by weight of compound enzyme was added. The mixture was pre-enzymatically hydrolyzed at 42°C for 2 hours, sterilized, and inoculated with seed cultures of Bifidobacterium adolescentis and Bifidobacterium infantis. The inoculation amounts of the seed cultures of Bifidobacterium adolescentis and Bifidobacterium infantis were 2v / v% and 3v / v%, respectively. Under anaerobic conditions, the mixture was fermented at 38°C and 110 r / min for 42 hours to obtain the first fermentation product.

[0122] The complex enzyme is a combination of hemicellulase and papain, with a mass ratio of 4:7.

[0123] Comparative Example 9

[0124] The difference from Example 3 is that no konjac gum enzymatic hydrolysate was added in step S4.

[0125] Specifically as follows:

[0126] S4. Re-fermentation: 10 parts by weight of the tilapia skin treatment product obtained in step S2 are added to 210 parts by weight of the first fermentation product in step S3. Under anaerobic conditions, fermentation is carried out at 38°C and 110 r / min for 30 h to obtain the second fermentation product.

[0127] Comparative Example 10

[0128] The difference from Example 3 is that no tilapia skin treatment product was added in step S4.

[0129] Specifically as follows:

[0130] S4. Re-fermentation: 10 parts by weight of the konjac gum enzymatic hydrolysate obtained in step S1 are added to 210 parts by weight of the first fermentation product in step S3. Under anaerobic conditions, fermentation is carried out at 38°C and 110 r / min for 30 h to obtain the second fermentation product.

[0131] Comparative Example 11

[0132] The difference from Example 3 is that no konjac gum hydrolysate and tilapia skin treatment product were added in step S4.

[0133] Specifically as follows:

[0134] S4. Re-fermentation: The first fermentation product from step S3 is fermented and cultured under anaerobic conditions at 38°C and 110 r / min for 30 h to obtain the second fermentation product.

[0135] Comparative Example 12

[0136] The difference from Example 3 is that step S5 was not performed.

[0137] Specifically as follows:

[0138] S1. Enzymatic hydrolysis of konjac gum: 100 parts by weight of konjac gum were added to 1000 parts by weight of water, 0.7 parts by weight of β-mannanase were added, and the mixture was enzymatically hydrolyzed at 42°C for 2 hours. After centrifugation and filtration, ethanol was added to the supernatant until the ethanol content in the system was 82 wt%. After precipitation for 4 hours, the mixture was dried to obtain the konjac gum enzymatic hydrolysis product.

[0139] S2. Processing of tilapia skin: 100 parts by weight of tilapia skin were washed, scales and flesh were removed, and the skin was added to 1000 parts by weight of water. 0.3 parts by weight of lipase were added, and the mixture was enzymatically hydrolyzed at 38°C for 1 hour. The mixture was filtered, and the residue was soaked in 0.12 mol / L NaOH solution for 1 hour. The residue was then washed, and 500 parts by weight of water were added to homogenize the mixture. Enzyme was added at 0.7 wt% of the total mass of the system. The mixture was enzymatically hydrolyzed at 40°C for 3 hours to inactivate the enzyme. Sodium chloride was added until the sodium chloride content in the system was 0.95 mol / L. Salting out was carried out at 4°C for 6 hours. Solid protein and supernatant were collected. Solid protein was dissolved in 1 wt% acetic acid solution and dialyzed for 5 hours. Then it was dialyzed in water for 5 hours and freeze-dried to obtain collagen. The supernatant was dialyzed for 2 hours and dried to obtain glycosaminoglycans. The collagen and glycosaminoglycans were mixed evenly to obtain the tilapia skin processed product.

[0140] The enzyme is a mixture of neutral protease and papain in a mass ratio of 4:2;

[0141] S3. Enzyme-assisted fermentation: 4 parts by weight of Ganoderma lucidum, 3 parts by weight of Poria cocos, 6 parts by weight of Ge Xian Mi, 4 parts by weight of oats, 0.7 parts by weight of lactoferrin and 1.5 parts by weight of ginseng were washed, dried and pulverized, and mixed to obtain a mixed powder. The mixed powder was added to 200 parts by weight of water, and 0.7 parts by weight of compound enzyme was added. The mixture was pre-enzymatically hydrolyzed at 42°C for 2 hours, sterilized, and inoculated with seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum. The inoculation amounts of the seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum were 2v / v%, 3v / v%, and 1.5v / v, respectively. Under anaerobic conditions, the mixture was fermented at 38°C and 110 r / min for 42 hours to obtain the first fermentation product.

[0142] The complex enzyme is hemicellulase and papain in a mass ratio of 4:7.

[0143] S4. Re-fermentation: Mix 4 parts by weight of the konjac gum enzymatic hydrolysate obtained in step S1 and 6 parts by weight of the tilapia skin treatment product obtained in step S2 evenly, add them to 210 parts by weight of the first fermentation product in step S3, and ferment under anaerobic conditions at 38°C and 110 r / min for 30 h to obtain the second fermentation product.

[0144] S5. Chelation of metal ions: Add 3 parts by weight of copper nitrate and 2 parts by weight of calcium nitrate to 1000 parts by weight of the second fermentation product obtained in step S4, stir and react for 17 min, filter, freeze dry, and obtain Cu-Ca chelated fermentation product.

[0145] S6. Encapsulation: 17 parts by weight of the Cu-Ca chelate fermentation product obtained in step S5 were added to 200 parts by weight of water, 20 parts by weight of sodium alginate and 0.7 parts by weight of Tween-80 were added, and the mixture was stirred for 10 min. The mixture was then added to 500 parts by weight of fish oil and emulsified at 10000 r / min for 15 min. 20 parts by weight of 3 wt% calcium ion solution were added dropwise, and the mixture was solidified at room temperature for 25 min. The mixture was then centrifuged, washed, and dried to obtain the Bifida ferment lysate.

[0146] Comparative Example 13

[0147] The difference from Example 3 is that copper nitrate was not added in step S6.

[0148] Specifically as follows:

[0149] S6. Chelation of metal ions: Add 5 parts by weight of calcium nitrate to 1000 parts by weight of the fermentation lysate obtained in step S5, stir for 17 min, filter, freeze dry to obtain Ca chelated fermentation lysate.

[0150] Comparative Example 14

[0151] The difference from Example 3 is that calcium nitrate was not added in step S6.

[0152] Specifically as follows:

[0153] S6. Chelation of metal ions: Add 5 parts by weight of copper nitrate to 1000 parts by weight of the fermentation lysate obtained in step S5, stir for 17 min, filter, freeze dry to obtain Cu chelated fermentation lysate.

[0154] Comparative Example 15

[0155] The difference from Example 3 is that step S6 was not performed.

[0156] Specifically as follows:

[0157] S1. Enzymatic hydrolysis of konjac gum: 100 parts by weight of konjac gum were added to 1000 parts by weight of water, 0.7 parts by weight of β-mannanase were added, and the mixture was enzymatically hydrolyzed at 42°C for 2 hours. After centrifugation and filtration, ethanol was added to the supernatant until the ethanol content in the system was 82 wt%. After precipitation for 4 hours, the mixture was dried to obtain the konjac gum enzymatic hydrolysis product.

[0158] S2. Processing of tilapia skin: 100 parts by weight of tilapia skin were washed, scales and flesh were removed, and the skin was added to 1000 parts by weight of water. 0.3 parts by weight of lipase were added, and the mixture was enzymatically hydrolyzed at 38°C for 1 hour. The mixture was filtered, and the residue was soaked in 0.12 mol / L NaOH solution for 1 hour. The residue was then washed, and 500 parts by weight of water were added to homogenize the mixture. Enzyme was added at 0.7 wt% of the total mass of the system. The mixture was enzymatically hydrolyzed at 40°C for 3 hours to inactivate the enzyme. Sodium chloride was added until the sodium chloride content in the system was 0.95 mol / L. Salting out was carried out at 4°C for 6 hours. Solid protein and supernatant were collected. Solid protein was dissolved in 1 wt% acetic acid solution and dialyzed for 5 hours. Then it was dialyzed in water for 5 hours and freeze-dried to obtain collagen. The supernatant was dialyzed for 2 hours and dried to obtain glycosaminoglycans. The collagen and glycosaminoglycans were mixed evenly to obtain the tilapia skin processed product.

[0159] The enzyme is a mixture of neutral protease and papain in a mass ratio of 4:2;

[0160] S3. Enzyme-assisted fermentation: 4 parts by weight of Ganoderma lucidum, 3 parts by weight of Poria cocos, 6 parts by weight of Ge Xian Mi, 4 parts by weight of oats, 0.7 parts by weight of lactoferrin and 1.5 parts by weight of ginseng were washed, dried and pulverized, and mixed to obtain a mixed powder. The mixed powder was added to 200 parts by weight of water, and 0.7 parts by weight of compound enzyme was added. The mixture was pre-enzymatically hydrolyzed at 42°C for 2 hours, sterilized, and inoculated with seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum. The inoculation amounts of the seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis and Bifidobacterium longum were 2v / v%, 3v / v%, and 1.5v / v, respectively. Under anaerobic conditions, the mixture was fermented at 38°C and 110 r / min for 42 hours to obtain the first fermentation product.

[0161] The complex enzyme is hemicellulase and papain in a mass ratio of 4:7.

[0162] S4. Re-fermentation: Mix 4 parts by weight of the konjac gum enzymatic hydrolysate obtained in step S1 and 6 parts by weight of the tilapia skin treatment product obtained in step S2 evenly, add them to 210 parts by weight of the first fermentation product in step S3, and ferment under anaerobic conditions at 38°C and 110 r / min for 30 h to obtain the second fermentation product.

[0163] S5. Co-treatment with modified lysozyme: Add 4 parts by weight of the modified lysozyme prepared in Preparation Example 3 to 1000 parts by weight of the second fermentation product obtained in step S4, heat to 42°C, enzymatically hydrolyze for 2 hours, inactivate the enzyme, centrifuge, collect the supernatant, freeze dry, and obtain the fermentation lysate product.

[0164] S6. Encapsulation: 17 parts by weight of the fermentation lysate obtained in step S5 were added to 200 parts by weight of water, 20 parts by weight of sodium alginate and 0.7 parts by weight of Tween-80 were added, and the mixture was stirred for 10 min. The mixture was then added to 500 parts by weight of fish oil and emulsified at 10000 r / min for 15 min. 20 parts by weight of 3 wt% calcium ion solution were added dropwise, and the mixture was solidified at room temperature for 25 min. The mixture was then centrifuged, washed, and dried to obtain the Bifida ferment lysate.

[0165] Test Example 1: In Vitro Cell Experiment

[0166] The Bifida ferment lysate obtained in the examples and comparative examples was added to the culture medium to prepare a 10 mg / mL solution. The solution was heated to 40°C, mechanically ruptured, centrifuged, and the supernatant was collected to obtain the sample solution.

[0167] 1. Resistance to light damage test

[0168] Human immortalized epidermal cells (HaCaT cells) were seeded in 96-well plates (2 × 10⁻⁶ cells / well). 4 / well), cultured until cell confluence reaches approximately 80%-90%. Discard the culture medium from the wells, add 100 μL of sample solution to each well to obtain the experimental group, positive control (add 100 μL of culture medium containing 0.05% vitamin C), and control and model groups, adding equal volumes of culture medium. Irradiate under UVB (280-320 nm) (dose 60 mJ / cm²). 2 The cells were incubated at 37°C for 24 hours, then 100 μL of CCK8-containing culture medium was added. After incubation for 30 minutes, the absorbance at 450 nm was measured. The control group was taken as 100%, and the relative cell viability of each group was calculated.

[0169] Relative cell viability = (OD value of experimental group / OD value of control group) × 100%

[0170] 2. Antioxidant test

[0171] HaCaT cells were seeded in 96-well plates (2 × 10⁻⁶ cells per well). 4 / well), cultured until cell confluence was approximately 80%-90%. The culture medium in each well was discarded, and 100 μL of sample solution was added to each well to obtain the experimental group and positive control (100 μL of medium containing 0.05% vitamin C was added). After incubation at 37°C for 24 h, the culture medium was discarded, and 100 μL of DMEM medium containing 2,7-dichlorofluorescein diacetate (DCFH-DA) was added. After incubation at 37°C for 20 min, the cells were washed, covered with buffer, and irradiated under UVA (dose 4.0 J / cm²). 2 After irradiation, fluorescence intensity was measured. The ROS level for each group was calculated, with the control group as 100%.

[0172] ROS level = (Experimental group fluorescence value / Control group fluorescence value) × 100%

[0173] 3. Test to promote the secretion of type I collagen in cells

[0174] Human skin fibroblasts (HDF cells) were seeded in 96-well plates (6 × 10⁶ cells / well). 3 / well), cultured until cell confluence was approximately 80%-90%. Discard the culture medium from each well, add 100 μL of sample solution to each well to obtain the experimental group and positive control (add 100 μL of culture medium containing 0.05% vitamin C). Incubate at 37°C for 6 hours, wash, cover cells with buffer, and irradiate under UVB (280-320 nm) (dose 20 mJ / cm²). 2 After irradiation, the culture medium was replaced with one-tenth serum solution for further culture. Irradiation was repeated every 12 hours for a total of 4 times. After the last irradiation, the culture medium was replaced with one-tenth serum solution containing the corresponding concentration of the test substance for further culture. After 72 hours, the culture medium was collected by centrifugation, and the content of type I collagen (COL-1) was measured using an ELISA kit. The control group was used as 100%, and the COL-1 level in each group was calculated.

[0175] COL-1 level = (OD value of experimental group / OD value of control group) × 100%

[0176] The results are shown in Table 1.

[0177] Table 1

[0178]

[0179]

[0180] Note: * indicates P < 0.05 compared to the model group.

[0181] As shown in the table above, the Bifida ferment lysate obtained in Examples 1-3 of this invention can effectively repair cell damage caused by UVB, effectively inhibit UVA-induced oxidative damage, and significantly promote collagen secretion.

[0182] Test Example 2

[0183] The Bifida ferment lysate obtained in the examples and comparative examples was added to deionized water to prepare a 10 mg / mL solution. The solution was heated to 40°C, mechanically broken up, centrifuged, and the supernatant was collected to obtain the sample solution.

[0184] One hundred and five female volunteers aged 22-47 years were selected on a voluntary basis and randomly divided into 21 groups of five each: Examples 1-5, Comparative Examples 1-15, and a control group. No medications or cosmetics unrelated to the experiment were applied during the trial. Samples were applied to both sides of the subjects' faces. The experimental groups used the corresponding sample solutions, while the control group used deionized water. The tests were conducted twice daily for two weeks. Before the test, subjects washed the test areas with clean water, dried them, and exposed them. They sat quietly for 20 minutes in an environment of 25±1℃ and 50±5% relative humidity. The stratum corneum moisture content of the test areas was measured using a skin moisture testing probe, and the average of five measurements was taken. The stratum corneum moisture content was measured before the start of the trial and again one hour after sample application on the evening of the 14th day. The percentage change in skin moisture content was calculated.

[0185] Similarly, skin elasticity and roughness were measured using a skin elasticity tester before the start of the test. On day 14, 1 hour after the sample was used, the skin elasticity and roughness of the test subjects were measured, and the rate of change (%) of skin elasticity and roughness was calculated.

[0186] Skin moisture content change rate (%) = (Moisture content after use - Moisture content before use) / Moisture content before use × 100%

[0187] Skin elasticity change rate (%) = (Skin elasticity after use - Skin elasticity before use) / Skin elasticity before use × 100%

[0188] Roughness change rate (%) = (Roughness before use - Roughness after use) / Roughness before use × 100%

[0189] The results are shown in Table 2.

[0190] Table 2

[0191] Group Change rate of skin moisture content (%) Change in skin elasticity (%) Roughness change rate (%) Model group 3.5 3.9 2.7 Example 1 30.2 19.5 24.9 Example 2 29.8 19.0 24.1 Example 3 30.9 20.9 25.3 Example 4 28.2 18.4 22.7 Example 5 27.9 18.0 23.1 Comparative Example 1 25.5 17.9 22.8 Comparative Example 2 26.0 17.2 22.1 Comparative Example 3 25.1 17.4 22.2 Comparative Example 4 24.8 16.8 21.8 Comparative Example 5 26.9 17.9 22.1 Comparative Example 6 26.4 17.0 21.1 Comparative Example 7 25.3 17.2 21.5 Comparative Example 8 25.8 16.8 21.7 Comparative Example 9 25.2 17.8 22.8 Comparative Example 10 26.8 15.9 19.9 Comparative Example 11 24.1 14.0 18.2 Comparative Example 12 23.2 16.1 20.4 Comparative Example 13 28.2 17.8 23.1 Comparative Example 14 27.9 17.2 23.6 Comparative Example 15 27.1 16.9 22.7

[0192] As shown in the table above, the Bifida ferment lysate obtained in Examples 1-3 of this invention significantly improves skin moisture retention, skin roughness, and skin elasticity.

[0193] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a Bifida ferment lysate, characterized in that, Includes the following steps: S1. Enzymatic hydrolysis of konjac gum: Add konjac gum to water, add β-mannanase for enzymatic hydrolysis, centrifuge, filter, add ethanol to the supernatant for precipitation, dry, and obtain konjac gum enzymatic hydrolysis product; S2. Processing of tilapia skin: Wash the tilapia skin, remove the scales and fish meat, add water, add lipase to deesterify, filter, soak the filter residue in alkaline solution, take it out and wash it, add water to homogenize, add enzyme to hydrolyze, inactivate the enzyme, add salt to precipitate, collect solid protein and supernatant, dissolve the solid protein in acetic acid solution and dialyze, freeze dry to obtain collagen, dialyze the supernatant and dry to obtain glycosaminoglycan, mix the obtained collagen and glycosaminoglycan evenly to obtain the tilapia skin processed product; S3. Enzyme-assisted fermentation: Ganoderma lucidum, Poria cocos, Ge Xianmi, oats, lactoferrin and ginseng are washed, dried and crushed, mixed to obtain a mixed powder, added to water, added compound enzyme, pre-enzymatic hydrolysis, sterilized, inoculated with fermentation bacteria, and anaerobic fermentation culture to obtain the first fermentation product. S4. Re-fermentation: Mix the konjac gum enzymatic hydrolysate obtained in step S1 and the tilapia skin treatment product obtained in step S2 evenly, add it to the first fermentation product in step S3, and continue anaerobic fermentation culture to obtain the second fermentation product. S5. Synergistic treatment with modified lysozyme: Add modified lysozyme to the second fermentation product obtained in step S4, heat for enzymatic hydrolysis, inactivate the enzyme, centrifuge, collect the supernatant, and obtain the fermentation lysate product. S6. Chelation of metal ions: Add copper and calcium salts to the fermentation lysate obtained in step S5, stir the reaction, filter, freeze dry, and obtain Cu-Ca chelated fermentation lysate. S7. Encapsulation: The Cu-Ca chelated fermentation lysate obtained in step S6 was added to water, along with sodium alginate and an emulsifier. The mixture was stirred and mixed thoroughly, then added to fish oil for emulsification. Calcium ion solution was added dropwise, and the mixture was allowed to solidify at room temperature. After centrifugation, washing, and drying, the Bifida ferment lysate was obtained. The modified lysozyme is prepared as follows: T1. Heat treatment of lysozyme: Add lysozyme to water, adjust the pH of the solution to 7.5-8, and heat for 10-15 minutes to obtain a heat-treated lysozyme system; T2. Modification treatment: Dithiothreitol and hexadecyltrimethylammonium bromide were added to the lysozyme system subjected to heat treatment in step T1, and the mixture was stirred at 30-35℃ for 1-3 hours. Small molecules were removed by dialysis, and the mixture was freeze-dried to obtain modified lysozyme. The compound enzyme is hemicellulase and papain, and the fermentation bacteria are seed liquids of Bifidobacterium adolescentis, Bifidobacterium infantis, and Bifidobacterium longum.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of konjac gum to β-mannanase is 100:0.5-1, the enzymatic hydrolysis temperature is 40-45℃, the time is 1-3 hours, ethanol is added until the ethanol content in the system is 80-85 wt%, and the precipitation time is 3-5 hours. In step S2, the mass ratio of tilapia skin to lipase is 100:0.2-0.4, the enzymatic hydrolysis temperature is 37-40℃, the time is 0.5-1 hours, and the alkaline solution is 0.1-0. The enzyme used in the enzymatic hydrolysis is a mixture of neutral protease and papain in a mass ratio of 3-5:2, with the amount of enzyme added being 0.5-1 wt% of the total mass of the system. The hydrolysis time is 2-4 h at a temperature of 35-45 °C. In the salting-out process, the salt is sodium chloride or ammonium sulfate, added until the salt content in the system is 0.9-1 mol / L. The salting-out time is 5-7 h at a temperature of 3-5 °C.

3. The preparation method according to claim 1, characterized in that, In step S3, the mass ratio of Ganoderma lucidum, Poria cocos, Ge Xian Mi (a type of herb), oats, lactoferrin, and ginseng is 3-5:2-4:5-7:3-5:0.5-1:1-2. The mass ratio of the mixed powder, water, and compound enzyme is 15-20:100-150:0.5-1. The pre-enzymatic hydrolysis temperature is 40-45℃, and the time is 1-3 hours. The anaerobic fermentation culture conditions are 37-39℃, 100-120 r / min, and fermentation culture for 36-48 hours under anaerobic conditions. In step S4, the mass ratio of the konjac gum enzymatic hydrolysis product, tilapia skin treatment product, and first fermentation product is 3-5:5-7:200-220. The continued anaerobic fermentation culture conditions are 37-39℃, 100-120 r / min, and fermentation culture for 24-36 hours under anaerobic conditions.

4. The preparation method according to claim 3, characterized in that, The mass ratio of the hemicellulase to the papain is 3-5:7; the bacterial count of the spore solution is 10. 8 -10 9 The inoculation amounts of the seed cultures of Bifidobacterium adolescentis, Bifidobacterium infantis, and Bifidobacterium longum were 1-3 v / v%, 2-4 v / v%, and 1-2 v / v, respectively.

5. The preparation method according to claim 1, characterized in that, In step S5, the mass ratio of the second fermentation product to the modified lysozyme is 1000:3-5, and the heating enzymatic hydrolysis temperature is 40-45℃ for 1-3 hours. In step S6, the mass ratio of the fermentation lysate, copper salt, and calcium salt is 1000:2-4:1-3, where the copper salt is copper chloride or copper nitrate, and the calcium salt is calcium chloride or calcium nitrate. The stirring reaction time is 15-20 minutes. In step S7, the mass ratio of the Cu-Ca chelated fermentation lysate, sodium alginate, and emulsifier is 15-20:17-22:0.5-1, where the emulsifier is selected from at least one of Tween-20, Tween-40, Tween-60, and Tween-80. The solidification time at room temperature is 20-30 minutes.

6. The preparation method according to claim 1, characterized in that, The temperature of the heat treatment in step T1 is 75-80℃, and the mass ratio of the lysozyme to water is 3-5:100; the mass ratio of the lysozyme system, dithiothreitol and hexadecyltrimethylammonium bromide in the heat treatment in step T2 is 150-200:0.5-1:0.2-0.

4.

7. A Bifida ferment lysate prepared by the preparation method according to any one of claims 1-6.

8. The application of the Bifida ferment lysate as described in claim 7 in the preparation of cosmetics with anti-aging, whitening, moisturizing, anti-inflammatory, anti-allergic, and skin barrier repair properties.

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