Bacillus fermentation product for repair and moisturization, and preparation method and application thereof

Abstract

The application belongs to the field of cosmetics, and particularly relates to a bacillus fermentation product for repairing and moisturizing, and a preparation method and application thereof. The preparation method of the bacillus fermentation product for repairing and moisturizing comprises the following steps: S1, culturing bacillus in a culture medium to obtain bacillus spores; S2, treating the bacillus spores obtained in step S1 with lysozyme and protease, and then treating the bacillus spores with an ultrahigh-pressure homogenizer to obtain a spore lysis product; and S3, centrifuging, concentrating and heating the spore lysis product obtained in step S2 to obtain the bacillus fermentation product for repairing and moisturizing. The spore lysis product is applied to the field of cosmetic raw material preparation, and the bacillus fermentation product obtained by the application has excellent repairing and moisturizing effects, can inhibit staphylococcus aureus, and has the effect of balancing skin microecology.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cosmetics, and particularly relates to a Bacillus fermentation product for repair and moisturizing, and a preparation method and application thereof. BACKGROUND

[0002] Bacillus belongs to Bacillaceae and Bacillus, is a kind of gram-positive bacteria that can produce resistant endospores, and the cells are rod-shaped and covered with a large number of calcium pyridinedicarboxylates. Spores, also known as spores, are the most resistant cells found in nature. The spores in the dormant state have strong resistance to heat, dryness, radiation, acid, alkali and organic solvents and other sterilization factors. The spores can survive for more than ten years or even thousands of years under adverse environmental conditions. When the environmental conditions are suitable, the spores can germinate to form cells that can divide and multiply.

[0003] The Bacillus fermentation product can be used as a cosmetic raw material for cosmetics. For example, Chinese Patent Application "Composition for preventing or improving aging comprising Bacillus pumilus strain or culture solution thereof" (Publication No. CN 110872568A) discloses a cosmetic composition comprising Bacillus pumilus strain or culture solution thereof, which is used as an effective ingredient to prevent or improve skin aging. Chinese Patent Application "Bacillus amyloliquefaciens BA-1, cytolysate, preparation method thereof, application in cosmetics and face cream" (Publication No. CN 115011531 A) discloses Bacillus amyloliquefaciens BA-1 and its cytolysate, which has low toxicity and certain protective effect on human skin fibroblasts. After UVA irradiation, the survival rate of human skin fibroblasts remains at a high level. Currently, the research and application of Bacillus fermentation product in the field of cosmetics are generally fermentation filtrate or cytolysate of nutrient cells, and very few involve spores and spore lysates. SUMMARY

[0004] In view of the deficiencies of the prior art, the purpose of the present application is to provide a Bacillus fermentation product for repair and moisturizing, and a preparation method and application thereof. The spore lysate is applied to the field of cosmetic raw material preparation for the first time. The Bacillus fermentation product obtained by the present application has excellent repair and moisturizing effects, can inhibit Staphylococcus aureus, has the effect of balancing skin microecology, and has very high commercial value.

[0005] The technical scheme of the present application is as follows:

[0006] A preparation method of a Bacillus fermentation product for repair and moisturizing, comprising the following steps:

[0007] S1 culture Bacillus in culture medium, obtain Bacillus spore;

[0008] S2 treat Bacillus spore obtained in step S1 with lysozyme and protease, then treat with ultra-high pressure homogenizer, obtain spore lysate;

[0009] S3 centrifuge, concentrate, heat spore lysate obtained in step S2, obtain Bacillus fermentation product for repair and moisturizing.

[0010] Further, Bacillus in step S1 includes Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium.

[0011] Further, the specific steps for culture Bacillus in culture medium, obtain Bacillus spore in step S1 are as follows:

[0012] (1) inoculate Bacillus strain into seed culture medium, culture aerobically at temperature of 30-37℃, culture time is 10-20h, obtain seed liquid;

[0013] (2) transfer seed liquid obtained in step (1) into fermentation culture medium containing rich carbon source, nitrogen source and inorganic salt, culture aerobically at temperature of 30-37℃, culture time is 36-72h, obtain fermentation liquid;

[0014] (3) centrifuge fermentation liquid obtained in step (2) at centrifugal speed of 4000-6000r / min, centrifugal time is 12-20min, collect precipitate as Bacillus spore.

[0015] Further, the carbon source in step (2) includes glucose, corn starch, corn flour, rice flour; the nitrogen source includes yeast extract, peptone, corn steep liquor, bran; the inorganic salt includes potassium dihydrogen phosphate, manganese sulfate, sodium chloride.

[0016] Further, the specific steps for treating Bacillus spore obtained in step S1 with lysozyme and protease, then treating with ultra-high pressure homogenizer, obtain spore lysate in step S2 are as follows:

[0017] (A) add pure water to Bacillus spore, so that mass fraction of Bacillus spore is 1%-10%, add lysozyme, treat at temperature of 50-55℃, treatment time is 6-12h;

[0018] (B) continue to add protease, treat at temperature of 45-50℃, treatment time is 4-8h, obtain material liquid;

[0019] (C) the obtained solution of step (B) is treated by an ultra-high pressure homogenizer, the homogenization pressure is 800bar-1500bar, and the homogenization time is 10min-30min, to obtain the spore lysis product.

[0020] Further, the added amount of the lysozyme in step (A) is 0.1-0.5% of the mass of the Bacillus spore.

[0021] Further, the added amount of the protease in step (B) is 0.1-0.5% of the mass of the Bacillus spore.

[0022] Further, the protease in step S2 is neutral protease.

[0023] Another object of the present application is to provide the Bacillus fermentation product for repair and moisturizing prepared by the preparation method of the Bacillus fermentation product for repair and moisturizing.

[0024] Still another object of the present application is to provide the use of the Bacillus fermentation product for repair and moisturizing in the preparation of the cosmetic for repair and moisturizing.

[0025] The Bacillus fermentation product for repair and moisturizing of the present application is prepared by culturing Bacillus in a culture medium to obtain Bacillus spores, treating the obtained Bacillus spores by lysozyme and protease, and then treating by an ultra-high pressure homogenizer to obtain a spore lysis product, and then centrifuging, concentrating and heating the obtained spore lysis product. The present application obtains qualified spore lysis product only by the specific combination of the three steps of treating the obtained Bacillus spores by lysozyme and protease, and then treating by an ultra-high pressure homogenizer, and it is difficult to obtain qualified spore lysis product without any step.

[0026] Compared with the prior art, the present application has the following advantages:

[0027] The spores of Bacillus have the characteristics of strong resistance, and are generally used as probiotics to replace vegetative cells and widely applied in the fields of feed and fertilizer, which can improve the immunity of the feeding animals and prevent and treat various plant diseases. The present application first applies the spore lysis product to the field of cosmetic raw material preparation, and the obtained Bacillus fermentation product has excellent repair and moisturizing effects, can inhibit Staphylococcus aureus, has the effect of balancing the skin microecology, and has extremely high commercial value. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 The preparation process flow chart of the Bacillus fermentation product for repair and moisturizing of the present application;

[0029] Figure 2Figure 1 is a microscope image of the seed liquid, fermentation broth, neutral protease hydrolysis liquid, and spore body lysis product after ultrahigh pressure homogenization in Example 1. Figure 2 a is a microscope image of the seed liquid in Example 1. Figure 2 b is a microscope image of the fermentation broth in Example 1. Figure 2 c is a microscope image of the neutral protease hydrolysis liquid in Example 1. Figure 2 d is a microscope image of the spore body lysis product after ultrahigh pressure homogenization in Example 1.

[0030] Figure 3 Figure 2 is a microscope image of the seed liquid, fermentation broth, neutral protease hydrolysis liquid, and spore body lysis product after ultrahigh pressure homogenization in Example 2. Figure 3 a is a microscope image of the seed liquid in Example 2. Figure 3 b is a microscope image of the fermentation broth in Example 2. Figure 3 c is a microscope image of the neutral protease hydrolysis liquid in Example 2. Figure 3 d is a microscope image of the spore body lysis product after ultrahigh pressure homogenization in Example 2.

[0031] Figure 4 Figure 3 is a picture of the Bacillus fermentation product for repair and moisturizing prepared in Example 2.

[0032] Figure 5 Figure 4 is a microscope image of the spore body lysis product after ultrahigh pressure homogenization in Comparative Example 1. Figure 6 Figure 5 is a microscope image of the spore body lysis product after treatment with lysozyme and ultrahigh pressure homogenization in Comparative Example 2. DETAILED DESCRIPTION

[0033] The present application is further described below through the description of specific embodiments, but this is not a limitation on the present application. Those skilled in the art can make various modifications or improvements to the present application according to the basic idea of the present application, but as long as they do not deviate from the basic idea of the present application, they are within the scope of the present application.

[0034] The raw materials used in the present application are commercially available unless otherwise specified. Lysozyme can be purchased from Nanning Pangbo Biological Engineering Co., Ltd.; protease is neutral protease with a specification of 130,000 U / g, which can be purchased from Nanning Dongheng Huadao Biological Technology Co., Ltd.; Bacillus megaterium is Bacillus megaterium microbial inoculant purchased from Beihai Yeshengwang Biological Technology Co., Ltd.; Bacillus subtilis can be purchased from Beihai Qiangxing Biological Technology Co., Ltd.; Bacillus licheniformis can be purchased from Guangdong Microbial Culture Collection Center, GDMCC No. 1.863.

[0035] The preparation process flow chart of the bacillus ferment product for repairing and moisturizing is shown in Figure 1 .

[0036] Example 1, a bacillus ferment product for repairing and moisturizing and a preparation method thereof

[0037] The preparation method of the bacillus ferment product for repairing and moisturizing comprises the following steps:

[0038] S1, culturing bacillus megaterium in a culture medium to obtain bacillus megaterium spores, the specific steps are as follows:

[0039] (1) inoculate bacillus megaterium into a seed culture medium (MRS culture medium, Guangdong HuanKai Microbial Technology Co., Ltd.) with an inoculation loop, and cultivate aerobically at a temperature of 30℃ (rotary shaker, rotation speed 180r / min) for 15h to obtain a seed liquid; the microscope examination picture of the seed liquid in Example 1 is shown in Figure 2 a, from which it can be seen that the bacterial cells under the microscope are in long chain shape and have no spores; Figure 2 a, from which it can be seen that the bacterial cells under the microscope are in long chain shape and have no spores;

[0040] (2) inoculate the seed liquid obtained in step (1) into a fermentation culture medium containing rich carbon source, nitrogen source and inorganic salt with an inoculation amount of 10%, and cultivate aerobically at a temperature of 30℃ (rotary shaker, rotation speed 220r / min) for 40h to obtain a fermentation liquid; the fermentation culture medium comprises the following components and mass percentages: rice flour 1%, peptone 1.5%, anhydrous manganese sulfate 0.02%, sodium chloride 0.5%, potassium dihydrogen phosphate 0.1%, and water 96.88%; the microscope examination picture of the fermentation liquid in Example 1 is shown in Figure 2 b, from which it can be seen that the bacterial cells under the microscope are in free spore form; Figure 2 b, from which it can be seen that the bacterial cells under the microscope are in free spore form;

[0041] (3) centrifuge the fermentation liquid obtained in step (2) at a speed of 4000r / min for 15min, and collect the precipitate as bacillus megaterium spores.

[0042] S2, treat the bacillus megaterium spores obtained in step S1 with lysozyme and protease, and then treat with an ultrahigh pressure homogenizer to obtain a bacillus megaterium spore lysate, the specific steps are as follows:

[0043] (A) add pure water to the bacillus megaterium spores to make the mass fraction of the bacillus megaterium spores 5%, and then add lysozyme, the addition amount of the lysozyme being 0.3% of the mass of the bacillus megaterium spores, and treat at a temperature of 52℃ for 10h;

[0044] (B) continue to add neutral protease, the amount of which is 0.3% of the mass of bacillus megaterium spores, and treat at a temperature of 48°C for 6 hours to obtain a liquid; the microscope image of the liquid obtained after the neutral protease hydrolysis in Example 1 is shown in Fig. Figure 2 c, from which it can be seen that the spores are partially broken and spore-lysing substances are precipitated; Figure 2 c, from which it can be seen that the spores are partially broken and spore-lysing substances are precipitated;

[0045] (C) treat the liquid obtained in step (B) with an ultrahigh-pressure homogenizer at a pressure of 800 bar for 30 minutes to obtain a spore-lysing product. The microscope image of the spore-lysing product obtained after the ultrahigh-pressure homogenization in Example 1 is shown in Fig. Figure 2 d, from which it can be seen that almost all the spores are broken and there are very few intact free spores; Figure 2 d, from which it can be seen that almost all the spores are broken and there are very few intact free spores;

[0046] S3 centrifuge the spore-lysing product obtained in step S2 at a speed of 5000 r / min for 15 minutes, collect the supernatant, concentrate the supernatant to a dry matter content of 3%, heat to 120°C, and keep the temperature for 20 minutes to inactivate the enzyme activity and residual viable bacteria, thereby obtaining a bacillus megaterium fermentation product for repair and moisturization.

[0047] Example 2: A bacillus megaterium fermentation product for repair and moisturization and a preparation method thereof

[0048] The preparation method of the bacillus megaterium fermentation product for repair and moisturization comprises the following steps:

[0049] S1 culture bacillus subtilis in a culture medium to obtain bacillus megaterium spores, and the specific steps are as follows:

[0050] (1) inoculate bacillus subtilis into a seed culture medium (nutrient broth, Guangdong HuanKai Microbial Technology Co., Ltd.) with an inoculation loop, and cultivate aerobically (rotary shaker, rotation speed 180 r / min) at a temperature of 37°C for 20 hours to obtain a seed liquid; the microscope image of the seed liquid in Example 2 is shown in Fig. Figure 3 a, from which it can be seen that the bacterial cells are observed under a microscope in a long chain shape without spores; Figure 3 a, from which it can be seen that the bacterial cells are observed under a microscope in a long chain shape without spores;

[0051] (2) The seed culture obtained in step (1) was inoculated at a 10% inoculum into a fermentation medium rich in carbon, nitrogen, and inorganic salts. It was then aerobically cultured at 37°C (using a rotary shaker at 220 rpm) for 48 hours to obtain the fermentation broth. The fermentation medium comprised the following components and their mass percentages: glucose 1%, yeast extract 0.8%, anhydrous manganese sulfate 0.02%, sodium chloride 0.5%, potassium dihydrogen phosphate 0.1%, and water 97.58%. A microscopic image of the fermentation broth in Example 2 is shown below. Figure 3 As shown in b, by Figure 3 b shows that the bacterial cells observed under the microscope appear as free spores;

[0052] (3) Centrifuge the fermentation broth obtained in step (2) at a speed of 5000 r / min for 15 minutes and collect the precipitate as Bacillus subtilis spores.

[0053] S2 involves treating the Bacillus sporosomes obtained in step S1 with lysozyme and protease, followed by ultra-high pressure homogenization to obtain the Bacillus subtilis sporosome lysis product. The specific steps are as follows:

[0054] (A) Add pure water to Bacillus subtilis sporosomes to make the mass fraction of Bacillus subtilis sporosomes 10%, add lysozyme, the amount of lysozyme added is 0.5% of the mass of Bacillus subtilis sporosomes, and treat at 55°C for 12 hours.

[0055] (B) Continue adding neutral protease, the amount of which is 0.5% of the mass of Bacillus subtilis sporosomes. Treat at 50°C for 4 hours to obtain a solution; the microscopic image of the solution obtained after neutral protease hydrolysis in Example 2 is shown below. Figure 3 As shown in c, by Figure 3 c shows that the sporosomes are partially ruptured, and sporolytic material is released.

[0056] (C) The solution obtained in step (B) is processed using an ultra-high pressure homogenizer at a pressure of 1200 bar for 20 minutes to obtain the sporosome lysis product. A microscopic image of the sporosome lysis product obtained after ultra-high pressure homogenization in Example 2 is shown below. Figure 3 As shown in d, by Figure 3 As can be seen from d, almost all the sporosomes were ruptured, and very few intact free sporosomes were found;

[0057] S3 centrifuging the spore lysate obtained in step S2 at a speed of 6000 r / min for 15 minutes, collecting the supernatant, concentrating the supernatant to a dry matter content of 3%, heating to 120℃ for 20 min to inactivate the enzyme activity and residual viable bacteria, thereby obtaining the Bacillus subtilis fermentation product for repair and moisturization.The picture of the Bacillus subtilis fermentation product for repair and moisturization prepared in Example 2 is shown in Figure 4 As can be seen, the Bacillus subtilis fermentation product prepared in the present application is colorless to light yellow, which is suitable for use in cosmetic formulation. Figure 4

[0058] Example 3, a Bacillus fermentation product for repair and moisturization and a preparation method thereof

[0059] The preparation method of the Bacillus fermentation product for repair and moisturization comprises the following steps:

[0060] S1 culturing Bacillus licheniformis in a culture medium to obtain Bacillus spores, the specific steps being:

[0061] (1) inoculating Bacillus licheniformis into a seed culture medium (nutrient broth, Guangdong HuanKai Microbial Technology Co., Ltd.) with an inoculation loop, and aerobically culturing (rotary shaker, rotation speed 180 r / min) at a temperature of 35℃ for 16 h to obtain a seed liquid;

[0062] (2) inoculating the seed liquid obtained in step (1) into a fermentation culture medium containing rich carbon source, nitrogen source and inorganic salt at an inoculation amount of 10%, and aerobically culturing (rotary shaker, rotation speed 220 r / min) at a temperature of 35℃ for 40 h to obtain a fermentation liquid; the fermentation culture medium comprises the following components and mass percentages: corn flour 2%, bran 0.3%, corn syrup 1.5%, anhydrous manganese sulfate 0.02%, sodium chloride 0.5%, potassium dihydrogen phosphate 0.1%, and water 95.58%;

[0063] (3) centrifuging the fermentation liquid obtained in step (2) at a speed of 6000 r / min for 15 minutes to collect the precipitate as Bacillus licheniformis spores.

[0064] S2 treating the Bacillus licheniformis spores obtained in step S1 with lysozyme and protease, and then treating with an ultrahigh pressure homogenizer to obtain a spore lysate, the specific steps being:

[0065] (A) adding pure water to the Bacillus licheniformis spores to obtain a mass fraction of 1%, and adding lysozyme at an amount of 0.1% of the mass of the Bacillus licheniformis spores, and treating at a temperature of 50℃ for 6 h;

[0066] (B) continue to add neutral protease, the amount of which is 0.1% of the mass of Bacillus licheniformis spores, and treat at a temperature of 45°C for 8h to obtain a liquid;

[0067] (C) treat the liquid obtained in step (B) with an ultrahigh pressure homogenizer at a pressure of 1500bar for 10min to obtain a spore lysis product.

[0068] S3 centrifuge the spore lysis product obtained in step S2 at a speed of 4000r / min for 15min, collect the supernatant, concentrate the supernatant to a dry matter content of 3%, heat to 120°C and maintain for 20min to inactivate the enzyme activity and residual viable bacteria, thereby obtaining a Bacillus subtilis fermentation product for repair and moisturization.

[0069] S1 cultivate Bacillus subtilis in a culture medium to obtain Bacillus subtilis spores, and the specific steps are as follows:

[0070] (1) inoculate Bacillus subtilis into a seed culture medium (nutrient broth, Guangdong HuanKai Microbial Technology Co., Ltd.) with an inoculation loop, and cultivate aerobically at a temperature of 37°C (rotary shaker, rotation speed 180r / min) for 20h to obtain a seed liquid;

[0071] (2) inoculate the seed liquid obtained in step (1) into a fermentation culture medium containing rich carbon sources, nitrogen sources and inorganic salts at an inoculation amount of 10%, and cultivate aerobically at a temperature of 37°C (rotary shaker, rotation speed 220r / min) for 48h to obtain a fermentation liquid; the fermentation culture medium comprises the following components and their mass percentages: glucose 1%, yeast extract 0.8%, anhydrous manganese sulfate 0.02%, sodium chloride 0.5%, potassium dihydrogen phosphate 0.1%, and water 97.58%;

[0072] (3) centrifuge the fermentation liquid obtained in step (2) at a speed of 5000r / min for 15min, and collect the precipitate as Bacillus subtilis spores.

[0073] S2 directly treat the Bacillus subtilis spores obtained in step S1 with an ultrahigh pressure homogenizer without lysozyme and protease treatment to obtain a spore lysis product, and the specific steps are as follows:

[0074] (A) add pure water to the Bacillus subtilis spores to make the mass fraction of the Bacillus subtilis spores 10%;

[0075] (B) The solution obtained in step (A) is treated with an ultra-high pressure homogenizer at a pressure of 1200 bar for 20 min to obtain the spore lysate.

[0076] The microscope image of the spore lysate obtained after ultra-high pressure homogenization in Comparative Example 1 is shown in FIG. 1. Figure 5 As can be seen from FIG. 1, a large number of spores still maintain the spore form, and a large number of spores survive after the spore lysate is cultured on nutrient agar, and are not killed by high pressure homogenization. Figure 5

[0077] S3 The spore lysate obtained in step S2 is centrifuged at a speed of 6000 r / min for 15 min, and the supernatant is collected. The supernatant is concentrated to a dry matter content of 3%, and heated to 120°C for 20 min to inactivate the enzyme activity and residual viable bacteria, thereby obtaining the product.

[0078] Comparative Example 2, the preparation method of the Bacillus subtilis fermentation product described in the comparative example comprises the following steps:

[0079] S1 Culturing Bacillus subtilis in a culture medium to obtain Bacillus subtilis spores, and the specific steps are as follows:

[0080] (1) The Bacillus subtilis is inoculated into a seed culture medium (nutrient broth, Guangdong HuanKai Microbial Technology Co., Ltd.) with an inoculation loop, and is aerobically cultured at a temperature of 37°C (rotary shaker, rotation speed 180 r / min) for 20 h to obtain a seed solution;

[0081] (2) The seed solution obtained in step (1) is inoculated into a fermentation medium containing rich carbon source, nitrogen source and inorganic salt at an inoculation amount of 10%, and is aerobically cultured at a temperature of 37°C (rotary shaker, rotation speed 220 r / min) for 48 h to obtain a fermentation solution; the fermentation medium comprises the following components and mass percentages: glucose 1%, yeast extract 0.8%, anhydrous manganese sulfate 0.02%, sodium chloride 0.5%, potassium dihydrogen phosphate 0.1%, and water 97.58%.

[0082] (3) The fermentation solution obtained in step (2) is centrifuged at a speed of 5000 r / min for 15 min, and the precipitate is collected as Bacillus subtilis spores.

[0083] S2 The Bacillus subtilis spores obtained in step S1 are only treated with lysozyme without protease treatment, and then treated with an ultra-high pressure homogenizer to obtain a spore lysate, and the specific steps are as follows:

[0084] ​(A) Add pure water to the Bacillus subtilis spores to make the mass fraction of the Bacillus subtilis spores 10%, and then add lysozyme, the added amount of the lysozyme being 0.5% of the mass of the Bacillus subtilis spores, and then treat at a temperature of 55°C for 12 hours;

[0085] (B) Treat the solution obtained in step (A) with an ultra-high pressure homogenizer, the homogenization pressure being 1200 bar and the homogenization time being 20 minutes, to obtain the Bacillus subtilis spore lysis product.

[0086] The microscope examination picture of the Bacillus subtilis spore lysis product treated with lysozyme and an ultra-high pressure homogenizer in Comparative Example 2 is shown in Figure 6 It can be found that a large number of spores still maintain the intact spore form, and a large number of spores survive when the solution is cultured on a nutrient agar medium and are not killed by the lysozyme and the ultra-high pressure homogenization treatment. Figure 6

[0087] S3 Centrifuge the Bacillus subtilis spore lysis product obtained in step S2 at a centrifugal speed of 6000 r / min for 15 minutes, collect the supernatant, concentrate the supernatant to a dry matter content of 3%, heat to 120°C, and keep the temperature for 20 minutes to kill the enzyme activity and residual viable bacteria, to obtain the Bacillus subtilis spore lysis product.

[0088] Test Example 1, Bacteriostatic Experiment

[0089] ① Preparation of inoculum: elute Staphylococcus aureus from a slope with 5ml TSB, collect the eluate in three 10ml centrifuge tubes, and adjust the concentration of the bacterial solution to 10 5 ~ 10 6 cfu / ml by turbidimetry, and count the viable bacteria by the plate method.

[0090] ② Plate coating: pour 18-20ml of agar medium-based culture medium into a disposable culture dish, and the pouring thickness is about 5-6mm. After the plate is solidified, aseptically take 100μl of the target bacterial solution on the surface of the culture medium, and quickly spread the bacterial solution evenly with a sterile coating rod.

[0091] ③ Oxford cup method for bacteriostatic operation: place a sterilized Oxford cup (a circular stainless steel tube with an inner diameter of 6nm, an outer diameter of 8nm, and a height of 10nm) on the coated culture medium, and press it gently to make it contact with the culture medium without gaps. Add about 200μl of the sample to be tested to the Oxford cup, and do not let it overflow. Each sample to be tested is done in triplicate. The bacteria are cultured at 37°C±1°C for 48-72h, and the results are observed.

[0092] ④ Result processing: observe the transparent ring bacteriostatic circle with the naked eye and measure the diameter of the bacteriostatic circle with a vernier caliper. After recording the data of the three parallel groups, take the average value. The bacteriostatic sensitivity determination standard is shown in Table 1, and the bacteriostatic experiment results are shown in Table 2. ​

[0093] Table 1 Bacteriostatic sensitivity determination criteria

[0094] Diameter of the inhibition zone (mm) Inhibition sensitivity 20 or more Very sensitive 15～20 Highly sensitive 10～14 Moderately sensitive 10 or less Lowly sensitive 0 Not sensitive

[0095] Table 2 Bacteriostatic experiment results

[0096] Sample to be tested Diameter of the inhibition zone (mm) Inhibition sensitivity Example 1 10.55 Moderately sensitive Example 2 12.13 Moderately sensitive Example 3 10.04 Moderately sensitive

[0097] As can be seen from Table 2, the Bacillus fermentation product for repairing and moisturizing prepared in Examples 1-3 of the present application can inhibit Staphylococcus aureus, and has the effect of balancing the skin microflora.

[0098] Test Example Two, efficacy experiment of repairing skin barrier

[0099] ① Test preparation before testing: selection of subjects: 12 healthy men and women, half each, and volunteers whose forearms had no scars, pigments, atrophy, fresh red macules or other blemishes affecting the determination of test results, aged between 18-50 years.

[0100] ② Other experimental instruments and materials: low-sensitizing external adhesive tape (3M Company, USA), transdermal water loss instrument VapoMeter (model SWL5648).

[0101] ③ Experimental method: the inner sides of the left and right forearms of the subjects were randomly marked with 3 areas of 3 cm x 3 cm in size as test areas, and were marked as Examples 1-3 groups, Comparative Examples 1-2 groups and a blank control group, respectively. The transdermal water loss value (TEWL value) of the skin was measured as the basis value before modeling, denoted as T0. The transparent adhesive tape was used to perform the tearing and pulling treatment in the test areas of the example groups, the comparative example groups and the blank control group, respectively. The transdermal water loss value (TEWL value) of the skin was measured again as the basis value after modeling, denoted as T1. The corresponding test examples, comparative example samples were used in the test areas, and no samples were used in the blank control group. After sitting in a constant temperature and humidity environment for 1 h, the transdermal water loss value (TEWL value) of the skin was measured again as the repair value after modeling, denoted as T2. The result was calculated: skin repair rate % = (T1-T2) / (T1-T0)*100%.

[0102] ④ Experimental results: the experimental results are shown in Table 3.

[0103] Table 3 Efficacy experiment results of repairing skin barrier

[0104]

[0105] As can be seen from Table 3, the skin repair rate of the subjects using the Bacillus fermentation product for repair and moisturizing prepared by Examples 1-3 of the present application is much higher than that of the blank control group, and is better than that of Comparative Examples 1-2, indicating that the Bacillus fermentation product for repair and moisturizing prepared by the present application has the effect of repairing damaged skin barrier and reducing epidermal water loss.

[0106] Test Example Three, Moisturizing Effect Experiment

[0107] Ten healthy men and women, five men and five women, were selected and randomly divided into five groups, one man and one woman in each group. Before the test, the subjects were required to enter a climate-controlled room (22±1℃, relative humidity 50%) for more than 20 min after washing for 2 h, and to keep a relaxed state, and the volunteers had no scars, pigments, atrophy, fresh red macules or other blemishes on the forearms that could affect the test results. The left and right medial forearms of the subjects were randomly marked with three areas of 3 cm x 3 cm in size as test areas, and the amount of Bacillus fermentation product prepared by Examples 1-3 and Comparative Examples 1-2 of the present application applied to each test area of each subject was (2.0±0.1) mg / cm 2 The skin moisture content was tested at time intervals of 1 h, 2 h, 4 h, and 6 h, using the skin moisture content Corneometer test probe of the multifunctional skin tester of Germany CK Company, and the average value of the change in skin moisture content was calculated.

[0108] The experimental results are shown in Table 4.

[0109] Table 4 Average Value Change Table of Skin Moisture Content

[0110] Group 1h(％) 2h(％) 4h(％) 6h(％) Example 1 group 15.37 12.63 8.28 5.90 Example 2 group 15.85 12.29 8.11 6.07 Example 3 group 15.25 12.21 7.85 5.82 Comparative Example 1 group 14.59 11.18 6.41 4.75 Comparative Example 2 group 14.35 11.29 6.88 4.93

[0111] As can be seen from Table 4, the average value of the skin moisture content of the subjects using the Bacillus fermentation product for repair and moisturizing prepared by Examples 1-3 of the present application is significantly higher than that of Comparative Examples 1-2, indicating that the Bacillus fermentation product for repair and moisturizing prepared by the present application has a stronger moisturizing effect.

[0112] The above detailed description further illustrates the purpose, technical solutions and beneficial effects of the present application, and it should be understood that the above is only a preferred embodiment of the present application and does not limit the protection scope of the present application in any way. Any person skilled in the art can make any form of equivalent replacement, modification or change to the technical solutions and technical content of the present application without departing from the scope of the technical solutions of the present application, and such changes still fall within the protection scope of the present application.

Claims

1. A method for preparing a Bacillus fermentation product for repair and moisturization, characterized by, The method comprises the following steps: S1: culturing Bacillus in a culture medium to obtain Bacillus spores; S2: treating the Bacillus spores obtained in step S1 with lysozyme and protease, and then treating the spores with an ultrahigh pressure homogenizer to obtain a spore lysate; S3: centrifuging, concentrating and heating the spore lysate obtained in step S2 to obtain a Bacillus fermentation product for repair and moisturizing; The specific steps of step S2 are as follows: (A) adding pure water to the Bacillus spores to obtain a mass fraction of 1% to 10%, adding lysozyme, and treating at a temperature of 50 to 55°C for 6 to 12 hours; (B) continuing to add protease, treating at a temperature of 45 to 50°C for 4 to 8 hours to obtain a liquid; (C) treating the liquid obtained in step (B) with an ultrahigh pressure homogenizer at a pressure of 800 to 1500 bar for 10 to 30 minutes to obtain the spore lysate; The addition amount of the lysozyme in step (A) is 0.1 to 0.5% of the mass of the Bacillus spores; The addition amount of the protease in step (B) is 0.1 to 0.5% of the mass of the Bacillus spores.

2. The method for preparing a Bacillus fermentation product for repairing and moisturizing according to claim 1, wherein, The Bacillus in step S1 includes Bacillus subtilis, Bacillus licheniformis and Bacillus megaterium.

3. The method for preparing a Bacillus fermentation product for repairing and moisturizing according to claim 1, wherein the Bacillus fermentation product is prepared by the method comprising the steps of: fermenting a culture medium containing a carbon source, a nitrogen source, and a metal ion source by a Bacillus strain; and recovering a fermentation product from the culture medium. The specific steps of step S1 are as follows: (1) inoculating Bacillus into a seed culture medium and culturing aerobically at a temperature of 30 to 37°C for 10 to 20 hours to obtain a seed liquid; (2) transferring the seed liquid obtained in step (1) into a fermentation culture medium containing rich carbon sources, nitrogen sources and inorganic salts, and culturing aerobically at a temperature of 30 to 37°C for 36 to 72 hours to obtain a fermentation liquid; (3) centrifuging the fermentation liquid obtained in step (2) at a speed of 4000 to 6000 r / min for 12 to 20 minutes, and collecting the precipitate as Bacillus spores.

4. The method for preparing Bacillus fermentation products for repair and moisturizing as described in claim 3, characterized in that, The carbon sources in step (2) include glucose, corn starch, corn flour and rice flour; the nitrogen sources include yeast extract, peptone, corn steep liquor and bran; and the inorganic salts include potassium dihydrogen phosphate, manganese sulfate and sodium chloride.

5. The method for preparing the Bacillus fermentation product for repairing and moisturizing according to claim 1, wherein the Bacillus fermentation product is prepared by the steps of: fermenting a culture medium containing a Bacillus strain in a culture vessel; and recovering the Bacillus fermentation product from the culture medium. The protease in step S2 is neutral protease.

6. The Bacillus fermentation product for repair and moisturizing prepared by the method of any one of claims 1 to 5.

7. Use of the Bacillus fermentation product for repair and moisturizing of claim 6 in the preparation of a cosmetic product for repair and moisturizing.

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