Black mulberry probiotic fermentation postbiotic with hair growth effect

Abstract

The invention discloses black mulberry probiotic fermentation post-biogen with a hair growth function, and belongs to the technical field of microorganisms. The black mulberry probiotic fermented prebiotics are obtained after black mulberries are fermented by probiotics, and comprise metabolites obtained after the black mulberries are fermented by the probiotics, inactivated probiotic thalli and / or thallus lysates of the probiotics; the probiotics comprise lactobacillus paracasei, lactobacillus reuteri, lactobacillus plantarum and bifidobacterium breve. Cell experiments and animal experiments prove that the black mulberry probiotics have a hair growth function after fermentation, and the black mulberry probiotics have a huge application prospect in preparation of anti-hair loss and hair care products.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a black mulberry probiotic fermented postbiotic with hair growth effect. Background Art

[0002] Hair plays a variety of roles in the human body, including protecting and maintaining the temperature of the head. Hair loss is defined as a decrease in hair density and is a common and frequently occurring disease in dermatology. The causes of hair loss are complex and varied, and can be roughly divided into the following categories: mental factors such as excessive mental stress and nervous tension; imbalance in sex hormone secretion or genetic factors; side effects of drugs such as immunosuppressants and chemotherapy drugs; nutritional metabolic factors such as partial eclipse and indigestion; physical or chemical factors such as environmental pollution and radiation damage; personal life factors such as alcoholism, staying up late, and not paying attention to hygiene. Although hair loss is not life-threatening, it can affect an individual's aesthetics, social activities, and quality of life.

[0003] To date, non-surgical treatment options for hair loss approved by the U.S. Food and Drug Administration (FDA) include drugs such as minoxidil and finasteride. Minoxidil is converted to minoxidil sulfate by sulfotransferases present in the scalp, thereby promoting the growth of hair follicle cells and reducing hair loss. Finasteride is a 5α-reductase inhibitor that blocks the conversion of testosterone to dihydrotestosterone (DHT), which causes androgenic alopecia. However, these drugs have side effects on patients. Therefore, there is a need to identify new, safe, and effective substances (derived from natural materials) to treat hair loss. Summary of the invention

[0004] The purpose of the present invention is to provide a black mulberry probiotic fermented postbiotic with a hair growth effect to solve the problems existing in the above-mentioned prior art. The present invention uses Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus plantarum and Bifidobacterium breve as a probiotic combination, and prepares plant-based postbiotics by fermenting black mulberries. Cell experiments and animal experiments confirm the hair growth effect of postbiotics, and it has great application prospects in the preparation of anti-hair loss and hair care products.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a black mulberry probiotic fermentation postbiotic with hair growth function, which is obtained by fermenting black mulberries with probiotics; the probiotics include Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus plantarum and Bifidobacterium breve.

[0007] Furthermore, it includes metabolites after probiotics ferment black mulberries, inactivated probiotic cells and / or probiotic cell lysates.

[0008] The present invention also provides a method for preparing the black mulberry fermentation postbiotics, comprising:

[0009] After black mulberries are added with water to make pulp, the pulp is filtered and sterilized, and the pulp is used as a black mulberry juice culture medium;

[0010] The Lactobacillus paracasei, the Lactobacillus reuteri, the Lactobacillus plantarum and the Bifidobacterium breve are activated and inoculated into the black mulberry juice culture medium, and a bacterial liquid is obtained after fermentation and culture.

[0011] Furthermore, during pulping, the solid-liquid ratio of the black mulberry to the water is 1 g:5 mL.

[0012] Furthermore, the filtration is performed twice through a 180-mesh sieve.

[0013] Furthermore, the fermentation culture condition is 37° C. for 48 hours.

[0014] Furthermore, after the fermentation culture was completed, the concentration of the bacterial solution was 1.8×10 9 CFU / mL.

[0015] The present invention also provides the use of the black mulberry probiotic fermentation postbiotics in the preparation of products with hair growth effects.

[0016] Furthermore, the product includes health care products, medicines or cosmetics.

[0017] Furthermore, the use of the product includes topical application or oral administration.

[0018] The present invention discloses the following technical effects:

[0019] The present invention uses Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus plantarum and Bifidobacterium breve as probiotic combinations, and prepares plant-based postbiotics by fermenting black mulberries. Black mulberries contain fatty acids such as linoleic acid, stearic acid and oleic acid. As a traditional Chinese medicine, they are oriented to the heart, liver and kidney meridians, and are a tonic medicine. Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus plantarum and Bifidobacterium breve promote the decomposition of active substances in black mulberries by fermenting black mulberries, and the metabolites produced by them have anti-inflammatory and antioxidant effects, and act together with the active ingredients in black mulberries to further improve hair follicle development and promote hair growth. At the same time, black mulberries are fermented using a probiotic combination of Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus plantarum and Bifidobacterium breve, which promotes the effect of tonifying the kidney and strengthening the liver, and can alleviate the hair loss caused by liver and kidney deficiency from the perspective of relieving liver qi and nourishing kidney essence.

[0020] The invention confirms the hair growth effect of black mulberry probiotic fermentation postbiotics through cell experiments and animal experiments, and has great application prospects in the preparation of anti-hair loss and hair care products. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] The Lactobacillus paracasei used in the embodiment of the present invention is strain numbered CICC6112; Lactobacillus reuteri is strain numbered CICC 6118; Lactobacillus acidophilus is strain numbered CICC 6082; Bifidobacterium breve is strain numbered CICC 6184, all of which are derived from the China Industrial Microbiological Culture Collection Administration Center; Lactobacillus plantarum is strain numbered CGMCC 1.9087, which is derived from the China General Microbiological Culture Collection Administration Center. The above strains can be purchased by the public from the above-mentioned collection institutions.

[0027] Example 1

[0028] 1. Preparation of black mulberry juice

[0029] Weigh 100g of black mulberry fruit, wash it repeatedly 2-3 times, drain it, add 500mL of deionized water at a liquid-to-solid ratio of 5:1 (mL / g), soak it overnight and then pulp it. The pulp is filtered twice through a 180-mesh sieve. After sterilization (121°C, 15min), the filtrate is cooled to room temperature and stored at 4°C for use as a black mulberry juice culture medium.

[0030] 2. Preparation of postbiotics

[0031] Postbiotics were prepared by fermenting black mulberry juice with Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus plantarum and Bifidobacterium breve.

[0032] All strains were stored in a -80℃ refrigerator with 30% glycerol before activation. A small amount of bacterial liquid was streaked on MRS solid medium with a sterile inoculation loop, cultured at 37℃ for 48h, and a single colony was picked and inoculated into MRS liquid medium, cultured at 37℃ for 18h, and then inoculated into 200mL black mulberry juice medium with a 3% inoculation amount, and finally the mixed bacterial inoculation amount was 9%. Continue to culture at 37℃ for 48h to obtain 1.8×10 9 CFU / mL of bacterial solution.

[0033] After the bacterial solution was centrifuged at 10000r / min for 10min, the supernatant was taken, which was the postbiotic.

[0034] Example 2

[0035] 1. Preparation of black mulberry juice

[0036] Weigh 100g of black mulberry fruit, wash it repeatedly 2-3 times, drain it, add 500mL of deionized water at a liquid-to-solid ratio of 5:1 (mL / g), soak it overnight and then pulp it. The pulp is filtered twice through a 180-mesh sieve. After sterilization (121°C, 15min), the filtrate is cooled to room temperature and stored at 4°C for use as a black mulberry juice culture medium.

[0037] 2. Preparation of postbiotics

[0038] Postbiotics were prepared by fermenting black mulberry juice with Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus plantarum and Bifidobacterium breve.

[0039] All strains were stored in a -80℃ refrigerator with 30% glycerol before activation. A small amount of bacterial liquid was streaked on MRS solid medium with a sterile inoculation loop, cultured at 37℃ for 48h, and a single colony was picked and inoculated into MRS liquid medium, cultured at 37℃ for 18h, and then inoculated into 200mL black mulberry juice medium with a 3% inoculation amount, and finally the mixed bacterial inoculation amount was 9%. Continue to culture at 37℃ for 48h to obtain 1.8×10 9 CFU / mL of bacterial solution.

[0040] The bacterial liquid was centrifuged at 10000 r / min for 10 min to obtain bacterial sludge, which was then homogenized under high pressure (800-1200 MPa, 3 times) to obtain bacterial lysate, which is the postbiotic.

[0041] Example 3

[0042] 1. Preparation of black mulberry juice

[0043] Weigh 100g of black mulberry fruit, wash it repeatedly 2-3 times, drain it, add 500mL of deionized water at a liquid-to-solid ratio of 5:1 (mL / g), soak it overnight and then pulp it. The pulp is filtered twice through a 180-mesh sieve. After sterilization (121°C, 15min), the filtrate is cooled to room temperature and stored at 4°C for use as a black mulberry juice culture medium.

[0044] 2. Preparation of postbiotics

[0045] Postbiotics were prepared by fermenting black mulberry juice with Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus plantarum and Bifidobacterium breve.

[0046] All strains were stored in -80℃ refrigerator with 30% glycerol before activation. A small amount of bacterial liquid was streaked on MRS solid medium with a sterile inoculation loop, cultured at 37℃ for 48h, and a single colony was picked and inoculated into MRS liquid medium, cultured at 37℃ for 18h, and then inoculated into 200mL black mulberry juice medium with 3% inoculation, and finally the mixed bacterial inoculation amount was 9%, and cultured at 37℃ for 48h to obtain 1.8×10 9CFU / mL of bacterial solution.

[0047] Heat the above bacterial solution at 95-105°C for 15-20 minutes to inactivate it. After cooling, take 200 μL and apply it to MRS solid culture medium, and culture it in a constant temperature incubator at 37°C for 48 hours. After observing the plate, no colonies are formed, which confirms that the bacteria have been completely inactivated. The inactivated bacterial solution is the postbiotic.

[0048] Test Example 1

[0049] The activity of human hair follicle dermal papilla cells (HDPC) and their interaction with hair follicle epithelial cells are key factors in initiating and maintaining hair growth. The migration ability of HDPC helps them to reposition within the hair follicles, and this dynamic distribution is essential for signal transduction to hair follicle epithelial cells and regulating hair growth. When the function of HDPC is affected, such as weakened migration ability, it may interfere with normal hair growth signals, thereby affecting the growth cycle and quality of hair. Therefore, the effect of the postbiotics prepared by Examples 1 to 3 on the migration of HDPC cells can reflect its regulation of hair growth.

[0050] 1. Cell recovery and culture

[0051] First, the frozen human hair follicle dermal papilla cells (HDPC) were taken out and quickly thawed in a 37°C water bath. Then, they were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed with 10% fetal bovine serum, 1×10 5 The cells were cultured in a DMEM high-glucose medium containing 100 mg / L penicillin and 100 mg / L streptomycin at 37°C in a cell culture incubator with 5% carbon dioxide. When the cells adhered to the wall and the confluence exceeded 80%, they were digested and passaged with trypsin containing 0.25% EDTA, and cells in the logarithmic growth phase were used for experiments.

[0052] 2. Cell migration assay

[0053] Digest and count HDPC cells in logarithmic growth phase, and adjust the cell suspension to 1.5×10 6 100 μL / mL, 2 mL per well was inoculated into a 6-well culture plate, and the cells were cultured in a 5% carbon dioxide, 37°C incubator until they were 90% confluent. The cells were drawn into a straight line with a 200 μL pipette tip, and then rinsed with PBS three times. The blank control group and treatment group were set up as follows.

[0054] Cell culture medium: 89% (v / v) DMEM medium, 10% fetal bovine serum, 1×10 5 U / L penicillin, 100mg / L streptomycin.

[0055] Blank control group: containing both cell culture medium and HDPC cells;

[0056] Treatment groups 1 to 3: The postbiotics prepared in Examples 1 to 3 were resuspended in cell culture medium (the resuspended solution contained 10 mg / L postbiotics), and the rest were the same as the blank control group.

[0057] According to the above settings of the blank control group and the treatment group, the fresh medium was replaced. After incubation for 15 hours, the width of the scratch was photographed using an optical microscope. The cell migration rate was analyzed as shown in Table 1.

[0058] Table 1 Cell migration rate in each group

[0059]

[0060] The results in Table 1 show that the cell migration rate of the blank control group was 28.64%, and the average cell migration rate of treatment groups 1 to 3 was 58.95%. The treatment group increased the migration rate of HDPC cells by 1.06 times, significantly promoting the migration ability of HDPCs cells, indicating that the postbiotics prepared in Examples 1 to 3 can improve hair follicle development and thus affect hair production.

[0061] Test Example 2

[0062] Since C57BL / 6J mice have melanocytes only in hair follicles, melanin synthesis is consistent with the hair growth cycle. Therefore, the hair growth cycle can be characterized by monitoring the transition of skin color from pink (no hair) to black (fully grown hair) and analyzing the effects of postbiotics on mouse hair growth.

[0063] Twenty-four male C57BL / 6J mice, 6 weeks old (about 20 g), were purchased and formally tested after one week of adaptation. First, the backs of all mice were shaved using an animal depilatory, and then a depilatory cream was applied to remove the hair in an area of ​​about 3 cm × 3 cm, so that the hair growth of C57BL / 6J mice was in the resting phase (the skin was pink), thus establishing the C57BL / 6J mouse hair loss model.

[0064] After the skin was stable for 24 hours, the telogen mice were randomly divided into 3 groups, 8 mice in each group, for a total of 24 mice. The groups were set as follows:

[0065] Blank group: physiological saline was used as control;

[0066] Yangshen group: 10mg / kg finasteride was used as a control;

[0067] Treatment groups 1-3: mice were gavaged with the postbiotics prepared in Examples 1-3 at a dose of 500 mg / kg mouse body weight; each mouse was gavaged with 0.2 mL / d for 3 weeks.

[0068] The back skin of mice was recorded at 7d, 14d and 21d, and the results are shown in Table 2.

[0069] Table 2 Changes in the back skin of mice in each group

[0070]

[0071] As shown in Table 2, at 7 days of administration, the back skin of the blank group mice was still pink, while the back skin of the treated group mice began to turn gray; at 14 days of administration, the back skin of the blank group mice began to turn gray, while the black area of ​​the back skin of the treated group mice increased significantly and hair shafts appeared; at 21 days of administration, compared with the blank group mice, the back skin of the treated group mice was almost completely covered with hair. This shows that oral postbiotics can significantly promote hair growth in mice.

[0072] Test Example 3

[0073] It can be seen from Experimental Example 2 that oral administration of the black mulberry probiotic fermented postbiotics prepared in Examples 1 to 3 has the effect of promoting hair growth in mice. The probiotic combination of fermented black mulberries was adjusted according to Table 3, and after the adjustment, the postbiotics were prepared in the manner of Example 3, and the dosage method was changed to verify the effect of black mulberry probiotic fermented postbiotics on hair growth again.

[0074] Table 3 Probiotic combinations of fermented black mulberries

[0075]

[0076] The test method is as follows:

[0077] 1. Hair removal for guinea pigs

[0078] 18 guinea pigs were randomly divided into 6 groups, each with 3 animals, corresponding to the postbiotics prepared in Example 3 and groups A to D, and 1 group was set as a blank control group. The animals in each group were depilated in advance at the same part of the back, with a depilatory area of ​​3 cm×3 cm, and the depilatory method was the same as in Experimental Example 2.

[0079] 2. Hair regrowth

[0080] The postbiotics prepared in Example 3 and groups A to D were applied to the depilated areas of the back skin of guinea pigs in the corresponding groups, twice a day, 1 mL each time, for 24 consecutive days.

[0081] On the 25th day, 10 new hairs were randomly taken from the hair removal area of ​​each mouse, and their lengths were measured under a microscope with a micrometer to calculate the average value and compare the hair growth differences among the groups. The test results are shown in Table 4.

[0082] Table 4 Results of hair regeneration on the back of guinea pigs in each group

[0083] Group New hair length (mm) A 7.6±1.26 B 7.4±1.74 C 8.6±1.58 D 8.1±1.35 Example 3 10.8±1.52 Blank control 7.1±1.52

[0084] As shown in Table 4, the best probiotic combination for fermenting black mulberries is Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus plantarum and Bifidobacterium breve. The black mulberry probiotic fermentation postbiotics prepared in the embodiment of the present invention can also promote hair growth by external application.

[0085] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A black mulberry probiotic fermentation postbiotic with hair growth effect, characterized in that: The invention is obtained by fermenting black mulberries with probiotics; the probiotics include Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus plantarum and Bifidobacterium breve.

2. The black mulberry fermentation postbiotic according to claim 1, characterized in that: It includes metabolites after probiotics ferment black mulberries, inactivated probiotic bacteria and / or probiotic bacteria lysates.

3. A method for preparing black mulberry fermentation postbiotics according to claim 1 or 2, characterized in that: include: After black mulberries are added with water to make pulp, the pulp is filtered and sterilized, and the pulp is used as a black mulberry juice culture medium; The Lactobacillus paracasei, the Lactobacillus reuteri, the Lactobacillus plantarum and the Bifidobacterium breve are activated and inoculated into the black mulberry juice culture medium, and a bacterial liquid is obtained after fermentation and culture.

4. The preparation method according to claim 3, characterized in that: During pulping, the solid-liquid ratio of the black mulberry to the water is 1 g:5 mL.

5. The preparation method according to claim 3, characterized in that: The filtration is performed twice through a 180-mesh sieve.

6. The preparation method according to claim 3, characterized in that: The fermentation culture condition is 37° C. for 48 h.

7. The preparation method according to claim 3, characterized in that: After the fermentation culture was completed, the concentration of the bacterial solution was 1.8×10 9 CFU / mL.

8. Use of the black mulberry probiotic fermentation postbiotics as claimed in claim 1 or 2 in the preparation of products having a hair growth effect.

9. The use according to claim 8, characterized in that: The products include health products, medicines or cosmetics.

10. The use according to claim 8, characterized in that: The product may be used topically or orally.

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