Probiotics that produce hyaluronic acid and help skin cells produce hyaluronic acid and prevent photoaging and use thereof

Abstract

The present invention belongs to the technical field of probiotic health care, and specifically provides a probiotic that produces hyaluronic acid and helps skin cells produce hyaluronic acid and prevent photoaging, and its application. The probiotic provided by the present invention is Bifidobacterium lactis nbk-BA83, and the deposit number is CGMCCNO.19255. The probiotic provided by the present invention can be fermented to produce hyaluronic acid, and its inactivated bacteria can promote the production of hyaluronic acid by skin keratinocytes; at the same time, its inactivated bacteria can inhibit skin aging by improving the antioxidant property of photoaging cells, prevent oxidative damage of photoaging skin keratinocytes induced by ultraviolet rays, and then improve skin condition, inhibit skin aging, improve skin water retention and moisture retention, and enhance skin barrier function.

Description

Technical Field

[0001] The invention relates to the technical field of probiotic health care, and in particular to a probiotic that helps skin produce hyaluronic acid and prevent photoaging, and an application thereof. Background Art

[0002] The skin is the largest organ in the human body and the first barrier separating the internal and external environments of the human body, protecting various organs and tissues in the body from mechanical, chemical, pathogenic microorganisms, etc. The epidermis of the skin is directly exposed to the external environment and is mainly composed of keratinocytes.

[0003] The structure of hyaluronic acid is a straight-chain high-molecular polysaccharide composed of glucuronic acid and N-acetylglucosamine. It has strong hygroscopicity and is soluble in water. It is a polymer with water-retaining function that exists naturally in human skin. Hyaluronic acid in the skin is mainly produced by skin keratinocytes and fibroblasts. Hyaluronic acid can form a viscoelastic water film in the epidermis to maintain skin moisture, strengthen the skin barrier, and prevent skin dryness. Its strong water absorption is conducive to regulating epidermal moisture, protecting cells from pathogens, preventing pathogenic bacteria from infecting, and maintaining the toughness and elasticity of the skin. When the level of hyaluronic acid in the skin decreases, the water-retaining function of the skin will decrease and wrinkles will occur. At present, hyaluronic acid is mostly added to skin care products or cosmetics to improve the water retention and moisturizing properties of the skin. However, high-content hyaluronic acid, as a high-nutrient substance, is prone to contamination by pathogens and affects skin health. On the other hand, when the molecular weight exceeds 400kDa, it cannot penetrate the skin barrier, and thus cannot play the role of improving skin health in the dermis.

[0004] Long-term exposure to ultraviolet rays will cause skin cells to produce a large number of reactive oxygen free radicals. Excessive reactive oxygen free radicals participate in the cellular oxidative stress process, which in turn breaks the balance of the antioxidant defense system, causing cell damage and aging, and leading to photoaging of the skin. Malondialdehyde is one of the end products of membrane lipid peroxidation, which can damage the biological membrane structure, lead to body aging, and reflect the degree of damage to cells and the body by free radicals. The body's antioxidant system is very important for maintaining cellular redox homeostasis. The human antioxidant system is composed of a variety of free enzymes, among which superoxide dismutase is the primary substance for scavenging free radicals, which can resist and block the damage caused by oxygen free radicals to cells, repair damaged cells, and delay aging. Collagen is a key factor in determining skin elasticity, and its degradation can cause skin damage. Hydroxyproline is a characteristic amino acid in collagen, reflecting the decomposition and metabolism of collagen. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Bifidobacterium lactis, which can be fermented to produce hyaluronic acid. The inactivated bacteria can promote the production of hyaluronic acid by skin keratinocytes, and can inhibit skin aging by improving the antioxidant activity of photoaging cells, thereby improving the water retention and moisture retention of the skin and enhancing the skin barrier function.

[0006] In a first aspect, the present invention claims protection for a probiotic, wherein the probiotic is Bifidobacterium lactis nbk-BA83, and the preservation number is CGMCC NO.19255.

[0007] More specifically, the present invention isolates a probiotic from the intestine of a healthy infant, which is identified as Bifidobacterium animalis lactis subspecies based on cell morphology, physiological and biochemical characteristics and 16S rRNA gene sequence determination results, and is named Bifidobacterium lactis nbk-BA83.

[0008] The Bifidobacterium lactis nbk-BA83 (classification name: Bifidobacterium lactis) provided by the present invention has been deposited in the China General Microbiological Culture Collection Center on December 30, 2019, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101; The deposit number is CGMCCNO.19255.

[0009] In a second aspect, the present invention claims protection for a probiotic powder, wherein the probiotic powder comprises live or inactivated bacteria of Bifidobacterium lactis nbk-BA83.

[0010] More specifically, the method for preparing inactivated bacteria of Bifidobacterium lactis nbk-BA83 comprises the following steps:

[0011] (1) Bifidobacterium lactis nbk-BA83 was inoculated into a seed culture medium for activation to obtain a primary seed solution.

[0012] (2) inoculating the primary seed solution obtained in step (1) into a seed culture medium and continuing to culture the culture medium to obtain a secondary seed solution.

[0013] (3) inoculating the secondary seed liquid obtained in step (2) into a fermentation medium for fermentation to obtain a fermentation liquid.

[0014] (4) After centrifuging and concentrating the fermentation liquid obtained in step (3), pure water is added, mixed evenly, and then inactivated under certain temperature and time conditions to obtain inactivated Bifidobacterium lactis nbk-BA83.

[0015] Preferably, the seed culture medium comprises, by mass fraction, 1-3% glucose, 1-3% peptone, 0.5-3% yeast extract, 0.2-1% anhydrous sodium acetate, 0.2-1% ammonium citrate, 0.02-0.08% magnesium sulfate, 0.02-0.08% manganese sulfate, 0.05-0.1% L-cysteine ​​hydrochloride, 0.05-0.1% Tween 80 and the remainder water, with a pH value of 6.2-6.8.

[0016] Preferably, the fermentation medium comprises, by mass fraction, 1-3% glucose, 1-3% peptone, 0.5-2% yeast powder, 0.2-1% beef powder, 0.2-1% anhydrous sodium acetate, 0.2-1% ammonium citrate, 0.02-0.08% magnesium sulfate, 0.02-0.08% manganese sulfate, 0.05%-0.1% L-cysteine ​​hydrochloride, 0.05-0.1% Tween 80 and the remainder water, with a pH value of 6.2-6.8.

[0017] After centrifugation and concentration, the fermentation broth of the present invention is inactivated at a temperature of 105-115° C. for 10-30 minutes.

[0018] According to the understanding of those skilled in the art, the present invention seeks protection for the use of the above-mentioned probiotics or the above-mentioned probiotic powder in the production of hyaluronic acid.

[0019] And, use of the above-mentioned probiotics or the above-mentioned probiotic powder in promoting the production of hyaluronic acid by skin keratinocytes.

[0020] And, use of the above-mentioned probiotics or the above-mentioned probiotic powder in reducing oxidative damage to skin keratinocytes.

[0021] The use of the invention in reducing oxidative damage to skin keratinocytes is performed by photoaging induced by ultraviolet rays.

[0022] The present invention also seeks to protect the use of the above-mentioned probiotics or the above-mentioned probiotic powder in increasing the expression level of hydroxyproline in photoaged cells.

[0023] And the use of the probiotics or the probiotic powder in preparing skin care products or cosmetics for preventing skin dryness and aging.

[0024] In a third aspect, the present invention seeks to protect a skin care product or cosmetic for preventing skin dryness and aging, wherein the skin care product or cosmetic for preventing skin dryness and aging contains the above-mentioned probiotics or the above-mentioned probiotic powder.

[0025] The beneficial effects of the present invention are:

[0026] The present invention provides a probiotic with beauty and anti-aging effects, Bifidobacterium lactis nbk-BA83, with a preservation number of CGMCC NO. 19255. The Bifidobacterium lactis nbk-BA83 provided by the present invention can be fermented to produce hyaluronic acid, and the inactivated Bifidobacterium lactis nbk-BA83 obtained by the preparation method of the present invention can promote skin keratinocytes to produce hyaluronic acid, prevent ultraviolet-induced photoaging cell oxidative damage, increase cell hydroxyproline levels, and thus play a beauty and anti-aging effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 This is the effect of Bifidobacterium lactis nbk-BA83 strain producing hyaluronic acid in Example 2 of the present invention. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1 Screening and identification of Bifidobacterium lactis nbk-BA83

[0031] This example provides the screening and identification of Bifidobacterium lactis nbk-BA83, and the steps are as follows:

[0032] The intestines of healthy infants were collected and diluted with sterile saline to an appropriate gradient, and then streaked on modified MRS agar plates for isolation. The plates were cultured anaerobically at 37°C for 72 hours, and single colonies were picked for microscopic examination and further streaked for purification. The bacteria were identified as animal Bifidobacterium lactis subspecies based on cell morphology, physiological and biochemical characteristics, and 16S rRNA gene sequence determination, and named Bifidobacterium lactis nbk-BA83.

[0033] The Bifidobacterium lactis nbk-BA83 provided by the present invention has been deposited in the China General Microbiological Culture Collection Center on December 30, 2019, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal Code: 100101; The deposit number is CGMCC NO.19255.

[0034] Example 2 Study on the production of hyaluronic acid by Bifidobacterium lactis nbk-BA83 strain

[0035] The preserved Bifidobacterium lactis nbk-BA83 glycerol tube strain was inoculated into the modified MRS medium and cultured at 37°C for 24 hours. The activated Bifidobacterium lactis nbk-BA83 was further inoculated into the modified MRS medium and cultured at 37°C for 12 hours. The culture was centrifuged and filtered, and the sterile fermentation supernatant was collected. The concentration of the fermentation supernatant was adjusted as the experimental group, and the modified MRS medium was used as the control group. The hyaluronic acid content in each group was determined using a hyaluronic acid ELISA kit. The results are shown in FIG. Figure 1 As shown, the hyaluronic acid level in the control group was (238.99±15.54) ng / mL, and the hyaluronic acid level in the experimental group was (266.01±9.46) ng / mL, which was significantly increased compared with the control group.

[0036] The present invention also conducts tests on other probiotic strains under the same conditions, and the hyaluronic acid production capacity of the Bifidobacterium lactis nbk-BA83 strain is better than that of the other strains.

[0037] Example 3 Preparation of inactivated bacteria of Bifidobacterium lactis nbk-BA83

[0038] The seed culture medium used in this embodiment is 2.5% glucose, 2% peptone, 1% yeast extract, 0.5% anhydrous sodium acetate, 0.5% ammonium citrate, 0.05% magnesium sulfate, 0.05% manganese sulfate, 0.05% L-cysteine ​​hydrochloride, 0.1% Tween 80 and the balance water, and the pH value is 6.8.

[0039] In terms of mass percentage, the fermentation medium used in this embodiment is 2.5% glucose, 0.5% beef extract powder, 2% yeast extract powder, 0.5% anhydrous sodium acetate, 0.5% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.05% manganese sulfate, 0.1% Tween 80 and the balance water, and the pH value is 6.8.

[0040] This embodiment provides a method for preparing inactivated bacteria of Bifidobacterium lactis nbk-BA83:

[0041] (1) Bifidobacterium lactis nbk-BA83 was inoculated into a seed culture medium for activation to obtain a primary seed solution.

[0042] (2) inoculating the primary seed solution obtained in step (1) into a seed culture medium and continuing to culture the culture medium to obtain a secondary seed solution.

[0043] (3) inoculating the secondary seed liquid obtained in step (2) into a fermentation medium for fermentation to obtain a fermentation liquid.

[0044] (4) The fermentation liquid obtained by culturing in step (3) is centrifuged and concentrated, and then inactivated at 115° C. for 20 min to obtain inactivated Bifidobacterium lactis nbk-BA83.

[0045] Example 4 Study on the promotion of hyaluronic acid production by keratinocytes by inactivated Bifidobacterium lactis nbk-BA83

[0046] This example verifies the effect of inactivated Bifidobacterium lactis nbk-BA83 on hyaluronic acid production by keratinocytes. The specific steps are as follows:

[0047] 1) The inactivated bacteria of Bifidobacterium lactis nbk-BA83 obtained in Example 3 were resuspended in DMEM medium. After resuspension, the Bifidobacterium lactis nbk-BA83 was 1×10 7 / mL, and used as the experimental group.

[0048] 2) Human skin keratinocytes HaCaT cells were cultured at a rate of 1×10 5 The cells were inoculated with 50 μg / mL of culture medium in a 96-well plate and cultured in an incubator containing 5% CO2 at 37°C for 24 h, followed by culture in serum-free DMEM medium for 24 h.

[0049] 3) 100 μL of DMEM medium and inactivated Bifidobacterium lactis nbk-BA83 bacteria (i.e., the inactivated Bifidobacterium lactis nbk-BA83 bacteria resuspended in step (1)) were added to each well of the above-cultured human skin keratinocytes HaCaT cells, respectively, as a blank control group and an experimental group.

[0050] 4) The hyaluronic acid content in each group was determined using a human hyaluronic acid ELISA kit.

[0051] The results are shown in Table 1. Compared with the blank control group, the hyaluronic acid production level of cells in the group supplemented with inactivated Bifidobacterium lactis nbk-BA83 was significantly increased.

[0052] Table 1 Hyaluronic acid production level of Bifidobacterium lactis nbk-BA83 inactivated bacteria group

[0053]

[0054] Note: * indicates P < 0.05 compared with the blank control group

[0055] Example 5 Effect of inactivated Bifidobacterium lactis nbk-BA83 on malondialdehyde levels in photoaged keratinocytes

[0056] This example verifies the effect of inactivated Bifidobacterium lactis nbk-BA83 on the malondialdehyde level of photoaged keratinocytes, and the specific steps are as follows:

[0057] 1) The inactivated bacteria of Bifidobacterium lactis nbk-BA83 obtained in Example 3 were resuspended in DMEM medium. After resuspension, the Bifidobacterium lactis nbk-BA83 was 1×10 7 / mL, and used as the experimental group.

[0058] 2) Human skin keratinocytes HaCaT cells were seeded on a 96-well plate and cultured at 37° C. in an incubator containing 5% CO 2 for 24 h.

[0059] 3) The cell culture medium was removed, and a blank control group, a model group, and an experimental group were set up respectively. 100uL of DMEM culture medium was added to the blank control group, 100uL of DMEM culture medium was added to the model group, and 100uL of resuspended inactivated Bifidobacterium lactis nbk-BA83 was added to the experimental group, and the cells were incubated for 24h.

[0060] 4) The cell culture medium was removed and the cells were rinsed twice with PBS buffer. Then, 100uL PBS buffer was added to each group. The cells in the model group and the experimental group were irradiated for 1 min at a distance of 10cm from the UV lamp.

[0061] 5) After irradiation, the PBS buffer was removed from each group, and DMEM culture medium was added for further incubation for 24 hours. The MDA level in each group was determined using a micro MDA kit according to the prescribed method.

[0062] The results are shown in Table 2. Compared with the blank control group, the MDA level in the model group increased significantly. Compared with the model group, the MDA level in the photoaged human immortalized keratinocytes in the Bifidobacterium lactis nbk-BA83 intervention group decreased significantly and was close to that in the normal control group.

[0063] Table 2 Effect of inactivated Bifidobacterium lactis nbk-BA83 on malondialdehyde levels in photoaged keratinocytes

[0064]

[0065]

[0066] Note: *** indicates P < 0.001 compared with the blank control group, ### indicates P < 0.001 compared with the model control group

[0067] Example 6 Effect of inactivated Bifidobacterium lactis nbk-BA83 on superoxide dismutase levels in photoaged keratinocytes

[0068] This example verifies the effect of inactivated Bifidobacterium lactis nbk-BA83 on the superoxide dismutase level of photoaged keratinocytes. The specific operation steps are the same as those in Example 5. After the experiment, the superoxide dismutase level of each group is detected using a superoxide dismutase detection kit according to the prescribed method.

[0069] The results are shown in Table 3. Compared with the blank control group, the SOD activity of the model group decreased significantly, while compared with the model group, the superoxide dismutase level of photoaged human keratinocytes in the Bifidobacterium lactis nbk-BA83 intervention group increased significantly.

[0070] Table 3 Effect of inactivated Bifidobacterium lactis nbk-BA83 on superoxide dismutase levels in photoaged keratinocytes

[0071]

[0072] Note: *** indicates P < 0.001 compared with the blank control group, ### indicates P < 0.001 compared with the model control group

[0073] Example 7 Effect of inactivated Bifidobacterium lactis nbk-BA83 on hydroxyproline levels in photoaged keratinocytes

[0074] This example verifies the effect of inactivated Bifidobacterium lactis nbk-BA83 on the hydroxyproline level of photoaged keratinocytes. The specific operation steps are the same as those in Example 5. After the experiment, the hydroxyproline level of each group is measured using a hydroxyproline detection kit according to the prescribed method.

[0075] The results are shown in Table 4. Compared with the blank control group, the hydroxyproline level in the model group was significantly decreased. Compared with the model group, the hydroxyproline level in the photoaged human keratinocytes in the Bifidobacterium lactis nbk-BA83 intervention group was increased.

[0076] Table 4 Effects of inactivated Bifidobacterium lactis nbk-BA83 on hydroxyproline levels in photoaged keratinocytes

[0077]

[0078]

[0079] Note: *** indicates P < 0.001 compared with the blank control group.

[0080] Example 8 Application of Bifidobacterium lactis nbk-BA83 in beauty and anti-aging solid beverage products

[0081] The present embodiment provides a probiotic solid beverage for beauty and anti-aging, which includes, by weight, 2.5 parts of Bifidobacterium lactis nbk-BA83, 1.5 parts of Lactobacillus paracasei nbk-LC16, 1 part of Lactobacillus helveticus nbk-LH308, 38 parts of lactitol, 26 parts of resistant dextrin, 15 parts of oligofructose, 8 parts of fish collagen peptides, 7 parts of fruit powder, and 1 part of Tremella polysaccharide.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of probiotics or probiotic powder in the production of hyaluronic acid; The probiotics are Bifidobacterium lactis nbk-BA83, with a preservation number of CGMCC NO.19255; The probiotic powder includes live or inactivated probiotics.

Images