CN115386513A - Probiotics capable of producing hyaluronic acid, helping skin cells to produce hyaluronic acid and preventing photoaging and application of probiotics - Google Patents
Probiotics capable of producing hyaluronic acid, helping skin cells to produce hyaluronic acid and preventing photoaging and application of probiotics Download PDFInfo
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- CN115386513A CN115386513A CN202210923363.7A CN202210923363A CN115386513A CN 115386513 A CN115386513 A CN 115386513A CN 202210923363 A CN202210923363 A CN 202210923363A CN 115386513 A CN115386513 A CN 115386513A
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- Prior art keywords
- probiotic
- skin
- hyaluronic acid
- nbk
- bifidobacterium lactis
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- Cosmetics (AREA)
Abstract
The invention belongs to the technical field of probiotic health care, and particularly provides a probiotic capable of producing hyaluronic acid, facilitating production of hyaluronic acid by skin cells and preventing photoaging and application of the probiotic. The probiotic provided by the invention is bifidobacterium lactis nbk-BA83 with the preservation number of CGMCC NO.19255. The probiotics provided by the invention can be fermented to produce hyaluronic acid, and the inactivated bacteria can promote skin keratinocytes to produce hyaluronic acid; meanwhile, the inactivated bacteria can inhibit skin aging by improving the oxidation resistance of photoaging cells, prevent the oxidative damage of photoaging skin keratinocytes induced by ultraviolet rays, further improve the skin state, inhibit skin aging, improve the water retention and moisture retention of the skin and enhance the barrier function of the skin.
Description
Technical Field
The invention relates to the technical field of probiotic health care, in particular to a probiotic for helping skin to produce hyaluronic acid and preventing photoaging and application thereof.
Background
The skin is the largest organ of the human body, is the first barrier for isolating the internal environment of the human body from the external environment, and protects various organs and tissues in the human body from invasion of mechanical, chemical, pathogenic microorganisms and the like. The epidermal layer of the skin is directly exposed to the external environment and is composed mainly of keratinocytes.
The hyaluronic acid structure is a straight chain high molecular polysaccharide composed of glucuronic acid and N-acetylglucosamine, has strong hygroscopicity, is soluble in water, and is a polymer with water retention effect naturally existing in human skin. Hyaluronic acid in the skin is mainly produced by skin keratinocytes and fibroblasts. Hyaluronic acid can form a viscoelastic water film on the epidermis, keep the moisture of the skin, strengthen the skin barrier and prevent the skin from drying. The strong water absorption of the skin care product is beneficial to regulating the moisture of epidermis, protecting cells from being invaded by pathogenic bacteria, preventing the infection of the pathogenic bacteria and maintaining the toughness and the elasticity of the skin. The level of hyaluronic acid in the skin decreases, the water retention function of the skin decreases, and wrinkles are generated. At present, the water retention and moisture retention of skin are improved by adding hyaluronic acid into skin care products or cosmetics, but on one hand, the high content of hyaluronic acid is taken as a high nutrient substance and is easy to cause pollution of pathogenic bacteria and influence on skin health, and on the other hand, when the molecular weight exceeds 400kDa, the hyaluronic acid cannot penetrate through a skin barrier and further cannot exert the effect of improving the skin health on a dermis layer.
Under long-term exposure to ultraviolet radiation, skin cells can generate a large amount of active oxygen free radicals, and the excessive active oxygen free radicals participate in the oxidative stress process of the cells, so that the balance of an antioxidant defense system is broken, cell damage and aging are caused, and the skin is subjected to a photo-aging phenomenon. Malondialdehyde is one of the final products of membrane lipid peroxidation, and can damage the structure of a biological membrane, cause aging of an organism and react free radicals to cells and damage degree of the organism. The antioxidant system of the body is very important for maintaining the redox homeostasis of cells, and the antioxidant system of the body consists of a plurality of free enzymes, wherein superoxide dismutase is the primary substance for clearing free radicals, can resist and block the damage of oxygen free radicals to the cells, repair damaged cells and delay aging. Collagen is a key factor determining skin elasticity, and its degradation can cause skin damage, and hydroxyproline is a characteristic amino acid in collagen and reflects the catabolic condition of collagen.
Disclosure of Invention
The invention aims to provide a strain of bifidobacterium lactis which can be fermented to produce hyaluronic acid, and an inactivated strain of the bifidobacterium lactis can promote skin keratinocytes to produce hyaluronic acid, and can inhibit skin aging by improving the oxidation resistance of photoaging cells, so that the bifidobacterium lactis can improve the water retention and moisture retention of skin and enhance the barrier function of skin.
In a first aspect, the invention claims a probiotic, wherein the probiotic is bifidobacterium lactis nbk-BA83 with the preservation number of CGMCC NO.19255.
More specifically, the invention separates a probiotic from healthy infant intestinal tracts, and the probiotic is identified as bifidobacterium lactis subspecies of animals through cell morphology, physiological and biochemical characteristics and 16S rRNA gene sequence determination results, and the lactobacillus subspecies is named as bifidobacterium lactis nbk-BA83.
The Bifidobacterium lactis nbk-BA83 (classification name: bifidobacterium lactis) provided by the invention is preserved in China general microbiological culture Collection center in 12-30 months in 2019, and the address is as follows: the institute of microbiology, national academy of sciences No. 3, xilu No.1, beijing, chaoyang, beijing; and E, postcode: 100101; the preservation number is CGMCC NO.19255.
In a second aspect, the invention claims a probiotic powder, which comprises live bacteria or inactivated bacteria of bifidobacterium lactis nbk-BA83.
More specifically, the preparation method of the bifidobacterium lactis nbk-BA83 inactivated bacteria comprises the following steps:
(1) Inoculating bifidobacterium lactis nbk-BA83 into a seed culture medium, and activating to obtain a first-stage seed solution.
(2) And (2) inoculating the primary seed solution obtained in the step (1) into a seed culture medium for continuous culture to obtain a secondary seed solution.
(3) And (3) inoculating the secondary seed liquid obtained in the step (2) into a fermentation culture medium, and performing fermentation culture to obtain a fermentation liquid.
(4) And (4) centrifugally concentrating the fermentation liquor obtained by culturing in the step (3), adding pure water, uniformly mixing, and inactivating under certain temperature and time conditions to obtain inactivated bifidobacterium lactis nbk-BA83.
Preferably, the seed culture medium comprises, by mass, 1-3% of glucose, 1-3% of peptone, 0.5-3% of yeast extract, 0.2-1% of anhydrous sodium acetate, 0.2-1% of ammonium citrate, 0.02-0.08% of magnesium sulfate, 0.02-0.08% of manganese sulfate, 0.05-0.1% of L-cysteine hydrochloride, 0.05-0.1% of Tween 80 and the balance of water, and the pH value is 6.2-6.8.
Preferably, the fermentation medium comprises, by mass, 1-3% of glucose, 1-3% of peptone, 0.5-2% of yeast powder, 0.2-1% of beef powder, 0.2-1% of anhydrous sodium acetate, 0.2-1% of ammonium citrate, 0.02-0.08% of magnesium sulfate, 0.02-0.08% of manganese sulfate, 0.05-0.1% of L-cysteine hydrochloride, 0.05-0.1% of Tween 80 and the balance of water, and the pH value is 6.2-6.8.
After centrifugal concentration, the fermentation liquor is inactivated for 10-30 min at 105-115 ℃.
According to the understanding of the person skilled in the art, the present invention claims the use of the probiotic bacteria described above or of the probiotic powder described above in the production of hyaluronic acid.
And the application of the probiotics or the probiotics powder in promoting the skin keratinocytes to produce hyaluronic acid.
And the application of the probiotics or the probiotic powder in reducing the oxidative damage of the skin keratinocytes.
The application of the composition in reducing the skin keratinocyte oxidation damage provided by the invention is that the skin keratinocyte oxidation damage is photoaging caused by ultraviolet induction.
The invention also claims the application of the probiotics or the probiotic powder in increasing the expression level of the hydroxyproline of the photoaged cells.
And the application of the probiotics or the probiotics powder in preparing skin care products, cosmetics, health care products, medicines or foods for preventing dry and aged skin.
In a third aspect, the invention provides a skin care product, cosmetic, health product, medicine or food for preventing skin dryness and aging, wherein the skin care product, cosmetic, health product, medicine or food for preventing skin dryness and aging contains the above probiotics or the above probiotic powder.
The invention has the beneficial effects that:
the invention provides a probiotic with beauty and anti-aging functions, namely bifidobacterium lactis nbk-BA83 with the preservation number of CGMCC NO.19255. The bifidobacterium lactis nbk-BA83 provided by the invention can be fermented to produce hyaluronic acid, and the killed bifidobacterium lactis nbk-BA83 obtained by the preparation method can promote skin keratinocytes to produce hyaluronic acid, prevent ultraviolet-induced photoaging cell oxidative damage, increase cellular hydroxyproline level and further play a role in beautifying and resisting aging.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings needed for the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.
FIG. 1 shows the effect of producing hyaluronic acid by Bifidobacterium lactis nbk-BA83 strain in example 2 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 screening and identification of Bifidobacterium lactis nbk-BA83
This example provides the screening and identification of bifidobacterium lactis nbk-BA83 by the following steps:
collecting intestinal tracts of healthy infants, diluting the intestinal tracts to a proper gradient by using sterile normal saline, performing streak separation on an improved MRS agar plate, performing anaerobic culture for 72 hours at 37 ℃, selecting a single colony for microscopic examination, further performing streak purification, and identifying the single colony as an animal bifidobacterium lactis subspecies named bifidobacterium lactis nbk-BA83 through cell morphology, physiological and biochemical characteristics and 16S rRNA gene sequence determination results.
The bifidobacterium lactis nbk-BA83 provided by the invention has been preserved in China general microbiological culture Collection center in 2019, 12 months and 30 days, and the addresses are as follows: the institute of microbiology, national academy of sciences No. 3, xilu No.1, beijing, chaoyang, beijing; and E, postcode: 100101; the preservation number is CGMCC NO.19255.
EXAMPLE 2 study of hyaluronic acid production by Bifidobacterium lactis nbk-BA83 Strain
Inoculating the preserved bifidobacterium lactis nbk-BA83 glycerol tube strain into an improved MRS culture medium, culturing for 24h at 37 ℃, continuously inoculating the activated bifidobacterium lactis nbk-BA83 into the improved MRS culture medium, culturing for 12h at 37 ℃, performing centrifugal filtration, collecting sterile fermentation supernatant, adjusting the concentration of the fermentation supernatant as an experimental group, taking the improved MRS culture medium as a control group, and measuring the content of hyaluronic acid in each group by using a hyaluronic acid ELISA kit, wherein the result is shown in figure 1, the hyaluronic acid in the control group is (238.99 +/-15.54) ng/mL, the hyaluronic acid in the experimental group is (266.01 +/-9.46) ng/mL, and the hyaluronic acid level is obviously increased compared with the control group.
The invention also tests other probiotic strains under the same conditions, and the capacity of the bifidobacterium lactis nbk-BA83 strain for producing hyaluronic acid is better than that of other strains.
Example 3 preparation of live killed Bifidobacterium lactis nbk-BA83
The seed medium used in this example was 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, in mass%, and the pH was 6.8.
The fermentation medium used in this example was, by mass, 2.5% glucose, 0.5% beef extract powder, 2% yeast extract powder, 0.5% anhydrous sodium acetate, 0.5% dibasic potassium phosphate, 0.05% magnesium sulfate, 0.05% manganese sulfate, 0.1% tween 80, and the balance water, and the pH was 6.8.
The embodiment provides a preparation method of bifidobacterium lactis nbk-BA83 inactivated bacteria, which comprises the following steps:
(1) Inoculating bifidobacterium lactis nbk-BA83 into a seed culture medium, and activating to obtain a first-stage seed solution.
(2) Inoculating the primary seed liquid obtained in the step (1) into a seed culture medium for continuous culture to obtain a secondary seed liquid.
(3) And (3) inoculating the secondary seed liquid obtained in the step (2) into a fermentation culture medium, and performing fermentation culture to obtain fermentation liquor.
(4) And (4) carrying out inactivation treatment on the fermentation liquor obtained by culture in the step (3) for 20min after centrifugal concentration at the temperature of 115 ℃ to obtain inactivated bifidobacterium lactis nbk-BA83.
Example 4 study of Bifidobacterium lactis nbk-BA83 inactivated bacteria on promotion of hyaluronic acid production by keratinocytes
This example demonstrates the effect of bifidobacterium lactis nbk-BA83 killed bacteria on hyaluronic acid production by keratinocytes. The method comprises the following specific steps:
1) The killed Bifidobacterium lactis nbk-BA83 obtained in example 3 was resuspended in DMEM medium until the volume of Bifidobacterium lactis nbk-BA83 was 1X 10 7 one/mL, as experimental group for use.
2) Human skin keratinocytes HaCaT cells were treated at 1X 10 5 seed/mL on a 96-well plate at 37 ℃ with 5% CO 2 After 24h, the cells were cultured in serum-free DMEM medium for 24h.
3) 100uLDMEM culture medium and bifidobacterium lactis nbk-BA83 inactivated bacteria (namely the bifidobacterium lactis nbk-BA83 inactivated bacteria after the resuspension in the step (1)) are respectively added into each hole of the cultured human skin keratinocyte HaCaT cells to be respectively used as a blank control group and an experimental group.
4) The hyaluronic acid content in each group was determined using a human hyaluronic acid ELISA kit.
The results are shown in table 1, and the hyaluronic acid production of cells of the inactivated lactobacillus bifidus nbk-BA83 group is significantly increased on average compared with that of the blank control group.
TABLE 1 Bifidobacterium lactis nbk-BA83 production of hyaluronic acid by killed bacterial cells
Note: * Indicates P < 0.05 compared to the blank control
Example 5 Effect of Bifidobacterium lactis nbk-BA83 Sterilization on the level of malondialdehyde in photoaged keratinocytes
This example demonstrates the effect of bifidobacterium lactis nbk-BA83 killed bacteria on the level of malondialdehyde in photoaged keratinocytes by the following specific steps:
1) The killed Bifidobacterium lactis nbk-BA83 obtained in example 3 was resuspended in DMEM medium until the volume of Bifidobacterium lactis nbk-BA83 was 1X 10 7 one/mL, as experimental group.
2) Inoculating human skin keratinocyte HaCaT cells on a 96-well plate, at 37 ℃ in a 5% CO content 2 The culture box is used for culturing for 24 hours.
3) And (3) removing the cell culture solution by suction, and respectively setting a blank control group, a model group and an experimental group, wherein the blank control group is added with 100uLDMEM culture medium, the model group is added with 100uLDMEM culture medium, the experimental group is added with 100uL of the killed bacteria of the bifidobacterium lactis nbk-BA83 after being resuspended, and the incubation is continued for 24h.
4) After the cell culture solution was aspirated and washed 2 times with PBS buffer, 100uLPBS buffer was added to each group, wherein the cells of the model group and the experimental group were irradiated at a distance of 10cm from the UV lamp for 1min, respectively.
5) After the irradiation is finished, each group absorbs PBS buffer solution, DMEM culture solution is added to continue incubation for 24 hours, and then the malonaldehyde level in each group is measured by a trace malonaldehyde kit according to a specified method.
As a result, as shown in table 2, the malondialdehyde level was significantly increased in the model group compared to the blank control group. Compared with the model group, the bifidobacterium lactis nbk-BA83 intervention group has obviously reduced photoaging human immortalized keratinocyte malondialdehyde level, and is close to a normal control group.
TABLE 2 Effect of Bifidobacterium lactis nbk-BA83 killed bacteria on photo-aged keratinocyte malondialdehyde levels
Note: * Denotes P < 0.001 in comparison with the blank control group, ## # denotes P < 0.001 in comparison with the model control group
Example 6 Effect of Bifidobacterium lactis nbk-BA83 Sterilization on the level of superoxide dismutase in photoaged keratinocytes
The present example verifies the effect of bifidobacterium lactis nbk-BA83 inactivated bacteria on the level of superoxide dismutase in photoaging keratinocytes, the specific operation steps are the same as example 5, and after the experiment is finished, the superoxide dismutase detection kit is used for detecting the level of superoxide dismutase in each group according to a specified method.
The results are shown in table 3, the SOD activity of the model group was significantly decreased compared to the blank control group, while the levels of superoxide dismutase of bifidobacterium lactis nbk-BA83 intervening group photoaged human keratinocytes were significantly increased compared to the model group.
TABLE 3 Effect of Bifidobacterium lactis nbk-BA83 inactivating bacteria on photoaged keratinocyte superoxide dismutase levels
Note: * Denotes P < 0.001 in comparison with the blank control group, ## # denotes P < 0.001 in comparison with the model control group
Example 7 Effect of Bifidobacterium lactis nbk-BA83 killed bacteria on photo-aged keratinocyte hydroxyproline levels
This example demonstrates the effect of bifidobacterium lactis nbk-BA83 killed bacteria on the hydroxyproline level of photoaging keratinocytes, the specific procedure is as in example 5, and the hydroxyproline level in each group is determined by hydroxyproline assay kit according to the specified method after the experiment is finished.
The results are shown in table 4, where the hydroxyproline levels were significantly reduced in the model group compared to the blank control group and increased in the photoaged human keratinocytes in the intervening group of bifidobacterium lactis nbk-BA83 compared to the model group.
TABLE 4 Effect of Bifidobacterium lactis nbk-BA83 killed bacteria on photo-aged keratinocyte hydroxyproline levels
Note: * Denotes P < 0.001 compared to the blank control group.
Example 8 application of Bifidobacterium lactis nbk-BA83 in beauty-treatment anti-aging solid beverage product
The embodiment provides a beauty and anti-aging probiotic solid beverage which comprises, 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 fructo-oligosaccharide, 8 parts of fish collagen peptide, 7 parts of fruit powder and 1 part of tremella polysaccharide.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (9)
1. The probiotic is characterized in that the probiotic is bifidobacterium lactis nbk-BA83 with the preservation number of CGMCC NO.19255.
2. A probiotic powder, characterized in that it comprises the live or inactivated bacteria of the probiotic of claim 1.
3. Use of a probiotic according to claim 1 or a probiotic powder according to claim 2 in the production of hyaluronic acid.
4. Use of the probiotic of claim 1 or the probiotic powder of claim 2 for promoting hyaluronic acid production by skin keratinocytes.
5. Use of the probiotic of claim 1 or the probiotic powder of claim 2 for reducing oxidative damage to skin keratinocytes.
6. Use according to claim 5, characterized in that said oxidative damage of skin keratinocytes is photoaging induced by UV light.
7. Use of a probiotic of claim 1 or a probiotic powder of claim 2 for increasing the expression level of photo-aged cell hydroxyproline.
8. Use of the probiotic of claim 1 or the probiotic powder of claim 2 for the preparation of a skin care, cosmetic, health care, pharmaceutical or food product for preventing skin dryness and aging.
9. A skin care product, cosmetic, health product, pharmaceutical product or food for preventing skin dryness and aging, wherein the skin care product, cosmetic, health product, pharmaceutical product or food for preventing skin dryness and aging contains the probiotic of claim 1 or the probiotic powder of claim 2.
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