Staphylococcus epidermidis fermentation liquor as well as preparation method and application thereof

Abstract

The invention relates to staphylococcus epidermidis fermentation liquor as well as a preparation method and application thereof, and belongs to the technical field of microbial fermentation. The invention provides a staphylococcus epidermidis fermentation broth with anti-inflammatory, anti-oxidation and whitening effects, staphylococcus epidermidis is a skin probiotic, a preparation method of the fermentation broth comprises the step of fermenting the staphylococcus epidermidis by using a fermentation medium containing glycerol or glucose, the content of the glycerol or the glucose is 10-50g / L, and the content of the staphylococcus epidermidis is 10-50g / L. The fermentation medium also contains beef extract, peptone and sodium chloride. The prepared staphylococcus epidermidis fermentation broth can effectively inhibit the expression level of inflammatory factors in cells, reduce ROS fluorescence intensity and MDA content, improve SOD enzyme activity, inhibit melanin synthesis and inhibit tyrosinase activity, can be applied to preparation of anti-inflammatory, anti-oxidation and whitening products, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, in particular to a Staphylococcus epidermidis fermentation liquid and a preparation method and application thereof. Background Art

[0002] Skin, as the largest organ of the human body, is not only a barrier to protect internal tissues from external damage, but also has multiple functions such as regulating body temperature, sensing touch, storing water and lipids, etc. However, despite the skin's strong self-repair and defense capabilities, it may still face a variety of problems and challenges including acne, oxidation and melanin synthesis.

[0003] Propionibacterium acnes is the main pathogen causing acne. After invading the human body from the skin, Propionibacterium acnes can release a variety of biological enzymes, such as lipase, protease and hyaluronidase. These biological enzymes can decompose triglycerides in sebum to produce low molecular weight polypeptides and free fatty acids, which in turn damage the sebaceous glands and cause local inflammation of the skin, i.e., acne. The secretion of sebum can also provide nutrition for the reproduction of Propionibacterium acnes, while Propionibacterium acnes reacts on the sebaceous glands, causing keratinization lesions in the hair follicle sebaceous gland ducts, obstructing the discharge of sebum, and aggravating the inflammatory reaction of acne. Commonly used drugs for the treatment of acne include antibiotics including tetracycline, erythromycin and clindamycin, and non-antibiotics including azelaic acid, benzoyl peroxide, selenium disulfide, niacinamide, etc. Long-term use of antibiotics or hormone drugs to treat acne is prone to adverse consequences such as bacterial resistance and drug dependence. Acidic substances can damage the stratum corneum of the skin, causing the keratin to loosen and the hair follicle sebaceous gland duct to keratinize abnormally. Therefore, seeking for mild, non-irritating and effective drugs for treating acne has become an urgent clinical need.

[0004] Oxidation is an important cause of skin damage. Excessive reactive oxygen species (ROS) causes the body to be in a state of oxidative stress, causing DNA damage, inducing skin inflammatory reactions, reducing the activity of antioxidant enzymes, inhibiting the production of collagen, and ultimately causing severe damage to cell function and resulting in wrinkles and sagging of the skin. Removing excess reactive oxygen species and free radicals from the body has become one of the most common anti-aging beauty methods. On the other hand, ROS acts as a second messenger in the pro-inflammatory signal cascade. When the body is in a state of oxidative stress, it can stimulate the production of pro-inflammatory cytokines and aggravate the inflammatory response. The body eventually shows a series of skin injuries such as redness, dryness, lack of elasticity, and deepening of wrinkles, which seriously affect people's confidence in their appearance.

[0005] Melanin is a high molecular weight biological pigment synthesized by melanocytes, distributed in the dermis of the skin, and determines the color of the skin. Proper melanin production is beneficial to skin health and can effectively absorb ultraviolet rays to protect the skin from photoaging damage. Excessive melanin synthesis not only simply darkens the skin, but can also directly or indirectly cause freckles, vitiligo, etc. on the skin, and even cause skin cancer, seriously damaging human health.

[0006] Microbial fermentation is a technology that uses culture media to accumulate bacteria and their metabolites. It has the advantages of mild experimental conditions, strong synergistic effects, and no introduction of external toxins. Most previous studies have focused on the fermentation of intestinal microorganisms. However, human skin is also a huge microbial reservoir. These microorganisms provide a physical barrier between the human body and the outside world, preventing the entry of irritants and pathogens, and informing the occurrence and development of immune responses. Staphylococcus epidermidis is the most widely distributed commensal bacterium on the human body surface, accounting for 65%-90% of surface staphylococci, and belongs to the category of skin probiotics. However, few studies have attempted to solve the above-mentioned skin problems by fermenting skin probiotics, especially Staphylococcus epidermidis. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a Staphylococcus epidermidis fermentation liquid and a preparation method and application thereof, wherein a fermentation medium containing glycerol or glucose is used to ferment Staphylococcus epidermidis, the content of glycerol or glucose is 10-50 g / L, and the fermentation medium also contains beef extract, peptone and sodium chloride. The prepared Staphylococcus epidermidis fermentation liquid is concentrated to obtain fermentation concentrates of different concentrations, which can be used for anti-inflammatory, anti-oxidation and whitening.

[0008] The first object of the present invention is to provide a method for preparing a Staphylococcus epidermidis fermentation broth having anti-inflammatory, antioxidant and whitening effects, wherein a fermentation medium containing glycerol or glucose is used to ferment Staphylococcus epidermidis to obtain the Staphylococcus epidermidis fermentation broth.

[0009] Furthermore, the fermentation medium includes 2-6 g / L beef extract, 12-18 g / L peptone, and 3-5 g / L sodium chloride.

[0010] Preferably, the fermentation medium comprises 3 g beef extract, 15 g peptone, 5 g sodium chloride, and 1 L of sterilized distilled water.

[0011] Furthermore, the amount of glucose added to the fermentation medium is 10-50 g / L.

[0012] Preferably, the amount of glucose added to the fermentation medium is 20 g / L.

[0013] Furthermore, the amount of glycerol added to the fermentation medium is 10-50 g / L.

[0014] Preferably, the amount of glycerol added to the fermentation medium is 20 g / L.

[0015] Furthermore, the fermentation temperature is 25-38°C.

[0016] Preferably, the fermentation temperature is 37°C.

[0017] Furthermore, the fermentation time is 24-72 hours.

[0018] Preferably, the fermentation time is 48 hours.

[0019] Furthermore, the Staphylococcus epidermidis is inoculated in a fermentation medium at a volume percentage of 2-10%.

[0020] Preferably, the Staphylococcus epidermidis is inoculated in the fermentation medium at a volume percentage of 5%.

[0021] The second object of the present invention is to provide the Staphylococcus epidermidis fermentation broth prepared by the above method.

[0022] Furthermore, the Staphylococcus epidermidis fermentation broth is concentrated.

[0023] The third object of the present invention is to provide the use of the above-mentioned Staphylococcus epidermidis fermentation broth in reducing the expression level of inflammatory factor IL-1β and / or inflammatory factor IL-6, wherein the Staphylococcus epidermidis fermentation broth is prepared from a fermentation medium containing glycerol and is a 0.5-fold fermentation dilution liquid.

[0024] The fourth object of the present invention is to provide the use of the above-mentioned Staphylococcus epidermidis fermentation broth in removing active oxygen, improving superoxide dismutase activity and / or reducing malondialdehyde content.

[0025] The fifth object of the present invention is to provide the use of the above-mentioned Staphylococcus epidermidis fermentation broth in inhibiting melanin synthesis and / or inhibiting tyrosinase activity, wherein the Staphylococcus epidermidis fermentation broth is prepared from a fermentation medium containing glucose and is a 2-fold concentrated fermentation broth.

[0026] Beneficial effects of the present invention:

[0027] Staphylococcus epidermidis is a common skin probiotic. The Staphylococcus epidermidis fermentation liquid prepared by the present invention can effectively inhibit the expression level of intracellular inflammatory factors, reduce the ROS fluorescence intensity and MDA content, increase the SOD enzyme activity, inhibit the synthesis of melanin and inhibit the activity of tyrosinase, and has the potential to be used in the preparation of anti-inflammatory, antioxidant and whitening products. Its antioxidant effect is better than that of vitamin C, and its whitening effect is better than that of kojic acid, and it has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0029] Figure 1 The results of determination of the relative expression of the intracellular inflammatory factor IL-1β under treatment with different Staphylococcus epidermidis fermentation broths in Example 2 of the present invention, wherein S is SFB, Y is Gly-SFB, U is Glu-SFB, and 1-3 are 0.5 times the concentration of the fermentation broth, the original concentration of the fermentation broth, and the 2 times concentrated fermentation broth, respectively;

[0030] Figure 2 The relative expression of the intracellular inflammatory factor IL-6 under different Staphylococcus epidermidis fermentation broth treatments in Example 2 of the present invention is measured, wherein S is SFB, Y is Gly-SFB, U is Glu-SFB, and 1-3 are 0.5 times the concentration of the fermentation broth, the original concentration of the fermentation broth, and the 2 times concentrated fermentation broth, respectively;

[0031] Figure 3 The results of measuring the fluorescence intensity of ROS under different treatments of Staphylococcus epidermidis fermentation broths in Example 3 of the present invention, wherein S is SFB, G is Gly-SFB, T is Glu-SFB, and 1-3 are the original concentration fermentation broth, 2-fold fermentation concentrate, and 4-fold fermentation concentrate, respectively;

[0032] Figure 4 The results of determination of intracellular SOD enzyme activity under different Staphylococcus epidermidis fermentation broth treatments in Example 3 of the present invention, wherein S is SFB, Y is Gly-SFB, U is Glu-SFB, and 1-3 are original concentration fermentation broth, 2-fold fermentation concentrate, and 4-fold fermentation concentrate, respectively;

[0033] Figure 5 The results of determination of intracellular MDA content under treatment with different Staphylococcus epidermidis fermentation broths in Example 3 of the present invention, wherein S is SFB, Y is Gly-SFB, U is Glu-SFB, and 1-3 are original concentration fermentation broth, 2-fold fermentation concentrate, and 4-fold fermentation concentrate, respectively;

[0034] Figure 6 The results of determination of intracellular melanin content under treatment with different Staphylococcus epidermidis fermentation broths in Example 4 of the present invention, wherein S is SFB, Y is Gly-SFB, U is Glu-SFB, and 1-3 are original concentration fermentation broth, 2-fold fermentation concentrate, and 4-fold fermentation concentrate, respectively;

[0035] Figure 7These are the results of determination of intracellular tyrosinase activity under treatment with different Staphylococcus epidermidis fermentation broths in Example 4 of the present invention, wherein S is SFB, Y is Gly-SFB, U is Glu-SFB, and 1-3 are original concentration fermentation broth, 2-fold fermentation concentrate, and 4-fold fermentation concentrate, respectively. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0037] The Staphylococcus epidermidis used in the following examples is BNCC330867.

[0038] The instruments used in the examples are as follows:

[0039] Instrument Name model Manufacturer Rotary evaporator RE-S2AA Yarong Biochemical Instrument Factory UV spectrophotometer UV-1800PC Aoyi Instrument Co., Ltd. Electronic balance AX224ZH / E Changzhou Ohaus Instruments Co., Ltd. Multifunctional ELISA reader Tecan Infinite 200PRO Shanghai Diken International Trading Co., Ltd. Heat collecting constant temperature heating magnetic stirrer BIONOON-10A Gongyi Yuhua Instrument Co., Ltd. Electric incubator FS-2 Nantong HuNan Scientific Instrument Co., Ltd. Clean bench SW-CJ-2FD Wuxi Capex Technology Co., Ltd. Ultra-low temperature refrigerator MDF-192(N) Japan Sanyo

[0040] The fermentation medium composition involved in the embodiment is as follows:

[0041] Original fermentation medium: 3.0 g beef extract, 15.0 g peptone, 5.0 g sodium chloride, 1.0 L distilled water, pH = 7.0;

[0042] Glucose fermentation medium: beef extract 3.0 g, peptone 15.0 g, sodium chloride 5.0 g, glucose 20.0 g, distilled water 1.0 L, pH = 7.0;

[0043] Glycerol fermentation medium: beef extract 3.0 g, peptone 15.0 g, sodium chloride 5.0 g, glycerol 20.0 g, distilled water 1.0 L, pH = 7.0;

[0044] 1.0L distilled water, 121℃, sterilize for 15min.

[0045] Example 1: Preparation of Staphylococcus epidermidis fermentation broth

[0046] (1) Seed culture: activating Staphylococcus epidermidis BNCC330867 and inoculating it into beef extract peptone medium to obtain seed culture solution;

[0047] (2) Fermentation culture: The seed culture solution was inoculated into the original fermentation medium, the glucose fermentation medium and the glycerol fermentation medium at an inoculation amount of 5%, and fermented at 37° C. for 48 hours to obtain fermentation broth. The fermentation broth obtained from the original fermentation medium was named SFB, the fermentation broth obtained from the glucose fermentation medium was named Glu-SFB, and the fermentation broth obtained from the glycerol fermentation medium was named Gly-SFB;

[0048] (3) Fermentation broth filtration: The three fermentation broths were centrifuged and filtered through a 0.22 μm filter membrane;

[0049] (4) Concentrating the fermentation broth: concentrating the filtered fermentation broth to prepare a 2-fold concentrated fermentation broth and a 4-fold concentrated fermentation broth;

[0050] (5) Dilution of fermentation broth: dilute the filtered fermentation broth to prepare a 0.5-fold concentration fermentation dilution broth.

[0051] Example 2: Application of Staphylococcus epidermidis fermentation broth in anti-inflammatory treatment

[0052] RAW264.7 cells were inoculated into DMEM complete medium and cultured for 24 hours until the cells were fully attached. Inactivated Propionibacterium acnes TI.P was added as a stimulating factor to stimulate inflammation. RAW264.7 cells were treated with three types of Staphylococcus epidermidis fermentation broth for 24 hours, and the cell pellets and culture supernatants of each treatment group were collected. The control group was only added with inactivated Propionibacterium acnes TI.P, without adding Staphylococcus epidermidis fermentation broth.

[0053] The relative expression of inflammatory factor IL-1β in the cell pellets of each treatment group was detected. Figure 1 As shown, S, Y and U represent SFB, Gly-SFB and Glu-SFB, respectively, and 1-3 represent 0.5 times fermentation concentrate, original concentration fermentation broth and 2 times concentrated fermentation broth, respectively.

[0054] The relative expression of inflammatory factor IL-6 in the cell pellets of each treatment group was detected. Figure 2 As shown, S, Y and U represent SFB, Gly-SFB and Glu-SFB, respectively, and 1-3 represent 0.5 times fermentation concentrate, original concentration fermentation broth and 2 times concentrated fermentation broth, respectively.

[0055] Compared with the original fermentation medium, the anti-inflammatory effect of Staphylococcus epidermidis fermentation broth obtained by treating with glucose fermentation medium and glycerol fermentation medium was significantly improved, and the relative expression of inflammatory factors in cells was significantly reduced. Among them, the fermentation broth Gly-SFB prepared using the fermentation medium containing glycerol had the most obvious inhibitory effect on the expression level of inflammatory factors, and after diluting Gly-SFB to 0.5 times the concentration, the anti-inflammatory effect was further improved.

[0056] Example 3: Application of Staphylococcus epidermidis fermentation broth in anti-oxidation

[0057] HaCat cells were inoculated into DMEM complete medium and cultured for 24 hours until the cells were fully attached. 2 O 2The cells were treated for 2 hours. HaCat cells were treated with three kinds of Staphylococcus epidermidis fermentation broth for 24 hours. The control group was treated with vitamin C for 24 hours without adding Staphylococcus epidermidis fermentation broth.

[0058] The fluorescence intensity of intracellular ROS was detected by flow cytometry. The detection channel was FITC channel, excitation was 488nm, emission was 530 / 30nm, and the detection voltage of the fluorescence channel was 350V. The detection results were as follows: Figure 3 As shown. Where S, G and T represent SFB, Gly-SFB and Glu-SFB respectively, and 1-3 represent original concentration fermentation broth, 2-fold fermentation concentrate and 4-fold concentrated fermentation broth respectively. Compared with the original fermentation medium, the intracellular ROS fluorescence intensity of the Staphylococcus epidermidis fermentation broth obtained by treating with glucose fermentation medium and glycerol fermentation medium was reduced, that is, the antioxidant effect was better. Among them, the antioxidant effect of 2-fold concentrated Gly-SFB and Glu-SFB was basically the same as that of vitamin C as a control.

[0059] The SOD enzyme detection kit was used to detect the changes in intracellular SOD enzyme activity. The results were as follows: Figure 4 As shown, S, Y and U represent SFB, Gly-SFB and Glu-SFB respectively, and 1-3 represent original concentration fermentation broth, 2-fold fermentation concentrate and 4-fold fermentation concentrate respectively. The fermentation broth of Staphylococcus epidermidis increased the activity of SOD enzyme in cells, and the effect of 4-fold concentration of Gly-SFB was the most prominent, which was much higher than that of vitamin C as a control.

[0060] The MDA detection kit was used to detect the changes in intracellular MDA content. The results were as follows Figure 5 As shown, S, Y and U represent SFB, Gly-SFB and Glu-SFB respectively, and 1-3 represent original concentration fermentation broth, 2-fold fermentation concentrate and 4-fold fermentation concentrate respectively. The Staphylococcus epidermidis fermentation broth significantly reduced the intracellular MDA content and had a good antioxidant effect.

[0061] Example 4: Application of Staphylococcus epidermidis fermentation broth in skin whitening

[0062] B16-F10 mouse melanoma cells were inoculated into RPMI 1640 medium and cultured for 24 hours until the cells were fully attached. Melanin α-MSH was added. B16-F10 cells were treated with three kinds of Staphylococcus epidermidis fermentation broth for 24 hours. The experimental group with kojic acid was used as a control.

[0063] Detect the melanin content in cells, the results are as follows Figure 6As shown, S, Y and U represent SFB, Gly-SFB and Glu-SFB, respectively, and 1-3 represent original concentration fermentation broth, 2-fold fermentation concentrate and 4-fold fermentation concentrate, respectively. The intracellular melanin content of B16 cells treated with Gly-SFB and Glu-SFB was the lowest compared with the control group and SFB treatment group, among which the 2-fold concentrated Glu-SFB fermentation broth had the most obvious effect.

[0064] Detection of intracellular tyrosinase activity, the results are as follows Figure 7 As shown, S, Y and U represent SFB, Gly-SFB and Glu-SFB, respectively, and 1-3 represent original concentration fermentation broth, 2-fold fermentation concentrate and 4-fold fermentation concentrate, respectively. After treatment with the same concentration of fermentation broth, Gly-SFB and Glu-SFB have better inhibitory effects on tyrosinase activity than SFB, among which the 2-fold concentrated Glu-SFB fermentation broth has the most obvious effect. In summary, the 2-fold concentrated Glu-SFB fermentation broth has good whitening effect, can inhibit intracellular tyrosinase activity, and inhibit the synthesis of intracellular melanin.

[0065] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing a Staphylococcus epidermidis fermentation broth having anti-inflammatory, antioxidant and whitening effects, characterized in that: A fermentation medium containing glycerol or glucose is used to ferment Staphylococcus epidermidis to obtain the Staphylococcus epidermidis fermentation liquid.

2. The preparation method according to claim 1, characterized in that: The fermentation medium comprises 2-6 g / L beef extract, 12-18 g / L peptone and 3-5 g / L sodium chloride.

3. The preparation method according to claim 1, characterized in that: The amount of glucose added to the fermentation medium is 10-50 g / L.

4. The preparation method according to claim 1, characterized in that: The amount of glycerol added to the fermentation medium is 10-50 g / L.

5. The preparation method according to claim 1, characterized in that: The fermentation time is 24-72 hours.

6. The preparation method according to claim 1, characterized in that: The Staphylococcus epidermidis is inoculated in a fermentation medium at a volume percentage of 2-10%.

7. Staphylococcus epidermidis fermentation broth prepared by any one of claims 1 to 6.

8. Use of the Staphylococcus epidermidis fermentation broth according to claim 7 in reducing the expression level of inflammatory factor IL-1β and / or inflammatory factor IL-6, characterized in that: The Staphylococcus epidermidis fermentation liquid is prepared from a fermentation medium containing glycerol and is a 0.5-fold fermentation dilution liquid.

9. Use of the Staphylococcus epidermidis fermentation broth according to claim 7 in scavenging active oxygen, increasing superoxide dismutase activity and / or reducing malondialdehyde content.

10. Use of the Staphylococcus epidermidis fermentation broth according to claim 7 in inhibiting melanin synthesis and / or inhibiting tyrosinase activity, characterized in that: The Staphylococcus epidermidis fermentation liquid is prepared from a fermentation medium containing glucose and is a 2-fold concentrated fermentation liquid.

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