CN114042031B

CN114042031B - Fermentation liquor extract of bacillus solitarius and application thereof

Info

Publication number: CN114042031B
Application number: CN202111552495.5A
Authority: CN
Inventors: 欧春凤; 周晗; 邹衡芳; 陈玉容
Original assignee: Guangzhou Yuanxiang Biotechnology Co ltd
Current assignee: Guangzhou Yuanxiang Biotechnology Co ltd
Priority date: 2021-12-17
Filing date: 2021-12-17
Publication date: 2022-10-21
Anticipated expiration: 2041-12-17
Also published as: CN114042031A

Abstract

The invention relates to an application of a fermentation liquor extract of Bacillus sonolavae in preparing an antioxidant product. The invention also provides a composite liquid of the extract of the fermentation liquor of the bacillus sonoralis, which comprises the following components in parts by weight: 80-90% of the extract of the fermentation liquor of the bacillus sonoralis, 0-20% of glycerol, 0-1% of p-hydroxyacetophenone, 0-1% of hexanediol and 0-1% of hydroxyethyl cellulose. In the research process, the obtained fermentation liquor extract of the Bacillus sorbierite has an antioxidation effect and can be used in antioxidative skin care products and medical and American products.

Description

Fermentation liquor extract of bacillus solitarius and application thereof

Technical Field

The invention relates to the field of microorganism application, in particular to a fermentation liquor extract of bacillus sonoralis and application thereof.

Background

Oxidation is one of the important threats of skin aging, and unhealthy diet, solarization, stress, environmental pollution and the like can cause free radicals of the skin to flood, so that the oxidation phenomena such as dark complexion, water shortage and the like are generated, and people need to pay attention to oxidation resistance in daily life no matter from a health level or a skin care level.

The antioxidation is not only environmental protection in the body, but also the antioxidation work is started on the face skin. The antioxidation of the face is to keep youth on the face and to prevent the damage of the external environment to the skin, such as the damage of ultraviolet rays, dirty air and the like to the skin, and the natural aging of the face can not leave obvious marks on the face all the year round, so the antioxidation and anti-aging skin care product has great attention.

In the anti-aging skin care products on the market, the skin is mainly protected against oxidation through chemically synthesized antioxidants, plant antioxidants and the like, however, the long-term use of chemically synthesized antioxidants such as BHA, BHT, TBHQ and the like is easy to cause poisoning accumulated in human bodies, and the chemically synthesized antioxidants have adverse effects on the liver, the spleen and the lung;

the bacillus sonoralis (Bacillus sonorensis) is a salt-tolerant bacillus, has strong capability of adapting to high-salt environment, keeps the osmotic pressure balance of cells through osmotic adjustment, has good physiological functions of sodium discharge and potassium absorption, and certain halophilic bacteria also contain retinal protein, thereby providing driving force for the movement of endoplasmic reticulum of the cells to maintain the normal metabolism of the cells. Some natural antibacterial substances are commercialized as biocontrol agents to be applied to agriculture, the salt-tolerant bacillus biocontrol agents are relatively stable, and compared with non-bacillus biocontrol agents and fungi biocontrol agents, the salt-tolerant bacillus antibacterial substances have great advantages in aspects of chemical compatibility, acid and alkali resistance, high temperature resistance and the like.

The research shows that the extract of the fermentation liquor of the Bacillus solitarian desert has excellent antioxidant effect, higher antioxidant effect than that of an artificially synthesized antioxidant, good safety, wide application in the field of cosmetics, high stability, and excellent synergistic antioxidant effect by scientific blending with various functional raw materials.

Disclosure of Invention

Based on the above, the invention aims to provide a fermentation liquor extract of the Bacillus sonoralis and application thereof.

The technical scheme for realizing the purpose comprises the following steps.

In a first aspect of the invention, the application of the fermentation liquor extract of the bacillus sonoralis is provided.

Application of fermentation liquor extract of Bacillus solitarius in preparing antioxidant product is provided.

The second aspect of the invention provides a skin care compound liquid with an antioxidant effect, which comprises the following components in parts by weight: 80-90% of the extract of the fermentation liquor of the bacillus sonoralis, 10-20% of glycerol, 0-1% of p-hydroxyacetophenone, 0-1% of hexanediol and 0-1% of hydroxyethyl cellulose.

In some preferred embodiments, the skin care compound liquid with the antioxidant effect comprises the following components in parts by weight: 80-85% of the extract of the fermentation liquor of the bacillus sonoralis, 10-15% of glycerol, 0.5-1% of p-hydroxyacetophenone, 0.5-1% of hexanediol, 0.5-1% of hydroxyethyl cellulose and the balance of water.

The skin care composite liquid with the antioxidation has the effects of improving the stability of the oxidation performance of the extract of the Bacillus sonolavae fermentation liquor and improving the activity effect.

In a third aspect of the invention, the product with antioxidation is provided, and the antioxidation active substance of the product contains the fermentation liquor extract of the bacillus sonneratidis or the skin care compound liquid with antioxidation.

In some embodiments, the product with the antioxidation effect is a cosmetic and comprises the following components, by weight, 2-20% of the composite liquid of the bacillus sonoralis fermentation liquid extract, 15-20% of a humectant, 0.1-0.25% of a thickening agent, 10-20% of grease, 5-8% of an emulsifier, 0.1-0.5% of a preservative, and the balance deionized water.

In some embodiments, the skin care compound liquid with the anti-oxidation effect is 8-12%, the small molecule sodium hyaluronate is 15-20%, the carbomer is 0.1-0.25%, the shea butter is 10-15%, the emulsifier is 5-8%, the preservative is 0.1-0.5%, and the balance is deionized water.

In some preferred embodiments, the skin care compound liquid with the antioxidant effect is 10%, the small molecule sodium hyaluronate is 15%, the carbomer is 0.1%, the shea butter is 10%, the fatty alcohol-polyoxyethylene ether is 5%, the potassium sorbate is 0.1%, and the balance is deionized water.

In some embodiments, the fermentation broth extract of bacillus sonolatus is obtained by the following preparation method, wherein the preparation method comprises the following steps:

(1) Activating the strain of the bacillus sonoralis desert;

(2) Culturing the seed solution of the bacillus sonoralis;

(3) And (3) fermentation:

inoculating the seed of the spore rod strain of the Sonola desert into a fermentation tank, and controlling the temperature to be 37 +/-0.5 ℃ and the dissolved oxygen to be 30 +/-3%; maintaining the pH value of the fermentation liquor in the tank at 7.0 +/-0.5 for fermentation;

(4) Centrifuging, collecting thallus, resuspending thallus, homogenizing, crushing, centrifuging, and collecting supernatant.

In one embodiment, the fermentation step comprises: sonola desert bacillus liquid seed OD ₆₀₀ Inoculating the strain with the strain accounting for 0.8 in a volume ratio of 1 to 9-11 into a fermentation tank; at the temperature of 37 +/-0.5 ℃, the initial stirring speed of 400 +/-50 rpm and the dissolved oxygen of 30 +/-3 percent; and (4) maintaining the pH value of the fermentation liquor in the tank to be 7.0 +/-0.5 for fermentation.

In some embodiments, the bacillus sonoralis is chinese Industrial microbial cultures Collection (CICC) CICC10847.

In some of these embodiments, the product is a pharmaceutical or cosmetic product.

In some of these embodiments, the cosmetic product is a medical, cosmetic, or beauty product.

In some of these embodiments, the cosmetic is a liquid formulation or cream or a lotion or a mask.

Compared with the prior art, the invention has the following beneficial effects:

the inventor of the invention discovers in research that the extract of the fermentation liquor of the bacillus solitarius has strong antioxidation function and skin care products for repairing skin tissue cells, and the skin care products can be prepared into skin care products in different dosage forms such as aqua, emulsion, cream and the like by using various rich SOD, vitamins, amino acids, mineral substances and the like contained in the extract of the fermentation liquor of the bacillus solitarius and blending the components with other added substances, and have strong antioxidation effect.

Drawings

FIG. 1 is a graph showing the results of the cytotoxicity assay in example 1.

FIG. 2 is a graph showing the results of the DPPH radical scavenging ability test experiment in example 2.

FIG. 3 is a graph showing the results of the antioxidant index test of cells in example 2.

FIG. 4 is a graph showing the results of the anti-oxidation experiment of zebrafish in example 4, wherein A and B are the effects of AAPH induced concentration in the control group on the survival rate of zebrafish embryos and the generation rate of active oxygen, and C and D are the effects of the concentration of Bacillus solitarius fermentation broth on the survival of zebrafish embryos.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The present invention will be described in further detail with reference to specific examples.

In some embodiments, the invention relates to a skin care product with antioxidant effect, which can be obtained by the following preparation method:

1) The preparation method of the Sonola desert bacillus fermentation liquor extract comprises the following steps: carrying out amplification culture on the bacillus sonoralis in an optimized proportion, fermenting through a fermentation tank, centrifugally collecting supernatant, filtering and storing;

2) The preparation method of the composite liquid of the extract of the fermentation liquid of the bacillus sonoralis desert comprises the following steps: adding 80-90% of the Bacillus solitarius fermentation broth extract, 10-20% of glycerol, 0-1% of p-hydroxyacetophenone, 0-1% of hexanediol and 0-1% of hydroxyethyl cellulose into water according to calculated amount, and stirring at 75-85 ℃ until no solid insoluble substances exist;

3) The preparation method of the antioxidant skin care product comprises the following steps: the antioxidant skin care product comprises, by mass, 15-20% of a humectant, 0.1-0.25% of a thickener, 10-20% of grease, 5-8% of an emulsifier, 0.1-0.5% of a preservative, and the balance deionized water, wherein the content of the composite liquid of the Bacillus Sonola desert bacillus fermentation liquor extract is 2-20%.

In the invention, the emulsifier is one or more of sodium dodecyl sulfate, fatty alcohol-polyoxyethylene ether and oliver 1000; the oil is one or more of shea butter, jojoba oil, simethicone and squalane.

The preservative is one or more of potassium sorbate and 1, 2-hexanediol.

The thickener is one or more of carbomer, acrylate copolymer, xanthan gum and hydroxyethyl cellulose.

The humectant is one or more of small molecular sodium hyaluronate, trehalose, dextran, tremella polysaccharide and allantoin.

Example 1

The method for extracting the fermentation liquor of the Bacillus Nolatani comprises the following steps:

(1) The strain (platform resource number: 1511C0005000010884, strain preservation number: CICC10847, chinese name: bacillus sonorensis, latin name: bacillus sonorensis) is streaked on LB solid medium, and cultured for 16h at 37 ℃. The single clone was picked up to 30mL of LB liquid medium, cultured at 37 ℃ and 220rpm, and the cultured strain was preserved.

(2) The fermentation is carried out by using a 5L fermentation tank, and the main fermentation steps are as follows:

(2.1) Strain activation

A tube of the preserved strain is taken from-80 ℃, frozen and thawed at normal temperature, 30 mu L of the strain is inoculated into 30mL of TB culture medium (the TB culture medium is taken as a basic culture medium, and the contents of the TB culture medium are respectively 10g/L of peptone, 24g/L of yeast powder, 12g/L of glucose, 1.2g/L of magnesium sulfate, 0.001g/L of ferrous sulfate and 0.01g/L of copper sulfate), and the strain is cultured at 37 ℃ for 8h (9-17.

(2.2) liquid culture of seeds

Transfer 300. Mu.L of activated bacteria to 330mL TB medium. The culture was carried out at 37 ℃ and 220rpm for 16 hours with shaking.

(2.3) setting of initial fermentation conditions of 5L fermentation tank

The fermentation culture adopts 5L fermentation tank with working volume of 3L and seed OD ₆₀₀ The fermenter was inoculated with an inoculum size of 1 (v/v) =0.8, and the seed solution concentration was measured. The initial pH was set at 7.0, the temperature at 37 deg.C, the initial stirring speed at 400rpm, and dissolved oxygen at 30%. And the acid, alkali, defoaming, temperature and dissolved oxygen are regulated to be automatic. 4mol/L NaOH solution and 2mol/L HCl solution are added in the whole fermentation process to adjust the pH value, and the pH value of the fermentation liquor in the tank is maintained to be about 7.0.

(2.4) feeding method

Feeding the residual sugar concentration feedback.

(2.5) centrifugation was carried out at 4200RPM for 30min, and the cells were collected, resuspended in PBS (1. Filtering with 0.22um filter membrane to obtain the fermentation product.

Cytotoxicity assays

The experimental process comprises the following steps: the cytotoxicity of the stock solution of the Bacillus solitarius fermentation broth extract described in example 1 was examined by measuring the absorbance value (OD value) at a wavelength of 540nm by neutral red uptake exposure (NRU) using BALB/3T3 cells, a mouse fibroblast cell line.

Incubation with 96-well plates, positive controls: sodium Lauryl Sulfate (SLS); negative control: ultrapure water; the test substance: the extract of the fermentation broth of the Bacillus sonoralis desert is prepared into 8 experimental concentrations (1000 ug/ml,100ug/ml,10ug/ml,1ug/ml,0.1ug/ml,0.01ug/ml,0.001 ug/ml) from the stock solution of the test substance by a logarithmic dilution method.

Referring to fig. 1, the experimental results show that when the concentration of the bacillus sonoralis is below 100ug/ml, the average value of relative cell activity is 83.3%, compared with the control group (SLS), the concentration of the bacillus sonoralis fermentation liquor extract is not more than 80%, and the extract has no cytotoxicity at the concentration.

Example 2

Experiment for testing scavenging capability of DPPH free radical

DPPH · solution: weighing 3mgDPPH with an electronic analytical balance, dissolving with absolute ethanol, fixing the volume in a 50ml volumetric flask with the concentration of 60 mug/ml, and shaking up for later use.

Preparing 5 fermentation liquor extracts of the Bacillus sonneraticus in example 1 with different concentrations, wherein the concentration is 1000 mug/ml, 800 mug/ml, 600 mug/ml, 400 mug/ml and 200 mug/ml respectively, sealing and shaking up for later use.

Preparing 5 vitamin E solutions with different concentrations of 1000. Mu.g/ml, 800. Mu.g/ml, 600. Mu.g/ml, 400. Mu.g/ml and 200. Mu.g/ml respectively, sealing, and shaking up for use.

Setting ddH ₂ O is a negative control.

90ul of DPPH solution and 10ul of test substance were added to a 96-well plate, mixed well and then the absorbance at 530nm was measured. Calculation of radical clearance SA (%) = (a) _{Control of} -A _{Experiment of} )/A _Control ×100％。

The results are shown in Table 2.1 and FIG. 2

TABLE 2.1

(II) cell antioxidant index test

1. Screening for H Using MTT ₂ O ₂ Concentration of mold

H at 1000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8, 3.9nmol/L, respectively ₂ O ₂ Treatment of 3T3 FineCell for 24 hr, detecting absorbance value with wavelength of 570nm with microplate reader, calculating inhibition rate, and IC 24 hr after administration ₅₀ =600nmol/L, the concentration is selected as H ₂ O ₂ Inducing the molding concentration of the oxidative stress model.

2. Screening for appropriate test substance concentrations Using MTT

And calculating the inhibition rate according to the absorbance value, and selecting the concentration of the test object as 100 mu g/ml for the next experiment.

3. Cellular antioxidant index test

(1) Inoculating cells into 6-well plate at certain cell density, culturing for 12 hr, replacing culture medium containing different medicines, establishing blank control group, and culturing ₂ O ₂ Group H ₂ O ₂ Adding Vc group, H ₂ O ₂ Adding the extract group of the Bacillus solitarius fermentation liquor;

(2) The medium was aspirated and rinsed 2 times with PBS;

(3) Adding 1ml PBS, scraping cells with a cell scraper, centrifuging to remove supernatant, adding 500ul homogenate medium according to the requirements of MDA, SOD, CAT and GSH-PX reagent kit, and crushing cells with a cell ultrasonic crusher in ice bath for 200W, 2s/time, 3s of gap and 10min of total time.

(4) The supernatant was centrifuged and assayed according to the kit instructions. And evaluating according to the relevant indexes of oxidative stress of the cells, such as MDA content, SOD enzyme activity, CAT enzyme activity and GSH enzyme activity.

The results are given in table 2.2 below and fig. 3:

the results show that H is compared to the blank control ₂ O ₂ The MDA content of the group is obviously increased, and the enzymatic activities of SOD, CAT and GSH are obviously reduced. And H ₂ O ₂ Compared with the Vc group and the Sonola desert bacillus fermentation liquor extract group, the content of MDA is reduced, and the activities of SOD, CAT and GSH are increased, so that the Sonola desert bacillus fermentation liquor extract is similar to the VC antioxidant mechanism, and the antioxidant effect of the Sonola desert bacillus fermentation liquor extract is superior to that of the VC antioxidant.

Example 3

A skin care composite liquid (antioxidant component) with antioxidant effect contains a composite liquid of a Sonola desert bacillus fermentation broth extract, and the mass ratio of each component is as follows: 80% of the extract of the fermentation liquor of the bacillus sonoralis, 10% of glycerol, 0.5% of p-hydroxyacetophenone, 0.5% of hexanediol and 0.9% of hydroxyethyl cellulose.

The preparation method comprises the following steps: heating the glycerol, p-hydroxyacetophenone, hexanediol, hydroxyethyl cellulose and a proper amount of water to 60-80 deg.C, stirring for dissolving, cooling to 40 deg.C, and adding into the extract of Bacillus somnosalae fermentation broth.

The composite liquid of the Bacillus somnoelans fermentation liquid extract is used for preparing an antioxidant skin care product and comprises the following components, by mass, 10% of the composite liquid with the antioxidant effect, 15% of small-molecule sodium hyaluronate, 0.1% of carbomer, 10% of shea butter, 5% of fatty alcohol-polyoxyethylene ether, 0.1% of potassium sorbate and the balance of deionized water.

The antioxidant skin care product is prepared by the following steps:

adding the micromolecular sodium hyaluronate, carbomer, potassium sorbate and deionized water in the above content into a water phase pot, heating to 85-90 ℃, and stirring to dissolve to obtain a water phase; adding the shea butter, fatty alcohol-polyoxyethylene ether and other materials into an oil phase pot, stirring and heating to 80-85 ℃, and pumping into an emulsifying pot after complete dispersion to obtain an oil phase; slowly pumping the materials in the water phase pot into an emulsifying pot through a filter screen for emulsification, stopping homogenizing after emulsifying for 15-40 minutes, starting circulating water for cooling, keeping stirring, vacuumizing and defoaming; cooling to 45 deg.C, adding the complex liquid of the Bacillus sorbierite fermentation broth extract, stirring, cooling to 36 deg.C, and discharging to obtain antioxidant emulsion.

Example 4

1. Acute transdermal toxicity test

1. Materials:

(1) Test substance example 3 skin Care composition (antioxidant emulsion).

(2) Animals including healthy SPF SD rat, 5 male animals and 5 female animals, and the weight range of the animals is 200g-300g.

2. Detection foundation GB7919-1987 (5.1)

3. The test method comprises the following steps:

(1) The hair is removed at both sides of the median line of the back of the animal 24 hours before the test, the surface area is more than 10 percent of the body surface area, and the skin is carefully inspected and is required to be complete and not damaged.

(2) The contamination method comprises uniformly coating the tested substance on the hair-removed area, covering with a layer of impermeable material, covering with 2 layers of porous gauze, and fixing with non-irritating adhesive plaster. After 24h of contact, the fixture and cover were removed and the skin was cleared of residual test substance with warm water.

(3) After the infection, the animals were observed for the toxic manifestation, the number of deaths and the death time, and the observation time was 14D. Body weights were weighed at the contaminated OD, 7D, 14D.

LD50 was calculated from the data and judged on a percutaneous toxicity grading Table.

4. The result of the detection

TABLE 4.1

5. Conclusion

The median lethal dose LD50 of the test substance to rats is more than 2000mg/kg.bw, and the acute toxicity dose is graded as low toxicity.

Second, single contact test

1. Materials:

(1) Test substance example 3 skin Care composition.

(2) Animals comprise 3 healthy adult female common-grade New Zealand rabbits, the weight range is 2-3 kg, and the animals are quarantined for 3 days before the test.

2. The detection basis is GB/T16886.10-2017/6.3

3. The test method comprises the following steps:

(1) The hair on both sides of the spine of the experimental animal is cut off or shaved 4-24h before the experiment, the epidermis cannot be damaged, and the hair removal range is about 10cm multiplied by 15cm respectively.

(2) On the next day, 0.5mL of each sample of the test substance is dripped on the unhaired skin of the corresponding area on both sides, then covered by a non-irritating plastic film, fixed and applied on a blank control area for 4 hours by using a non-irritating adhesive tape, the treatment method and the treatment time are the same as those of the test area, and the test is carried out by using the same amount of sterilized normal saline. The skin local reactions were observed for 1h, 24h, 48h and 72h after removal of the test article, respectively, and the irritation response scores were performed.

4. The result of the detection

TABLE 4.2

Test group animal weights: 1.2.1099kg 2. 2.1135kg 3. 2.0958kg

body weight of positive control group animals: 1.2.1121kg 2. 2.1098kg 3. 2.1310kg

5. and (4) detection conclusion: the skin care product composition has the skin irritation index of 0 to New Zealand rabbits, and has very slight skin irritation strength.

Anti-oxidation experiment of zebra fish

(1) The tested substance is the extract of the fermentation liquor of the Bacillus sonoralis in example 1.

(2) Selecting healthy zebra fish (average body length is 3.46cm, average weight is 161.37 mg) with similar size, changing water every 2d during the test period, feeding basic bait for 2 times every day, dark period ratio is 14h/10h, water temperature is 27 ℃, pH value is 7.2, dissolved oxygen is 7.8mg/L, and hardness is 250mg/L calculated by CaCO 3. Zebrafish were placed in the proportion of 2 male and female fish.

(3) Constructing an AAPH-induced zebra fish oxidative stress model: selecting fish embryos which normally develop 8-9 hpf, and placing the fish embryos in a 6-hole cell culture plate with 20 fish embryos per hole. Adding 2ml of embryo culture fluid per well (10% NaCl,0.3% KCl,0.3% ₂ ，0.79％MgSO ₄ ) Or embryo culture solutions (5, 10, M) containing AAPH-inducing solutions,15. 20 and 25 mmol/l), arranging three parallel control holes in each group, culturing in a constant-temperature incubator at 27 ℃, and detecting the survival rate of zebra fish embryos and the generation rate of active oxygen.

(4) Evaluation of the toxicity of the extract of the fermentation liquor of the bacillus sonoralis: selecting a certain concentration (1, 10, 100, 1000, 2000 mug/mL) of the extract of the Bacillus Sonolaris fermentation liquor to carry out toxicity evaluation experiments, adding a sample into 8-9 hpf of embryo development, then placing the sample into an incubator at 27 ℃ to culture, and carrying out detection on the survival rate of the zebra fish embryos.

Survival rate of zebra fish: and (4) counting the development condition of embryos observed on time every day, removing dead embryos in time, and finally counting the survival rate of the juvenile fishes of each experimental group in the period from development to 72 hpf.

(5) The oxidation resistance evaluation of the fermentation liquor extract of the Bacillus sonolania in the zebra fish is as follows: according to the toxicity evaluation result, selecting the nontoxic concentration range of the extract of the Bacillus sonoralis fermentation liquor to carry out an antioxidant activity evaluation experiment, carrying out embryo administration on 8-9 hpf of embryo development, carrying out pre-incubation in a constant temperature incubator at 27 ℃, and carrying out oxidation induction by using AAPH induction concentration (15 mmol/L) after 1 h. The experiment is divided into a blank control group, an AAPH model group and an AAPH induction + Bacillus somnophilus fermentation liquor extract group, each group is provided with three parallel holes, and the experiment system is 2mL. Blank control group: 2mL of blank embryo culture solution; AAPH model group: 2mL of an AAPH inducer (final concentration 15 mmol/L) prepared from an embryo culture solution; AAPH induction + bacillus sonola fermentation broth extract group: 1.9mL of the sample solution of the Bacillus Desmophilus fermentation broth extract with no toxicity concentration range prepared by embryo culture solution is added into each well, and 0.1mL of AAPH inducer (final concentration 15 mmol/L) is added after pre-incubation for 1 h. After the completion of the administration, the zebra fish embryos were transferred to an incubator at 27 ℃ for further incubation for 72 hours, 100. Mu.L of 1g/mL tricaine solution was added to each well to anesthetize the zebra fish, and photographs were taken under a fluorescence microscope, and the ROS production rate (%) of each group was expressed as the ratio of the fluorescence intensity of the sample group to that of the blank group. Statistical treatment results are expressed in (± s). See table below and figure 4 for results.

TABLE 4.3 Effect of AAPH inducing concentration on zebrafish embryo survival and reactive oxygen species production

TABLE 4.4 Effect of the concentration of the Bacillus somnophilus fermentation broth on the survival of zebrafish embryos

TABLE 4.5 Effect of Bacillus somnophilus fermentation broth extract on ROS levels in zebrafish embryos

Excessive accumulation of ROS in cells can deplete cellular organelles and biomolecules, and the sample set decreases ROS production rate in a dose-dependent manner over a range of concentrations, indicating that the antioxidant properties of the sample increase in a dose-dependent manner.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The application of the fermentation liquor extract of the bacillus solitarian desert in preparing the antioxidant product is characterized in that the fermentation liquor extract of the bacillus solitarian desert is prepared by the following steps:

(1) Activating the strain of the bacillus sonoralis desert;

(2) Culturing the seed solution of the bacillus sonoralis;

(3) And (3) fermentation: inoculating the seed of the spore rod strain of the Sonola desert into a fermentation tank, and controlling the temperature to be 37 +/-0.5 ℃ and the dissolved oxygen to be 30 +/-3%; maintaining the pH value of the fermentation liquor in the tank at 7.0 +/-0.5 for fermentation;

(4) Centrifuging, collecting thallus, resuspending, homogenizing, crushing, centrifuging, collecting supernatant,

the strain preservation number of the Bacillus sonolaraensis is CICC10847 of China Industrial microbiological culture Collection center, and the antioxidant product is a cosmetic.

2. Use according to claim 1, wherein the fermentation comprises: sonola desert bacillus liquid seed OD ₆₀₀ =0.8, inoculating into a fermentation tank according to the inoculation amount of 1; at the temperature of 37 +/-0.5 ℃, the initial stirring speed of 400 +/-50 rpm and the dissolved oxygen of 30 +/-3 percent; and (5) maintaining the pH value of the fermentation liquor in the tank to be 7.0 +/-0.5 for fermentation.

3. The skin care compound liquid with the antioxidation function is characterized by comprising the following components in parts by weight: the extract of fermentation broth of Bacillus sonoralis of claim 1, which comprises 80-90% glycerol, 10-20% p-hydroxyacetophenone, 0-1% hexylene glycol, 0-1% hydroxyethyl cellulose, and the balance deionized water.

4. The skin care compound liquid with the antioxidant effect as claimed in claim 3, which is characterized by comprising the following components in parts by weight: the extract of fermentation broth of Bacillus solitarius of claim 1, 80-85%, glycerol 10-15%, p-hydroxyacetophenone 0.5-1%, hexanediol 0.5-1%, hydroxyethyl cellulose 0.5-1%, and deionized water in balance.

5. A product having an antioxidant effect, characterized in that the antioxidant active substance of the product comprises the fermentation broth extract of bacillus sonotrodestris of claim 1, and the product having an antioxidant effect is a cosmetic.

6. The product with antioxidant effect as claimed in claim 5, characterized by comprising the following components by weight: the skin care compound liquid with antioxidant effect of claim 3, which comprises 2-20% of humectant, 15-20% of thickener, 0.1-0.25% of grease, 5-8% of emulsifier, 0.1-0.5% of preservative and the balance of deionized water.

7. The product with antioxidant effect as claimed in claim 5, which comprises the following components by weight: the skin care complex liquid with antioxidant effect of claim 3, 8-12%, small molecule sodium hyaluronate 15-20%, carbomer 0.1-0.25%, shea butter 10-15%, emulsifier 5-8%, preservative 0.1-0.5%, and the balance of deionized water.

8. The product with antioxidant effect as claimed in any one of claims 5 to 7, wherein the cosmetic is a medical and cosmetic product.

9. The product having antioxidant effect as claimed in any one of claims 5 to 7, characterized in that the cosmetic is a skin care product.

10. Product with antioxidant action according to any of claims 5 to 7, characterized in that the cosmetic product is a cosmetic product.

11. The product having antioxidant effect as claimed in any one of claims 5 to 7, wherein the cosmetic is a liquid preparation or a cream or a mask.

12. Product with antioxidant action according to any of claims 5 to 7, characterized in that the cosmetic product is a lotion.