CN115637236A - Keratinase-producing strain TC5 and application thereof - Google Patents

Abstract

The invention discloses a keratinase-producing strain TC5 and application thereof. The strain can produce keratinase and has the function of hydrolyzing keratin. Through determination, after the strain is cultured for 72 hours, the activity of the keratinase reaches 103.69U/mL, the keratinase produced by the strain still has higher activity in a medium-temperature environment, and the strain has stronger feather degradation effect, and has important value on the research of the keratinase and the application in industrial production.

Description

Keratinase-producing strain TC5 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, and in particular relates to a strain of keratinase-producing bacteria Bacillus sp.TC5 intersecting mountain and lake sediment sources and application thereof.

Background

The sediment microorganisms are important components of the ecosystem of the high mountain lake. As a special fresh water ecosystem, the high mountain lake has less animal and plant activities relative to lakes with low altitude, so that microorganisms play an important role in the material circulation and energy conversion processes of the ecosystem. The mountain lakes provide special living environments for microorganisms due to the special geographical positions of the mountain lakes. In order to adapt to the special habitat of the sediments in the high mountains and lakes, microorganisms gradually evolve various physiological and biochemical characteristics, so that a large amount of novel bioactive substances and genetic substances with potential application values are generated.

The keratin is insoluble structural protein widely existing in hair, skin, feather, claw and the like, contains more disulfide bonds, hydrogen bonds and hydrophobic acting force, and has the characteristics of stable chemical structure, insolubility in water and difficult degradation. As a big livestock and poultry breeding country in China, about 70 million tons of high-quality feather protein is produced every year, more than 80 percent of the feather protein is keratin, but most of the feather protein is not reasonably utilized, and even serious environmental pollution is caused by improper treatment. Tens of thousands of feathers are degraded slowly when naturally accumulated, and attached nitrogen, phosphorus and sulfur-containing substances such as excrement, blood and the like are also easy to become nutrient sources of other pathogenic bacteria, so that the environment pollution is caused by improper treatment. These protein-rich wastes were previously made into feeds by high-pressure heat hydrolysis, but this method not only consumes a lot of energy, but also loses a lot of nutrients because of too high temperature. Therefore, there has been a lot of interest in alternatively biologically transforming waste feathers. The biological method mainly adopts microorganisms to degrade the keratin, and has the advantages of mild conditions, energy conservation and no damage to nutrient substances in the keratin. Keratinase degrades feather keratin and produces low-quality peptides with antioxidant activity, providing an alternative to efficient bioconversion and increasing its nutritional value.

Keratinase (Keratinase) is a class of enzymes that specifically hydrolyze insoluble keratin such as feathers, hair, etc. to soluble proteins or polypeptides. The keratinase has wide sources, bacteria, fungi and actinomycetes can secrete the keratinase, and the physicochemical properties are greatly different due to different sources. At present, various keratinases are separated and purified from various wild strains and genetic engineering bacteria for producing the keratinases, and the physicochemical properties of the keratinases are researched. The keratinase with different properties has wide application field and good application prospect and commercial value. For example, the application of the depilatory in leather production can improve the quality of fur production and reduce the environmental pollution generated in the production process. Keratinase can also be used in detergent to enhance the effect of removing different stains, or in skin care products to remove cutin on the surface of skin, help the skin care products to reach the deep layer of skin, and help the skin to absorb nutrients in the skin care products. The application range of the keratinase is very wide, and the application range has great significance for realizing the high-efficiency utilization and high-valued application of the keratin resources, so that the research of the keratinase shows great value.

Disclosure of Invention

The invention aims to provide a strain of keratinase-producing bacteria Bacillus sp.TC5 from sediments in transected mountains and lakes and application thereof.

A strain of keratinase-producing bacteria, which is classified and named as Bacillus sp.TC5, wherein the Bacillus sp.TC5 strain is preserved in China Center for Type Culture Collection (CCTCC) at 8 months and 6 days in 2021, and the address is as follows: the preservation number is CCTCC M2021975.

The keratinase-producing bacteria (Bacillus sp.TC5) are separated from sediments of mountains and lakes crossing mountains.

The bacterial colony of the strain cultured on an LB flat plate for 24 hours is white and round, the surface is smooth and semitransparent, and the edge is neat; gram staining is positive, the thallus is rod-shaped, and spores are present. The keratinase-producing bacterium Bacillus sp.TC5 is subjected to physiological and biochemical determination, and has negative oxidase test, negative catalase test and negative lecithin enzyme test, can hydrolyze gelatin and casein, and has negative methyl red test and H ₂ The S test is positive, and the nitrate reduction test is positive.

The keratinase-producing bacterium (Bacillus sp.) TC5 can produce keratinase and has the function of hydrolyzing keratin.

The application of the keratinase-producing bacteria (Bacillus sp.) TC5 is used for producing keratinase and hydrolyzing keratin.

Specifically, keratinase-producing bacteria TC5 keratinase are used for feather degradation, skin depilation or skin exfoliation.

Further, the fermentation liquor obtained by the keratinase-producing bacteria TC5 is matched with calcium thioglycolate to be applied to skin depilation.

Further, keratinase-producing fermentation medium (g/L): 10g of duck feather and Na ₂ HPO ₄ 1 g，KH ₂ PO ₄ 0.3g， Na ₂ CO ₃ 1 g，MgSO ₄ ·7H ₂ O 1g，CaCl ₂ 1 g,pH 7.8；

Further, inoculating the keratinase-producing strain TC5 in an LB liquid culture medium, culturing at 30 ℃,180rpm for 24h, inoculating a fermentation culture medium according to 2% of the inoculum size, performing shaking culture at 30 ℃,200rpm for 120h, taking fermentation liquor every 24h, centrifuging at 10000rpm to remove thalli, and obtaining a supernatant which is a crude enzyme solution.

When the keratinase-producing bacteria TC5 produce keratinase, the keratinase is activated by manganese ions, calcium ions, magnesium ions, potassium ions, cobalt ions, sodium ions and barium ions and is inhibited by high-concentration aluminum ions, iron ions, copper ions and zinc ions.

Further, the keratinase-producing strain TC5 reached the optimum reaction temperature at 55 ℃.

Further, keratinase-producing bacteria TC5 keratinase reach the optimum reaction pH at a pH of 9.5.

The strain can produce keratinase and has the function of hydrolyzing keratin. Through determination, after the strain is cultured for 72 hours, the activity of the keratinase reaches 103.69U/mL, the keratinase has higher activity in a medium-temperature environment, and has stronger feather degradation effect, and the strain has important value on the research of the keratinase and the application in industrial production.

Drawings

FIG. 1 screening of keratinase-producing strains;

left panel: no inoculation, right panel: inoculating strain TC5 and culturing for 72 hours;

FIG. 2. Keratinase-producing bacterium Bacillus sp.TC5 and system constructed by part of related strains according to 16S rDNA sequence

Developing the tree;

FIG. 3 shows the activity of the keratinase-producing bacteria Bacillus subtilis TC5 in fermentation culture at different times;

FIG. 4 shows the enzyme activity of the protease Bacillus sp.TC5 of keratinase-producing bacteria under different metal ion conditions;

FIG. 5. Relative activity and thermal stability of Bacillus sp.TC5 keratinase at different temperatures;

FIG. 6 relative activity and acid-base stability of Bacillus sp.TC5 keratinase at different pH;

left panel: keratinase optimum reaction pH right diagram: acid-base stability;

FIG. 7.Bacillus sp.TC5 feather degrading ability by keratinase;

FIG. 8 is an HE staining electron microscope image of rat skin treated with Bacillus sp.TC5 fermentation broth for 10 h;

wherein A is a photograph of a dehairing skin mirror of rat skin treated by Bacillus sp.TC5 fermentation liquor, and B is an HE staining electron microscope image of the dehaired rat skin treated by the Bacillus sp.TC5 fermentation liquor.

FIG. 9 is an HE staining electron micrograph of Bacillussp.TC5 fermentation broth treated Bama corium Sus domestica for 12 h;

wherein A is a photograph of a skin mirror for dehairing Bama miniature pig skin treated by Bacillus sp.TC5 fermentation liquor, and B is an HE dyeing electron microscope image of the Bama miniature pig skin treated by the Bacillus sp.TC5 fermentation liquor after dehairing.

FIG. 10 shows an electron microscope for HE staining of rat skin treated by Bacillussp.TC5 fermentation broth lyophilized powder mixed with calcium thioglycolate depilatory cream;

wherein A is a photograph of a depilatory skin mirror treated by Bacillus sp.TC5 fermentation broth freeze-dried powder mixed with calcium thioglycolate depilatory cream, and B is an HE dyeing electron microscope image of the depilatory skin treated by Bacillus sp.TC5 fermentation broth freeze-dried powder mixed with calcium thioglycolate depilatory cream.

FIG. 11 is an electron microscope image of HE staining of rat skin treated with a commercial depilatory cream;

wherein A is the photograph of depilatory skin mirror of rat skin treated by the commercial depilatory cream, and B is the photograph of HE staining electron microscope after depilatory of rat skin treated by the commercial depilatory cream.

FIG. 12 is HE staining electron microscopy images of Bacillussp.TC5 fermentation broth treated Bama fragrant pigskin for 1,2, 3, 4, 5 h.

Detailed Description

The invention is further explained below with reference to the drawings and the specific examples, without limiting the invention in any way. The following examples are provided by conventional reagents and method steps in the art, unless otherwise indicated.

Example 1 treatment of isolated samples of bacteria TC5

Weighing 10g of sediment sample stored at 4 ℃, placing the sediment sample into a 100ml sterilized normal saline conical flask to prepare a sample suspension with the mass fraction of 10%, oscillating the sample suspension at the rotating speed of 180rpm for 30min, standing the sample suspension for 30s, and performing gradient dilution on the suspension, wherein the dilution times are respectively 10 ^-1 ，10 ^-2 ，10 ^-3 ，10 ^-4 ，10 ^-5 ，10 ^-6 ，10 ^-7 From dilution factor 10 ^-5 ，10 ^-6 ，10 ^-7 The liquid of (2) was applied to an LB solid medium plate in an amount of 200. Mu.l each, three replicates were set for each dilution, and then the plate was subjected to static culture in an incubator at 37 ℃ and 16 ℃. After each colony grows well, the plate streaking is repeated, and separation and purification are carried out. After thatRepeatedly streaking the strain separated from LB on a milk flat plate, placing the milk flat plate in a thermostat at 16 ℃ and 37 ℃ for static culture for 12-36h, and selecting the strain with an obvious degradation loop to obtain the strain producing protease. Then, after the protease-producing strain is inoculated into an LB culture medium and cultured to a logarithmic growth phase, a bacterium solution is sucked and inoculated into a feather fermentation culture medium at 2% (v/v), fermentation culture is carried out for 72 hours at 16 ℃,30 ℃ and 200rpm, the state of the feather in the culture medium is observed, and the feather is completely degraded and disappears to obtain the keratinase-producing strain TC5 (figure 1). Wherein, the left graph: no inoculation, right panel: the inoculated strain TC5 was cultured for 72 hours.

Example 2 biological Properties and physiological, biochemical and molecular characterization of Strain TC5

S1, morphological characteristics: the bacterial colony morphology of the strain cultured on an LB solid medium plate for 96h is as follows: the bacterial colony is white and round, the surface is smooth and semitransparent, and the edge is neat; gram staining is positive, the thallus is rod-shaped and has spores.

S2, physiological and biochemical characteristics: according to physiological and biochemical determination, the bacterial strain TC5 is negative in an oxidase test, negative in a catalase test and negative in an lecithinase test, can hydrolyze gelatin and casein, is negative in a methyl red test, and is H ₂ The S test is positive, and the nitrate reduction test is positive.

See table 1.

TABLE 1 physiological and biochemical characteristics of keratinase-producing bacteria TC5

Note: positive; -, negative; w, white; r, rod shape

S3, sequence determination and analysis of 16S rDNA of strain TC5

S31, extracting bacterial genome DNA: biospin bacterial genome extraction kit (BioFlux, hangzhou)

PCR amplification of S32.16S rDNA gene

The PCR primers were synthesized by Suzhou Hongxn Biotechnology Ltd.

Primer A：5’-AGAGTTTGATCCTGGCTCAG-3’

Primer B：5’-ACGGCTACCTTGTTACGACTT-3’

The PCR reaction system is as follows:

genomic DNA	0.5μL
		Primer A	0.5μL
Primer B	0.5μL
		2×Taq Master Mix	10μL
Nuclease-free water	8.5μL

PCR amplification conditions: at 95 ℃ for 5min, at 95 ℃ for 30s, at 55 ℃ for 1min, at 72 ℃ for 90s, for 30 cycles; 10min at 72 ℃.

And (3) sequence determination: after the PCR product is purified, it is sent to the engine biology sequencing, and it is compared with the known sequence in GenBank database for BLAST analysis, and the 16S rDNA sequence of the related species is obtained from the database, and the phylogenetic tree is constructed, as shown in figure 2. Through comparison and analysis, the homology of the keratinase-producing strain TC5 and the strain Bacillus stratosphericus JN179 is the highest, and the 16S rDNA sequence of the strain TC5 has 99 percent of homology with the strain Bacillus stratosphericus JN 179.

The 16S rDNA sequence analysis and the physiological and biochemical characteristic analysis are integrated, which shows that the strain belongs to the keratinase producing strain, namely the keratinase producing strain Bacillus TC5, the strain Bacillus TC5 is preserved in China Center for Type Culture Collection (CCTCC) at 8 months and 6 days in 2021, and the preservation number is as follows: CCTCC M2021975.

Example 3 determination of the Activity of the keratinase enzyme produced by the keratinase-producing bacterium Bacillus sp.TC5

Inoculating keratinase-producing strain TC5 into an LB liquid culture medium, culturing at 30 ℃ and 180rpm for 24 hours, inoculating a fermentation culture medium according to the inoculum size of 2%, performing shaking culture at 30 ℃ and 200rpm for 120 hours, taking fermentation liquor every 24 hours, centrifuging at 10000rpm to remove thalli, obtaining supernatant as crude enzyme liquid, and measuring the activity of keratinase by adopting a Gradisar method: and adding 40 mul of diluted enzyme solution into 40 mul of 0.5 percent soluble keratin substrate, carrying out water bath reaction at 50 ℃ for 10min, and adding 80 mul of 0.4mol/l TCA solution to terminate the reaction. The control group was terminated by adding the same volume of TCA solution in advance, and the rest conditions were the same. After the reaction is finished, centrifuging at 10000r/min for 2min, and absorbing the supernatant to determine the absorbance at the wavelength of 280 nm.

Definition of the unit of keratinase activity:

the amount of enzyme (U) required to make OD at 280nm higher than that of the control group by 0.01 per minute in a mixed reaction system at 50 ℃. Test results show that the activity of the keratinase can reach 103.69U/mL after the keratinase-producing bacteria Bacillus sp.TC5 is cultured for 72 hours, and the figure is shown in figure 3.

Feather fermentation medium (g/L): 10g of duck feather and Na ₂ HPO ₄ 1 g，KH ₂ PO ₄ 0.3g，Na ₂ CO ₃ 1 g，MgSO ₄ ·7H ₂ O 1g，CaCl ₂ 1 g,pH 7.8；

Example 4 Activity of keratinase enzyme produced by Strain Bacillus mucilaginosus sp.TC5 producing keratinase under different Metal ion conditions

Al at 50mM concentration ³⁺ ,Ba ²⁺ ,Mn ²⁺ ,Co ²⁺ ,Fe ³⁺ ,Mg ²⁺ ,Zn ²⁺ ,K ⁺ ,Fe ²⁺ ,Cu ²⁺ ,Na ⁺ And Ca ²⁺ And (3) solution. Obtaining the enzyme after fermentationThe solutions were mixed with each ion solution, the enzyme solution of the control group was diluted by the same fold with Tris-HCl buffer, the keratinase activity was measured at the final ion concentrations of 2.5mM and 10.0mM, and the effect of each metal ion on the keratinase activity was observed (see FIG. 4).

In FIG. 4, it can be seen that: when the keratinase-producing bacteria TC5 produce keratinase, the keratinase is activated by manganese ions, calcium ions, magnesium ions, potassium ions, cobalt ions, sodium ions and barium ions and is inhibited by high-concentration aluminum ions, iron ions, copper ions and zinc ions.

Example 5 optimum reaction temperature and thermal stability of keratinase produced by keratinase-producing bacterium Bacillus sp.TC5

Carrying out keratinase activity determination on a reaction system of keratinase at 30, 40, 45, 50, 55, 60 and 70 ℃ respectively, and determining the optimal reaction temperature of the keratinase; the enzyme solutions were incubated in 37, 50 and 60 ℃ water baths for 15, 30, 45 and 60min, respectively, and the enzyme solutions stored at 4 ℃ were used as controls to determine the activity of keratinase at the optimum temperature, respectively, and to analyze the thermostability of keratinase.

The test results show that the influence of temperature on the activity of the keratinase is firstly promoted to be inhibited, and the activity of the keratinase reaches the highest value at about 55 ℃ and then gradually decreases. The keratinase still retains 70 percent of enzyme activity after being warm-bathed for 1 hour at medium temperature (37-50 ℃) and has good thermal stability.

Example 6 optimum pH and acid-base stability of keratinase produced by keratinase producing bacterium Bacillus sp.TC5

Mu.l of keratinase was mixed with 20. Mu.l of buffers of 50mM at different pH (4.0-11.0) using the following buffer systems: citric acid-Na ₂ HPO ₄ Carrying out enzymatic reaction on a buffer solution (pH 4.0-7.0), a Tris-HCl buffer solution (pH 7.0-9.0) and a Gly-NaOH buffer solution (pH 9.0-11.0) at the optimal temperature of 55 ℃, comparing absorbance and determining the optimal pH value; diluting the enzyme solution in buffer solutions with different pH values in equal proportion, standing at 4 ℃ for 60min, performing enzymatic reactions at the optimal temperature by taking untreated enzyme solution (namely fermented enzyme solution which is not diluted by the buffer solution and adjusted by the pH value) as a control, comparing absorbance, and analyzing the acid-base stability of the keratinase.

The results show that the activity of keratinase is inhibited under acidic conditions, and the activity of keratinase gradually increases with increasing pH, the optimum reaction pH is about 9.5, and then the activity decreases. After the enzyme is incubated in acid or alkaline buffer solution for 1h, the enzyme activity is reduced to a certain extent, but the enzyme activity can still be maintained above 60% in the pH value range of 7-9.5, and the enzyme is typical alkaline protease (see figure 6).

Example 7 feather degrading ability of keratinase produced by keratinase-producing bacterium Bacillus sp.TC5

The duck feathers are washed by water, sprayed with 75% alcohol, dried and weighed, then subjected to enzymolysis by keratinase produced by Bacillus sp.TC5, and enzyme solutions with different dosages are added among different groups. Meanwhile, a glycine-sodium hydroxide buffer group is used as a control group, and the degradation capability of the keratinase produced by Bacillus sp.TC5 on duck feathers is calculated by the following formula (see figure 7).

Adding 0.1g of duck feather into each group, simultaneously adding 30ml of Gly-NaOH buffer solution into the first group, adding 5ml of crude enzyme solution and 25ml of Gly-NaOH buffer solution into the second group, and repeating the steps until 30ml of crude enzyme solution is added into the seventh group. Along with the increase of the addition amount of the enzyme solution, the feather degradation rate is obviously improved, and when 30ml of crude enzyme solution is added, the degradation rate reaches nearly 40 percent.

Example 8 application of Strain TC5 keratinase fermentation broth in skin depilation

(1) Depilatory effect on rat skin

(1) Preparation of mouse skin

A rat of 8 weeks old is selected as a sample, after the sample is sacrificed, the skin of the back is peeled off by a scalpel, a fat layer is removed, and after water filling and washing, the skin is cut into small pieces of 2.5cm multiplied by 1.5cm to be used as experimental materials.

(2) Conditions for depilation

Fermentation medium (g/L) for producing keratinase by optimizing fermentation conditions and considering skin safety: 2g of soybean meal, 1g of corn flour,2g of bran, na ₂ HPO ₄ 1g，KH ₂ PO ₄ 0.3g，CaCl ₂ 1g，Na ₂ CO ₃ 1g，MgSO ₄ ·7H ₂ O1 g, pH 7.8. 2 percent of TC5 bacterial liquid inoculation amount, placing a fermentation culture bottle in a combined type full-temperature constant incubator, fermenting for 72 hours at 37 ℃ and 180rpm.

(3) Method for determining the activity of a keratinase enzyme with soluble keratins as substrates: adding 40 mul of diluted enzyme solution into 40 mul of 0.5 percent soluble keratin substrate, carrying out water bath reaction at 50 ℃ for 10min, and adding 80 mul of 0.4mol/L TCA solution to terminate the reaction. In the control group, 40. Mu.L of diluted enzyme solution was added to 80. Mu.L of 0.4mol/LTCA solution to terminate the reaction, and 40. Mu.L of 0.5% soluble keratin substrate was added after 10min of water bath reaction at 50 ℃. After the reaction is finished, centrifuging at 10000r/min for 2min, absorbing the supernatant, and measuring the light absorption temperature at the wavelength of 280 nm. The keratinase activity unit (U) is defined as: the OD280 value of the mixed reaction system was 0.01 higher per liter than that of the control group per minute and was defined as 1U. KerTC5 has a keratinase activity unit of 256U/mL as measured by the above method. The rat skin was placed in an EP tube containing KerTC5 crude fermentation broth, placed in a combined full-temperature constant incubator, and shaken at a constant speed for 10h. The incubator was set at 40 ℃ and 180rpm.

Depilatory effect

The experimental result shows that the fermentation liquor has remarkable unhairing effect on the raw material skin; as shown in FIG. 8A, the dehaired rat skin was observed with a skin mirror, with intact skin lines, clear pores, and no stubble left on the skin. The dehaired rat skin was fixed with 4% paraformaldehyde for 24h, paraffin sectioned, and HE stained. HE staining Electron micrograph is shown in FIG. 8B, where the collagen layer of the skin is not destroyed

(2) Depilatory effect of back skin of four-month-old Bama miniature pig

The back skin (with hair) of a four-month-old Bama miniature pig is selected as an experimental object, the back skin of the miniature pig is peeled off by a scalpel, the skin is cut into small pieces of 2.5cm multiplied by 1.5cm, and the skin pieces covered with the hair of the pig are taken as experimental materials. Depilatory conditions are as follows: the enzyme activity unit is 256U/mL at 40 ℃ and 180rpm. The unhairing condition of the pigskin is observed regularly, and the result shows that the hard bristles and the villi on the pigskin are loosened after 12 hours under the condition, and the pigskin can be easily pulled out with the roots by using tweezers; after being fully washed, the hair completely falls off, the unhairing effect is excellent, as shown in figure 9A, when the bama xiang pigskin after unhairing is observed by using a skin mirror, the skin has complete grains, clear pores and no stubble on the skin. Taking unhaired Bama miniature pig skin, fixing the unhaired Bama miniature pig skin with 4% paraformaldehyde for 24 hours, carrying out paraffin section, and then carrying out HE dyeing. HE staining electron micrograph is shown in FIG. 9B, which shows that the pores are clean and the collagen layer of the skin is not damaged

Application of fermentation broth obtained in example 9 in combination with calcium thioglycolate in skin depilation

The depilatory effect of the fermentation liquor mixed with the calcium thioglycolate on rat skin is compared with that of the depilatory cream sold on the market

(1) Preparation of raw hide

A8-week-old rat is selected as a sample, after sacrifice, the skin on the back is peeled off by a scalpel, a fat layer is removed, and after water filling and washing, the skin is cut into small pieces of 2.5cm multiplied by 1.5cm to be used as experimental materials.

(2) Keratinase mixed calcium thioglycolate formula

The component A comprises the following raw materials in parts by weight: 0.5 part of hydroxyethyl cellulose, 0.2 part of allantoin, 5 parts of glycerol and 0.1 part of metal ion chelating agent;

the component B comprises the following raw materials in parts by weight: 3 parts of caprylic/capric triglyceride, 5 parts of isononyl isononanoate, 3 parts of cetostearyl alcohol, 0.5 part of tocopherol acetate and 1 part of polydimethylsiloxane;

the component C comprises the following raw materials in parts by weight: 1 part of 1, 2-hexanediol;

the component D comprises the following raw materials in parts by weight: 0.1 part of Ker TC5 crude enzyme liquid freeze-dried powder (the enzyme activity is 90U/mg), 3 parts of calcium thioglycolate and 1 part of essence are put into a freeze dryer at the temperature of 70 ℃ below zero.

1) Putting hydroxyethyl cellulose into 40wt% of purified water to be soaked until the hydroxyethyl cellulose is wet for later use;

2) Placing allantoin and glycerol into 40wt% purified water for use;

3) Uniformly mixing the mixture obtained in the step 1) and the mixture obtained in the step 2), and adding a metal ion chelating agent;

4) Mixing 1, 2-hexanediol, enzyme solution freeze-dried powder, calcium thioglycollate and essence with 20wt% of purified water, and stirring for later use;

5) Evenly stirring caprylic capric triglyceride, isononyl isononanoate, cetearyl alcohol, tocopherol acetate and polydimethylsiloxane for later use;

6) Heating the mixture obtained in the step 3) in a water bath kettle at 85 ℃, and uniformly stirring;

7) Heating the mixture obtained in the step 5) in a pot to 85 ℃, and uniformly stirring;

8) Putting the solution obtained in the step 6) into an emulsifying stirrer, stirring at a medium speed for 3min, adding the raw materials obtained in the step 7) while stirring, and stirring at a high speed for 8min;

9) Cooling the raw materials obtained in the step 8) to 40 ℃, adding the raw materials obtained in the step 4), stirring at a high speed until the materials are fully and uniformly stirred, and obtaining the depilatory cream.

(3) Depilatory effect

The depilation conditions were: the prepared keratinase depilatory cream is evenly smeared on rat skin at room temperature (25 ℃), and is kept stand. And compared with the depilatory cream sold on the market, the operating experimental conditions are kept consistent.

The experiment result shows that the mixed calcium thioglycolate after the fermentation liquor is freeze-dried has remarkable unhairing effect on rat skin, and after 25min, all rat hairs fall off; the skin mirror is used for observing that the depilated skin has complete lines, clear pores and no stubble residue on the skin, and HE dyeing of rat skin sections shows that the horny layer is well preserved (figure 10). The surface of the rat skin after depilation by the commercial depilatory cream is smooth, HE dyeing of skin sections shows that the cuticle is kept intact, but the cuticle is thinner than the keratinase depilatory cream of the invention (figure 11), because the calcium thioglycolate content of the cuticle is 2 times higher than that of the keratinase depilatory cream, the pH value is also up to 12.333, and the application of a large amount of harmful chemicals in depilation is avoided by the enzyme depilatory method due to the biological characteristics of the enzyme depilatory method, the enzyme depilatory cream is generally recognized as the cleanest depilatory method, thereby being an environment-friendly and resource-saving technology and having great application potential.

Application of keratinase fermentation broth obtained in example 10 to skin exfoliation

(1) Preparation of raw hide

The back skin of a four-month-old Bama miniature pig is selected as a sample (the barrier effect of the four-month-old miniature pig is closest to the skin of the human body) to serve as an experimental object, the back skin of the miniature pig is peeled off by a scalpel, the skin is cut into small pieces of 2.5cm multiplied by 1.5cm, and the skin pieces covered with the pig hair are taken as experimental materials. Depilatory conditions are as follows: at 40 ℃,180rpm, and the unit of enzyme activity is 256U/mL. Sampling is carried out every 1 hour, paraffin sections are made after the tissue fixing solution is fixed for 24 hours, and HE staining is carried out. As shown in FIG. 12, the epidermis and the horny layer of the pigskin remained intact within 5 hours, and the results showed that the keratinase had a mild exfoliating effect.

Claims (10)

1. A strain of keratinase-producing bacteria (Bacillus sp.) TC5 with the preservation number of CCTCC M2021975.

2. The keratinase-producing bacterium (Bacillus sp.) TC5 of claim 1, wherein the oxidase test is negative, the catalase test is negative, the lecithinase test is negative, the gelatin and casein can be hydrolyzed, the methyl red test is negative, and H is positive according to physiological and biochemical determination ₂ The S test is positive, and the nitrate reduction test is positive.

3. The keratinase-producing bacterium (Bacillus sp.) TC5 of claim 1, which is capable of producing keratinase and hydrolyzing keratinase.

4. Use of the keratinase-producing bacterium (Bacillus sp.) TC5 according to any of claims 1 to 3 for producing keratinase to hydrolyze keratin.

5. The use according to claim 4, characterized in that the keratinase-producing bacteria TC5 keratinase is used for feather degradation, skin depilation or skin exfoliation.

6. Use according to claim 4, characterized in that the fermentation broth obtained from keratinase-producing bacteria TC5 is used in combination with calcium thioglycolate for the depilation of skin.

7. Use according to claim 4, characterized in that the keratinase-producing fermentation medium (g/L): 10g of duck feather and Na ₂ HPO ₄ 1 g，KH ₂ PO ₄ 0.3g，Na ₂ CO ₃ 1 g，MgSO ₄ ·7H ₂ O 1g，CaCl ₂ 1 g,pH 7.8；

Further, inoculating the keratinase-producing strain TC5 in an LB liquid culture medium, culturing at 30 ℃,180rpm for 24h, inoculating a fermentation culture medium according to the inoculation amount of 2%, performing shaking culture at 32 ℃,200rpm for 120h, taking fermentation liquor every 24h, centrifuging at 10000rpm to remove thalli and substrate impurities, and obtaining a supernatant which is a crude enzyme solution.

8. The use according to claim 4, wherein the activation by manganese ions, calcium ions, magnesium ions, potassium ions, cobalt ions, sodium ions, barium ions is inhibited by high concentrations of aluminum ions, iron ions, copper ions, zinc ions.

9. The use according to claim 4, wherein the keratinase-producing bacteria TC5 reaches an optimum reaction temperature at 55 ℃.

10. The use according to claim 4, wherein the keratinase-producing bacteria TC5 keratinase reaches an optimum reaction pH at a pH of 9.5.

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