Method for preparing tea bran enzyme through bacillus fermentation and application of tea bran enzyme

Abstract

The invention relates to the technical field of cosmetic hair washing and caring, in particular to a method for preparing tea bran enzyme through bacillus fermentation and application of the tea bran enzyme. The method comprises the steps that 1, tea bran is crushed and then squeezed at low temperature, a composite enzymolysis agent with a specific proportion is adopted for treatment, and pretreatment is conducted under the steam explosion condition containing sodium bicarbonate; (2) bacillus subtilis is inoculated for directional fermentation, and the tea bran matrix is decomposed by utilizing the high enzyme activity characteristic of bacillus subtilis; and (3) removing the polysaccharide-protein compound through selective precipitation of glycosaminoglycan, and filtering and purifying to obtain the high-activity tea bran enzyme. Through whole chain innovation of process-strain-purification, the technical bottlenecks of low component extraction rate and poor stability of the natural tea bran are solved, efficient and low-consumption green preparation is realized, and the method has remarkable industrial application value in the field of cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic hair washing and hair care, and in particular to a method for preparing tea bran enzyme by utilizing Bacillus fermentation and application thereof. Background Art

[0002] Existing shampoo products commonly use chemically synthesized surfactants (such as AES, AESA, SLS, and SLES), which have strong degreasing properties and the risk of damaging the scalp barrier, leading to dandruff, dermatitis, and hair loss. Natural alternatives such as tea saponin, while milder, suffer from low extraction yields (<40%), poor foaming, and insufficient stability. Tea saponin and tea polyphenols are derived from tea bran, a widely available raw material with high annual production and very low cost. Tea bran is rich in tea saponin, protein, and amino acids. As a natural surfactant, tea saponin has the ability to cleanse and degrease, preventing over-degreasing while providing antibacterial and repairing properties. Tea saponin also regulates the balance of scalp flora, reduces dandruff formation, and relieves scalp inflammation, with no toxic side effects. The amino acids in tea bran nourish the hair cuticle, repair damaged hair scales, and enhance hair toughness.

[0003] Therefore, existing technologies often use tea bran to prepare shampoo products, including fermenting the tea bran and then compounding it with other ingredients. However, traditional tea bran fermentation processes have low release efficiency of active ingredients (tea saponins and antimicrobial peptides) and fail to address the issue of reduced utilization due to precipitation of polysaccharide-protein complexes.

[0004] In order to solve the above problems, it is necessary to propose a method for preparing tea bran enzyme by using Bacillus fermentation and its application. Summary of the Invention

[0005] In view of the above, it is necessary to provide a method for preparing tea bran enzyme using Bacillus fermentation and its application, which solves the technical bottleneck of low extraction rate and poor stability of natural tea bran components, realizes high-efficiency, low-consumption green preparation, and has significant industrial application value in the field of cosmetics.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A method for preparing tea bran enzyme by fermenting with Bacillus sp., the method comprising the following steps:

[0008] (1) Pretreatment: The tea bran is crushed and then pressed at 55-60°C until the oil residue is ≤30%. Then, a composite enzymatic hydrolyzer is added and the mixture is treated at 50-55°C and pH 5.5-7.0 for 2-3 h. Finally, the mixture is steam exploded to obtain the pretreated tea bran for use.

[0009] (2) Fermentation: Cooling the pretreated tea bran to 30-50°C, inoculating 0.2-0.8% Bacillus subtilis, stirring and fermenting for 6-8 hours to obtain a fermentation liquid;

[0010] (3) Precipitation: Filter the fermentation broth, then add 0.5-0.8% aminopolysaccharide and stir for 5-20 minutes, let it settle for 8-10 hours, and set aside;

[0011] (4) Filtration: Add 2% diatomaceous earth to the fermentation liquid, stir, adsorb, filter, and sterilize to obtain tea bran enzyme.

[0012] In the present invention, further, in the step (1), the tea bran is crushed to 0.5-1 mm.

[0013] In the present invention, further, the compound enzyme preparation in step (1) is composed of lipase: cellulase: xylanase: pectinase = 1:2:1:0.5.

[0014] In the present invention, the lipase is derived from Aspergillus oryzae, with an enzyme activity of ≥10,000 U / g. Cellulase: enzyme activity 1,000-1,100 U / g; xylanase: enzyme activity 8,000-9,000 U / g; pectinase: enzyme activity 3,000-4,000 U / g.

[0015] In the present invention, further, the steam explosion treatment in step (1) is performed at 0.8-1.0 MPa for 3-5 minutes and then the pressure is instantly released to normal pressure.

[0016] In the present invention, further, during the steam explosion process, 0.1-0.3% sodium bicarbonate solution is dissolved in the steam.

[0017] In the present invention, further, the Bacillus in step (2) is Bacillus subtilis, and the cellulase activity and protease activity secreted by the Bacillus subtilis during the fermentation process are ≥50 U / mL and ≥80 U / mL, respectively.

[0018] The Bacillus fermentation broth was prepared by inoculating the strain into LB medium and culturing at 30-37° C. for 12-16 hours until OD600 was ≥2.0; collecting the bacteria by centrifugation and resuspending them in physiological saline until the number of viable bacteria was ≥1×10 9 CFU / mL.

[0019] In the present invention, further, in step (3), the fermentation liquid is filtered through 200 mesh.

[0020] The present invention also provides a tea bran enzyme prepared according to the above method, and the tea bran enzyme is used in shampoo products.

[0021] The present invention also provides a shampoo composition, comprising 1-15 wt% of the tea bran enzyme according to claim 9 and a cosmetically acceptable carrier.

[0022] The present invention has at least the following beneficial effects:

[0023] The present invention proposes a method for preparing tea bran enzyme using Bacillus fermentation and its application. Through a three-step synergistic technology of combined enzymatic hydrolysis with steam explosion pretreatment, Bacillus directional fermentation, and amino polysaccharide precipitation purification, the efficient release and stabilization of tea bran active ingredients are achieved, achieving the following multiple breakthroughs:

[0024] (1) A breakthrough improvement in the yield and purity of active ingredients. Through the synergistic effect of composite enzymatic hydrolysis (lipase: cellulase: xylanase: pectinase = 1:2:1:0.5) and sodium bicarbonate-assisted steam explosion, the lignin-hemicellulose composite structure of the tea bran cell wall was significantly destroyed, the porosity of the tea bran was increased to 92% (only 78% in the control group), the tea saponin yield was increased to 76.8% (an increase of 102% compared to the traditional process), and the enzyme turbidity was reduced by 71%. In addition, this application further limits the lipase (derived from Aspergillus oryzae, enzyme activity ≥10,000 U / g) to specifically hydrolyze tea seed oil triglycerides, controlling the oil residue rate to ≤3%, solving the problem of tea saponin dissolution caused by oil encapsulation.

[0025] (2) Synergistic enhancement of fermentation efficiency and active ingredient function: Directed fermentation was performed using Bacillus subtilis (cellulase activity ≥50 U / mL, protease activity ≥80 U / mL). The enzyme system secreted by the fermented tea bran synergistically interacts with the porous structure of the pretreated tea bran, increasing the content of small molecule peptides (MW <1000 Da) by 32% (to 32.5 mg / g). This significantly enhances the scalp permeability of the active ingredients and directly acts on hair follicle stem cells. The aminopolysaccharide precipitation method selectively removes polysaccharide-protein complexes, avoiding the co-precipitation loss of active ingredients in traditional processes and increasing filtration speed.

[0026] (3) Significant application advantages of the terminal product: This application further uses the prepared tea saponin in a shampoo composition. After the tea bran enzyme (25% addition amount) is compounded with natural surfactants, it reduces chemical synthetic surfactants and reduces the amount of hair chemical conditioners. The dandruff reduction rate reaches 82% (commercially available products are only 48%), the scalp erythema index is reduced to 0.2 (non-irritating), and the hair breaking force is increased by 35%. It is confirmed that its effect of repairing the scalp barrier and regulating the balance of the bacterial flora through the synergistic mechanism of small molecule peptides and tea saponins far exceeds that of similar products.

[0027] In summary, the present invention solves the technical bottlenecks of low extraction rate and poor stability of natural tea bran ingredients through full-chain innovation of process-strain-purification, realizes high-efficiency, low-consumption green preparation, and has significant industrial application value in the field of cosmetics. DETAILED DESCRIPTION

[0028] All features disclosed in this specification, or all steps in the disclosed methods or processes, except mutually exclusive features and / or steps, can be combined in any manner.

[0029] Any feature disclosed in this specification (including any accompanying claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features.

[0030] Example 1:

[0031] This embodiment provides a method for preparing tea bran enzyme by fermentation with Bacillus sp., the method comprising the following steps:

[0032] (1) Pretreatment: The tea bran was crushed into 0.7 mm and then pressed at 57 °C until the oil residue was 27%. Then, a composite enzymatic agent was added and the mixture was treated at 52 °C and pH 6.0 for 2.5 h. Finally, the mixture was steam-exploded at 0.9 MPa for 4 min and then the pressure was instantly released to normal pressure. During the steam explosion process, 0.2% sodium bicarbonate solution was dissolved in the steam to obtain the pretreated tea bran for use. The composite enzyme preparation was composed of lipase: cellulase: xylanase: pectinase = 1:2:1:0.5. The lipase was derived from Aspergillus oryzae with an enzyme activity of 12,000 U / g.

[0033] (2) Fermentation: The pretreated tea bran was cooled to 40°C, and then inoculated with 0.5% Bacillus subtilis for fermentation. The fermentation was stirred for 6-8 hours to obtain a fermentation broth. The Bacillus subtilis was used, and the cellulase activity and protease activity secreted during the fermentation process were 58 U / mL and 96 U / mL, respectively. The Bacillus subtilis fermentation broth was prepared by the following method: the strain was inoculated into LB medium and cultured at 35°C for 14 hours until OD600 ≥ 2.0; the cells were collected by centrifugation and resuspended in physiological saline until the number of viable cells was 1.2 × 10 9 CFU / mL;

[0034] (3) Precipitation: Filter the fermentation broth, then add 0.6% amino polysaccharide and stir for 10 minutes, let it settle for 9 hours, and set aside;

[0035] (4) Filtration: Add 2% diatomaceous earth to the fermentation liquid, stir, adsorb, filter, and sterilize to obtain tea bran enzyme.

[0036] Example 2:

[0037] This embodiment provides a method for preparing tea bran enzyme by fermentation with Bacillus sp., the method comprising the following steps:

[0038] (1) Pretreatment: The tea bran was crushed into 0.5 mm and then pressed at 55 °C until the oil residue was 26%. Then, a composite enzymatic agent was added and the mixture was treated at 50 °C and pH 5.5 for 2 h. Finally, the mixture was steam-exploded at 0.8 MPa for 3 min and then the pressure was instantly released to normal pressure. During the steam explosion process, 0.1% sodium bicarbonate solution was dissolved in the steam to obtain the pretreated tea bran for use. The composite enzyme preparation was composed of lipase: cellulase: xylanase: pectinase = 1:2:1:0.5. The lipase was derived from Aspergillus oryzae with an enzyme activity of 12,000 U / g.

[0039] (2) Fermentation: The pretreated tea bran was cooled to 30°C, then inoculated with 0.2% Bacillus subtilis, and stirred for fermentation for 6 hours to obtain a fermentation broth; the Bacillus subtilis was selected, and the cellulase activity and protease activity secreted during the fermentation process were 60 U / mL and 95 U / mL, respectively; the Bacillus subtilis fermentation broth was prepared by the following method: the strain was inoculated into LB medium, and cultured at 30°C for 12 hours until OD600 ≥ 2.0; the cells were collected by centrifugation, and resuspended in physiological saline until the number of viable cells was 1.5 × 10 9 CFU / mL;

[0040] (3) Precipitation: Filter the fermentation broth, then add 0.5% amino polysaccharide and stir for 5 minutes, let it settle for 8 hours, and set aside;

[0041] (4) Filtration: Add 2% diatomaceous earth to the fermentation liquid, stir, adsorb, filter, and sterilize to obtain tea bran enzyme.

[0042] Example 3:

[0043] This embodiment provides a method for preparing tea bran enzyme by fermentation with Bacillus sp., the method comprising the following steps:

[0044] (1) Pretreatment: The tea bran was crushed into 1 mm and then pressed at 60 °C until the oil residue was 28%. Then, a composite enzymatic agent was added and the mixture was treated at 55 °C and pH 7.0 for 3 h. Finally, the mixture was steam-exploded at 1.0 MPa for 5 min and then the pressure was instantly released to normal pressure. During the steam explosion process, 0.3% sodium bicarbonate solution was dissolved in the steam to obtain the pretreated tea bran for use. The composite enzyme preparation was composed of lipase: cellulase: xylanase: pectinase = 1:2:1:0.5. The lipase was derived from Aspergillus oryzae with an enzyme activity of 12,000 U / g.

[0045] (2) Fermentation: The pretreated tea bran was cooled to 50°C, and then inoculated with 0.8% Bacillus subtilis for fermentation. The fermentation was stirred for 6 h to obtain a fermentation broth. The Bacillus subtilis was used, and the cellulase activity and protease activity secreted during the fermentation process were 59 U / mL and 94 U / mL, respectively. The Bacillus subtilis fermentation broth was prepared by inoculating the strain into LB medium and culturing at 37°C for 16 h until OD600 ≥ 2.0. The cells were collected by centrifugation and resuspended in physiological saline until the viable count was 1.3 × 10 9 CFU / mL;

[0046] (3) Precipitation: Filter the fermentation broth, then add 0.8% amino polysaccharide and stir for 20 minutes, let it settle for 10 hours, and set aside;

[0047] (4) Filtration: Add 2% diatomaceous earth to the fermentation liquid, stir, adsorb, filter, and sterilize to obtain tea bran enzyme.

[0048] Test data:

[0049] Test 1:

[0050] The applicant set up multiple groups for comparative experiments to compare the tea saponin yield, antimicrobial peptide content, and filtration speed of the fermentation broth obtained under different pretreatment methods. The groups are as follows:

[0051] Experimental group: tea bran enzyme obtained by the method of Example 1 of the present application;

[0052] Control group A: no pretreatment, other methods are the same as Example 1;

[0053] Control group B: The pretreatment was only pressing, i.e. the tea bran was crushed to 0.7 mm and then pressed at 57°C until the oil residue was 4.6%. Other pretreatment methods were removed. Other methods were the same as in Example 1.

[0054] Control group C: the enzymatically hydrolyzed composite enzyme preparation was composed of lipase: cellulase: xylanase: pectinase = 1:1:1:1; other methods were the same as in Example 1;

[0055] Control group D: The xylanase in the complex enzyme preparation was removed, and the other methods were the same as those in Example 1.

[0056] Comparing the effects of the above group treatments, the results are shown in Table 1:

[0057] Table 1 Comparison of some indicators under different pretreatment processes

[0058] Group Tea saponin yield Antimicrobial peptides (mg / g) Filtration speed (L / h) Control group A 38.2% 12.1 125 Control group B 54.3% 18.5 185 Control group C 61.7% 23.9 265 Control group D 49.5% 25.1 228 Experimental group 76.8% 32.5 475

[0059] According to the results in Table 1, the pretreatment method described in this application increased filtration speed by 280% and tea saponin yield by 102% (vs. Control A), far exceeding the industry average. Furthermore, the tea saponin yield in the group without xylanase dropped dramatically, and filtration speed was also slow. This is likely due to the formation of a three-dimensional barrier by undegraded hemicellulose, resulting in a high viscosity in the fermentation broth, which hinders the dissolution of tea saponin. These experiments demonstrate that the raw materials in this application's complex enzyme preparation are essential, and their proportions cannot be adjusted.

[0060] Test 2:

[0061] The applicant continued to conduct criticality verification tests on different lipase sources and enzyme activities, including setting up a control group EG and comparing it with the experimental group. Among them, the other conditions of the control group EG and the experimental group were fixed, and only the lipase type and enzyme activity were adjusted. The grouping and test results are shown in Table 2:

[0062] Table 2 Lipase source and criticality verification of enzyme activity

[0063] Group Lipase type Enzyme activity (U / g) Grease residue rate Tea saponin yield Control group E Candida lipase 12,000 4.5% 55.1% Control group F Aspergillus oryzae lipase 8,000 3.7% 63.2% Experimental group Aspergillus oryzae lipase 12,000 2.7% 76.8% Control group G No lipase added - 7.2% 38.5%

[0064] According to the test results, the experimental group of the present application (Example 1) used Aspergillus oryzae lipase with an enzyme activity of 12,000 U / g. Under the treatment, the oil residue rate was low and the tea saponin yield was high. It is considered that this may be because the Aspergillus oryzae lipase of the present application specifically hydrolyzes tea seed oil triglycerides. When the enzyme activity is ≥10,000 U / g, the oil residue is ≤3%, which further increases the tea saponin yield.

[0065] Test 3:

[0066] This experiment verifies the effect of steam explosion in synergy with sodium bicarbonate. The method is the same as the above experiment except for the differences emphasized. The grouping and verification results are shown in Table 3:

[0067] Table 3 Verification of the effect of steam explosion synergistic with sodium bicarbonate

[0068] Group Steam explosion conditions Porosity of tea bran Tea saponin yield Enzyme turbidity (NTU) Control group H <![CDATA[Without NaHCO3]]> 78% 68.5% 120 Experimental group <![CDATA[Containing 0.2% NaHCO3]]> 92% 76.8% 42 Control group I No steam explosion 35% 52.6% 480

[0069] According to the test results, it was concluded that adding NaHCO3 to steam explosion can promote the dissolution of lignin, increase the porosity, and double the efficiency of subsequent fermentation, ultimately reducing the turbidity of the final product by 71%. It can be seen that steam explosion synergistically with sodium bicarbonate has a very outstanding and unpredictable effect.

[0070] Test 4:

[0071] This experiment continued to verify the synergistic effect of the enzyme activity of the fermentation strains. Specifically, the same pretreated tea bran (Example 1) was used to compare different strains. The results are shown in Table 4:

[0072] Table 4 Verification of synergistic effect of enzyme activity of strains

[0073] strain type Cellulase activity (U / mL) Protease activity (U / mL) Small molecule peptide content (mg / g) Bacillus licheniformis (not up to standard) 42 75 23.7 Bacillus subtilis 58 96 32.5

[0074] According to the results in Table 4, the qualified strain increased the content of small molecule peptides (MW < 1000Da) by 32%, which can effectively enhance scalp permeability (penetrating the scalp stratum corneum and directly acting on hair follicle stem cells). It is considered that the application of this strain in shampoo products can achieve deep penetration and exert its effect.

[0075] Test 5:

[0076] In order to verify the key role of aminopolysaccharide precipitation in the purification of tea bran enzyme, the applicant further designed a comparative experiment to examine the effects of different precipitation methods on the recovery rate of active ingredients, the removal effect of polysaccharide-protein complexes and the stability of the final product.

[0077] Experimental group: using the method of Example 1 of the present invention (0.6% aminopolysaccharide precipitation);

[0078] Control group J: traditional ethanol precipitation method (60% ethanol, 4°C for 12 h);

[0079] Control group K: no precipitant was added, only natural sedimentation;

[0080] Control group L: Glycosaminoglycan (0.6%) was used instead of aminopolysaccharide.

[0081] Except for the above-mentioned contents, the other methods are the same as those in Example 1. The test results are shown in Table 5:

[0082] Table 5 Verification data of aminopolysaccharide precipitation effect

[0083] Group Antimicrobial peptide retention rate Polysaccharide residue (mg / g) Precipitation rate after storage at 4℃ for 14 days (%) Experimental group 93.8% 1.2 2.1 Control group J 76.5% 8.7 15.3 Control group K 54.2% 25.6 32.8 Control group L 80.1% 3.5 9.4

[0084] According to test results, traditional precipitation methods (such as ethanol and glycosaminoglycans) have difficulty achieving both high recovery rates and low impurity residues. However, the present invention, through the precise precipitation of aminopolysaccharides, simultaneously achieves in a single step: high recovery of active ingredients (>93%), efficient removal of polysaccharide-protein complexes (removal rate >90%), and long-term stability of the final product (precipitation rate <3%).

[0085] Application examples:

[0086] Based on the above content, the applicant further conducted terminal product performance verification (shampoo application), specifically preparing a shampoo composition comprising: tea bran enzyme: 25%, cocamidopropyl betaine: 8%, glycerin: 3%, and the balance being deionized water.

[0087] In addition, a control group was set up with a commercially available natural surfactant shampoo.

[0088] Sixty volunteers were divided into two groups. Thirty volunteers tested the shampoo composition, while the other 30 tested a commercially available natural surfactant shampoo. Both groups tested for 28 days. Aside from the shampoo used, the cleansing method (frequency and duration) and testing procedures were identical. The test results are shown in Table 6.

[0089] Table 6 Human trial report

[0090] index Shampoo composition containing the enzyme of the present invention Commercially available natural surfactant shampoo Dandruff reduction rate 82% 48% Scalp erythema index 0.2 1.8 (mild irritation) Improved hair breakage resistance 35% 12%

[0091] According to the above test results, the shampoo composition of the present application has excellent effects, which is believed to be due to the synergy between the small molecule peptides in the enzyme and the tea saponin, which effectively repairs the scalp barrier and improves the health of the scalp and hair.

[0092] In summary, the present invention solves the technical bottlenecks of low extraction rate and poor stability of natural tea bran ingredients through full-chain innovation of process-strain-purification, realizes high-efficiency, low-consumption green preparation, and has significant industrial application value in the field of cosmetics, especially shampoo products.

[0093] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for preparing tea bran enzyme by using Bacillus fermentation, characterized in that: The method comprises the following steps: (1) Pretreatment: The tea bran is crushed and then pressed at 55-60°C until the oil residue is ≤30%. Then, a composite enzymatic hydrolyzer is added and the mixture is treated at 50-55°C and pH 5.5-7.0 for 2-3 h. Finally, the mixture is steam exploded to obtain the pretreated tea bran for use. (2) Fermentation: Cooling the pretreated tea bran to 30-50°C, inoculating 0.2-0.8% Bacillus subtilis for fermentation, stirring and fermenting for 6-8 hours to obtain a fermentation liquid; (3) Precipitation: Filter the fermentation broth, then add 0.5-0.8% aminopolysaccharide and stir for 5-20 minutes, let it settle for 8-10 hours, and set aside; (4) Filtration: Add 2% diatomaceous earth to the fermentation liquid, stir, adsorb, filter, and sterilize to obtain tea bran enzyme.

2. The method according to claim 1, characterized in that In the step (1), the tea bran is crushed to 0.5-1 mm.

3. The method according to claim 1, characterized in that The composite enzyme preparation in step (1) is composed of lipase: cellulase: xylanase: pectinase in a ratio of 1:2:1:0.

5.

4. The method according to claim 3, characterized in that The lipase is derived from Aspergillus oryzae, and the enzyme activity is ≥10,000 U / g.

5. The method according to claim 1, wherein The steam explosion treatment in step (1) is performed at 0.8-1.0 MPa for 3-5 minutes and then the pressure is instantly released to normal pressure.

6. The method according to claim 5, characterized in that During the steam explosion process, 0.1-0.3% sodium bicarbonate solution is dissolved in the steam.

7. The method according to claim 1, characterized in that The Bacillus in step (2) is Bacillus subtilis, and the cellulase activity and protease activity secreted by the Bacillus subtilis during the fermentation process are ≥50 U / mL and ≥80 U / mL, respectively.

8. The method according to claim 1, characterized in that In step (3), the fermentation liquid is filtered through 200 mesh.

9. A tea bran enzyme prepared according to the method according to any one of claims 1 to 8, characterized in that: The tea bran enzyme is used in shampoo products.

10. A shampoo composition, characterized in that The shampoo composition comprises 1-15 wt% of the tea bran enzyme according to claim 9, and a cosmetically acceptable carrier.