Method for fermenting cacumen biotae with compound bacteria and application

Abstract

The invention relates to the field of biological fermentation, in particular to a method for fermenting cacumen biotae with compound bacteria and application of the cacumen biotae, and the compound bacteria comprise lactococcus lactis and pichia pastoris. The method comprises the following steps: (1) pretreating cacumen biotae; (2) performing enzyme treatment on the cacumen biotae; (3) fermenting the cacumen biotae; and (4) treating fermentation liquor. The invention has the following beneficial effects: (1) enzymolysis parameters are optimized, fermentation parameters are optimized, a fermentation liquor extraction method is optimized, and a better fermentation method is obtained; (2) the Chinese arborvitae twigs are fermented, so that compared with fermentation of various traditional Chinese medicine raw materials in the prior art, the components are simplified, and the method is more convenient; and (3) a mouse experiment proves that more flavones and a better anti-hair loss effect can be obtained by using the optimized fermentation method, and the application of the cacumen biotae is more facilitated.

Description

Technical Field

[0001] The invention relates to the field of biological fermentation, and in particular to a method and application of compound bacteria fermenting Platycladus orientalis leaves. Background Art

[0002] Platycladus orientalis is an evergreen tree with a broad ovate crown and flat twigs arranged in a plane. It is monoecious and has unisexual flowers. The male cones are yellow and consist of microsporophyll leaves that are arranged in pairs. Each microsporophyll has three pollen sacs, and the cone scales and bract scales are completely fused. The cones mature in the same year, the seed scales are lignified and cracked, and the seeds are wingless or have ridges. The leaves are small and scale-like, with many branches, and the twigs are flat and close to the twigs, arranged in pairs, with glandular grooves in the middle of the back of the leaves, dark green or yellow-green.

[0003] Platycladus orientalis leaf is a commonly used traditional Chinese medicine for the treatment of hair loss. It is recorded in the Chinese Materia Medica that 160 cases were treated by applying ethanol extract of Platycladus orientalis leaf to the hair loss area, with a total effective rate of 77.5%. Platycladus orientalis leaf has a complex chemical composition, including flavonoids, volatile oils, tannins, etc., which have the effects of antibacterial and anti-inflammatory, vasodilating, anti-dandruff and anti-hair loss, and hair growth. Modern medical research has found that the flavonoid components in Platycladus orientalis leaf can activate hair matrix cells, promote blood circulation, improve hair follicle development and growth, revive hair follicles with declining hair growth ability, and supplement nutrients for the revived hair follicles to play a role in hair care and hair growth. In addition, cedrol, quercetin and sedge biflavonoids in Platycladus orientalis leaf have the activity of promoting cell autophagy.

[0004] Studies have shown that autophagy disorders in hair papilla cells may be a potential mechanism of androgenic alopecia, and inducing autophagy in hair papilla cells is sufficient to prevent hair loss. Platycladus orientalis leaf extracts are usually extracted with water, ethanol, etc. as solvents by heating and reflux extraction, and then the extract is concentrated to dryness. Platycladus orientalis extracts have inhibitory effects on Staphylococcus aureus, Escherichia coli, etc. There are many methods for extracting flavonoids from Platycladus orientalis leaf extracts. Ning Na et al. reviewed the extraction process of total flavonoids from Platycladus orientalis leaves, including leaching, ethanol reflux extraction, ethanol ultrasonic extraction, ethanol microwave extraction, enzymatic extraction under ultrasonic conditions, and methanol homogenization extraction. By comparison, the extraction rate of total flavonoids from Platycladus orientalis leaves was the highest, at 5.905%, under the conditions of ethanol volume fraction of 60%, solid-liquid ratio of 1:20, microwave time of 4 min, and microwave power of 385 W.

[0005] Prior art CN117530907B discloses a plant fermentation product for oil control, soothing and scalp care. The raw materials for preparation include 1-5 parts of Platycladus orientalis leaves, 1-3 parts of licorice roots, 1-3 parts of rose bud powder, 1-3 parts of rice, 1-2 parts of Chuanxiong, 1-2 parts of ginseng roots, 1-2 parts of wheat germ, 1-2 parts of ginger, 1-3 parts of fir-leaf fern algae, 0.5-2 parts of oligofructose, and 70-80 parts of water. The composition containing plant and probiotic fermentation is all natural plants and biological strains, has good antibacterial effect, and each component synergizes and is green and safe. Through scientific proportioning, it can effectively remove dandruff and oil, improve scalp ecology; by repairing the scalp barrier, controlling scalp sebum secretion, soothing and anti-inflammatory, removing dandruff and relieving itching, and helping to restore the scalp to a healthy state. At the same time, it can also significantly increase hair density, promote hair health, and make hair smooth.

[0006] Prior art CN117511827B discloses a fermented product of Platycladus orientalis leaves for scalp care and oil control and dandruff removal, and its preparation method and application. The method first inoculates Pichia pastoris and Bacillus subtilis in a fermentation medium at a mass ratio of 4.7-5.3:0.7-1.3, sterilizes after fermentation, and then inoculates a specific lactococcus lactis flora for anaerobic fermentation to obtain the fermented product of Platycladus orientalis leaves. The fermented product of Platycladus orientalis leaves has excellent scalp care and oil control and dandruff removal effects, and is suitable as and / or for preparing cosmetic or medical compositions.

[0007] Prior art CN115414301B discloses a plant anti-dandruff composition, characterized in that it is composed of glycolipids and plant extracts, wherein the mass ratio of glycolipids to plant extracts is (1 to 4): (2 to 5); the glycolipid is a sophorolipid; the preparation method of the sophorolipid comprises: culturing Candida aurea on a plate, picking a single colony to ferment in a liquid culture medium, standing and stratifying after the fermentation, and collecting the bottom sophorolipid; the preparation method of the plant extract comprises: mixing raw materials rosemary leaves and Platycladus orientalis leaves in a mass ratio of 96:4, adding water 4 times the mass of the raw materials, heating and refluxing at 95°C for extraction for 2.5 hours, filtering, collecting the filtrate, and obtaining a plant extract.

[0008] However, the above-mentioned prior arts all added other traditional Chinese medicine components or other additional components during the fermentation of Platycladus orientalis leaves. The present application will simplify the above-mentioned components, use Platycladus orientalis leaves alone for fermentation, and optimize the fermentation strains and parameters, in order to explore a simpler and more efficient method and application of composite bacteria fermentation of Platycladus orientalis leaves. Summary of the invention

[0009] In order to solve the above problems, the present invention first provides a method for fermenting Platycladus orientalis leaves with a composite bacteria, wherein the composite bacteria comprises Lactococcus lactis and Pichia pastoris.

[0010] In some embodiments, the steps include: (1) Pretreatment of Platycladus orientalis leaves; (2) Enzyme treatment of Platycladus orientalis leaves; (3) Fermentation of Platycladus orientalis leaves.

[0011] In certain embodiments, the method further comprises the following steps: (4) fermentation broth treatment.

[0012] In certain embodiments, the method comprises the following steps: the fermentation broth is treated by extraction with n-butanol and ethyl acetate.

[0013] The present invention also provides an application of the method for fermenting Platycladus orientalis leaves with composite bacteria, and the application is to prepare daily chemical products containing Platycladus orientalis leaves active ingredients.

[0014] In certain embodiments, the application is that the active ingredient of Platycladus orientalis leaves is flavonoids or Platycladus orientalis leaf polysaccharides.

[0015] In certain embodiments, the daily chemical product is a hair care product.

[0016] Finally, the present invention provides a product of Platycladus orientalis leaves fermented by composite bacteria, and the product is prepared by fermentation using the above method.

[0017] In certain embodiments, the application is to prepare daily chemical products containing active ingredients from Platycladus orientalis leaves.

[0018] In certain embodiments, the daily chemical product is a hair care product.

[0019] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The present invention uses enzymatic hydrolysis before fermentation and uses n-butanol and ethyl acetate for extraction after fermentation. The present invention optimizes the enzymatic hydrolysis parameters, the fermentation parameters, and the fermentation broth extraction method to obtain a better fermentation method; (2) Compared with the fermentation of multiple Chinese medicinal materials in the prior art, the fermentation of Platycladus orientalis leaves of the present invention simplifies the components, making the invention more convenient; (3) The mouse experiment of the present invention proves that the use of the optimized fermentation method can obtain more flavonoids, better anti-hair loss effect, and is more conducive to the application of Platycladus orientalis leaves. DETAILED DESCRIPTION

[0020] In order to make the technical problems to be solved, technical solutions and advantages of the present invention more clearly understood, a detailed description will be given below in conjunction with specific embodiments.

[0021] Lactococcus lactis and Pichia pastoris were purchased from commercial sources.

[0022] Embodiment 1 The method of compound bacteria fermentation Platycladus orientalis leaf Step (1): 10 g of Platycladus orientalis leaves were washed, impurities removed, freeze-dried, and ground into powder; Step (2): add 200 mL of deionized water, add 10 mg of complex enzyme (200,000 U / g cellulase and 200,000 U / g pectinase, weight ratio of 1:1), enzymolysis in a 37°C water bath for 4 h, and then add 10 mg of hydrolyzing protease (200,000 U / g) for 4 h to obtain an enzymolysis solution, and boil for 15 minutes to inactivate; Step (3): inoculating 2% Pichia pastoris and 2% Lactococcus lactis by mass for fermentation, wherein the fermentation starting temperature is 30° C., the fermentation starting pH is 5.5, the fermentation starting oxygen content is 18, the fermentation stirring speed is 45 r / min, the fermentation time is 16 h, and a fermentation liquid is obtained.

[0023] Step (4): After filtering the fermentation liquid with sterile gauze, mix it with n-butanol and ethyl acetate in a ratio of 1 / 3 (volume ratio), leave it to stand at room temperature for 72 hours, separate the liquids and remove the lower aqueous phase filtrate, and remove the upper organic solvent by vacuum rotary evaporation at 60°C and 40°C to obtain a solid sample.

[0024] Example 2 Method for fermenting Platycladus orientalis leaves with composite bacteria Step (1): 10 g of Platycladus orientalis leaves were washed, impurities removed, freeze-dried, and ground into powder; Step (2): add 200 mL of deionized water, add 10 mg of complex enzyme (200,000 U / g cellulase and 200,000 U / g pectinase, weight ratio of 1:1), enzymolysis in a 37°C water bath for 4 h, and then add 10 mg of hydrolyzing protease (200,000 U / g) for 4 h to obtain an enzymolysis solution, and boil for 15 minutes to inactivate; Step (3): inoculating 2% Pichia pastoris and 2% Lactococcus lactis by mass for fermentation, wherein the fermentation starting temperature is 30° C., the fermentation starting pH is 5.5, the fermentation starting oxygen content is 18, the fermentation stirring speed is 45 r / min, the fermentation time is 16 h, and a fermentation liquid is obtained.

[0025] Step (4): After filtering the fermentation liquid with sterile gauze, the solvent is removed by vacuum rotary evaporation to obtain a solid sample.

[0026] Comparative Example 1: Method for fermenting Platycladus orientalis leaves with composite bacteria Step (1): 10 g of Platycladus orientalis leaves were washed, impurities removed, freeze-dried, and ground into powder; Step (2): add 200 mL of deionized water, add 10 mg of cellulase (200,000 U / g), and enzymolyze in a 37°C water bath for 4 h, then add 10 mg of protease (200,000 U / g) and enzymolyze for 4 h to obtain an enzymatic solution, and boil for 15 minutes to inactivate; Step (3): inoculating 2% Pichia pastoris and 2% Lactococcus lactis by mass for fermentation, wherein the fermentation starting temperature is 30° C., the fermentation starting pH is 5.5, the fermentation starting oxygen content is 18, the fermentation stirring speed is 45 r / min, the fermentation time is 16 h, and a fermentation liquid is obtained.

[0027] Step (4): After filtering the fermentation liquid with sterile gauze, mix it with n-butanol and ethyl acetate in a ratio of 1 / 3 (volume ratio), leave it to stand at room temperature for 72 hours, separate the liquids and remove the lower aqueous phase filtrate, and remove the upper organic solvent by vacuum rotary evaporation at 60°C and 40°C to obtain a solid sample.

[0028] Comparative Example 2: Method for fermenting Platycladus orientalis leaves with composite bacteria Step (1): 10 g of Platycladus orientalis leaves were washed, impurities removed, freeze-dried, and ground into powder; Step (2): Add 200 mL of deionized water, add 10 mg of pectinase (200,000 U / g), and enzymolyze in a 37°C water bath for 4 h, then add 10 mg of hydrolyzing protease (200,000 U / g) and enzymolyze for 4 h to obtain an enzymatic solution, and boil for 15 minutes to inactivate; Step (3): inoculating 2% Pichia pastoris and 2% Lactococcus lactis by mass for fermentation, wherein the fermentation starting temperature is 30° C., the fermentation starting pH is 5.5, the fermentation starting oxygen content is 18, the fermentation stirring speed is 45 r / min, the fermentation time is 16 h, and a fermentation liquid is obtained.

[0029] Step (4): After filtering the fermentation liquid with sterile gauze, mix it with n-butanol and ethyl acetate in a ratio of 1 / 3 (volume ratio), leave it to stand at room temperature for 72 hours, separate the liquids and remove the lower aqueous phase filtrate, and remove the upper organic solvent by vacuum rotary evaporation at 60°C and 40°C to obtain a solid sample.

[0030] Comparative Example 3: Method for fermenting Platycladus orientalis leaves with composite bacteria Step (1): 10 g of Platycladus orientalis leaves were washed, impurities removed, freeze-dried, and ground into powder; Step (2): add 200 mL of deionized water, add 20 mg of compound enzyme (200,000 U / g cellulase and 200,000 U / g pectinase, weight ratio of 1:1), perform enzymolysis in a 37°C water bath for 8 h, obtain enzymolysis solution, and boil for 15 minutes to inactivate; Step (3): inoculating 2% Pichia pastoris and 2% Lactococcus lactis by mass for fermentation, wherein the fermentation starting temperature is 30° C., the fermentation starting pH is 5.5, the fermentation starting oxygen content is 18, the fermentation stirring speed is 45 r / min, the fermentation time is 16 h, and a fermentation liquid is obtained.

[0031] Step (4): After filtering the fermentation liquid with sterile gauze, mix it with n-butanol and ethyl acetate in a ratio of 1 / 3 (volume ratio), leave it to stand at room temperature for 72 hours, separate the liquids and remove the lower aqueous phase filtrate, and remove the upper organic solvent by vacuum rotary evaporation at 60°C and 40°C to obtain a solid sample.

[0032] Comparative Example 4: Method for fermenting Platycladus orientalis leaves with composite bacteria Step (1): 10 g of Platycladus orientalis leaves were washed, impurities removed, freeze-dried, and ground into powder; Step (2): add 200 mL of deionized water, add 10 mg of complex enzyme (200,000 U / g cellulase and 200,000 U / g pectinase, weight ratio of 1:1), enzymolysis in a 37°C water bath for 4 h, and then add 10 mg of hydrolyzing protease (200,000 U / g) for 4 h to obtain an enzymolysis solution, and boil for 15 minutes to inactivate; Step (3): inoculating 4% Pichia pastoris by mass for fermentation, wherein the fermentation starting temperature is 30° C., the fermentation starting pH is 5.5, the fermentation starting oxygen content is 18, the fermentation stirring speed is 45 r / min, the fermentation time is 16 h, and a fermentation liquid is obtained.

[0033] Step (4): After filtering the fermentation liquid with sterile gauze, mix it with n-butanol and ethyl acetate in a ratio of 1 / 3 (volume ratio), leave it to stand at room temperature for 72 hours, separate the liquids and remove the lower aqueous phase filtrate, and remove the upper organic solvent by vacuum rotary evaporation at 60°C and 40°C to obtain a solid sample.

[0034] Comparative Example 5: Method for fermenting Platycladus orientalis leaves with composite bacteria Step (1): 10 g of Platycladus orientalis leaves were washed, impurities removed, freeze-dried, and ground into powder; Step (2): add 200 mL of deionized water, add 10 mg of complex enzyme (200,000 U / g cellulase and 200,000 U / g pectinase, weight ratio of 1:1), enzymolysis in a 37°C water bath for 4 h, and then add 10 mg of hydrolyzing protease (200,000 U / g) for 4 h to obtain an enzymolysis solution, and boil for 15 minutes to inactivate; Step (3): inoculating 4% by mass of Lactococcus lactis for fermentation, wherein the fermentation starting temperature is 30° C., the fermentation starting pH is 5.5, the fermentation starting oxygen content is 18, the fermentation stirring speed is 45 r / min, the fermentation time is 16 h, and a fermentation liquid is obtained.

[0035] Step (4): After filtering the fermentation liquid with sterile gauze, mix it with n-butanol and ethyl acetate in a ratio of 1 / 3 (volume ratio), leave it to stand at room temperature for 72 hours, separate the liquids and remove the lower aqueous phase filtrate, and remove the upper organic solvent by vacuum rotary evaporation at 60°C and 40°C to obtain a solid sample.

[0036] Example 3 Determination of component indexes of Platycladus orientalis leaf fermentation products Determination of total flavonoids Determination of total flavonoids content: 0.5 mL of methanol extract of Platycladus orientalis leaf fermentation product and 1.5 mL of 5% sodium nitrite were mixed, shaken and allowed to stand for 6 min, 0.3 mL of 10% aluminum nitrate was added, shaken and allowed to stand for 6 min, 1 mL of 4% sodium hydroxide was added, 95% ethanol was added to make the volume 5 mL, shaken and allowed to stand for 15 min, and the absorbance was measured at 510 nm. The rutin solution was used as the standard solution, and the solution without the standard solution was used as the control to draw a standard curve.

[0037] (2) Oil control efficacy test (2.1) Preparation of reagents: Testosterone (T) solution: Dissolve 36 mg of testosterone in anhydrous ethanol to prepare a testosterone solution with a concentration of 5 mmol / L; NADPH (reduced coenzyme II) solution: Dissolve 42.5 mg of NADPH tetrasodium salt in water to prepare a 2 mmol / L NADPH solution; Reaction solution: Dissolve DTT (dithiothreitol) and sodium phosphate in water to make the final concentrations of DTT and sodium phosphate 1 mmol / L and 20 mmol / L, respectively, to obtain a reaction solution.

[0038] (2.2) Determination of 5α-reductase activity: The reaction components were mixed in order (reaction solution 155 μL, constant temperature 37 ℃; testosterone solution 10 μL; NADPH solution 10 μL; 5α-reductase 20 μL), and the absorbance change of the reaction system at 340 nm was continuously monitored within 4 min. The rate of change reflected the activity of 5α-reductase (in the reaction system at a temperature of 37 ℃, the amount of enzyme that causes the NADPH concentration to decrease by 1 μmol / L per minute is one enzyme activity unit), which was recorded as the initial enzyme activity.

[0039] (2.3) Determination of 5α-reductase inhibition rate of fermentation products: The reaction components were mixed in order (reaction solution 155 μL, constant temperature 37 ° C; testosterone solution 10 μL; NADPH solution 10 μL; 15 μL of the fermentation product of Platycladus orientalis leaves of Example 1 to Comparative Example 5, respectively; 5α-reductase 20 μL), and the change in the absorbance of the reaction system at 340 nm was continuously monitored within 4 min. The enzyme activity of 5α-reductase inactivation was calculated with reference to the NADPH standard curve.

[0040] Formula: 5α-reductase inhibition rate = inactivated enzyme activity / initial enzyme activity × 100% The components of the fermented product of Platycladus orientalis leaves prepared above were tested, and the results are shown in Table 1 below.

[0041] Table 1 Component indexes of fermentation products of Platycladus orientalis leaves Total flavonoids mg / g 5α-reductase inhibition rate% Example 1 42.6 91 Example 2 26.6 59 Comparative Example 1 29.1 65 Comparative Example 2 26.4 58 Comparative Example 3 31.2 69 Comparative Example 4 35.4 79 Comparative Example 5 37.6 81 The results are shown in Table 1: the total flavonoids content of the fermented product of Platycladus orientalis leaves in Example 1 was the highest, and the 5α-reductase inhibition effect was the most significant (p<0.05).

[0042] Experimental Example 4 Inflammatory Factor Inhibition Test in Mice RAW264.7 cells grown to the logarithmic phase were cultured at 1.5×10 5 Cells were inoculated at a density of 100 μg / mL in a 24-well plate, with 800 μL of cell suspension per well, and cultured in a 37°C, 5% CO2 incubator for 24 h. When the cells grew to near fusion, 50 μL of the sample concentration of 10 mg / L was administered, with 3 replicates per group. The positive control was 50 μL of 10 μg / mL dexamethasone, and 1 μg / mL lipopolysaccharide (LPS) was used for stimulation 1 h later. After 24 h of incubation in a cell culture incubator, the cell culture medium was transferred to a centrifuge tube, centrifuged at 5000 r / min for 10 min, and the cell supernatant was collected and tested according to the instructions of the mouse TNF-α enzyme-linked immunosorbent assay kit.

[0043] ×100% Table 2. Results of TNF-α secretion by RAW Experimental Group Inhibition rate Positive group 100% Blank Group 0.00% Example 1 97.31% Example 2 63.99% Comparative Example 1 67.41% Comparative Example 2 63.48% Comparative Example 3 75.17% Comparative Example 4 82.14% Comparative Example 5 84.37% From the results in Table 2, it can be seen that the Platycladus orientalis leaf extract prepared by the fermentation method provided in Example 1 of the present invention has a good TNF-α inhibitory effect (p<0.05).

[0044] Example 5 Hair loss experiment in mice Thirty 10-week-old male C3H / HeJ mice of equivalent weight were randomly divided into a normal group, a model control group, and an experimental group, with 10 mice in each group. Normal group: no treatment; Model control group: melted rosin and paraffin were used for back depilation, and 150 mg / kg cyclophosphamide was intraperitoneally injected once a day for 7 consecutive days. Experimental group: Take a 5% aqueous solution of the fermented product of Platycladus orientalis leaves of Example 1-Comparative Example 5 and gently massage the depilatory area for 5 minutes, once a day. The relative expression of IFN-γ in mice was detected using a commercial kit, and the specific results are shown in Table 3.

[0045] Table 3 IFN-γ expression Grouping Relative expression of IFN-γ Normal group 1.0 Model control group 3.4 Example 1 1.5 Example 2 2.7 Comparative Example 1 2.5 Comparative Example 2 2.7 Comparative Example 3 2.3 Comparative Example 4 2.1 Comparative Example 5 1.9 Since IFN-γ can induce the follicular expression of major histocompatibility complex class I and class II molecules, it leads to the collapse of hair follicle tissue immune immunity and induces autoimmune alopecia. The results in Table 3 show that the fermented extract of Platycladus orientalis leaves of the present application can well inhibit the expression of IFN-γ, thereby having the function of treating or improving alopecia. Among them, the effect of Example 1 is the most significant (p<0.05).

[0046] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for fermenting Platycladus orientalis leaves with composite bacteria, characterized in that: The composite bacteria comprises Lactococcus lactis and Pichia pastoris.

2. The method according to claim 1, characterized in that The following steps are involved: (1) Pretreatment of Platycladus orientalis leaves; (2) Enzyme treatment of Platycladus orientalis leaves; (3) Fermentation of Platycladus orientalis leaves.

3. The method according to claim 1, characterized in that The method also includes the following steps: (4) fermentation liquid treatment.

4. The method according to claim 3, characterized in that The fermentation liquid is treated by extraction with n-butanol and ethyl acetate.

5. Application of a method for fermenting Platycladus orientalis leaves with a composite bacteria, characterized in that: The application is to prepare daily chemical products containing Platycladus orientalis leaf active ingredients.

6. The use according to claim 5, characterized in that The application is that the active ingredient of Platycladus orientalis leaves is flavonoids or Platycladus orientalis leaf polysaccharide.

7. The use according to claim 5, characterized in that The daily chemical product is a hair care product.

8. A product of composite bacteria fermentation of Platycladus orientalis leaves, characterized in that: The product is prepared by fermentation according to any one of claims 1 to 4.

9. Use of the product according to claim 8, characterized in that The application is to prepare daily chemical products containing Platycladus orientalis leaf active ingredients.

10. The use according to claim 9, characterized in that The daily chemical product is a hair care product.