Method and application of fermenting Platycladus orientalis leaves with compound bacteria

Abstract

The present invention relates to the field of biological fermentation. Specifically, it relates to a method and application for fermenting Platycladus orientalis leaves with a composite bacterium, and the composite bacterium includes Lactococcus lactis and Pichia pastoris. The method includes the following steps: (1) pretreatment of Platycladus orientalis leaves; (2) enzymatic treatment of Platycladus orientalis leaves; (3) fermentation of Platycladus orientalis leaves; (4) treatment of the fermentation broth. The present invention has the following beneficial effects: (1) The present invention optimizes the enzymatic hydrolysis parameters, fermentation parameters, and the method for extracting the fermentation broth, and obtains a better fermentation method; (2) The fermentation of Platycladus orientalis leaves in the present invention simplifies the components compared with the fermentation of various traditional Chinese medicine raw materials in the prior art, making the invention more convenient; (3) The mouse experiments of the present invention prove that by using the optimized fermentation method, more flavonoids can be obtained, the anti-hair loss effect is better, and it is more beneficial to the application of Platycladus orientalis leaves.

Description

Technical Field

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

[0002] Platycladus orientalis is an evergreen tree with a broad ovoid crown, flattened branchlets arranged in a single plane. It is monoecious with unisexual flowers. The male cone is yellow, composed of alternately opposite microsporophylls, each microsporophyll bearing 3 pollen sacs, and the ovuliferous scale and bract scale are completely fused. The cone matures in the same year, the seed scales are lignified and split, and the seeds have no wings or have ridges. The leaves are small and scale-like, much branched, the branchlets are flattened and closely attached to the branchlets, arranged in opposite pairs in a cross pattern, with a glandular groove in the middle of the leaf back, dark green or yellowish green.

[0003] Platycladus orientalis leaves are a commonly used traditional Chinese medicine for treating hair loss. It is recorded in "Chinese Herbal Medicine" that applying the ethanol extract of Platycladus orientalis leaves to the hair loss area and treating 160 cases achieved a total effective rate of 77.5%. The chemical composition of Platycladus orientalis leaves is complex, including flavonoids, volatile oils, tannins, etc., and has effects such as antibacterial and anti-inflammatory, blood vessel dilation, dandruff removal and hair loss prevention, and hair growth and proliferation. Modern medical research has found that the flavonoid component in Platycladus orientalis leaves can activate hair matrix cells, promote blood circulation, improve hair follicle development and growth, revive hair follicles with declining hair growth ability, and play a role in nourishing and growing hair by supplementing nutritional components for the revived hair follicles. In addition, cedrol, quercitrin and sciadopitysin in Platycladus orientalis leaves have the activity of promoting autophagy.

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

[0005] The prior art CN117530907B discloses a plant fermentation product for controlling oil, soothing and nursing the scalp. The preparation raw materials include, by weight, 1-5 parts of Platycladus orientalis leaves, 1-3 parts of liquorice roots, 1-3 parts of rosebud powder, 1-3 parts of rice, 1-2 parts of Ligusticum wallichii, 1-2 parts of ginseng roots, 1-2 parts of wheat sprouts, 1-2 parts of ginger, 1-3 parts of Caulerpa taxifolia, 0.5-2 parts of fructooligosaccharide, and 70-80 parts of water. The composition contains a combination of plants and probiotic fermentation, all of which are natural plants and biological strains, having good antibacterial effects. Each component synergistically enhances the effect and is green and safe. Through scientific proportioning, it can effectively remove dandruff and oil, improve the scalp ecology; through repairing the scalp barrier, controlling the secretion of scalp sebum, soothing and anti-inflammatory, removing dandruff and relieving itching, it helps to restore the healthy state of the scalp. At the same time, it can also significantly increase hair density, promote the health of hair quality, and make hair smooth and compliant.

[0006] The prior art CN117511827B discloses a Platycladus orientalis fermentation product for scalp care, oil control and dandruff removal, its preparation method and application. In this method, Pichia pastoris and Bacillus subtilis are first inoculated into a fermentation medium according to a mass ratio of 4.7-5.3:0.7-1.3, fermented and then sterilized, and then a specific Lactococcus lactis flora is inoculated for anaerobic fermentation to obtain the Platycladus orientalis fermentation product. The Platycladus orientalis fermentation product has excellent scalp care, oil control and dandruff removal effects and is suitable for being used as and / or preparing cosmetics or medical compositions.

[0007] The prior art CN115414301B discloses a plant dandruff-removing composition, which is characterized in that it is composed of glycolipid and plant extract, wherein the mass ratio of glycolipid to plant extract is (1-4):(2-5); the glycolipid is sophorolipid; the preparation method of the sophorolipid includes: performing plate culture on Candida sp., picking single colonies into a liquid medium for fermentation, standing and separating layers after fermentation is completed, and collecting the bottom-layer sophorolipid; the preparation method of the plant extract includes: mixing raw materials of rosemary leaves and Platycladus orientalis leaves according to a mass ratio of 96:4, adding 4 times the mass of water of the raw materials, heating and refluxing at 95°C for 2.5 h, filtering, and collecting the filtrate to obtain the plant extract.

[0008] However, in the fermentation of Platycladus orientalis in the above prior art, other traditional Chinese medicine components or other additional components are added. In this application, the above components will be simplified, and Platycladus orientalis will be fermented alone, and its fermentation strains and parameters will be optimized to explore a simpler, more efficient method and application of fermenting Platycladus orientalis with a composite bacterium. Summary of the Invention

[0009] To solve the above problems, the present invention first provides a method for fermenting Platycladus orientalis with a composite bacterium, and the composite bacterium includes Lactococcus lactis and Pichia pastoris.

[0010] In some embodiments, it includes the following steps:

[0011] (1)Pre-treatment of Platycladus orientalis leaves;

[0012] (2)Enzymatic treatment of Platycladus orientalis leaves;

[0013] (3)Fermentation of Platycladus orientalis leaves.

[0014] In some embodiments, the following steps are further included: (4) Treatment of the fermentation broth.

[0015] In some embodiments, the following steps are included: The treatment of the fermentation broth is extraction using n-butanol and ethyl acetate.

[0016] The present invention also provides an application of a method for fermenting Platycladus orientalis leaves with a composite bacterium, and the application is to prepare a daily chemical product containing the active ingredients of Platycladus orientalis leaves.

[0017] In some embodiments, the active ingredient of Platycladus orientalis leaves in the application is flavonoid or Platycladus orientalis polysaccharide.

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

[0019] The present invention finally provides a product obtained by fermenting Platycladus orientalis leaves with a composite bacterium, and the product is prepared by fermentation using the above method.

[0020] In some embodiments, the application is to prepare a daily chemical product containing the active ingredients of Platycladus orientalis leaves.

[0021] In some embodiments, the daily chemical product is a hair care product.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects:

[0023] (1)In the present invention, enzymatic hydrolysis is used before fermentation, and extraction using n-butanol and ethyl acetate is used after fermentation. The present invention optimizes the enzymatic hydrolysis parameters, optimizes the fermentation parameters, and optimizes the fermentation broth extraction method, and obtains a better fermentation method;

[0024] (2)The fermentation of Platycladus orientalis leaves in the present invention simplifies the components compared with the fermentation of various traditional Chinese medicine raw materials in the prior art, making the invention more convenient;

[0025] (3)The mouse experiment of the present invention proves that by using the optimized fermentation method, more flavonoids can be obtained, better anti-hair loss effects can be achieved, and it is more beneficial to the application of Platycladus orientalis leaves. Detailed implementation manners

[0026] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to specific embodiments.

[0027] Lactococcus lactis and Pichia pastoris are both purchased from commercial channels.

[0028] Example 1 Method for Fermenting Platycladus orientalis Leaves with Compound Bacteria

[0029] Step (1): Wash 10 g of Platycladus orientalis leaves, remove impurities, freeze-dry, and grind them into powder;

[0030] Step (2): Add 200 mL of deionized water, add 10 mg of compound enzyme (200,000 U / g cellulase and 200,000 U / g pectinase, weight ratio 1:1), enzymatically hydrolyze in a 37°C water bath for 4 h, then add 10 mg of hydrolytic protease (200,000 U / g) and enzymatically hydrolyze for 4 h to obtain an enzymolysis solution, and boil for 15 minutes to inactivate;

[0031] Step (3): Inoculate 2% Pichia pastoris and 2% Lactococcus lactis by mass for fermentation. The initial fermentation temperature is 30°C, the initial fermentation pH is 5.5, the initial fermentation dissolved oxygen content is 18, the fermentation stirring speed is 45 r / min, and the fermentation duration is 16 h to obtain a fermentation broth.

[0032] Step (4): After filtering the fermentation broth with sterile gauze, mix it with n-butanol and ethyl acetate at a ratio of 1 / 3 (volume ratio) respectively, let it stand at room temperature for 72 h, separate the lower aqueous phase filtrate by liquid separation, and vacuum rotary evaporate the upper organic solvents at 60°C and 40°C respectively to remove the solvents to obtain solid samples.

[0033] Example 2 Method for Fermenting Platycladus orientalis Leaves with Compound Bacteria

[0034] Step (1): Wash 10 g of Platycladus orientalis leaves, remove impurities, freeze-dry, and grind them into powder;

[0035] Step (2): Add 200 mL of deionized water, add 10 mg of compound enzyme (200,000 U / g cellulase and 200,000 U / g pectinase, weight ratio 1:1), enzymatically hydrolyze in a 37°C water bath for 4 h, then add 10 mg of hydrolytic protease (200,000 U / g) and enzymatically hydrolyze for 4 h to obtain an enzymolysis solution, and boil for 15 minutes to inactivate;

[0036] Step (3): Inoculate 2% Pichia pastoris and 2% Lactococcus lactis by mass for fermentation. The initial fermentation temperature is 30°C, the initial fermentation pH is 5.5, the initial fermentation dissolved oxygen content is 18, the fermentation stirring speed is 45 r / min, and the fermentation duration is 16 h to obtain a fermentation broth.

[0037] Step (4): After filtering the fermentation broth with sterile gauze, vacuum rotary evaporate to remove the solvent to obtain solid samples.

[0038] Comparative Example 1 Method for Fermenting Platycladus orientalis Leaves with Compound Bacteria

[0039] Step (1): Wash 10 g of Platycladus orientalis leaves, remove impurities, freeze-dry, and grind them into powder;

[0040] Step (2): Add 200 mL of deionized water and 10 mg of cellulase (200,000 U / g), enzymatically hydrolyze at 37 °C in a water bath for 4 h, then add 10 mg of protease (200,000 U / g) and enzymatically hydrolyze for 4 h to obtain an enzymatic hydrolysate, and inactivate by boiling for 15 minutes;

[0041] Step (3): Inoculate 2% Pichia pastoris and 2% Lactococcus lactis by mass for fermentation. The initial fermentation temperature is 30 °C, the initial fermentation pH is 5.5, the initial fermentation dissolved oxygen content is 18, the fermentation stirring speed is 45 r / min, and the fermentation duration is 16 h to obtain a fermentation broth.

[0042] Step (4): After filtering the fermentation broth with sterile gauze, mix it with n-butanol and ethyl acetate at a ratio of 1 / 3 (volume ratio) respectively, let it stand at room temperature for 72 h, separate and remove the lower aqueous phase filtrate, and vacuum rotary evaporate the upper organic solvents at 60 °C and 40 °C respectively to remove the solvents and obtain solid samples.

[0043] Method for fermenting Platycladus orientalis leaves by compound bacteria in Comparative Example 2

[0044] Step (1): Wash 10 g of Platycladus orientalis leaves, remove impurities, freeze-dry, and grind them into powder;

[0045] Step (2): Add 200 mL of deionized water and 10 mg of pectinase (200,000 U / g), enzymatically hydrolyze at 37 °C in a water bath for 4 h, then add 10 mg of protease (200,000 U / g) and enzymatically hydrolyze for 4 h to obtain an enzymatic hydrolysate, and inactivate by boiling for 15 minutes;

[0046] Step (3): Inoculate 2% Pichia pastoris and 2% Lactococcus lactis by mass for fermentation. The initial fermentation temperature is 30 °C, the initial fermentation pH is 5.5, the initial fermentation dissolved oxygen content is 18, the fermentation stirring speed is 45 r / min, and the fermentation duration is 16 h to obtain a fermentation broth.

[0047] Step (4): After filtering the fermentation broth with sterile gauze, mix it with n-butanol and ethyl acetate at a ratio of 1 / 3 (volume ratio) respectively, let it stand at room temperature for 72 h, separate and remove the lower aqueous phase filtrate, and vacuum rotary evaporate the upper organic solvents at 60 °C and 40 °C respectively to remove the solvents and obtain solid samples.

[0048] Method for fermenting Platycladus orientalis leaves by compound bacteria in Comparative Example 3

[0049] Step (1): Wash 10 g of Platycladus orientalis leaves, remove impurities, freeze-dry, and grind them into powder;

[0050] Step (2): Add 200 mL of deionized water and 20 mg of a composite enzyme (200,000 U / g cellulase and 200,000 U / g pectinase, with a weight ratio of 1:1), enzymatically hydrolyze in a 37°C water bath for 8 h to obtain a hydrolyzate, and boil for 15 minutes to inactivate the enzyme;

[0051] Step (3): Inoculate 2% Pichia pastoris and 2% Lactococcus lactis by mass for fermentation. The initial fermentation temperature is 30°C, the initial fermentation pH is 5.5, the initial fermentation dissolved oxygen content is 18, the fermentation stirring speed is 45 r / min, and the fermentation duration is 16 h to obtain a fermentation broth.

[0052] Step (4): After filtering the fermentation broth with sterile gauze, mix it with n-butanol and ethyl acetate at a ratio of 1 / 3 (volume ratio) respectively, let it stand at room temperature for 72 h, separate and remove the lower aqueous phase filtrate, and vacuum rotary evaporate the upper organic solvents at 60°C and 40°C respectively to remove the solvents and obtain solid samples.

[0053] Method for fermenting Platycladus orientalis leaves by composite bacteria in Comparative Example 4

[0054] Step (1): Wash 10 g of Platycladus orientalis leaves, remove impurities, freeze-dry, and grind into powder;

[0055] Step (2): Add 200 mL of deionized water and 10 mg of a composite enzyme (200,000 U / g cellulase and 200,000 U / g pectinase, with a weight ratio of 1:1), enzymatically hydrolyze in a 37°C water bath for 4 h, and then add 10 mg of protease (200,000 U / g) for enzymatic hydrolysis for 4 h to obtain a hydrolyzate, and boil for 15 minutes to inactivate the enzyme;

[0056] Step (3): Inoculate 4% Pichia pastoris by mass for fermentation. The initial fermentation temperature is 30°C, the initial fermentation pH is 5.5, the initial fermentation dissolved oxygen content is 18, the fermentation stirring speed is 45 r / min, and the fermentation duration is 16 h to obtain a fermentation broth.

[0057] Step (4): After filtering the fermentation broth with sterile gauze, mix it with n-butanol and ethyl acetate at a ratio of 1 / 3 (volume ratio) respectively, let it stand at room temperature for 72 h, separate and remove the lower aqueous phase filtrate, and vacuum rotary evaporate the upper organic solvents at 60°C and 40°C respectively to remove the solvents and obtain solid samples.

[0058] Method for fermenting Platycladus orientalis leaves by composite bacteria in Comparative Example 5

[0059] Step (1): Wash 10 g of Platycladus orientalis leaves, remove impurities, freeze-dry, and grind into powder;

[0060] Step (2): Add 200 mL of deionized water and 10 mg of a composite enzyme (200,000 U / g cellulase and 200,000 U / g pectinase, with a weight ratio of 1:1). Enzymatically hydrolyze at 37 °C in a water bath for 4 h, then add 10 mg of a protease (200,000 U / g) and enzymatically hydrolyze for 4 h to obtain an enzymatic hydrolysate. Boil for 15 minutes to inactivate the enzymes.

[0061] Step (3): Inoculate 4% Lactococcus lactis by mass for fermentation. The initial fermentation temperature is 30 °C, the initial fermentation pH is 5.5, the initial oxygen content for fermentation is 18, the fermentation stirring speed is 45 r / min, and the fermentation duration is 16 h to obtain a fermentation broth.

[0062] Step (4): After filtering the fermentation broth with sterile gauze, mix it with n-butanol and ethyl acetate at a ratio of 1 / 3 (volume ratio) respectively. Let it stand at room temperature for 72 h, then separate the liquid to remove the lower aqueous filtrate. The upper organic solvents are respectively vacuum rotary evaporated at 60 °C and 40 °C to remove the solvents and obtain solid samples.

[0063] Example 3: Determine the component indicators of the fermented product of Platycladus orientalis leaves

[0064] Determination of total flavonoids

[0065] Determination of total flavonoid content: Mix 0.5 mL of the methanol extract of the fermented product of Platycladus orientalis leaves with 1.5 mL of 5% sodium nitrite, shake well and let stand for 6 min. Add 0.3 mL of 10% aluminum nitrate, shake well and let stand for 6 min, then add 1 mL of 4% sodium hydroxide, and make up the volume to 5 mL with 95% ethanol. Shake well and let stand for 15 min, and measure the absorbance at 510 nm. Use rutin solution as the standard solution and the solution without adding the standard solution as the control to draw the standard curve.

[0066] (2) Oil control efficacy test

[0067] (2.1) Preparation of reagents:

[0068] Testosterone (T) solution: Dissolve 36 mg of testosterone in absolute ethanol to prepare a testosterone solution with a concentration of 5 mmol / L;

[0069] NADPH (reduced coenzyme II) solution: Dissolve 42.5 mg of NADPH tetrasodium salt in water to prepare a NADPH solution with a concentration of 2 mmol / L;

[0070] 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 the reaction solution.

[0071] (2.2) Determination of 5α-reductase activity:

[0072] Mix the reaction components in the following order (155 μL of reaction solution, maintained at 37 °C; 10 μL of testosterone solution; 10 μL of NADPH solution; 20 μL of 5α-reductase), and continuously monitor the change in the absorbance of the reaction system at 340 nm within 4 min. The activity of 5α-reductase is reflected by the change rate (in a reaction system at 37 °C, the amount of enzyme that causes the NADPH concentration to decrease by 1 μmol / L per minute is defined as one enzyme activity unit), which is recorded as the initial enzyme activity.

[0073] (2.3)Determination of the 5α-reductase inhibition rate of the fermented product:

[0074] Mix the reaction components in the following order (155 μL of reaction solution, maintained at 37 °C; 10 μL of testosterone solution; 10 μL of NADPH solution; 15 μL of the Platycladus orientalis fermented product from Example 1 to Comparative Example 5 respectively; 20 μL of 5α-reductase), and continuously monitor the change in the absorbance of the reaction system at 340 nm within 4 min. Calculate the inactivated enzyme activity of 5α-reductase with reference to the NADPH standard curve.

[0075] Formula: 5α-reductase inhibition rate = inactivated enzyme activity / initial enzyme activity × 100%

[0076] Detect the components of the prepared Platycladus orientalis fermented product, and the results are shown in Table 1 below.

[0077] Table 1 Component indicators of the Platycladus orientalis fermented product

[0078] 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

[0079] The results are shown in Table 1: The total flavonoid content of the Platycladus orientalis fermented product in Example 1 is the highest, and the 5α-reductase inhibition effect is the most significant (p < 0.05).

[0080] Experimental Example 4 Inhibitory test of mouse inflammatory factors

[0081] Seed RAW264.7 cells in the logarithmic growth phase at a density of 1.5×10 5 cells / mL in a 24-well plate, with 800 μL of cell suspension in each well. Culture in an incubator at 37 °C and 5% CO2 for 24 h. When the cells are nearly confluent, administer the drug at a sample concentration of 10 mg / L and 50 μL. Set 3 replicate wells for each group. The positive control is 50 μL of dexamethasone at 10 μg / mL. After 1 h, stimulate with 1 μg / mL lipopolysaccharide (LPS). After incubating in the cell culture incubator for 24 h, transfer the cell culture medium into a centrifuge tube, centrifuge at 5000 r / min for 10 min to collect the cell supernatant, and detect according to the instructions of the mouse TNF-α enzyme-linked immunosorbent assay kit.

[0082] × 100%

[0083] Table 2. Results of RAW secreting TNF-α

[0084] Test 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%

[0085] Analysis of the results in Table 2: The Platycladus orientalis extract prepared by the fermentation method provided in Example 1 of the present invention has a good TNF-α inhibitory effect (p < 0.05).

[0086] Example 5 Mouse hair loss experiment

[0087] Thirty 10-week-old male C3H / HeJ mice with comparable body weights 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: The back was depilated by applying melted rosin and paraffin, and 150 mg / kg of cyclophosphamide was intraperitoneally injected once a day for 7 consecutive days. Experimental group: An aqueous solution of the Platycladus orientalis fermentation product of Examples 1 - Comparative Example 5 with a concentration of 5% was gently massaged on the depilated area for 5 min once a day. A commercial kit was used to detect the relative expression level of IFN-γ in the mice. The specific results are shown in Table 3.

[0088] Table 3 IFN-γ expression level

[0089] Grouping Relative expression level 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

[0090] Since IFN-γ can induce the follicular expression of major histocompatibility complex class I and class II molecules, leading to the breakdown of immune privilege in follicular tissues and inducing autoimmune alopecia. And the results in Table 3 show that the Platycladus orientalis fermentation extract of the present application can well inhibit the expression of IFN-γ, thus having the function of treating or improving hair loss. Among them, the effect of Example 1 is the most significant (p < 0.05).

[0091] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for fermenting Platycladus orientalis leaves by compound bacteria, characterized in that, It includes the following steps: Step (1): Wash 10 g of Platycladus orientalis leaves, remove impurities, freeze-dry, and grind them into powder; Step (2): Add 200 mL of deionized water and 10 mg of complex enzyme, enzymatically hydrolyze in a 37°C water bath for 4 h, then add 10 mg of protease for enzymatic hydrolysis for 4 h to obtain an enzymatic hydrolysate, and boil for 15 minutes to inactivate; The complex enzyme is composed of cellulase and pectinase, and the weight ratio of the cellulase to the pectinase is 1:1; the enzyme activities of the cellulase, pectinase, and protease are all 200,000 U / g; Step (3): Inoculate 2% of Pichia pastoris and 2% of Lactococcus lactis by mass for fermentation to obtain a fermentation broth; Step (4): After filtering the fermentation broth with sterile gauze, mix it with n-butanol and ethyl acetate respectively in a volume ratio of 1 / 3, let it stand at room temperature for 72 h, then separate the lower aqueous phase filtrate by liquid separation, and vacuum rotary evaporate the upper organic solvents at 60°C and 40°C respectively to remove the solvents to obtain solid samples.

2. A product of fermenting Platycladus orientalis leaves with a composite bacterium, characterized in that, The product is prepared by fermentation according to the method described in claim 1, wherein the total flavonoid content is 42.6 mg / g and the 5α-reductase inhibition rate is 91%.

3. Use of the product according to claim 2, characterized in that, The application is to prepare a daily chemical product containing the active ingredients of Platycladus orientalis leaves, and the daily chemical product is a hair care product.