CN117158579A - Application of a fermented black wolfberry product with anti-hair loss effect - Google Patents

Abstract

The invention belongs to the field of microbial technology, and particularly relates to the application of a black wolfberry fermented product with the effect of preventing hair loss. The application is the black wolfberry enzyme stock solution and the application of the black wolfberry enzyme in the preparation of products to prevent hair loss, and also includes the preparation of kidney-improving kidney products. Applications in functional and/or immunity-enhancing products. The present invention adopts unique cold process technology, which not only fully utilizes the efficacy of raw material ingredients, but also fully exerts the efficacy of beneficial ingredients produced by fermentation. The black wolfberry enzyme stock solution and its products (black wolfberry fermentation products) prepared by applying the cold process technology of the present invention It can balance yin and yang, nourish the liver and kidneys and prevent hair loss. It can also enhance immunity.

Description

Application of a fermented black wolfberry product with anti-hair loss effect

This application is for the application number 202210660831.6, the application date is 2022.06.13, and the name is "One

A divisional application for an invention patent application with "multifunctional black wolfberry enzyme stock solution and its preparation process".

Technical field

The invention belongs to the field of microbial technology, and particularly relates to the application of a black wolfberry fermented product with anti-hair loss effect, specifically a black wolfberry enzyme stock solution with anti-hair loss effect and its products in the preparation of improved kidney function, prevention of hair loss and/or Applications in immunity-boosting products.

Background technique

Modern life is fast-paced, people's lives are stressful, irregular eating habits and irregular lifestyles such as staying up late leave most of the body in a sub-healthy state with low immunity. Relevant research results show that kidney deficiency can lead to immune dysfunction, and kidney-tonifying traditional Chinese medicine can regulate various indicators related to humoral immunity and cellular immunity, and improve the immune function of patients with kidney deficiency.

Hair loss has a great impact on people's lives, seriously affecting people's normal life and mood. Currently, most of the products for hair loss are for external use and target hair loss caused by seborrheic dermatitis, folliculitis and other diseases. There are few products that treat hair loss from within. The outer angle adjusts the body and solves the problem of hair loss.

The current enzyme production process generally involves hot water leaching, extraction, and fermentation to obtain a fermentation mixture. Enzymes are mostly sterilized by high-temperature sterilization. High temperatures cause the components in enzyme products to be destroyed, active ingredients such as anthocyanins to degrade, and the utilization rate of active ingredients is greatly reduced.

Contents of the invention

In order to solve the above problems, the present invention provides a black wolfberry enzyme stock solution that can balance yin and yang, nourish the liver and kidneys, prevent hair loss, and enhance immunity. Enzymes are made from animals, plants, fungi, etc., with or without the addition of auxiliary materials, and contain specific biologically active ingredients (including polysaccharides, oligosaccharides, proteins and peptides, amino acids, and vitamins) obtained through microbial fermentation. The product.

In order to improve the efficacy of black wolfberry enzyme products, the present invention adopts a unique cold process technology, which not only fully utilizes the efficacy of raw material ingredients, but also fully utilizes the efficacy of beneficial ingredients produced by fermentation.

The technical solution adopted by the present invention is as follows. A multifunctional black wolfberry enzyme stock solution includes the following raw materials in parts by weight: water 82-95g/L, black wolfberry 3.0-6.0g/L, mulberry 1.0-4.0g/L, Isomaltooligosaccharide 1.0-5.0g/L, fructooligosaccharide 0.1-3.0g/L.

The preparation method of the above-mentioned multifunctional black wolfberry enzyme stock solution includes the following steps:

1) Raw material pretreatment: Put isomaltooligosaccharide and fructooligosaccharide into water. After all the materials are dissolved, sterilize at 115°C for 30 minutes and cool to 37°C;

2) Feeding: according to the weight parts described in claim 1, put sterile black wolfberry and mulberry into the sterilized feed liquid under aseptic conditions, stir evenly to ensure that all raw materials are soaked;

3) Strain activation: Inoculate Lactobacillus plantarum HCS03-001, Lactobacillus reuteri HCS02-001, and Lactobacillus rhamnosus HCS01-013 into the activation medium at an inoculum volume of 0.06%, and the liquid volume is 50 to 80 %, culture;

4) Fermentation: Inoculate the activated bacterial liquid obtained in step 3) into the feed liquid obtained in step 2), with an inoculation amount of 5% and a liquid filling amount of 50-75%; fermentation is carried out with stirring; fermentation starts at 45 hours, each time Monitor the pH value of the fermentation broth every hour. When the difference between the two consecutive pH values is <0.10, the fermentation ends; at the end of the fermentation, the pH value is <3.40;

5) Rough filtration of fermentation broth: Centrifuge the fermentation broth obtained in step 4) to collect the filtrate;

6) Centrifugation of fermentation broth: Centrifuge the fermentation broth obtained in step 5);

7) Filtration and impurity removal from the fermentation broth: Pour the feed liquid obtained in step 6) into a cross-flow membrane filter to filter and remove impurities;

8) Filtration and sterilization of the fermentation liquid: The fermentation liquid in step 7) is sterilized at room temperature using polymer material microporous membrane sterilization equipment to obtain black wolfberry enzyme stock solution.

Preferably, in the above-mentioned method for preparing a multifunctional black wolfberry enzyme stock solution, in step 2), the order of feeding is black wolfberry and mulberry.

Preferably, in the above-mentioned method for preparing a multifunctional black wolfberry enzyme stock solution, in step 3), according to the mass ratio, Lactobacillus plantarum HCS03-001: Lactobacillus reuteri HCS02-001: Lactobacillus rhamnosus HCS01- 013=10:9:1.

Preferably, in the above-mentioned method for preparing a multifunctional black wolfberry enzyme stock solution, in step 3), the activation medium: anhydrous glucose 40g/L, yeast extract 15g/L, yeast peptone 10g/L, L-malic acid 3g/L, citric acid monohydrate 2g/L, potassium dihydrogen phosphate 2g/L, calcium chloride 0.5g/L, magnesium sulfate 0.06g/L, manganese sulfate 0.01g/L, adjust the pH to 6.60 ;115℃, 30min sterilization.

Preferably, in the above-mentioned method for preparing a multifunctional black wolfberry enzyme stock solution, in step 3), the culture conditions are 37°C for 16±1 h, and the culture is stopped when the number of viable bacteria in the bacterial solution reaches 1.0×10 ⁹ CFU/mL or more.

Preferably, in the above-mentioned method for preparing a multifunctional black wolfberry enzyme stock solution, in step 4), the stirring speed during fermentation: 60-80r/min; the ventilation volume is 0; the initial air pressure is 0.02-0.03MPa; at 37°C Ferment for 47±2h.

A multi-functional black wolfberry enzyme is prepared by placing the above-mentioned black wolfberry enzyme stock solution in a container, adding maltitol liquid, xylitol, apple powder, and sucralose, stirring, letting it stand, and sterilizing to obtain the black wolfberry enzyme.

Preferably, the above-mentioned multifunctional black wolfberry enzyme contains, in parts by weight, 947.86 parts of black wolfberry enzyme stock solution, 25 parts of maltitol solution, 25 parts of xylitol, 2 parts of apple powder, and 0.14 parts of sucralose.

Black wolfberry is a functional raw material that is both medicinal and edible. It contains protein, fat, organic acids, sugars, vitamins and a variety of trace elements. In addition, it is rich in a variety of functional ingredients, such as flavonoids, anthocyanins, proanthocyanidins, anthocyanins and polysaccharides, which have high nutrients and health care functions. The use of wolfberry as medicine was first recorded in "Shen Nong's Materia Medica" and is listed as top grade. It nourishes the liver, nourishes the kidneys and moistens the lungs. "Compendium of Materia Medica": "moistens the lungs and promotes body fluids, nourishes the kidneys and adds essence". Lycium barbarum belongs to the liver, kidney, and lung meridians. It not only nourishes the liver and kidneys, but also moistens the lungs and promotes body fluids. If the essence, blood, and body fluids are sufficient, the hair will be nourished and moisturized and will not fall off easily.

Mulberry, the fruit of the mulberry tree, is rich in a variety of nutrients. It is rich in active proteins, vitamins, minerals, amino acids, anthocyanins and other ingredients. It can significantly improve human immunity, nourish the liver and kidneys, nourish yin and blood, and blacken the body. Invention and other effects. "Dictionary of Traditional Chinese Medicine": "Mulberry enters the liver and kidney meridian, and its main functions are to nourish the liver, nourish the kidneys, extinguish wind, and nourish liquid." "Southern Yunnan Materia Medica": "Benefit the kidneys and solidify essence, and long-term use can blacken the eyes." " "Suixiju Diet Spectrum": "Nourishes the liver and kidneys, replenishes blood, dispels rheumatism, strengthens walking, calms deficient wind, and clears deficient fire."

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

1. The enzyme raw materials of the present invention use medicinal and food homologous raw materials, and the selected lactic acid bacteria are all isolated from human feces, so the application safety is high.

2. The enzyme of the present invention applies cold process preparation technology and uses specific probiotics for fermentation, which can effectively retain the active ingredients and fully utilize the beneficial ingredients produced by fermentation.

3. The enzyme of the present invention can nourish the liver and kidneys, regulate the body from the inside out, prevent hair loss and enhance human immunity, and is effective quickly. The amount of hair loss can be reduced after one week of consumption.

4. The enzyme raw material of the present invention uses black wolfberry. Black wolfberry is sweet in taste and neutral in nature, and is rich in protein, wolfberry polysaccharide, amino acids, vitamins, minerals, trace elements and other nutrients. It is also rich in natural proanthocyanidins. Proanthocyanidin OPC is the most effective natural water-soluble free radical scavenger and has strong antioxidant capacity.

5. The enzyme raw material of the present invention uses mulberry, which contains a variety of trace elements and functional ingredients, such as rutin, anthocyanins, resveratrol, etc., and has good anti-cancer, anti-aging, fatigue relief, and black hair. Effects such as eye function.

Detailed ways

Embodiment 1 A multifunctional black wolfberry enzyme stock solution and its preparation process

A multifunctional black wolfberry enzyme stock solution, including the following raw materials in parts by weight: water 82g/L, black wolfberry 6.0g/L, mulberry 4.0g/L, isomaltooligosaccharide 5.0g/L, and fructooligosaccharide 3.0g /L.

The preparation process is as follows:

1. Raw material pretreatment: Weigh the raw materials according to the formula, put isomaltooligosaccharide and fructooligosaccharide into water with 105% of the fermentation weight. After all the materials are dissolved, sterilize at 115°C for 30 minutes and cool to 37°C;

2. Feeding: Put the sterile black wolfberry and mulberry into the sterilized feed liquid under aseptic conditions, stir evenly to ensure that all the raw materials are soaked;

Preferably, the order of feeding is: black wolfberry, mulberry;

3. Bacteria activation: Inoculate Lactobacillus plantarum HCS03-001, Lactobacillus reuteri HCS02-001, and Lactobacillus rhamnosus HCS01-013 (the mass ratio of the three bacteria is 10:9:1) at an inoculation volume of 0.06% Inoculate into the activation medium, fill the liquid volume with 50-80%, and culture at 37°C for 16±1h. The number of viable bacteria in the bacterial solution reaches more than 1.0×10 ⁹ CFU/mL;

Described activation medium: anhydrous glucose 40g/L, yeast extract 15g/L, yeast peptone 10g/L, L-malic acid 3g/L, citric acid monohydrate 2g/L, potassium dihydrogen phosphate 2g/L , calcium chloride 0.5g/L, magnesium sulfate 0.06g/L, manganese sulfate 0.01g/L, adjust pH to 6.60; sterilize at 115°C for 30 minutes.

4. Fermentation: Inoculate the activated bacteria into the feed liquid obtained in step 2, the inoculation amount is 5%, and the liquid filling amount is 50-75%; stir culture, stirring speed: 60-80r/min; ventilation volume is 0 ; Initial air pressure 0.02-0.03MPa; Cultivate at 37°C for 47±2h; Fermentation starts at 45h, monitor the pH value of the fermentation liquid every hour, when the difference between two consecutive pH values is <0.10, the fermentation ends; the fermentation endpoint pH value is <3.40 (fermentation The liquid temperature is normal temperature); the sensory state of the fermentation liquid: dark purple (diluted 10 times to light purple), with the unique flavor of black wolfberry, a slight aroma of mulberry, and a sour taste.

5. Coarse filtration of fermentation liquid: centrifugally filter the fermentation liquid in step 4 to collect the filtrate, including the residual fermentation liquid at the bottom of the fermentation tank, to reduce losses. The filtrate has no obvious residue, is dark purple, has a sour taste, and has a slight mulberry aroma, and the yield reaches more than 93%.

The yield calculation formula is:

6. Centrifugation of fermentation broth: Centrifuge the fermentation broth obtained in step 5. The centrifuge speed is 13000r/min (frequency 43.95Hz), and the peristaltic pump flow rate is 300r/min (flow rate is about 1.5L/min); the appearance of the product remains dark after centrifugation. Purple liquid, the solution is homogeneous, without impurities, and the yield is over 96%.

The yield calculation formula is:

7. Fermentation liquid membrane filtration and impurity removal: Pour the feed liquid obtained in step 6 into a cross-flow membrane filter for filtration and impurity removal; the filter membrane cleaning and replacement time is adjusted according to the smoothness of the discharge until it is clear and dark purple ( The color is lighter than that of crude filtration, diluted 10 times to light purple), the solution is uniform, no impurities, and the yield is more than 95%.

The yield calculation formula is:

8. Filtration and sterilization of the fermentation liquid: The fermentation liquid in step 7 is sterilized at room temperature using microporous membrane sterilization equipment with a membrane pore size of 0.22 μm and made of organic polymer materials to obtain the black wolfberry fermentation stock solution. .

Example 2 Comparison of different processes for a multifunctional black wolfberry enzyme stock solution

Thermal process preparation: use the raw material formula described in Example 1 to produce black wolfberry enzyme stock solution. Immerse black wolfberry and mulberry in water and cook at 100°C for 30-50 minutes, then put them into the sterilized fructooligosaccharide and isomaltooligosaccharide liquid. After probiotic fermentation and centrifugation, use 131°C ultra-high temperature sterilization for 15 seconds. Sterilize and pasteurize after aseptic filling.

Detection of some beneficial components of the enzyme stock solution: Comparative analysis of the beneficial components of the black wolfberry enzyme prepared by the thermal process and the black wolfberry enzyme prepared by the cold process in Example 1 is shown in Table 1. The contents of organic acids, crude polysaccharides, and polyphenols in the black wolfberry enzyme prepared by the cold process are higher than those of the enzyme prepared by the hot process. Crude polysaccharides can improve the body's immune function. Polyphenols can block the covalent binding of carcinogens to cellular DNA, prevent DNA single-strand breaks, and improve the body's cellular immune function. Organic acids have antibacterial, choleretic, anti-inflammatory, hypoglycemic, antioxidant, and immune-regulating effects. They can soften blood vessels and promote the absorption of calcium and iron.

Table 1 Comparative analysis of beneficial components of black wolfberry enzyme stock solution prepared by different processes

Effect on renal function of kidney deficiency model rats: After 1 week of adaptive feeding of SPF grade healthy male adult SD rats, experimental animals were randomly divided into 2 groups according to body weight: 20 rats in the normal group and 50 rats in the model group. The normal group was given a 0.5% carboxymethylcellulose sodium solution 10mL·kg ^-1 d ^-1 by gavage, and the modeling group was given a 150mg·kg ^-1 adenine suspension by gavage for 2 weeks. After that, the modeling group was randomly divided into 4 groups. : There are 20 rats in the model group, 10 rats in the hot process enzyme stock solution group, 10 rats in the cold process enzyme stock solution group, and 10 rats in the positive control group of which Guiluerxian ointment 10g·kg ^-1 was administered through the stomach. The model group and the enzyme stock solution group were given 150 mg·kg ^-1 adenine by gavage in the morning every other day to maintain the model, while the enzyme stock solution group was given 20 mg·kg ^-1 by gavage every afternoon for 4 weeks.

After the test, the serum creatinine (CREA), urea nitrogen (BUN), adrenocorticotropic hormone (ACTH), testosterone (T), and triiodothyronine (T3) contents of rats in each group are shown in Table 2. After 6 weeks of modeling, the serum creatinine and urea nitrogen contents of rats in the model group were significantly higher than those of the normal group, and the contents of adrenocorticotropic hormone, testosterone, and triiodothyronine were significantly lower than those of the normal group, indicating that the renal function of rats and weakened functions of the adrenal glands, gonads, and thyroid glands. After 4 weeks of oral administration of black wolfberry enzyme stock solution, compared with the model group, the contents of urea nitrogen and creatinine in the cold process black wolfberry enzyme stock solution group were significantly reduced, and the contents of ACTH, T and T3 were significantly increased. The hot process black wolfberry enzyme stock solution group The urea nitrogen of the group increased significantly, creatinine increased significantly, ACTH content increased significantly, and T and T3 levels increased. This shows that the black wolfberry enzyme stock solution has a good regulatory effect on the renal function and glandular hormone levels of kidney deficiency model rats, and the cold process group has better effects.

Table 2 Rat renal function indicators of black wolfberry enzyme stock solution prepared by different processes

Example 3 Preparation of a multifunctional black wolfberry enzyme stock solution product

The black wolfberry enzyme stock solution obtained in Example 1 was prepared. 947.86g of the black wolfberry enzyme was poured into the preparation tank. 25g of maltitol solution, 25g of xylitol, 2g of apple powder and 0.14g of sucralose were weighed and put into the preparation. In the jar, stir until completely dissolved, stir for 50 minutes, and let stand for 10 minutes. Filter and sterilize the material liquid. In the early stage of sterilization, part of the material liquid can be discarded. After sterilization, the material liquid state is: clear, dark purple (lighter than before sterilization, diluted 10 times to light purple), and the solution is uniform. No impurities. It has the unique flavor of black wolfberry, with a slight aroma of mulberry. The smell is lighter than before sterilization, and the taste is sour and sweet. The sampling test results show that there are no foreign bacteria. It is filled aseptically, 180mL/bottle.

Embodiment 4 A multifunctional black wolfberry enzyme anti-hair loss functional test

The trial screened a total of 61 people with hair loss problems due to insufficient liver and kidney function, with an average age of 31 years old. According to the traditional Chinese medicine diagnostic standards of the "Guiding Principles for Clinical Research of New Traditional Chinese Medicines", the criteria for selecting candidates are formulated, as shown in Table 3.

Table 3 Criteria for selection of candidates for the trial

Trial patient exclusion criteria:

(1) Patients with serious primary or secondary diseases such as endocrine, heart, brain, liver, kidney and hematopoietic function, or mental illness;

(2) Those who have participated in other clinical trials for the treatment of androgenic alopecia in the past two months;

(3) Those who have undergone surgical hair transplantation.

Criteria for elimination and dropout of trial patients:

(1) Patients who use other drugs related to this disease without authorization during treatment;

(2) Those who cannot follow the doctor’s instructions due to poor compliance, causing obstacles to the judgment of efficacy and safety assessment;

(3) This product was not consumed as required during the experiment;

(4) Those who are unwilling to continue participating in clinical trials.

The test subjects took black wolfberry enzyme products, one bottle per day (180mL/bottle), and the test period was one month. Based on clinical practice, the number of hair loss, greasiness, itching, dandruff, and new hair regeneration are scored, and the treatment effect is judged by the effectiveness after the test. The judgment criteria are shown in Table 4.

Table 4 Symptom score

Rating results:

0-10 points: poor effect;

11-20 points: The effect is average;

21-30 points: The effect is significant.

A multi-functional black wolfberry enzyme solution product was tested for its anti-hair loss efficacy. The specific results are shown in Table 5.

Table 5 Anti-hair loss efficacy test of a multi-functional black wolfberry enzyme stock solution product

Note: Effective rate = (number of people with significant effects + number of people with average effects) / number of participants * 100%

The test results are shown in Table 4. The experimenters had a significant hair loss reduction effect after consuming black wolfberry enzyme products for one week. As the period of taking black wolfberry enzyme is extended, the effectiveness of preventing hair loss increases. During the test, it appeared that the test subjects did not consume the product as required, and part of the data was eliminated. There were 45 valid data in the fourth week. At this time, the anti-hair loss effective rate reached 93.33%. The black wolfberry enzyme of the present invention has obvious anti-hair loss function.

Example 5 A multifunctional black wolfberry enzyme liquid to enhance immunity function test

1. Experimental mouse modeling

Forty SD male rats were randomly divided into control group, model group, low-dose group, medium-dose group, and high-dose group, with a total of 5 groups, 10 rats in each group. The control group was given 10 mL/kg of normal saline every day; the other groups were given Tripterygium wilfordii polyglycoside tablets (20 mg/kg) for 30 consecutive days to create a rat kidney essence deficiency model. The model group was given Tripterygium wilfordii polyglycoside tablets daily for 4 hours. The low-dose black wolfberry enzyme liquid group was given 10 mL/kg of physiological saline by intragastric administration every day, the medium-dose group was intragastrically administered with 15 mL/kg every day, and the high-dose group was intragastrically administered with 20 mL/kg every day for 4 consecutive weeks.

2. Determination of serum immunoglobulin G (IgG), interleukin VI (IL-6), and interleukin II (IL-2) levels

2 hours after the last intragastric administration, 1.5 mL of blood was taken from the femoral artery, left to stand for 30 minutes, and then centrifuged at 3000 r·min ^-1 for 15 minutes. The upper serum was taken and stored at 4°C for testing. Use an ELISA kit and measure the absorbance value at 450nm with a microplate reader.

3. Whole blood CD ₃ ⁺ , CD ₃ ⁺ CD ₄ ⁺ , CD ₃ ⁺ CD ₈ ⁺ , CD ₄ /CD ₈ percentage determination

Two hours after the last intragastric administration, 2 mL of blood was taken from the femoral artery and added to a blood collection tube containing sodium heparin and shaken well. Add 100 μL of anticoagulant blood to the flow tube, add 5 μL of each of Mouse Anti-Rat CD4, Mouse Anti-Rat CD8a, and Mouse Anti-RatCD3 antibodies respectively, and stain for 30 min in the dark at 4°C; add 2 ml of 1X hemolysin and keep at room temperature. 10 min; discard the supernatant after centrifugation for 5 min and repeat twice. After adding 400 μL of PBS solution, vortex and mix, the sample was analyzed using CytoFlex flow cytometer, and the results were analyzed using Kaluza software.

4. Measurement results

The measurement results of serum immunoglobulin G (IgG), interleukin VI (IL-6), and interleukin II (IL-2) are shown in Table 6. Compared with the model group, the results of those taking black wolfberry herbal enzyme There were changes in the measured values in different dose groups. The IgG content in the high-dose group was significantly increased to 3204.49 μg/mL, the IL-6 content was significantly reduced to 11.18pg/mL, and the IL-2 content was significantly increased to 18.84pg/mL. Comparing three groups of different doses of black wolfberry enzyme, the high-dose group had the most significant effect.

Table 6 Effects of black wolfberry enzyme on serum IL-2, IL-6, and IgG contents in kidney essence deficiency model rats

After mice took black wolfberry enzyme, the effects on T lymphocytes are shown in Table 7. Compared with the model group, the CD ₃ ⁺ , CD ₃ ⁺ CD ₄ ⁺ , and CD ₄ /CD ₈ ratios of mice in each dose group were significantly increased, and the CD ₃ ⁺ CD ₈ ⁺ ratio was significantly decreased, and the higher the dose, the more significant the effect.

Table 7 Effects of black wolfberry enzyme on CD ₃ ⁺ , CD ₃ ⁺ CD ₄ ⁺ , CD ₃ ⁺ CD ₈ ⁺ , and CD ₄ /CD ₈ ratios in whole blood samples of kidney essence deficiency model rats

Black wolfberry enzyme can restore the above indicators to normal levels, improve the immune function of the kidney essence deficiency model group, nourish the kidney and qi, regulate cellular immunity and humoral immunity and other related indicators, thereby improving the immune function of patients with kidney deficiency.

Claims (10)

1. The application of the black matrimony vine enzyme stock solution with the alopecia preventing effect in preparing the alopecia preventing product is characterized in that the black matrimony vine enzyme stock solution comprises the following raw materials in parts by weight: 82-95g/L of water, 3.0-6.0g/L of lycium ruthenicum, 1.0-4.0g/L of mulberry, 1.0-5.0g/L of isomaltooligosaccharide and 0.01-3.0g/L of fructooligosaccharide,

the preparation method of the black matrimony vine enzyme stock solution comprises the following steps:

1) Pretreatment of raw materials: putting isomaltooligosaccharide and fructooligosaccharide into water, sterilizing at 115 ℃ for 30min after all materials are dissolved, and cooling to 37 ℃;

2) Feeding: according to the weight portion of claim 1, aseptic lycium ruthenicum and mulberry are put into the sterilized feed liquid under the aseptic condition, and the materials are stirred uniformly, so that the materials are soaked completely;

3) Activating strains: inoculating lactobacillus plantarum HCS03-001, lactobacillus reuteri HCS02-001 and lactobacillus rhamnosus HCS01-013 into an activated culture medium according to an inoculum size of 0.06%, and culturing the lactobacillus plantarum HCS03-001 and the lactobacillus reuteri HCS01-013 with a liquid loading amount of 50-80%;

4) Fermentation: inoculating the activated bacterial liquid obtained in the step 3) into the feed liquid obtained in the step 2), wherein the inoculation amount is 5%, and the liquid loading amount is 50-75%; stirring for fermentation; the fermentation starts for 45 hours, the pH value of the fermentation liquid is monitored every hour, and when the pH value difference between two successive times is less than 0.10, the fermentation ends; the pH value at the fermentation end point is less than 3.40;

5) Coarse filtration of fermentation liquor: centrifuging the fermentation broth obtained in the step 4), and filtering to collect filtrate;

6) And (3) centrifuging fermentation liquor: centrifuging the fermentation broth obtained in step 5);

7) Filtering and removing impurities from fermentation liquor: pumping the feed liquid obtained in the step 6) into a cross-flow membrane filter, and filtering and removing impurities;

8) Filtering and sterilizing fermentation liquor: and (3) performing normal-temperature sterilization on the fermentation broth in the step (7) by adopting high-molecular material microporous membrane sterilization equipment to obtain a black matrimony vine enzyme stock solution.

2. The application of the black matrimony vine enzyme stock solution with the alopecia preventing effect in preparing a product for improving kidney function and/or improving immunity is characterized in that the black matrimony vine enzyme stock solution comprises the following raw materials in parts by weight: 82-95g/L of water, 3.0-6.0g/L of lycium ruthenicum, 1.0-4.0g/L of mulberry, 1.0-5.0g/L of isomaltooligosaccharide and 0.01-3.0g/L of fructooligosaccharide,

the preparation method of the black matrimony vine enzyme stock solution comprises the following steps:

1) Pretreatment of raw materials: putting isomaltooligosaccharide and fructooligosaccharide into water, sterilizing at 115 ℃ for 30min after all materials are dissolved, and cooling to 37 ℃;

2) Feeding: according to the weight portion of claim 1, aseptic lycium ruthenicum and mulberry are put into the sterilized feed liquid under the aseptic condition, and the materials are stirred uniformly, so that the materials are soaked completely;

3) Activating strains: inoculating lactobacillus plantarum HCS03-001, lactobacillus reuteri HCS02-001 and lactobacillus rhamnosus HCS01-013 into an activated culture medium according to an inoculum size of 0.06%, and culturing the lactobacillus plantarum HCS03-001 and the lactobacillus reuteri HCS01-013 with a liquid loading amount of 50-80%;

4) Fermentation: inoculating the activated bacterial liquid obtained in the step 3) into the feed liquid obtained in the step 2), wherein the inoculation amount is 5%, and the liquid loading amount is 50-75%; stirring for fermentation; the fermentation starts for 45 hours, the pH value of the fermentation liquid is monitored every hour, and when the pH value difference between two successive times is less than 0.10, the fermentation ends; the pH value at the fermentation end point is less than 3.40;

5) Coarse filtration of fermentation liquor: centrifuging the fermentation broth obtained in the step 4), and filtering to collect filtrate;

6) And (3) centrifuging fermentation liquor: centrifuging the fermentation broth obtained in step 5);

7) Filtering and removing impurities from fermentation liquor: pumping the feed liquid obtained in the step 6) into a cross-flow membrane filter, and filtering and removing impurities;

8) Filtering and sterilizing fermentation liquor: and (3) performing normal-temperature sterilization on the fermentation broth in the step (7) by adopting high-molecular material microporous membrane sterilization equipment to obtain a black matrimony vine enzyme stock solution.

3. The use according to any one of claims 1-2, wherein in step 2) the order of administration is lycium ruthenicum and mulberry.

4. Use according to any one of claims 1-2, characterized in that in step 3) the lactobacillus plantarum HCS03-001 is in mass ratio: lactobacillus reuteri HCS02-001: lactobacillus rhamnosus HCS 01-013=10:9:1.

5. The use according to any one of claims 1-2, wherein in step 3) the activation medium: 40g/L of anhydrous glucose, 15g/L of yeast extract, 10g/L, L of yeast peptone, 3g/L of malic acid, 2g/L of citric acid monohydrate, 2g/L of potassium dihydrogen phosphate, 0.5g/L of calcium chloride, 0.06g/L of magnesium sulfate and 0.01g/L of manganese sulfate, and regulating the pH value to 6.60; sterilizing at 115 deg.C for 30 min.

6. The use according to any one of claims 1-2, wherein in step 3) the culture conditions are 37℃for 16.+ -. 1h, the viable count of the bacterial liquid is up to 1.0X10 @ ⁹ Culture was stopped above CFU/mL.

7. Use according to any one of claims 1-2, characterized in that in step 4) the stirring speed during fermentation is: 60-80r/min; ventilation is 0; the initial air pressure is 0.02-0.03MPa; fermenting at 37deg.C for 47+ -2 h.

8. The application of the black matrimony vine enzyme with the alopecia preventing function in preparing a product for preventing alopecia is characterized in that the black matrimony vine enzyme is prepared by placing the black matrimony vine enzyme stock solution in the application of claim 1 in a container, adding maltitol solution, xylitol, apple powder and sucralose, stirring, standing and sterilizing.

9. The application of the black matrimony vine enzyme with alopecia preventing function in preparing products for improving kidney function and/or improving immunity is characterized in that the black matrimony vine enzyme is prepared by placing black matrimony vine enzyme stock solution in the application of claim 2 in a container, adding maltitol solution, xylitol, apple powder and sucralose, stirring, standing and sterilizing.

10. The use according to any one of claims 8 to 9, wherein the black matrimony vine enzyme comprises 947.86 parts by weight of black matrimony vine enzyme stock solution, 25 parts by weight of maltitol solution, 25 parts by weight of xylitol, 2 parts by weight of apple powder and 0.14 part by weight of sucralose.