Chickpea fermentation liquor as well as preparation method and application thereof

Abstract

The invention discloses high-safety and high-activity chickpea fermentation liquor as well as a preparation method and application thereof. The fermentation liquor is prepared by fermenting chickpeas through specific kefir grain symbiotic flora, the phytic acid content in the raw materials is remarkably reduced to be smaller than or equal to 0.4%, the oxalic acid content is remarkably reduced to be smaller than or equal to 0.07%, the phytic acid content and the oxalic acid content are far lower than those of a water extraction method, an enzymolysis method and a single-strain fermentation method, and potential damage of anti-nutritional factors to scalp is effectively eliminated. Meanwhile, rich active ingredients such as chickpea essence A, small molecular protein peptide and kefir grain lysate are generated in the fermentation process. Experiments show that the fermentation liquor can efficiently inhibit 5alpha-reductase (the inhibition rate is larger than or equal to 80%) and remarkably promote regeneration of mouse hair follicles, has excellent oxidation resistance and malassezia activity inhibition, and can be used for preparing hair products for preventing hair loss and maintaining hair follicles.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a fermentation liquid for preventing hair loss, nourishing hair follicles, scalp, and strengthening hair roots, as well as its preparation method and application. Background Technology

[0002] With the fast pace of modern life, increased environmental pressure, and changes in lifestyle, hair loss has become a significant health issue affecting people worldwide. Statistics show that the number of people experiencing hair loss in my country has exceeded 250 million, with alopecia areata, androgenetic alopecia, and seborrheic alopecia accounting for a significant proportion, seriously impacting patients' physical and mental health and quality of life.

[0003] Currently, the mainstream methods for preventing hair loss on the market mainly cover two areas: drug treatment and surgical treatment. In terms of drug treatment, chemical drugs are typically represented by minoxidil and finasteride. Minoxidil, as a vasodilator, stimulates hair follicle growth by promoting local blood circulation; finasteride, as a 5α-reductase inhibitor, inhibits the conversion of testosterone to dihydrotestosterone, reducing the damage of dihydrotestosterone to hair follicles. Although these chemical drugs have shown some efficacy in treating hair loss, long-term use poses many safety risks. Clinical studies have shown that some users have experienced adverse skin reactions such as hirsutism and allergic dermatitis, and some patients have even experienced serious side effects such as sexual dysfunction. Furthermore, once medication is discontinued, hair loss is highly likely to recur, making a complete cure difficult to achieve. Plant-based hair loss prevention products (such as those with traditional ingredients like ginger, arborvitae leaves, and Polygonum multiflorum) are favored by consumers due to their natural properties. However, the active ingredients in these plant extracts are mostly macromolecular structures, which are limited by the physiological barrier of human skin, resulting in extremely low transdermal absorption rates. This leads to weak and unstable anti-hair loss effects, making it difficult to meet the needs of clinical treatment.

[0004] In the field of surgical treatment, hair transplantation technology has made significant progress in recent years, and the surgical procedure has become increasingly mature. However, there are still many technical bottlenecks that need to be addressed. During hair follicle transplantation, the survival rate of hair follicles is low due to factors such as individual differences and surgical procedures. At the same time, the donor area has limited hair follicle resources, making it difficult to meet the treatment needs of patients with large areas of hair loss. A single hair transplantation surgery is time-consuming, expensive, and has a long postoperative recovery period, increasing the time and financial burden on patients.

[0005] Given the aforementioned shortcomings of existing hair loss prevention methods, developing a new type of hair loss prevention product derived from natural substances, possessing high safety, and capable of targeting multiple points to address the pathological mechanisms of hair loss has become a pressing technical challenge and an inevitable trend in the current field of hair loss treatment. Summary of the Invention

[0006] To address the technical problems in the prior art, this invention provides a fermentation liquid for preventing hair loss, nourishing hair follicles, scalp, and strengthening hair roots, as well as its preparation method and application.

[0007] This invention provides a fermentation liquid, especially a chickpea fermentation liquid that prevents hair loss, nourishes hair follicles, moisturizes the scalp, and strengthens hair roots, which can be used as a hair care product.

[0008] This invention first provides a method for preparing chickpea fermented liquid that prevents hair loss, nourishes hair follicles, moisturizes the scalp, and strengthens hair roots, including fermenting chickpeas with kefir grains.

[0009] The fermentation broth of this invention achieves comprehensive multi-target maintenance of hair loss prevention and scalp health through the synergistic effect of four pathways: "inhibiting 5α-reductase, nourishing hair follicles, scavenging free radicals, and inhibiting Malassezia".

[0010] Chickpeas contain abundant protein, chickpea protein A, isoflavones, gentianin, copper, zinc, iron, and other active ingredients, as well as anti-nutritional factors such as phytic acid and oxalic acid. This invention, through kefir grain fermentation, significantly reduces the high concentrations of phytic acid and oxalic acid in the chickpea raw material, directly reducing the chelating effect on essential minerals (such as Zn and Fe) on the scalp surface and around hair follicles. These minerals are key factors in maintaining the integrity of the scalp stratum corneum and participating in energy metabolism and enzymatic reactions in hair follicle cells. The increased bioavailability helps strengthen the scalp's physical barrier function, maintain a healthy ion balance, reduce scalp inflammation, and inhibit harmful microorganisms.

[0011] Kefir grains, as a natural symbiotic microbial community (including lactic acid bacteria, yeast, and acetic acid bacteria), produce fermentation products with a high diversity of highly active metabolites.

[0012] The inventors have discovered that fermenting chickpeas using kefir grains not only fully preserves the main active ingredients of chickpeas but also hydrolyzes and dissolves many beneficial components in the fermentation broth. Through the powerful metabolic capabilities of microorganisms, chickpea components are effectively transformed, key anti-nutritional factors—phytic acid and oxalic acid—are degraded, and chelated minerals (Fe, Zn, Mg, Ca), small peptides, and amino acids are released and activated. This enhances the bioavailability of anti-hair loss components such as chickpea extract A and highly active isoflavones, and produces highly active metabolites unique to kefir, including organic acids, polysaccharides, and vitamins, enriching the functional components of the fermentation broth. Studies have found that the synergistic effect of the original active ingredients of chickpeas, the secondary metabolites produced during fermentation, and the lysates from kefir grains can better nourish the scalp and prevent hair loss.

[0013] This invention has found that the fermentation broth can reduce the content of phytic acid and oxalic acid, release chelated minerals (Fe, Zn, Mg, Ca), repair the scalp microenvironment, create conditions for healthy hair follicles, strengthen the scalp barrier function, reduce the proliferation of Malassezia, and reduce dandruff and scalp inflammation.

[0014] Furthermore, the fermentation broth of this invention degrades key anti-nutritional factors phytic acid and oxalic acid, with a significantly better degradation effect than water extraction, enzymatic hydrolysis, and fermentation with a single Lactobacillus (Streptococcus thermophilus), effectively reducing the damage of phytic acid to the scalp barrier. The polypeptide content in the fermentation broth of this invention is higher than that obtained by enzymatic hydrolysis, water extraction, and fermentation with a single Lactobacillus (Streptococcus thermophilus).

[0015] Furthermore, the fermentation broth of the present invention has a phytic acid content of ≤0.4% (w / w) and an oxalic acid content of ≤0.07% (w / w).

[0016] The fermentation broth described in this invention significantly inhibits 5α-reductase and reduces the androgen-induced hair loss pathway.

[0017] Specifically, the fermentation broth described in this invention is made from chickpeas as the base material through kefir grain fermentation.

[0018] The kefir grains described in this invention can be conventional kefir grains. In the embodiments of this invention, the kefir grains refer to kefir grains that have been artificially preserved through generations and have the structure and characteristics of kefir grains.

[0019] In some embodiments of the present invention, the raw materials also include sugar, which may be inulin, specifically one or more of glucose, fructose, galactose, sucrose, brown sugar, white sugar, and black sugar; its main function is to provide a carbon source for the kefir grains, promote the growth and reproduction of fermentation strains, and accelerate the fermentation process.

[0020] In some embodiments of the present invention, the preparation method of the above-mentioned fermentation broth includes:

[0021] In some embodiments, 5 to 10 parts by weight of chickpeas are soaked for 12 to 16 hours in advance to allow them to fully expand.

[0022] In some embodiments, during pulping, the ratio of chickpeas to distilled water is 1:(2-5) (g / mL).

[0023] In some embodiments, the carbon source content is 0.5% to 2%.

[0024] In some embodiments, the viable count of the kefir granules used is (2.0–4.0) × 10⁻⁶. 8 Approximately CFU / mL.

[0025] In some embodiments, the kefir grain inoculum is 5% to 10%.

[0026] In some embodiments, the fermentation temperature of the chickpea fermentation broth is 25℃~35℃, the fermentation time is 1~3 days, and it is stirred regularly, 1~2 times / day, 5~10 minutes / time.

[0027] In some embodiments of the present invention, the fermentation broth is centrifuged at 4000-6000 r / min for 15-20 min to remove the precipitate, the supernatant is collected and sterilized (120℃, 15 min) to obtain chickpea fermentation broth.

[0028] On the one hand, the chickpea fermented liquid of the present invention can inhibit 5α-reductase that attacks hair follicle growth and cut off the pathway of seborrheic alopecia; on the other hand, it can balance the scalp microecology, nourish hair follicles, and promote hair growth; and furthermore, it can scavenge free radicals, resist oxidation, protect the scalp environment, and resist scalp aging.

[0029] Compared with existing technologies, the significant advancement of this invention lies in the fact that it is the first to discover and verify that fermenting chickpeas using the complex symbiotic system of kefir grains can specifically and efficiently degrade anti-nutritional factors such as phytic acid and oxalic acid, which are difficult to remove by conventional methods. Its degradation efficiency far exceeds that of water extraction, enzymatic hydrolysis, or single lactic acid bacteria fermentation. This technological effect is unexpected; it not only greatly improves the scalp safety of the fermentation liquid but also achieves a synergistic unity of "degrading harmful substances" and "enriching beneficial components" in preventing hair loss by releasing chelated minerals, producing small-molecule active peptides and unique metabolites, resulting in a "1+1>2" technological effect.

[0030] Attached image:

[0031] Appendix Figure 1 This is a graph showing the test results of the effect of preparation method on peptide content;

[0032] Appendix Figure 2 This is a graph showing the test results of the effect of preparation method on phytic acid content;

[0033] Appendix Figure 3 This is a graph showing the test results of the effect of preparation method on oxalic acid content;

[0034] Appendix Figure 4 This is a graph showing the test results of the antibacterial effect of the present invention against Malassezia;

[0035] Appendix Figure 5 This is a graph showing the test results of the DPPH free radical scavenging rate of this invention;

[0036] Appendix Figure 6 This is a diagram showing the inhibitory effect of the present invention on 5α-reductase;

[0037] Appendix Figure 7 This is a comparison chart of hair growth in mice on days 2, 11, and 18. Appendix Figure 8 This is a comparison image of the condition of the mouse's skin and hair follicles on day 6. Detailed Implementation

[0038] The present invention will now be described in detail with reference to the embodiments.

[0039] The kefir grains used below and the viable bacterial count in the bacterial solutions prepared in the following examples are approximately 3.0 × 10⁸ CFU / mL.

[0040] Example 1

[0041] 1) Inoculate kefir grains into MRS liquid medium and incubate at 25°C.

[0042] Activate for 18–24 hours before use.

[0043] 2) Select 10 kg of high-quality, mold-free chickpeas, rinse them 3-5 times with clean water to remove surface dust and impurities. Soak the washed chickpeas in clean water for 16 hours to allow them to fully absorb water and swell.

[0044] 3) Drain the soaked chickpeas and add an appropriate amount of distilled water, with a material-to-liquid ratio of 1:3.

[0045] (g / mL), use a high-speed tissue homogenizer to grind at a speed of 10000-12000 r / min for 3-5 min to make a uniform chickpea milk liquid, and add 0.5% inulin to the filtrate as a carbon source.

[0046] 4) Pour the prepared chickpea filtrate into a fermenter and sterilize it using high-temperature steam sterilization at 121°C for 20–30 minutes to kill any contaminating bacteria in the filtrate. After the filtrate cools to 32–35°C, inoculate it with activated kefir granules at a rate of 10%.

[0047] 5) Place the fermentation tank at a constant temperature of 35℃ for fermentation. The fermentation time is 2 days. Stir regularly, once a day for 5 minutes each time.

[0048] 6) After fermentation, centrifuge the fermentation broth at 4000 r / min for 20 min to remove the precipitate.

[0049] Collect the supernatant and sterilize it (120℃, 15 min) to obtain chickpea fermentation broth.

[0050] Example 2

[0051] 1) Inoculate kefir grains into MRS liquid medium and incubate at 25°C for 18–24 h to activate them for later use.

[0052] 2) Select 10 kg of high-quality, mold-free chickpeas, rinse them 3-5 times with clean water to remove surface dust and impurities. Soak the washed chickpeas in clean water for 12 hours to allow them to fully absorb water and swell.

[0053] 3) Drain the soaked chickpeas and add an appropriate amount of distilled water, with a material-to-liquid ratio of 1:5.

[0054] (g / mL), use a high-speed tissue homogenizer to grind at a speed of 10000-12000 r / min for 3-5 min to make a uniform chickpea milk, and add 1% inulin as a carbon source to the filtrate.

[0055] 4) Pour the prepared chickpea filtrate into a fermenter and sterilize it using high-temperature steam sterilization at 121°C for 20–30 minutes to kill any contaminating bacteria in the filtrate. After the filtrate cools to 22–25°C, inoculate it with activated kefir granules at a rate of 5%.

[0056] 5) Place the fermentation tank at a constant temperature of 25℃ for fermentation. The fermentation time is 3 days. Stir regularly, twice a day for 5 minutes each time.

[0057] 6) After fermentation, centrifuge the fermentation broth at 5000 r / min for 15 min to remove the precipitate.

[0058] Collect the supernatant and sterilize it (120℃, 15 min) to obtain chickpea fermentation broth.

[0059] Example 3

[0060] 1) Inoculate kefir grains into MRS liquid medium and incubate at 25°C for 18–24 h to activate them for later use.

[0061] 2) Select 10 kg of high-quality, mold-free chickpeas, rinse them 3-5 times with clean water to remove surface dust and impurities. Soak the washed chickpeas in clean water for 12 hours to allow them to fully absorb water and swell.

[0062] 3) Drain the soaked chickpeas and add an appropriate amount of distilled water, with a material-to-liquid ratio of 1:5.

[0063] (g / mL), use a high-speed tissue homogenizer to grind at a speed of 10000-12000 r / min for 3-5 min to make a uniform chickpea milk, and add 1% inulin as a carbon source to the filtrate.

[0064] 4) Pour the prepared chickpea filtrate into a fermenter and sterilize it using high-temperature steam sterilization at 121°C for 20–30 minutes to kill any contaminating bacteria in the filtrate. After the filtrate cools to 25–28°C, inoculate it with activated kefir granules at an inoculation rate of 8%.

[0065] 5) Place the fermentation tank at a constant temperature of 28℃ for fermentation. The fermentation time is 3 days. Stir regularly, once a day for 5 minutes each time.

[0066] 6) After fermentation, centrifuge the fermentation broth at 6000 r / min for 15 min to remove the precipitate.

[0067] Collect the supernatant and sterilize it (120℃, 15 min) to obtain chickpea fermentation broth.

[0068] Comparative Example 1

[0069] This comparative example provides a fermentation broth whose preparation method differs from that of Example 1 only in that chickpeas are replaced with soybeans.

[0070] Comparative Example 2

[0071] This comparative example provides a fermentation broth, the preparation method of which differs from Example 1 only in that kefir grains (Lentilactobacillus skefiri) are replaced with Streptococcus thermophilus, and the fermentation temperature is 37°C; wherein the viable cell count in the bacterial broth used is 3.0 × 10⁻⁶. 8 Approximately CFU / mL.

[0072] Comparative Example 3

[0073] This comparative example provides a chickpea extract, the preparation method of which differs from Example 1 only in that it uses water extraction. The steps are as follows:

[0074] Hot water extraction method: Grind chickpeas through a 60-mesh sieve using a Chinese medicine pulverizer. Take an appropriate amount of chickpea powder, add it to a certain proportion of water, mix thoroughly, heat to 55℃, and extract. Stir once every 10 minutes during the process. Centrifuge at 3000r / min for 10 minutes and take the supernatant for later use.

[0075] Comparative Example 4

[0076] This comparative example provides a chickpea enzymatic hydrolysate, the preparation method of which differs from that of Example 1 only in that it uses ultrasonic enzymatic hydrolysis for extraction. The steps are as follows:

[0077] Ultrasonic enzymatic hydrolysis: Chickpeas were pulverized through a 60-mesh sieve using a traditional Chinese medicine pulverizer. 300 U / g of cellulase was added to a 35% ethanol solution as the enzymatic hydrolysate. The mixture was ultrasonically hydrolyzed at 55°C for 1 hour. After the reaction was completed, a certain amount of anhydrous ethanol was added until the final ethanol concentration was 65% to terminate the enzymatic hydrolysis reaction. The mixture was then extracted in an ultrasonic cleaner for 20 minutes and then filtered.

[0078] Experimental Example 1: Determination of Peptide Content

[0079] Take an appropriate amount of chickpea kefir fermentation broth and centrifuge at 10,000 rpm for 10 minutes. Collect the supernatant and dilute 1 ml of the clear supernatant 10 times. Add 0.1–0.6 mL of 2% BSA reference solution to 10 mL test tubes, add water to the 1 mL mark, add 4 mL of biuret reagent, vortex mix, and incubate at room temperature for 30 minutes. Use distilled water as a blank control. Measure the absorbance at 540 nm to prepare a standard curve: Y = y = 0.1839x + 0.0143, R0. 2 =0.9986 (Y is the absorbance of the reference standard, X is the concentration). Take 1 ml of sample solution, add 4 ml of biuret reagent, vortex mix, and develop color at room temperature for 30 min. Repeat 3 times. Use distilled water as a blank control and measure the absorbance at 540 nm.

[0080] Table 1. Effect of preparation method on peptide content (x±SD)

[0081]

[0082]

[0083] The example groups showed significant differences compared to the comparative examples (*p<0.001 compared to Comparative Example 2; &p<0.001 compared to Comparative Example 3; #p<0.001 compared to Comparative Example 4). Peptide content: chickpea fermentation broth (this invention) > chickpea enzymatic hydrolysate (Comparative Example 4) > Lactobacillus chickpea fermentation broth (Comparative Example 2) > chickpea water extract (Comparative Example 3). These results indicate that the peptide content of the fermentation broth of this invention is significantly higher than that of other treatment methods, suggesting that kefir grain fermentation can more effectively decompose chickpea protein, releasing small molecule active peptides, which are beneficial for scalp absorption.

[0084] Experimental Example 2: Determination of Phytic Acid and Oxalic Acid Content

[0085] (1) Phytic acid content determination

[0086] Take 5 mL of the sample solution into a 10 mL centrifuge tube, add 0.63 mL of 2 mg / mL FeCl3 solution, and incubate in a boiling water bath for 45 min. After cooling, centrifuge at 12000 rpm for 15 min, discard the supernatant, wash three times with 5 mL of 3% trichloroacetic acid solution, and then wash once with 5 mL of distilled water. Add 6 mL of 0.5 mol / L NaOH solution to the above precipitate, incubate in a boiling water bath for 30 min, cool, centrifuge at 12000 rpm for 15 min, discard the supernatant, wash the precipitate with 5 mL of water, add 5 mL of 0.5 mol / L HCl solution, and incubate in a boiling water bath for 25 min to completely dissolve the Fe(OH)3 precipitate. Dilute the resulting FeCl3 solution to 25 mL with water. Use a pipette to take 3 mL of the test solution into a test tube, add 5 mL of 0.2% hydroquinone, then add 5 mL of 0.05% o-phenanthroline, mix well, then add 0.18 mL of 25% sodium citrate solution, mix well, let stand for 5 min, and measure the absorbance at a wavelength of 510 nm.

[0087] A standard curve was prepared using FeCl3·6H2O. An appropriate amount of HCl was added during solution preparation to prevent the formation of Fe(OH)3 precipitate. The regression equation for the standard curve was Y = 0.0048X + 0.0641 (where Y is the absorbance at 510 nm and X is the mass concentration of iron (μg / mL)). The phytic acid content was then calculated based on the assumption that one molecule of phytic acid is bound to every four iron atoms. The calculation formula is as follows.

[0088] Phytic acid content / % = (ρ1×M1×V1) / (M2×4×m1×10) 6 )×100

[0089] In the formula: ρ1 is the iron concentration (μg / mL) obtained from the standard curve;

[0090] M1 represents the molar mass of phytic acid (g / mol); M2 represents the molar mass of iron (g / mol).

[0091] V1 is the volume of the extraction solution in mL; m1 is the mass of the sample in g.

[0092] Table 2. Effect of preparation method on phytic acid content (x±SD)

[0093] Group Phytic acid content / % Example 1 0.38±0.02*&# Example 2 0.33±0.05*&# Example 3 0.3±0.04*&# Comparative Example 2 0.96±0.06 Comparative Example 3 1.24±0.09 Comparative Example 4 1.04±0.06

[0094] The phytic acid content of the chickpea fermentation broth of the present invention (Examples 1-3) is significantly lower than that prepared by Comparative Examples 2-4, with significant differences (*p<0.001 compared to Comparative Example 2; &p<0.001 compared to Comparative Example 3; #p<0.001 compared to Comparative Example 4). Specifically, the phytic acid content is as follows: chickpea water extract (Comparative Example 3) > chickpea enzymatic hydrolysate (Comparative Example 4) > Lactobacillus chickpea fermentation broth (Comparative Example 2) > chickpea fermentation broth (the present invention).

[0095] (2) Determination of oxalic acid content

[0096] Take 5 mL of sample solution into a 10 mL centrifuge tube, add an equal volume of 2.0 mol / L hydrochloric acid solution, heat in an 80℃ water bath for 10 minutes, remove and cool to room temperature, then centrifuge at 12,000 rpm for 15 minutes. Take the supernatant, filter it through a 0.22 μm aqueous microporous membrane into a vial for analysis. Use an Agilentzorbax SB-Aq column (4.6 mm × 250 mm, 5 μM), detection wavelength 210 nm, column temperature 25℃, mobile phase 0.1% phosphoric acid solution, flow rate 0.5 mL / min, injection volume 10 μL, run for 12 min. Accurately weigh 0.2 g of oxalic acid standard, dissolve in water and dilute to 100 mL to prepare a standard stock solution, obtaining the standard curve Y = 13.271X + 0.0023 (Y is the peak area on the x-axis, X is the mass concentration of oxalic acid standard solution on the x-axis).

[0097] Table 3. Effect of preparation method on oxalic acid content (x±SD)

[0098] Group Oxalic acid content / % Example 1 0.07±0.02**&## Example 2 0.064±0.02**&## Example 3 0.066±0.01*&## Comparative Example 2 0.114±0.01 Comparative Example 3 0.154±0.01 Comparative Example 4 0.106±0.01

[0099] The oxalic acid content of the chickpea fermentation broth of the present invention (Examples 1-3) is significantly lower than that prepared by Comparative Examples 2-4, showing a significant difference (compared to Comparative Example 2, *p<0.001, **p<0.05; compared to Comparative Example 3, &p<0.001; compared to Comparative Example 4, ##p<0.05). Specifically, the oxalic acid content is as follows: chickpea water extract (Comparative Example 3) > chickpea Lactobacillus fermentation broth (Comparative Example 2) > chickpea enzymatic hydrolysate (Comparative Example 4) > chickpea fermentation broth (the present invention).

[0100] The above data demonstrate that the fermentation method of this invention can significantly reduce the content of phytic acid and oxalic acid. This lays a solid foundation for the fermentation broth to exert excellent scalp care functions: it effectively eliminates the potential damage to the scalp barrier caused by high concentrations of anti-nutritional factors, releases chelated minerals, and thus promotes hair follicle health by improving the scalp microenvironment.

[0101] Experiment Example 3: Antioxidant and antibacterial effects

[0102] (1) Evaluation of Malassezia inhibitory effect

[0103] The standard strain of Malassezia (Malasseziafurfur) (strain number: BNCC337308) was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (China General Microbiological Culture Preservation Center).

[0104] Preparation of culture medium:

[0105] Solid culture medium: Take 40g of malt extract powder, 20g of ox bile powder, 2.5g of glyceryl monooleate, 10g of Tween 40, 15g of agar, and 1.0L of distilled water, put them into a beaker and let them dissolve completely, then sterilize at 121℃ for 15min.

[0106] Liquid culture medium: Take 20g of maltose, 2g of yeast extract, 20g of glucose, 20g of olive oil, and 1.0L of distilled water, put them into a beaker and let them dissolve completely, then sterilize at 121℃ for 15min.

[0107] Preparation of bacterial suspension: Dissolve Malassezia standard Candida powder in 0.5 mL of sterile physiological saline according to the instructions. Spread evenly on a solid culture medium and incubate at 30°C. Subculture twice, once every 72 hours, to prepare a bacterial suspension with a bacterial content of approximately (1.0–2.0) × 10⁻⁶. 6 CFU / mL.

[0108] Pour the heat-sterilized culture medium into sterile petri dishes with a diameter of 9 cm, ensuring each dish contains 15–20 mL of liquid culture medium. After cooling, add 0.2 mL of Malassezia bacterial suspension (concentration 1.0 x 10⁻⁶). 6 (Cells / mL), spread the culture medium evenly on the surface using a spreader. Place the Oxford cup vertically on the corresponding culture medium surface using tweezers, gently pressing to ensure seamless contact. Place three tubes on each plate, adding 0.1 ml of the test solution to each tube and labeling them carefully to prevent overflow. Malassezia: After anaerobic incubation at 37°C for 2 days, observe the results. Assess the inhibitory effect using the diameter d of the inhibition zone. Use an uninoculated bacterial suspension as a blank control group.

[0109] Table 4. Antibacterial effect of samples against Malassezia (x±SD)

[0110]

[0111]

[0112] Examples 1-3 all showed antibacterial effects against Malassezia, with Example 1 showing the best effect. The antibacterial effect of chickpea fermentation broth against Malassezia was significantly higher than that of Comparative Examples 1-4 (Δp<0.001 compared to Comparative Example 1; *p<0.001 compared to Comparative Example 2; &p<0.001 compared to Comparative Example 3; #p<0.001 compared to Comparative Example 4).

[0113] (2) DPPH free radical scavenging experiment

[0114] Prepare a 0.2 mmol / L DPPH solution (ready to use), then pipette 250 μL of the test solution into an EP tube, add 750 μL of DPPH solution, and react in the dark at room temperature for 30 min. Measure the absorbance (A value) at 517 nm, n = 3.

[0115] DPPH free radical scavenging rate = 1 - (A sample - A blank) / A control × 100%

[0116] Table 5. DPPH radical scavenging rate of the samples (x±SD)

[0117]

[0118]

[0119] The DPPH free radical scavenging rate of chickpea fermentation broth in Examples 1-3 was higher than 80%, which was significantly higher than that of Comparative Examples 1-4 (Δp<0.001 compared with Comparative Example 1; *p<0.001 compared with Comparative Example 2; &p<0.001 compared with Comparative Example 3; #p<0.001 and ##p<0.05 compared with Comparative Example 4), indicating that chickpea fermentation broth has the function of scavenging DPPH free radicals and thus playing an antioxidant role.

[0120] Inhibition rate of 5α-reductase in fermentation broth of Experiment Example 4

[0121] Six male SD rats were euthanized after fasting for 12 hours. The prostate glands were removed from the rats on a low-temperature dissection table, minced, and weighed. Pre-cooled buffer (containing 0.32 mol / L sucrose, 0.1 mmol / L dithiothreitol, 1 mmol / L EDTA, and 0.2 mol / L phosphate buffer) was added at a mass ratio of 1:5 and homogenized rapidly in a homogenizer. The homogenate was then centrifuged (13000 r / min, 10 min) using a refrigerated centrifuge. The supernatant (discarding the uppermost fat layer) was collected and diluted to 20 mL with buffer. The mixture was then aliquoted into EP tubes and stored at -80°C for later use.

[0122] 5α-Reductase Activity Assay: 0.5 mL phosphate buffer, 0.2 mL test solution, 200 μL testosterone solution (300 mg / L), and 200 μL NADPH solution (0.8 g / L) were added sequentially to a stoppered test tube. Finally, 0.5 mL of 5α-reductase extract was added. The reaction was carried out at 37 °C for 30 min. After the reaction, 3 mL of dichloromethane was added to stop the reaction and extract testosterone. 0.25 mL of propylparaben (100 mg / L) was added as an internal standard, and the mixture was centrifuged (5000 r / min, 10 min). The upper aqueous phase was discarded, and approximately 1 mL of the organic phase was transferred, evaporated to dryness, and the residue was dissolved in 1.5 mL of methanol. 10 μL of the residue was measured using high-performance liquid chromatography (HPLC) to determine the residual testosterone content. A positive control was a 0.05 mg / L finasteride group. 5α-reductase reaction tubes and 5α-reductase blank tubes were also prepared.

[0123] 5α-Reductase inhibition rate = (R sample - R reaction) / (R blank - R reaction) × 100%

[0124] In the formula, R is the ratio of the target peak area to the target peak area.

[0125] Table 6. Inhibitory effect of samples on 5α-reductase (x±SD)

[0126]

[0127] The inhibition rate of 5α-reductase in the fermentation broth of this invention was close to that of the positive control group. The inhibition rate of the example groups was better than that of the comparative groups (Δp<0.001 compared with comparative example 1; *p<0.001 compared with comparative example 2; &p<0.001 compared with comparative example 3; #p<0.001 compared with comparative example 4), with example 1 showing the highest inhibition rate. This indicates that chickpea fermentation broth can effectively inhibit 5α-reductase (inhibition rate ≥80%), blocking the key pathway of androgenetic alopecia.

[0128] Experiment Example 5: Effect of Fermentation Broth on Hair Growth in Mice

[0129] Grouping: Thirty male C57BL / 6 (SPF grade) mice were quarantined in the quarantine room for 3 days before being transferred to the breeding room. The hair on the backs of the mice (3×4cm) was removed the day before the experiment to avoid skin damage. The experimental animals were randomly divided into 3 groups: a blank group (physiological saline), a 2% minoxidil positive group, and a 4% fermentation broth group, with 10 mice in each group. They were stored in separate cages and numbered.

[0130] Each group of mice was given 0.3 mL of the corresponding test solution on the back hair area once a day for 18 days. During the administration period, the growth of the back hair of the mice was observed and recorded daily.

[0131] On days 2, 11, and 18 after administration, 10 hairs were randomly plucked from each mouse, and the hair length was measured using a ruler.

[0132] Table 7. Hair length (x±SD) in different groups of mice

[0133] Group Day 2 (mm) Day 11 (mm) Day 18 (mm) Blank group - 4.68±0.02 11.23±0.45 Positive control group - 9.34±0.15*** 24.11±0.38*** Fermentation broth group - 9.02±0.24*** 26.5±0.63***

[0134] Regarding the length of mouse hair, there were significant differences compared to the control group and the other two groups. On day 11, the 2% minoxidil group had the longest hair length (P<0.001), and on day 18, the 4% fermentation broth group had the longest hair length (P<0.001). In terms of total hair length, both the 2% minoxidil group and the 4% chickpea fermentation broth group accelerated hair growth.

[0135] On days 2, 11, and 18 after administration, the backs of mice were photographed, and the area of ​​hair coverage on the backs of mice was calculated using ImageJ software. The hair coverage rate was calculated according to the formula.

[0136] Hair coverage (%) = Area of ​​newly grown hair / Area of ​​hair removal × 100%

[0137] Table 8. Number of days of skin changes in different groups of mice (x±SD)

[0138] Group Time it takes for skin to change from pink to gray / d Time from gray skin to hair growth / d Blank group 7.18±1.03 10.02±0.81 Positive control group 5.64±1.52** 8.34±0.24* Fermentation broth group 6.18±0.54* 8.91±0.5*

[0139] Note: "*" indicates that P<0.05 compared with the blank control group.

[0140] Regarding the time it took for mouse hair to transition from the telogen phase to the anagen phase, compared to the 7.18 days in the blank control group, the other two groups showed significant differences. The 2% minoxidil group entered the anagen phase the fastest (5.64 days, P<0.01), followed by the 4% fermentation broth group (6.18 days, P<0.05), both showing significant effects in inducing or promoting hair growth (P<0.05). There was no significant difference in total hair growth time among the blank control group, the 2% minoxidil group, and the 4% chickpea fermentation broth group. Results are as follows... Figure 7 As shown in the figure (a: blank group; b: positive control group; c: fermentation broth group).

[0141] Table 9. Hair coverage of mice in different groups (x±SD)

[0142] Group Day 2 (%) Day 11 (%) Day 18 (%) Blank group - 51.3±0.6 100 Positive control group - 79.1±3.9* 100 Fermentation broth group - 75.4±4.1* 100

[0143] Note: "*" indicates that P<0.05 compared with the blank control group.

[0144] On day 6 (when hair grew on the back of mice in the fermentation broth group), one mouse from each group was randomly selected and sacrificed by cervical dislocation. Intact skin specimens were taken from the same area of ​​the shaved area, fixed with 4% paraformaldehyde, prepared into paraffin sections, stained with hematoxylin and eosin (HE), and the number of hair follicles was analyzed under a 10X optical microscope. Five fields of view were randomly selected from each slide for analysis.

[0145] Table 10. Number of hair follicles in mouse skin on day 6

[0146] Group Number of hair follicles / each Blank group 138±22 Positive control group 407±17** Fermentation broth group 555±28**#

[0147] Note: "**" indicates P < 0.01 compared to the blank control group; "#" indicates P < 0.05 compared to the positive control group. Results are as follows... Figure 7 As shown in the figure (a: blank group; b: positive control group; c: fermentation broth group).

[0148] In terms of hair growth density, the 4% fermentation liquid group showed the best hair growth effect, followed by 4% fermentation liquid > 2% minoxidil >> blank control group.

[0149] In summary, chickpea fermented liquid can accelerate hair growth, with effects comparable to the positive control minoxidil in terms of hair length increase and coverage improvement; chickpea fermented liquid can shorten the telogen phase of hair follicles and significantly promote the transformation of hair follicles from the telogen phase to the anagen phase, with an initiation efficiency close to that of minoxidil; chickpea fermented liquid can enhance the regeneration capacity of hair follicles, with a significantly higher number of hair follicles than the positive control group, demonstrating its unique advantages in hair follicle maintenance and regeneration.

[0150] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A chickpea fermentation broth, characterized in that, The fermentation liquid is prepared by fermenting chickpea raw material with Kefir granules, and the content of phytic acid is less than or equal to 0.4%, and the content of oxalic acid is less than or equal to 0.07%.

2. The chickpea fermentation broth according to claim 1, characterized in that, The fermentation liquid further comprises the following components produced by fermenting the Kefir granules: biochanin A, formononetin, Kefir granule lysate, and protein peptides with a molecular weight less than 10 kDa.

3. A method of preparing the chickpea fermentation broth of claim 1 or 2, characterized by, The method comprises the following steps: (1) After soaking the chickpea for 12-16 hours, drain the water, add water at a material-to-liquid ratio of 1:(2-5) (g / mL), and crush to prepare a slurry; (2) After adding a carbon source to the slurry, sterilize at 121 DEG C for 20-30 minutes, and cool to 25-37 DEG C; (3) Inoculate Kefir granule bacterial solution at an inoculation amount of 5%-10%; (4) Ferment for 1-3 days, stir 1-2 times a day, and each time for 5-10 minutes; (5) Centrifuge the fermentation liquid at a speed of 4000-6000 r / min for 15-20 minutes, collect the supernatant, and obtain the finished product after sterilization.

4. The method of claim 3, wherein In step 1), the crushing is performed at a high speed of 10000-12000 r / min for 3-5 minutes.

5. The method of claim 3, wherein In step 2), the carbon source is one or more of inulin, glucose, fructose, galactose, sucrose, brown sugar, white sugar, and black sugar.

6. The method of claim 3, wherein Step 3) the viable cell count of the kefir granules is (2.0-4.0) x 10 8 CFU / mL.

7. The method of claim 3, wherein In step 4), the fermentation temperature is 25-35 DEG C.

8. A fermentation product, which is a cell-free sterile liquid, a sterile filtrate of the liquid, or a sterile supernatant, or a concentrate or dry product thereof, prepared from the fermentation liquid of any one of claims 1-7 for preventing hair loss, maintaining hair follicles, nourishing the scalp, strengthening hair growth, and rooting.

9. A hair composition characterized in that The fermentation liquid of claim 1 or 2, and a cosmetically or pharmaceutically acceptable adjuvant.

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