Gastrodia elata microbial fermentation product as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and discloses a gastrodia elata microbial fermentation product as well as a preparation method and application thereof. According to the invention, three aspergillus oryzae are utilized to successfully carry out microbial fermentation on gastrodia elata, the gastrodia elata microbial fermentation product of which the content of active ingredients gastrodin and p-hydroxybenzyl alcohol is obviously increased is obtained, and the pharmacological action of the gastrodia elata microbial fermentation product on treating or improving oligoasthenospermia is verified through an oligoasthenospermia mouse model; results show that the gastrodia elata microbial fermentation product has better effects of improving sperm concentration, enhancing sperm motility and rapidly moving forwards, can be used as an active ingredient of a medicine for treating or improving oligospermia and asthenospermia, and has wide application.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a microbial fermentation product of Gastrodia elata, a preparation method thereof, and an application thereof in treating or improving oligoasthenospermia. Background Art

[0002] Gastrodia elata BI., also known as Red Arrow, Motherless, Wind-Stilling Herb, Gastrodia elata Tomorrow, etc., is a plant of the genus Gastrodia in the Orchidaceae family. Its dried tuber is used as medicine and is a traditional precious Chinese medicinal material in China. It has a sweet and flat taste and is non-toxic, with the effects of calming endogenous wind and stopping convulsions, suppressing hyperactive liver-yang, and dispelling wind and dredging collaterals. It is commonly used to treat dizziness, headache due to wind pathogen, insomnia and other symptoms. Since November 2023, Gastrodia elata has been officially added to the list of "medicinal and edible homologous" varieties, with triple values of medicine, health care and food. Modern research shows that the main active ingredients of Gastrodia elata are gastrodin and its aglycone p-hydroxybenzyl alcohol. The Chinese Pharmacopoeia uses gastrodin and its aglycone as the quality control components of Gastrodia elata, and they are also the material basis for Gastrodia elata to exert its medicinal effects, with activities such as hypnotic sedation, anti-convulsion, anti-vertigo, analgesia, depression, anxiety, cognitive impairment, protecting myocardial cells, reducing blood pressure, anti-thrombosis and anti-platelet aggregation. The health care effects of Gastrodia elata are extensive. Referring to the functional catalog of health foods, the main health care functions of Gastrodia elata are: assisting in reducing blood pressure, reducing blood lipid, reducing blood sugar, improving sleep, enhancing immunity, assisting in improving memory, having an auxiliary protective effect on chemical liver injury, antioxidant, regulating intestinal flora and promoting digestion, etc., and the mechanism of exerting effects on the human body is also relatively complex. At present, there is no report on the pharmacological effects of Gastrodia elata and its processed products in treating or improving oligoasthenospermia.

[0003] Oligoasthenospermia is one of the most common diseases of male infertility. Oligoasthenospermia includes oligospermia and asthenospermia. Clinically, oligospermia often coexists with low sperm motility, and at this time it is called oligoasthenospermia. Oligoasthenospermia can be caused by various factors, such as lifestyle, genetic factors, diseases and drugs, etc., ultimately resulting in a decrease in sperm quantity or a decline in sperm quality. Due to the unclear pathogenesis of oligoasthenospermia, it brings many difficulties to clinical treatment. At present, although there are many traditional treatment methods such as drug treatment, surgical treatment, assisted reproductive technology and intracytoplasmic sperm injection, there are problems such as large side effects, high costs and unstable effects. Traditional Chinese medicine in China has a history of thousands of years in treating diseases. Its theoretical system is extensive and profound, and the resources of Chinese herbal medicines are extremely rich, playing an important role in the clinical treatment of oligoasthenospermia. Research shows that traditional Chinese medicine has definite curative effects in increasing sperm concentration, improving sperm motility and improving sperm quality.

[0004] Traditional Chinese medicine fermentation uses traditional Chinese medicine materials or medicinal residues as substrates, and through the metabolic activities of specific microorganisms (such as bacteria, fungi, yeasts, etc.), and through enzymatic reactions, biotransformations or symbiotic fermentations, it is a modern biotechnology that can directionally change the composition, structure and efficacy characteristics of the medicinal materials. Microbial fermentation secretes enzyme systems such as cellulase and protease to decompose the cell walls and macromolecular components (such as polysaccharides and proteins) of traditional Chinese medicine materials, converting insoluble substances into small molecule active components that are easily absorbed, thereby improving the efficacy release efficiency. For example, after the fermentation of Astragalus membranaceus, the extraction rates of polysaccharides and astragaloside are significantly increased, and the bioavailability is increased by 3.2 times. Therefore, selecting suitable microorganisms is the key technology for traditional Chinese medicine fermentation. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a preparation method of Gastrodia elata microbial fermentation products. At the same time, this Gastrodia elata microbial fermentation product can be used as an active ingredient for treating or improving oligoasthenospermia.

[0006] The Gastrodia elata microbial fermentation product provided by the present invention is obtained by biochemically fermenting Gastrodia elata with Aspergillus oryzae. The contents of the active ingredients gastrodin and p-hydroxybenzyl alcohol in the fermentation product are significantly increased.

[0007] The present invention also provides a preparation method of Gastrodia elata microbial fermentation products, including solid-state fermentation and liquid fermentation.

[0008] The solid-state fermentation includes the following steps:

[0009] 1) After fresh Gastrodia elata is washed and cut into small pieces or thin slices (Gastrodia elata slices or Gastrodia elata powder are soaked in an appropriate amount of distilled water), it is subjected to high-pressure moist heat sterilization;

[0010] 2) After the Aspergillus oryzae spore powder is mixed and evenly stirred with flour stir-fried at a weight ratio of 1:1 to 10, it is inoculated into the pretreated Gastrodia elata at an inoculation amount of 5-15% (preferably 10%) of the weight of Gastrodia elata;

[0011] 3) Ferment at 30 °C and 70% humidity in an incubator for 2-30 days;

[0012] 4) After the fermentation is completed, it is subjected to moist heat sterilization to obtain the Gastrodia elata microbial fermentation product.

[0013] The liquid fermentation includes the following steps:

[0014] 1) Gastrodia elata powder or Gastrodia elata extract is added to a culture solution with a weight-volume ratio of 1:5 to 50 g / L and then subjected to high-pressure moist heat sterilization as a culture medium;

[0015] 2) The Aspergillus oryzae liquid culture pre-cultured in a potato liquid medium is inoculated into the culture medium in step (1) at a ratio of 1-10% by volume (preferably 1%);

[0016] 3) Fermentation in an incubator at 30°C and 160 rpm for 3-20 days;

[0017] 4) After the fermentation is completed, high pressure wet heat sterilization and freeze drying are performed to obtain the Gastrodia elata microbial fermentation product.

[0018] The present invention also provides the use of the above-mentioned Gastrodia elata microbial fermentation product as an active ingredient for treating or improving oligoasthenospermia.

[0019] Compared with the prior art, the present invention utilizes the catalytic effect of microbial enzymes to perform enzymatic conversion on Gastrodia elata, thereby obtaining a Gastrodia elata microbial fermentation product with a higher content of active ingredients. The in vivo animal model experiment confirmed that the fermentation product has significant activity in treating or improving oligoasthenospermia, can be used as an active ingredient for treating oligoasthenospermia, and has a wide range of uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 HPLC chart of the active ingredient content in Gastrodia elata (A is the HPLC chart of the standard product, and B is the HPLC chart of sample 1).

[0021] Figure 2 Statistical analysis of Gastrodia elata microbial fermentation products improving sperm concentration and sperm motility in oligoasthenospermia mice. DETAILED DESCRIPTION

[0022] To further illustrate the present invention, the preparation method of the Gastrodia elata microbial fermentation product provided by the present invention and its application in treating or improving oligoasthenospermia are described in detail below in conjunction with the examples.

[0023] The invention screens out three kinds of Aspergillus oryzae from commonly used food fermentation strains Aspergillus niger, Aspergillus oryzae, Bacillus subtilis and Lactobacillus plantarum, namely Aspergillus oryzae Shanghai Niang 3.042, Aspergillus oryzae CGMCC 3.2073 and Aspergillus oryzae CGMCC 3.407, which have good conversion ability to Gastrodia elata. The contents of active ingredient gastrodin and its aglycone p-hydroxybenzyl alcohol in Gastrodia elata products after fermentation are significantly increased, and the content of active ingredient in Gastrodia elata products fermented by Aspergillus oryzae CGMCC 3.407 is the most significantly increased.

[0024] Example 1: Screening of Gastrodia elata microbial fermentation strains

[0025] 1) Preparation of Gastrodia elata extract

[0026] Referring to the preparation method of Gastrodia elata reference solution in the Chinese Pharmacopoeia, the Gastrodia elata tubers were crushed and passed through an 80-mesh sieve, placed in a stoppered conical flask, 50% methanol in a weight-to-volume ratio of 20 times was added, ultrasonic extraction was performed for 30 minutes, filtered, and the solvent was recovered from the filtrate to obtain the Gastrodia elata extract.

[0027] 2) Biofermentation of Gastrodia elata

[0028] The Gastrodia elata extract was fermented and transformed using common liquid fermentation technology. Aspergillus niger and Aspergillus oryzae were cultured in a potato liquid medium (1000.0 mL of potato extract, 20.0 g of glucose, natural pH) at 30 °C and 160 rpm; Bacillus subtilis was cultured in a nutrient broth liquid medium (5.0 g of peptone, 3.0 g of beef extract powder, 5.0 g of NaCl, 1000.0 mL of distilled water, pH 7.0) at 30 °C and 160 rpm; Lactobacillus plantarum was cultured statically in an MRS liquid medium (10.0 g of casein peptone (trypsin digested), 10.0 g of beef extract powder, 5.0 g of yeast extract powder, 20.0 g of glucose, 1.0 g of Tween 80, 5.0 g of sodium acetate, 2.0 g of ammonium citrate, 2.0 g of K2HPO4, 0.2 g of MgSO4·7H2O, 0.05 g of MnSO4·H2O, 1000.0 mL of distilled water, pH 6.2 - 6.5) at 37 °C for liquid fermentation culture. Each strain was inoculated in duplicate in parallel. The blank control group did not add the Gastrodia elata extract, and the biological fermentation group contained 0.1 mg / mL of the Gastrodia elata extract. After 7 days of fermentation culture, the fermentation broth was freeze-dried to obtain the Gastrodia elata fermentation powder.

[0029] 3) Determination of the contents of gastrodin and p-hydroxybenzyl alcohol

[0030] Referring to the content determination method in the Chinese Pharmacopoeia, the contents of the active ingredients gastrodin and p-hydroxybenzyl alcohol in the Gastrodia elata fermentation powder were detected by HPLC. Comparing with the contents of gastrodin and p-hydroxybenzyl alcohol in the original extract, the strains with significantly increased contents were determined as strains with fermentation activity. The results are shown in Table 1.

[0031] Table 1. Screening results of strains

[0032]

[0033]

[0034] Note: "+" indicates good fermentation activity

[0035] The results showed that Aspergillus oryzae Huniang 3.042, Aspergillus oryzae CGMCC 3.2073, and Aspergillus oryzae CGMCC 3.407 had good transformation ability for Gastrodia elata.

[0036] Example 2: Preparation of Gastrodia elata microbial fermentation products by solid-state fermentation method

[0037] Taking the fermentation of fresh Gastrodia elata by Aspergillus oryzae CGMCC 3.407 as an example, the process of preparing Gastrodia elata microbial fermentation products by solid-state fermentation method was described.

[0038] 1) Preparation of Gastrodia elata culture medium

[0039] After washing fresh Gastrodia elata, cut it into small pieces about 10mm×10mm, take it out after autoclaving at 121℃ for 20min, cool it to 35 - 40℃, and avoid caking. Add stir-fried flour with a weight ratio of 1:1 to fresh Gastrodia elata, stir evenly, and adjust the moisture content to 40%.

[0040] 2) Seed liquid preparation and inoculation

[0041] Inoculate Aspergillus oryzae CGMCC 3.407 into a potato slant medium (1000.0mL of potato extract, 20.0g of glucose, 15.0g of agar, pH natural), place it in an incubator at 30℃ for 5 days until the mycelia grow vigorously and form spores. Wash the spores of the slant culture with sterile normal saline, filter to remove the mycelia to obtain a high-concentration spore suspension. Inoculate the spore suspension into a potato liquid medium, and culture it in a shaker at 30℃ and 160rpm for 24 hours to form an active seed liquid. Spray the seed liquid evenly on the surface of Gastrodia elata at a weight-to-volume ratio of 5%, and stir well to ensure uniform distribution of the strain.

[0042] 3) Fermentation

[0043] Carry out fermentation in an incubator at a temperature of 30℃ and a humidity of 70%, keep the cotton plug for natural ventilation, and continue fermentation for 15 days until the mycelia cover the surface of Gastrodia elata.

[0044] 4) Post-treatment

[0045] After fermentation, subject Gastrodia elata to high-pressure autoclaving, dry and cool it to obtain a fresh Gastrodia elata solid-state fermentation product (Sample 1).

[0046] Using the above fresh Gastrodia elata solid-state fermentation process, end the fermentation after 5 days, 10 days, 20 days, 25 days, and 30 days of fermentation respectively, carry out high-pressure autoclaving, dry and cool it to obtain fresh Gastrodia elata solid-state fermentation products at different times, namely Sample 1-A, Sample 1-B, Sample 1-C, Sample 1-D, and Sample 1-E.

[0047] If the raw material is Gastrodia elata cut pieces or Gastrodia elata powder, just add an appropriate amount of distilled water to soak it during the preparation of the Gastrodia elata medium, then carry out autoclaving at 121℃ for 20min, and keep the subsequent steps the same to obtain a Gastrodia elata cut pieces solid-state fermentation product (Sample 2) and a Gastrodia elata powder solid-state fermentation product (Sample 3).

[0048] Example 3: Preparation of Gastrodia elata microbial fermentation product by liquid fermentation method

[0049] Taking the fermentation of Gastrodia elata powder by Aspergillus oryzae CGMCC 3.407 as an example, the process of preparing Gastrodia elata microbial fermentation product by liquid fermentation method is described.

[0050] 1) Preparation of seed liquid

[0051] The Aspergillus oryzae CGMCC 3.407 preserved on a slant in a test tube was inoculated into a 250 mL conical flask (containing 100 mL of potato liquid medium). After culturing with shaking at 160 rpm and 30 °C for 24 hours on a shaker, it was used as the seed solution.

[0052] 2) Preparation of Gastrodia elata fermentation broth

[0053] 10 g of dried Gastrodia elata tuber or decoction pieces were pulverized and passed through an 80-mesh sieve, placed in a conical flask, 90 mL of potato liquid medium was added, and then autoclaved at 121 °C for 20 min. After cooling to room temperature, 1 mL of the seed solution was added.

[0054] 3) Fermentation

[0055] Liquid fermentation was carried out in a shaker at 30 °C and 160 rpm, and the cotton plug was kept for natural ventilation, and continuous fermentation was carried out for 7 days.

[0056] 4) Post-treatment

[0057] The Gastrodia elata fermentation broth was subjected to high-pressure moist heat sterilization and then vacuum freeze-dried to obtain the Gastrodia elata powder liquid fermentation product (sample 4).

[0058] Using the above Gastrodia elata powder liquid fermentation process, fermentation was terminated after 3 days, 5 days, 10 days, 15 days and 20 days of fermentation respectively. The fermentation broth was subjected to high-pressure moist heat sterilization and then vacuum freeze-dried to obtain the products of Gastrodia elata powder liquid fermentation at different times, namely sample 4-A, sample 4-B, sample 4-C, sample 4-D and sample 4-E.

[0059] If the raw material is fresh Gastrodia elata, it can be first ground into Gastrodia elata original pulp with a juicer. Weigh 15 g of Gastrodia elata original pulp, add 90 mL of potato liquid medium, then make Gastrodia elata fermentation broth, and keep the subsequent steps the same to obtain the fresh Gastrodia elata liquid fermentation product (sample 5).

[0060] If the raw material is Gastrodia elata extract, weigh 0.5 g of Gastrodia elata extract, add 100 mL of potato liquid medium, then make Gastrodia elata fermentation broth, and keep the subsequent steps the same to obtain the Gastrodia elata extract liquid fermentation product (sample 6).

[0061] Example 4: Preparation of Gastrodia elata enzyme hydrolysis product by mixed enzymatic hydrolysis

[0062] 10 g of dried Gastrodia elata tuber or decoction pieces were pulverized and passed through an 80-mesh sieve, placed in a conical flask, 300 mL of water, 0.75 g of cellulase and 0.75 g of pectinase were added, and then stirred into a homogeneous slurry. It was placed in a water bath at 68 °C and heated with stirring for enzymatic hydrolysis for 2 hours. Vacuum freeze-dried to obtain the Gastrodia elata enzyme hydrolysis product (sample 7).

[0063] Example 6: Determination of the Content of Active Ingredients in the Microbial Fermentation Products of Gastrodia elata

[0064] 1) Preparation of the Test Solution of Gastrodia elata

[0065] The dried Gastrodia elata tuber was crushed and passed through an 80-mesh sieve to obtain the unfermented Gastrodia elata sample powder, which was the Gastrodia elata group. The Gastrodia elata fermentation products in the examples were crushed and passed through an 80-mesh sieve to obtain the powder of each Gastrodia elata fermentation product.

[0066] Approximately 0.5 g of the Gastrodia elata group and the powder of the Gastrodia elata fermentation products were taken and placed in a stoppered conical flask. 25 mL of 50% methanol was added, and ultrasonic extraction was carried out for 30 minutes (500 W, 40 KHz). After cooling, it was shaken well, filtered, and the subsequent filtrate was taken as the test solution of Gastrodia elata.

[0067] 2) Preparation of the Reference Solution

[0068] An appropriate amount of reference substances of gastrodin, p-hydroxybenzyl alcohol, parishin A, parishin B, parishin C, and parishin E was accurately weighed and dissolved in a 3% acetonitrile aqueous solution to prepare a mixed reference solution containing 0.85 mg of gastrodin, 0.48 mg of p-hydroxybenzyl alcohol, 0.53 mg of parishin A, 0.49 mg of parishin B, 0.52 mg of parishin C, and 0.45 mg of parishin E per 1 mL.

[0069] 3) Chromatographic Conditions

[0070] An YMC ODS-C8 chromatographic column (4.6 mm×250 mm, 5 μm) was used; acetonitrile - 0.1% phosphoric acid solution was used as the mobile phase, the detection wavelength was 220 nm, and the flow rate was 0.8 mL·min -1 , the column temperature was 30 °C, and the gradient elution program was 3% - 10% acetonitrile for 0 - 10 min, 10% - 12% acetonitrile for 10 - 15 min, 12% - 18% acetonitrile for 15 - 25 min, 18% acetonitrile for 25 - 40 min, and 18% - 95% acetonitrile for 40 - 42 min.

[0071] 4) Results

[0072] The results of the content of active ingredients in the control group and each Gastrodia elata fermentation product are shown in Table 2. The results of the content of active ingredients after different fermentation times of Gastrodia elata are shown in Table 3.

[0073] Table 2. Content of Active Ingredients in the Microbial Fermentation Products of Gastrodia elata (mg·g -1 )

[0074] Group Gastrodin p-Hydroxybenzyl alcohol Ginsenoside A Ginsenoside B Ginsenoside C Ginsenoside E Gastrodia group 1.947±0.125 0.465±0.104 8.744±0.116 1.367±0.204 0.926±0.135 6.524±0.067 Sample 1 18.531±0.108 3.922±0.216 2.254±0.203 1.221±0.132 1.775±0.237 2.028±0.102 Sample 2 32.162±0.231 8.737±0.193 1.208±0.293 1.073±0.169 2.824±0.193 3.643±0.085 Sample 3 40.516±0.257 12.984±0.261 1.085±0.137 2.384±0.117 1.537±0.128 2.206±0.118 Sample 4 29.758±0.343 4.862±0.277 2.719±0.226 1.505±0.148 1.442±0.205 1.987±0.091 Sample 5 22.914±0.226 9.506±0.301 2.577±0.302 2.102±0.214 1.609±0.171 2.031±0.146 Sample 6 43.802±0.264 20.385±0.185 1.803±0.259 1.318±0.153 1.713±0.154 1.865±0.134 Sample 7 4.348±0.325 1.836±0.137 7.327±0.128 1.462±0.169 1.058±0.115 5.631±0.122

[0075] Table 3. Content of Active Ingredients at Different Fermentation Times of Gastrodia elata (mg·g -1 )

[0076] Group Gastrodin p-Hydroxybenzyl alcohol Ginsenoside A Ginsenoside B Ginsenoside C Ginsenoside E Sample 1 18.531±0.108 3.922±0.216 2.254±0.203 1.221±0.132 1.775±0.237 2.028±0.102 Sample 1-A 1.982±0.136 0.507±0.115 9.167±0.129 1.338±0.193 1.274±0.251 7.388±0.142 Sample 1-B 5.327±0.301 0.998±0.173 9.085±0.137 1.321±0.201 1.219±0.101 6.242±0.154 Sample 1-C 17.905±0.265 3.151±0.108 1.912±0.304 1.006±0.167 1.547±0.238 1.964±0.193 Sample 1-D 10.621±0.182 2.376±0.159 1.824±0.135 1.055±0.193 1.143±0.158 1.256±0.124 Sample 1-E 3.776±0.235 0.926±0.237 1.127±0.164 0.915±0.328 1.049±0.172 1.033±0.267 Sample 4 29.758±0.343 4.862±0.272 2.719±0.226 1.505±0.148 1.442±0.205 1.987±0.091 Sample 4-A 12.253±0.175 2.891±0.134 7.251±0.182 1.475±0.186 1.267±0.204 5.836±0.228 Sample 4-B 20.523±0.214 3.467±0.235 4.036±0.127 1.381±0.194 1.183±0.152 3.041±0.175 Sample 4-C 28.618±0.126 3.924±0.281 2.873±0.155 1.257±0.125 1.208±0.184 2.557±0.193 Sample 4-D 17.652±0.233 2.425±0.166 2.156±0.176 1.109±0.122 1.130±0.151 1.925±0.174 Sample 4-E 8.284±0.207 1.354±0.152 2.088±0.153 1.116±0.171 0.984±0.239 1.245±0.146

[0077] The results showed that the content of gastrodin in the microbial fermentation products of Gastrodia elata Bl. (Samples 1 - 6) of the present invention was 10 - 40 mg·g -1 which was 10 - 20 times higher than that of the Gastrodia elata Bl. group (before fermentation), and the content of p - hydroxybenzyl alcohol was 3 - 20 mg·g -1 which was 8 - 50 times higher than that of the Gastrodia elata Bl. group. Except for the significant decrease in the content of balisongoside A, the contents of other active ingredients did not show an obvious decrease. Compared with the enzymatically hydrolyzed products of Gastrodia elata Bl. (Sample 7), the content of gastrodin in the microbial fermentation products of Gastrodia elata Bl. of the present invention was 4 - 10 times that of the enzymatically hydrolyzed products, and the content of p - hydroxybenzyl alcohol was 2 - 10 times that of the enzymatically hydrolyzed products.

[0078] Meanwhile, there were significant differences in the contents of gastrodin and p - hydroxybenzyl alcohol in the fermentation products of Gastrodia elata Bl. at different fermentation times (Samples 1 - A to 1 - E and Samples 4 - A to 4 - E). The contents of gastrodin and p - hydroxybenzyl alcohol were the highest when solid - state fermentation was carried out for 15 days (Sample 1) and liquid - state fermentation was carried out for 7 days (Sample 4).

[0079] Example 7: Effect of the fermentation products of Gastrodia elata Bl. on treating oligoasthenospermia

[0080] Take about 100 g of Gastrodia elata Bl., enzymatically hydrolyzed products of Gastrodia elata Bl., and fermentation products of Gastrodia elata Bl. respectively, crush them and pass through an 80 - mesh sieve, place them in a stoppered conical flask, add 500 mL of 50% methanol, extract ultrasonically for 30 minutes (500 W, 40 KHz), let it cool, shake well, filter, and recover methanol from the filtrate to obtain the extract of the fermentation products of Gastrodia elata Bl. The extract is mixed and swirled with an appropriate amount of physiological saline for subsequent animal administration.

[0081] Adult 8 - week - old male ICR mice were used to prepare an oligoasthenospermia model by single intraperitoneal injection of busulfan (30 mg / kg). The animals were randomly divided into a blank control group, a model group, a Gastrodia elata Bl. group, an enzymatically hydrolyzed products of Gastrodia elata Bl. (Sample 7) treatment group, and a fermentation products of Gastrodia elata Bl. (Samples 1 - 6) treatment group (20 mg / kg), with 10 mice in each group. The treatment groups of the fermentation products of Gastrodia elata Bl. were given intragastric administration once a day for 4 consecutive weeks. The control group and the model group were given the same volume of physiological saline. After the administration, the mice were sacrificed and both epididymides were taken. The cauda epididymis was chopped in a small plate containing 2 mL of Tyrode's solution, incubated at 37°C for 20 minutes, and gently shaken to discharge sperm. The sperm concentration, sperm motility, and percentage of forward - moving sperm were detected using a CASA computer - assisted sperm quality analysis system. The results are as Figure 2 shown.

[0082] Compared with the control group, the sperm concentration, sperm motility, and percentage of sperm with forward motility in the model group of mice were all significantly decreased, and the differences were statistically significant (P<0.001). Compared with the model group, the sperm concentration, sperm motility, and percentage of sperm with forward motility in each group administered with fermented Gastrodia elata products were all significantly increased, and the differences were statistically significant (P<0.001). However, the Gastrodia elata group and the enzymatically hydrolyzed Gastrodia elata product group did not significantly improve the sperm concentration, sperm motility, and percentage of sperm with forward motility in mice. The experimental results indicate that fermented Gastrodia elata products can significantly increase sperm concentration and improve spermatogenesis disorders, enhance sperm motility and improve asthenospermia symptoms, and can be used as active ingredients for the treatment or improvement of oligoasthenospermia.

[0083] Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of the present invention claimed is defined by the appended claims and their equivalents.

Claims

1. A preparation method of a Gastrodia elata microbial fermentation product, characterized in that, The gastrodia elata is prepared by fermenting gastrodia elata with Aspergillus oryzae. The gastrodia elata is selected from fresh gastrodia elata, gastrodia elata decoction pieces, gastrodia elata powder or gastrodia elata extract. The fermentation method is solid-state fermentation or liquid-state fermentation.

2. The preparation method according to claim 1, characterized in that, The solid-state fermentation comprises the following steps: 1) pre-sterilizing fresh Gastrodia elata, Gastrodia elata slices or Gastrodia elata powder; 2) Aspergillus oryzae spore powder and fried flour are mixed at a weight ratio of 1:1-10, and then inoculated into the pretreated Gastrodia elata at an inoculum amount of 5-15% by weight of Gastrodia elata; 3) Fermentation at 30°C, 70% humidity for 2-30 days; 4) After the fermentation is completed, wet heat sterilization is performed to obtain the Gastrodia elata microbial fermentation product; The liquid fermentation comprises the following steps: 1) adding gastrodia elata powder or gastrodia elata extract to a potato liquid culture medium at a weight volume ratio of 1:5-50 g / L and sterilizing by high pressure moist heat to prepare the culture medium; 2) inoculating the Aspergillus oryzae liquid pre-cultured in a potato liquid culture medium into the culture medium of step (1) at a ratio of 1 to 10% by volume; 3) Temperature 30°C, 160r / min, fermentation 3-20 days; 4) After the fermentation is completed, high pressure wet heat sterilization and freeze drying are performed to obtain the Gastrodia elata microbial fermentation product.

3. The solid-state fermentation time according to claim 2 is preferably 15 days.

4. The liquid fermentation time according to claim 2 is preferably 7 days.

5. The preparation method according to claim 1, characterized in that, The Aspergillus oryzae is selected from Aspergillus oryzae Shanghai Niang 3.042, Aspergillus oryzae CGMCC 3.2073 or Aspergillus oryzae CGMCC 3.

407.

6. A microbial fermentation product of Gastrodia elata, characterized in that The invention is prepared by the method according to any one of claims 1 to 5.

7. The Gastrodia elata microbial fermentation product according to claim 6, wherein The content of gastrodin in the microbial fermentation product of Gastrodia elata is 10-40 mg·g -1 , and the content of p-hydroxybenzyl alcohol is 3-20 mg·g -1 .

8. A functional food, health food or medicine, characterized in that, A product containing the Gastrodia elata microbial fermentation product according to claim 6 or 7.

9. The functional food, health food or medicine according to claim 8, wherein The functional food or health food or medicine is an oral preparation, injection, granule, pill, capsule, powder, sustained-release preparation, controlled-release preparation, or targeted preparation.

10. Use of the Gastrodia elata microbial fermentation product according to claim 6 or 7 in the preparation of food or medicine for improving or treating oligospermia or asthenospermia.

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