Method for enhancing nutritional quality of complete soybeans through oyster mushroom mycelia and application

Abstract

The invention discloses a method for enhancing the nutritional quality of complete soybeans through oyster mushroom mycelia and application. The invention belongs to the field of food technology and microbial fermentation, and particularly relates to a method for enhancing the nutritional quality of complete soybeans through oyster mushroom mycelia and application. The method provided by the invention comprises the following steps: fermenting complete soybeans by using oyster mushroom mycelia to obtain oyster mushroom mycelium fermented soybeans, and the oyster mushroom is the oyster mushroom registered in China General Microbiological Culture Collection Center (CGMCC) with the registration number of CGMCC No.5.784. After solid fermentation of the oyster mushroom mycelium, the nutritional quality of the oyster mushroom mycelium fermented soybean powder is remarkably enhanced, the bioavailability of soybeans is improved, and the product is expected to become a source of a natural antioxidant and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of food technology and microbial fermentation, and specifically relates to a method and application of enhancing the nutritional quality of whole soybeans by using oyster mushroom mycelium. Background Art

[0002] Plant protein is an important source of dietary protein for humans, especially soybeans, which have a protein content of about 40%, providing high-quality protein for humans. At the same time, soybeans are rich in phenolic active substances, especially soy isoflavones in the form of aglycones, which have strong free radical scavenging ability and antioxidant effects. Regular consumption of soybeans has a preventive or regulatory effect on aging, hypertension, obesity, type 2 diabetes, cancer, Alzheimer's disease and cardiovascular diseases. However, compared with animal protein, soy protein has an unbalanced amino acid and lacks methionine. In addition, soybeans contain allergenic proteins and anti-nutritional factors, such as trypsin inhibitors, soybean lectins, phytic acid, etc., which affect the bioavailability of soybeans in the human body.

[0003] Oyster mushroom is a common edible fungus. Its life cycle includes the mycelium growth stage, the fruiting body differentiation stage and the fruiting body growth and development stage. The oyster mushrooms we usually eat are mature fruiting bodies. Oyster mushrooms are not only rich in protein nutrition, but also have complementary essential amino acids with soybeans. For example, the content of methionine and cystine is low in most bean proteins, but sufficient in edible fungal proteins. In addition, oyster mushrooms are low in fat and high in fiber, and contain vitamins B1, B2, C, D2, minerals and 8 essential amino acids for the human body to meet the normal metabolic needs of the human body. Oyster mushrooms are rich in natural active ingredients, such as peptides, triterpenes, phenolic compounds, polysaccharides, glycoproteins, etc., and have antioxidant, anti-diabetic, anti-obesity, immunomodulatory, anti-cancer and anti-tumor properties.

[0004] Microbial solid fermentation of soybeans, such as natto, soy sauce and fermented black beans, has long been favored by Asian countries such as China, Japan and South Korea due to its unique flavor and nutritional value. Microorganisms contain rich enzyme systems. During the fermentation of soybeans, macromolecules in soybeans are converted into small molecules, and beany substances and anti-nutritional factors in soybeans are degraded to varying degrees, thereby improving the digestion and absorption characteristics and bioavailability of fermented soybeans. At the same time, a variety of functional active substances are produced during the fermentation process, such as peptides, isoflavones, saponins, oligosaccharides, etc. Edible fungi secrete a large amount of enzymes during the mycelium growth stage, such as cellulase, protease, pectinase, polyphenol oxidase, etc. Therefore, using the mycelium of edible fungi for solid fermentation of soybeans can not only obtain fermented soybeans with improved functional active ingredients, but also give the product the active substances of edible fungi, which has gradually attracted people's attention. The European Food Safety Authority conducted a safety evaluation on the mixed product (NF) of pea and rice protein fermented with shiitake mushroom mycelium. The protein content of the product was increased, and the content of phytic acid, protease inhibitors and lectins was reduced. The solid fermentation of seven kinds of grains (wheat, rice, oats, corn, millet, millet and sorghum) using Agaricus blazei mycelium has been found to increase the content of nutrients in grains. There is no report on the solid fermentation of soybeans using Pleurotus ostreatus mycelium. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to improve the quality of soybeans, increase the nutritional value of soybeans, simplify the fermentation process, and increase the utilization rate of soybeans.

[0006] In order to solve the above problems, the present invention provides a method for preparing soybean fermented with Pleurotus ostreatus mycelium.

[0007] The method for preparing soybeans fermented with oyster mushroom mycelium provided by the invention comprises the following steps: fermenting soybeans with oyster mushroom mycelium to obtain soybeans fermented with oyster mushroom mycelium; the oyster mushroom is oyster mushroom (Pleurotus ostreatus) and its registration number in the General Microorganism Center of China Microorganism Culture Collection Committee is CGMCC No.5.784.

[0008] In the above method, the soybeans are whole soybeans.

[0009] In the above method, the method comprises the following steps:

[0010] 1) Preparing Pleurotus ostreatus mycelium: activating the Pleurotus ostreatus strains described above and inoculating them into a PDA solid culture medium to obtain Pleurotus ostreatus mycelium;

[0011] 2) Preparing soybean culture medium: soaking soybeans in water, filtering water, and sterilizing to obtain soybean culture medium;

[0012] 3) Solid-state fermentation: inoculating the mycelium of Pleurotus ostreatus into soybean culture medium and fermenting to obtain the mycelium fermented soybeans.

[0013] In the above method, in step 2), the soybeans are soaked in water at a solid-liquid ratio of 1:1.5-3.5, and the soaking time is 6-10 hours.

[0014] In the above method, in step 2), the ratio of the material to the liquid in the water used for soaking the soybeans is preferably 1:2, and the soaking time is 8 hours.

[0015] In the above method, the fermentation conditions in step 3) are 22-28° C., 50-80% humidity, and dark culture for 15-40 days.

[0016] In the above method, the fermentation conditions in step 3) are preferably 25° C., 70% humidity and dark culture for 33 days.

[0017] In the above method, the ratio of the above-mentioned oyster mushroom and the above-mentioned soybean culture medium meets the requirement of 1 to 5 cm 2 The mycelium of Pleurotus ostreatus: 100-500 g of the soybean culture medium.

[0018] The invention provides a soybean product fermented by Pleurotus ostreatus mycelium prepared by the method described above.

[0019] In the above, the product is a medicine, food, health product, animal feed or animal feed additive.

[0020] The present invention also provides the application of the method described above in the following:

[0021] 1) preparing the aforementioned soybean product fermented with Pleurotus ostreatus mycelium;

[0022] 2) Increase the protein content in soybeans;

[0023] 3) Increase methionine content;

[0024] 4) reducing the starch and / or crude fiber content in soybeans;

[0025] 5) Reduce the content of allergenic proteins and / or anti-nutritional factors in soybeans;

[0026] 6) converting soybean glycoside isoflavones into aglycone isoflavones;

[0027] 7) Increase the total phenolic content and antioxidant activity of soybeans or scavenge free radicals.

[0028] In the above, the soybean glycoside soy isoflavones can be daidzein, glycitin and genistin.

[0029] In the above, the daidzein-type soy isoflavones may be daidzein, glycitein and genistein.

[0030] In the above, the allergenic proteins in soybean are glycinin and β-conglycinin, and the anti-nutritional factors are trypsin inhibitor and soybean lectin.

[0031] In the above, the free radical scavenging is scavenging DPPH free radical or scavenging ABTS free radical.

[0032] The present invention has shown that solid fermentation of Pleurotus ostreatus mycelium is expected to be an effective means to increase the active ingredients of edible fungi and improve the nutritional properties of soybeans, degrade soybean anti-nutritional factors and improve the bioavailability of soybeans. The technical culture medium has simple components and simple technology, low cost and low energy consumption. In the fermentation production process, no waste water or waste gas is generated, and environmental pollution is reduced. The soybean fermentation product obtained by this method will become a food base material beneficial to human health and used in the field of food processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the growth of Pleurotus ostreatus mycelium on the PDA plate.

[0034] Figure 2 The growth of Pleurotus ostreatus mycelium cultured on soybean medium at different time periods.

[0035] Figure 3 These are samples of dried and crushed soybeans fermented with Pleurotus ostreatus mycelium at different fermentation times.

[0036] Figure 4 The SDS-PAGE images of soybeans before and after solid fermentation. The molecular weight of the protein marker ranges from 11 to 180 kDa; RAW: raw soybeans; SF: unfermented soybeans; FSF: soybeans fermented with Pleurotus ostreatus mycelium.

[0037] Figure 5 The HPLC chromatograms before and after soybean solid fermentation. A: standard chromatogram; B: HPLC chromatogram before soybean solid fermentation; C: HPLC chromatogram after soybean solid fermentation. 1: daidzein; 2: glycitein; 3: genistin; 4: daidzein; 5: glycitein; 6: genistein. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0039] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0040] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0041] The edible fungi of Pleurotus ostreatus in the following examples were purchased from the General Microbiological Center of China Microorganism Culture Collection Committee with a registration number of CGMCC No. 5.784, and the website address is: https: / / cgmcc.net / directory / detail?cgmccid=5.784&number=&genus=Pleurotus&species=&yiming=&page=1. The culture method was to inoculate in PDA slant medium and store at 4°C.

[0042] The soybeans in the following examples are soybeans (Glycine max), produced in Mudanjiang City, Heilongjiang Province, with a product number of 100019456052. The public can obtain the biological material from the applicant, and the biological material is only used to repeat the experiment of the present invention and cannot be used for other purposes.

[0043] The following examples were processed using SPSS11.5 statistical software, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

[0044] Example 1: Preparation of soybeans fermented with Pleurotus ostreatus mycelium

[0045] 1. Activation of Pleurotus ostreatus mycelium

[0046] 1) PDA solid medium: potato powder 3 g / L, KH2PO4 3 g / L, glucose 20 g / L, MgSO4·7H2O 1.5 g / L, agar 15 g / L, natural pH.

[0047] 2) Take a small piece of Pleurotus ostreatus mycelium with culture medium from the PDA slant culture medium with an inoculation spatula, inoculate it on a PDA solid plate, and culture it upright at 25°C in the dark for 10 days until the mycelium is fully grown.

[0048] 2. Preparation of soybean culture medium

[0049] 1) Soybean pretreatment: Soybeans are first washed with clean water to remove impurities such as spoiled seeds and stones. Soaked in deionized water at 20°C with a material-liquid ratio of 1:2 for 8 hours.

[0050] 2) Bottle sterilization: First, filter the soaked soybeans with gauze to remove moisture, and then wipe the surface of the soybean seeds dry. Put the soaked soybeans into 340mL tissue culture bottles, 120g per bottle (wet basis). After filling, seal the cap and put it into the autoclave, 121℃, sterilize for 90 minutes.

[0051] 3. Vaccination

[0052] After the soybean solid culture medium is naturally cooled to room temperature, the mycelium of Pleurotus ostreatus on the PDA solid culture medium is cut into 1 cm × 1 cm square pieces according to the requirements of sterile operation, and inoculated into the soybean culture medium, with 3 small pieces inoculated into each culture medium.

[0053] 4. Fermentation

[0054] After the inoculation was completed, the culture medium was placed in an artificial incubator at 25°C and 70% humidity in the dark for 33 days.

[0055] 5. Save

[0056] The fermented mixture of Pleurotus ostreatus and soybeans was placed in an oven at 60°C and dried for 24 hours, then crushed with a grinder and passed through a 40-mesh sieve to obtain Pleurotus ostreatus mycelium fermented soybean powder, which was placed in a ziplock bag and stored in a ventilated and dry place.

[0057] Example 2: Determination of nutritional components of soybeans fermented with Pleurotus ostreatus mycelium

[0058] 1. Determination of basic nutritional components of soybeans fermented with Pleurotus ostreatus mycelium

[0059] 1) Determine the moisture content of soybeans fermented with Pleurotus ostreatus mycelium according to the national standard GB / T 5009.3-2016.

[0060] 2) The ash content in soybeans fermented with Pleurotus ostreatus mycelium was determined according to the national standard GB / T 5009.4-2016.

[0061] 3) The protein content in soybeans fermented with Pleurotus ostreatus mycelium was determined according to the national standard GB / T 5009.5-2016.

[0062] 4) Determine the fat content in soybeans fermented with Pleurotus ostreatus mycelium according to the national standard GB / T 5009.6-2016.

[0063] 5) The content of reducing sugar in soybean fermented with Pleurotus ostreatus mycelium was determined according to the national standard GB / T 5009.7-2016.

[0064] 6) Determine the starch content in soybeans fermented with Pleurotus ostreatus mycelium according to the national standard GB / T 5009.9-2016.

[0065] 7) The content of soluble dietary fiber and insoluble dietary fiber in soybean fermented with Pleurotus ostreatus mycelium was determined according to the national standard GB / T 5009.88-2016.

[0066] Unfermented soybeans were used as the control group. The test results are shown in Table 1: Compared with the control group, the ash, protein and reducing sugar contents of soybeans fermented with Pleurotus ostreatus mycelium increased by 22.07%, 27.80% and 322%, respectively. The contents of starch, soluble dietary fiber and insoluble dietary fiber decreased by 32.43%, 50% and 28.05%, respectively. It can be seen that Pleurotus ostreatus mycelium consumed starch and dietary fiber during growth, but increased the protein content in the fermented product.

[0067] Table 1 Basic nutritional content of soybeans fermented with Pleurotus ostreatus mycelium

[0068]

[0069] 2. Essential amino acid score of soybeans fermented with Pleurotus ostreatus mycelium

[0070] 1) The contents of 16 kinds of amino acids including aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine in soybeans fermented with Pleurotus ostreatus mycelium were determined according to the national standard GB / T 5009.124-2016. The contents of tryptophan and cystine in soybeans fermented with Pleurotus ostreatus mycelium were determined according to the national standard GB / T 18246-2019.

[0071] 2) Calculate amino acid scores according to the method of Bano (1992)

[0072] Soybeans are considered to be a source of high plant protein content. Solid fermentation of Pleurotus ostreatus mycelium can not only increase the protein content, but also improve the amino acid structure, making it more suitable for human digestion and utilization. Table 2 shows the amino acid composition of soybeans fermented with Pleurotus ostreatus mycelium. A total of 18 amino acids were determined, including 10 essential amino acids and 8 non-essential amino acids. The total amount of essential amino acids in soybeans fermented with Pleurotus ostreatus mycelium was 277.73 mg / g, and the proportion of essential amino acids was 40%, which was higher than the WHO / FAO recommended value (36%) and close to the recommended value of whole egg protein (48.9%). It is reported that sulfur-containing amino acids (methionine and cysteine) in soybean protein are limiting amino acids. The Met+Cys content in soybeans fermented with Pleurotus ostreatus mycelium was greater than that in the control group.

[0073] In addition, the essential amino acid score of soybeans fermented with Pleurotus ostreatus mycelium was 59.41 (Table 3). The results showed that fermentation with Pleurotus ostreatus mycelium increased the amino acid score and changed the composition of essential amino acids in soybeans.

[0074] Table 2 Essential amino acid content of soybeans fermented with Pleurotus ostreatus mycelium

[0075]

[0076]

[0077] Note: “-” means FAO / WHO has no recommended value for this indicator.

[0078] Table 3 Essential amino acid scores of soybeans fermented with Pleurotus ostreatus mycelium

[0079]

[0080]

[0081] Note: The amino acid score refers to the essential amino acid pattern recommended by FAO / WHO (1973).

[0082] 3. Determination of allergenic proteins and anti-nutritional factors in soybeans fermented with Pleurotus ostreatus mycelium

[0083] Samples to be tested: Raw soybean group: soybeans were not sterilized under high pressure and directly crushed; Unfermented soybean group: soybeans were sterilized under high pressure but not fermented, then dried and crushed; Pleurotus ostreatus mycelium fermented soybeans: soybeans were sterilized under high pressure and then inoculated with Pleurotus ostreatus mycelium for fermentation, then dried and crushed.

[0084] 1) With raw soybean as control, the contents of glycinin, β-conglycinin, trypsin inhibitor and soybean lectin in the raw soybean group, unfermented soybean group and soybean fermented with Pleurotus ostreatus mycelium were detected by enzyme-linked immunosorbent assay. The analysis method and operation steps were determined according to the kit instructions of Tianjin Longke Xinyu Biotechnology Co., Ltd. Soybean globulin rapid detection kit (Cat. No.: 11S-EA), β-conglycinin rapid detection kit (Cat. No.: 7S-EA), trypsin inhibitor detection kit (Cat. No.: KTI-EA), soybean lectin detection ELISA kit (Cat. No.: SBA-EA).

[0085] 2) SDS-PAGE

[0086] Soluble protein was extracted from soybeans fermented with Pleurotus ostreatus mycelium using a soybean globulin extraction kit (Tianjin Longke Xinyu Biotechnology Co., Ltd., catalog number: 11S-EA). Soluble protein content was determined using a Bradford protein quantification kit (Beijing Tiangen Biotechnology Co., Ltd., catalog number: PA102). After mixing with 5× protein loading buffer, boil in a boiling water bath for 10 min. After cooling, centrifuge at 3000 rpm / min for 3 min for electrophoresis analysis. The sample loading volume was 10 μL, and the marker loading volume was 6 μL. Soluble protein was separated using a 12% polyacrylamide separation gel at 120 V for 55 min. After electrophoresis, the gel was stained with Coomassie Brilliant Blue Rapid Staining Solution for 2 h, then decolorized in distilled water, and then photographed and preserved.

[0087] Firstly, SDS-PAGE was performed to verify the glycinin (11S) and β-conglycinin (7S) in soybeans fermented with Pleurotus ostreatus mycelium. Figure 4 As shown in the figure: The main allergenic proteins in soybeans are glycinin and β-conglycinin. After high-pressure sterilization of soybeans, the basic subunit of glycinin (22kDa), the acidic subunit of glycinin (35-40kDa), the β-conglycinin β subunit (50kDa), the β-conglycinin α subunit (67kDa) and the β-conglycinin α subunit (71kDa) all disappeared, and no protein bands were identified in the SDS-PAGE graph, indicating that high-temperature sterilization has a great impact on soybean allergenic proteins. After fermentation, glycinin (11S) and β-conglycinin (7S) were not detected again.

[0088] With reference to the results of SDS-PAGE, the contents of allergenic proteins such as glycinin and β-conglycinin, and the contents of anti-nutritional factors including trypsin inhibitor and soybean lectin in soybeans fermented with Pleurotus ostreatus mycelium were determined by a kit. The results are shown in Table 4. After high-temperature sterilization and fermentation, glycinin, β-conglycinin, trypsin inhibitor and soybean lectin in soybeans fermented with Pleurotus ostreatus mycelium were all degraded by more than 99%.

[0089] Table 4 Contents of allergenic proteins and anti-nutritional factors in soybeans fermented with Pleurotus ostreatus mycelium

[0090]

[0091] Example 3: Qualitative and quantitative analysis of soybean isoflavones in soybeans fermented with Pleurotus ostreatus mycelium

[0092] 1. Extraction of soy isoflavones from soybeans fermented with Pleurotus ostreatus mycelium

[0093] 1) Degreasing: Take 30g of dried and crushed soybean samples fermented with mycelium of Pleurotus ostreatus and put them into a 500mL beaker, add 150mL of n-hexane (Pleurotus ostreatus mycelium fermented soybean powder: n-hexane = 1:5, w / v) and stir continuously overnight with a magnetic stirrer at room temperature. After standing for 30 minutes, after the fermented soybean powder of Pleurotus ostreatus mycelium settles and separates from the upper organic solvent, pour out the organic solvent for recovery and collect the residue. Add 150mL of n-hexane and stir for 3h. Repeat this step twice. Place the collected residue in a fume hood to dry.

[0094] 2) Extraction: Weigh 3 g of defatted Pleurotus ostreatus mycelium fermented soybean powder, add 60 mL of 80% methanol and extract at 25°C, 55 rpm / min for 2 h. Then, centrifuge the sample at 20°C, 10000 rpm / min for 20 min, and collect the supernatant. The residue is extracted twice with 80% methanol, the residue is discarded, and the supernatant is combined. The supernatant is used for subsequent qualitative and quantitative analysis of soybean isoflavones, determination of polyphenol content and antioxidant activity.

[0095] 2. Qualitative and quantitative analysis of soybean isoflavones in soybeans fermented with Pleurotus ostreatus mycelium

[0096] The methanol extract of soybean powder fermented with Pleurotus ostreatus mycelium was detected by HPLC–UV method.

[0097] 1) Chromatographic conditions

[0098] Chromatographic system: Waters 2596

[0099] UV detector: Waters 2996

[0100] Chromatographic column: C18 reverse phase column (4.6mm×250mm, 5μm)

[0101] Column temperature: 40°C

[0102] Flow rate: 1mL / min

[0103] Mobile phase: Liquid A is acetic acid aqueous solution (pH 3.0), Liquid B is 100% acetonitrile

[0104] Gradient elution: 0-17 min, 5% acetonitrile → 45% acetonitrile; 17-27 min, 45% acetonitrile → 90% acetonitrile; 27-33 min, 90% acetonitrile; 33-34 min, 90% acetonitrile → 100% acetonitrile; 34-49 min, 100% acetonitrile; 49-50 min, 100% acetonitrile → 5% acetonitrile; 50-65 min, 5% acetonitrile

[0105] UV detection wavelength: 285nm

[0106] Injection volume: 10 μL

[0107] 2) Configuration of standard products

[0108] Soy isoflavone standard stock solution: weigh 5 mg of 6 standards (Shanghai Yuanye Biotechnology Co., Ltd., product numbers B20226, B20554, B21038, B20227, B20555, B21039, respectively), add 5 mL of dimethyl sulfoxide-acetonitrile-methanol (1:2:2, v / v / v), fully dissolve, and prepare 1 mg / mL single-label standard stock solution.

[0109] Soy isoflavones single-label standard working solution: dilute the soy isoflavones standard stock solution 10 times with 80% methanol to prepare a 0.1 mg / mL single-label standard working solution.

[0110] Soy isoflavone mixed standard working solution: Take 0.5mL of each of the six soybean isoflavone standard stock solutions, dilute to 5mL with 80% methanol, and prepare a soybean isoflavone mixed standard working solution with a concentration of 100μg / mL for each component. Then continue to dilute with 80% methanol to a series of soybean isoflavone mixed standard working solutions with concentrations of 0.5μg / mL, 1μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 30μg / mL, 40μg / mL, and 50μg / mL. Draw a standard curve with the peak area as the ordinate and the concentration of each component as the abscissa.

[0111] 3) Qualitative and quantitative analysis of soybean isoflavones in soybeans fermented with Pleurotus ostreatus mycelium

[0112] Qualitative analysis: 6 different soy isoflavone single-label working standard solutions and the soy isoflavone extracts of soy powder fermented with Pleurotus ostreatus mycelium were subjected to HPLC detection according to chromatographic conditions, and qualitative analysis was performed based on the retention time of the single-label working standard solutions.

[0113] Quantitative analysis: The mixed standard working solution of soybean isoflavones with different concentration gradients is subjected to HPLC detection according to chromatographic conditions. The sample is appropriately diluted to ensure that the concentration of each component is within the detection range of the standard curve.

[0114] Soy isoflavones are a special type of phenolic substance naturally present in soybeans, which are divided into glycosides and aglycones. Glycoside soy isoflavones are usually combined with sugar, which reduces bioavailability and also affects human digestion and absorption. However, the aglycone form of soy isoflavones is not only easier to be absorbed by the body due to its simple structure and low molecular weight, but also has strong free radical scavenging ability and the ability to inhibit reactive oxygen species (ROS).

[0115] The composition of soybean isoflavones before and after solid fermentation of soybeans with Pleurotus ostreatus mycelium was identified by HPLC. Figure 5 As shown in the figure, six main soybean isoflavone components were detected according to retention time, namely daidzein, glycitein, genistin, daidzein, glycitein and genistein. Solid fermentation of oyster mushroom mycelium promoted the hydrolysis of daidzein into daidzein. Neither daidzein nor genistin was detected in soybeans fermented with oyster mushroom mycelium.

[0116] By quantitative analysis (Table 5), the total amount of isoflavones in the unfermented soybean group is 18.75mg / 10g, while the total amount of soy isoflavones in the Pleurotus ostreatus mycelium fermented soybean obtained in Example 1 is 16.10mg / 10g. Although fermentation reduces the total content of soy isoflavones, there is biotransformation of isoflavones. Among them, the total amount of 3 kinds of soy isoflavone aglycones of daidzein, glycitein and genistein in the unfermented soybean accounts for about 17.83% of the total amount of soy isoflavones, while the total amount of soy isoflavone aglycones in the Pleurotus ostreatus mycelium fermented soybean accounts for about 96.48%. Although the contents of glycitein and glycitein have been reduced by 72.26% and 76.18% respectively. But the two aglycones of daidzein and genistein have increased significantly.

[0117] Therefore, solid fermentation using Pleurotus ostreatus mycelium promoted the biotransformation of soybean isoflavone glycosides into aglycones. It also showed that soybean fermented with Pleurotus ostreatus mycelium had potential antioxidant effects and had important application value for human dietary health.

[0118] Table 5 The content of soybean isoflavones in soybeans fermented with Pleurotus ostreatus mycelium

[0119] Content mg / 10g Control group Fermented soybeans with oyster mushroom mycelium Soybean glycoside 13.23±0.79 Not detected Daidzein <![CDATA[0.60±0.08 b ]]> <![CDATA[6.76±0.25 a ]]> Glycitin <![CDATA[2.04±0.07 a ]]> <![CDATA[0.57±0.07 b ]]> Glycitein <![CDATA[1.79±0.00 a ]]> <![CDATA[0.43±0.02 b ]]> Genistin 0.14±0.01 Not detected Genistein <![CDATA[0.95±0.06 b ]]> <![CDATA[8.34±0.31 a ]]>

[0120] Example 4: Determination of polyphenol content and antioxidant activity of soybeans fermented with Pleurotus ostreatus mycelium

[0121] 1. Determination of polyphenols (TPC) content in soybeans fermented with Pleurotus ostreatus mycelium

[0122] (1) Extraction of polyphenols (TPC) from soybeans fermented with Pleurotus ostreatus mycelium: The method is the same as that in Example 3.

[0123] (2) Draw a standard curve: Accurately weigh 25 mg of gallic acid standard, dissolve it in methanol and dilute it in a 25 mL volumetric flask to obtain a 1 mg / mL standard stock solution. Prepare standard solutions of certain mass concentrations (0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30 mg / mL) and draw a standard curve with different concentrations of gallic acid as the horizontal axis and absorbance as the vertical axis.

[0124] (3) Determination of TPC content: 20 μL of the sample solution and different concentrations of gallic acid standard solution were added to a 96-well plate in sequence, and then 20 μL of 50% Folin-Ciocalteu reagent was added to each well. After 30 minutes, 120 μL of distilled water and 40 μL of 7.5% Na2CO3 solution were added in sequence, and the reaction was carried out at room temperature in the dark for 2 hours. 80% methanol solution was used as a blank control, and its absorbance was measured at a wavelength of 765 nm. The results are expressed as mg gallic acid (GAE) / g (DW).

[0125] 2. DPPH free radical scavenging ability of soybean fermented with Pleurotus ostreatus mycelium

[0126] (1) Extraction of DPPH free radicals from soybeans fermented with Pleurotus ostreatus mycelium: The method is the same as that in Example 3.

[0127] (2) Draw a standard curve: Use different concentrations of water-soluble vitamin E standards (0-100 μmol / L) as the horizontal axis and DPPH free radical scavenging rate as the vertical axis to make a standard curve.

[0128] (3) Determination of DPPH free radical scavenging ability: 100 μL of water-soluble vitamin E standard solution of different concentrations and sample solution were mixed with 100 μL DPPH-methanol (0.2 mM) and reacted in the dark for 30 min at room temperature. The absorbance Ax was measured at a wavelength of 517 nm. Methanol was used to replace the DPPH-methanol solution as the control system and the absorbance Ac was measured; 80% methanol was used to replace the sample solution as the blank system and the absorbance Ao was measured. The DPPH free radical scavenging rate was calculated according to the following formula, and the result was expressed as μmol water-soluble vitamin E (TE) / g (DW).

[0129] 3. ABTS free radical scavenging ability of soybean fermented with Pleurotus ostreatus mycelium

[0130] (1) Extraction of ABTS free radicals from soybeans fermented with Pleurotus ostreatus mycelium: The method is the same as that in Example 3.

[0131] (2) Draw a standard curve: Use different concentrations of water-soluble vitamin E standards (0-150 μmol / L) as the horizontal axis and the ABTS free radical scavenging rate as the vertical axis to make a standard curve.

[0132] (3) Determination of ABTS free radical scavenging ability: 7mmol / L ABTS solution and 4.9mmol / L potassium persulfate solution were mixed in a volume ratio of 2:1, placed in a dark place for 16 hours, and diluted with distilled water until the absorbance at a wavelength of 734nm was (0.7±0.02), thus obtaining the ABTS working solution. 40μL of water-soluble vitamin E standard solution and sample solution of different concentrations were mixed with 200μL ABTS working solution, respectively, and after standing in the dark for 6 minutes, the absorbance Ax at a wavelength of 734nm was measured. Distilled water was used to replace the ABTS working solution as the control system and the absorbance Ac was measured; 80% methanol was used to replace the sample solution as the blank system and the absorbance Ao was measured. The ABTS free radical scavenging rate was calculated according to the following formula, and the result was expressed as μmol water-soluble vitamin E (TE) / g (DW).

[0133] The total phenol content (TPC) and antioxidant activity results determined by the present invention are shown in Table 6. The polyphenol content of the control group was 4.62 mg GAE / g. After solid fermentation, the TPC of soybeans fermented with Pleurotus ostreatus mycelium was 20.72 mg GAE / g, which was 4.47 times that of the control group. The antioxidant activity was positively correlated with the water-soluble vitamin E equivalent.

[0134] Regarding the scavenging ability of DPPH free radicals, the DPPH free radical scavenging ability of the control group was 5.04μmol TE / g, and the DPPH free radical scavenging ability of soybeans fermented with Pleurotus ostreatus mycelium was 28.36μmo TE / g, which was 5.62 times that of the control group. Regarding the scavenging ability of ABTS free radicals, the ABTS free radical scavenging ability of the control group was 171.2μmol TE / g, and the ABTS free radical scavenging ability of soybeans fermented with Pleurotus ostreatus mycelium was 500μmol TE / g, which was 2.92 times that of the control. Therefore, after solid fermentation of Pleurotus ostreatus mycelium, the antioxidant activity of soybeans fermented with Pleurotus ostreatus mycelium was significantly enhanced. This product is expected to become a source of natural antioxidants and be developed into a food base material beneficial to human health in the form of products or food ingredients for use in the field of food processing.

[0135] Table 6 Polyphenol content and antioxidant activity of soybeans fermented with Pleurotus ostreatus mycelium

[0136] Comparison Fermented soybeans with oyster mushroom mycelium TPC (mg GAE / g) <![CDATA[4.62±0.04 b ]]> <![CDATA[20.72±0.33 a ]]> DPPH (μmol TE / g) <![CDATA[5.04±0.12 b ]]> <![CDATA[28.36±1.63 a <!-- 10 -->]]> ABTS (μmol TE / g) <![CDATA[171.2±15.50 b ]]> <![CDATA[500.00±2.77 a ]]>

[0137] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for preparing soybean fermented with Pleurotus ostreatus mycelium, characterized in that: The method comprises fermenting soybeans with oyster mushroom mycelium to obtain oyster mushroom mycelium fermented soybeans, wherein the oyster mushroom is oyster mushroom (Pleurotus ostreatus) and its registration number in the General Microorganism Center of China Microorganism Culture Collection Committee is CGMCC No.5.

784.

2. The method according to claim 1, characterized in that The soybeans are whole soybeans.

3. The method according to claim 1 or 2, characterized in that: The method comprises the following steps: 1) Preparing Pleurotus ostreatus mycelium: activating the Pleurotus ostreatus strain described in claim 1 and inoculating it into a PDA solid culture medium to obtain Pleurotus ostreatus mycelium; 2) Preparing soybean culture medium: soaking soybeans in water, filtering water, and sterilizing to obtain soybean culture medium; 3) Solid-state fermentation: inoculating the mycelium of Pleurotus ostreatus into soybean culture medium and fermenting to obtain the mycelium fermented soybeans.

4. The method according to claim 3, characterized in that In step 2), the soybeans are soaked in water at a solid-liquid ratio of 1:1.5-3.5, and the soaking time is 6-10 hours.

5. The method according to claim 4, characterized in that The fermentation conditions in step 3) are culturing at 22-28° C., 50-80% humidity and in the dark for 15-40 days.

6. The method according to any one of claims 1 to 5, characterized in that: The ratio of the above-mentioned oyster mushroom and the above-mentioned soybean culture medium meets the following requirements: 1-5 cm 2 The mycelium of Pleurotus ostreatus: 100-500 g of the soybean culture medium.

7. A soybean product fermented with Pleurotus ostreatus mycelium prepared by the method according to any one of claims 1 to 6.

8. The product according to claim 7, characterized in that The product is a medicine, food, health product, animal feed or animal feed additive.

9. Use of the method according to any one of claims 1 to 6 in: 1) preparing the soybean product according to claim 7; 2) Increase the protein content in soybeans; 3) Increase methionine content; 4) reducing the starch and / or crude fiber content in soybeans; 5) Reduce the content of allergenic proteins and / or anti-nutritional factors in soybeans; 6) converting soybean glycoside isoflavones into aglycone isoflavones; 7) Increase the total phenolic content and antioxidant activity of soybeans or scavenge free radicals.

10. The use according to claim 9, characterized in that The allergenic proteins in soybean are soybean globulin and beta-conglycinin, the anti-nutritional factors in soybean are trypsin inhibitor and soybean lectin; the free radical scavenging method includes scavenging DPPH free radical or scavenging ABTS free radical.

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