Application of Agaricus sphaerocephala in producing gamma-aminobutyric acid and cultivation method thereof

Abstract

The invention discloses an application of Agaricus punctatus in producing gamma-aminobutyric acid and a cultivation method thereof. The Agaricus punctatus and Agaricus cyprinus strains are named Finc-W-247 and Finc-B-6, respectively. The Agaricus punctatus and Agaricus cyprinus strains obtained by the invention have high production capacity of gamma-aminobutyric acid and are good sources for manufacturing various functional foods rich in gamma-aminobutyric acid.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungus cultivation, and specifically relates to an application of Agaricus punctatus in producing gamma-aminobutyric acid and a cultivation method thereof. Background Art

[0002] The scientific names of both white jade mushroom and oyster mushroom are Hypsizigus marmoreus, which are different strains of the commercially cultivated edible fungus. Hypsizigus marmoreus, also known as jade mushroom, is one of the new edible fungi with rapid development in the Chinese market. It is also a rare edible fungus variety with both food and medicinal value. Due to its unique aroma and taste, it has been favored by consumers in recent years. The main materials for Hypsizigus marmoreus cultivation include sawdust, corn cobs, bran, rice bran and cottonseed hulls.

[0003] Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system, which has good water solubility and thermal stability. It has been confirmed that GABA, as a small molecular weight non-protein amino acid, is safe for consumption and can be used in the production of beverages and other foods. Studies have shown that consuming a certain amount of GABA has physiological effects such as improving sleep quality, lowering blood pressure, and beautifying the skin. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0005] As one aspect of the present invention, the present invention overcomes the deficiencies in the prior art and provides an application of Agaricus spp. in producing gamma-aminobutyric acid, wherein the Agaricus spp. is Pleurotus eryngii strain Finc-W-247 or Pleurotus ostreatus strain Finc-B-6.

[0006] The Pleurotus eryngii strain Finc-W-247 was deposited in the China Center for Type Culture Collection on September 24, 2012, located at Wuhan University, No. 16, Luojiashan Road, Wuchang District, Wuhan City, Hubei Province, China, with the deposit number being CCTCC NO: 2012378.

[0007] The Oyster mushroom strain Finc-B-6 was deposited on November 28, 2016 at the General Microbiology Center of China Microorganism Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC No.13194.

[0008] As another aspect of the present invention, the present invention provides a method for culturing the Agaricus spp.: the Agaricus spp. is used to produce gamma-aminobutyric acid, and the culturing method comprises the following steps:

[0009] (1) inoculating the Agaricus spp. strain onto a potato dextrose agar solid plate medium and culturing until the mycelium fills the plate;

[0010] (2) Mix the culture material evenly, put it into the culture bottle, and sterilize it at high temperature;

[0011] (3) The Agaricus spp. strain obtained in step (1) is inoculated into the cultivation bottle, and the bottle is transferred to a mushroom house for mycelial culture, and the mycelium is scratched. After continued culture, Agaricus spp. fruiting bodies are obtained.

[0012] As a preferred embodiment of the method for culturing Agaricus variegata of the present invention: in step (1), Agaricus variegata strains are inoculated onto a potato dextrose agar solid plate culture medium and cultured at 23° C. for 14 days until the mycelium covers the plate.

[0013] As a preferred embodiment of the method for cultivating Agaricus variegata of the present invention: in step (2), the cultivation material has a formula of: 40-50% (weight percentage, the same below) of corn cobs, 20-30% of sawdust, 10-15% of rice bran, 15-20% of bran, and 5% of corn flour, wherein the water content is 64-66%.

[0014] As a preferred embodiment of the method for cultivating Agaricus punctatus of the present invention: in step (2), 4 g / L of glutamic acid aqueous solution is further added to the cultivation material.

[0015] As a preferred embodiment of the method for cultivating Agaricus punctatus of the present invention: in step (2), 8 g / L of sodium glutamate aqueous solution is further added to the cultivation material.

[0016] As a preferred embodiment of the method for culturing Agaricus oleifera of the present invention: in step (2), the high temperature sterilization is carried out at 121° C. for 4 hours.

[0017] As a preferred embodiment of the method for cultivating Agaricus punctatus of the present invention: in step (3), the transferring to the mushroom house for mycelium cultivation is to cultivate mycelium in the mushroom house at 23°C.

[0018] Beneficial effects of the present invention: The present invention provides a method for increasing the content of γ-aminobutyric acid in the fruiting bodies of white jade mushroom and shimeji, and the present invention screens out white jade mushroom and shimeji strains with high γ-aminobutyric acid production, including adding glutamic acid and sodium glutamate to the cultivation materials of white jade mushroom and shimeji. The present invention has low addition cost, is convenient and easy to operate, can be well combined with the existing factory-based white jade mushroom and shimeji cultivation system, and realizes the production of white jade mushroom and shimeji rich in GABA. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0020] Figure 1 This is a photo of the fruiting body.

[0021] Figure 2 is the GABA content at different growth stages.

[0022] Figure 3 These are the morphological diagrams of fruiting bodies at different growth stages. DETAILED DESCRIPTION

[0023] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0024] Embodiment 1:

[0025] The main reagents and culture medium are prepared as follows:

[0026] The enoki mushroom cultivation material includes: 40-50% (weight percentage, the same below) of corn cobs, 20-30% of sawdust, 10-15% of rice bran, 15-20% of bran, 5% of corn flour, wherein the water content is 64-66%.

[0027] In order to study the differences in GABA content in the fruiting bodies of different varieties of Pleurotus eryngii and Pleurotus ostreatus, 10 strains were selected from the germplasm resource bank for cultivation experiments, which were numbered Finc-W-247, H3, B3, Finc-B-6, X3, Z42, Z111, Z124, Z156 and Z162, see Figure 1 10 strains of bacteria successfully produced mushrooms. Figure 1 a is the growth form of Pleurotus eryngii fruiting body, Figure 1 b is the growth morphology of the fruiting body of Oyster mushroom.

[0028] The methods for cultivating Pleurotus eryngii and Pleurotus ostreatus and detecting the content of γ-aminobutyric acid are as follows:

[0029] (1) Inoculate the fungus of Pleurotus eryngii or Pleurotus ostreatus onto potato dextrose agar (PDA) and culture at 23°C for 14 days until the mycelium fills a 90 mm culture dish.

[0030] (2) According to the formula of the cultivation material of Pleurotus eryngii and Pleurotus ostreatus, mix the cultivation material evenly, bottle it at a volume of 560±10g / bottle, and sterilize it at high temperature at 121°C for 4h.

[0031] (3) The white jade mushroom and oyster mushroom strains prepared in (1) were inoculated into cultivation bottles respectively, and transferred to a mushroom house at 23°C for mycelium culture. The mycelium was cultured for 30 days until the bottles were full. The culture was continued for 40 days to allow the mycelium to fully mature. The mycelium was scratched and cultured for another 20 days to obtain fresh white jade mushroom and oyster mushroom fruiting bodies.

[0032] (4) Take 100 g of each of the fruiting bodies of Pleurotus eryngii and Pleurotus ostreatus, dry them with hot air at 55°C to constant weight, grind them with a high-speed grinder, and pass them through a 60-mesh sieve. The sample powder is sealed and stored in a cool and dry place.

[0033] Accurately weigh 70-80 mg of the sample powder to be tested, add 6 mol / L hydrochloric acid solution for acid hydrolysis, and determine the γ-aminobutyric acid content using Hitachi 835-50 amino acid automatic analyzer.

[0034] The GABA dry weight content (DW) of the fruiting bodies of three strains of Pleurotus eryngii and seven strains of Pleurotus ostreatus was determined using an amino acid analyzer, and it was found that the GABA content of different varieties had significant differences, as shown in Table 1.

[0035] Table 1 GABA content in the fruiting bodies of 10 strains of Pleurotus eryngii and Pleurotus ostreatus

[0036]

[0037] Temporal and spatial changes of GABA content during fruiting body development:

[0038] The GABA content of the stipes and caps of Pleurotus eryngii and Pleurotus ostreatus at different growth stages was determined by amino acid analyzer. Figure 2 The results showed that the GABA content in the caps of the fruiting bodies of Pleurotus eryngii and Pleurotus ostreatus showed a trend of first decreasing and then increasing. The GABA content accumulated to the maximum in the third stage, but the GABA content in the stipe showed a trend of gradual decrease. During the entire fruiting body development process, the GABA content in the cap was higher than that in the stipe in both high-yield and low-yield GABA strains. Among them, the GABA content in the cap of the mature fruiting body of Finc-W-247 was as high as 1272 mg / kg DW, which was about 16 times that of the stipe of Finc-W-247 at the same period; the GABA content in the cap of the mature fruiting body of Finc-B-6 was as high as 1284 mg / kg DW, which was about 7.5 times that of the stipe of Finc-B-6 at the same period.

[0039] Figure 3 The different growth stages of Finc-W-247 and Finc-B-6 proton entities. Figure 3 (a) shows the three stages of growth of Finc-W-247 strain, and (b) shows the three stages of growth of Finc-B-6 strain.

[0040] Pleurotus eryngii: Finc-W-247 (preservation number is CCTCC NO: 2012378), Oyster mushroom: Finc-B-6 (preservation number is CGMCC No.13194). Both are public strains.

[0041] Embodiment 2:

[0042] The main reagents and culture medium are configured as follows:

[0043] The enoki mushroom cultivation material includes: 40-50% (weight percentage, the same below) of corn cobs, 20-30% of sawdust, 10-15% of rice bran, 15-20% of bran, 5% of corn flour, wherein the water content is 64-66%.

[0044] 4g / L glutamic acid solution: Weigh 4g of glutamic acid, dissolve it in 1L of distilled water, mix well and store in a sealed container.

[0045] 8g / L sodium glutamate solution: Weigh 8g of sodium glutamate, dissolve it in 1L of distilled water, mix well and store in a sealed container.

[0046] In order to study the difference in GABA content in the fruiting bodies of different strains of Pleurotus eryngii and Pleurotus ostreatus after adding glutamate or sodium glutamate, Pleurotus eryngii Finc-W-247 and Pleurotus ostreatus Finc-B-6 with the highest GABA content were screened out from 10 strains for cultivation experiments.

[0047] The cultivation and detection methods of Pleurotus eryngii and Pleurotus ostreatus with added glutamic acid or sodium glutamate are as follows:

[0048] (1) The strains of Pleurotus eryngii and Pleurotus ostreatus were inoculated onto potato dextrose agar (PDA) and cultured at 23°C for 14 days until the mycelium filled the 90 mm plate.

[0049] (2) According to the formula of cultivation materials for Pleurotus eryngii and Oyster mushroom, 4 g / L glutamic acid solution or 8 g / L sodium glutamate solution was mixed with the cultivation materials, bottled at 560 ± 10 g, and sterilized at 121 °C for 4 h.

[0050] (3) The white jade mushroom and oyster mushroom strains prepared in (1) were inoculated into cultivation bottles respectively, and transferred to a mushroom house at 23°C for mycelium culture. The mycelium filled the bottles after 30 days. The mycelium was ripened for 40 days to fully ripen. The mycelium was scratched and cultured for another 20 days to obtain fresh white jade mushroom and oyster mushroom fruiting bodies.

[0051] (4) Take 100 g of each of the fruiting bodies of Pleurotus eryngii and Pleurotus ostreatus, and dry them with hot air at 55°C to constant weight. After being crushed by a high-speed grinder, pass through a 60-mesh sieve, and store the sample powder in a sealed container in a cool and dry place. Accurately weigh 70-80 mg of the sample powder to be tested, add 6 mol / L hydrochloric acid solution for acid hydrolysis, and determine the γ-aminobutyric acid content using a Hitachi 835-50 amino acid automatic analyzer.

[0052] Results Statistics:

[0053] The GABA content of the white jade mushroom measured in step (4) was 321.50 mg / kg, the GABA content of the white jade mushroom added with 4g / L glutamate solution was 245.3 mg / kg (DW), and the GABA content of the white jade mushroom added with 8g / L sodium glutamate solution was 415 mg / kg (DW); the GABA content of the oyster mushroom was 256.90 mg / kg, the GABA content of the oyster mushroom added with 4g / L glutamate solution was 242.6 mg / kg (DW), and the GABA content of the oyster mushroom added with 8g / L sodium glutamate solution was 329.7 mg / kg (DW). The above experimental results show that the addition of sodium glutamate is beneficial to increase the content of GABA in white jade mushroom and oyster mushroom.

[0054] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. An application of Agaricus sphaerocephala in producing gamma-aminobutyric acid, characterized in that: The agaricus is Pleurotus eryngii strain Finc-W-247 or Pleurotus ostreatus strain Finc-B-6; The cultivation method of the described species of Agaricus sphaerocephala consists of the following steps: (1) Inoculate the Agaricus spp. strain onto a potato dextrose agar solid plate medium and culture until the mycelium fills the plate; (2) Mix the cultivation material evenly, put it into the cultivation bottle, and sterilize it at high temperature; the formula of the cultivation material is: 40-50% corn cobs, 20-30% miscellaneous wood chips, 10-15% rice bran, 15-20% bran, 5% corn flour, and add 8 g / L sodium glutamate aqueous solution; the water content of the cultivation material is 64-66%; (3) inoculating the Agaricus spp. strain obtained in step (1) into the cultivation bottle, transferring it to a mushroom house for mycelial culture, scratching the fungus, and continuing to culture to obtain Agaricus spp. fruiting bodies; The Pleurotus eryngii strain Finc-W-247 was deposited in the China Center for Type Culture Collection (CCTCC) on September 24, 2012, with a deposit number of CCTCC NO: 2012378; the Oyster mushroom strain Finc-B-6 was deposited in the China General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on November 28, 2016, with a deposit number of CGMCC No.13194.

2. The use according to claim 1, characterized in that: In step (1), the Agaricus spp. strain is inoculated onto a potato dextrose agar solid plate medium and cultured at 21-23° C. for 14-15 days until the mycelium fills the plate.

3. The use according to claim 1 or 2, characterized in that: In step (2), the high temperature sterilization is carried out at 120-121°C for 4-5 hours.

4. The use according to claim 1 or 2, characterized in that: In step (3), the step of transferring the spawn to the mushroom house for mycelium cultivation is to carry out mycelium cultivation in the mushroom house at 20-23°C.

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