TWI510615B - Solid medium for culturing ganoderma strains and application thereof - Google Patents

Description

Ganoderma lucidum solid medium and its application

The invention relates to a solid medium of Ganoderma lucidum, in particular to a solid medium of Ganoderma lucidum added with wheat germ.

With the changes of society, the pace of life is becoming more and more compact, and the pressure is accompanied. The proportion of people suffering from depression and insomnia is gradually increasing, which affects the quality of life and health of human beings.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in mammals. It has many physiological activities such as anti-depression, improving insomnia, and improving autonomic nervous system dysfunction. Many microorganisms are known to contain γ-aminobutyric acid, and therefore, a sufficient amount of γ-aminobutyric acid can be ingested by eating or drinking a fermented food to achieve anti-depression and an effect of improving symptoms of insomnia.

Although gamma-aminobutyric acid can be chemically synthesized today, gamma-aminobutyric acid produced by chemical methods has safety concerns. On the other hand, although natural γ-aminobutyric acid is produced by microbial fermentation, the current microbial fermentation method still cannot produce high concentration of γ-aminobutyric acid, which makes anti-depression and anti-insomnia of fermented foods and drinks. The effect is limited, and it is urgent to develop a microbial culture medium capable of producing high γ-aminobutyric acid content and a fermentation method thereof.

In view of the defects of the prior art, the present invention provides a solid medium of Ganoderma lucidum which can be fermented by Ganoderma lucidum to produce a large amount of γ-aminobutyric acid.

The invention further provides a use of a solid medium of Ganoderma lucidum for fermentation to produce a large amount of γ-aminobutyric acid.

The invention further provides a use of a solid medium of Ganoderma lucidum for producing a food rich in γ-aminobutyric acid.

The present invention further provides a method for producing a large amount of high-concentration γ-aminobutyric acid, which comprises at least 20 mg of γ -aminobutyric acid per gram of Ganoderma lucidum solid medium.

In a preferred embodiment, the present invention provides a solid medium of Ganoderma lucidum, which is a solid medium of Ganoderma lucidum prepared by a plurality of preparation steps, wherein the plurality of preparation steps comprises: (a) mixing a plurality of medium components to obtain a mixture of medium components. The medium component mixture includes: wheat germ, 10% to 60% by weight; and water, 20% to 50% by weight; and (b) high temperature and sterilization treatment to obtain the ganoderma lucidum A solid medium, wherein the high temperature means 100 ° C to 150 ° C.

In a preferred embodiment, the concentration of the wheat germ in the medium component mixture is 30% to 40% by weight.

In a preferred embodiment, the medium component mixture further comprises germ rice, and the concentration of the germ rice is 10% to 40% by weight.

In a preferred embodiment, the concentration of the wheat germ is 30% to 40%, the weight percentage of the germ rice is 10% to 20%, and the weight concentration of the water is 50%.

In a preferred embodiment, the medium component mixture further comprises casein, soy protein, or soybean.

In a further preferred embodiment, the present invention provides a solid medium of Ganoderma lucidum, which is a solid medium prepared from a plurality of preparation steps, wherein the plurality of preparation steps comprises: (a) mixing a plurality of medium components to obtain a medium component. a mixture, the medium component mixture includes: wheat germ; and water; and (b) the high temperature and sterilization treatment to obtain the ganoderma lucidum solid medium, wherein the high temperature means 100 ° C ~ 150 ° C; in step (a), The composition of the wheat germ includes gliadin, and the concentration of the gliadin in the mixture of the components of the medium is 0.4% to 2.6%.

In another preferred embodiment, the present invention provides a solid medium of Ganoderma lucidum, which is a solid medium prepared from a plurality of preparation steps, wherein the plurality of preparation steps comprises: (a) mixing a plurality of medium components to obtain a medium component. a mixture, the medium component mixture includes: wheat germ; and water; and (b) high temperature and sterilization treatment to obtain the ganoderma lucidum solid medium, wherein the high temperature Refers to 100 ° C ~ 150 ° C; in step (a), the components of wheat germ include wheat glutinous protein, and the concentration of wheat gluten in the mixture of the components of the medium is 0.007% to 0.07% by weight.

In a preferred embodiment, in step (b), it is applied at 121 ° C for at least 15 minutes to simultaneously achieve high temperature cooking and sterilization.

In a preferred embodiment, it is used to culture Ganoderma lucidum to produce a high concentration of γ -aminobutyric acid; wherein, the high concentration of γ -aminobutyric acid means at least 20 mg of γ -amine per gram of Ganoderma lucidum solid medium. Butyric acid.

In a preferred embodiment, it is used for cultivating Ganoderma lucidum to produce a food containing a high concentration of γ -aminobutyric acid; wherein a high concentration of γ -aminobutyric acid means at least 20 mg γ per gram of Ganoderma lucidum solid medium. - Aminobutyric acid.

In still another preferred embodiment, the present invention provides a method of producing γ -aminobutyric acid, the steps comprising:

(a) mixing a plurality of medium components to obtain a medium component mixture comprising: wheat germ, 10% to 60% by weight; and water, 20% to 50% by weight ;as well as

(b) A high temperature medium and a sterilization process are used to prepare a solid medium of Ganoderma lucidum, wherein the high temperature means 100 ° C to 150 ° C.

(c) Adding a Ganoderma lucidum strain to produce γ -aminobutyric acid by fermentation.

In still another preferred embodiment, after the fermentation in step (c), the ganoderma lucidum solid medium comprises at least 20 mg of γ -aminobutyric acid per gram.

Fig. 1A: The amount of hyphae obtained by culturing A strain of Ganoderma lucidum for 7 days under different conditions of solid rice medium at 30 °C.

Fig. 1B: The amount of hyphae obtained by culturing C ganoderma lucidum strains for 7 days under different rice solid medium cultures at 30 °C.

Fig. 2A: The amount of mycelium obtained by culturing A strain of Ganoderma lucidum for 7 days at 30 ° C after adding different concentrations of nitrogen source to the bottom of the solid medium of the germ rice.

Fig. 2B: The amount of hyphae obtained by culturing C ganoderma lucidum strain at 7 ° C for 7 days after adding different concentrations of nitrogen source to the bottom of the solid medium of the germ rice.

Fig. 3A: The amount of mycelium obtained by culturing Ganoderma lucidum strain at 7 ° C for 7 days after adding 1% glutamate at the bottom of the solid medium of the germ rice.

Fig. 3B: γ-aminobutyric acid yield obtained by culturing Ganoderma lucidum strain for 7 days at 30 ° C after adding 1% glutamate at the bottom of the solid medium of the germ rice.

Figure 4: γ-aminobutyric acid content of the manufactured Ganoderma lucidum solid medium after mixing different proportions of germ rice and wheat germ.

Fig. 5A: The amount of hyphae obtained by culturing A ganoderma lucidum strains in different proportions of germ rice and wheat germ at 7 ° C for 7 days.

Fig. 5B: The amount of γ-aminobutyric acid obtained by culturing A ganoderma lucidum strains at different ratios of germ rice and wheat germ for 7 days at 30 °C.

Fig. 6A: The amount of hyphae obtained by culturing C. ganoderma strains in different proportions of germ rice and wheat germ at 7 °C for 7 days.

Figure 6B: Different ratios of germ rice and wheat germ culture at 30 °C The amount of γ-aminobutyric acid obtained by raising C strain of Ganoderma lucidum for 7 days.

Figure 7: 2nd Ganoderma lucidum solid medium (containing 20% wheat) at 32 °C Germ, 50% water, and 30% germ rice) The amount of mycelium obtained by culturing A strain of Ganoderma lucidum for 21 days and the amount of γ-aminobutyric acid.

Figure 8A: In the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% After adding 0.1% of different nitrogen sources to water and 30% of germ rice, the amount of mycelium obtained by culturing A strain of Ganoderma lucidum for 7 days at 30 ° C was obtained.

Figure 8B: In the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% The amount of γ-aminobutyric acid obtained by culturing A ganoderma lucidum strain for 7 days at 30 ° C after adding 0.1% of different nitrogen sources to water and 30% of germ rice.

Figure 9A: In the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% After adding 0.1% of different nitrogen sources to water and 30% of germ rice, the amount of mycelium obtained by culturing C ganoderma lucidum strain at 7 ° C for 7 days was obtained.

Figure 9B: In the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% The amount of γ-aminobutyric acid obtained by culturing C ganoderma lucidum strain for 7 days at 30 ° C after adding 0.1% of different nitrogen sources to water and 30% of germ rice.

In view of the shortcomings of the prior art, the inventors believe that based on the research results of many years, it is believed that a solid medium of Ganoderma lucidum can be developed, and a high concentration of γ -aminobutyric acid is produced by fermentation of Ganoderma lucidum.

The following is a detailed description of embodiments of the invention, as well as the techniques and features of the invention. However, the present embodiment is not intended to limit the present invention, and any person familiar with the technology does not take off. Various changes and modifications made within the spirit and scope of the present invention are intended to be included in the scope of the present invention.

Ganoderma spp. is a genus of Ganoderma lucidum, an annual white rot fungus mainly distributed in the tropics, subtropics and temperate zones. At present, there are about 150 to 200 kinds of natural ganoderma lucidum in the world, and there are 17 species in Taiwan, including G.australe , G.tropicum , G.weberianum , G.boninense , G.lucidum (Ganoderma lucidum), G.japonicum ( Zizhizhi ). With G.formosana . Ganoderma lucidum sold in the market is dominated by G. lucidum (Ganoderma lucidum).

The components in the fruit body of the ganoderma lucidum or the mycelium have physiological activities such as blood sugar lowering, blood pressure lowering, anti-oxidation, anti-tumor, anti-coagulation, immune regulation, liver protection, etc. The physiological active substances in the mycelium include: polysaccharide (polysaccharide), Triterpenoids, superoxide dismutase (SOD), protein, germanium, adenosine, and steroid.

The two strains of Ganoderma lucidum strains A and C used in the present invention are all classified as Ganoderma lucidum , and are purchased from the Bioresource Conservation and Research Center of Hsinchu Food Industry Development Research Institute. According to the relevant provisions of the Patent Law, no deposit is required (A strain product number BCRC36111; C strain product number BCRC36674). Other Ganoderma lucidum strains, such as other Ganoderma lucidum strains in the Center for Bioresource Conservation and Research of the Food Industry Development Institute, may also be used, but are not limited thereto.

Oryza has 24 species, including 2 cultivars and 22 wild species, distributed in tropical and subtropical regions of the world. The cultivars include rice ( Oryza sativa ) and West African cultivated rice ( Oryza glaberrima ). Seeds of rice and West African cultivated rice, commonly known as rice. Among them, rice includes different subspecies such as indica ( Oryza sativa indica ) and indica ( Oryza sativa japonica ).

The rice is processed to remove the chaff and then the brown rice is obtained. The brown rice of the present invention is purchased from Aibu (product name: organic organic brown rice; brand: Chihshang, Taiwan), but not limited thereto. The brown rice is ground to remove the rice bran layer and retain the germ, which is the embryo rice. The germ rice of the present invention is purchased from Aibu (product name: germ rice; label: Zhongxing Rice, Taiwan), but not limited thereto. .

Japanese japonica rice is improved in Taiwan and has a new line of japonica rice. The white rice obtained by processing the rice, the rice bran layer and the germ after processing is called Pon Lai rice (also known as Japonica rice). The Penglai Mi Aibu of the invention (product name: three good rice; label: three good rice, Taiwan), but not limited to this.

The white rice obtained by processing the rice in the indica rice and removing the chaff, rice bran layer and germ is called indica rice. The glutinous rice of the invention is purchased from the Dongli rice milling factory (product name: Yuhuangchang glutinous rice; brand: Yuhuangmi, Taiwan), but not limited thereto.

The casein used in the present invention was purchased from Sigma-Aldrich (Sigma-Aldrich, Saint Louis, MO, USA), and the soy protein was purchased from Sino-US Union Industrial Co., Ltd. (Taiwan), soybean. (Soybean) was purchased from Aibu (Taiwan), and monosodium glutamate (MSG) was purchased from Sigma-Aldrich (Sigma-Aldrich, Saint Louis, MO, USA), but not limited thereto.

Experiment 1: Effect of the solid medium of various rice on the growth of Ganoderma lucidum

(1) Preparation of rice solid medium bottom

Mix 40g of porphyria, 40g of glutinous rice, 40g of brown rice, or 40g of germ rice with 40mL of primary water, respectively, so that the weight percentage of the plenum, glutinous rice, brown rice, or germ rice is 50%, placed in a pickles bottle (referring to a glass bottle containing a metal lid), after high temperature and sterilization treatment, respectively, to obtain a solid medium bottom of Penglai rice, a solid medium bottom of glutinous rice, a solid medium bottom of brown rice, or a solid of germ rice The substrate is cultured.

The high temperature refers to 100 ° C to 150 ° C. Preferably, the high temperature means 121 ° C. Preferably, it is allowed to act at 121 ° C for at least 15 minutes to simultaneously achieve high temperature cooking and sterilization. In the preferred embodiment of the present invention, it is applied at 121 ° C for 20 minutes to simultaneously achieve high temperature cooking and sterilization effects.

(2) Inoculation of Ganoderma lucidum strains and observation of mycelial growth

In the bottom of the solid medium of Penglai rice, the bottom of the solid medium of glutinous rice, brown rice The rice solid medium bottom or the germ rice solid medium bottom is inoculated with A ganoderma lucidum strain or C ganoderma lucidum strain, and cultured at 30 ° C for 7 days in the dark, to obtain a Penglai rice and hyphae mixture, a glutinous rice and hyphae mixture, Brown rice and hyphae mixture, or a mixture of germ rice and hyphae (hereinafter collectively referred to as rice and hyphae mixture). The amount of Ganoderma lucidum mycelium in various rice and hyphae mixtures was measured.

The inventor detects the content of ergosterol in the mixture of rice and hyphae to indirectly obtain the amount of ganoderma lucidum, and the steps include:

A. Making Ganoderma lucidum mycelium standard calibration curve

A strain of Ganoderma lucidum or C. ganoderma lucidum strain was inoculated in a liquid medium, cultured at 30 ° C, 110 rpm for 7 days, and the mycelium was collected by centrifugation, washed, and lyophilized. The liquid medium may be, for example, potato dextrose medium (product name, Potato Dextrose Broth, PDB; brand BD), but is not limited thereto. Next, the freeze-dried mycelium powder was formulated into 10, 25, 50, and 100 mg/L mycelium powders with absolute alcohol, and extracted at room temperature for 1 hour. The supernatant was collected by centrifugation (8000 x g, 10 min). Quantify to 5mL with absolute alcohol, filter through 0.22μm filter, and then The ergosterol content was analyzed by high performance liquid chromatography (HPLC). Finally, using the relationship between the mycelial concentration of mycelial powder and the ergosterol content, a standard calibration curve for Ganoderma lucidum mycelium was prepared.

B. Measuring the amount of Ganoderma lucidum mycelium in the mixture of rice and hyphae

After the rice and hyphae mixture was lyophilized, 1 g of the lyophilized sample was added to 2 mL of absolute alcohol, and extracted at room temperature for 1 hour. The supernatant was collected by centrifugation (8000 x g, 10 min). The residue was diluted to 5 mL with absolute alcohol, filtered through a 0.22 μm filter, and the ergosterol content was analyzed by high performance liquid chromatography. The amount of ergosterol is converted into the amount of mycelium by using the Ganoderma lucidum mycelium standard calibration curve.

See Figure 1A and Table 1. Fig. 1A shows the amount of hyphae obtained by culturing A strain of Ganoderma lucidum for 7 days under different conditions of solid rice medium at 30 °C. The horizontal coordinates from left to right are the solid medium bottom of the germ rice, the solid medium bottom of the brown rice, the bottom of the solid medium of the Penglai rice, and the bottom of the solid medium of the glutinous rice. The ordinate is the amount of hyphae. If the letters between the two groups are the same, it means that there is no significant difference between the two groups; otherwise, there is a significant difference. In Fig. 1A, the amount of hyphae obtained by culturing A strain of Ganoderma lucidum in the bottom of solid medium of gram of germ rice is 0.242±0.060 g, and the amount of hyphae obtained by culturing in the bottom of solid medium per gram of brown rice is 0.143±0.006 g, and culture is carried out. The amount of hyphae obtained in the bottom of the solid medium per gram of lentil rice was 0.099±0.001 g, and the amount of hyphae obtained in the bottom of the solid medium per gram of glutinous rice was 0.139±0.003 g. Among them, the A ganoderma lucidum strain is cultured on the bottom of the solid medium of the germ rice to obtain the highest amount of mycelium.

See Figure 1B and Table 1. Fig. 1B shows the amount of hyphae obtained by culturing C ganoderma lucidum strains for 7 days under different rice solid medium cultures at 30 °C. The horizontal coordinates from left to right are the solid medium bottom of the germ rice, the solid medium bottom of the brown rice, the bottom of the solid medium of the Penglai rice, and the bottom of the solid medium of the glutinous rice. The ordinate is the amount of hyphae. If the letters between the two groups are the same, it means that there is no significant difference between the two groups; otherwise, there is a significant difference. In Figure 1B, C strain of Ganoderma lucidum is cultured in each The amount of hyphae obtained in the bottom of the solid medium of the gram germ rice is 0.181±0.007 g, and the amount of the hyphae obtained in the bottom of the solid medium per gram of brown rice is 0.170±0.006 g, and is cultivated in the bottom of the solid medium per gram of porphyria rice. The amount of hyphae was 0.022±0.003 g, and the amount of hyphae cultured in the bottom of the solid medium per gram of glutinous rice was 0.064±0.004 g. Among them, the C ganoderma lucidum strain is cultured on the bottom of the solid medium of the germ rice to obtain the highest amount of mycelium.

The experimental results of Figs. 1A, 1B and Table 1 show that the highest mycelial amount can be obtained when the A or C Ganoderma lucidum strain is cultured at the bottom of the solid medium of the germ rice, so the subsequent experiment will be carried out based on the solid medium of the germ rice.

Each of the above experiments was triple-covered, and the data representation was mean ± standard deviation.

Experiment 2: Effect of different nitrogen sources on the growth of Ganoderma lucidum strains in the solid medium of germ rice

(1) Preparing a solid medium for germ rice containing different nitrogen sources

Mix 40g of germ rice with 40mL of primary water, add 0.1% or 1% by weight of different nitrogen sources, put in the pickles bottle, and after high temperature and sterilization, make the inclusion Germ rice solid medium with different nitrogen sources.

In the preferred embodiment of the present invention, it is applied at 121 ° C for 20 minutes to simultaneously achieve high temperature cooking and sterilization effects.

Among them, different nitrogen sources refer to casein, soy protein or soybean.

(2) Inoculation of Ganoderma lucidum strain A or C, the steps and methods are the same as in Experiment 1.

Please refer to FIG. 2A , which is a mycelial amount obtained by culturing A strain of Ganoderma lucidum for 7 days at 30 ° C after adding different nitrogen sources at the bottom of the solid medium of the germ rice. The horizontal coordinates from left to right are control group (no protein added), casein group (addition of 0.1% or 1% casein), soy protein group (addition of 0.1% or 1% soy protein) and soybean group ( Add 0.1% or 1% soy). The ordinate is the amount of hyphae. 2A shows that the growth condition of the A ganoderma lucidum strain was not significantly increased after the A-Ganoderma lucidum strain was cultured for 7 days in the solid medium containing the different protein.

Please refer to FIG. 2B , which is a mycelial amount obtained by culturing C ganoderma lucidum strain at 7 ° C for 7 days after adding different nitrogen sources at the bottom of the solid medium of the germ rice. Both the abscissa and the ordinate are the same as in Fig. 2A. 2B shows that after the G. lucidum strain cultured in the solid medium of the germ rice containing 0.1% different proteins for 7 days, the amount of mycelium contained per gram of rice and mycelium mixture was significantly higher than that of the control group, and The amount of hyphae obtained from the medium supplemented with 0.1% casein was the highest, 0.323 ± 0.004 g (1.76 times the control group). In addition, the addition of 1% did not significantly increase the amount of hyphae of the C. ganoderma strain.

Experiment 3: Effect of adding glutamic acid sodium on the growth of Ganoderma lucidum strain and the yield of γ -aminobutyric acid in the solid medium of germ rice

Glutamate is a good source of amino acid nitrogen, so whether the monosodium glutamate (MSG) can increase the production of mycelium and γ -aminobutyric acid.

(1) Preparing a solid medium for germ rice containing 1% sodium glutamate

Mix 40g of germ rice with 40mL of primary water, add 1% by weight of sodium glutamate, put it into the pickle bottle, and after high temperature and sterilization, obtain 1% sodium glutamate. Germ rice solid medium.

(2) Inoculation of Ganoderma lucidum strain A or C, the steps and methods are the same as in Experiment 1.

(III) Analysis of γ -aminobutyric acid content

The rice and hyphae mixture was mixed with 100 mM hydrochloric acid and shaken at room temperature for 45 minutes. The membrane was filtered through a membrane having a pore size of 0.45 mm, and the filtrate was further reacted with o-phthaladehyde to prepare a γ -aminobutyric acid derivative. The content of γ -aminobutyric acid derivatives was analyzed by high performance liquid chromatography with UV 345 nm wavelength.

Referring to FIG. 3A, the amount of mycelium obtained by culturing Ganoderma lucidum strain for 7 days at 30 ° C after adding 1% glutamate at the bottom of the solid medium of the germ rice is used. Figure 3A shows A and C Ganoderma lucidum strains from left to right; the ordinate is the amount of hyphae. If the letters between the two groups are the same, it means that there is no significant difference between the two groups; otherwise, there is a significant difference. Fig. 3A shows that the A ganoderma lucidum strain was cultured in a solid medium containing 1% glutamate or in a control group, and the amount of hyphae obtained by the two was not significantly different. The C. lucidum strain was cultured in a solid medium containing 1% glutamate or in a control group, and the amount of hyphae obtained by the two was also not significantly different.

Referring to FIG. 3B, the γ -aminobutyric acid yield of the Ganoderma lucidum strain was cultured at 30 ° C for 7 days after adding 1% glutamate at the bottom of the solid medium of the germ rice. Figure 3B shows A and C Ganoderma lucidum strains from left to right; the ordinate is γ -aminobutyric acid content. Fig. 3B shows that the A-Ganoderma lucidum strain was cultured in a solid medium containing 1% glutamate in a germ rice, and the yield of γ -aminobutyric acid was significantly higher than that of the control group (p<0.01). The G. ganoderma strain was cultured in a solid medium containing 1% glutamate in the germ rice, and the yield of γ -aminobutyric acid was also significantly higher than that of the control group (p<0.01).

Experiment 4: Analysis of the general composition of wheat germ

Wheat is the grain of the Triticum spp. plant. The wheat germ is the germ in the wheat grain, accounting for about 2.5% of the whole grain of wheat.

The protein content in wheat germ is extremely high, and the protein content is about 29% to 30%. Among the protein components of wheat germ, globulin accounts for about 18.9%, Gliadin accounts for about 14.0%, and wheat glutenin (Glutenin) accounts for about 0.3% to 0.37%.

The wheat germ system used in the present invention was purchased from Life Sea Biotechnology Co., Ltd. (Shandong, China).

The wheat germ used in the present invention is obtained by cutting a germ from a wheat grain, grinding it into a powder, forming a sheet, or refining and drying. However, it is not limited thereto.

In order to understand the general composition of wheat germ, it is analyzed by the commonly used A.O.A.C. (1980) method.

Please refer to Table 2 for the general composition of the wheat germ used in the present invention. In Table 2, the water content in the wheat germ powder was 1.65%, the crude protein was 29.26%, the crude fat was 7.50%, the ash was 5.10%, and the carbohydrate was 56.38%.

Each of the above experiments was performed in triplicate, and the data representation was mean ± standard deviation.

Experiment 5: Preparation of Ganoderma lucidum solid medium and its concentration of γ -aminobutyric acid

(1) Preparation of Ganoderma lucidum solid medium

Step 1: mixing a plurality of medium components to obtain a 0th medium component mixture, a first medium component mixture, a second medium component mixture, a third medium component mixture, a fourth medium component mixture, or a fifth medium component mixture; and a step 2: After the high temperature and sterilization treatment, the 0th Ganoderma lucidum solid medium, the 1st Ganoderma lucidum solid medium, the 2nd Ganoderma lucidum solid medium, the 3rd Ganoderma lucidum solid medium, the 4th Ganoderma lucidum solid medium, or the 5th Ganoderma lucidum solid medium were respectively prepared. The high temperature refers to 100 ° C to 150 ° C.

In step 1, the 0th medium component mixture includes 0% wheat germ, 50% water, and 50% germ rice. The first medium component mixture includes 10% wheat germ, 50% water, and 40% germ rice. 20% of the second medium ingredient mixture Wheat germ, 50% water, and 30% germ rice. The third medium component mixture includes 30% wheat germ, 50% water, and 20% germ rice. The fourth medium component mixture includes 40% wheat germ, 50% water, and 10% germ rice. The fifth medium component mixture includes 50% wheat germ, 50% water, and 50% germ rice. The concentration percentage of each component in the above 0 to 5 medium component mixture is a weight percent concentration.

In step 2, it is applied at 121 ° C for at least 15 minutes to simultaneously achieve high temperature cooking and sterilization.

Furthermore, the 0th medium of the Ganoderma lucidum solid medium in the step 2 is obtained by high temperature sterilization treatment; the first medium of the Lingzhi solid medium is prepared by high temperature and sterilization; the second solid medium of Ganoderma lucidum The second medium mixture is prepared by high temperature sterilization; the third medium of the Lingzhi solid medium is prepared by high temperature sterilization; the fourth medium of the Lingzhi solid medium is prepared by high temperature and sterilization. The fifth mixture of the 5th Ganoderma lucidum solid medium is prepared by high temperature and sterilization treatment.

(II) Analysis of γ -aminobutyric acid content

The Ganoderma lucidum solid medium was mixed with 100 mM hydrochloric acid and shaken at room temperature for 45 minutes. The membrane was filtered through a membrane having a pore size of 0.45 mm, and the filtrate was further reacted with o-phthaladehyde to prepare a γ -aminobutyric acid derivative. The content of γ -aminobutyric acid derivatives was analyzed by high performance liquid chromatography with UV 345 nm wavelength.

Please refer to FIG. 4, which is a γ -aminobutyric acid content of the solid medium of Ganoderma lucidum prepared by mixing different proportions of germ rice and wheat germ. The horizontal coordinates are from left to right: 0th Ganoderma lucidum solid medium, 1st Ganoderma lucidum solid medium, 2nd Ganoderma lucidum solid medium, 3rd Ganoderma lucidum solid medium, 4th Ganoderma lucidum solid medium, and 5th Ganoderma lucidum solid medium. The ordinate is the concentration of γ -aminobutyric acid contained in the Ganoderma lucidum solid medium. Figure 4 shows that the higher the proportion of wheat germ, the higher the content of γ -aminobutyric acid in the solid medium of Ganoderma lucidum. Among them, in the 0th Ganoderma lucidum solid medium containing no wheat germ, the concentration of γ -aminobutyric acid per gram of the medium is 7.567±0.376 mg; in the 5th Ganoderma lucidum solid medium containing 50% wheat germ, γ per gram of the medium The aminobutyric acid concentration was 11.379 ± 0.550 mg.

Experiment 6: Effect of various solid mediums of Ganoderma lucidum on the amount of mycelial strain and the yield of γ -aminobutyric acid

A strain of Ganoderma lucidum was inoculated into the above-mentioned 0~5 Ganoderma lucidum solid medium, and cultured at 30 ° C for 7 days, and the amount of mycelium and the content of γ-aminobutyric acid were measured.

The rice and hyphae mixture obtained after the cultivation was mixed with 100 mM hydrochloric acid, and shaken at room temperature for 45 minutes. The membrane was filtered through a membrane having a pore size of 0.45 mm, and the filtrate was further reacted with o-phthaladehyde to prepare a γ -aminobutyric acid derivative. The content of γ -aminobutyric acid derivatives was analyzed by high performance liquid chromatography with UV 345 nm wavelength.

Please refer to Figure 5A and Table 3. Fig. 5A shows the amount of hyphae obtained by culturing A ganoderma lucidum strains in different proportions of germ rice and wheat germ at 7 °C for 7 days. From the left to the right, the horizontal coordinate is 0th Ganoderma lucidum solid medium, the first Ganoderma lucidum solid medium, the 2nd Ganoderma lucidum solid medium, the 3rd Ganoderma lucidum solid medium, the 4th Ganoderma lucidum solid medium, and the 5th Ganoderma lucidum solid medium. The ordinate is the amount of hyphae contained per gram of rice and hyphae mixture after 7 days of culture. Fig. 5A and Table 3 show that after the A-Ganoderma lucidum strain is cultured in the 0~5 Ganoderma lucidum solid medium, the amount of hyphae per gram of rice and hyphae mixture is 0.292±0.060 g, 0.579±0.018 g, 0.745±. 0.032 g, 0.665 ± 0.002 g, 0.573 ± 0.004 g, and 0.335 ± 0.020 g.

The experimental results show that the amount of mycelium obtained by the first to fourth Ganoderma lucidum solid medium culture More. Therefore, it is preferred that the Ganoderma lucidum solid medium contains both germ rice and wheat germ. Preferably, the concentration of the wheat germ is 10% to 40%, and the concentration of the germ rice is 10% to 40%.

Please refer to Figure 5B and Table 3. Fig. 5B is the amount of γ-aminobutyric acid obtained by culturing A ganoderma lucidum strains in different proportions of germ rice and wheat germ at 7 ° C for 7 days. From the left to the right, the horizontal coordinate is 0th Ganoderma lucidum solid medium, the first Ganoderma lucidum solid medium, the 2nd Ganoderma lucidum solid medium, the 3rd Ganoderma lucidum solid medium, the 4th Ganoderma lucidum solid medium, and the 5th Ganoderma lucidum solid medium. The ordinate is the concentration of γ -aminobutyric acid, wherein the content of γ -aminobutyric acid in the original medium is represented by the bottom of the mesh; the γ -aminobutyric acid contained in the mixture of the rice and the hyphae after 7 days of culture is cultured. The total amount is presented in the form of a mesh bottom and a twill overlay. Fig. 5B and Table 3 show that the concentration of γ -aminobutyric acid per gram of rice and mycelium mixture was 11.187±0.134 g and 14.607± after the A-Ganoderma lucidum strain was cultured in the 0~5 Ganoderma lucidum solid medium. 0.271 g, 20.373 ± 0.044 g, 25.514 ± 0.129 g, 30.616 ± 0.089 g, and 25.151 ± 0.214 mg.

The experimental results showed that the amount of γ -aminobutyric acid obtained by the culture of the first to fifth Ganoderma lucidum solid medium was large. Therefore, it is preferred that the Ganoderma lucidum solid medium contains 10% to 50% wheat germ. Preferably, the concentration of wheat germ is 30-40%. Preferably, the concentration of the germ rice is from 0% to 40%. Preferably, the concentration of the germ rice is 10% to 40%. Preferably, the concentration of the germ rice is 10% to 20%.

Experiment 7: Effect of various solid mediums of Ganoderma lucidum on the amount of mycelial strain of C and Ganoderma lucidum and the yield of γ -aminobutyric acid

The G. lucidum strain was inoculated into the above-mentioned 0~5 Ganoderma lucidum solid medium, and cultured at 30 ° C for 7 days, and the amount of mycelium and the content of γ-aminobutyric acid were measured.

Please refer to Figure 6A and Table 3. Figure 6A is at 30 ° C, in different proportions The amount of hyphae obtained from germ rice and wheat germ culture C ganoderma strain for 7 days. From the left to the right, the horizontal coordinate is 0th Ganoderma lucidum solid medium, the first Ganoderma lucidum solid medium, the 2nd Ganoderma lucidum solid medium, the 3rd Ganoderma lucidum solid medium, the 4th Ganoderma lucidum solid medium, and the 5th Ganoderma lucidum solid medium. The ordinate is the amount of hyphae contained per gram of rice and mycelium mixture after 7 days of culture. 6A shows that the amount of hyphae per gram of rice and mycelium mixture is 0.185±0.007 g, 0.479±0.014 g, 0.635±0.002 g after the C. lucidum strain is cultured in the 0~5 Ganoderma lucidum solid medium. , 0.608 ± 0.007 g, 0.635 ± 0.014 g, and 0.451 ± 0.009 g.

The experimental results show that the amount of hyphae obtained from the first to the fifth Ganoderma lucidum solid medium is higher. many.

Please refer to Figure 6B and Table 3. Fig. 6B is the amount of γ-aminobutyric acid obtained by culturing C. ganoderma strains in different proportions of germ rice and wheat germ at 7 °C for 7 days. From the left to the right, the horizontal coordinate is 0th Ganoderma lucidum solid medium, the first Ganoderma lucidum solid medium, the 2nd Ganoderma lucidum solid medium, the 3rd Ganoderma lucidum solid medium, the 4th Ganoderma lucidum solid medium, and the 5th Ganoderma lucidum solid medium. The ordinate is the concentration of γ -aminobutyric acid, wherein the content of γ -aminobutyric acid in the original medium is represented by the bottom of the mesh; the γ -aminobutyric acid contained in the mixture of the rice and the hyphae after 7 days of culture is cultured. The total amount is presented in the form of a mesh bottom and a twill overlay. Figure 6B and Table 3 show that the concentration of γ -aminobutyric acid per gram of rice and mycelium mixture is 9.108±0.243 mg, 20.629± after the C. lucidum strain is cultured in the 0~5 Ganoderma lucidum solid medium. 0.099 mg, 23.775 ± 0.167 mg, 29.351 ± 0.120 mg, 22.901 ± 0.111 mg, and 22.533 ± 0.068 mg.

The experimental results showed that the amount of γ -aminobutyric acid obtained from the first to fifth Ganoderma lucidum solid medium was large. Therefore, it is preferred that the Ganoderma lucidum solid medium contains 10% to 50% wheat germ. Preferably, the concentration of wheat germ is 20-40%. Preferably, the concentration of the germ rice is from 0% to 40%. Preferably, the concentration of the germ rice is 10% to 40%. Preferably, the concentration of the germ rice is 10% to 30%.

Each of the above experiments was performed in triplicate, and the data representation was mean ± standard deviation.

Experiment 8: Effect of culture time on the yield of γ -aminobutyric acid

Mixing 20% by weight of wheat germ, 5% by weight of water, and 30% of germ rice into a pickle bottle, and after high temperature sterilization, the second solid of Ganoderma lucidum containing different nitrogen sources is obtained. Medium.

In the preferred embodiment of the present invention, it is applied at 121 ° C for 20 minutes to simultaneously achieve high temperature cooking and sterilization effects.

Culture A strain of Ganoderma lucidum for 21 days at 32 ° C, on the 4th, 7th, 10th, 14th, The amount of hyphae and the content of γ-aminobutyric acid were measured on days 18 and 21.

Referring to Figure 7, the amount of mycelium obtained by culturing A ganoderma lucidum strain for 21 days in the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% water, and 30% germ rice) at 32 ° C and The amount of γ-aminobutyric acid. Figure 7 shows that the Ganoderma lucidum strain was cultured in Ganoderma lucidum solid medium until the 21st day, and the γ -aminobutyric acid concentration per gram of rice and mycelium mixture was increased to 119 mg, which was significantly higher than that obtained only for 7 days. The amount of γ -aminobutyric acid (see Table 3, culturing A ganoderma lucidum strain for 7 days in the second Ganoderma lucidum solid medium, containing 20.373 mg of γ -aminobutyric acid per gram of rice and mycelium mixture).

Experiment 9: Effect of nitrogen source on the yield of γ -aminobutyric acid

Mix 20% by weight of wheat germ, 50% water, and 30% of germ rice, and add 0.1% or 1% by weight of different nitrogen sources into the pickles bottle. After the sterilization treatment, the second solid medium of Ganoderma lucidum containing different nitrogen sources is prepared.

In the preferred embodiment of the present invention, it is applied at 121 ° C for 20 minutes to simultaneously achieve high temperature cooking and sterilization effects.

Among them, different nitrogen sources refer to casein, soy protein or soybean.

The A or C Ganoderma lucidum strain was cultured for 7 days at 30 ° C, and the γ-aminobutyric acid content was measured.

Please refer to Figures 8A, 8B, 9A, 9B. 8B is a strain of the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% water, and 30% germ rice), and then culturing A ganoderma lucidum strain 7 at 30 ° C. The amount of γ-aminobutyric acid obtained in days. Figure 9B is After adding 0.1% different nitrogen sources to the second Ganoderma lucidum solid medium (containing 20% wheat germ, 50% water, and 30% germ rice), the γ- obtained by culturing C ganoderma strain for 7 days at 30 ° C Amount of aminobutyric acid.

In Fig. 8B and Fig. 9B, the control group is the second Ganoderma lucidum solid medium in the present case, and the other group is the medium obtained by adding the different nitrogen sources to the second Ganoderma lucidum solid medium of the present case. As can be seen from FIG. 8B and FIG. 9B, the addition of 0.1% casein, 0.1% soy protein, or 0.1% soybean can make the γ-amine obtained by culturing the Ganoderma lucidum strain using the Ganoderma lucidum solid medium in the present case. The yield of butylbutyrate was significantly increased (p<0.05).

The above-mentioned FIG. 5B, FIG. 6B and Table 3 show that the Ganoderma lucidum strain is cultured in the solid medium of the Ganoderma lucidum in the present case, and a high concentration of γ -aminobutyric acid can be produced. Accordingly, the solid medium of Ganoderma lucidum can be used to culture Ganoderma lucidum to produce a food containing a high concentration of γ -aminobutyric acid. Among them, the high concentration of γ -aminobutyric acid means that at least 20 mg of γ -aminobutyric acid is contained per gram of the Ganoderma lucidum solid medium. Figure 7 above shows that, preferably, a high concentration of gamma -aminobutyric acid means at least 30 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, the high concentration of gamma -aminobutyric acid means at least 50 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, the high concentration of gamma -aminobutyric acid means at least 60 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, the high concentration of gamma -aminobutyric acid means at least 100 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, the high concentration of gamma -aminobutyric acid means at least 200 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium.

Preferably, the step of preparing the solid medium of the ganoderma lucidum in the present case comprises: (a) mixing a plurality of medium components to obtain a mixture of medium components, the medium component mixture comprising: Wheat germ (wheat germ, weight percent concentration of 10% to 60%; and water, weight percent concentration of 20% to 50%; and (b) high temperature and sterilization treatment, the ganoderma lucidum solid medium is prepared, wherein The high temperature means 100 ° C ~ 150 ° C.

When the medium component mixture contains 10% to 60% wheat germ, it may, for example, correspond to 0.4% to 2.6% of gliadin in the mixture of the medium components, or may be, for example, equivalent to 0.007% to 0.07 in the mixture of the medium components. % wheat prion protein.

Preferably, the concentration of the wheat germ in the medium component mixture is 30% to 40% by weight.

Preferably, the medium component mixture further comprises embryo rice, and the concentration of the germ rice is 10% to 40% by weight.

In another preferred embodiment of the present invention, the concentration of the wheat germ is 30% to 40%, the weight percentage of the germ rice is 10% to 20%, and the weight of the water is 100% by weight. The concentration is 50%.

When the medium component mixture contains 30% to 40% wheat germ, it may be, for example, 1.0% to 2.0% of gliadin in the mixture of the medium components, or may be, for example, 0.02% to 0.05 in the mixture of the medium components. % wheat prion protein.

Preferably, the medium component mixture further comprises casein, soy protein, and soybean.

In another preferred embodiment of the present invention, the preparation step of the Ganoderma lucidum solid medium comprises: (a) mixing a plurality of medium components to obtain a medium component mixture, the culture The nutrient component mixture includes: wheat germ (wheat germ; and water; and (b) high temperature and sterilization treatment to obtain the ganoderma lucidum solid medium, wherein the high temperature means 100 ° C ~ 150 ° C; and the step (a In the wheat germ component, Gliadin is included, and the concentration of the gliadin in the mixture of the components of the medium is 0.4% to 2.6%.

Preferably, the concentration of the gliadin in the medium mixture is from 1.0% to 2.0% by weight.

In a further preferred embodiment of the present invention, the preparation step of the Ganoderma lucidum solid medium comprises: (a) mixing a plurality of medium components to obtain a medium component mixture, the medium component mixture comprising: wheat germ (wheat germ); And (b) obtaining the solid medium of the ganoderma lucidum by high temperature and sterilization, wherein the high temperature means 100 ° C ~ 150 ° C; and in the step (a), the components of the wheat germ include wheat gluten (Glutenin) And the concentration of the wheat gluten protein in the mixture of the medium components is 0.007% to 0.07% by weight.

Preferably, the concentration of the wheat gluten protein in the mixture of the medium components is 0.02% to 0.05% by weight.

Preferably, it is applied at 121 ° C for at least 15 minutes to simultaneously achieve high temperature cooking. And sterilization effect.

Preferably, it is used to culture Ganoderma lucidum to produce a high concentration of γ -aminobutyric acid; wherein, a high concentration of γ -aminobutyric acid means at least 20 mg of γ -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, a high concentration of gamma -aminobutyric acid means at least 30 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, the high concentration of gamma -aminobutyric acid means that at least 50 mg of gamma -aminobutyric acid is produced per gram of Ganoderma lucidum solid medium. Preferably, a high concentration of gamma -aminobutyric acid means at least 60 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, a high concentration of gamma -aminobutyric acid means at least 100 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium. Preferably, a high concentration of gamma -aminobutyric acid means at least 200 mg of gamma -aminobutyric acid per gram of Ganoderma lucidum solid medium.

The present invention further provides a method for producing high concentration γ -aminobutyric acid by microbial fermentation to solve the industrial problem that only γ -aminobutyric acid can be prepared by chemical methods. The step of producing the γ -aminobutyric acid by the microbial fermentation method comprises:

(a) mixing a plurality of medium components to obtain a mixture of medium components comprising: wheat germ (by weight, concentration of 10% to 60%; and water, weight percent concentration of 20) %~50%; and

(b) A high temperature medium and a sterilization process are used to prepare a solid medium of Ganoderma lucidum, wherein the high temperature means 100 ° C to 150 ° C.

(c) Adding a Ganoderma lucidum strain to produce γ -aminobutyric acid by fermentation.

Wherein, the high concentration of γ -aminobutyric acid means that the ganoderma lucidum solid medium contains at least 20 mg of γ -aminobutyric acid per gram. Preferably, the high concentration of γ -aminobutyric acid means that the ganoderma lucidum solid medium contains at least 30 mg of γ -aminobutyric acid per gram. Preferably, the high concentration of γ -aminobutyric acid means that the ganoderma lucidum solid medium contains at least 100 mg of γ -aminobutyric acid per gram. Preferably, the high concentration of γ -aminobutyric acid means that the ganoderma lucidum solid medium contains at least 200 mg of γ -aminobutyric acid per gram.

In each of the examples of the present invention, the concentration percentages of the components of the 0 to 5 medium component mixture are all weight percent concentrations. Each concentration percentage in the present invention is a weight percent concentration.

The above is only the preferred embodiment of the present invention, and is not intended to limit the scope of the claims of the present invention. Therefore, various other modifications or retouchings, etc., which are not departing from the spirit of the present invention, should be included in the present invention. Within the scope of the patent application.

Claims (13)

A method for producing γ -aminobutyric acid, the method comprising the steps of: (a) mixing a plurality of medium components to obtain a mixture of medium components, the mixture comprising: wheat germ (wheat germ), having a concentration of 10 by weight %~50%; and water, the concentration by weight is 20%~50%; and (b) obtaining a solid medium of Ganoderma lucidum by high temperature and sterilization, wherein the high temperature means 100 ° C ~ 150 ° C; c) Adding a Ganoderma lucidum strain to produce γ -aminobutyric acid by fermentation. The method of claim 1, wherein the medium component mixture of the step (a) has a concentration of 50% by weight of the wheat germ, and after the step (c) is fermented at 30 ° C, per gram. The Ganoderma lucidum solid medium includes at least 20 mg of γ -aminobutyric acid. The method according to claim 1, wherein the concentration of the wheat germ in the medium component mixture of the step (a) is 30% to 40% by weight. According to the method of claim 1, the medium component mixture of the step (a) further comprises embryo rice, and the concentration of the germ rice is 10% to 40% by weight. According to the method of claim 4, in the medium component mixture of the step (a), the weight concentration of the wheat germ is 30% to 40%, and the weight concentration of the germ rice is 10% to 20%. And the weight percentage of water is 50%. The method of claim 4, wherein the mixture of the medium components of the step (a) further comprises casein, soy protein or soybean. For example, in the method described in claim 1, 3, 4, 5 or 6, the step (b) is carried out at 121 ° C for at least 15 minutes to simultaneously achieve high temperature cooking and sterilization effects. According to the method of claim 4 or 6, in the medium component mixture of the step (a), the weight concentration of the wheat germ is 20% to 40%, and the weight concentration of the germ rice is 10%. ~30%, in the step (c), the Ganoderma lucidum strain is cultured at 30 ° C for 7 days to produce a high concentration of γ -aminobutyric acid; wherein, the high concentration of γ -aminobutyric acid refers to each gram of the gram Ganoderma lucidum solid medium produces at least 20 mg of γ -aminobutyric acid. The method of claim 4 or 6, wherein the medium component mixture of the step (a) has a weight percentage of wheat germ of 20% and a concentration of germ rice of 30% by weight. In (c), the Ganoderma lucidum strain is cultured at 32 ° C for 21 days to produce a high concentration of γ -aminobutyric acid; wherein, the high concentration of γ -aminobutyric acid means at least 100 per gram of the Ganoderma lucidum solid medium. Mg γ -aminobutyric acid. According to the method of claim 4 or 6, in the medium component mixture of the step (a), the weight concentration of the wheat germ is 20% to 40%, and the weight concentration of the germ rice is 10%. ~30%, in the step (c), the Ganoderma lucidum strain is cultured at 30 ° C for 7 days to produce a food containing a high concentration of γ -aminobutyric acid; wherein, a high concentration of γ -aminobutyric acid means The gram of Ganoderma lucidum solid medium contains at least 20 mg of γ -aminobutyric acid. The method of claim 4 or 6, wherein the medium component mixture of the step (a) has a weight percentage of wheat germ of 20% and a concentration of germ rice of 30% by weight. In (c), the Ganoderma lucidum strain is cultured at 32 ° C for 21 days to produce a food containing a high concentration of γ -aminobutyric acid; wherein, the high concentration of γ -aminobutyric acid means at least one gram of the Ganoderma lucidum solid medium per gram. Contains 50 mg of gamma -aminobutyric acid. The method of claim 4 or 6, wherein the medium component mixture of the step (a) has a weight percentage of wheat germ of 20% and a concentration of germ rice of 30% by weight. In (c), the Ganoderma lucidum strain is cultured at 32 ° C for 21 days to produce a food containing a high concentration of γ -aminobutyric acid; wherein, the high concentration of γ -aminobutyric acid means at least one gram of the Ganoderma lucidum solid medium per gram. Contains 60 mg of gamma -aminobutyric acid. The method of claim 4 or 6, wherein the medium component mixture of the step (a) has a weight percentage of wheat germ of 20% and a concentration of germ rice of 30% by weight. In (c), the Ganoderma lucidum strain is cultured at 32 ° C for 21 days to produce a food containing a high concentration of γ -aminobutyric acid; wherein, the high concentration of γ -aminobutyric acid means at least one gram of the Ganoderma lucidum solid medium per gram. Contains 100 mg of gamma -aminobutyric acid.