CN114790426B - Application of desmodium Chaetomium strain in production of gamma aminobutyric acid and cultivation method thereof - Google Patents
Application of desmodium Chaetomium strain in production of gamma aminobutyric acid and cultivation method thereof Download PDFInfo
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Abstract
The invention discloses an application of desmodium trix strain in producing gamma aminobutyric acid and a cultivation method thereof, the desmodium trix strain is named Fv-HL, and the desmodium trix strain obtained by the invention has high gamma-aminobutyric acid capacity and is a good source for manufacturing various functional foods rich in gamma-aminobutyric acid.
Description
Technical Field
The invention belongs to the technical field of desmodium triumphane strain breeding, and particularly relates to application of desmodium triumphane strain in production of gamma aminobutyric acid and a cultivation method thereof.
Background
Gamma-aminobutyric acid (gamma-aminobutyric acid, GABA) is a naturally occurring four-carbon non-protein amino acid, widely distributed in nature. The research proves that GABA is the main amino acid neurotransmitter involved in the inhibitory synaptic transmission, and has wide physiological functions, such as lowering blood pressure, lowering blood sugar, regulating arrhythmia, resisting anxiety, protecting liver, etc. In the human brain, GABA is converted from glutamic acid in the brain by glutamate decarboxylase with stronger specificity. However, accumulation of GABA becomes extremely difficult with age and increased mental stress. The health of human body can be promoted by supplementing daily diet.
GABA content was found and measured in almost all commercial and model crops, except bacteria, fungi and animals. GABA in common crops such as tomatoes, spinach, broccoli and the like is 300-400 mg/kg, and the GABA content in wheat germs is accumulated to 1630mg/kg. Less research is conducted on GABA in edible fungi.
Common preparation methods of GABA at present are a chemical synthesis method, a plant enrichment method and a microbial fermentation method. The chemical synthesis method has the defects of high cost, low yield, harsh reaction conditions, easiness in chemical component residue, easiness in environmental pollution and the like, and is not suitable for the aspects of foods, medicines and the like. The plant enrichment method has high cost, low yield and good safety; the microbial fermentation method has low cost and high yield, is not limited by space, environment and resources, and therefore, the plant enrichment method and the microbial fermentation method are more selected for enriching GABA. At present, no document report exists on a breeding method of high-yield gamma-aminobutyric acid trichodesmium and enrichment of gamma-aminobutyric acid content of trichodesmium by a microbial fermentation method.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
As one of the aspects of the invention, the invention provides an application of a desmodium Chaetomium strain in the production of gamma aminobutyric acid, which is characterized in that: the desmodium trichosanthis strain is named Fv-HL.
As another aspect of the invention, the invention provides a cultivation method of the desmodium tridymium strain, which comprises the following steps:
(1) Inoculating the desmodium tridymium strain to a potato dextrose agar solid culture medium PDA, and collecting hypha after culturing;
(2) Picking the desmodium tridymite cultured in the step (1), inoculating the desmodium tridymite into potato dextrose broth PDB, and culturing in a dark place to obtain desmodium tridymite hyphae;
(3) Inoculating the mycelium in the step (1) into a sterile cultivation bottle for cultivation, and performing fruiting management and light-shielding cultivation to obtain the desmodium triquetrum fruiting body.
As a preferable scheme of the cultivation method of the desmodium triquetrum strain, the invention has the following advantages: the potato dextrose agar solid culture medium PDA has the mass percentage of 3.9-4.5 percent.
As a preferable scheme of the cultivation method of the desmodium triquetrum strain, the invention has the following advantages: the potato dextrose broth liquid culture medium PDB comprises 2.4-3.0% of potato dextrose broth powder by mass percent.
As a preferable scheme of the cultivation method of the desmodium triquetrum strain, the invention has the following advantages: in the step (1), hyphae are collected after the culture, and the culture time is 10-12 d.
As a preferable scheme of the cultivation method of the desmodium triquetrum strain, the invention has the following advantages: in the step (1), hyphae are collected after the culture, and the culture temperature is 22-25 ℃.
As a preferable scheme of the cultivation method of the desmodium triquetrum strain, the invention has the following advantages: in the step (2), the light-shielding culture is carried out for 10 to 12 days at the temperature of 22 to 25 ℃ and then hyphae are collected.
As a preferable scheme of the cultivation method of the desmodium triquetrum strain, the invention has the following advantages: in the step (3), the light-shielding culture is performed at 25 ℃ for 50-70d.
As a preferable scheme of the cultivation method of the desmodium triquetrum strain, the invention has the following advantages: in the step (2), 0.5-1 g/L of L-glutamic acid is added into the potato dextrose broth PDB.
As a preferable scheme of the cultivation method of the desmodium triquetrum strain, the invention has the following advantages: in the step (3), 2.4-3.2 g/L of L-glutamic acid is added into the cultivation bottle.
The invention has the beneficial effects that: the high-yield strain Fv-HL of the desmodium trichosanthis has GABA content reaching 28431.47mg/kg (figure 4) in mycelium and GABA content reaching 4303.63mg/kg (figure 5) in fruiting body, has high gamma-aminobutyric acid capacity and is a good source for manufacturing various functional foods rich in gamma-aminobutyric acid.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a diagram showing the shape of mycelia and fruiting bodies of Desmodium styracifolium.
FIG. 2 shows GABA content variation during growth of Desmodium makinsonii.
FIG. 3 is a diagram showing different growth periods of the proton entity of the Desmodium styracifolium strain.
FIG. 4 is a graph showing GABA content peaks detected by culturing the mycelium of Desmodium styracifolium strain Fv-HL.
FIG. 5 is a graph showing the peak GABA content of the Desmodium album strain Fv-HL fruiting body culture detection.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1:
the main reagents and the culture medium are as follows:
PDA solid medium: 39g of potato dextrose agar powder, 1000mL of distilled water, and natural pH value, and is used for mycelium culture.
PDB liquid medium: 24g of potato dextrose broth powder, 1000ml of distilled water, and natural pH value, is used for mycelium culture.
Seed liquid medium: 20g of white granulated sugar, 3.3g of puffed soybean meal, 0.66g of magnesium sulfate, 0.51g of monopotassium phosphate and 0.15g of dipotassium phosphate, and double distilled water is used for culturing hyphae by constant volume to 1L and regulating pH to 5.8-6.2.
0.1M hydrochloric acid: taking 860 mu L of hydrochloric acid, adding distilled water to a volume of 100mL, uniformly mixing, and storing in a sealed container.
10% trichloroacetic acid solution: 10g of trichloroacetic acid was weighed, dissolved in 100mL of distilled water, and stored in a sealed container after being mixed uniformly.
Amino acid extract: preparing a solution from 0.1M hydrochloric acid and 10% trichloroacetic acid solution according to a volume ratio of 1:2, uniformly mixing, and sealing and preserving in a dark place.
In order to explore the difference of GABA content of different types of desmodium triquetrum, 26 industrial production strains were screened from germplasm resource libraries for cultivation experiments, and the serial numbers are Fv-WC, ENOKI-J, J-3, ×3E, J5011, WH25, SCY1-2, fv-SY, ENOKI-H, WX01, ENOKI-I, W1638, XH, FM, fv-FM, 2345, fv-GF, fv-RYJ, fv-KL, fv-BY, fv-HTC, ENOKI-G, fv-GR, fv-CYS, fv-YH and Fv-HL respectively, see FIG. 1. Finally 22 strains successfully fruiting. The a of FIG. 1 is the growth form of the mycelium of Desmodium falciparum, and the b of FIG. 1 is the growth form of the fruiting body of Desmodium falciparum.
The cultivation and detection method of the desmodium trijuga is as follows:
(1) The desmodium tridymite strain is inoculated on Potato Dextrose Agar (PDA) solid culture medium, cultured for 10 days at 25 ℃ and then mycelium is collected.
(2) Picking up 10 pieces of cultured 8mm on the PDA flat plate in the step (1) 2 The desmodium pubescens fungus blocks are inoculated in potato dextrose broth liquidMycelia were collected after 10d incubation in medium (PDB) at 25℃in the absence of light.
(3) Accurately weighing 1g of mycelium obtained in the step (2) after filtering, adding 5mL of amino acid extracting solution (0.1M hydrochloric acid and 10% trichloroacetic acid solution are prepared according to the volume ratio of 1:2), carrying out ice bath ultrasonic treatment for 10min, wherein the working condition is that the ultrasonic power is 200W, the working time is 3.0s, and the stop time is 3.0s. After the ultrasonic treatment, centrifuging for 10min at 4 ℃ and 8000rpm, collecting supernatant, storing in a refrigerator at-20 ℃, and measuring the content of gamma-aminobutyric acid by using a Hitachi 835-50 type amino acid automatic analyzer.
(4) Inoculating the desmodium trijuglandis activated in the step (1) into a sterile cultivation bottle for cultivation, and performing fruiting management. After 50-70d of light-shielding culture at 25 ℃, fresh desmodium triquetrum fruiting bodies are obtained.
(5) 150g of fresh desmodium trijuglandis fruiting body in mature period is weighed, placed at 55 ℃ and dried to constant weight by hot air. Pulverizing with high-speed pulverizer, sieving with 60 mesh sieve, and sealing and storing the sample powder in shade and dry place.
(6) Accurately weighing 70-80mg of sample powder to be measured, adding 6mol/L hydrochloric acid solution for acid hydrolysis, and measuring the content of gamma-aminobutyric acid by using a Rili 835-50 type amino acid automatic analyzer.
GABA content (DW) of fruiting bodies of 22 strains of Desmodium styracifolium was measured by using an amino acid analyzer, and the GABA content of Desmodium styracifolium between different varieties was significantly different, as shown in Table 1.
TABLE 1 GABA content (DW) of the fruiting body of Desmodium styracifolium strain 22
Spatiotemporal changes in GABA content during sporophore development: the GABA content of the stipe and the pileus of the desmodium pileus in different growth periods is measured by an amino acid analyzer, and is shown in a figure 2, and the results show that the GABA content in the stipe and the pileus of the fruiting body of the desmodium pileus shows a trend of descending first and then ascending, and the GABA content accumulation reaches the maximum value in the third stage. During the whole fruiting body development process, the GABA content in the pileus is higher than that in the stigmas, both in the high-yield or low-yield GABA strains. Wherein the GABA content in the fruiting body pileus of Fv-HL mature period is up to 5056.88mg/kg DW, which is about 1.9 times of that of Fv-HL pileus of the same period. FIG. 3 shows different growth periods of the Desmodium styracifolium proton entity, and FIGS. 3 (a) - (c) show the first, second and third growth stages of Desmodium styracifolium, respectively, and 1,2 represent Desmodium styracifolium germplasm ENOKI-J, fv-HL, respectively.
Fv-HL strain is a published strain described in "establishing core germplasm population and molecular identity card of Flammulina velutipes based on genetic diversity", journal of fungus theory, gao Lihui, et al, 2021.
Example 2:
(1) The desmodium tridymite Fv-HL strain is inoculated on potato dextrose agar solid medium (PDA), and mycelia are collected after 10d of culture at 25 ℃.
(2) Picking up 10 pieces of cultured 8mm on the PDA flat plate in the step (1) 2 The desmodium tridymite blocks are inoculated into Potato Dextrose Broth (PDB), and mycelia are collected after being cultured for 10 days at 25 ℃ in a dark place.
(3) Accurately weighing 1g of mycelium obtained in the step (2) after filtering, adding 5mL of amino acid extracting solution (0.1M hydrochloric acid and 10% trichloroacetic acid solution are prepared according to the volume ratio of 1:2), carrying out ice bath ultrasonic treatment for 10min, wherein the working condition is that the ultrasonic power is 200W, the working time is 3.0s, and the stop time is 3.0s. After the ultrasonic treatment, centrifuging for 10min at 4 ℃ and 8000rpm, collecting supernatant, storing in a refrigerator at-20 ℃, and measuring the content of gamma-aminobutyric acid by using a Hitachi 835-50 type amino acid automatic analyzer.
(4) Inoculating the desmodium trijuglandis activated in the step (1) into a sterile cultivation bottle for cultivation, and performing fruiting management. After 50-70d of light-shielding culture at 25 ℃, fresh desmodium triquetrum fruiting bodies are obtained.
(5) 150g of fresh desmodium trijuglandis fruiting body in mature period is weighed, placed at 55 ℃ and dried to constant weight by hot air. Pulverizing with high-speed pulverizer, sieving with 60 mesh sieve, and sealing and storing the sample powder in shade and dry place.
(6) Accurately weighing 70-80mg of sample powder to be measured, adding 6mol/L hydrochloric acid solution for acid hydrolysis, and measuring the content of gamma-aminobutyric acid by using a Rili 835-50 type amino acid automatic analyzer.
And (3) result statistics: the amino acid analyzer measured GABA content of the Desmodium styracifolium strain Fv-HL mycelium culture was 28431.47mg/kg (FIG. 4), GABA content of the fruiting body culture was 4303.63mg/kg dry weight (FIG. 5), GABA content of the fruiting body culture was 645.54mg/kg fresh weight.
Study example:
and comparing different strains to perform laboratory liquid culture on different culture mediums with different components, and measuring GABA content in the different culture mediums. GABA content data showed that mycelia were cultivated in seed liquid medium to obtain GABA content of 1018.72mg/kg, whereas mycelia were cultivated in PDB liquid to obtain GABA content of 28431.47mg/kg, which was much higher than seed liquid medium. Therefore, PDB medium was chosen as the laboratory mycelium liquid medium.
L-glutamic acid is added into a PDB culture medium, and the influence of the L-glutamic acid on the GABA content of hypha level is observed, so that the result shows that the GABA production capability of the strain can be improved by adding low-concentration L-glutamic acid of 0.5-1 g/L, and the inhibition effect of the addition of high-concentration glutamic acid on the GABA synthesis of the desmodium tridactylum mycelium is achieved. When the glutamic acid addition concentration was 0.5g/L, the GABA content enriched in the mycelium reached the highest, which was 41.9% higher than that in the control group (the method of example 2, which used PDB medium, without adding nutrient substances). However, when the L-glutamic acid addition concentration was 1.5g/L, GABA yield in the mycelium was lower than that in the control group. With the continuous increase of the L-glutamic acid addition concentration, the ability of mycelia to enrich GABA is rather lowered, probably because the ability of mycelia to biologically convert GABA by using L-glutamic acid is limited, and the high concentration of substrate can enhance the osmotic pressure of cell membranes, so that the growth of mycelia is inhibited, which is unfavorable for the accumulation of GABA.
L-glutamic acid is added into a solid culture medium of a desmodium triquetrum culture bottle, the solid culture medium without the L-glutamic acid is used as a control, the influence of the L-glutamic acid on the GABA content of the fruiting body is observed, and the result shows that when the L-glutamic acid addition concentration is 3.2g/L, the GABA yield enriched by the desmodium triquetrum fruiting body reaches the maximum and is 1.95 times of that of the control group. The reason why the GABA yield is not increased but decreased is probably because GAD is limited in GABA synthesis pathway and the L-glutamic acid content of too high concentration may saturate the L-glutamic acid in the fruiting body and cannot promote GAD decarboxylation enrichment GABA reaction.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (10)
1. An application of desmodium Chaetomium strain in the production of gamma aminobutyric acid is characterized in that: the desmodium trichosanthis strain is named Fv-HL.
2. The use according to claim 1, characterized in that: the cultivation method of the desmodium triquetrum strain comprises the following steps:
(1) Inoculating the desmodium tridymium strain to a potato dextrose agar solid culture medium PDA, and collecting hypha after culturing;
(2) Picking the desmodium tridymite cultured in the step (1), inoculating the desmodium tridymite into potato dextrose broth PDB, and culturing in a dark place to obtain desmodium tridymite hyphae;
(3) Inoculating the mycelium in the step (1) into a sterile cultivation bottle for cultivation, and performing fruiting management and light-shielding cultivation to obtain the desmodium triquetrum fruiting body.
3. The use according to claim 2, characterized in that: the potato dextrose agar solid medium PDA comprises 3.9-4.5% of potato dextrose agar by mass.
4. A use according to claim 2 or 3, characterized in that: the potato dextrose broth liquid culture medium PDB comprises 2.4-3.0% of potato dextrose broth powder by mass.
5. A use according to claim 2 or 3, characterized in that: in the step (1), mycelium is collected after the culturing, and the culturing time is 10-12 d.
6. A use according to claim 2 or 3, characterized in that: in the step (1), mycelia are collected after the culture, and the culture temperature is 22-25 ℃.
7. A use according to claim 2 or 3, characterized in that: in the step (2), the light-shielding culture is carried out at 22-25 ℃ for 10-12 d, and then hyphae are collected.
8. A use according to claim 2 or 3, characterized in that: in the step (3), the light-shielding culture is performed at 25 ℃ for 50-70d.
9. A use according to claim 2 or 3, characterized in that: in the step (2), 0.5-1 g/L of L-glutamic acid is added into the potato dextrose broth liquid culture medium PDB.
10. A use according to claim 2 or 3, characterized in that: in the step (3), 2.4-3.2 g/L of L-glutamic acid is added into the cultivation bottle.
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| JP2012187068A (en) * | 2011-03-11 | 2012-10-04 | Hokkaido Research Organization | γ-AMINO BUTYRIC ACID-CONTAINING FOOD, AND METHOD FOR PRODUCING THE SAME |
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