Application of natural mixed culture YakQH5 in fermentation substrate methane production and cordyceps militaris cultivation

Abstract

The invention belongs to the technical field of biology, and particularly provides application of a natural mixed culture YakQH5 in methane production of a fermentation substrate and cultivation of cordyceps militaris, a natural mixed culture YakQH5 enzyme solution with an optimized process can obviously promote methane production, and the accumulated methane production amount is increased by 2.26 times compared with that of a control group; the invention further provides fermentation wastewater of the natural mixed culture YakQH5, starch wastewater and bean dregs, the fermentation wastewater is compounded with the starch wastewater and the bean dregs to be used for cultivating cordyceps militaris, by optimizing a matrix formula, the fresh weight of sporocarp, the biological conversion rate and the cordycepin content of the sporocarp are remarkably increased, and compared with a traditional cultivation matrix, the cultivation matrix has obvious advantages.

Description

Technical Field

[0001] The invention belongs to the field of microbial new energy and environmental protection, and relates to the application of natural mixed culture YakQH5 in fermentation matrix methane production and cultivation of Cordyceps militaris. Background Art

[0002] my country has a large amount of livestock and poultry manure emissions, about 3 billion tons per year, which is the main source of agricultural non-point source pollution. Cow manure accounts for about 37% of it, and it is urgent to utilize it as a resource. Cow manure is usually used as biofuel, feed, and organic fertilizer, but the utilization rate is generally less than 50-60%. Corn stalks are also a large amount of waste, and they all face the problem of resource development and utilization.

[0003] Although the carbon-nitrogen ratio of cow dung is suitable for anaerobic digestion and methane production, and hydrolysis and acidification are important rate-limiting steps in the anaerobic digestion of livestock and poultry manure, the high lignocellulose content of cow dung and corn straw limits the improvement of methane production efficiency. The pretreatment of cow dung and corn straw has become an effective method to promote hydrolysis and acidification and effectively increase methane production. A large number of studies have used different pretreatment methods to improve the anaerobic digestion efficiency of cow dung and corn straw, such as hydrothermal pretreatment, addition of nanoparticles, addition of biochar, microwave-alkali treatment, and ultrasonic pretreatment. These methods have the problems of high cost, complex pretreatment methods, and low efficiency. Compared with these pretreatment methods, although the research on enzymatic pretreatment to improve the methane production of anaerobic digestion of cow dung and corn straw is limited, this method has the characteristics of high efficiency, low cost and low energy consumption, and will become a hot research direction in the future.

[0004] Cordyceps militaris is a large fungus that can be used as both medicine and food. It specifically synthesizes cordycepin, a quality-controlled product, and has good prospects for food and medicine. The fruiting bodies of Cordyceps militaris are mainly cultivated using oatmeal or rice culture media, which leads to high production costs for Cordyceps militaris. The development of alternative culture media has become a common key issue in Cordyceps militaris production. The large-scale discharge of methane-producing slurry and sludge from anaerobic digestion of cow dung causes environmental pollution problems, and the low carbon-nitrogen ratio makes it impossible to directly use slurry and sludge in Cordyceps militaris culture media. Starch wastewater is a waste from potato processing and can provide a rich carbon source. The mixture of starch wastewater and cow dung slurry and sludge can effectively improve the carbon-nitrogen ratio of slurry and sludge. At the same time, the introduction of bean dregs (soy product processing waste) can increase the voids in the cultivation matrix, which is more conducive to the growth of Cordyceps militaris mycelium and the formation of primordia. The mixture of cow dung slurry and sludge, starch wastewater and bean dregs has become a good substitute for the cultivation matrix of Cordyceps militaris. Relevant research has used culture media prepared from potatoes and filter ceramics to cultivate Cordyceps militaris fruiting bodies (patent number: 201410632217.4); when 10-66% bean dregs are added to the culture media, the yield of Cordyceps militaris fruiting bodies is significantly higher than that of wheat or rice culture media (patent number: 201210206174.4). At present, there are few studies on the cultivation of Cordyceps militaris fruiting bodies using alternative culture media such as starch wastewater, cow dung slurry and bean dregs. The effects of alternative culture media based on these raw materials on the biomass and cordycepin content of Cordyceps militaris fruiting bodies are still unclear. Summary of the invention

[0005] The primary purpose of the present invention is to provide an application of a natural mixed culture of anaerobic fungi (Neocallimastix frontalis) and methanogens (Methanobrevibactergottschalkii) YakQH5 in fermentation substrate methane production, wherein the natural mixed culture YakQH5 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on March 9, 2020, with a deposit number of CGMCC No.19299, and the substrate is made of cow dung and corn straw powder.

[0006] Preferably, the matrix is ​​prepared by mixing cow dung and corn stalk powder in a mass ratio of 5:1.

[0007] The second object of the present invention is to provide an application of a substrate fermented by a natural mixed culture of anaerobic fungi (Neocallimastix frontalis) and methanogens (Methanobrevibactergottschalkii) YakQH5 in the cultivation of Cordyceps militaris, wherein the natural mixed culture YakQH5 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on March 9, 2020, with a deposit number of CGMCC No.19299, and the substrate is made of cow dung and corn stalk powder.

[0008] The third object of the present invention is to provide a method for cultivating Cordyceps militaris, wherein the method comprises the following steps:

[0009] (1) The natural mixed culture YakQH5 was inoculated into corn stalks, and the fermentation liquid after pretreatment for 24 to 120 hours was inoculated into a liquid anaerobic fungal culture medium, and the supernatant prepared by centrifugation was the natural lignocellulose degrading enzyme fermentation liquid;

[0010] (2) mixing cow dung and corn stalk powder to form a fermentation matrix, adding the fermentation liquid prepared in step (1) to the fermentation matrix at a rate of 1% (w / v), reacting to obtain an enzyme solution and wastewater;

[0011] (3) inoculating Cordyceps militaris spawn into a sterilized seed culture medium cooled to room temperature, and culturing to prepare a seed solution; mixing the starch wastewater and the wastewater obtained in step (2) in a volume ratio of 6:1 to 1:1, respectively, adding nutrients and 100 to 500 g / L of bean dregs, and packaging them into cans sealed with polypropylene film, sterilizing, cooling to room temperature, inoculating 10% of the seed solution, sealing, and culturing at 22° C. in the dark until they are covered with mycelium;

[0012] (4) The mycelium obtained in step (3) is cultured at 22-25°C / 12h and 16-18°C / 12h under conditions of light intensity and humidity of 40-60% until primordia are formed, and then cultured under conditions of 22°C, light intensity and humidity of 80-95% until the fruiting body grows to 8-10cm.

[0013] Preferably, the corn stalks added in step (1) are added at a concentration of 0.5% to 3% w / v, and the treatment time is 24h to 120h.

[0014] Preferably, the mass ratio of cow dung to corn stalk powder in the fermentation matrix in step (2) is 5:1, and the reaction time is 1 to 6 days.

[0015] Preferably, the volume ratio of the starch wastewater described in step (3) to the wastewater obtained in step (2) is 6:1 to 3:1.

[0016] Preferably, the amount of dregs added in step (3) is 300 g / L.

[0017] The fourth object of the present invention is to provide Cordyceps militaris cultivated by the method.

[0018] The beneficial effects of the present invention are as follows: (1) The present invention provides a new use of the natural mixed culture YakQH5 of anaerobic fungi (Neocallimastix frontalis) and methanogens (Methanobrevibactergottschalkii) in the production of methane from fermentation substrates. The experimental results show that the cumulative methane production of the natural mixed culture YakQH5 enzyme solution pretreatment group on the 10th day is significantly higher than that of the xylanase solution and Bacillus subtilis solution pretreatment groups and the control group (P<0.01), and the cumulative methane production of the xylanase pretreatment group is close to but still significantly higher than that of the Bacillus subtilis pretreatment group (P<0.05). Therefore, the use of the YakQH5 enzyme solution with an optimized process can significantly promote methane production, which is 2.26 times higher than the control.

[0019] (2) The present invention also provides a fermentation wastewater of a natural mixed culture of anaerobic fungi (Neocallimastixfrontalis) and methanogens (Methanobrevibacter gottschalkii) YakQH5, which is compounded with starch wastewater and bean dregs for use in cultivating Cordyceps militaris. By optimizing the matrix formula, the fresh weight of the fruiting body, the biological conversion rate and the cordycepin content of the fruiting body are significantly improved, which has obvious advantages over traditional cultivation matrices. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Optimization of YakQH5 enzyme solution conditions

[0021] Figure 2 Optimizing the time of pretreatment of fermentation substrate with different enzyme solutions

[0022] Figure 3 Screening for the best pretreatment method Note: CT represents the control group, BS represents the Bacillus subtilis solution pretreatment group, XYL represents the xylanase pretreatment group, and YAK represents the optimized YakQH5 enzyme solution pretreatment group

[0023] Figure 4 Results of the experimental group when the amount of okara added was 300g / L DETAILED DESCRIPTION

[0024] In order to better illustrate the present invention, the following is further described in conjunction with specific embodiments. Unless otherwise specified, the operations used in the following embodiments are all conventional operations well known to the technicians of the present embodiments.

[0025] Unless otherwise specified, the reagents and consumables described in the following examples can be purchased from the market.

[0026] In the following examples, the seed culture medium includes: 34 g / L glucose, 17 g / L peptone, 2 g / L potassium dihydrogen phosphate, and 2 g / L magnesium sulfate.

[0027] In the following examples, the rice culture medium comprises: 300 g / L brown rice, 3 g / L silkworm pupa powder, 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate, and 0.1 g / L vitamin B1;

[0028] In the following examples, the oat culture medium comprises: 300 g / L oats, 3 g / L silkworm pupa powder, 2 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate, and 0.01 g / L vitamin B1;

[0029] Embodiment 1

[0030] 1.1 Enzyme preparations

[0031] 1.1.1 YakQH5 enzyme solution: We isolated the natural mixed culture of anaerobic fungi (Neocallimastix frontalis) and methanogens (Methanobrevibacter gottschalkii) YakQH5 (hereinafter referred to as mixed culture YakQH5) from the rumen fluid of grazing yaks in Xinghai County, Qinghai Province. The mixed culture YakQH5 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 9, 2020, with the deposit number CGMCC No.19299. The mixed culture YakQH5 was inoculated into corn stover with different mass concentrations (0.5%, 1%, 2%, 3%, w / v), and 25 mL of the fermentation broth after pretreatment for 24, 48, 72, 96, and 120 h was inoculated into 225 mL of liquid anaerobic fungal culture medium. The supernatant prepared after centrifugation at 1000×g for 10 min was the natural lignocellulose degrading enzyme fermentation broth (hereinafter collectively referred to as YakQH5 enzyme broth).

[0032] 1.1.2 Xylanase solution: Take the commercial powdered xylanase (enzyme activity of 600,000 U / g) from Baiyin Sainuo Biotechnology Co., Ltd., dissolve it in distilled water until saturated, and take the supernatant as the xylanase solution.

[0033] 1.1.3 Bacillus subtilis solution: Bacillus subtilis product purchased from Hebi Xingwang Biotechnology Co., Ltd. was prepared into saturated bacterial solution according to the instructions.

[0034] 1.2 Cow dung, corn stalks and inoculants

[0035] Cow dung was obtained from Lanzhou Yili Dairy Co., Ltd. The total solids (TS) and volatile solids (VS) of the cow dung were 27.69 and 16.21%, respectively.

[0036] Corn stalks were obtained from a farm in Yuzhong Township, Lanzhou City, dried naturally, and crushed to a particle size of 0.5-0.8 cm. The TS and VS were 81.69 and 70.19%, respectively, and stored at room temperature for later use.

[0037] The sludge was taken from a long-term stable biogas digester in a township farm in Yuzhong, Lanzhou, and the anaerobic granular sludge was taken from a long-term stable biogas tank in the laboratory. The sludge and anaerobic granular sludge were mixed in a volume ratio of 1:1 to make inoculum. The TS and VS of the inoculum were 6.33 and 2.37%, respectively.

[0038] 1.3 Effects of different enzyme preparations on methane production from anaerobic digestion of cow dung and corn straw

[0039] 1.3.1 Optimization of YakQH5 enzyme solution

[0040] Cow dung and corn straw powder were mixed at a mass ratio of 5:1 to prepare a fermentation matrix. The YakQH5 enzyme solution of different processes in step 1.1.1 was added to the fermentation matrix at 1% (w / v), and placed in a 500mL serum bottle anaerobic digestion system with a reaction volume of 200mL. After reacting for 48h, 200mL of inoculum was added, and the anaerobic system was rebuilt with nitrogen. Anaerobic digestion was carried out at 39°C for 10d. During the anaerobic digestion process, the system was shaken at a speed of 120r / min for 12h every 2d, and each group of gases was collected using a 5L gas collection bag. The methane volume fraction in the gas was detected using BIOGAS5000, and the cumulative gas production was recorded by the water displacement method. The cumulative methane production was the product of the cumulative gas production and the methane volume fraction.

[0041] The results are as follows Figure 1 As shown, after adding 2% corn stover and pretreatment for 72 hours, the optimized YakQH5 enzyme solution was obtained, which maximized the methane production by anaerobic digestion. Therefore, the optimized YakQH5 enzyme solution obtained under this condition was used in the subsequent process.

[0042] 1.3.2 Optimizing the time of pretreatment of fermentation substrate with different enzyme solutions

[0043] Using the optimized YakQH5 enzyme solution obtained in 1.3.1, according to the same method as 1.3.1, different enzyme solutions were set to pre-treat for 1, 2, 3, 4, 5, and 6 days respectively, and Bacillus subtilis solution and xylanase solution were added to the fermentation matrix according to the dosage in the commercial enzyme instructions. According to the above results, the optimized YakQH5 enzyme solution was added to the fermentation matrix, and the different enzyme solution pretreatment times that resulted in the highest cumulative methane production on the 10th day were screened.

[0044] The results are as follows Figure 2As shown in the figure, with the increase of pretreatment time of different enzyme solutions, the cumulative methane amount of each group on the 10th day showed an inverted U-shaped trend. The highest cumulative methane amount was obtained when the YakQH5 enzyme solution group, xylanase solution group and Bacillus subtilis solution group pretreated the fermentation substrate for 3, 4 and 5 days, respectively. This process was used for subsequent research.

[0045] 1.3.3 Screening the best pretreatment method

[0046] Using the optimized process obtained in the previous step, the differences in cumulative methane production on the 10th day between the pretreatment groups of optimized YakQH5 enzyme solution, xylanase solution and Bacillus subtilis solution were compared, and the experiment without enzyme addition and pretreatment was set as the control group.

[0047] The results are as follows Figure 3 As shown in the figure, the cumulative methane production of the group pretreated with the optimized YakQH5 enzyme solution on the 10th day was significantly higher than that of the groups pretreated with the xylanase solution and the Bacillus subtilis solution and the control group (P<0.01), and the cumulative methane production of the group pretreated with the xylanase solution was close to but still significantly higher than that of the group pretreated with the Bacillus subtilis solution (P<0.05). Therefore, the use of the optimized YakQH5 enzyme solution can significantly promote methane production, which is 2.26 times higher than the control.

[0048] In this step, the fermentation liquid of the 10th day of methanogenesis in the YakQH5 enzyme solution pretreatment group optimized in the previous step (hereinafter collectively referred to as YakQH5 wastewater) was used for Cordyceps militaris cultivation research.

[0049] 1.4 Cultivation of Cordyceps militaris based on YakQH5, starch wastewater and bean dregs

[0050] 1.4.1 Preparation of culture medium

[0051] After the YakQH5 enzyme liquid optimization process, most of the carbon source of the above fermentation matrix (cow dung and corn straw powder mixed in a mass ratio of 5:1) was converted into methane. There are two major problems when YakQH5 wastewater is used to cultivate Cordyceps militaris: 1) odor; 2) the carbon-nitrogen ratio is significantly reduced.

[0052] Aiming at the odor of YakQH5 wastewater, the present invention uses the bacterial liquid of Bacillus subtilis, lactic acid bacteria, and Saccharomyces cerevisiae (the number of live bacteria is about 260 million / g) and the enzyme liquid of lipase and protease (the enzyme activity is about 200,000 U / g) and other raw materials, and mixes the biological deodorant with the same mass ratio to form a biological deodorant, mixes the biological deodorant, glucose (or molasses) and water in a mass ratio of 1:2:30, and stands at room temperature for 3 days to prepare an active deodorant for deodorizing YakQH5 wastewater, wherein the Bacillus subtilis in the biological deodorant is stored in the China General Microbiological Culture Collection Management Center, and the strain number is CGMCC 1.1849. After mixing the active deodorant and YakQH5 wastewater in a volume ratio of 1:100 and standing at room temperature for 5 days, the removal rates of ammonia and hydrogen sulfide in the YakQH5 wastewater reach 93.6% and 87.2% respectively, which is used for subsequent Cordyceps militaris cultivation research.

[0053] In order to solve the problem of low carbon-nitrogen ratio of YakQH5 wastewater, introducing starch wastewater (carbon-nitrogen ratio of more than 20) into YakQH5 wastewater can effectively improve the carbon-nitrogen ratio of YakQH5 wastewater. At the same time, adding bean dregs to increase the voids in the cultivation matrix is ​​beneficial to the growth of Cordyceps militaris mycelium and the formation of primordium.

[0054] 1.4.2 Optimization of cultivation medium

[0055] Cordyceps militaris CICC 14014 (purchased from China Industrial Microbiological Culture Collection Center) was used as the experimental material. Six bacterial blocks were inoculated from a PDA test tube into a seed culture medium that had been sterilized and cooled to room temperature. The seed solution was prepared after culturing at 25°C and 120 r / min for 5 days. Starch wastewater (taken from Junchi Hongfa Vermicelli Factory, Qinzhou District, Tianshui City) and YakQH5 wastewater with volume ratios of 6:1, 5:1, 4:1, 3:1, 2:1, and 1:1 were mixed respectively, and then nutrients were added to reach appropriate concentrations (silkworm pupa powder 3g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate 2g / L, vitamin B1 0.05 g / L) to make 6 groups of culture media, and the bean dregs (taken from Wang's Jiangshui Tofu Processing Factory, Huaniu Town, Maiji District, Tianshui City) of each group of culture media were added with a gradient of 100, 200, 300, 400 and 500g / L, and rice culture medium and oat culture medium were set as control groups, packaged into canning bottles sealed with polypropylene film, sterilized at 121°C for 30min and naturally cooled to room temperature, inoculated with 10% seed solution and sealed, and then cultured at 22°C in the dark until covered with mycelium. Then, the culture is carried out at 22-25° C. (12 hours) and 16-18° C. (12 hours) under the conditions of light and humidity of 40-60% until primordium is formed, and then the culture is carried out under the conditions of 22° C., light and humidity of 80-95% until the fruiting bodies grow to 8-10 cm and then harvested, and the biological conversion rate is calculated, and the cordycepin content of the control and the first 6 groups of fruiting bodies with the highest biomass is detected by liquid chromatography. Wherein the biological efficiency=(fresh weight of Cordyceps militaris fruiting bodies / dry weight of culture medium)×100%.

[0056] 100-500 g / L of dregs were added to the culture medium with the same other ingredients. The experimental results showed that the various indicators were better when 300 g / L of dregs was added, so the culture medium with 300 g / L of dregs was selected for subsequent experiments.

[0057] The research results are as follows Figure 4 As shown, using Cordyceps militaris CICC 14014 as the research material, when 300g / L bean dregs were added, the fruiting body yield was significantly higher than that of other groups. The study found that when the wastewater ratio was 5:1, the fruiting body fresh weight, biological efficiency, biological efficiency growth rate and cordycepin content were significantly higher than those of the rice and oat matrix groups. The culture period at this time was 49 days, which was lower than the control group.

[0058] In summary, the present invention provides a new use of the natural mixed culture YakQH5 of anaerobic fungi (Neocallimastixfrontalis) and methanogens (Methanobrevibacter gottschalkii) in fermentation substrate methane production. The experimental results show that the cumulative methane production of the natural mixed culture YakQH5 enzyme solution pretreatment group on the 10th day is significantly higher than that of the xylanase solution and Bacillus subtilis solution pretreatment group and the control group (P<0.01), and the cumulative methane production of the xylanase pretreatment group is close to but still significantly higher than that of the Bacillus subtilis pretreatment group (P<0.05). Therefore, the use of the YakQH5 enzyme solution with an optimized process can significantly promote methane production, which is 2.26 times higher than the control. The present invention also provides fermentation wastewater of a natural mixed culture of anaerobic fungi (Neocallimastix frontalis) and methanogens (Methanobrevibactergottschalkii) YakQH5, which is compounded with starch wastewater and bean dregs for cultivating Cordyceps militaris. By optimizing the matrix formula, the fresh weight of the fruiting body, the biological conversion rate and the cordycepin content of the fruiting body are significantly improved. Compared with traditional cultivation matrices, the present invention has obvious advantages and broad application prospects.

Claims

1. Application of natural mixed culture YakQH5 of anaerobic fungi (Neocallimastix frontalis) and methanogens (Methanobrevibactergottschalkii) in methanogenesis from fermentation substrates, characterized in that: The natural mixed culture YakQH5 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 9, 2020, with the deposit number CGMCC No.19299, and the matrix is ​​made of cow dung and corn straw powder.

2. The use according to claim 1, characterized in that The matrix is ​​prepared by mixing cow dung and corn stalk powder in a mass ratio of 5:

1.

3. The use of a substrate fermented by a natural mixed culture of anaerobic fungi (Neocallimastixfrontalis) and methanogens (Methanobrevibactergottschalkii) YakQH5 in the cultivation of Cordyceps militaris, characterized in that the natural mixed culture YakQH5 was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on March 9, 2020, with a deposit number of CGMCC No.19299, and the substrate is made of cow dung and corn straw powder.

4. A method for cultivating Cordyceps militaris, characterized in that: The method comprises the following steps: (1) The natural mixed culture YakQH5 was inoculated into corn stalks, and the fermentation liquid after pretreatment for 24 to 120 hours was inoculated into a liquid anaerobic fungal culture medium, and the supernatant prepared by centrifugation was the natural lignocellulose degrading enzyme fermentation liquid; (2) mixing cow dung and corn stalk powder to form a fermentation matrix, adding the fermentation liquid prepared in step (1) at a rate of 1% (w / v) to the fermentation matrix, and reacting to obtain an enzyme solution and wastewater; (3) inoculating Cordyceps militaris spawn into a sterilized seed culture medium cooled to room temperature, and culturing to prepare a seed solution; mixing the starch wastewater and the wastewater obtained in step (2) in a volume ratio of 6:1 to 1:1, respectively, adding nutrients and 100 to 500 g / L of bean dregs, and packaging them into cans sealed with polypropylene film, sterilizing, cooling to room temperature, inoculating 10% of the seed solution, sealing, and culturing at 22° C. in the dark until they are covered with mycelium; (4) The mycelium obtained in step (3) is cultured at 22-25°C / 12h and 16-18°C / 12h under conditions of light intensity and humidity of 40-60% until primordia are formed, and then cultured under conditions of 22°C, light intensity and humidity of 80-95% until the fruiting body grows to 8-10cm.

5. The method according to claim 4, characterized in that The addition concentration of the corn stalks in step (1) is 0.5% to 3% w / v, and the treatment time is 24h to 120h.

6. The method according to claim 4, characterized in that The mass ratio of cow dung to corn stalk powder in the fermentation matrix described in step (2) is 5:1, and the reaction time is 1 to 6 days.

7. The method according to claim 4, characterized in that The volume ratio of the starch wastewater described in step (3) to the wastewater obtained in step (2) is 6:1 to 3:

1.

8. The method according to claim 4, characterized in that The amount of okara added in step (3) is 300 g / L.

9. Cordyceps militaris cultivated by the method according to any one of claims 4 to 8.

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