A method for cultivating oyster mushrooms using white wine lees

Abstract

The invention discloses a method for cultivating oyster mushrooms by using white wine lees. The method comprises the following steps: stirring and mixing white wine lees, corn cobs and quicklime, adjusting the water content, inoculating a composite microbial agent and fermenting the mixture, adjusting the pH value of the fermented material to 8-10 and the water content to 60%-70%, bagging and sterilizing the fermented material, inoculating 5%-20% of oyster mushroom cultivation seeds, and culturing the mixture for 20-30 days under the conditions of controlling the temperature at 20-25°C and the relative humidity at 60%-70%. The method can effectively reduce the cost of oyster mushroom cultivation materials and improve the economic benefits of oyster mushroom cultivation.

Description

Technical Field

[0001] The invention belongs to the technical field of edible fungus cultivation, and particularly relates to a method for cultivating Pleurotus ostreatus by utilizing white wine lees. Background Art

[0002] Oyster mushroom is one of the most cultivated edible fungi in my country, belonging to the order Agaricales, family Pleurotus, genus Pleurotus. Oyster mushroom is popular among people because of its high nutritional value and delicious taste. At present, the main material commonly used for oyster mushroom cultivation is cottonseed hull, but the price of cottonseed hull has been rising in recent years, reaching 2,400 to 2,800 yuan per ton. The increase in the cost of cultivation materials has reduced the benefits of cultivating oyster mushrooms, becoming one of the constraints on the development of the edible fungi industry.

[0003] Liquor lees are the residues left after fermentation and distillation of grains such as sorghum and wheat. In recent years, the output of liquor lees has declined, but there are still at least 20 million tons of output each year. Compared with cottonseed hulls, the market price of liquor lees is only 100 to 200 yuan per ton.

[0004] Prior art 1, proposes a method for cultivating Pleurotus ostreatus using distiller's grains as the main raw material and a formula for cultivating Pleurotus ostreatus using distiller's grains, which includes 100-400 parts of sauce-flavored distiller's grains, 40-150 parts of any one of cottonseed hulls, bran or corn flour; 10-300 parts of any one of sawdust or cottonseed hulls, 2-6 parts of superphosphate, 0.5-7 parts of quicklime, and the rest is water, and the distiller's grains must be dried and pre-treated by electric heating.

[0005] Prior art 2 also discloses a method for cultivating edible fungi using distiller's grains as the main raw material, wherein the pH value of the whole batch is adjusted to 7.4-7.8 by acid-base titration of distiller's grains liquid, and the materials include: (1) 1600 g fresh distiller's grains, (2) 20 g sucrose, (3) 180 g sawdust, (4) 50 g corn cobs, (5) 70 g cotton husks, (6) 50 g urea, (7) 10 g quicklime, (8) 5 g potassium hydroxide, (9) 5 g magnesium sulfate decahydrate, (10) 10 g superphosphate, and (11) 800 ml water, and the distiller's grains must be pretreated by sun drying and high temperature.

[0006] Prior art 3 discloses culturing Coprinus comatus using distiller's grains as the main raw material, using corn cobs and distiller's grains as the matrix for fermentation, and using the fermented material as the cultivation material. The volume ratio of corn cobs to distiller's grains is 2:1, but the corn cobs and distiller's grains after fermentation are not acid-base adjusted, and are directly covered with soil to adjust the moisture content before cultivation.

[0007] The existing technology has a clear intention to cultivate edible fungi by replacing cottonseed hulls with distiller's grains to reduce the production cost of edible fungi. However, the mainstream method of processing distiller's grains is still washing, sun drying, and drying. These methods consume resources and consume a lot of energy. There is still a need to explore mature methods of processing distiller's grains through fermentation of bacterial strains to obtain cultivation substrates and form stable and high-quality cultivation methods.

[0008] Existing technology:

[0009] Chinese patent document 1, publication number CN113396775A;

[0010] Chinese patent document 2, publication number CN110447462A;

[0011] Chinese patent document 3, publication number is CN107637387A. Summary of the invention

[0012] In view of the problems of the prior art, the present invention provides a method for cultivating Pleurotus ostreatus using white wine lees, and the specific technical scheme is as follows:

[0013] A method for cultivating Pleurotus ostreatus using white wine lees, the method comprising:

[0014] The white wine lees, corn cobs and quicklime are stirred and mixed and the water content is adjusted, and after inoculation with a composite microbial agent for fermentation, the pH of the fermentation material is adjusted to 8-10 and the water content is adjusted to 60%-70%;

[0015] The fermentation materials are bagged and sterilized, and then inoculated with 5% to 20% of oyster mushroom cultivation species, and cultured for 20 to 30 days under the conditions of a controlled temperature of 20 to 25° C. and a relative humidity of 60 to 70%.

[0016] Preferably, the water content of the white wine lees, corn cobs and quicklime is adjusted to 50% to 70% after being stirred and mixed (preferably water flows out when the fermented material is squeezed by hand).

[0017] Preferably, the white wine lees, corn cobs, and quicklime are stirred and mixed, piled into a cone shape, and naturally fermented until the germination index reaches more than 60% (the germination index is determined by referring to the method in NY / T 525-2021 "Organic Fertilizer"), and the carbon-nitrogen ratio is between 20 and 40 (the determination of carbon refers to the determination method in NY / T 525-2021 "Organic Fertilizer", and the determination of nitrogen refers to GB / T 6432-2018 "Determination of crude protein content in feed by Kjeldahl method").

[0018] Preferably, after the mushrooms are fully grown, the incision is made, the room temperature is maintained at 15-20°C, the relative humidity is maintained at 80-90%, the light is maintained at 300-500 lux, and the mushroom caps are harvested when they grow to 5-10 cm and the edges of the mushroom caps are not completely opened. The mushroom harvesting conditions are the common conditions for harvesting mushrooms.

[0019] The beneficial effects of the present invention are:

[0020] (1) Effectively reduce the cost of oyster mushroom cultivation materials and improve the economic benefits of oyster mushroom cultivation;

[0021] (2) The process of pre-treating wine lees by microbial fermentation and using it as a cultivation medium for Pleurotus ostreatus has the advantages of being simpler to operate and requiring less energy than the traditional methods of washing, sun drying, and drying. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the temperature change trend of the mixture after pretreatment with bacterial agent;

[0023] Figure 2 It is the changing trend of germination index of the mixture after pretreatment with bacterial agent;

[0024] Figure 3 It is the changing trend of the carbon-nitrogen ratio of the mixture after pretreatment with bacterial agent. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments.

[0026] The inventors have consulted the prior art materials and found that Chinese Patent Document 1 with Publication No. CN112029681A separately cultured Bacillus velezensis UN-5 and the fungus Trichoderma reesei and mixed them in proportion to prepare a composite microbial agent. The bacteria and fungi in the composite microbial agent work together at different stages of wine lees fermentation to improve substrate utilization, shorten the wine lees fermentation cycle, and improve product quality. Chinese Journal Document 2: Ren Haiyan, Metabolic pathway of 17α-ethynyl estradiol degradation by Sphingobacterium sp. JCR5 [J], Environmental Science, 2006, 27(9), 1835-1839, which publicly discovered that strain JCR5 (identified as thermophilic Sphingobacterium sp.) can use steroidal estrogens (sterone, 17β-estradiol, estriol and ethinyl estradiol), contraceptive production intermediates and some aromatic compounds as the sole carbon source for growth.

[0027] Combined with other consulted information, a composite microbial agent composed of Sphingobacteriumthermophilum, Pseudoxanthomonas suwonensis and Bacillus velezensis was designed to pretreat wine lees and eliminate the adverse effects of aldehydes, alcohols, acids and other substances in the wine lees on the growth of Pleurotus ostreatus.

[0028] In the embodiments of the present invention, the strains described herein can be obtained through commercial channels, for example:

[0029] The thermophilic Sphingobacterium may be Sphingobacterium thermophilum. Alternatively, the strain may be obtained from Shanghai Xuanya Biotechnology Co., Ltd. (address: Room 11546, Building 14, No. 3265, Jinhai Road, Shanghai, Postal Code: 201499);

[0030] The Pseudoxanthomonas suwonensis may be Pseudoxanthomonas suwonensis. Alternatively, the strain may be obtained from China Marine Microbial Culture Collection Center (address: No. 178, Daxue Road, Xiamen, zip code: 361005).

[0031] The Bacillus velezensis may be Bacillus velezensis. Alternatively, the strain may be obtained from Shanghai Guyan Industrial Co., Ltd. (address: No. 1155, Xinzhuan Road, Songjiang District, Shanghai, zip code: 201600).

[0032] In an embodiment of the present invention, the preparation method of the composite microbial agent includes at least one of the following methods or steps:

[0033] (1) Weigh 10 g / L molasses and 15 g / L yeast peptone;

[0034] (2) Add 800-900 mL / L of water and stir evenly to fully dissolve the molasses and yeast peptone;

[0035] (3) Adjust the pH of the culture medium to 6.0-8.0 and make up the volume;

[0036] (4) sterilizing with high pressure steam at 115° C. for 20 to 30 min to obtain a fermentation medium;

[0037] (5) inoculating the thermophilic sphingobacterium seed solution into the fermentation medium at an inoculum amount of 1%, and fermenting and culturing for 28 hours based on the stable period of the thermophilic sphingobacterium;

[0038] (6) inoculating the Bacillus Velez seed solution into the fermentation medium at an inoculum rate of 1%, and fermenting and culturing for 30 hours based on the stable period of Bacillus Velez;

[0039] (7) The seed solution of Pseudomonas suwanensis was inoculated into the fermentation medium at an inoculum rate of 1%, and the fermentation culture was carried out for 30 hours based on the stable period of Pseudomonas suwanensis.

[0040] More specifically, experimental groups of CK, T1 to T7 pretreated white wine grains, corn cobs and quicklime mixtures were designed, among which CK (Control) was a sterile water pretreatment group, T1 (11A), T2 (30C) and T3 (15F) were single-bacteria microbial agent pretreatment groups of thermophilic Sphingobacillus, Pseudomonas suwanensis and Bacillus velezii, respectively, T4 (11A / 30C) was a double-bacteria microbial agent pretreatment group of thermophilic Sphingobacillus and Pseudomonas suwanensis, T5 (11A / 15F) was a double-bacteria microbial agent pretreatment group of thermophilic Sphingobacillus and Bacillus velezii, T6 (30C / 15F) was a double-bacteria microbial agent pretreatment group of Pseudomonas suwanensis and Bacillus velezii, and T7 (11A / 30C / 15F) was a composite microbial agent pretreatment group of thermophilic Sphingobacillus, Pseudomonas suwanensis and Bacillus velezii.

[0041] Figure 1 to Figure 3 They are the temperature change trends of pretreated liquor lees, corn cobs, and quicklime mixtures in different test groups (using an electronic thermometer to measure the center temperature of the mixture), the germination index change trends (refer to the determination method in NY / T525-2021 "Organic Fertilizer"), and the carbon-nitrogen ratio (the determination of carbon refers to the determination method in NY / T 525-2021 "Organic Fertilizer", and the determination of nitrogen refers to the determination method in GB / T 6432-2018 "Determination of crude protein content in feed by Kjeldahl method"). Temperature can reflect the strength of microbial metabolic activity. Figure 1 As shown in the figure, microorganisms use the easily degradable organic matter in the white wine lees to metabolize and produce heat, causing the temperature of the pile to rise. The composite microbial agent group has the highest temperature in the early stage of fermentation, which also reflects that the microbial metabolic activity of this group is the strongest. The germination index can reflect the toxicity of the sample. The larger the germination index, the weaker the toxicity of the sample. Figure 2 As shown, the germination index of the composite microbial agent group was the highest, indicating that this group had a higher ability to degrade toxic substances in white wine grains than other groups. Figure 3 The results showed that the composite microbial agent group had a stronger ability to degrade organic matter, which was one of the reasons why the pile temperature was higher in the early stage of fermentation.

[0042] After verification and comparison, it was found that the composite microbial agent composed of thermophilic Sphingobacterium, Pseudomonas suwanensis and Bacillus velez was the best for the pretreatment of the mixture of white wine grains, corn cobs and quicklime. Therefore, the present invention uses a composite microbial agent composed of thermophilic Sphingobacterium, Pseudomonas suwanensis and Bacillus velez to pretreat the mixture of white wine grains, corn cobs and quicklime, wherein the number of viable bacteria of thermophilic Sphingobacterium (Sphingobacterium thermophilum) is ≥10 7CFU / mL, Pseudoxanthomonas suwonensis viable count ≥ 10 7 CFU / mL, Bacillus velezensis viable count ≥ 10 7 CFU / mL, and the ratio of the viable bacteria counts of the three was 1:1:1.

[0043] It should be noted that the bacterial agent used for the pretreatment of the mixture of white wine grains, corn cobs and quicklime disclosed in the present invention is not the only one. The proposed composite microbial agent composed of thermophilic Sphingobacillus, Pseudomonas suwanensis and Bacillus velezensis is only one of the bacterial agent combinations that have been verified to be usable and have relatively obvious effects.

[0044] Based on viable count of Sphingobacterium thermophilum ≥ 10 7 CFU / mL, Pseudoxanthomonas suwonensis viable count ≥ 10 7 CFU / mL, Bacillus velezensis viable count ≥ 10 7 CFU / mL, and the ratio of the number of live bacteria among the three is 1:1:1. The process of pretreating the mixture of white wine lees, corn cobs and quicklime is as follows:

[0045] (1) Use quicklime to neutralize the acid in the white wine lees and adjust the pH value of the white wine lees to about 7;

[0046] (2) After the white wine lees are treated in step (1), corn cobs are added to adjust the carbon-nitrogen ratio to 20-45;

[0047] (3) After the mixture is treated in step (2), tap water is added to control the water content to 65%, and the mixture is mixed evenly;

[0048] (4) Inoculate 5% of the composite microbial agent into the mixture after treatment in step (3), mix evenly, stack, ferment at an ambient temperature above 15° C. for 24 hours (24 hours is not a definite reference and may fluctuate according to the actual operation process, for example, the deviation may be 0.5 hours before and after).

[0049] In the above mixture, there are 70-80 parts of white wine lees, 15-30 parts of corn cobs, and 2-4 parts of quicklime. Except for the wine lees which are measured by fresh weight, the remaining materials are measured by dry weight. Quicklime is mainly used to neutralize and adjust the acid in the white wine lees so that the pH of the white wine lees meets the requirements. The amount of quicklime added is modified appropriately according to the amount of white wine lees added.

[0050] Based on the above-mentioned method for pretreating the mixture, 70 kg of white wine lees and 25 kg of corn cobs were selected and mixed to obtain a pretreated fermentation mixture. An orthogonal experiment was designed, and the factors of the orthogonal experiment were the adjusted pH and water content.

[0051] The test method is:

[0052] Adjusting the pH and water content of the fermented mixture to obtain a cultivation material;

[0053] The culture material was placed in a polyethylene bag of 45-55 cm in length and 22-26 cm in width and sterilized by high pressure steam at 120°C for 8 hours;

[0054] After the culture medium is cooled to room temperature, 5% of the culture medium is inoculated with Pleurotus ostreatus and cultured at a temperature of 25°C and a relative humidity of 65% until the mycelium is fully grown;

[0055] When the mushrooms are fully grown, cut them open, keep the room temperature at 20℃, the relative humidity at 85%, and the light at 400lux, and harvest when the mushroom cap grows to 5-10cm and the edge of the mushroom cap is not completely opened;

[0056] Calculate the fresh weight yield of Pleurotus ostreatus harvested per kilogram of culture medium.

[0057] Table 1 is the data of the orthogonal test, factor one is the adjusted pH, ranging from 6 to 8, factor two is the adjusted water content, ranging from 60 to 70%, a total of 66 groups of test data.

[0058]

[0059] Table 1

[0060] According to Table 1, the adjusted moisture content factor has an impact on the fresh weight yield of the Pleurotus ostreatus harvested from the cultivation material, which first increases and then decreases within the range of 60-70%, and the optimal moisture content is about 64%; the adjusted pH also has an impact on the fresh weight yield of the Pleurotus ostreatus harvested from the cultivation material. Referring to the experience of the prior art, the pH adjustment is also set to 6-8. Under this condition, it is found that the fresh weight yield of the Pleurotus ostreatus harvested from the cultivation material also presents an approximate normal distribution, that is, as the pH increases, the fresh weight yield of the harvested Pleurotus ostreatus first increases and then decreases, and the optimal pH is about 7. However, the inventors found that since the difference between different pH levels is only 0.2, when the pH is adjusted to about 8, the fresh weight yield of the harvested Pleurotus ostreatus basically has a not very obvious recovery trend.

[0061] Based on the above findings, the inventors took pH 8 as the starting point to further explore the effect of pH values ​​between 8 and 10 on the fresh weight yield of harvested Pleurotus ostreatus.

[0062] Based on the above-mentioned method for pretreatment of the mixture, 70 kg of white wine lees and 25 kg of corn cobs were selected and mixed to obtain a pretreated fermentation mixture. An orthogonal experiment was designed, and the factors of the orthogonal experiment were the adjusted pH and water content.

[0063] The test method is:

[0064] Adjusting the pH and water content of the fermented mixture to obtain a cultivation material;

[0065] The culture material was placed in a polyethylene bag of 45-55 cm in length and 22-26 cm in width and sterilized by high pressure steam at 120°C for 8 hours;

[0066] After the culture medium is cooled to room temperature, 5% of the culture medium is inoculated with Pleurotus ostreatus and cultured at a temperature of 25°C and a relative humidity of 65% until the mycelium is fully grown;

[0067] When the mushrooms are fully grown, cut them open, keep the room temperature at 20℃, the relative humidity at 85%, and the light at 400lux, and harvest when the mushroom cap grows to 5-10cm and the edge of the mushroom cap is not completely opened;

[0068] Calculate the fresh weight yield of Pleurotus ostreatus harvested per kilogram of culture medium.

[0069] Table 2 shows the data of orthogonal test. Factor 1 is the adjusted pH, ranging from 8 to 10, and factor 2 is the adjusted water content, ranging from 60 to 70%. There are 66 sets of test data in total, of which pH 8 is used.

[0070] The data results for the same pH values ​​in Table 1.

[0071]

[0072] Table 2

[0073] According to Table 2, when the adjusted pH is 8-10, the fresh weight yield of Pleurotus ostreatus harvested from the cultivation material has a rapid increase stage after the pH exceeds 8. The optimal pH of this stage is about 8.6, and the fresh weight yield of Pleurotus ostreatus harvested from the cultivation material reaches a maximum of 0.78kg, which is significantly better than 0.64kg when the pH is 7. Moreover, when the pH is 8.6, the optimal water content is increased to about 68%.

[0074] The cost of the oyster mushroom cultivation material of the present invention is significantly reduced, and the economic benefits of oyster mushroom cultivation are improved. The market price of cottonseed hulls is 2400 to 2800 yuan / ton, while the price of white wine lees is only 100 to 200 yuan / ton. The formula for making oyster mushroom cultivation material using white wine lees provided by the present invention greatly saves the cost of cultivation material. At the same time, the lees are pretreated by microbial fermentation and used as oyster mushroom cultivation material, which has the advantages of simpler operation and less energy consumption compared with the traditional water washing, sun drying and drying methods.

[0075] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them.

Claims

1. A method for cultivating Pleurotus ostreatus using white wine lees, characterized in that: Stir and mix the white wine lees, corn cobs and quicklime and adjust the water content to 50%-70%, 70-80 parts of white wine lees, 15-30 parts of corn cobs and 2-4 parts of quicklime. After inoculating the composite microbial agent for fermentation, adjust the pH of the fermentation material to 8.4-9 and the water content to 60%-70%; After the fermentation materials are bagged and sterilized, 5% to 20% of Pleurotus ostreatus cultivars are inoculated and cultured for 20 to 30 days at a controlled temperature of 20 to 25°C and a relative humidity of 60 to 70%. The composite microbial agent is composed of thermophilic sphingobacillus, pseudoxanthomonas suwanensis and Bacillus velez, and the number of viable thermophilic sphingobacillus is ≥10 7 CFU / mL, Pseudomonas suwanensis live bacteria count ≥ 10 7 CFU / mL, number of viable Bacillus Velezii ≥ 10 7 CFU / mL, the ratio of the number of live bacteria among the three is 1:1:

1.

2. The method for cultivating Pleurotus ostreatus using white wine lees according to claim 1, characterized in that: When the mushrooms are fully grown, make a cut, keep the room temperature at 15-20℃, the relative humidity at 80-90%, and the light at 300-500 lux, and harvest when the cap grows to 5-10 cm and the edge of the cap is not fully opened.

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