Method for cultivating edible mushrooms by utilizing distillers' grains

Abstract

The invention discloses a method for cultivating edible mushrooms by utilizing vinasse, which comprises the following steps: S1, pretreating Maotai-flavor vinasse: crushing the Maotai-flavor vinasse until the particle size is 1-3mm, and removing impurities to obtain vinasse coarse powder; s2, tannin degradation: adding a tannin degradation agent into the vinasse coarse powder for fermentation to obtain tannin degraded vinasse; s3, crude fiber degradation: adding a crude fiber degradation aid into the tannin degraded vinasse, adjusting the water content of the system to 55-60%, and performing enzymolysis at 32-36 DEG C for 12-24 hours to obtain enzymolysis soy sauce flavor type vinasse; s4, preparation of a culture medium: uniformly mixing 65-75 parts by weight of the enzymolysis Maotai-flavor vinasse, 0.3-1.0 part by weight of monopotassium phosphate, 0.9-1.3 parts by weight of magnesium sulfate, 0.8-2.2 parts by weight of gypsum, 1.0-1.9 parts by weight of calcium superphosphate and 0.5-1.5 parts by weight of a pH buffer agent, and adjusting the pH value to 5-8 to obtain the edible mushroom culture medium; and S5, inoculating the culture medium with edible fungi.

Description

Technical Field

[0001] This invention relates to the field of distiller's grains recycling, and more specifically to a method for cultivating edible fungi using distiller's grains. Background Technology

[0002] Distiller's grains are solid residues produced during the brewing process, mainly composed of fiber, protein, yeast cells, and incompletely decomposed starch remaining after saccharification and fermentation of grains (such as sorghum, glutinous rice, and barley). Depending on the brewing raw materials and processes, distiller's grains can be categorized into baijiu (Chinese white liquor) grains, beer grains, huangjiu (yellow wine) grains, and rice wine grains, among which the grains from the sauce-aroma type have the highest nutritional value. The brewing process for sauce-aroma baijiu is the most complex and time-consuming of all aroma types, and its core processes directly affect the nutritional composition of the grains, giving them characteristics such as high crude protein content, easily absorbed dietary fiber, and a comprehensive and rich concentration of minerals and vitamins.

[0003] Currently, the uses of soy sauce-flavored distiller's grains include: direct use as feed, simple fermentation as organic fertilizer, and drying as feed or fertilizer. However, there are no reports of using soy sauce-flavored distiller's grains for cultivating edible fungi.

[0004] However, due to the special nature of the brewing process, the lees of sauce-flavored liquor have unique characteristics that cannot be ignored compared with those of light-flavored and strong-flavored liquors. Specifically, they are: (1) High tannin content and strong toxicity: The tannin content of sauce-flavored liquor is 2 to 3 times that of light-flavored liquor. As a polyphenol, tannin is easy to combine with enzyme proteins (such as cellulase and amylase) in the mycelium of edible fungi to form irreversible precipitation, which damages the integrity of the cell membrane and leads to the obstruction of mycelial metabolism; (2) Poor pH stability: Due to the large amount of organic acids (mainly lactic acid and acetic acid) remaining after multiple rounds of fermentation, the initial pH of sauce-flavored liquor is only 3.5-4.0. Although traditional single-adjustment (such as adding lime) can temporarily adjust the pH to 5.0-6.0, the continuous release of organic acids during the cultivation process will cause the pH to drop sharply to below 3.8 within 7-10 days, which is lower than the suitable pH range (4.0-9.0) for the colonization of edible fungi mycelium; (3) Poor flavor synergy: Sauce-flavored lees contain unique sauce-flavored substances (ethyl hexanoate, 4-ethylguaiacol, etc.). If not specifically regulated, these substances will antagonize the metabolites of edible fungi (such as oxalic acid and succinic acid), resulting in a bitter taste in the fruiting body. Therefore, if the above defects can be overcome, sauce-flavored lees can be applied to the cultivation of edible fungi to turn waste into treasure. Summary of the Invention

[0005] The present invention aims to provide a method for cultivating edible fungi using distiller's grains, thereby turning waste into treasure by applying soy sauce-flavored distiller's grains to the cultivation of edible fungi.

[0006] A method for cultivating edible fungi using distiller's grains includes: S1. Pretreatment of soy sauce-flavored lees: Crush the soy sauce-flavored lees to a particle size of 1-3 mm, remove impurities, and obtain coarse lees powder; S2, Tannin Degradation: Tannin-degrading agent is added to the coarse lees powder and fermented to obtain tannin-degraded lees; S3. Crude fiber degradation: Add a crude fiber degradation aid to the tannin-degraded lees, adjust the water content of the system to 55-60%, and enzymatically hydrolyze at 32-36℃ for 12-24 hours to obtain enzymatically hydrolyzed sauce-flavored lees. S4: Culture medium preparation: By weight, take 65-75 parts of enzymatically hydrolyzed soy sauce-flavored distiller's grains, 0.3-1.0 parts of potassium dihydrogen phosphate, 0.9-1.3 parts of magnesium sulfate, 0.8-2.2 parts of gypsum, 1.0-1.9 parts of superphosphate, and 0.5-1.5 parts of pH buffer. Mix them evenly and adjust the pH to 5-8 to obtain the edible fungus culture medium. S5: Inoculate the culture medium with edible fungi.

[0007] Working principle and beneficial effects of the present invention: Due to the high-temperature fermentation process during the brewing of soy sauce-flavored liquor, rice husk clumps and impurities (such as pebbles and aggregated liquor particles) are easily formed. Crushing these clumps to 1-3 mm increases the specific surface area, allowing subsequent tannin-degrading agents and crude fiber-degrading aids to fully contact the liquor particles, thus improving reaction efficiency. Removing impurities also prevents uneven physical properties of the culture medium and inhibits the growth of unwanted microorganisms on the surface of impurities. Tannins, as polyphenols, bind to the enzyme proteins of edible fungi mycelia, inhibiting metabolic activity. By adding tannin-degrading agents, the synergistic effect of microbial fermentation and enzymatic hydrolysis is utilized to specifically break down the ester and glycosidic bonds of tannins, decomposing large tannin molecules into smaller molecules (such as gallic acid and glucose) that can be utilized by edible fungi.

[0008] The crude fiber content of fermented grains in soy sauce-flavored liquor is 18-25%, and the lignin and cellulose form a tight complex that is difficult for edible fungi to degrade with their own enzyme systems. By adding crude fiber degradation aids, cellulase decomposes cellulose, destroys the complex structure, and releases usable carbon sources. The enzymatically hydrolyzed grains are used as a basic raw material, supplemented with inorganic nutrients such as potassium dihydrogen phosphate and magnesium sulfate to supplement the phosphorus and magnesium elements necessary for the growth of edible fungi; gypsum and superphosphate adjust the permeability and calcium content of the culture medium; and pH buffers stabilize the pH of the system, avoiding a sudden drop in pH caused by residual organic acids in the fermented grains, and maintaining the pH at 5-8 to meet the growth needs of edible fungi.

[0009] In the optimized form, the tannin degrading agent is a compound of Aspergillus niger and tannin enzyme preparation, and the amount added is 0.5-2% of the weight of the crude distiller's grains; the weight ratio of Aspergillus niger to tannin enzyme preparation is 1:1, and the enzyme activity of the tannin enzyme preparation is ≥50U / g.

[0010] During fermentation, Aspergillus niger can secrete endogenous tanninases, which form a synergistic effect with exogenous tanninase preparations. The exogenous enzymes rapidly initiate tannin degradation, while the endogenous enzymes continuously catalyze the reaction. An addition amount of 0.5–2% can ensure the optimal ratio of enzyme concentration to substrate, and an enzyme activity ≥50 U / g ensures catalytic efficiency.

[0011] Optimized, the crude fiber degradation aid is a compound of cellulase and xylanase, and the addition amount is 0.2-0.8% of the weight of the crude distiller's grains; the weight ratio of cellulase to xylanase is 0.5-1, the enzyme activity of cellulase is ≥1000U / g, and the enzyme activity of xylanase is ≥800U / g.

[0012] Cellulase mainly breaks down cellulose, while xylanase mainly breaks down hemicellulose. A ratio of 0.5 to 1 can match the natural ratio of cellulose to hemicellulose in the mash of soy sauce-flavored liquor. Adding 0.2 to 0.8% can avoid cost waste due to excessive enzymes or incomplete degradation due to insufficient enzymes.

[0013] Optimized, the pH buffer is a compound of sodium bicarbonate and potassium dihydrogen phosphate in a weight ratio of 0.3 to 0.5.

[0014] Sodium bicarbonate and potassium dihydrogen phosphate form a buffer pair, and a ratio of 0.3–0.5 can be used to precisely control the acidic environment of the fermented grains in soy sauce-flavored liquor: when the acidity of the system increases, sodium bicarbonate neutralizes H+. + When alkalinity increases, potassium dihydrogen phosphate releases H₂. + Maintain a stable pH level between 5 and 8.

[0015] The optimized culture medium further includes 0.2-1.3 parts by weight of Astragalus membranaceus and 1.2-2.2 parts by weight of Polygonum multiflorum, wherein both Astragalus membranaceus and Polygonum multiflorum are pulverized to 80-100 mesh.

[0016] The optimized edible fungi species include shiitake mushrooms, oyster mushrooms, king oyster mushrooms, or button mushrooms. Detailed Implementation

[0017] The following detailed description illustrates the specific implementation method: A method for cultivating edible fungi using distiller's grains includes: S1. Pretreatment of soy sauce-flavored lees: Crush the soy sauce-flavored lees to a particle size of 1-3 mm, remove impurities, and obtain coarse lees powder; S2, Tannin Degradation: Tannin-degrading agent is added to the coarse lees powder and fermented to obtain tannin-degraded lees; The tannin degrading agent is a compound of Aspergillus niger and tannin enzyme preparation, and the amount added is 0.5-2% of the weight of the crude distiller's grains; the weight ratio of Aspergillus niger to tannin enzyme preparation is 1:1, and the enzyme activity of the tannin enzyme preparation is ≥50U / g.

[0018] S3. Crude fiber degradation: Add a crude fiber degradation aid to the tannin-degraded lees, adjust the water content of the system to 60%, and enzymatically hydrolyze at 36℃ for 24 hours to obtain enzymatically hydrolyzed sauce-flavored lees; The crude fiber degradation aid is a compound of cellulase and xylanase, and the amount added is 0.2-0.8% of the weight of the crude distiller's grains powder; the weight ratio of cellulase to xylanase is 0.5-1, the enzyme activity of cellulase is ≥1000U / g, and the enzyme activity of xylanase is ≥800U / g.

[0019] S4: Culture medium preparation: By weight, take 75 parts of enzymatically hydrolyzed soy sauce-flavored distiller's grains, 1.0 part of potassium dihydrogen phosphate, 0.9-1.3 parts of magnesium sulfate, 2.2 parts of gypsum, 1.0-1.9 parts of superphosphate, and 0.5-1.5 parts of pH buffer. Mix them evenly and adjust the pH to 5-8 to obtain the edible fungus culture medium. The pH buffer is a compound of sodium bicarbonate and potassium dihydrogen phosphate with a weight ratio of 0.3-0.5.

[0020] S5: Inoculate the culture medium with edible fungi.

[0021] The following embodiments and comparative examples were designed based on the above method. See Table 1 below:

[0022] Sauce-flavored distiller's grains: purchased from a winery in Guizhou Province, with initial physicochemical indicators of tannin content 3.2%, crude fiber content 22%, pH value 3.8, and moisture content 62%, free from mold and off-odors; Shiitake mushroom spawn: Shiitake mushroom strain L808, purchased from a provincial edible fungi research institute, with qualified spawn vitality test (mycelial germination rate ≥98%).

[0023] Microbial preparations and enzyme preparations: Aspergillus niger preparation (live count ≥10) 8 CFU / g), tannin enzyme preparation (enzyme activity 50U / g), cellulase (enzyme activity 1000U / g), xylanase (enzyme activity 800U / g), all are food grade. Nutritional excipients and buffers: potassium dihydrogen phosphate, magnesium sulfate, gypsum, superphosphate (all analytical grade); sodium bicarbonate (food grade), used to prepare pH buffer systems.

[0024] Inoculation procedure: In a clean bench, use a sterile inoculation needle to pick up the L808 shiitake mushroom spawn and inoculate it into the culture bottle at an inoculation rate of 6% of the culture medium weight. After inoculation, quickly seal the bottle with a sealing film and mark the group and inoculation date. Mycelial growth stage: Place the inoculated culture bottle in a constant temperature and humidity incubator and set the culture parameters as follows: temperature 25℃, relative humidity 70%, CO2 concentration 1500ppm. Incubate in the dark and observe the mycelial growth daily, recording the number of days it takes for the mycelium to fill the bottle. Fruiting body formation stage: When the mycelium fills the bottle, adjust the culture parameters to: temperature 20℃, relative humidity 80%, CO2 concentration 1000ppm, light intensity 250 lux (12 hours of light per day). Continue to cultivate until the fruiting bodies mature (cap diameter 3-5cm, stipe thick and without hollows). Record the cultivation period, harvest the fruiting bodies and weigh them.

[0025] Tannin residue: The tannin residue in the culture medium was determined by the Folin-Ciocalteu method; Crude fiber residue: The crude fiber residue was determined by the acid detergent method (GB / T5009.10-2003); pH fluctuation value: The pH value of the culture medium is measured daily, and the difference between the initial pH (6.5) and the lowest pH during the culture period is recorded as the pH fluctuation value. Fresh weight of fruiting bodies: After harvesting mature fruiting bodies, weigh them using an electronic balance and calculate the average fresh weight of each bottle (average of 3 replicates).

[0026] The following data was obtained, as shown in Table 2:

[0027] Conclusion Analysis: (I) The role and optimal dosage of tannin degrading agents 1. Necessity of Tannin Degrading Agent: Compared with control group 1 (without tannin degrading agent), Example 5 showed that the tannin residue decreased from 1.85% to 0.40%, a reduction of 78.4%; the number of days for mycelium to fill the bottle was shortened from 45 days to 24 days, a reduction of 46.7%; and the fresh weight of fruiting bodies increased from 180g / bottle to 242g / bottle, an increase of 34.4%. This indicates that the tannin degrading agent can effectively eliminate the toxic inhibition of high tannins in soy sauce-flavored liquor lees on shiitake mushroom mycelium, and significantly promote mycelial growth and fruiting body development.

[0028] 2. The tannin residue levels in control groups 4 (tannin enzyme preparation only) and 5 (Aspergillus niger fungicide only) (0.65% and 0.70%) were higher than those in Example 5 (0.40%), while the fresh weight of fruiting bodies (215g / bottle and 212g / bottle) was lower than that in Example 5 (242g / bottle). This demonstrates that the combination of Aspergillus niger fungicide and tannin enzyme preparation has a synergistic effect. Exogenous tannin enzymes rapidly initiate tannin degradation, while endogenous tannin enzymes secreted by Aspergillus niger continuously catalyze the reaction, increasing the degradation efficiency by more than 35%.

[0029] 3. Optimal addition amount: Comparing Examples 1 (0.5%), 5 (1%), and 6 (2%), the tannin residue decreased from 0.75% to 0.39% with increasing addition amount, but the difference in tannin residue between 1% and 2% addition amounts was only 0.01%, and the difference in fresh weight of fruiting bodies was <3%. Considering both cost and effect, the optimal addition amount of tannin degrading agent is 1%.

[0030] (II) The role and optimal parameters of crude fiber degradation aids 1. Necessity of crude fiber degradation aid: Compared with Example 5, control group 2 (without crude fiber degradation aid) showed that the crude fiber residue decreased from 16.5% to 8.0%, a decrease of 51.5%; the fresh weight of fruiting bodies increased from 200g / bottle to 242g / bottle, an increase of 21%. This indicates that it can destroy the lignin and cellulose complex in soy sauce-flavored liquor lees, release usable carbon sources, and solve nutritional problems.

[0031] 2. Optimal Dosage: Comparing Examples 7 (0.2%), 5 (0.5%), and 9 (0.8%), the crude fiber residue decreased from 11.2% to 7.7% with increasing dosage. However, the difference in crude fiber residue between 0.5% and 0.8% dosages was only 0.3%, and the difference in fresh fruiting body weight was <2% (242g / bottle vs. 237g / bottle). Considering the cost of enzyme preparations, the optimal dosage of the crude fiber degradation aid is 0.5%.

[0032] 3. Optimal Enzyme Ratio: The crude fiber residue (8.2%, 8.0%) in Examples 2 (enzyme ratio 0.5, 10.5%) and 5 (enzyme ratio 0.8) was lower than that in Examples 1 (enzyme ratio 0.5, 7.8%) and 3 (enzyme ratio 1, 7.8%), and the fresh weight of the fruiting bodies was also higher (238g / bottle, 242g / bottle). Considering the natural ratio of cellulose to hemicellulose in the mash of soy sauce-flavored liquor (approximately 1:1.2), a weight ratio of cellulase to xylanase of 0.8 is optimal.

[0033] (III) The role and optimal ratio of pH buffer 1. Necessity of pH buffer: Compared with Example 5, control group 3 (without pH buffer) showed a decrease in pH fluctuation from 1.20 to 0.30, a reduction in the number of days for mycelium to fully colonize the bottle from 30 days to 24 days, and an increase in the fresh weight of fruiting bodies from 210g / bottle to 242g / bottle. This indicates that the buffer can inhibit the sudden drop in pH caused by the continuous release of organic acids from the fermented grains of soy sauce-flavored liquor, maintain a stable culture environment, and prevent mycelial metabolism from being hindered.

[0034] 2. Optimal component ratio: The pH fluctuation values ​​(0.30) of Examples 2 (ratio 0.4) and 5 (ratio 0.4) were less than those of Examples 1 (ratios 0.3, 0.45) and Examples 3 (ratios 0.5, 0.25), and the fresh weight of the fruiting bodies was also higher (238g / bottle, 242g / bottle). Although Example 3 had the smallest pH fluctuation, an excessively high proportion of sodium bicarbonate could easily lead to excessively alkaline culture medium, affecting mycelial respiration. Therefore, the optimal weight ratio of sodium bicarbonate to potassium dihydrogen phosphate was 0.4.

[0035] (iv) Optimal combination of various technical parameters Example 5 (1% tannin degrading agent, 0.5% crude fiber degradation aid, cellulase to xylanase ratio 0.8, 1 part pH buffer, buffer ratio 0.4) showed the best performance in all indicators: shortest time for mycelium to fill the bottle (24 days), highest fresh weight of fruiting bodies (242g / bottle), lowest tannin and crude fiber residues (0.40% and 8.0%, respectively), and smallest pH fluctuation (0.30). This combination can achieve a synergistic effect of "tannin degradation, fiber decomposition, and pH stabilization," maximizing the utilization efficiency of soy sauce-flavored distiller's grains and the yield and quality of edible fungi.

[0036] (v) Verification of the blank group and the overall effect The blank group (without any treatment) had mycelium filling the bottle for up to 52 days, and the fresh weight of the fruiting bodies was only 150g / bottle. The tannin and crude fiber residues were as high as 2.90% and 20.5%, respectively, proving that the untreated soy sauce-flavored lees could not meet the growth requirements of edible fungi. In contrast, the fresh weight of the fruiting bodies in Example 5 was 61.3% higher than that of the blank group.

[0037] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for cultivating edible fungi using distiller's grains, characterized in that, include: S1. Pretreatment of soy sauce-flavored lees: Crush the soy sauce-flavored lees to a particle size of 1-3 mm, remove impurities, and obtain coarse lees powder; S2, Tannin Degradation: Tannin-degrading agent is added to the coarse lees powder and fermented to obtain tannin-degraded lees; S3. Crude fiber degradation: Add a crude fiber degradation aid to the tannin-degraded lees, adjust the water content of the system to 55-60%, and enzymatically hydrolyze at 32-36℃ for 12-24 hours to obtain enzymatically hydrolyzed sauce-flavored lees. S4: Culture medium preparation: By weight, take 65-75 parts of enzymatically hydrolyzed soy sauce-flavored distiller's grains, 0.3-1.0 parts of potassium dihydrogen phosphate, 0.9-1.3 parts of magnesium sulfate, 0.8-2.2 parts of gypsum, 1.0-1.9 parts of superphosphate, and 0.5-1.5 parts of pH buffer. Mix them evenly and adjust the pH to 5-8 to obtain the edible fungus culture medium. S5: Inoculate the culture medium with edible fungi.

2. The method for cultivating edible fungi using distiller's grains according to claim 1, characterized in that, The tannin degrading agent is a compound of Aspergillus niger and tannin enzyme preparation, and the amount added is 0.5-2% of the weight of the crude distiller's grains; the weight ratio of Aspergillus niger to tannin enzyme preparation is 1:1, and the enzyme activity of the tannin enzyme preparation is ≥50U / g.

3. The method for cultivating edible fungi using distiller's grains according to claim 2, characterized in that, The crude fiber degradation aid is a compound of cellulase and xylanase, and the amount added is 0.2-0.8% of the weight of the crude distiller's grains powder; the weight ratio of cellulase to xylanase is 0.5-1, the enzyme activity of cellulase is ≥1000U / g, and the enzyme activity of xylanase is ≥800U / g.

4. The method for cultivating edible fungi using distiller's grains according to claim 3, characterized in that, The pH buffer is a compound of sodium bicarbonate and potassium dihydrogen phosphate, with a weight ratio of 0.3 to 0.

5.

5. The method for cultivating edible fungi using distiller's grains according to claim 4, characterized in that, The culture medium also includes 0.2-1.3 parts by weight of Astragalus membranaceus and 1.2-2.2 parts by weight of Polygonum multiflorum, wherein both Astragalus membranaceus and Polygonum multiflorum are pulverized to 80-100 mesh.

6. The method for cultivating edible fungi using distiller's grains according to claim 5, characterized in that, The edible fungi species include shiitake mushrooms, oyster mushrooms, king oyster mushrooms, or button mushrooms.

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