Mushroom bran fermentation and amino acid extraction process

Abstract

The invention provides a mushroom bran fermentation and amino acid extraction process, and belongs to the technical field of microorganisms. According to the invention, the mushroom bran fermentation material after fruiting is taken as a substrate, the amino acid liquid is extracted by utilizing an enzymolysis method, and the amino acid liquid can be used as an organic component of a water-soluble fertilizer in agricultural production to provide a required nutrient source for plant growth. In addition, the bacillus amyloliquefaciens JK-2 is used as a research object to carry out salt tolerance domestication, and the domesticated strain is used as a microbial component of the water-soluble fertilizer to prepare the microbial agent, so that the application effect of the novel composite water-soluble fertilizer can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a process for fermenting fungus dregs and extracting amino acids thereof. Background Art

[0002] In the mushroom cultivation industry, used mushroom substrates are usually considered as a waste material, namely, mushroom bran. Except for a small part of them being recycled as mushroom culture materials, the general treatment method is to discard or burn them, which not only wastes resources, but also breeds harmful organisms and pollutes the environment. This phenomenon has attracted the attention of scientific researchers.

[0003] At present, many studies have shown that mushroom bran can be used as a sustainable resource. Mushroom bran has rich active ingredients. Compared with the initial fresh matrix, its crude fiber content is greatly reduced, crude protein content is significantly increased, and it is rich in various trace elements. For example, the crude fiber content of vinegar lees and mushroom bran is 30%, crude protein is 9.85%, ash is 16.35%, calcium is 1.69%, and phosphorus is 0.49%. After fermentation, the crude fiber of vinegar lees and mushroom bran dropped to 5.07%, crude protein increased to 24.94%, crude fat was 1.52%, ash was 13.04%, calcium was 1.50%, phosphorus was 0.83%, and the total content of various amino acids was 16.61%.

[0004] Through exploration, it has been found that mushroom bran can be used in promoting the transition to a circular economy. For example, it can be used as compost, a substrate for the growth of other edible fungi, animal feed, promoting animal health, and producing packaging and building materials, biofertilizer and soil improvement. This series of applications can make agricultural production more sustainable and efficient.

[0005] Water-soluble amino acid fertilizer is a new type of functional organic fertilizer that can provide plants with an easily absorbed nitrogen source, promote protein synthesis and photosynthesis, and improve soil structure better than traditional fertilizers. Since amino acid fertilizer is completely soluble in water, it can be easily applied through sprayers, irrigation systems, etc. It is non-toxic and residue-free, meeting the requirements of developing environmentally friendly sustainable agriculture. Summary of the invention

[0006] The present invention uses the fermented material of mushroom husk after mushroom production as a substrate, and uses enzymatic hydrolysis to extract amino acid liquid, which can be used as an organic component of water-soluble fertilizer in agricultural production to provide the required nutrient source for plant growth. In addition, the laboratory-preserved Bacillus amyloliquefaciens JK-2 was used as a research object, and salt tolerance was domesticated. After domestication, the strain was used as a microbial component of water-soluble fertilizer to prepare a bacterial agent, which can increase the application effect of the new composite water-soluble fertilizer. Explore the effect of the new amino acid microbial water-soluble fertilizer on plant growth and development.

[0007] The invention aims to provide a process for extracting amino acids from fungus husks.

[0008] Common amino acid extraction processes include acid hydrolysis, alkaline hydrolysis and enzymolysis. Among them, the acid-base hydrolysis amino acid extraction method is low in cost, simple, rapid and thorough, but it easily leads to the destruction of the tryptophan structure, produces waste acid to pollute the environment, has a long reaction time, and easily destroys the amino acid structure. The enzymolysis method has a short reaction time, mild conditions, does not require special conditions and equipment, does not cause racemization, and does not destroy the amino acid structure. Therefore, the present invention uses enzymatic hydrolysis to extract amino acids.

[0009] The overall process of extracting amino acids by enzymatic hydrolysis of the invention comprises the following steps: crushing of fungus husks - screening - water bathing - enzymolysis - enzyme inactivation - centrifugation - pigment removal - concentration - purification.

[0010] The invention provides changes in temperature, pH, water content, C / N, amino acid content and other indicators during the processing of mushroom husks and the fermentation process, and in particular designs different enzyme types and proportions, enzymatic reaction pH, temperature and time to screen the best amino acid enzymatic hydrolysis process: when flavor enzyme: papain: cellulase are compounded and extracted in a ratio of 1:1:1 and the total addition amount is 0.4%, the amino acid content is the highest; and the amino acid content is the highest when the reaction is carried out at 50°C for 5 hours.

[0011] The present invention performs salt tolerance acclimation on strain JK-2 disclosed in Chinese patent CN202311702691.5.

[0012] The strain JK-2 used in the present invention is Bacillus amyloliquefaciens JK-2 disclosed in Chinese patent CN202311702691.5. In the present invention, the strain is salt-tolerant and then compounded with the extracted amino acid solution for co-application, wherein the salt-tolerant concentration gradient is set to 1%, 2%, 3%, 5%, 7% and 10%. The specific salt-tolerant acclimation operation is as follows: the salt concentration is gradually increased. LB culture medium with the above 6 salt concentrations, i.e., 6 NaCl concentrations, is prepared. First, the bacterial lawn of the JK-2 strain is inoculated into an LB culture medium containing 1% NaCl, placed in a shaking table at 37°C and 200r / min to culture until the bacterial solution is turbid, as a seed solution, and a plate count is performed; the bacterial solution in the 1% NaCl culture medium is inoculated into an LB culture medium containing 2% NaCl at a 10% inoculation amount again, and placed in a shaking table at 37°C and 200r / min to culture until the bacterial solution is turbid, and a plate count is performed; and so on. Observe its growth under different salt concentrations.

[0013] The invention uses a novel amino acid microbial water-soluble fertilizer compounded with a domesticated salt-tolerant strain and an amino acid liquid phase as a compounding agent for habitat stress. After being gradually domesticated at a salt concentration of 1%-10%, the amyloliquefaciens Bacillus JK-2 of the invention can tolerate 10% salt stress and can grow relatively stably under 1%-10% salt stress, indicating that JK-2 can resist a salt stress environment, providing an important basis for subsequent experiments.

[0014] Instruction Manual

[0015] Figure 1 This is the temperature change diagram of the pile during the fermentation process;

[0016] Figure 2 This is the change diagram of the moisture content of the pile;

[0017] Figure 3 is the pH change diagram;

[0018] Figure 4 It is the EC value change diagram;

[0019] Figure 5 It is the graph of organic matter changes;

[0020] Figure 6 It is the N content change diagram;

[0021] Figure 7 This is the trend diagram of carbon-nitrogen ratio (C / N);

[0022] Figure 8 This is the trend chart of seed germination index changes;

[0023] Fig. 9 It is the trend chart of amino acid content;

[0024] Fig.10 This is a color change trend chart during the fermentation of the pile;

[0025] Fig.11 It is the amino acid content diagram under different enzyme types and ratios;

[0026] Fig.12 It is the amino acid content diagram under different enzymatic reaction temperatures;

[0027] Fig.13 It is the amino acid content diagram under different enzymatic reaction time;

[0028] Fig.14 The figure shows the change of effective viable bacterial count under different salt concentrations of the culture medium. DETAILED DESCRIPTION

[0029] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] Example 1 Mushroom husk treatment and fermentation

[0031] The test fungus residues were collected from a mushroom shed for edible fungi cultivation in Hebei Province, and the discarded mushroom sticks after picking were crushed by a crusher. The strip-type aerobic composting was adopted, and the pre-treatment steps were as follows: the strip-type aerobic composting was conducted, and the strip-type aerobic composting was conducted, and the pre-treatment steps were as follows: the strip-type aerobic composting was conducted, and the strip-type aerobic composting was conducted, and the pre-treatment steps were as follows: the strip-type aerobic composting was conducted, and the crushed strip-type aerobic composting was conducted, and the moisture content was adjusted to about 50%, the C / N content was 28:1, the amount of the decomposing agent added was 0.5‰-1‰, the pH value was about 6, and the strip-type aerobic composting was conducted first, and the strip-type aerobic composting was conducted after 24 hours (flat-topped trapezoidal composting), and the strip-type aerobic composting was conducted after 24 hours, ...

[0032] Sample collection and determination: The five-point sampling method was used to take samples from the top, center and around the mushroom bran pile. After mixing evenly, 500g of the sample was taken to determine the changing trends of indicators such as moisture content, pH, organic matter, total nitrogen content, C / N, EC value, seed germination index, amino acid content, color and odor, and to determine whether it was fully decomposed.

[0033] (1) Temperature

[0034] The average value of the readings at different positions is taken as the temperature of the pile body for this measurement. The data is read once at 10 a.m. and 4 p.m., and the average value is taken as the temperature value for that day.

[0035] The results are as follows Figure 1 As shown in the figure, the temperature of the pile body during the fermentation process showed a rapid temperature rise period, a stable middle period, and then a temperature drop and maintenance period. The temperature rose to above 60℃ 25 days after the pile was built, and the highest temperature during the composting process was 61.2℃. After each turning of the pile, the temperature dropped and then rose again, and the temperature of the pile body showed a downward trend on the 61st day.

[0036] (2) Water content

[0037] Use a moisture meter. Moisture is an important parameter that affects the rate of compost maturity, and appropriate moisture is a necessary condition for maintaining optimal microbial activity.

[0038] The results are as follows Figure 2 As shown in the figure, the moisture content of the pile showed an overall downward trend, from the initial 40% to the final 18.4%. The temperature increase and microbial activity during the composting period will lead to the loss of moisture, so the moisture content of the pile dropped rapidly.

[0039] (3) pH and EC determination methods

[0040] After each sampling, the material was air-dried and sieved (0.1 mm). The material and distilled water were weighed into a beaker at a ratio of material: water = 1 g: 10 ml. After mixing, the mixture was allowed to stand. The pH was measured with a glass electrode pH meter, and the EC value was determined with a DDSJ-319L conductivity meter.

[0041] The results are as follows Figure 3 As shown, the pH change is generally on an upward trend. As the composting progresses, organic matter such as protein in the pile continues to decompose, releasing alkaline substances and NH3, which increases the pH of the material. The pH is 8.5 at the end of composting.

[0042] In actual research, the degree of compost maturity can be evaluated by regularly monitoring the changes in EC values. Figure 4 It can be seen that the EC value trend during the composting period first increased and then decreased, and finally tended to be stable.

[0043] (4) Organic matter and total nitrogen content

[0044] Determined according to the Ministry of Agriculture and Rural Affairs standard NY / T525-2021 method.

[0045] Organic matter is an important source of microbial life activities. On the one hand, it is decomposed by microorganisms to generate carbon dioxide and water, releasing energy for microorganisms to use, and on the other hand, it exists in the material in the form of stable organic matter. As shown in the figure, after fermentation, the content of organic matter generally shows a downward trend. After the composting is completed, the organic matter content is 26.18%. The organic matter is getting lower and lower because the easily decomposable carbon-containing organic matter in the pile is decomposed, absorbed and utilized by microorganisms, and converted into gases such as CO2 through respiration and discharged from the pile, so the carbon content gradually becomes lower.

[0046] The total nitrogen content during composting increased rapidly in the early stage and stabilized in the middle and late stages, with the highest N content being 1.72% ( Figure 6 ).

[0047] (5) Carbon-nitrogen ratio (C / N)

[0048] Organic matter was determined according to the standard NY525-2021 of the Ministry of Agriculture and Rural Affairs. The amount of pure C was converted from organic matter, and the amount of pure N was converted from crude protein, and the C / N ratio was calculated.

[0049] C / N is often used as one of the indicators to evaluate the maturity of organic waste composting. The decrease of C / N can reflect the humification degree and decomposition process of organic matter. Figure 7 As shown in the figure, the C / N ratio of the bran material decreased with the increase of composting time until it stabilized. The C / N value at the end of fermentation was 15.2.

[0050] (6) Seed germination index

[0051] Seed germination index (GI) is an effective method to evaluate whether a material is phytotoxic. The determination method refers to the seed germination index technical standard in the "New Standard for Commercial Organic Fertilizers NY 525-2021". When the GI reaches 70%, it can be considered to be fully decomposed and non-toxic to plants.

[0052] The results are as follows Figure 8 As shown in the figure, at the end of the fermentation of the fungus bran and the composting, the influence of the fungus bran at different composting periods on the germination index of cucumber seeds was measured. The germination index continued to rise in the early stage and began to decline in the later stage, but it was never lower than 80%.

[0053] (7) Amino acid content

[0054] The amino acid content was determined according to the method of GB / T18246-20193. Samples were taken at each compost turning and at the end of fermentation.

[0055] The results are as follows Fig. 9 As shown in the figure, the amino acid content in the pile increases with the fermentation time, showing an upward trend. At the end of fermentation, the amino acid content in the pile reaches 3.3%.

[0056] (8) Color and smell

[0057] When the color of the pile changes from light to dark during fermentation, and the smell becomes more and more like the smell of soil, it means that the composting is more complete ( Fig.10 ).

[0058] Example 2 Extraction of amino acid solution from decomposed fungus husk

[0059] Common amino acid extraction processes include acid hydrolysis, alkaline hydrolysis and enzymolysis. Among them, the acid-base hydrolysis amino acid extraction method is low in cost, simple, rapid and thorough, but it easily leads to the destruction of the tryptophan structure, produces waste acid to pollute the environment, has a long reaction time, and easily destroys the amino acid structure. The enzymolysis method has a short reaction time, mild conditions, does not require special conditions and equipment, does not cause racemization, and does not destroy the amino acid structure. Therefore, the present invention uses enzymatic hydrolysis to extract amino acids.

[0060] Overall process: mushroom bran crushing - screening - water bath - enzymatic hydrolysis - enzyme inactivation - centrifugation - pigment removal - concentration - purification.

[0061] Dry the decomposed fungus bran at 105°C to constant weight, crush, and pass through a 40-mesh sieve to obtain uniform fungus bran powder and remove impurities. Accurately weigh 50g of fungus bran powder, mix it evenly with distilled water at a solid-liquid ratio of 1:25g / mL, soak it at 70°C for 3h (pay attention to stirring), separate the soaking liquid and microwave it for 3min, take the filter residue and repeat the above steps again, wait for the 2 soaking liquids to cool to room temperature, perform enzymolysis, then heat to 95°C to inactivate the enzyme for 15min, centrifuge (10000r / min) for 6min, collect the filtrate, pass it through 101 macroporous resin to remove pigments, and concentrate the filtrate on a rotary evaporator at a concentration ratio of 1:10g / mL. After cooling, add anhydrous ethanol to precipitate and remove impurities, leave overnight, centrifuge, take the supernatant, and obtain amino acid liquid.

[0062] The enzymatic hydrolysis process was designed with different enzyme types and proportions, enzymatic reaction pH, temperature and time to screen the optimal amino acid enzymatic hydrolysis process.

[0063] (1) Screening of optimal enzyme types and ratios

[0064] Flavor enzyme (endoprotease cuts the peptide bond inside the polypeptide to form short-chain peptides), papain (has a wide range of proteolytic functions on short-chain polypeptides, proteins, esterified amino acids and amide bonds) and cellulase (can decompose cellulose into oligosaccharides or monosaccharides) are selected for the ratio of single enzyme and compound enzyme, the total amount of enzyme added is 0.4%, and the mixture is heated at a constant temperature of 50°C for 5 hours. Taking the amino acid content as an indicator, the single enzyme or compound enzyme with the best extraction effect is selected to screen the ratio of the compound enzyme.

[0065] The results show that flavor enzyme and papain have better extraction effects. When the three enzymes are combined in a ratio of 1:1:1 for extraction, the amino acid content is the highest. Therefore, it is most appropriate to select a composite enzyme with a ratio of flavor enzyme: papain: cellulase = 1:1:1 and a total addition amount of 0.4%.

[0066] Table 1 Enzyme types and proportions

[0067]

[0068] (2) Screening of the optimal enzymatic reaction temperature

[0069] After determining the dosage of the complex enzyme, the reaction temperature was optimized. The constant temperature water bath was set to different temperatures (35°C, 40°C, 45°C, 50°C, 55°C), the water bath time was 5h, and the optimal enzymatic temperature was determined by measuring the amino acid content. Fig.12 As shown in the figure, when the enzymatic reaction temperature is 50℃, the amino acid content is the highest. Therefore, the optimal reaction temperature is 50℃.

[0070] (3) Screening of the optimal enzymatic reaction time: After determining the amount of complex enzyme and reaction temperature, screen the optimal water bath time. Set the constant temperature water bath to different times (2h, 3h, 4h, 5h, 6h), and determine the optimal reaction time by measuring the amino acid content. The results are as follows Fig.13 As shown in the figure, when the enzymatic reaction time is 5h for extraction, the amino acid content is the highest. Therefore, the optimal reaction temperature is 5h.

[0071] Example 3 Salt tolerance acclimation of strain JK-2

[0072] (1) Using Bacillus amyloliquefaciens JK-2 disclosed in Chinese invention patent CN202311702691.5 for salt tolerance acclimation:

[0073] (2) Compounding the salt-tolerant domesticated strain with the amino acid solution extracted under the optimal conditions screened in Example 2:

[0074] Bacterial solution:amino acid solution=1:1.

[0075] (3) Application was conducted in indoor potted plant experiments. The test plants were radish, chili pepper, and Chinese cabbage.

[0076] Different treatments were designed, including application of bacterial agent alone, application of amino acid solution alone, application of bacterial agent + amino acid solution compound solution and CK. The application was performed once a week. After 2 months, the growth of the plants was observed and it was found that the application of bacterial agent + amino acid solution compound solution had the best effect and the best growth.

[0077] The results are as follows Fig.14 As shown, after gradual acclimation to 1%-10% salt concentration, Bacillus amyloliquefaciens JK-2 can tolerate 10% salt stress and can grow relatively stably under 1%-10% salt stress, indicating that JK-2 can resist the salt stress environment, providing an important basis for subsequent experiments.

[0078] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting amino acids from fungus husks, characterized in that: The method comprises the following steps: mixing the mushroom dregs with auxiliary materials, adjusting the C / N ratio to 24:1-30:1, and then starting aerobic composting to obtain decomposed mushroom dregs; The decomposed mushroom bran is dried to a constant weight, then crushed, soaked in water, and the extract is separated and then subjected to microwave treatment; The liquid treated by microwaves is subjected to enzymatic hydrolysis, and the filtrate is collected by centrifugation after the enzyme is inactivated; the enzyme used for the enzymatic hydrolysis comprises at least one of the following: flavor enzyme, papain and cellulase; The pigment in the filtrate is removed and then concentrated. The impurities in the concentrated solution are removed by using anhydrous ethanol and then the supernatant is collected to obtain an amino acid solution.

2. The method according to claim 1, characterized in that: The auxiliary materials include C / N regulating materials, a composting agent and a pH regulator. The amount of the composting agent added is 0.5-1‰ based on the mass of the mushroom husk, and the pH value is 5.5-6.

5.

3. The method according to claim 1, characterized in that: The water content of the mixture of the mushroom bran and the auxiliary materials is 45% to 50%.

4. The method according to claim 3, characterized in that: During the aerobic composting, the compost is turned every 4 to 5 days, and the total composting period is not less than 80 days.

5. The method according to claim 1, characterized in that: The material-liquid ratio during the soaking is 1:25 g / mL, the soaking temperature is 70° C., and the soaking time is 3 hours.

6. The method according to claim 1, characterized in that: The power of the microwave treatment is 600W and the time is 3min.

7. The method according to claim 1, characterized in that: The centrifugal speed is 10000 r / min, the time is 6 min, the filtrate is collected, and the pigment is removed by 101 macroporous resin. The filtrate is concentrated by rotary evaporator at a concentration ratio of 1:10 g / mL.

8. The method according to claim 1, characterized in that: The temperature of the enzymolysis is 45-55° C., and the time is 4-6 hours.

9. Fungus bran amino acids obtained by the method according to any one of claims 1 to 8.

10. Use of the fungus bran amino acid according to claim 9 in preparing a compound agent for habitat stress.

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