Enzymolysis biomass feed, method for preparing feed by utilizing edible fungus enzymolysis biomass and application

Abstract

The invention relates to the technical field of fermented feeds, in particular to an enzymolysis biomass feed, a method for preparing a feed by utilizing edible fungus enzymolysis biomass and application. According to the method disclosed by the invention, the beta-mannan in the biomass (palm meal, vinasse and corn straw) is successfully degraded by screening the edible fungi and optimizing an enzyme-producing culture medium by utilizing the characteristic that the large edible fungi produce the beta-mannanase, so that anti-nutritional factors in the biomass are reduced, and the yield of the beta-mannanase is increased. Effective development of agricultural and sideline product wastes is realized, the rumination digestibility of the obtained enzymolysis biomass feed is also improved, and the problem of shortage of feed resources is relieved. The method for preparing the feed by using the edible fungus enzymolysis biomass provided by the invention is simple and convenient to operate, free of environmental pollution problems, suitable for industrial development and utilization of agricultural and sideline product wastes, wide in application prospect, and also has important significance on sustainable development of animal husbandry.

Description

Technical Field

[0001] The present invention relates to the technical field of fermented feed, and in particular to an enzymatically hydrolyzed biomass feed, a method for preparing feed by enzymatically hydrolyzing biomass using edible fungi, and application thereof. Background Art

[0002] With the continuous development of animal husbandry, the demand for animal feed is increasing day by day, and the animal husbandry industry is facing the severe test of feed resource shortage. In order to alleviate the shortage of feed resources, especially the shortage of energy feed and protein feed resources, the development and utilization of agricultural and sideline product waste is a good way.

[0003] Agricultural and sideline product waste refers to waste from agricultural and sideline product processing, such as corn stalks, rice bran, cassava residue, rapeseed meal, palm meal, distiller's grains, etc. Among them, palm meal is the residue after palm fruit oil extraction. At present, the global production of palm kernel meal is about 7 million tons per year. Due to its huge output, low price and suitable nutritional content, it has the potential for wide application in the livestock and poultry feed industry. Although palm meal contains high nutrients and rich mineral elements, it also contains extremely high anti-nutritional factors, such as β-mannan. Other waste from agricultural and sideline product processing, such as corn stalks and distiller's grains, also contain β-mannan. In the process of feed utilization, β-mannan can easily cause nutritional diarrhea in livestock and poultry, limit its nutrient digestibility, and even cause the death of young animals. Therefore, how to degrade β-mannan in agricultural and sideline product waste, so as to achieve the effective development of agricultural and sideline product waste, improve feed digestibility, and alleviate the problem of feed resource shortage, is of great significance to the utilization of agricultural and sideline product waste and the sustainable development of animal husbandry. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing feed by enzymatically hydrolyzing biomass using edible fungi in view of the deficiencies of the prior art.

[0005] The technical problem that the present invention needs to solve is to provide an enzymatic biomass feed prepared by the above method.

[0006] The final technical problem to be solved by the present invention is to provide the application of the above enzymatically hydrolyzed biomass feed in feeding ruminants.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] A method for preparing feed by enzymatically hydrolyzing biomass using edible fungi, wherein the edible fungi producing beta-mannanase are activated and placed in an enzyme production medium for enzyme production culture, and then the cultured edible fungi are inoculated into a mixed medium containing biomass for enzymatic hydrolysis culture to prepare enzymatically hydrolyzed biomass feed;

[0009] Wherein, the edible fungus is an edible fungus of the genus Ganoderma.

[0010] Specifically, the edible fungi of the genus Ganoderma include but are not limited to any one or a combination of Ganoderma lucidum, Ganoderma rubrum and Ganoderma rubrum. Edible fungi belonging to the genus Ganoderma and capable of producing β-mannan in the prior art are all within the scope of protection of the present invention.

[0011] In some embodiments of the present invention, the edible fungi are GXGD-EF-2 (Ganoderma lucidum), GXGD-EF-3 (Ganoderma lucidum-22), GXGD-EF-4 (Ganoderma lucidum-33), GXGD-EF-5 (Red Ganoderma), GXGD-EF-6 (Sessile Ganoderma), GXGD-EF-7 (Taishan Red Ganoderma), GXGD-EF-61 (White Ganoderma), GXGD-EF-65 (Ganoderma lucidum), GXGD-EF-66 (Red Ganoderma), and GXGD-EF-81 (Red Ganoderma).

[0012] The enzyme production culture medium has a formula of: 150-300 g / L potato, 25-35 g / L carbon source, 3-13 g / L enzymatic substrate, and 1-5 g / L KH2PO4.

[0013] Specifically, the carbon source is any one of glucose, brown sugar, and molasses, or a combination of them, preferably brown sugar.

[0014] Specifically, the enzymatic hydrolysis substrate is any one or a combination of konjac flour, locust bean gum, and locust bean gum, preferably konjac flour.

[0015] Preferably, the enzyme production culture medium has a formula of: 150 g / L potato, 30 g / L brown sugar, 8 g / L konjac flour, and 3 g / L KH2PO4.

[0016] The culture conditions for the enzyme production culture are: 24-35°C, 100-200 rpm for 3 to 10 days. The preferred culture conditions are: 26°C, 150 rpm for 6 days.

[0017] Wherein, the inoculation amount is 2-15% v / v, and the preferred inoculation amount is 10% v / v.

[0018] The mixed culture medium containing biomass, based on the dry weight of raw materials, has a formula of: 0-50wt% palm meal, 0-50wt% Luzhou-flavor distiller's grains, 0-50wt% corn straw, 0-2wt% KH2PO4, and water is added to adjust the water content to 50-60wt% of the total weight. Specifically, the total weight is the sum of the weight of water and the dry weight of raw materials.

[0019] In some embodiments of the present invention, the formula is: 33wt% palm meal, 33wt% Luzhou-flavor distiller's grains, 33wt% corn straw, 1wt% KH2PO4, and water is added to adjust the water content to 50wt% of the total mass.

[0020] The enzymatic hydrolysis culture has the following culture conditions: constant temperature of 24-35°C and constant humidity of 80-90% for 20-30 days.

[0021] The β-mannanase activity is defined as: under the conditions of 37° C. and pH 5.5, the amount of enzyme required to release 1 micromole of reducing sugar from a 3 mg / mL mannan solution per minute is one β-mannanase activity unit (U).

[0022] Among them, the detection method of β-mannanase activity refers to "GBT36861-2018 Feed Additives-Determination of β-mannanase activity". Principle: β-mannanase can degrade mannan into oligosaccharides and monosaccharides, and reducing oligosaccharides and monosaccharides produce a color reaction with 3,5-dinitrosalicylic acid (DNS) reagent in a boiling water bath. The color depth of the reaction solution is proportional to the amount of reducing sugar produced by enzymatic hydrolysis, and the amount of reducing sugar produced is proportional to the enzyme activity of β-mannanase in the reaction solution.

[0023] Specifically, the 3,5-dinitrosalicylic acid (DNS) reagent is prepared as follows: weigh 3.15 g of 3,5-dinitrosalicylic acid (chemically pure), add 500 mL of water, and stir in a 45°C water bath for 5 s. Then slowly add 100 mL of 200 g / L sodium hydroxide solution and stir continuously until the solution is clear and transparent (during the addition of sodium hydroxide, the solution temperature should not exceed 48°C). Then gradually add 91.00 g of potassium sodium tartrate tetrahydrate, 2.50 g of phenol and 2.50 g of anhydrous sodium sulfite, continue heating in a 45°C water bath, add 300 mL of water, and stir continuously until completely dissolved. After cooling to room temperature, dilute to 1000 mL with water. Filter, take the filtrate, store in a brown plastic bottle, and keep away from light. Use after 7 days of storage at room temperature, with a shelf life of 6 months.

[0024] The enzymatic biomass feed prepared by the method of preparing feed by enzymatically hydrolyzing biomass using edible fungi is also within the scope of protection of the present invention.

[0025] The application of the enzymatically hydrolyzed biomass feed in feeding ruminants is also within the scope of protection of the present invention.

[0026] Specifically, by testing the in vitro ruminant digestibility before and after enzymatic hydrolysis, it was found that the ruminant digestibility after 15 days of enzymatic hydrolysis was 49.180%, which was 77.4% higher than that of the raw material. The anti-nutritional factor of β-mannan was greatly reduced, which improved the nutrient digestion of enzymatic biomass feed.

[0027] Beneficial effects:

[0028] (1) The present invention utilizes the property of large edible fungi to produce β-mannanase, screens edible fungi, and optimizes the enzyme-producing culture medium to successfully degrade β-mannan in biomass (palm meal, distiller's grains, corn stalks). This not only reduces the anti-nutritional factors in the biomass and realizes the effective development of agricultural and sideline product waste, but also improves the ruminant digestibility of the feed, alleviating the problem of feed resource shortage.

[0029] (2) The method for preparing feed by enzymatically hydrolyzing biomass using edible fungi provided by the present invention is simple and convenient to operate, has no environmental pollution problem, is suitable for the industrial development and utilization of agricultural and sideline product waste, has broad application prospects, and is of great significance to the sustainable development of animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more clear.

[0031] Figure 1 The standard curve of β-mannanase.

[0032] Figure 2 This is the enzyme activity curve of β-mannanase in GXGD-EF-6.

[0033] Figure 3 This is the flat plate image of GXGD-EF-6.

[0034] Figure 4 This is the mycelium image of GXGD-EF-6 under a 40× eyepiece.

[0035] Figure 5 This is a state diagram of GXGD-EF-6 in enzyme-producing liquid culture medium.

[0036] Figure 6 This is the β-mannanase activity curve of GXGD-EF-6 in the fourth enzyme production medium.

[0037] Figure 7 This shows the changes of the three elements in the raw material before enzymatic hydrolysis and after 15 days of enzymatic hydrolysis.

[0038] Figure 8 The ruminant digestibility of the raw material before enzymatic hydrolysis and after 15 days of enzymatic hydrolysis is shown in the figure. The dry matter digestibility in the figure is the ruminant digestibility. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0040] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0041] Example 1: Screening of large edible fungi producing β-mannan

[0042] 10 strains of large edible fungi of the genus Ganoderma (Ganoderma Karst.) were retrieved from the laboratory strain library of the High-Tech University of Technology Biotechnology Research Institute Co., Ltd. The sources of the 10 strains of large edible fungi of the genus Ganoderma are shown in Table 1 below.

[0043] Table 1 Basic information of 10 large edible fungi

[0044] Laboratory No. name Full board cycle (days) source GXGD-EF-2 Lingzhi 8 1 GXGD-EF-3 Lingzhi-22 12 2 GXGD-EF-4 Lingzhi-33 6 2 GXGD-EF-5 Red Ganoderma 6 1 GXGD-EF-6 Ganoderma resinaceum 6 2 GXGD-EF-7 Taishan Ganoderma 12 3 GXGD-EF-61 Ganoderma leucocontextum 10 1 GXGD-EF-65 Lingzhi 8 1 GXGD-EF-66 Chizhi 10 3 GXGD-EF-81 Chizhi 6 3

[0045] Note: 1. Donated by Kunming Edible Fungi Research Institute

[0046] 2. Purchased at the Lijiang Ancient Town Farmers Market

[0047] 3. Purchased from Guangzhou Longdong Edible Fungi Plant

[0048] The 10 strains were activated on plates and the β-mannanase activity was tested. The specific process is as follows: After the 10 strains were activated with edible fungus mother culture medium, the edible fungus blocks that grew vigorously on the edible fungus mother culture medium were selected. The activated fungus blocks with a diameter of 6 mm were cut with an Oxford cup and inoculated into the first enzyme production medium for β-mannanase. 3 to 5 fungus blocks were inoculated in each culture medium, and cultured at 26°C and 150rpm for 10 days. The β-mannanase of different Ganoderma lucidum fungi was tested every day, and the Ganoderma lucidum with the highest β-mannanase activity was screened out.

[0049] Among them, the formula of the edible fungus mother culture medium is: 20% potato boiled juice, 20g sucrose, 20g agar, add water to 1000mL. Specifically, wash and peel the potatoes, and cut them into small pieces. Weigh 200g of potato pieces, add 1000mL of water, boil for 20 minutes, and filter. Add water to the filtered juice to 1000mL to make 20% potato boiled juice. Add agar and sucrose to the potato boiled juice, boil it, dissolve it, add water, package it, sterilize it at natural pH, 118℃ for 20 minutes, and set aside.

[0050] The formula of enzyme production culture medium is: potato 200g / L, glucose 30g / L, konjac flour 8g / L, KH2PO4 3g / L, natural pH, sterilized at 118℃ for 20 minutes.

[0051] The definition of β-mannanase activity is: the amount of enzyme required to release 1 micromole of reducing sugar from a 3 mg / mL mannan solution per minute at 37°C and pH 5.5 is one β-mannanase activity unit (U).

[0052] The detection method of β-mannanase activity refers to "GBT36861-2018 Feed Additives-Determination of β-mannanase activity". Principle: β-mannanase can degrade mannan into oligosaccharides and monosaccharides. Reducing oligosaccharides and monosaccharides produce a color reaction with DNS reagent in a boiling water bath. The color depth of the reaction solution is proportional to the amount of reducing sugar produced by enzymatic hydrolysis, and the amount of reducing sugar produced is proportional to the enzyme activity of β-mannanase in the reaction solution.

[0053] The specific detection method is as follows: take an appropriate amount of enzyme solution, dilute and fix the volume with acetic acid-sodium acetate buffer solution (0.1mol / L, pH5.5), and control the β-mannanase activity in the diluted enzyme solution to be tested between 0.04U / mL and 0.08U / mL. If the pH of the enzyme solution deviates from 5.5 after dilution, adjust it to 5.5 with 0.1mol / L acetic acid solution or 0.1mol / L sodium acetate solution, and then dilute it appropriately with acetic acid-sodium acetate buffer solution and fix the volume.

[0054] Pipette 4.0mL of acetic acid-sodium acetate buffer solution, add 5.0mL of DNS reagent, and heat in a boiling water bath for 5min. After cooling to room temperature with water, dilute to 25mL with water to prepare a reagent blank solution. Pipette 2mL of each D-mannose standard series solution, add to a 25mL graduated test tube (make 2 parallels for each concentration), then add 2mL of acetic acid-sodium acetate buffer and 5mL of DNS reagent respectively, shake slightly by hand, heat in a boiling water bath for 5min, take out, quickly cool to room temperature with water, and then dilute to 25mL with water. Adjust to zero with the reagent blank solution, and measure the absorbance (A value) at 540nm. Draw a standard curve with D-mannose concentration as the X-axis and absorbance A value as the Y-axis to obtain the linear regression equation. The data for drawing the standard curve are shown in Table 2, and the corresponding DNS standard curve is shown in Figure 1 According to the enzyme activity calculation formula, the β-mannanase activity of 10 large edible fungi was calculated, and the specific results are shown in Table 3.

[0055] Enzyme activity calculation formula: Enzyme activity (U / mL) = 1000*n*((A-A0)*k+b) / (30*M)

[0056] In the formula: A-sample absorbance; A0-blank absorbance; k-standard curve slope; b-standard curve intercept; M-180.2g / mol; 30-enzyme hydrolysis time; 1000-conversion factor; n-sample dilution multiple.

[0057] Table 2 DNS standard curve drawing data

[0058] OD540 absorbance Concentration (g / L) OD540 absorbance Concentration (g / L) 0.048 0.1 0.306 0.5 0.108 0.2 0.374 0.6 0.176 0.3 0.44 0.7 0.243 0.4

[0059] Table 3 β-mannanase activity of 10 large edible fungi

[0060]

[0061] From the β-mannanase activity of these 10 large edible fungi, it can be seen that the β-mannanase activity of the 10 strains showed a trend of increasing first and then decreasing, but the enzyme production capacity was significantly different. On the 6th day, the enzyme activity of the 10 strains reached the highest, among which GXGD-EF-6 had the highest enzyme activity of 2.19U / mL. Figure 2 This is the activity curve of β-mannanase in GXGD-EF-6 strain.

[0062] By observing the morphology of GXGD-EF-6, we can find that ( Figure 3 ), GXGD-EF-6 showed white color on the edible fungus mother culture medium, diffused radially outward, and had a flocculent texture; no exudate was produced, no soluble pigment was produced, and the growth rate was 11.25mm / d. Through the stereomicroscope, the mycelium, spores and other microscopic morphologies of GXGD-EF-6 on the edible fungus solid culture medium were observed ( Figure 4 ), GXGD-EF-6 had thick hyphae at 40 times the pressure, with a hyphae width of 2.42-5.43 μm, diverging in a tree-like shape, and its conidia were round, small in size, and grew in a spherical shape at the top of the branched hyphae. GXGD-EF-6 showed a white hyphae ball shape in the enzyme-producing liquid culture medium, with different sizes ( Figure 5 ).

[0063] Furthermore, the mycelial plate of GXGD-EF-6 was sent to a third-party company (Beijing Ruiboxingke Beijing Ruiboxingke Biotechnology Co., Ltd.) for gene sequence comparison. Its ITS sequence information is shown in SEQ ID NO.1, and the gene sequence was compared by NCBI (www.ncbi.nlm.nih.gov), which was indeed Ganoderma resinaceum.

[0064] SEQ ID NO.1:

[0065] .

[0066] Example 2: Optimization of enzyme production culture

[0067] 1. Optimization of degradation substrate in enzyme production medium

[0068] The degradation substrate konjac flour in the first enzyme production medium was replaced with locust bean gum or carob gum to investigate the enzyme activity of β-mannanase produced by GXGD-EF-6 in the first 6 days of the enzyme production medium. The specific process is as follows:

[0069] Use an Oxford cup to cut off the activated GXGD-EF-6 strain blocks with a diameter of 6 mm, and inoculate them into the second enzyme-producing medium or the third enzyme-producing medium for producing β-mannanase. Inoculate 3 to 5 blocks in each enzyme-producing medium, and culture them at 26°C and 150rpm for 10 days to examine the enzyme activity of GXGD-EF-6 in the enzyme-producing medium for the first 6 days.

[0070] Among them, the formula of the second enzyme-producing culture medium is: 200g / L potato, 30g / L glucose, 8g / L locust bean gum, 3g / L KH2PO4, natural pH, and sterilized at 118°C for 20 minutes.

[0071] The third enzyme production culture medium formula is: 200g / L potato, 30g / L glucose, 8g / L bean gum, 3g / L KH2PO4, natural pH, and sterilized at 118°C for 20 minutes.

[0072] 2. Carbon source optimization

[0073] The carbon source glucose in the first enzyme production medium was replaced with brown sugar or molasses to investigate the enzyme activity of β-mannanase produced by GXGD-EF-6 in the first 6 days of the enzyme production medium. The specific process is as follows:

[0074] Use an Oxford cup to cut off the activated GXGD-EF-6 strain blocks with a diameter of 6 mm, and inoculate them into the third enzyme-producing medium or the fourth enzyme-producing medium for producing β-mannanase. Inoculate 3 to 5 blocks in each enzyme-producing medium, and culture them at 26°C and 150rpm for 10 days to examine the enzyme activity of GXGD-EF-6 in the enzyme-producing medium for the first 6 days.

[0075] The fourth enzyme-producing culture medium formula is: 200 g / L potato, 30 g / L brown sugar, 8 g / L konjac flour, 3 g / L KH2PO4, natural pH, and sterilized at 118°C for 20 minutes.

[0076] The fifth enzyme production culture medium formula is: 200g / L potato, 30g / L molasses, 8g / L konjac flour, 3g / L KH2PO4, natural pH, and sterilized at 118°C for 20 minutes.

[0077] The production of β-mannanase by the GXGD-EF-6 strain under the above different enzyme production medium formulas is shown in Table 4. The β-mannanase activity curve of the GXGD-EF-6 strain using the fourth enzyme production medium is shown in Figure 6 shown.

[0078] Table 4 β-mannanase production of GXGD-EF-6 under different enzyme production medium formulas

[0079]

[0080] From the enzyme activity of β-mannanase produced by GXGD-EF-6 strain in these four groups of enzyme production medium formulas, it can be seen that the enzyme production peaks are different in different formulas. The second and third enzyme production mediums have the highest enzyme activity on the 6th day, and the fourth and fifth enzyme production mediums have the highest enzyme activity on the 5th day. Among them, the fourth enzyme production medium has the highest enzyme activity, reaching 2.39U / mL.

[0081] Example 3: Orthogonal test optimization of enzyme production medium

[0082] The optimal enzyme production medium and enzyme production time of GXGD-EF-6 were obtained by Example 2. This example further optimizes the addition amount of potato, brown sugar, konjac flour and KH2PO4 in the fourth enzyme production medium by orthogonal test method, and takes the enzyme activity value of β-mannanase as the investigation index. The culture process and conditions refer to Example 1 or Example 2. Specifically, the component optimization parameters in the fourth enzyme production medium are shown in Table 5, and the optimization results are shown in Table 6.

[0083] Table 5 Optimization parameters of enzyme production medium components

[0084]

[0085] Table 6 Optimization results of enzyme production medium components

[0086]

[0087] According to the results of the orthogonal experiment, the optimal group is the A1B2C2D2 group, namely: 150g / L potatoes, 30g / L brown sugar, 8g / L konjac flour, 3g / L KH2PO4, natural pH, and sterilization at 118℃ for 20 minutes.

[0088] Example 4: Degradation of raw materials by GXGD-EF-6

[0089] The GXGD-EF-6 strain was inoculated into the optimal enzyme production medium (formula: 150 g / L potato, 30 g / L brown sugar, 8 g / L konjac flour, 3 g / L KH2PO4), 3 to 5 bacterial lumps were inoculated into the enzyme production medium, and cultured at 26°C and 150 rpm for 5 days.

[0090] The cultured GXGD-EF-6 was inoculated into a mixed medium containing palm meal, Luzhou-flavor distiller's grains and corn straw at 10% v / v, mixed evenly, and cultured in a constant temperature and 85% constant humidity incubator at 26°C. The changes in cellulose, hemicellulose and lignin in the samples were detected every 5 days for 30 days.

[0091] The mixed culture medium containing palm meal, Luzhou-flavor distiller's grains and corn straw has a formula of: 33wt% palm meal, 33wt% Luzhou-flavor distiller's grains, 33wt% corn straw, 1wt% KH2PO4, and water is added to adjust the water content to 50wt% of the total mass.

[0092] The steps for determining the content of cellulose, hemicellulose and lignin in the sample are as follows:

[0093] (1) Weigh 0.3000 ± 0.001 g of absolute dry sample into a hydrolysis bottle, mark it, and make replicate samples;

[0094] (2) Add 3.00±0.01mL of 72% sulfuric acid and stir until the sample is fully mixed. After mixing, place in a 30±3℃ water bath for 60±5min, stirring every 5-10min;

[0095] (3) Take out the stirred material, add 84±0.04 mL of deionized water to dilute it to 4%, tighten the knob, invert to mix, treat at 121°C for 1 h, take out, cool, and then open;

[0096] (4) After acid hydrolysis, vacuum filtration was performed using G3 glassware, and 50 mL of the filtrate was taken to measure the contents of lignin, cellulose, and xylan;

[0097] (5) Wash the filtered residue with hot deionized water until it is neutral, dry the residue in an oven at 105±3°C until it is absolutely dry, and weigh and record. After drying, transfer it to a muffle furnace and burn it at 575±25°C for 24±6h to measure the ash content. Take it out and cool it to room temperature, then weigh and record. Take a parallel control for each sample. In this way, the content of acid-insoluble lignin is obtained;

[0098] (6) Take 20 mL of the liquid obtained after filtration in step (5) and place it in a conical flask, adjust the pH to 5-6 with NaOH, dilute it appropriately and filter it with a 0.2 μm filter membrane, and analyze the sugar content by HPLC to obtain the sugar concentrations of glucose, arabinose, and xylose. Take a parallel control for each sample.

[0099] The steps for preparing the standard curve are as follows:

[0100] Weigh 0.1 mg of various sugars (glucose, xylose and arabinose, the closer the concentration is to the sugar concentration in the acid hydrolysis sample, the better) and place them in a hydrolysis bottle, add 10 mL of deionized water, add 348 μL of 72% sulfuric acid, and tighten the knob. Treat at 121°C for 1 hour, take out and cool, open and dilute by multiples, filter with a 0.2 μm filter membrane, and draw a liquid phase. With the sugar concentration as the X-axis and the liquid peak as the Y-axis, draw a standard curve to obtain a linear regression equation.

[0101] Lignin content (AIL%) = (G2-G1) / W×100%

[0102] Where: G2 is the absolute dry weight of the residue after filtration and the glass filter, G1 is the weight of the ash after calcination and the glass filter, and W is the absolute dry weight of the sample.

[0103] Cellulose content (%) = HPLCglu / Rglu×0.9×V / W×100%

[0104] Wherein: HPLCglu is the glucose value read by HPLC; Rglu is the glucose correction factor; 0.9 is the conversion factor of glucose and cellulose; V is the total volume of the filtrate, 0.087 L; W is the absolute dry weight of the sample in g.

[0105] Hemicellulose content (%) = (HPLCxyl / Rxyl+HPLCara / Rara) × 0.88 × V / W × 100%

[0106] Wherein: 0.88 is the conversion coefficient of xylose, arabinose and hemicellulose; V is the total volume of the filtrate, 0.087 L; W is the absolute dry weight of the sample in g; HPLCxyl and HPLCara are the values ​​of xylose and arabinose read by HPLC; Rxyl and Rara are the correction coefficients of xylose and arabinose.

[0107] The high performance liquid chromatography (HPLC) parameters were set as follows: using a differential refractometer, a 300 mm × 7.8 mm chromatographic column, a 5 mM dilute sulfuric acid solution as the mobile phase, a flow rate of 0.6 mL / min, a column temperature of 60 °C, and a detection cell temperature of 35 °C.

[0108] The changes of cellulose, hemicellulose and lignin in the samples before and after enzymatic hydrolysis are shown in Table 7. Figure 7 shown.

[0109] Table 7 Changes of three elements before and after enzymatic hydrolysis of raw materials

[0110] Cellulose(%) Hemicellulose (%) Lignin(%) Comparison 22.66 23.85 19.94 Experimental group - 5 days 19.58 8.31 19.53 Experimental group - 10 days 14.52 9.63 19.20 Experimental group - 15 days 10.72 8.56 19.90 Experimental group - 20 days 15.88 9.49 19.71 Experimental group - 25 days 14.33 9.54 19.37 Experimental group - 30 days 14.01 8.93 19.01

[0111] From the analysis of the changes of the three elements over 30 days, the degradation effect of cellulose was the strongest on the 15th day, followed by the 30th day, 25th day, and 10th day; the degradation effect of hemicellulose was the strongest on the 5th day, followed by the 15th day and 30th day; the enzymatic hydrolysis effect of lignin was average. Due to the high proportion of cellulose, the degradation effect of the three elements after 15 days of enzymatic hydrolysis was the best. Compared with before enzymatic hydrolysis, the cellulose and hemicellulose in the experimental group after 15 days of enzymatic hydrolysis decreased by 58.6% in total.

[0112] In summary, the following conclusions can be drawn: the enzyme activity of GXGD-EF-6 strain was highest when it was cultured in enzyme-producing medium (formula: 150 g / L potato, 30 g / L brown sugar, 8 g / L konjac flour, 3 g / L KH2PO4) on the 5th day. The cultured GXGD-EF-6 strain was inoculated into a mixed medium containing palm meal, Luzhou-flavor distiller's grains and corn straw, and the degradation effect of the three elements in the raw materials was best on the 15th day.

[0113] Example 5: In vitro ruminant digestibility

[0114] The in vitro ruminant digestibility of the raw material before enzymatic hydrolysis and the sample after 15 days of enzymatic hydrolysis was tested as follows:

[0115] (1) Preparation of pepsin solution: 8.4 mL of concentrated hydrochloric acid was added to about 500 mL of deionized water, and then the volume was adjusted to 1 L to prepare a 0.1 mol / L hydrochloric acid solution. 2 g of 1:10000 pepsin (purchased from Beijing Solebow Technology Co., Ltd., model CAS: 9001-75-6) was added to the 0.1 mol / L hydrochloric acid solution to prepare a 0.2% pepsin solution.

[0116] (2) Preparation of cellulase solution: 6.8 g CH3COONa·3H2O or 4.15 g CH3COONa was added to 2.9 mL glacial acetic acid, the pH was adjusted to 4.6, 10 g 1:10000 cellulase was added, and then the volume was adjusted to 1 L to obtain 1 L of buffer solution. (Cellulase was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., model CAS number: 9012-54-8) to fully dissolve it.

[0117] (3) Weigh 0.5 g of the sample and place it in a 50 mL conical flask. Add 50 mL of pepsin solution and seal the flask with plastic wrap (otherwise the hydrochloric acid will evaporate). Incubate the flask at 39°C for 16 h and shake the flask every 4 h.

[0118] (4) After filtering with qualitative filter paper, rinse the filtered residue into a conical flask without loss with 50 mL of cellulase solution, seal with plastic wrap, and incubate at 39°C for 48 hours, shaking every 12 hours. Before filtering, inactivate the cellulase at 90°C for 30 minutes.

[0119] (5) Filter with weighed and numbered quantitative filter paper, rinse the residue with distilled water at least three times, dry the filter paper and residue at 100°C and weigh them.

[0120] The results of in vitro ruminant digestibility of the raw materials before enzymatic hydrolysis and the samples after 15 days of enzymatic hydrolysis are shown in Table 8 and Figure 5 As shown. Wherein, ruminant digestibility (%) = 100 × (M3-(M2-M1)) / M1

[0121] Table 7 Ruminant digestibility of raw materials and enzymatic hydrolysis for 15 days

[0122] Sample name M1 sample / g M2 filter paper / g M3 drying total weight / g Ruminant digestibility / % raw material 0.5010 0.7957 1.1578 27.725 Enzymatic hydrolysis for 15 days 0.5002 0.7865 1.0407 49.180

[0123] According to Table 7 and Figure 8The results of ruminant digestibility show that the rumen digestibility after 15 days of enzymatic hydrolysis is 49.180%, which is 77.4% higher than that of the raw material. This shows that the anti-nutritional factor of β-mannan in this product is greatly reduced, and the nutrient digestion of the palm meal-vinasse-corn straw combination is improved. It can be used as an energy feed raw material for ruminants, alleviate the shortage of feed resources, and reduce feed costs.

[0124] The present invention provides an enzymatic hydrolysis of biomass feed, a method for preparing feed by enzymatic hydrolysis of biomass using edible fungi, and an idea and method for application. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing feed by enzymatically hydrolyzing biomass using edible fungi, characterized in that: The edible fungi producing beta-mannanase are activated and placed in an enzyme production medium for enzyme production culture, and then the cultured edible fungi are inoculated into a mixed medium containing biomass for enzymatic hydrolysis culture to prepare enzymatic biomass feed; Wherein, the edible fungus is an edible fungus of the genus Ganoderma.

2. The method according to claim 1, characterized in that The enzyme production culture medium has a formula of: 150-300 g / L potato, 25-35 g / L carbon source, 3-13 g / L enzymatic substrate, and 1-5 g / L KH2PO4.

3. The method according to claim 2, characterized in that The carbon source is any one of glucose, brown sugar, and molasses, or a combination thereof, preferably brown sugar.

4. The method according to claim 2, characterized in that: The enzymatic hydrolysis substrate is any one or a combination of konjac flour, locust bean gum, and locust bean gum, preferably konjac flour.

5. The method according to claim 1, characterized in that: The enzyme production culture has the following culture conditions: culture at 24-35°C and 100-200 rpm for 3-10 days.

6. The method according to claim 1, characterized in that The inoculation has an inoculation amount of 2 to 15% v / v.

7. The method according to claim 1, characterized in that The mixed culture medium containing biomass has a formula of 0-50wt% palm meal, 0-50wt% Luzhou-flavor distiller's grains, 0-50wt% corn straw, and 0-2wt% KH2PO4 based on the dry basis of raw materials, and water is added to adjust the water content to 50-60wt% of the total mass.

8. The method according to claim 1, characterized in that The enzymatic hydrolysis culture has the following culture conditions: constant temperature of 24-35° C. and constant humidity of 80-90% for 20-30 days.

9. Enzymatically hydrolyzed biomass feed prepared by the method according to any one of claims 1 to 8.

10. Use of the enzymatically hydrolyzed biomass feed according to claim 9 in feeding ruminants.

Images