Straw bacterium enzyme compound preparation as well as preparation method and application thereof

Abstract

The invention provides a straw bacterium enzyme compound preparation as well as a preparation method and application thereof. The straw bacterium enzyme compound preparation comprises a microbial compound preparation and an enzyme preparation, the weight ratio of the microbial compound preparation to the enzyme preparation is 19: 1, and the microbial compound preparation comprises a lactic acid bacteria preparation prepared from lactobacillus plantarum, a bacillus preparation prepared from bacillus subtilis and a saccharomycetes preparation prepared from candida utilis. The enzyme preparation is prepared from cellulase. After the corn straw is treated by the bacterial enzyme compound preparation, the neutral detergent fiber content of the corn straw is reduced from 75.64% to 64.32%, the cellulose component is reduced from 40.11% to 17.98%, the fiber component in the corn straw is effectively degraded, and the nutritional quality of feed is improved. The crude protein content of the corn straw is increased from 3.84% to 8.31%, and the acid soluble protein content is increased from 0.90% to 2.13%. The protein component of the corn straw is obviously improved, more free amino acids and small peptides are released, and the absorption rate of the protein component is improved.

Description

Technical Field

[0001] This application relates to the field of biological feed, and specifically relates to a straw enzyme complex preparation, its preparation method and application. Background Art

[0002] As one of the three major food crops in the world, corn has strong adaptability and is widely planted. Straw returning to the field is the main utilization method. Although it can improve soil fertility and carbon content, long-term straw returning may exacerbate soil acidification and increase nitrogen source input. In addition, straw burning not only wastes resources but also pollutes the environment and increases the risk of cardio-pulmonary diseases. Therefore, improving the utilization efficiency of straw resources has become a current research hotspot.

[0003] With the increasing demand for meat products, the overgrazing of beef and mutton sheep has led to the deterioration of the grassland ecological environment. Therefore, it is particularly important to develop and utilize crop wastes such as corn straw as a source of roughage. By reasonably developing and utilizing these resources, not only can the contradiction between feed supply and demand be effectively alleviated, the cost of animal husbandry be reduced, but also environmental protection and sustainable development can be promoted. Therefore, the feed utilization of straw resources has broad development prospects.

[0004] Corn straw is rich in cellulose and hemicellulose, but has a low protein content. Moreover, the fiber components of air-dried and yellowed corn straw have poor quality, and the lignin content is significantly increased, resulting in poor palatability. At the same time, the content of soluble carbohydrates is low and the nutritional value is low. These characteristics limit its application as high-quality feed. Therefore, further treatment is needed to improve the nutritional quality of corn straw, increase the feed utilization level of corn straw, solve the dilemma of corn straw in the application of roughage for ruminants, and provide support for the sustainable development of animal husbandry.

[0005] Patent CN119081967A provides "a highly efficient and safe lignocellulose-degrading composite microbial agent and its application". This method aims to degrade the lignocellulose components of straw crops, release the monosaccharide components contained therein, and improve the problems of low nutritional quality and difficult utilization of straw crops. The content of crude protein and acid-soluble protein in corn straw is low. The current treatment methods mainly focus on improving the fiber components of corn straw, and generally lack the ability to improve the protein components.

[0006] Patent CN118901870A, a general-purpose enzyme complex feed fermenting agent, performs well in the modification of corn straw fibers and also has a certain improvement on the protein components. However, the enzymes involved in this method are more complex, the addition amount is higher, and the economic cost is higher.

[0007] The enzymatic treatment method is an effective way to improve the release ability of nutrients in corn straw. Glycosidase can break down components such as cellulose and hemicellulose in the cell wall of rice bran, converting most of the crude fiber into monosaccharides that are easy to absorb; protease can also degrade some of the released proteins. The microbial fermentation method is also a reliable way to improve the nutritional value of corn straw. Some studies have shown that the microbial cell protein accumulated during growth can be used to improve the protein component of straw feed, thus greatly improving the utilization rate of corn straw. Summary of the Invention

[0008] To overcome the deficiencies in the prior art, such as the insufficient degradation of crude fiber in straw feed and the general lack of ability to improve protein components, the main object of the present invention is to provide a bacterial enzyme complex preparation for straw, its preparation method and application, which solves the problems of low utilization rate of crude fiber in straw feed and lack of protein nutrition.

[0009] To achieve the aforementioned invention object, the technical solutions adopted by the present invention include:

[0010] A Bacillus subtilis N3, deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC NO.29518, the deposit date being January 4, 2024, and the taxonomic name being Bacillus subtilis.

[0011] The application of the Bacillus subtilis N3 in the preparation of a bacterial enzyme complex preparation for straw.

[0012] A bacterial enzyme complex preparation for straw, comprising a microbial complex preparation and an enzyme preparation. The weight ratio of the microbial complex preparation to the enzyme preparation is 19:1. The microbial complex preparation includes a lactic acid bacteria preparation prepared from Lactobacillus plantarum, a bacillus preparation prepared from Bacillus subtilis N3, and a yeast preparation prepared from Candida utilis. The enzyme preparation is obtained from cellulase.

[0013] The volume ratio of the lactic acid bacteria preparation, the bacillus preparation, and the yeast preparation is (2 - 6):(1 - 2):(2 - 4).

[0014] The volume ratio of the lactic acid bacteria preparation, the bacillus preparation, and the yeast preparation is 2:1:2.

[0015] A preparation method of a bacterial enzyme complex preparation for straw, comprising the following steps:

[0016] (1) Prepare a lactic acid bacteria preparation using Lactobacillus plantarum, prepare a bacillus preparation using the Bacillus subtilis described in claim 1, and prepare a yeast preparation using Candida utilis.

[0017] (2) Mix the lactic acid bacteria preparation, bacillus preparation, and yeast preparation according to a volume ratio of (2-6):(1-2):(2-4) to obtain a microbial complex preparation;

[0018] (3) Mix the microbial complex preparation and the enzyme preparation according to a weight ratio of 19:1 to obtain a bacteria-enzyme complex preparation.

[0019] In the step (2), the lactic acid bacteria preparation, bacillus preparation, and yeast preparation are mixed according to a volume ratio of 2:1:2.

[0020] Application of a straw bacteria-enzyme complex preparation in the preparation of straw fermented feed.

[0021] Mix the bacteria-enzyme complex preparation with the straw fermentation raw materials evenly to obtain a fermentation material, and anaerobic fermentation can obtain the fermented feed. The bacteria-enzyme complex preparation is 5% of the dry weight of the fermentation raw materials.

[0022] The water content of the fermented feed is 70-75%; the anaerobic fermentation conditions are 30°C for 15-20 days.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. The bacteria-enzyme complex preparation described in this method is easy to prepare, and the added enzymes are all common enzymes and are easy to purchase.

[0025] 2. After being treated with the bacteria-enzyme complex preparation of the present invention, the neutral detergent fiber content of corn straw is reduced from 75.64% to 64.32%, and the cellulose component is reduced from 40.11% to 17.98%. The fiber components in corn straw can be effectively degraded by the bacteria-enzyme complex preparation of the present invention, improving the nutritional quality of the feed.

[0026] 3. After being treated with the bacteria-enzyme complex preparation of the present invention, the crude protein content of corn straw is increased from 3.84% to 8.31%, and the acid-soluble protein content is increased from 0.90% to 2.13%. After being treated with the bacteria-enzyme complex preparation of the present invention, the protein components of corn straw are significantly improved, and more free amino acids and small peptides are released, which is beneficial to improving the absorption rate of protein components.

[0027] Using the bacteria-enzyme complex preparation can greatly improve the utilization rate of corn straw, improve the nutritional value of feed mainly composed of corn straw, not only improve the food supply problem, but also contribute to the healthy development of the corn straw processing industry. Description of the Drawings

[0028] Figure 1Analysis diagram of the fiber component modification ability in Examples 2 - 6; a is the diagram of the dry matter content change in Examples 2 - 6, b is the diagram of the hemicellulose content change in Examples 2 - 6, c is the diagram of the neutral detergent fiber content change in Examples 2 - 6, d is the diagram of the cellulose content change in Examples 2 - 6, e is the diagram of the acid detergent fiber content change in Examples 2 - 6, f is the diagram of the lignin content change in Examples 2 - 6;

[0029] Figure 2 Analysis diagram of the protein component modification ability in Examples 2 - 6; a is the diagram of the crude protein content change in Examples 2 - 6, b is the diagram of the acid-soluble protein content change in Examples 2 - 6;

[0030] Figure 3 Analysis diagram of the fermentation quality modification ability in Examples 2 - 6. Detailed implementation manners

[0031] In the following, the examples are exemplary descriptions of the main experimental evidences, rather than limiting the core content and application scope of the present invention disclosed by the quantity of evidences. It should be noted that in all these drawings and corresponding descriptions, only the concepts, principles and representative experimental evidences of the disclosed implementation manners of the present invention are shown exemplarily. When the evidence chain is complete, it is not necessary to show all the specific detailed information and extended details of each example listed in the present invention.

[0032] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified.

[0033] The lactic acid bacteria preparation, bacillus preparation, and yeast preparation involved in the present invention are prepared from Lactiplantibacillus plantarum, Bacillus subtilis, and Candida utilis respectively. Among them, Lactiplantibacillus plantarum and Candida utilis are common strains on the market, and Bacillus subtilis N3 is screened by this laboratory and preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number of CGMCC NO. 29518.

[0034] Example 1

[0035] A screening process for Bacillus subtilis. The strains in the present invention are soil-screened strains.

[0036] 1. Soil source: The soil in the present invention is all taken from corn straw farmland.

[0037] 2. Strain screening process:

[0038] (1) Preparation of soil bacterial suspension: Add 1% (w / v) soil sample into 100 mL of 0.9% (w / v) NaCl solution, and shake the bacteria suspension for 30 min at 30 °C and 130 rpm. After standing for 30 min, the supernatant obtained is the bacterial suspension of the feed sample.

[0039] (2) Screening and isolation of strains: Add the soil bacterial suspension into 50 mL of LB medium at 10% (v / v). In an aerobic environment, incubate at 30 °C, 220 rpm for 48 h. Then take the bacterial liquid, and dilute it by a factor of 10 6 ~10 9 and coat it on the LB solid medium, and culture it at 30 °C for 48 h. Select the solid medium with good strain growth, pick different single colonies on it for separate streak purification. After 3 - 4 purification processes, the obtained stable single colonies are regarded as pure bacteria.

[0040] (3) Re - screening of strains: Use the basal salt medium with sodium carboxymethyl cellulose and skim milk as the sole carbon source as the re - screening medium respectively. Pick the purified strains in step (2), evenly coat them on the re - screening medium, and culture at 30 °C for 48 h. Select the medium with good strain growth, stain it with 0.1% congo red reagent for 15 min, and then elute it with 0.1 M NaCl solution twice. Finally, select the strains with cellulose degradation circles, that is, the target cellulose - degrading bacteria.

[0041] (4) Identification of strains: Use the 16sRNA method to identify the genus of the strains, and then determine the species of the strains through ecological methods (colony morphology, microscopic detection of strain morphology). The identified strain is Bacillus subtilis, named Bacillus subtilis N3.

[0042] Example 2

[0043] A bacterial - enzyme complex preparation for corn straw is composed of lactic acid bacteria preparation, bacillus preparation, yeast preparation and enzyme preparation. The volume ratio of the lactic acid bacteria preparation, bacillus preparation and yeast preparation is 2:1:2.

[0044] 1. The preparation method of the lactic acid bacteria preparation is as follows:

[0045] (1) Primary culture: Take the Lactobacillus plantarum preserved in a glycerol tube, inoculate it into 5 mL of MRS medium, and place it in a shaker at 37 °C and 220 rpm for 12 h.

[0046] (2) Secondary culture: Take the above-activated primary seed liquid and inoculate it into 50 mL of MRS medium at an inoculation amount of 5% (OD is 0.3), and culture it in a shaker at 37 °C and 220 rpm for 12 h.

[0047] (3) Preparation of lactic acid bacteria preparation: Take the above secondary seed liquid and inoculate it into 50 mL of brown sugar medium at an inoculation amount of 5% (OD is 0.3), and culture it in a shaker at 37 °C and 220 rpm for 12 h. The obtained fermentation broth is the lactic acid bacteria preparation.

[0048] (4) The MRS medium includes 20 g / L of glucose, 10 g / L of peptone, 4 g / L of yeast extract powder, 5 g / L of beef extract powder, 1 mL of Tween-80, 2 g / L of dipotassium hydrogen phosphate, 0.2 g / L of magnesium sulfate, 2 g / L of ammonium citrate, 0.05 g / L of manganese sulfate, 5 g / L of sodium acetate, 1 g / L of L-cysteine, pH 7.0 - 7.2. Dissolve each component except agar in distilled water, adjust the pH value. For solid medium, add 2% agar powder and sterilize it at 115 °C under high pressure for 15 min for standby.

[0049] (5) The brown sugar medium includes 30 g / L of brown sugar, pH 7.0 - 7.2. Dissolve each component except agar in distilled water, adjust the pH value, and sterilize it at 115 °C under high pressure for 15 min for standby.

[0050] 2. The preparation method of the bacillus preparation is as follows:

[0051] (1) Primary culture: Take the glycerol tube-preserved Bacillus subtilis N3 and inoculate it into 5 mL of LB medium, and culture it in a shaker at 30 °C and 220 rpm for 12 h.

[0052] (2) Secondary culture: Take the above-activated primary seed liquid and inoculate it into 50 mL of LB medium at an inoculation amount of 5% (OD is 0.3), and culture it in a shaker at 30 °C and 220 rpm for 12 h.

[0053] (3) Preparation of bacillus preparation: Take the above secondary seed liquid and inoculate it into 50 mL of brown sugar medium at an inoculation amount of 5% (OD is 0.3), and culture it in a shaker at 30 °C and 220 rpm for 12 h. The obtained fermentation broth is the bacillus preparation.

[0054] (4) The LB medium includes 10 g / L of tryptone, 5 g / L of yeast extract powder, 10 g / L of sodium chloride, pH 7.0 - 7.2. Dissolve each component except agar in distilled water, adjust the pH value. For solid medium, add 1.5% agar powder and sterilize it at 121 °C under high pressure for 15 min for standby.

[0055] (5) The brown sugar culture medium includes 30 g / L of brown sugar and has a pH of 7.0 - 7.2. Dissolve the components except agar in distilled water, adjust the pH value, and autoclave at 115°C for 15 min for standby.

[0056] 3. The preparation method of the yeast preparation is as follows:

[0057] (1) Primary culture: Take the Candida utilis preserved in a glycerol tube and inoculate it into 5 mL of YPD medium, and place it in a shaker at 30°C and 220 rpm for 12 h.

[0058] (2) Secondary culture: Take the above-activated primary seed liquid and inoculate it into 50 mL of LB medium at an inoculation amount of 5% (OD is 0.3), and culture it in a shaker at 30°C and 220 rpm for 12 h.

[0059] (3) Preparation of the Bacillus preparation: Take the above secondary seed liquid and inoculate it into 50 mL of brown sugar culture medium at an inoculation amount of 5% (OD is 0.3), and culture it in a shaker at 30°C and 220 rpm for 12 h. The obtained fermentation broth is the yeast preparation.

[0060] (4) The YPD medium includes 20 g / L of glucose, 20 g / L of peptone, 10 g / L of yeast extract powder, and has a pH of 7.0 - 7.2. Dissolve the components except agar in distilled water, adjust the pH value. For the solid medium, add 2% agar powder and autoclave at 115°C for 15 min for standby.

[0061] (5) The brown sugar culture medium includes 30 g / L of brown sugar and has a pH of 7.0 - 7.2. Dissolve the components except agar in distilled water, adjust the pH value, and autoclave at 115°C for 15 min for standby.

[0062] 4. The preparation method of the enzyme preparation is as follows:

[0063] (1) The enzyme preparation only includes cellulase. Inoculate the cellulase into 50 mL of brown sugar culture medium, stir evenly to obtain the enzyme preparation.

[0064] 5. Preparation of corn straw fermentation raw materials

[0065] (1) The corn straw for fermentation is washed repeatedly with water to remove dust and impurities;

[0066] (2) Dry it at 60°C until the moisture content of the straw is less than 10%;

[0067] (3) After the dried straw is crushed by a high-speed crusher, sieve out the components between 20 mesh and 80 mesh;

[0068] (4) Sterilize the obtained corn straw at 121°C for 30 min to obtain the corn straw fermentation raw material.

[0069] 6. Preparation of Fermented Corn Straw Samples

[0070] (1) Mix the above lactic acid bacteria preparation, Bacillus subtilis preparation, and yeast preparation in a ratio of 2:1:2 to obtain a microbial compound preparation;

[0071] (2) Mix the microbial compound preparation in step (1) with the above enzyme preparation to obtain a microbial-enzyme compound preparation;

[0072] (3) Mix the microbial-enzyme compound preparation and the above fermentation raw materials evenly, and supplement water to 70-75% of the total mass to obtain a fermentation material;

[0073] (4) The addition amounts of the microbial preparation and the enzyme preparation are 4.75% and 0.25% of the total weight of the corn straw fermentation raw materials respectively, and the total addition amount is 5% of the total weight of the fermentation raw materials;

[0074] (5) Pack the fermentation material obtained in step (3) into vacuum bags at a rate of 100 g / bag, and place it in the dark at room temperature for fermentation for 20 d, repeating three biological parallels.

[0075] (6) After the fermentation of the fermentation material in step (5) is completed, a fermented corn straw sample is obtained.

[0076] 7. Collection and Determination of Fermented Corn Straw Samples

[0077] (1) Nutritional quality determination: The fermented sample is dried in an oven at 60 °C for 24 h, pulverized, and sealed in a self-sealing bag for determination of dry matter, crude protein, neutral detergent fiber, and acid detergent fiber. The dry matter content is determined with reference to the national standard GB / T 6435-2002 "Determination Method of Feed Moisture"; neutral detergent fiber and acid detergent fiber are determined using a Haineng F2000 fully automatic fiber analyzer; crude protein is determined using a Haineng K1100 Kjeldahl nitrogen analyzer. The nutritional index data after fermentation are shown in Table 1, and the change rate of nutritional quality after fermentation (compared with the non-added fermentation in Example 8) is shown in Table 2.

[0078] (2) Fermentation quality determination: Take 10 g of the straw sample, add 100 mL of distilled water, stir well for 1 h, and immediately measure the pH value using a pH meter. Filter with a 0.22 μm filter membrane, and the filtrate is used for determination of acetic acid and lactic acid. The contents of acetic acid and lactic acid are detected by a Shimadzu RID-20A high performance liquid chromatograph. Take 1 mL of the suspension, dilute and coat it at six dilution degrees of 10 -4 -10 -9 to count the viable bacteria count. The index data of fermentation quality (compared with the non-added fermentation in Example 8) are shown in Table 3. The change in viable bacteria count is shown in Table 4.

[0079] Example 3

[0080] A microbial enzyme composite preparation for corn straw consists of lactic acid bacteria preparation, bacillus preparation, yeast preparation, and enzyme preparation. The volume ratio of the lactic acid bacteria preparation, bacillus preparation, and yeast preparation is 6:1:2. The preparation methods of the lactic acid bacteria preparation, bacillus preparation, yeast preparation, enzyme preparation, and corn straw fermentation raw materials are the same as those in Example 2.

[0081] 1. Preparation of corn straw fermentation samples

[0082] (1) Mix the above-mentioned lactic acid bacteria preparation, bacillus subtilis preparation, and yeast preparation in a ratio of 6:1:2 to obtain a microbial composite preparation;

[0083] (2) Mix the microbial composite preparation in step (1) with the above-mentioned enzyme preparation to obtain a microbial enzyme composite preparation;

[0084] (3) Mix the microbial enzyme composite preparation and the above-mentioned fermentation raw materials evenly, and supplement water to 70 - 75% of the total mass to obtain a fermentation material;

[0085] (4) The addition amounts of the microbial preparation and the enzyme preparation are 4.75% and 0.25% of the total weight of the corn straw fermentation raw materials respectively, and the total addition amount is 5% of the total weight of the fermentation raw materials;

[0086] (5) Pack the fermentation material obtained in step (3) into vacuum bags at a rate of 100 g / bag, and place it in the dark at room temperature for fermentation for 20 d, repeating three biological parallels.

[0087] (6) After the fermentation of the fermentation material in step (5) is completed, a corn straw fermentation sample is obtained.

[0088] 2. Collection and determination of corn straw fermentation samples

[0089] (1) Determination of nutritional quality: The determination method is the same as that in Example 2. The nutritional index data after fermentation are shown in Table 1, and the change rate of nutritional quality after fermentation (compared with the fermentation without addition in Example 8) is shown in Table 2.

[0090] (2) Determination of fermentation quality: The determination method is the same as that in Example 2. The index data of fermentation quality (compared with the fermentation without addition in Example 8) are shown in Table 3. The change in viable count is shown in Table 4.

[0091] Example 4

[0092] A microbial enzyme composite preparation for corn straw consists of lactic acid bacteria preparation, bacillus preparation, yeast preparation, and enzyme preparation. The volume ratio of the lactic acid bacteria preparation, bacillus preparation, and yeast preparation is 2:2:2. The preparation methods of the lactic acid bacteria preparation, bacillus preparation, yeast preparation, enzyme preparation, and corn straw fermentation raw materials are the same as those in Example 2.

[0093] 1. Preparation of Fermented Corn Straw Samples

[0094] (1) Mix the above lactic acid bacteria preparation, Bacillus subtilis preparation, and yeast preparation in a ratio of 2:2:2 to obtain a microbial compound preparation;

[0095] (2) Mix the microbial compound preparation in step (1) with the above enzyme preparation to obtain a microbial-enzyme compound preparation;

[0096] (3) Mix the microbial-enzyme compound preparation and the above fermentation raw materials evenly, and supplement water to 70 - 75% of the total mass to obtain a fermentation material;

[0097] (4) The addition amounts of the microbial preparation and the enzyme preparation are 4.75% and 0.25% of the total weight of the corn straw fermentation raw materials respectively, and the total addition amount is 5% of the total weight of the fermentation raw materials;

[0098] (5) Pack the fermentation material obtained in step (3) into vacuum bags at a rate of 100 g / bag, and place it in the dark at room temperature for fermentation for 20 d, repeating three biological parallels.

[0099] (6) After the fermentation of the fermentation material in step (5) is completed, a fermented corn straw sample is obtained.

[0100] 2. Collection and Determination of Fermented Corn Straw Samples

[0101] (1) Nutritional quality determination: The determination method is the same as that in Example 2. The nutritional index data after fermentation are shown in Table 1, and the change rate of nutritional quality after fermentation (compared with the non-added fermentation in Example 8) is shown in Table 2.

[0102] (2) Fermentation quality determination: The determination method is the same as that in Example 2. The index data of fermentation quality (compared with the non-added fermentation in Example 8) are shown in Table 3. The change in viable count is shown in Table 4.

[0103] Example 5

[0104] A microbial-enzyme compound preparation for corn straw is composed of a lactic acid bacteria preparation, a bacillus preparation, a yeast preparation, and an enzyme preparation. The volume ratio of the lactic acid bacteria preparation, the bacillus preparation, and the yeast preparation is 2:1:4. The preparation methods of the lactic acid bacteria preparation, the bacillus preparation, the yeast preparation, the enzyme preparation, and the corn straw fermentation raw materials are the same as those in Example 2.

[0105] 1. Preparation of Fermented Corn Straw Samples

[0106] (1) Mix the above lactic acid bacteria preparation, Bacillus subtilis preparation, and yeast preparation in a ratio of 2:1:4 to obtain a microbial compound preparation;

[0107] (2) Mix the microbial compound preparation in step (1) with the above enzyme preparation to obtain a microbial-enzyme compound preparation;

[0108] (3) Mix the enzyme-bacteria complex preparation and the above fermentation raw materials evenly, and supplement water to 70-75% of the total mass to obtain the fermentation material.

[0109] (4) The addition amounts of the microbial preparation and the enzyme preparation are 4.75% and 0.25% of the total weight of the corn straw fermentation raw materials respectively, and the total addition amount is 5% of the total weight of the fermentation raw materials.

[0110] (5) Pack the fermentation material obtained in step (3) into vacuum bags at a quantity of 100 g / bag, and place it in the dark at room temperature for fermentation for 20 d, repeating three biological parallels.

[0111] (6) After the fermentation of the fermentation material described in step (5) is completed, a corn straw fermentation sample is obtained.

[0112] 2. Collection and determination of corn straw fermentation samples

[0113] (1) Nutritional quality determination: The determination method is the same as that in Example 2. The nutritional index data after fermentation are shown in Table 1, and the change rate of nutritional quality after fermentation (compared with fermentation without addition) is shown in Table 2.

[0114] (2) Fermentation quality determination: The determination method is the same as that in Example 2. The index data of fermentation quality (compared with fermentation without addition in Example 8) are shown in Table 3. The change in viable bacteria count is shown in Table 4.

[0115] Example 6

[0116] A microbial complex preparation for corn straw is composed of a lactic acid bacteria preparation, a bacillus preparation, and a yeast preparation, and the volume ratio of the lactic acid bacteria preparation, the bacillus preparation, and the yeast preparation is 2:1:2. The preparation methods of the lactic acid bacteria preparation, the bacillus preparation, the yeast preparation, and the corn straw fermentation raw materials are the same as those in Example 2.

[0117] 1. Preparation of corn straw fermentation samples

[0118] (1) Mix the above lactic acid bacteria preparation, bacillus subtilis preparation, and yeast preparation in a ratio of 2:1:2 to obtain a microbial complex preparation.

[0119] (2) Mix the microbial complex preparation and the above corn straw fermentation raw materials evenly, and supplement water to 70-75% of the total mass to obtain the fermentation material.

[0120] (3) The addition amount of the microbial preparation is 4.75% of the total weight of the corn straw fermentation raw materials.

[0121] (4) Pack the fermentation material obtained in step (2) into vacuum bags at a quantity of 100 g / bag, and place it in the dark at room temperature for fermentation for 20 d, repeating three biological parallels.

[0122] (5) After the fermentation of the fermented material described in step (4), a corn straw fermentation sample is obtained.

[0123] 2. Collection and determination of corn straw fermentation samples

[0124] (1) Nutritional quality determination: The determination method is the same as that in Example 2. The nutritional index data after fermentation are shown in Table 1, and the change rate of nutritional quality after fermentation (compared with the non-added fermentation in Example 8) is shown in Table 2.

[0125] (2) Fermentation quality determination: The determination method is the same as that in Example 2. The index data of fermentation quality (compared with the non-added fermentation in Example 8) are shown in Table 3. The viable count data are shown in Table 4.

[0126] Example 7

[0127] A biological preparation for corn straw consists of an enzyme preparation. The preparation methods of the enzyme preparation and the corn straw fermentation raw materials are the same as those in Example 2.

[0128] 1. Preparation of corn straw fermentation samples

[0129] (1) Mix the enzyme preparation and the above-mentioned corn straw fermentation raw materials evenly, and supplement water to 70 - 75% of the total mass to obtain a fermented material;

[0130] (2) The addition amount of the enzyme preparation is 0.25% of the corn straw fermentation raw materials;

[0131] (3) Pack the fermented material obtained in step (1) into vacuum bags at a rate of 100 g / bag, and place it in the dark at room temperature for fermentation for 20 d, repeating three biological parallels.

[0132] (4) After the fermentation of the fermented material described in step (3), a corn straw fermentation sample is obtained.

[0133] 2. Collection and determination of corn straw fermentation samples

[0134] (1) Nutritional quality determination: The determination method is the same as that in Example 2. The nutritional index data after fermentation are shown in Table 1, and the change rate of nutritional quality after fermentation (compared with the non-added fermentation in Example 8) is shown in Table 2.

[0135] (2) Fermentation quality determination: The determination method is the same as that in Example 2. The index data of fermentation quality (compared with the non-added fermentation in Example 8) are shown in Table 3. The viable count data are shown in Table 4.

[0136] Example 8

[0137] Corn straw without biological agent treatment, and the preparation method of the corn straw fermentation raw materials is the same as that in Example 2.

[0138] 1. Preparation of fermentation samples

[0139] (1) Mix the fermentation raw material prepared from corn straw with the brown sugar culture medium evenly, and supplement water to 70 - 75% of the total mass to obtain the fermentation material, repeating three biological parallels;

[0140] (2) The addition amount of the brown sugar culture medium is 5% of the fermentation raw material;

[0141] (3) After the fermentation of the fermentation material in step (1) ends, a corn straw fermentation sample is obtained.

[0142] 2. Collection and determination of fermentation samples

[0143] (1) Determination of nutritional quality: The determination method is the same as that in Example 2. The nutritional index data after fermentation are shown in Table 1.

[0144] (2) Determination of fermentation quality: The determination method is the same as that in Example 2. The change in the viable count is shown in Table 4.

[0145] Example 9: Comparative Example 1

[0146] A general - type bacterial - enzyme compound feed fermenting agent is composed of a yeast preparation, a lactic acid bacteria preparation, and an enzyme preparation. The preparation methods of the yeast preparation, the lactic acid bacteria preparation, and the corn straw fermentation raw material are the same as those in Example 1.

[0147] 1. The preparation method of the enzyme preparation is as follows:

[0148] (1) The enzyme preparation is composed of a mixture of β - glucanase, xylanase, mannanase, and laccase. Mix β - glucanase, xylanase, mannanase, and laccase in a ratio of 1:1:1:1, and add them to 50 mL of the brown sugar culture medium, stirring evenly to obtain the enzyme preparation.

[0149] 2. Preparation of corn straw fermentation samples

[0150] (1) Mix the above - mentioned yeast preparation, lactic acid bacteria preparation, and enzyme preparation in a ratio of 2:2:4 to obtain a bacterial - enzyme compound feed fermenting agent;

[0151] (2) Mix the bacterial - enzyme compound feed fermenting agent obtained in step (1) and the above - mentioned corn straw fermentation raw material evenly, and supplement water to 70 - 75% of the total mass to obtain the fermentation material;

[0152] (3) The addition amount of the bacterial - enzyme compound feed fermenting agent is 0.6% of the corn straw fermentation raw material;

[0153] (4) Pack the fermentation material obtained in step (1) into vacuum bags at a rate of 100 g / bag, and place it in the dark at room temperature for fermentation for 20 d, repeating three biological parallels.

[0154] (5) After the fermentation of the fermented material described in step (4), a corn straw fermentation sample is obtained.

[0155] 2. Collection and determination of corn straw fermentation samples

[0156] (1) Nutritional quality determination: The determination method is the same as that in Example 2. The nutritional index data after fermentation are shown in Table 1, and the change rate of nutritional quality after fermentation (compared with Example 10: Comparative Example 2 without added fermentation) is shown in Table 2.

[0157] (2) Fermentation quality determination: The determination method is the same as that in Example 2. The index data of fermentation quality (compared with Example 10: Comparative Example 2 without added fermentation) are shown in Table 3.

[0158] Example 10: Comparative Example 2

[0159] Corn straw was not treated with a general-purpose bacterial enzyme complex feed fermenter. The preparation method of the corn straw fermentation raw material is the same as that in Example 2.

[0160] 1. Preparation of fermentation samples

[0161] (1) Mix the fermented raw material prepared from corn straw with the brown sugar culture medium evenly, and supplement water to 70 - 75% of the total mass to obtain the fermented material, repeating three biological parallels;

[0162] (2) The addition amount of the brown sugar culture medium is 2% of the fermentation raw material;

[0163] (3) After the fermentation of the fermented material described in step (1), a corn straw fermentation sample is obtained.

[0164] 2. Collection and determination of fermentation samples

[0165] (1) Nutritional quality determination: The determination method is the same as in Example 2. The nutritional index data after fermentation are shown in Table 1. (2) Fermentation quality determination: The determination method is the same as in Example 2. The index data of fermentation quality are shown in Table 3.

[0166] Table 1 Nutritional index data of each example after fermentation (dry matter %)

[0167]

[0168]

[0169] Table 2 Change rate of nutritional quality of each example after fermentation (%)

[0170]

[0171]

[0172] Table 3 Index data of fermentation quality of each example after fermentation (%)

[0173]

[0174] Table 4 Changes in viable cell counts of Examples 2 to 8 during fermentation (×10 10 CFU / g)

[0175]

[0176]

[0177] Conclusion

[0178] 1. In Examples 2 - 5, the corn straw was treated with a bacterial enzyme complex preparation, and the nutritional quality and fermentation quality of the treated corn straw were significantly improved. Among them, the comprehensive corn straw modification ability of Example 1 was the best. In Examples 6 and 7, the microbial preparation and the enzyme preparation were used separately.

[0179] (1) As shown in Table 4, compared with Example 6 (single microbial preparation), the viable cell count of yeast in Examples 2 - 5 was significantly increased. Combining Tables 1, 2, 4 and Figure 2 it can be seen that yeast is the key component for modifying the protein component of corn straw. Therefore, the bacterial enzyme complex preparation is better than using the microbial preparation alone.

[0180] (2) As shown in Table 3, compared with Example 7 (enzyme preparation), Examples 2 - 5 were more excellent in improving the fermentation quality. Therefore, the bacterial enzyme complex preparation is better than using the enzyme preparation alone.

[0181] (3) In Examples 2 - 5, the differences in the modification abilities of the nutritional quality and fermentation quality of each group were not significant. There were only certain obvious differences in the fermentation quality at the initial stage of fermentation. At the end of fermentation, the differences among the groups were not obvious; combining Table 4 and Figure 3 it can be seen that the differences in the fermentation quality were consistent with the changes in the viable cell count.

[0182] (4) Example 2 showed the best performance in modifying the nutritional quality and fermentation quality;

[0183] (5) As Figure 3 shown in Table 3, in Example 2, the contents of lactic acid, acetic acid, and ethanol were significantly increased, and the pH value was reduced to 3.54, which could effectively inhibit the growth of miscellaneous bacteria;

[0184] (6) As Figure 1 shown in c, e and Tables 1 and 2, in Example 2, the contents of neutral detergent fiber and acid detergent fiber were reduced by 12.09% and 10.00% respectively, indicating that the fiber components of the treated corn straw were significantly degraded, effectively increasing the release of pentose and hexose in the corn straw;

[0185] (7) As shown in Figure 1 b and d in Tables 1 and 2, in Example 2, the contents of cellulose and hemicellulose decreased by 49.96% and 10.58% respectively, which verified the release of pentose and hexose in (3), increased the content of soluble carbohydrates in corn stover, and contributed to promoting the digestion of corn stover by animals.

[0186] (8) As shown in Figure 2 a and b in Tables 1 and 2 of Example 2, the contents of crude protein and acid-soluble protein increased by 64.41% and 137.33% respectively, significantly improving the problem of lack of protein components in corn stover and enhancing the nutritional value of corn stover.

[0187] In summary, the enzyme-microorganism complex preparation can significantly improve the nutritional composition of corn stover, degrade fiber components, increase the content of soluble carbohydrates, reduce the digestion difficulty of corn stover, increase the content of the overall protein components in corn stover, and solve the problem of lack of protein nutrition in corn stover.

[0188] 2. Comparison between Example 2 and Comparative Example 1 (general enzyme-microorganism complex feed fermenter).

[0189] (1) In terms of the preparation composition, the main difference between Example 2 and Comparative Example 1 lies in the composition of the enzyme preparation. The enzyme preparation composition of Comparative Example 1 is more complex and difficult to prepare, while the composition of Example 2 is simple, easy to prepare, and more economical.

[0190] (2) In terms of the addition amount of the preparation, Example 2 has a larger addition amount of the microbial preparation and a similar addition amount of the enzyme preparation compared with Comparative Example 1. The reason is that the enzyme-microorganism complex preparation in Example 2 relies on microorganisms to achieve the effects of multiple enzymes in Comparative Example 1. Considering the secretion efficiency of microbial enzymes, it is necessary to increase the initial addition amount.

[0191] (3) In terms of the fermentation quality (i.e., the contents of lactic acid, acetic acid, and ethanol), as shown in Table 3, compared with Comparative Example 1, after fermentation with the enzyme-microorganism complex preparation in Example 2, the contents of lactic acid, acetic acid, and ethanol are significantly better than those in Comparative Example 1.

[0192] (4) In terms of the fiber components (i.e., neutral detergent fiber, acid detergent fiber, cellulose, hemicellulose, and lignin), as shown in Tables 1 and 2, Example 2 and Comparative Example 1 have similar degradation rates of neutral detergent fiber, which are 9.67% and 9.52% respectively. However, Example 1 has a higher degradation rate of acid detergent fiber.

[0193] (5) In terms of the protein components (i.e., crude protein and acid-soluble protein), as shown in Tables 1 and 2, Example 2 has higher promotion rates of crude protein and acid-soluble protein.

[0194] In summary, compared with the general-purpose bacterial enzyme composite feed fermenter, the bacterial enzyme composite preparation has more advantages in terms of economy, improvement of feed fermentation quality, modification of feed fiber components, and modification of feed protein components. It can better improve the nutritional composition of feed and enhance the feeding value of feed.

[0195] The content of the above embodiments only details a part of the specific implementation manners of the present invention, but is not limited to the embodiments currently disclosed in the present invention. In addition, the substantial content protected by the present invention is not limited thereto. Without departing from the design scope of the present invention, any other modifications, equivalent replacements, improvements, etc. made according to the thinking principle and technical means of the present invention all fall within the protection scope of the present invention.

Claims

1. A Bacillus subtilis N3, deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC NO.29518.

2. Use of the Bacillus subtilis N3 described in claim 1 in the preparation of a straw-bacterium-enzyme composite preparation.

3. A straw-bacteria-enzyme composite preparation, characterized in that: The invention comprises a microbial composite preparation and an enzyme preparation, wherein the weight ratio of the microbial composite preparation to the enzyme preparation is 19:1, the microbial composite preparation comprises a lactic acid bacteria preparation prepared by Lactobacillus plantarum, a bacillus preparation prepared by Bacillus subtilis N3 as claimed in claim 1, and a yeast preparation prepared by Candida utilis, and the enzyme preparation is prepared by cellulase.

4. The straw-bacterium-enzyme composite preparation according to claim 3, characterized in that: The volume ratio of the lactic acid bacteria preparation, the bacillus preparation and the yeast preparation is (2-6):(1-2):(2-4).

5. The straw-bacterium-enzyme composite preparation according to claim 3, characterized in that: The volume ratio of the lactic acid bacteria preparation, the bacillus preparation and the yeast preparation is 2:1:

2.

6. A method for preparing a straw-bacteria-enzyme composite preparation, characterized in that: The steps include: (1) using Lactobacillus plantarum to prepare a lactic acid bacteria preparation, using the Bacillus subtilis described in claim 1 to prepare a bacillus preparation, and using Candida utilis to prepare a yeast preparation; (2) mixing the lactic acid bacteria preparation, the bacillus preparation, and the yeast preparation in a volume ratio of (2-6):(1-2):(2-4) to obtain a microbial composite preparation; (3) The microbial compound preparation and the enzyme preparation are mixed in a weight ratio of 19:1 to obtain a bacterial-enzyme compound preparation.

7. The method for preparing the straw-bacteria-enzyme composite preparation according to claim 6, characterized in that: In the step (2), the lactic acid bacteria preparation, the bacillus preparation and the yeast preparation are mixed in a volume ratio of 2:1:

2.

8. Application of a straw-bacteria-enzyme composite preparation in the preparation of straw fermented feed.

9. The use of the straw-bacteria-enzyme composite preparation in the preparation of straw fermented feed according to claim 8, characterized in that: The bacterial enzyme compound preparation according to claim 3 is mixed evenly with the straw fermentation raw material to obtain fermentation material, and fermentation feed can be obtained by anaerobic fermentation. The bacterial enzyme compound preparation accounts for 5% of the dry weight of the fermentation raw material.

10. The use of the straw-bacteria-enzyme composite preparation according to claim 9 in preparing straw fermented feed, characterized in that: The water content of the fermented feed is 70-75%; the anaerobic fermentation conditions are 30°C for 15-20 days.

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