Fermented rapeseed meal as well as preparation method and application thereof

Abstract

The invention relates to the technical field of animal feed, in particular to fermented rapeseed meal as well as a preparation method and application thereof. The present invention provides a fermented rapeseed meal preparation method, which comprises: mixing a rapeseed meal-containing fermentation raw material, an enzyme preparation and a first fermentation bacterial agent, carrying out aerobic fermentation, and after the aerobic fermentation is completed, adding a second fermentation bacterial agent to carry out anaerobic fermentation, the first fermentation inoculant comprises aspergillus niger, aspergillus oryzae, bacillus subtilis and bacillus amyloliquefaciens; the second fermentation microbial agent comprises candida utilis and lactobacillus plantarum. According to the method, the content of toxins and antinutritional factors in the rapeseed meal is effectively reduced, meanwhile, the content of crude protein in the fermented rapeseed meal and the digestion efficiency and metabolizable protein content of nutrient substances such as dry matter and protein in rumen are remarkably improved, and the fermented rapeseed meal is good in palatability, can be used as a good soybean meal substitute and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of animal feed, and in particular to fermented rapeseed meal and a preparation method and application thereof. Background Art

[0002] The most commonly used plant-based protein feed in feed is soybean meal, but with the sharp rise in soybean meal prices in recent years, other protein feeds are urgently needed to replace soybean meal in animal farming to reduce feed costs. Rapeseed meal is a by-product of extracting oil from rapeseed. The protein level is 35% to 40%, and the amino acid types are complete and balanced. Among them, essential amino acids account for 37% to 38%, and the ratio of essential amino acids to non-essential amino acids is 0.56 to 0.61 (Shi Anning, Wang Yajun. Research progress on the application of fermented rapeseed meal in aquaculture [J]. Scientific Fish Farming, 2023, (06): 74-76; Peng Shuang, Xiang Qian, Song Jinqiu. Effects of fermented rapeseed meal on nutrient digestibility, growth performance and serum parameters of growing pigs [J]. China Feed, 2020, (08): 116-120.), as a protein feed to replace soybean meal is a cost-effective choice. However, rapeseed meal contains anti-nutritional factors such as phenolic compounds, tannins, phytic acid, glucosinolates (hereinafter referred to as glucosinolates) and their derivatives. These anti-nutritional factors can affect the production performance and health of animals, seriously restricting the application of rapeseed meal in animal husbandry.

[0003] In existing research, rapeseed meal is treated mainly in three ways: physical, chemical and biological. Physical methods include: high temperature sterilization, steam stripping, water extraction, extrusion and baking. Among them, high pressure sterilization can effectively reduce the level of oxazolidinone in rapeseed meal; using a twin-screw extruder to extrude high-glucosinolate rapeseed meal can reduce the concentration of glucosinolates in rapeseed meal to a lower level. Although physical treatment can effectively reduce the glucosinolate content in rapeseed meal, it will cause problems such as the destruction of amino acids such as lysine and methionine and the loss of solids, resulting in a decrease in the nutritional value of rapeseed meal. In chemical treatment, glucosinolates react with iron, copper or nickel salts and are catalytically decomposed by heating. However, in the decomposition products, toxic 1-cyano-2-hydroxy-3-butane still remains. Chemical treatment also includes ethanol treatment, sodium carbonate treatment and other methods. The rapeseed meal was treated with sodium carbonate or autoclaved, and 0.10 mol / L NaOH was added to methanol, with an alcohol-oil molar ratio of 180:1. The reaction was carried out at 40°C for 3 hours, and the conversion rate reached 98%, and the glucosinolate content was reduced to 0.07% (Qian J, Yang Q, Sun F, et al. Cogeneration of biodiesel and nontoxic rapeseedmeal from rapeseed through in-situ alkalinetransesterification [J]. Bioresource technology, 2013, 128: 8-13.). However, chemical treatment methods are prone to reduce the amino acid level of rapeseed meal and chemical residues. Both physical and chemical treatment methods are difficult to guarantee or improve the nutritional value of rapeseed meal.

[0004] Biological treatment mainly refers to solid fermentation technology. Solid fermentation simulates the natural environment of most microorganisms and is not easily contaminated by bacteria. In addition, since it is not easily inhibited by substrates, the enzyme production efficiency of microorganisms is higher. The existing rapeseed meal solid fermentation technology mainly uses rapeseed meal or adds cornmeal, bran, etc. as raw materials, dissolves composite bacteria (yeast, Aspergillus niger, Aspergillus oryzae, etc.) in water to make a suspension and adds it to it, adds molasses, sucrose, etc. to provide energy for the bacteria, puts it into a sealed bag and ferments it at a constant temperature for 4 to 40 hours to obtain fermented rapeseed meal (patent applications CN113073056A, CN104397347A, CN108260710A, CN109452449A, CN106819359A; Wu Ying, Fan Zhen, Ma Guijun. Research and application progress of fermented protein feed in animal husbandry industry [J]. Feed Research, 2018(06):1-3.).

[0005] Studies have shown that adding fermented rapeseed meal to broiler feed can improve their production performance and increase body weight (Konkol, D., Jonuzi, E., Popiela, E., Sierżant, K., Korzeniowska, M., Leicht, K., Gumowski, M., Krasowska, A., Łukaszewicz, M., & Korczyński, M. 2023. Influence of solid state fermentation with Bacillus subtilis 67 strain on the nutritional value of rapeseed meal and its effects on performance and meatquality of broiler chickens. Poultry Science, 102(7), 102742.). Replacing 8.50% soybean meal with 11.00% fermented rapeseed meal not only did not have a negative impact on the production performance of fattening pigs, but also promoted the growth of fattening pigs and improved feed conversion rate to a certain extent (patent application CN106819359A). Fermented rapeseed meal was used to replace a certain amount of soybean meal in the diet of Nile tilapia, white shrimp, silver carp and red sea bream. After a period of feeding, it was found that it is completely feasible to use rapeseed meal as a partial replacement for soybean meal as a protein feed raw material for aquatic animals after fermentation and detoxification (Shi Anning, Wang Yajun. Research progress on the application of fermented rapeseed meal in aquaculture [J]. Scientific Fish Farming, 2023, (06): 74-76.). However, the existing rapeseed meal biofermentation technology is mostly applied to monogastric animals such as pigs, poultry, and aquatic products, and there is very little research and application as a ruminant feed. Summary of the invention

[0006] The invention provides a fermented rapeseed meal and a preparation method and application thereof.

[0007] The digestive system structure of ruminants such as cattle is significantly different from that of monogastric animals such as pigs, which makes their digestion of feed and absorption and utilization of nutrients also significantly different from those of monogastric animals. Therefore, feed and nutrients suitable for monogastric animals such as pigs may not necessarily be well utilized by ruminants, and there is often a problem of low nutrient absorption and utilization rate. In the prior art, there are few rapeseed meal fermentation methods developed for the digestive characteristics of ruminant stomachs, and even fewer focus on the digestion and absorption efficiency of fermented rapeseed meal in the special stomach structure of ruminants. The present invention develops a preparation method for fermented rapeseed meal for ruminants, which significantly improves the digestion efficiency of nutrients such as protein in the fermented rapeseed meal and the content of metabolizable protein therein by ruminants while ensuring that the content of toxins and anti-nutritional factors in the fermented rapeseed meal is effectively reduced.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] The present invention provides a method for preparing fermented rapeseed meal, the method comprising: mixing a fermentation raw material containing rapeseed meal with an enzyme preparation and a first fermentation bacterial agent and performing aerobic fermentation, and after the aerobic fermentation is completed, adding a second fermentation bacterial agent and performing anaerobic fermentation; The first fermentation bacterial agent comprises Aspergillus niger, Aspergillus oryzae, Bacillus subtilis and Bacillus amyloliquefaciens; The second fermentation bacterial agent comprises Candida utilis and Lactobacillus plantarum.

[0010] Rapeseed meal fermentation in the prior art mostly adopts a one-step fermentation method. The present invention finds that, in view of the digestive characteristics of the stomach of ruminants, the rapeseed meal is first subjected to aerobic fermentation and then to anaerobic fermentation, and the fermentation agent composed of the above-mentioned specific bacterial species determined through screening and optimization is used respectively in the aerobic fermentation and the anaerobic fermentation, and the aerobic fermentation stage and the anaerobic fermentation stage can work well in synergy, which can not only significantly reduce the content of toxins and anti-nutritional factors in the fermented rapeseed meal and significantly increase the content of crude protein in the fermented rapeseed meal, but also the fermented rapeseed meal is particularly suitable for rumen digestion of ruminants, and the protein and dry matter contained therein have significantly improved rumen digestion efficiency, and the content of metabolizable protein is significantly increased.

[0011] In the first fermentation bacterial agent described above, the ratio of the effective live bacteria count of Aspergillus niger, Aspergillus oryzae, Bacillus subtilis and Bacillus amyloliquefaciens is (1.5-20): (0.96-1.6): 3:30.

[0012] Controlling the inoculation amount of Aspergillus niger, Aspergillus oryzae, Bacillus subtilis and Bacillus amyloliquefaciens within the above range is more conducive to reducing the content of toxins and anti-nutritional factors in the fermented rapeseed meal, increasing the content of crude protein in the fermented rapeseed meal, the rumen digestion efficiency of the fermented rapeseed meal and the content of metabolizable protein.

[0013] Preferably, in the aerobic fermentation stage, the addition amount of the first fermentation agent is 3.6-5.4 billion cfu / kg fermented rapeseed meal.

[0014] In the second fermentation bacterial agent described above, the ratio of the effective viable counts of Candida utilis and Lactobacillus plantarum is 1:(1.2-1.8).

[0015] In order to make the aerobic fermentation stage and the anaerobic fermentation stage work better together, by adding the above-mentioned ratio of Candida utilis and Lactobacillus plantarum in the anaerobic fermentation stage, the growth of miscellaneous bacteria and aerobic bacteria added in the aerobic fermentation stage can be suppressed to the utmost, and the nutrients in the fermented rapeseed meal obtained after aerobic fermentation can be better retained; in addition, the fermented rapeseed meal obtained by the above-mentioned anaerobic fermentation also has a sour aroma, which can increase the palatability of animals and has an appetite-inducing effect.

[0016] Preferably, during the anaerobic fermentation stage, the amount of the second fermentation agent added is 750-800 million cfu / kg fermented rapeseed meal.

[0017] The enzyme preparation described above comprises: cellulase 2100-2200 U / g, xylanase 1400-1500 U / g, β-mannanase 1200-1300 U / g, pectinase 5400-5500 U / g, amylase 320-330 U / g, saccharifying enzyme 8100-8200 U / g, bromelain 3450-3550 U / g, papain 3550-3650 U / g, and acid protease 1700-1800 U / g.

[0018] The role of the enzyme preparation is mainly to hydrolyze the macromolecular substances such as cellulose, polysaccharides, proteins in the rapeseed meal into small molecules, on the one hand, to provide available small molecule nutrients for the fermentation strains, and on the other hand, to cooperate with the fermentation to increase the content of metabolizable nutrients in the fermented rapeseed meal. Microorganisms have the highest efficiency in taking up monosaccharides. The present invention specifically introduces saccharifying enzymes, which are rarely used in the preparation of fermented rapeseed meal, to generate heat by decomposing carbohydrates during fermentation, and to increase the internal temperature of the fermentation system, so that the temperature is close to the optimal reaction temperature of saccharifying enzymes, to accelerate the conversion of polysaccharides in the rapeseed meal to monosaccharides, to provide more monosaccharide substrates for microbial growth, and then to promote the growth of fermented strains and the fermentation process. The protease, cellulase, polysaccharide hydrolase, etc. in the above-mentioned enzyme preparation cooperate with each other, can better provide available small molecule nutrients for the fermentation strains, promote the growth and metabolism of the fermentation strains, and cooperate with the fermentation strains to increase the content of metabolizable nutrients in the fermented rapeseed meal.

[0019] Preferably, the enzyme preparation is added in an amount of 3-5 g / kg fermented rapeseed meal.

[0020] The above-mentioned aerobic fermentation is first fermented at 29-31°C for 20-28h, and then fermented at 27-28°C for 44-52h. The present invention finds that the bacteria in the first fermentation agent can grow faster at 29-31°C (especially 30°C), and can produce enzymes more efficiently at 27-28°C (especially 28°C). Therefore, the aerobic fermentation is set with the above temperature changes, so that the bacteria in the first fermentation agent grow at the optimal temperature first, and then produce enzymes at the optimal temperature.

[0021] The above-mentioned anaerobic fermentation is carried out at 29-31°C for 90-110 hours.

[0022] During aerobic fermentation, stir the fermentation materials every 6-10 hours. During anaerobic fermentation, turn over and shake the fermentation bag every 6-10 hours.

[0023] In the present invention, the fermentation is solid fermentation.

[0024] The fermentation raw materials mentioned above include not only rapeseed meal, but also nutritional additives and one or more selected from cornmeal and bran.

[0025] Preferably, the fermentation raw materials include rapeseed meal, cornmeal, bran and nutritional additives.

[0026] The above-mentioned nutritional additives include additive A and additive B, wherein additive A includes the following components: 30-40 g / L NH4Cl, 7-10 g / L KH2PO4, 5-10 g / L NaH2PO4, 5-10 g / L MgSO4, and 0.1-0.4 g / L MnCl2·4H2O; and additive B includes the following components: 10-20 g / L calcium pantothenate and 60-80 g / L tricalcium citrate.

[0027] Preferably, in the fermentation raw materials, the mass ratio of rapeseed meal, cornmeal and bran is (1500-2500): (50-100): (50-100); the addition amount of auxiliary agent A is 18-22 mL / kg fermented rapeseed meal, and the addition amount of auxiliary agent B is 8-12 mL / kg fermented rapeseed meal.

[0028] Preferably, the fermentation raw material further comprises water, and the added amount of water is preferably in a ratio of (2-4):7 to the total mass of rapeseed meal, cornmeal and bran.

[0029] During the fermentation process, in order to ensure the water supply of the bacteria, water can be added as needed.

[0030] The method for preparing the fermented rapeseed meal is preferably a method for preparing fermented rapeseed meal for ruminants.

[0031] Based on the above method, the present invention provides a fermented rapeseed meal, which is prepared by the above-mentioned method for preparing fermented rapeseed meal.

[0032] The fermented rapeseed meal prepared by the above method has the characteristics of low toxin content, low anti-nutritional factor content, high crude protein content, high rumen digestion efficiency, high metabolizable protein content, good palatability, etc., and is particularly suitable as a protein feed for ruminants.

[0033] The present invention also provides the use of the fermented rapeseed meal described above in the preparation of ruminant feed or the breeding of ruminants.

[0034] Preferably, the ruminant is a cattle.

[0035] The present invention provides a ruminant feed, which comprises the fermented rapeseed meal mentioned above.

[0036] The beneficial effects of the present invention include at least: the preparation method of the fermented rapeseed meal provided by the present invention, by adding specific fermentation bacteria and enzyme preparations and combining aerobic fermentation and anaerobic fermentation, effectively reduces the content of toxins and anti-nutritional factors in the rapeseed meal, and significantly improves the crude protein content in the fermented rapeseed meal, the digestion efficiency of dry matter, protein and other nutrients in the rumen, and the metabolizable protein content after rumen digestion, and the fermented rapeseed meal has good palatability and can be used as a good soybean meal substitute; in addition, the method has the advantages of simple operation, low cost, high efficiency, safety, greenness, environmental protection, etc., and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 and Figure 2 They are the DM and CP degradation curves of the fermented rapeseed meal and rapeseed meal raw materials in the experimental examples of the present invention, respectively. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The fermentation strains used in the following examples are described below.

[0041] Aspergillus niger: Manufacturer: Shandong Hezhong Kangyuan Biotechnology Co., Ltd.; Trade mark: 3.324; Number of viable bacteria per unit mass: 10 billion cfu / g.

[0042] Aspergillus oryzae: Manufacturer: Jining Yuyuan Biotechnology Co., Ltd.; Trademark number: JY309; Number of viable bacteria per unit mass: 16 billion cfu / g.

[0043] Bacillus subtilis: Manufacturer: Orian (Hubei) Bioengineering Co., Ltd.; Number of viable bacteria per unit mass: 1 billion cfu / g.

[0044] Bacillus amyloliquefaciens: Manufacturer: Beihai Yiqiang Biotechnology Co., Ltd.; Number of viable bacteria per unit mass: 100 billion cfu / g.

[0045] Candida utilis: Manufacturer: Anhui Quanmin Environmental Protection Technology Co., Ltd.; Number of viable bacteria per unit mass: 100 million cfu / g.

[0046] Lactobacillus plantarum: Manufacturer: Xi'an Mixianer Biotechnology Co., Ltd.; Number of viable bacteria per unit mass: 10 billion cfu / g.

[0047] The information of the enzyme preparations used in the following examples is as follows.

[0048] Cellulase: Manufacturer: Nanning Dongheng Huadao Biotechnology Co., Ltd.; Enzyme activity: 20000U / g.

[0049] Xylanase: Manufacturer: Nanning Dongheng Huadao Biotechnology Co., Ltd.; Enzyme activity: 10000 U / g.

[0050] β-Mannanase: Manufacturer: Shengda Food Additive Co., Ltd.; Enzyme activity: 50000U / g.

[0051] Pectinase: Manufacturer: Nanning Dongheng Huadao Biotechnology Co., Ltd.; Enzyme activity: 31300 U / g.

[0052] Amylase: Manufacturer: Nanning Dongheng Huadao Biotechnology Co., Ltd.; Enzyme activity: 1000 U / g.

[0053] Glucoamylase: Manufacturer: Shandong Longkote Enzyme Preparation Co., Ltd.; Enzyme activity: 100,000 U / g.

[0054] Bromelain: Manufacturer: Nanning Dongheng Huadao Biotechnology Co., Ltd.; Enzyme activity: 50,000 U / g.

[0055] Papain: Manufacturer: Nanning Dongheng Huadao Biotechnology Co., Ltd.; Enzyme activity: 102000 U / g.

[0056] Acid protease: Manufacturer: Nanning Dongheng Huadao Biotechnology Co., Ltd.; Enzyme activity: 50,000 U / g.

[0057] Example 1

[0058] This embodiment provides a method for preparing fermented rapeseed meal, and the steps are as follows: Taking a 5 kg fermentation bag with a one-way valve as an example, the finished product of fermented rapeseed meal is about 3 kg.

[0059] 1. Disinfect the inside of the incubator with 75% alcohol and 15-fold diluted Sanisol to kill bacteria and fungi. It can be used after air drying.

[0060] 2. First, mix 1950g rapeseed meal, 75g cornmeal and 75g bran evenly and put them into a fermentation bag with a one-way valve, then add 900 mL of distilled water.

[0061] 3. Add enzyme preparation, first fermentation bacteria, auxiliary agent A and auxiliary agent B, mix the above preparations evenly with the raw materials in step 2 in a fermentation bag, put it into a constant temperature box without sealing, and ferment aerobically at 30°C for 24 hours, stirring the raw materials every 8 hours.

[0062] 4. After 24 hours of aerobic fermentation, lower the temperature of the constant temperature box to 28°C and continue aerobic fermentation for 48 hours, stirring the raw materials every 8 hours.

[0063] 5. After a total of 72 hours of aerobic fermentation, add the second fermentation agent. Because aerobic fermentation produces a lot of heat and consumes a certain amount of water, insufficient water will seriously affect the growth of bacteria and fungi, so it is necessary to supplement water during the fermentation process. Place the fermentation bag on the scale, add distilled water to a total weight of 3050g (including the weight of the fermentation bag, the average bag weight is 32g), seal the bag with a sealing machine, and squeeze out the air in the bag through the one-way exhaust valve of the fermentation bag. Place it in a constant temperature box and ferment it anaerobically at 30℃ for 96 hours, turning the fermentation bag over and shaking it every 8 hours. After the anaerobic fermentation is completed, the fermented rapeseed meal product is obtained.

[0064] The formulation used in the above method is as follows: (1) Preparation of nutritional supplements

[0065] The following additives are added to each kilogram of fermented rapeseed meal product: Auxiliary agent A: NH4Cl 0.7 g, KH2PO4 0.175 g, NaH2PO4 0.14 g, MgSO4 0.14 g, MnCl2·4H2O 0.0046 g, distilled water 20 mL.

[0066] Additive B: 0.14 g calcium pantothenate, 0.7 g tricalcium citrate, 10 mL distilled water.

[0067] (2) Preparation of enzyme preparations

[0068] The following enzyme preparations are added to each kilogram of fermented rapeseed meal product: Cellulase 0.43 g (8600U), xylanase 0.58 g (5800U), β-mannanase 0.10 g (5000U), pectinase 0.70 g (21910U), amylase 1.306 g (1306U), saccharifying enzyme 0.324 g (32400U), bromelain 0.28 g (14000U), papain 0.14 g (14280U), acid protease 0.14 g (7000U), totaling 4.00 g; add 10 mL of distilled water to dissolve and mix.

[0069] (3) Preparation of microbial agents

[0070] The following fermentation agents are added to each kilogram of fermented rapeseed meal product: First fermentation agent: 0.015 g Aspergillus niger (150 million cfu), 0.01 g Aspergillus oryzae (160 million cfu), 0.3 g Bacillus subtilis (300 million cfu), and 0.03 g Bacillus amyloliquefaciens (3 billion cfu), 20 mL distilled water. Add 3 kg brown sugar per kilogram of agent. After adding brown sugar, activate for 1 hour and use immediately.

[0071] Second fermentation agent: 3g of Candida utilis (300 million cfu) and 0.045g of Lactobacillus plantarum (450 million cfu), 7 mL of distilled water. Add 0.32 kg of brown sugar per kilogram of agent. After adding brown sugar, activate it for 1 hour and use it immediately.

[0072] The above method is for making 3 kg of fermented rapeseed meal. In the actual laboratory test, a fermentation bag with a maximum capacity of 5 kg is used. In order to avoid the measurement error of trace bacterial enzyme preparations, it is recommended to make 3 kg of fermented rapeseed meal in each bag each time.

[0073] In the above method, additives A and B can be added to the mixture of rapeseed meal, cornmeal and bran in any order, but if the two prepared solutions are mixed in advance, they need to be fully stirred without precipitation before adding.

[0074] In the above method, during the aerobic fermentation stage of solid fermentation, there is a concentration gradient of oxygen and water, and there is also a gradient of solutes containing nutrients, which leads to vigorous metabolic activity of the bacteria in the upper layer of the fermentation bag and is not easy to stir evenly. However, after entering the anaerobic fermentation, the fermentation bag is inflated due to the continuous production of CO2 by yeast, the pressure in the fermentation bag increases, the viscosity between the rapeseed meal decreases, and it is easy to stir evenly after turning it over. During aerobic fermentation, the bacteria have vigorous respiration and metabolism, and produce a lot of heat. The part of the fermentation bag with a high density of bacteria produces too much heat, which may cause local Maillard reaction and reduce the nutritional value of the fermented rapeseed meal. The number of stirring times can be increased appropriately to speed up the heat dissipation of the fermentation bag, while ensuring that the room temperature is at a lower temperature to prevent the thermostat from high temperature alarm.

[0075] Example 2

[0076] This embodiment provides a method for preparing fermented rapeseed meal, which differs from the preparation method of embodiment 1 only in that: the first fermentation bacterial agent used is: Aspergillus niger 0.02 g (2 billion cfu), Aspergillus oryzae 0.006 g (96 million cfu), Bacillus subtilis 0.3 g (300 million cfu) and Bacillus amyloliquefaciens 0.03 g (3 billion cfu); the second fermentation bacterial agent is: Candida utilis 3 g (300 million cfu) and Lactobacillus plantarum 0.045 g (450 million cfu); The composition of the enzyme preparation used was as follows: cellulase 0.43 g (8600U), xylanase 0.58 g (5800U), β-mannanase 0.10 g (5000U), pectinase 0.70 g (21910U), amylase 1.306 g (1306U), saccharifying enzyme 0.324 g (32400U), bromelain 0.28 g (14000U), papain 0.14 g (14280U), and acid protease 0.14 g (7000U).

[0077] Comparative Example 1

[0078] This comparative example provides a method for preparing fermented rapeseed meal, which differs from the preparation method of Example 1 only in that the first fermentation bacteria used are: 0.02 g (2 billion cfu) of Aspergillus niger, 0.013 g (208 million cfu) of Aspergillus oryzae, 0.2 g (200 million cfu) of Bacillus subtilis and 0.03 g (3 billion cfu) of Bacillus amyloliquefaciens.

[0079] Experimental example

[0080] The fermented rapeseed meal prepared in the above-mentioned embodiments and comparative examples and the preparation method thereof were subjected to effect verification tests. The test was divided into two parts: Test 1 and Test 2. Test 1 was the determination of changes in the content of nutrients, pH, anti-nutritional factors and toxins of the fermented rapeseed meal. Test 2 was a nylon bag throwing test, in which the sample was put into the rumen of a dairy cow through a fistula to detect its rumen degradation characteristics.

[0081] The data were preliminarily analyzed using Excel software, and the results were presented as mean ± standard deviation. The NLIN program in SAS 9.4 software was used to calculate the rumen degradation parameters a, b, and c, and the ANOVA in SPSS statistical software was used to perform one-way analysis of variance for each index. Tukey's method was used for multiple comparisons. P The difference was significant when <0.05.

[0082] 1. Determination of the composition of fermented rapeseed meal

[0083] The control group I was rapeseed meal raw material, and 2 repetitions were set; the control group II was a mixture of rapeseed meal, cornmeal and bran mixed in proportion before fermentation, and 2 repetitions were set; the experimental group I was the fermented rapeseed meal prepared by the method of Example 1, the experimental group II was the fermented rapeseed meal prepared by the method of Example 2, and the experimental group III was the fermented rapeseed meal prepared by the method of Comparative Example 1. Each experimental group was set with 5 repetitions, and the specification of the fermented rapeseed meal prepared was 3000 g, and the total weight of the raw materials, bacterial enzyme preparations and brown sugar was about 3045 g.

[0084] The dry matter was determined by drying the sample at 105℃ for 4 h, cooling it at room temperature for 2 h, and then weighing it; the crude fat was determined in accordance with GB / T 6433-2006; the crude ash was determined in accordance with GB / T 6438-2007; the crude protein was determined by the Dumas combustion method; the starch was determined by the AOAC 996.11 method; the pH was determined as follows: 5 g of the sample was weighed and placed in a 50 mL conical flask, 10 mL of distilled water was added, the sample was shaken and allowed to stand for 24 h, and the pH was determined using a pH meter; the zearalenone and aflatoxin B1 were determined by fluorescent immunocompetitive method; the detection of oxazolidinone was carried out in accordance with GB / T 13089-2020; the detection of isothiocyanates was carried out in accordance with NY / T 1596-2008.

[0085] The results are shown in Table 1. The crude protein content of the fermented rapeseed meal products of Examples 1 and 2 is extremely significant ( P <0.01) was higher than before fermentation (control group II), and zearalenone and aflatoxin B1 were significantly lower than before fermentation ( P <0.01), oxazolidinethione was lower than before fermentation, but the difference was not significant ( P=0.54). Compared with the rapeseed meal raw material, the crude protein of the fermented rapeseed meal of Comparative Example 1 has a significantly lower trend, and compared with the fermented rapeseed meals of Examples 1 and 2, the crude protein is significantly reduced, compared with Example 1, the aflatoxin B1 content is increased, and compared with Example 2, the dry matter content is significantly reduced. It can be seen that the fermented rapeseed meal of the present invention has the characteristics of high protein content and low toxin content.

[0086] Table 1 Nutrient composition, toxins and anti-nutritional factors of rapeseed meal before and after fermentation

[0087] Note: Different lowercase letters in the same row indicate significant differences ( P <0.05)、 P The values ​​are the results of significant difference analysis among the control group I, II, and the experimental group I, II, and III.

[0088] 2. Nylon bag throwing test

[0089] The experiment adopted the rumen in situ nylon bag method, and the time point when the nylon bag was put into the rumen of the dairy cow was recorded as 0 h. The rapeseed meal (control) and fermented rapeseed meal (fermented rapeseed meal prepared by the methods of Examples 1 and 2, respectively) were set at 2, 6, 8, 12, 16, 24, 36 and 48 h, a total of 7 sampling time points, and 2 parallels were set for each cow at each sampling time point, and 3 healthy Holstein cows in mid-lactation with similar body conditions were used as replicates. Drying at 65℃ for 48 h, weighing, and drying at 105℃ for 4 h to determine the dry matter content; the crude protein content was determined using the Dumas combustion method.

[0090] The degradation parameter was calculated based on the degradation rate at each time point using the exponential model proposed by Φrskov et al. (Φrskoy ER, Mcdonald I, The estimation of protein degradation in the rumen from incubation measurements weighted according to rate of passage. Journal of Agricultural Science, 1979, 92(2): 499-503). The calculation formula is as follows: P = a+b (1 - e -ct )

[0091] Where: P (%) is the degradation rate of a nutrient at time point t; a (%) is the fast degradation part; b (%) is the slow degradation part; c is the degradation rate of the slow degradation part (%·h -1After obtaining the a, b and c values ​​of each sample, the effective degradation rate (ED) was calculated by entering the following formula: ED = a+b×c / (c+K)×100%

[0092] Where: K is the rumen outflow rate constant of the feed to be tested, and the value in this test is 0.0528 (%·h -1 ).

[0093] The contents of rumen degradable protein (RDP) and rumen undegradable protein (RUP) in the samples were calculated according to the following formula: RDP={a+b×[K d / (K d +K p )]}×CP / DM; RUP={b×[K p / (K d +K p )]+c}×CP / DM; Where: a is the rapidly degradable part of crude protein in each sample; b is the slowly degradable part of crude protein; c is the non-degradable part of crude protein; K d K is the degradation rate of the slowly degradable part of crude protein; p is the rumen circulation rate of the sample, and the value in this test is 0.0528 (%·h -1 ).

[0094] The content of metabolizable protein provided by the sample is calculated according to the following formula: MP = RDP × 65.9% + RUP × 80%; In the formula: MP is the metabolizable protein provided by the feed, 65.9% is the efficiency of rumen degradable protein being finally converted into metabolizable protein after digestion and absorption in the small intestine, and 80% is the efficiency of rumen non-degradable protein being finally converted into metabolizable protein after digestion and absorption in the small intestine.

[0095] The results are shown in Tables 2, 3 and Figure 1 , Figure 2 As shown in Table 2, the disappearance rates of dry matter (DM) and crude protein (CP) of the fermented rapeseed meal of Examples 1 and 2 at each time point were higher than those of the rapeseed meal raw material (Table 2 and Figure 1 , Figure 2 ), and the metabolizable protein of the fermented rapeseed meal of Examples 1 and 2 was significantly higher than that of the rapeseed meal ( P <0.05) (Table 3). It can be seen that the fermented rapeseed meal prepared by the method of the present invention can provide more metabolizable protein for dairy cows after digestion and absorption.

[0096] Table 2 DM and CP disappearance rates of rapeseed meal raw material and fermented rapeseed meal at different time points

[0097] Table 3 CP rumen degradation parameters of rapeseed meal raw material and fermented rapeseed meal at different time points

[0098] Note: P The value of rapeseed meal, Example 1, and 2 P value.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing fermented rapeseed meal, characterized in that: The method comprises: mixing a fermentation raw material containing rapeseed meal with an enzyme preparation and a first fermentation bacterial agent for aerobic fermentation, and after the aerobic fermentation is completed, adding a second fermentation bacterial agent for anaerobic fermentation; The first fermentation bacterial agent comprises Aspergillus niger, Aspergillus oryzae, Bacillus subtilis and Bacillus amyloliquefaciens; The second fermentation bacterial agent comprises Candida utilis and Lactobacillus plantarum; Among them, in the first fermentation bacterial agent, the ratio of the effective live bacteria count of Aspergillus niger, Aspergillus oryzae, Bacillus subtilis and Bacillus amyloliquefaciens is (1.5-20): (0.96-1.6): 3:

30.

2. The method for preparing fermented rapeseed meal according to claim 1, characterized in that: The addition amount of the first fermentation agent is 3.6-5.4 billion cfu / kg fermented rapeseed meal.

3. The method for preparing fermented rapeseed meal according to claim 1, characterized in that: In the second fermentation bacterial agent, the ratio of the effective viable counts of Candida utilis and Lactobacillus plantarum is 1:(1.2-1.8); And / or, the added amount of the second fermentation agent is 750-800 million cfu / kg fermented rapeseed meal.

4. The method for preparing fermented rapeseed meal according to any one of claims 1 to 3, characterized in that: The enzyme preparation comprises: cellulase 2100-2200 U / g, xylanase 1400-1500 U / g, β-mannanase 1200-1300 U / g, pectinase 5400-5500 U / g, amylase 320-330 U / g, saccharifying enzyme 8100-8200 U / g, bromelain 3450-3550 U / g, papain 3550-3650 U / g, and acid protease 1700-1800 U / g; And / or, the enzyme preparation is added in an amount of 3-5 g / kg fermented rapeseed meal.

5. The method for preparing fermented rapeseed meal according to any one of claims 1 to 3, characterized in that: The aerobic fermentation is firstly carried out at 29-31°C for 20-28h, and then at 27-28°C for 44-52h; And / or, the anaerobic fermentation is carried out at 29-31° C. for 90-110 h.

6. The method for preparing fermented rapeseed meal according to any one of claims 1 to 3, characterized in that: The fermentation raw material further comprises a nutritional additive and one or more selected from cornmeal and bran; The nutritional additives include additive A and additive B, wherein additive A includes the following components: NH4Cl 30-40 g / L, KH2PO4 7-10 g / L, NaH2PO4 5-10 g / L, MgSO4 5-10 g / L, MnCl2·4H2O 0.1-0.4 g / L; The auxiliary agent B includes the following components: 10-20 g / L of calcium pantothenate and 60-80 g / L of tricalcium citrate.

7. The method for preparing fermented rapeseed meal according to claim 6, characterized in that: In the fermentation raw materials, the mass ratio of rapeseed meal, cornmeal and bran is (1500-2500): (50-100): (50-100); the addition amount of auxiliary agent A is 18-22 mL / kg of fermented rapeseed meal, and the addition amount of auxiliary agent B is 8-12 mL / kg of fermented rapeseed meal.

8. A fermented rapeseed meal, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the fermented rapeseed meal according to claim 8 in the preparation of ruminant feed or in ruminant breeding.

10. A ruminant feed, characterized in that: It comprises the fermented rapeseed meal according to claim 8.

Images