Preparation method of fermented soybean meal feed

Abstract

The invention discloses a preparation method of a fermented soybean meal feed. The preparation method comprises the following steps: preparing a fermentation inoculant and a soybean meal fermentation material; inoculating an aspergillus fermentation inoculant into the soybean meal fermentation material to carry out first-stage aerobic fermentation; then inoculating a bacillus fermentation inoculant to carry out second-stage aerobic fermentation; then inoculating an anaerobic probiotic fermentation inoculant, and carrying out third-stage anaerobic fermentation; and finally, drying, crushing and sieving to obtain the fermented soybean meal feed. According to the method, the problems of single bacterium advantage and strain antagonism in the fermentation process can be effectively avoided; the parameter regulation and control process in the fermentation process is greatly simplified; the obtained fermented soybean meal feed is good in palatability and has acid fragrance, the content of acid soluble protein and crude protein in the fermented soybean meal feed is obviously increased, the content of crude fiber and antinutritional factors is obviously reduced, and the fermented soybean meal feed does not contain mycotoxin and is high in safety.

Description

Technical Field

[0001] The invention relates to feed preparation, in particular to a method for preparing fermented soybean meal feed. Background Art

[0002] With the rapid development of the feed industry and livestock and poultry farming, soybean meal, as the main by-product of soybean processing, has become one of the most widely used plant protein feed raw materials with its high protein content of 40-50%, 1-2% fat content and about 10-15% carbohydrate content. However, the presence of a variety of anti-nutritional factors in soybean meal (such as trypsin inhibitors, urease, lectins, phytic acid, soybean antigen protein and soybean lectin, etc.) significantly reduces its feeding value. In addition, the soybean protein in soybean meal has a complex molecular structure and a large molecular weight, which leads to its digestibility and biological value in animals being lower than that of animal proteins such as fish meal, which greatly limits the application of soybean meal in feed.

[0003] At present, the main deep processing methods to improve the utilization efficiency of soybean meal for feed include physical methods, chemical methods and biological fermentation methods: physical methods include high temperature heating, microwave, extrusion, ultrafiltration and other processing technologies, which can reduce the content of anti-nutritional factors in soybean meal to a certain extent. However, physical methods usually cannot significantly improve the quality of soybean meal protein, and may even cause the loss of nutrients due to improper processing conditions; chemical methods include ammonia treatment, soda ash treatment, metal salt catalytic treatment and alcohol dissolution treatment. These methods have a certain effect on removing anti-nutritional factors, but due to the complex operation, high cost, and the possible introduction of residual chemicals, their wide application in the feed industry is limited; biological fermentation methods use microorganisms or enzyme preparations to ferment or enzymatically hydrolyze soybean meal, which can significantly increase the content of polypeptides and free amino acids in soybean meal, effectively remove anti-nutritional factors, and are rich in active substances such as probiotics, organic acids and multifunctional peptides. It has shown significant advantages in alleviating weaning stress of suckling pigs, reducing the diarrhea rate of suckling pigs, improving the carcass quality of livestock and poultry, and improving animal production performance and immunity.

[0004] Although the biofermentation method has shown great potential in improving the feed value of soybean meal, the following problems still exist in its application: (1) Single bacterial advantage and strain antagonism of fermentation strains: In a mixed bacterial fermentation system, some strains may overproliferate due to metabolic advantages, thereby inhibiting the activity of other strains and causing a decrease in system diversity. In addition, resource competition between strains and the accumulation of metabolites may lead to strain antagonism, further weakening the fermentation effect and product consistency. (2) Process parameters are difficult to determine: Different strains have different requirements for environmental conditions (such as temperature, pH value, oxygen concentration, etc.), which increases the difficulty of regulating fermentation process parameters and easily causes an imbalance in the proportion of bacterial populations, thereby affecting the quality and stability of fermentation products. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a method for preparing high-quality fermented soybean meal feed by staged fermentation.

[0006] Technical solution: The method for preparing fermented soybean meal feed of the present invention comprises:

[0007] (1) preparing fermentation agents of Aspergillus, Bacillus and anaerobic probiotics respectively;

[0008] (2) preparing soybean meal fermentation feed;

[0009] (3) inoculating the soybean meal fermentation material with an Aspergillus fermentation agent in an amount not exceeding 12% of the mass of the soybean meal fermentation material to carry out the first stage of aerobic fermentation;

[0010] (4) After the first stage of aerobic fermentation is completed, a Bacillus fermentation agent of no more than 12% of the mass of the initial soybean meal fermentation material is inoculated to carry out the second stage of aerobic fermentation;

[0011] (5) After the second stage of aerobic fermentation is completed, an anaerobic probiotic fermentation agent of no more than 12% of the mass of the initial soybean meal fermentation material is inoculated to carry out the third stage of anaerobic fermentation;

[0012] (6) Drying, crushing and sieving to obtain fermented soybean meal feed.

[0013] Preferably, in step 1, the Aspergillus fermentation agent is Aspergillus niger liquid, the Bacillus fermentation agent is Bacillus cereus liquid, and the anaerobic probiotic fermentation agent is Bifidobacterium-Lactobacillus liquid.

[0014] Preferably, the Aspergillus niger liquid contains the CICC2238 strain, the Bacillus cereus liquid contains the CMCC63305 strain, and the Bifidobacterium-Lactobacillus liquid contains the CCFM16, CCFM683, CCFM202, CCFM1068, and CN1566 strains.

[0015] Preferably, step 2 comprises weighing soybean meal, adding 1.8-2.2 times the mass of water, mixing thoroughly and then sterilizing.

[0016] Preferably, the soybean meal is soybean meal 46.

[0017] Preferably, step 3 comprises:

[0018] (31) inoculating 8-12% of the mass of soybean meal fermentation material with Aspergillus niger liquid and spreading it flat to a thickness of 2.5-4 cm;

[0019] (32) 24-26°C, static fermentation for 5-7 days;

[0020] Among them, the concentration of the inoculated Aspergillus niger strain is not less than 150 mg / mL.

[0021] Preferably, step 4 comprises:

[0022] (41) The fermentation product obtained in step 3 is inoculated with 8-12% of the mass of the initial soybean meal fermentation material of Bacillus cereus liquid and spread to a thickness of 2.5-4 cm;

[0023] (42) 34-38°C, static fermentation for 1-2 days;

[0024] Among them, the concentration of inoculated Bacillus cereus strains is not less than 10 8 cfu / mL.

[0025] Preferably, step 5 comprises:

[0026] (51) The fermentation product obtained in step 4 is inoculated with a bifidobacterium-lactobacillus culture solution of 8-12% of the mass of the initial soybean meal fermentation material, and transferred into a sealed container;

[0027] (52)30-37℃, static fermentation for 2-3 days.

[0028] Among them, the concentration of inoculated Bifidobacterium-Lactobacillus strains is not less than 10 8 cfu / mL.

[0029] Preferably, in step 6, the drying temperature is 50-60° C., and the powder is sieved through a 30-40 mesh screen after being crushed.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. It can effectively avoid the problems of single bacteria dominance and strain antagonism in the fermentation process; 2. It simplifies the parameter control process in the fermentation process; 3. The acid-soluble protein and crude protein contents in the obtained fermented soybean meal feed are significantly improved, and the crude fiber and anti-nutritional factor contents are significantly reduced, and it does not contain mycotoxins and has high safety; 4. It has good palatability and a sour aroma. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below.

[0032] Example 1: Preparation of fermented soybean meal feed 1

[0033] 1. Prepare fermentation agent:

[0034] (1) Aspergillus niger liquid: Aspergillus niger CICC2238 was inoculated on a PDA medium plate and cultured at 25°C. After the mycelium covered the entire plate, a culture medium block (0.5×0.5×0.5 cm) containing bacteria and spores was scratched and inoculated into PDB medium at a ratio of 3 blocks per 170 mL. The culture was carried out at 25°C, 200 rpm, and cultured for 2 days. The Aspergillus niger concentration was 200 mg / mL.

[0035] (2) Bacillus cereus culture liquid: Bacillus cereus CMCC63305 was inoculated on LB solid culture medium plates and cultured at 36°C. After colonies were formed, a single colony was picked up using an inoculation loop and inoculated into LB liquid culture medium at a ratio of 1 colony per 100 mL. The culture was incubated at 36°C, 200 rpm, for 10 h. The concentration of Bacillus cereus was 1.4×10 8 cfu / mL;

[0036] (3) Bifidobacterium-Lactobacillus culture liquid: Dayixi powder package (containing CCFM16, CCFM683, CCFM202, CCFM1068, and CN1566 strains) was inoculated into MRS liquid culture medium and cultured anaerobically at 33°C, 200 rpm for 20 h. The concentration of Bifidobacterium-Lactobacillus strains was 1.6×10 8 cfu / mL.

[0037] 2. Prepare soybean meal fermentation feed:

[0038] Weigh soybean meal, add twice the mass of water, mix thoroughly, and sterilize at high temperature.

[0039] 3. The first stage of aerobic fermentation

[0040] The soybean meal fermented material obtained in step 2 was added with Aspergillus niger CICC2238 culture liquid of 10% of the mass of the soybean meal fermented material, spread in a shallow dish with a thickness of 3 cm, and statically fermented at 25° C. for 6 days.

[0041] 4. Second stage of aerobic fermentation

[0042] The soybean meal fermented material obtained in step 3 was added with 10% of the mass of the initial soybean meal fermented material of Bacillus cereus CMCC63305 bacterial liquid, spread in a shallow dish with a thickness of 3 cm, and statically fermented at 25° C. for 6 days.

[0043] 5. The third stage of anaerobic fermentation

[0044] The soybean meal fermentation material obtained in step 4 was added with 10% of the mass of the Bifidobacterium-Lactobacillus culture solution of the initial soybean meal fermentation material, and the mixture was transferred into a sealed conical flask and statically fermented at 33° C. for 3 days.

[0045] 6. The fermented soybean meal obtained in step 5 is dried at 55° C., crushed, and sieved through a 35-mesh sieve to obtain a fermented soybean meal feed.

[0046] Example 2: Preparation of fermented soybean meal feed 2

[0047] 1. Prepare fermentation agent:

[0048] (1) Aspergillus niger liquid: Aspergillus niger CICC2238 was inoculated on a PDA medium plate and cultured at 26°C. After the mycelium covered the entire plate, a culture medium block (0.5×0.5×0.5 cm) containing the mycelium and spores was scratched and inoculated into PDB medium at a ratio of 3 blocks per 100 mL. The culture was carried out at 25°C, 200 rpm, for 1 day. The Aspergillus niger concentration was 150 mg / mL.

[0049] (2) Bacillus cereus culture liquid: Bacillus cereus CMCC63305 was inoculated on LB solid medium plates and cultured at 38°C. After colonies were formed, a single colony was picked up using an inoculation loop and inoculated into LB liquid medium at a ratio of 1 colony per 100 mL. The culture was carried out at 38°C, 200 rpm, and the strain concentration of Bacillus cereus was 1×10 8 cfu / mL;

[0050] (3) Bifidobacterium-Lactobacillus culture liquid: Dayixi powder package (containing CCFM16, CCFM683, CCFM202, CCFM1068, and CN1566 strains) was inoculated into MRS liquid culture medium and cultured anaerobically at 37°C, 200 rpm for 18 h. The concentration of Bifidobacterium-Lactobacillus strains was 1.3×10 8 cfu / mL.

[0051] 2. Prepare soybean meal fermentation feed:

[0052] Weigh soybean meal, add 1.8 times the mass of water, mix thoroughly, and sterilize at high temperature.

[0053] 3. The first stage of aerobic fermentation

[0054] The soybean meal fermented material obtained in step 2 was added with 12% of the mass of the soybean meal fermented material, and the Aspergillus niger CICC2238 culture liquid was spread in a shallow dish with a thickness of 2.5 cm, and statically fermented at 25° C. for 5 days.

[0055] 4. Second stage of aerobic fermentation

[0056] The soybean meal fermented material obtained in step 3 was added with 12% of the mass of the initial soybean meal fermented material of Bacillus cereus CMCC63305 bacterial liquid, spread in a shallow dish with a thickness of 3 cm, and statically fermented at 25° C. for 6 days.

[0057] 5. The third stage of anaerobic fermentation

[0058] The soybean meal fermented material obtained in step 4 was added with 10% of the mass of the Bifidobacterium-Lactobacillus culture solution of the initial soybean meal fermented material, and the mixture was transferred into a sealed conical flask and statically fermented at 37° C. for 2 days.

[0059] 6. The fermented soybean meal obtained in step 5 is dried at 55° C., crushed, and sieved through a 35-mesh sieve to obtain a fermented soybean meal feed.

[0060] Example 3: Preparation of fermented soybean meal feed 3

[0061] 1. Prepare fermentation agent:

[0062] (1) Aspergillus niger liquid: Aspergillus niger CICC2238 was inoculated on a PDA medium plate and cultured at 26°C. After the mycelium covered the entire plate, a culture medium block (0.5×0.5×0.5 cm) containing the mycelium and spores was scratched and inoculated into PDB medium at a ratio of 3 blocks per 100 mL. The culture was incubated at 24°C, 200 rpm, for 2 days. The Aspergillus niger concentration was 200 mg / mL.

[0063] (2) Bacillus cereus culture liquid: Bacillus cereus CMCC63305 was inoculated on LB solid medium plates and cultured at 38°C. After colonies were formed, a single colony was picked up using an inoculation loop and inoculated into LB liquid medium at a ratio of 1 colony per 100 mL. The culture was incubated at 35°C, 200 rpm, for 12 h. The concentration of Bacillus cereus was 1.8×10 8 cfu / mL;

[0064] (3) Bifidobacterium-Lactobacillus culture liquid: Dayixi powder package (containing CCFM16, CCFM683, CCFM202, CCFM1068, and CN1566 strains) was inoculated into MRS liquid culture medium and cultured anaerobically at 30°C, 200 rpm for 24 h. The concentration of Bifidobacterium-Lactobacillus strains was 1.9×10 8 cfu / mL.

[0065] 2. Prepare soybean meal fermentation feed:

[0066] Weigh soybean meal, add 1.8 times the mass of water, mix thoroughly, and sterilize at high temperature.

[0067] 3. The first stage of aerobic fermentation

[0068] The soybean meal fermented material obtained in step 2 was added with 8% of the mass of the soybean meal fermented material, and the Aspergillus niger CICC2238 culture liquid was spread in a shallow dish with a thickness of 2.5 cm, and statically fermented at 24° C. for 7 days.

[0069] 4. Second stage of aerobic fermentation

[0070] The soybean meal fermented material obtained in step 3 was added with 8% of the mass of the initial soybean meal fermented material of Bacillus cereus CMCC63305 bacterial liquid, spread in a shallow dish with a thickness of 3 cm, and statically fermented at 35° C. for 1 day.

[0071] 5. The third stage of anaerobic fermentation

[0072] The soybean meal fermentation material obtained in step 4 was added with 8% of the mass of the initial soybean meal fermentation material, and the mixture was transferred into a sealed conical flask and statically fermented at 30° C. for 3 days.

[0073] 6. The fermented soybean meal obtained in step 5 is dried at 55° C., crushed, and sieved through a 35-mesh sieve to obtain a fermented soybean meal feed.

[0074] Example 4: Component Analysis of Fermented Soybean Meal Feeds 1-3

[0075] According to the detection method specified in the industry standard NY / T 2218-2022, the fermented soybean meal and ordinary soybean meal obtained in the above three examples were tested, and each example was repeated three times. The test data were subjected to one-way analysis of variance, and the data results were expressed as "mean ± standard deviation", with P < 0.05 as the criterion for judging the significance of the difference. The results are shown in Tables 1 and 2:

[0076] Table 1 Component test results of fermented soybean meal feed 1-3

[0077] Detection indicators Example 1 Example 2 Example 3 Soybean meal 46 Crude protein (%) 52.9±2.1 60.9±3.3 55.4±1.9 46.1±1.2 Crude fiber (%) 6.3±0.5 2.2±0.2 3.9±0.3 15.7±1.6 Crude ash (%) 5.8±0.2 6.2±0.2 5.7±0.1 6.4±0.2 Acid soluble protein (%) 42.1±0.9 45.3±0.8 43.6±0.6 2.8±0.3 Lysine(%) 2.6±0.1 2.7±0.1 2.5±0.1 2.4±0.1 Stachyose(%) ND ND ND 35.7±2.5 Urease Urease activity (U / g) 0.09±0.01 0.07±0.01 0.07±0.01 0.18±0.01 β-conglycinin (mg / g) 28.3±1.4 15.5±1.2 32.2±2.3 112.3±9.2 Coliform group (MPN / g) <3.0 <3.0 <3.0 <30

[0078] Among them, ND means not detected.

[0079] Table 2 Mycotoxin detection results in fermented soybean meal and ordinary soybean meal

[0080]

[0081] Among them, ND means not detected.

[0082] The results showed that the crude protein content (46.1%) of fermented soybean meal in Examples 1-3 was significantly higher (P<0.05) than that of soybean meal 46, which were 52.9%, 60.9% and 55.4% respectively; the crude fiber content (15.7%) of fermented soybean meal in Examples 1-3 was significantly lower (P<0.05) than that of soybean meal 46, which were 6.3%, 2.2% and 3.9% respectively; the acid-soluble protein content (2.8%) of fermented soybean meal in Examples 1-3 was significantly higher (P<0.05) than that of soybean meal 46, which were 42.1%, 45.3% and 43.6% respectively; the anti-nutritional factor stachyose of fermented soybean meal in Examples 1-3 was reduced to below the detection limit compared with soybean meal 46, and the urease activity and β-conglycinin were significantly decreased (P<0.05). Further, none of the 6 common mycotoxins were detected in fermented soybean meal Examples 1-3.

[0083] Example 5: Oral toxicity test of fermented soybean meal feed 1-3

[0084] 1. The testing environment is SPF level, temperature 20-26℃, humidity 40-70%;

[0085] 2. The experimental animals were SPF-grade KM mice, weighing 18-22 g, half male and half female, a total of 20 mice, of which the female mice were unmated and non-pregnant, and were provided by Jinan Pengyue Experimental Animal Breeding Co., Ltd.;

[0086] 3. Weigh 10g of fermented soybean meal feed 1, 2 or 3 respectively, add pure water to prepare 40mL of test solution, mix well, mark and set aside;

[0087] 4. KM mice were fasted for 4 h, with free access to water, and given oral administration at a dose of 10,000 mg / kg·bw, after which the mice continued to fast for 1 h;

[0088] 5. Observe for 14 days, including the skin, fur, eyes, mucous membranes, respiratory system, urogenital system, digestive system and nervous system. Comprehensively observe and record the time, extent and duration of changes in the animals, evaluate possible target organs of toxic effects, weigh and record the animal weight at the beginning and end of the experiment, and weigh the animals once a week during the observation period.

[0089] The results are shown in Table 2. After the KM mice were given the fermented soybean meal diet for 1, 2 or 3 days, no abnormal symptoms and no death were observed within 14 days.

[0090] Table 2 Body weight and mortality of KM mice after being fed with fermented soybean meal diet for 1, 2 or 3 days

[0091]

[0092] After the experimental observation, all animals were killed and pathological examination was performed. No abnormalities were found. The acute oral toxicity of fermented soybean meal feed 1, 2 or 3 to KM mice was LD 50 ≥10000mg / kg·bw.

Claims

1. A method for preparing fermented soybean meal feed, characterized in that: include: (1) preparing fermentation agents of Aspergillus, Bacillus and anaerobic probiotics respectively; (2) preparing soybean meal fermentation feed; (3) inoculating the soybean meal fermentation material with an Aspergillus fermentation agent in an amount not exceeding 12% of the mass of the soybean meal fermentation material to carry out the first stage of aerobic fermentation; (4) After the first stage of aerobic fermentation is completed, a Bacillus fermentation agent of no more than 12% of the mass of the initial soybean meal fermentation material is inoculated to carry out the second stage of aerobic fermentation; (5) After the second stage of aerobic fermentation is completed, an anaerobic probiotic fermentation agent of no more than 12% of the mass of the initial soybean meal fermentation material is inoculated to carry out the third stage of anaerobic fermentation; (6) Drying, crushing and sieving to obtain fermented soybean meal feed.

2. The method for preparing fermented soybean meal feed according to claim 1, characterized in that: In the step 1, the Aspergillus fermentation agent is Aspergillus niger liquid, the Bacillus fermentation agent is Bacillus cereus liquid, and the anaerobic probiotic fermentation agent is Bifidobacterium-Lactobacillus liquid.

3. The method for preparing fermented soybean meal feed according to claim 2, characterized in that: The Aspergillus niger liquid contains the CICC2238 strain, the Bacillus cereus liquid contains the CMCC63305 strain, and the Bifidobacterium-Lactobacillus liquid contains the CCFM16, CCFM683, CCFM202, CCFM1068, and CN1566 strains.

4. The method for preparing fermented soybean meal feed according to claim 1, characterized in that: The step 2 comprises: weighing soybean meal, adding 1.8-2.2 times the mass of water, mixing thoroughly and sterilizing.

5. The method for preparing fermented soybean meal feed according to claim 4, characterized in that: The soybean meal is soybean meal 46.

6. The method for preparing fermented soybean meal feed according to claim 1, characterized in that: The step 3 comprises: (31) inoculating 8-12% of the mass of soybean meal fermentation material with Aspergillus niger liquid and spreading it flat to a thickness of 2.5-4 cm; (32) 24-26°C, static fermentation for 5-7 days; Among them, the concentration of the inoculated Aspergillus niger strain is not less than 150 mg / mL.

7. The method for preparing fermented soybean meal feed according to claim 1, characterized in that: The step 4 comprises: (41) The fermentation product obtained in step 3 is inoculated with 8-12% of the mass of the initial soybean meal fermentation material of Bacillus cereus liquid and spread to a thickness of 2.5-4 cm; (42) 34-38°C, static fermentation for 1-2 days; Among them, the concentration of inoculated Bacillus cereus strains is not less than 10 8 cfu / mL.

8. The method for preparing fermented soybean meal feed according to claim 1, characterized in that: The step 5 comprises: (51) The fermentation product obtained in step 4 is inoculated with a bifidobacterium-lactobacillus culture solution of 8-12% of the mass of the initial soybean meal fermentation material, and transferred into a sealed container; (52)30-37℃, static fermentation for 2-3 days. Among them, the concentration of inoculated Bifidobacterium-Lactobacillus strains is not less than 10 8 cfu / mL.

9. The method for preparing fermented soybean meal feed according to claim 1, characterized in that: In step 6, the drying temperature is 50-60°C.

10. The method for preparing fermented soybean meal feed according to claim 1, characterized in that: In the step 6, the sieving is performed through a 30-40 mesh sieve.

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