A fermented feed mainly containing rice bran and a preparation method thereof

Abstract

The present application belongs to the technical field of fermented feed, and particularly relates to a fermented feed taking rice bran as main material and a preparation method thereof. High-quality protein and other nutritional components in rice bran are usually combined with substances such as phytic acid in rice bran, and are not easy to be digested and absorbed by the gastrointestinal tract of animals. In view of the above problems, the present application provides a fermented feed taking rice bran as main material, which comprises fermentation raw materials, fermentation seed liquid, phytase and acid protease. Research shows that, for the degradation of phytate in rice bran, in addition to the phytase in the fermented feed playing a role, the type and combination of strains in the fermented feed also promote the degradation of phytic acid (salt) in rice bran. The bacillus licheniformis, lactobacillus plantarum and saccharomyces cerevisiae preferred by the present application can further improve the degradation rate of phytic acid (salt) and protein in rice bran, improve the release amount of inorganic phosphorus and polypeptide in the fermented feed, and then improve the utilization rate of nutritional components in rice bran.

Description

Technical Field

[0001] This invention belongs to the field of fermented feed technology, specifically relating to a fermented feed with rice bran as the main ingredient and its preparation method. Background Technology

[0002] The livestock industry accounts for 34% of my country's total agricultural output. One of the major problems facing the development of my country's livestock industry is the low utilization rate of feed ingredients. Currently, the proportion of grain used for feed has increased from 20% in the 1950s to 40% of total grain production; it is estimated that by 2030, my country's demand for feed grain may reach 56.5% of total grain demand. Solving the problem of secure feed grain supply will be key to ensuring food security. In addition to finding new sources of feed ingredients, improving the utilization rate of existing feed ingredients is the main way to alleviate the above problems.

[0003] Rice bran is a major byproduct of rice milling, accounting for nearly 10% of total rice processing, with an annual output of approximately 110,000 to 150,000 tons. Rice bran contains 11-15% crude protein, mainly composed of high-quality albumin, globulin, and other proteins. However, rice bran has a high phytic acid content, reaching up to 58 mg / g. Phytic acid readily combines with proteins, calcium, and other minerals to form insoluble phytates, significantly affecting the digestion and absorption of high-quality protein, calcium, and other nutrients in the animal's gastrointestinal tract, thus reducing the utilization rate of rice bran feed ingredients. Furthermore, phytic acid readily complexes with digestive enzymes in the animal's gastrointestinal tract, reducing enzyme activity and further decreasing the digestion and absorption of high-quality protein, calcium, and other nutrients, further reducing the utilization rate of rice bran feed ingredients.

[0004] Microbial fermentation is a reliable method to improve the nutritional value of feed ingredients and reduce anti-nutritional factors. Microbial fermentation technology can use extracellular hydrolytic enzymes secreted by microorganisms to degrade feed ingredients, remove potential anti-nutritional factors (such as phytic acid and phytates) in feed ingredients, and degrade hydrophobic proteins in feed ingredients into small molecule peptides. On the one hand, it improves the utilization rate of high-quality protein and other nutrients in feed ingredients, and on the other hand, it endows the feed with good antioxidant and anti-inflammatory effects.

[0005] By mixing rice bran with corn gluten meal and wheat bran in a mass ratio of 6:3:1, and adding molasses at 1% of the total mass of the fermentation raw materials, a fermented feed with rice bran as the main ingredient is formed. This not only effectively alleviates the shortage of fermented feed in my country, but also helps to increase the added value of rice bran and promotes the healthy development of the rice processing industry. Summary of the Invention

[0006] The problem with existing technologies is that the high-quality protein and other nutrients in rice bran are usually tightly bound to substances such as phytic acid, making them difficult for animals to digest, absorb, and utilize. To address this problem, this invention provides a fermented feed with rice bran as the main ingredient, comprising fermentation raw materials, fermentation seed liquid, phytase, and acidic protease. The fermentation seed liquid includes Bacillus licheniformis seed liquid, Lactobacillus plantarum seed liquid, and Saccharomyces cerevisiae seed liquid.

[0007] The Bacillus licheniformis seed culture, Lactobacillus plantarum seed culture, and Saccharomyces cerevisiae seed culture were obtained by seed propagation of Bacillus licheniformis (accession number CGMCC 13109), Lactobacillus plantarum (accession number CGMCC 24691), and Saccharomyces cerevisiae (accession number CGMCC 21353), respectively.

[0008] The volume ratio of Bacillus licheniformis seed liquid to Lactobacillus plantarum seed liquid and Saccharomyces cerevisiae seed liquid in the fermentation seed liquid is (3-10):(1-6):(1-3);

[0009] The fermentation raw materials include rice bran, corn gluten meal, wheat bran, and molasses. The mass ratio of rice bran to corn gluten meal and wheat bran is 6:3:1, and the amount of molasses added is 1% of the total mass of the fermentation raw materials.

[0010] The fermentation seed liquid includes compound microbial seed liquid and sterile water. The inoculation amount of the compound microbial seed liquid is 4-12% of the total weight of the fermentation raw materials. The moisture content of the fermented feed is 25-45% (excluding the moisture in the compound microbial seed liquid).

[0011] The amount of phytase added to the fermented feed is 10-50 U / g.

[0012] The acidic protease is added to the fermented feed at a rate of 10-90 U / g.

[0013] Specifically, the preparation method of the fermented feed mainly composed of rice bran includes the following steps:

[0014] (1) Bacillus licheniformis, Lactobacillus plantarum and Saccharomyces cerevisiae were cultured separately to obtain seed culture liquids, and then mixed thoroughly according to the formula volume ratio to obtain compound seed culture liquids;

[0015] (2) According to the formula amount, the compound bacterial seed liquid obtained in step (1) is thoroughly mixed with sterile water to obtain fermentation seed liquid;

[0016] (3) Add rice bran, corn gluten powder, wheat bran and molasses into a mixer according to the formula and mix thoroughly to obtain raw material premix;

[0017] (4) According to the formula, add phytase, acidic protease and the fermented seed liquid obtained in step (2) to the raw material premix obtained in step (3), mix thoroughly, and then seal and anaerobic ferment to obtain fermented feed.

[0018] Specifically, the method for propagating Bacillus licheniformis seeds includes the following steps:

[0019] (1) Slant culture of strains

[0020] Single strains of Bacillus licheniformis were inoculated onto slant agar and cultured at 30-35℃ for 1-3 days to obtain primary seed culture. The slant agar was LB solid medium.

[0021] (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 30-35℃ to obtain Bacillus licheniformis seed liquid with an OD value of 0.55-0.60. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10g tryptone, 5g yeast powder, 10g sodium chloride, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making the volume up to 1000mL. The solution is then sterilized at 121℃ for 30min to obtain Bacillus licheniformis seed culture medium with pH=6.8-7.0.

[0022] Specifically, the method for propagating Lactobacillus plantarum seeds includes the following steps:

[0023] (1) Slant culture of strain

[0024] Single strains of Lactobacillus plantarum were inoculated onto slant agar and cultured at 30-35℃ for 1-3 days to obtain primary seed culture. The slant agar was MRS solid medium.

[0025] (2) The primary seeds obtained in step (1) are inoculated into a seed culture medium and cultured at 30-35℃ to obtain a Lactobacillus plantarum seed solution with an OD value of 0.55-0.60. The inoculation amount is one loopful of bacteria. The seed culture medium is composed of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution. The solution is dissolved in water and the volume is adjusted to 1000mL. The solution is then sterilized at 121℃ for 30min to obtain a Lactobacillus plantarum seed culture medium with pH = 6.2-6.6.

[0026] Specifically, the method for propagating brewer's yeast seeds includes the following steps:

[0027] (1) Slant culture of strains

[0028] Single strains of Saccharomyces cerevisiae were inoculated onto slant culture medium and cultured at 28-30℃ for 1-3 days to obtain primary seed culture. The slant culture medium was YPD solid medium.

[0029] (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 28-30℃ for 18h to obtain a brewer's yeast seed liquid with an OD value of 0.55-0.60. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10g of yeast extract, 20g of tryptone and 20g of glucose in water and making the volume up to 1000mL, and sterilizing at 121℃ for 30min to obtain the brewer's yeast seed culture medium.

[0030] Specifically, the LB solid culture medium is a medium with pH = 6.8-7.0 obtained by dissolving 10g tryptone, 5g yeast powder, 10g sodium chloride, 20g agar powder, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making up to 1000mL, and then sterilizing at 121℃ for 30min.

[0031] Specifically, the MRS solid culture medium is a medium with a pH of 6.2-6.6 obtained by dissolving 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, 20g agar, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making up to 1000mL, and then sterilizing at 121℃ for 30min.

[0032] Specifically, the YPD solid culture medium is obtained by dissolving 10g yeast extract, 20g tryptone, 20g glucose, and 20g agar powder in water to a final volume of 1000mL, and then sterilizing at 121℃ for 30min.

[0033] Specifically, the fermentation method is bag fermentation, the fermentation temperature is 25-37℃, and the fermentation time is 3-7 days.

[0034] The present invention has the following beneficial effects:

[0035] (1) The fermented feed of the present invention contains phytase, which can degrade phytic acid (salt) in rice bran into inositol and inorganic phosphorus. After the phytic acid (salt) is degraded, the high-quality protein and other nutrients in rice bran can be better digested, absorbed and utilized by the animal's gastrointestinal tract.

[0036] (2) The present invention has found that, in addition to the role of phytase in fermented feed in the degradation of phytic acid (salt) in rice bran, the type and combination of microorganisms in fermented feed also promote the degradation of phytic acid (salt) in rice bran. The research results show that, based on the fermentation raw materials, fermentation methods and conditions of the present invention, using Bacillus licheniformis, Lactobacillus plantarum and Saccharomyces cerevisiae as the preferred composite microorganisms can further improve the degradation rate of phytic acid (salt) in rice bran, further increase the release of inorganic phosphorus in fermented feed, and consequently, further improve the utilization rate of nutrients in rice bran.

[0037] (3) The present invention has found that the preferred combination of Bacillus licheniformis (accession number CGMCC 13109), Lactobacillus plantarum (accession number CGMCC 24691), and Saccharomyces cerevisiae (accession number CGMCC 21353) can better transform the rice bran-based fermented raw material of the present invention into a fermented feed with high water-soluble protein and high small molecule polypeptide content through microbial fermentation.

[0038] (4) The present invention has found that, in addition to the role of acidic protease in the fermented feed, the type and combination of microorganisms in the fermented feed also promote the degradation of protein in the fermented feed, which is mainly composed of rice bran. The research results show that, for the fermented feed, fermentation method and conditions of the present invention, using Bacillus licheniformis, Lactobacillus plantarum and Saccharomyces cerevisiae as the preferred composite microorganisms can further improve the degradation rate and water solubility of protein in the feed, further increase the content of small molecule polypeptides in the fermented feed, and consequently, further improve the utilization rate of nutrients in rice bran. Detailed Implementation

[0039] The present invention will be described in detail below with reference to embodiments. However, it should be understood that the following embodiments are merely illustrative examples of implementation of the present invention and are not intended to limit the scope of the present invention.

[0040] The YPD solid culture medium described in the following embodiments of the present invention is obtained by dissolving 10g yeast extract, 20g tryptone, 20g glucose, and 20g agar powder in water to a final volume of 1000mL, and then sterilizing at 121°C for 30min.

[0041] Example 1

[0042] A fermented feed primarily composed of rice bran comprises fermentation raw materials, fermentation seed liquid, phytase (CAS: 37288-11-2), and acidic protease (CAS: 9025-49-4). The fermentation seed liquid consists of Bacillus licheniformis seed liquid, Saccharomyces cerevisiae seed liquid, and Lactobacillus plantarum seed liquid.

[0043] The Bacillus licheniformis seed culture, Lactobacillus plantarum seed culture, and Saccharomyces cerevisiae seed culture were obtained by seed propagation of Bacillus licheniformis (accession number CGMCC 13109), Lactobacillus plantarum (accession number CGMCC 24691), and Saccharomyces cerevisiae (accession number CGMCC 21353), respectively.

[0044] The volume ratio of Bacillus licheniformis seed liquid to Lactobacillus plantarum seed liquid and Saccharomyces cerevisiae seed liquid in the fermentation seed liquid is 6:2:1.

[0045] The fermentation raw materials consist of rice bran, corn gluten powder, wheat bran, and molasses. The mass ratio of rice bran to corn gluten powder and wheat bran is 6:3:1, and the amount of molasses added is 1% of the total mass of the fermentation raw materials.

[0046] The fermentation seed liquid is composed of compound bacterial seed liquid and sterile water. The inoculation amount of the compound bacterial seed liquid is 8% of the total weight of the fermentation raw materials, and the moisture content of the fermented feed is 35%.

[0047] The phytase is added to the fermented feed at a rate of 30 U / g.

[0048] The acidic protease was added to the fermented feed at a rate of 70 U / g.

[0049] The method for propagating Bacillus licheniformis seeds comprises the following steps:

[0050] (1) Slant culture of strains

[0051] A single Bacillus licheniformis strain was inoculated onto an slant culture medium and cultured at 35°C for 1 day to obtain primary seed culture. The slant culture medium was LB solid medium.

[0052] (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 35°C to obtain a seed liquid with an OD value of 0.55. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10g tryptone, 5g yeast powder, 10g sodium chloride, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making the volume up to 1000mL. The solution is then sterilized at 121°C for 30min to obtain Bacillus licheniformis seed culture medium with pH=6.8.

[0053] The method for propagating Lactobacillus plantarum seeds comprises the following steps:

[0054] (1) Slant culture of strain

[0055] Single strains of Lactobacillus plantarum were inoculated onto slant agar and cultured at 35°C for 1 day to obtain primary seed culture. The slant agar was MRS solid medium.

[0056] (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 35°C to obtain a seed liquid with an OD value of 0.55. The inoculation amount is one loopful of bacteria. The seed culture medium is composed of 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution. The solution is dissolved in water and the volume is adjusted to 1000mL. The solution is then sterilized at 121°C for 30min to obtain a Lactobacillus plantarum seed culture medium with pH=6.2.

[0057] The method for propagating the brewer's yeast seed includes the following steps:

[0058] (1) Slant culture of strains

[0059] A single strain of Saccharomyces cerevisiae was inoculated onto an agar slant and cultured at 30°C for 1 day to obtain primary seed culture. The agar slant culture medium was YPD solid medium.

[0060] (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 30°C to obtain a seed liquid with an OD value of 0.55. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10g of yeast extract, 20g of tryptone and 20g of glucose in water and making the volume up to 1000mL, and sterilizing at 121°C for 30min to obtain the Saccharomyces cerevisiae seed culture medium.

[0061] The LB solid medium is a medium with pH = 6.8 obtained by dissolving 10g tryptone, 5g yeast powder, 10g sodium chloride, 20g agar powder, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making up to 1000mL, and then sterilizing at 121℃ for 30min.

[0062] The MRS solid culture medium was prepared by dissolving 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, 20g agar, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and bringing the volume to 1000mL, then sterilizing at 121℃ for 30min to obtain a culture medium with a pH of 6.2.

[0063] The fermented feed with rice bran as the main ingredient is prepared as follows:

[0064] (1) Bacillus licheniformis, Lactobacillus plantarum and Saccharomyces cerevisiae were cultured separately to obtain seed culture liquids, and then mixed thoroughly according to the formula volume ratio to obtain compound seed culture liquids;

[0065] (2) According to the formula amount, the compound bacterial seed liquid obtained in step (1) is thoroughly mixed with sterile water to obtain fermentation seed liquid;

[0066] (3) Add rice bran, corn gluten powder, wheat bran and molasses into a mixer according to the formula and mix thoroughly to obtain raw material premix;

[0067] (4) Add the formulated amount of phytase, acidic protease and the fermented seed liquid obtained in step (2) to the raw material premix obtained in step (3), mix thoroughly, and then carry out sealed anaerobic fermentation. The fermentation method is bag fermentation, the fermentation temperature is 30℃, and the fermentation time is 5 days to obtain fermented feed.

[0068] Example 2

[0069] A fermented feed primarily composed of rice bran comprises fermentation raw materials, fermentation seed liquid, phytase, and acidic protease. The fermentation seed liquid consists of Bacillus licheniformis seed liquid, Lactobacillus plantarum seed liquid, and Saccharomyces cerevisiae seed liquid.

[0070] The Bacillus licheniformis seed culture, Lactobacillus plantarum seed culture, and Saccharomyces cerevisiae seed culture were obtained by seed propagation of Bacillus licheniformis (accession number CGMCC 13109), Lactobacillus plantarum (accession number CGMCC 24691), and Saccharomyces cerevisiae (accession number CGMCC 21353), respectively.

[0071] The volume ratio of Bacillus licheniformis seed liquid to Lactobacillus plantarum seed liquid and Saccharomyces cerevisiae seed liquid in the fermentation seed liquid is 10:6:3.

[0072] The fermentation raw materials consist of rice bran, corn gluten powder, wheat bran, and molasses. The mass ratio of rice bran to corn gluten powder and wheat bran is 6:3:1, and the amount of molasses added is 1% of the total mass of the fermentation raw materials.

[0073] The fermentation seed liquid is composed of compound bacterial seed liquid and sterile water. The inoculation amount of the compound bacterial seed liquid is 12% of the total weight of the fermentation raw materials, and the moisture content of the fermented feed is 45%.

[0074] The phytase is added to the fermented feed at a rate of 50 U / g.

[0075] The acidic protease was added to the fermented feed at a rate of 90 U / g.

[0076] The method for propagating Bacillus licheniformis seeds comprises the following steps:

[0077] (1) Slant culture of strains

[0078] Single strains of Bacillus licheniformis were inoculated onto slant agar and cultured at 33°C for 3 days to obtain primary seed culture. The slant agar was LB solid medium.

[0079] (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 33°C to obtain a seed liquid with an OD value of 0.60. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10g tryptone, 5g yeast powder, 10g sodium chloride, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making the volume up to 1000mL. The solution is then sterilized at 121°C for 30min to obtain Bacillus licheniformis seed culture medium with pH=7.0.

[0080] The method for propagating Lactobacillus plantarum seeds comprises the following steps:

[0081] (1) Slant culture of strain

[0082] Single strains of Lactobacillus plantarum were inoculated onto slant agar and cultured at 33°C for 3 days to obtain primary seed culture. The slant agar was MRS solid medium.

[0083] (2) The primary seeds obtained in step (1) were inoculated into the seed culture medium and cultured at 33°C to obtain a seed liquid with an OD value of 0.60. The inoculation amount was one loopful of bacteria. The seed culture medium was made by dissolving 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making up to 1000mL. The solution was then sterilized at 121°C for 30min to obtain a Lactobacillus plantarum seed culture medium with pH=6.6.

[0084] The method for propagating the brewer's yeast seed includes the following steps:

[0085] (1) Slant culture of strain

[0086] A single strain of Saccharomyces cerevisiae was inoculated onto an agar slant and cultured at 30°C for 3 days to obtain primary seed culture. The agar slant culture medium was YPD solid medium.

[0087] (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 30°C to obtain a seed liquid with an OD value of 0.60. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10g of yeast extract, 20g of tryptone and 20g of glucose in water and making the volume up to 1000mL, and sterilizing at 121°C for 30min to obtain the Saccharomyces cerevisiae seed culture medium.

[0088] The MRS solid culture medium is prepared by dissolving 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, 20g agar, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and bringing the volume to 1000mL, then sterilizing at 121℃ for 30min to obtain a culture medium with pH=6.6.

[0089] The LB solid medium is a medium with pH = 7.0 obtained by dissolving 10g tryptone, 5g yeast powder, 10g sodium chloride, 20g agar powder, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making up to 1000mL, and then sterilizing at 121℃ for 30min.

[0090] The fermented feed with rice bran as the main ingredient is prepared as follows:

[0091] (1) Bacillus licheniformis, Lactobacillus plantarum and Saccharomyces cerevisiae were cultured separately to obtain seed culture liquids, and then mixed thoroughly according to the formula volume ratio to obtain compound seed culture liquids;

[0092] (2) According to the formula amount, the compound bacterial seed liquid obtained in step (1) is thoroughly mixed with sterile water to obtain fermentation seed liquid;

[0093] (3) Add rice bran, corn gluten powder, wheat bran and molasses into a mixer according to the formula and mix thoroughly to obtain raw material premix;

[0094] (4) Add the formulated amount of phytase, acidic protease and the fermented seed liquid obtained in step (2) to the raw material premix obtained in step (3), mix thoroughly, and then carry out sealed anaerobic fermentation. The fermentation method is bag fermentation, the fermentation temperature is 37℃, and the fermentation time is 7 days to obtain fermented feed.

[0095] Example 3

[0096] A fermented feed primarily composed of rice bran comprises fermentation raw materials, fermentation seed liquid, phytase, and acidic protease. The fermentation seed liquid consists of Bacillus licheniformis seed liquid, Lactobacillus plantarum seed liquid, and Saccharomyces cerevisiae seed liquid.

[0097] The Bacillus licheniformis seed culture, Lactobacillus plantarum seed culture, and Saccharomyces cerevisiae seed culture were obtained by seed propagation of Bacillus licheniformis (accession number CGMCC 13109), Lactobacillus plantarum (accession number CGMCC 24691), and Saccharomyces cerevisiae (accession number CGMCC 21353), respectively.

[0098] The volume ratio of Bacillus licheniformis seed liquid to Lactobacillus plantarum seed liquid and Saccharomyces cerevisiae seed liquid in the fermentation seed liquid is 3:1:1.

[0099] The fermentation raw materials consist of rice bran, corn gluten powder, wheat bran, and molasses. The mass ratio of rice bran to corn gluten powder and wheat bran is 6:3:1, and the amount of molasses added is 1% of the total mass of the fermentation raw materials.

[0100] The fermentation seed liquid is composed of compound bacterial seed liquid and sterile water. The inoculation amount of the compound bacterial seed liquid is 4% of the total weight of the fermentation raw materials, and the moisture content of the fermented feed is 25%.

[0101] The phytase is added to the fermented feed at a rate of 10 U / g.

[0102] The acidic protease is added to the fermented feed at a rate of 10 U / g.

[0103] The method for propagating Bacillus licheniformis seeds comprises the following steps:

[0104] (1) Slant culture of strains

[0105] Single strains of Bacillus licheniformis were inoculated onto slant agar and cultured at 30°C for 2 days to obtain primary seed culture. The slant agar was LB solid medium.

[0106] (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 30°C to obtain a seed liquid with an OD value of 0.55. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10g tryptone, 5g yeast powder, 10g sodium chloride, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making the volume up to 1000mL. The solution is then sterilized at 121°C for 30min to obtain Bacillus licheniformis seed culture medium with pH=6.9.

[0107] The method for propagating Lactobacillus plantarum seeds comprises the following steps:

[0108] (1) Slant culture of strains

[0109] Single strains of Lactobacillus plantarum were inoculated onto slant agar and cultured at 30°C for 2 days to obtain primary seed culture. The slant agar was MRS solid medium.

[0110] (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 30°C to obtain a seed liquid with an OD value of 0.60. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making up to 1000mL. The solution is then sterilized at 121°C for 30min to obtain a Lactobacillus plantarum seed culture medium with pH=6.4.

[0111] The method for propagating the brewer's yeast seed includes the following steps:

[0112] (1) Slant culture of strain

[0113] A single strain of Saccharomyces cerevisiae was inoculated onto an agar slant and cultured at 28°C for 2 days to obtain primary seed culture. The agar slant culture medium was YPD solid medium.

[0114] (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 28°C to obtain a seed liquid with an OD value of 0.58. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10g of yeast extract, 20g of tryptone and 20g of glucose in water and making the volume up to 1000mL, and sterilizing at 121°C for 30min to obtain the brewing yeast culture medium.

[0115] The MRS solid culture medium is prepared by dissolving 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, 20g agar, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and bringing the volume to 1000mL, then sterilizing at 121℃ for 30min to obtain a culture medium with pH=6.5.

[0116] The LB solid medium is a medium with pH = 7.0 obtained by dissolving 10g tryptone, 5g yeast powder, 10g sodium chloride, 20g agar powder, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making up to 1000mL, and then sterilizing at 121℃ for 30min.

[0117] The fermented feed with rice bran as the main ingredient is prepared as follows:

[0118] (1) Bacillus licheniformis, Lactobacillus plantarum and Saccharomyces cerevisiae were cultured separately to obtain seed culture liquids, and then mixed thoroughly according to the formula volume ratio to obtain compound seed culture liquids;

[0119] (2) According to the formula amount, the compound bacterial seed liquid obtained in step (1) is thoroughly mixed with sterile water to obtain fermentation seed liquid;

[0120] (3) Add rice bran, corn gluten powder, wheat bran and molasses into a mixer according to the formula and mix thoroughly to obtain raw material premix;

[0121] (4) Add the formulated amount of phytase, acidic protease and the fermented seed liquid obtained in step (2) to the raw material premix obtained in step (3), mix thoroughly, and then carry out sealed anaerobic fermentation. The fermentation method is bag fermentation, the fermentation temperature is 25℃, and the fermentation time is 3 days to obtain fermented feed.

[0122] Comparative Example 1 is the same as Example 1, except that Comparative Example 1 uses Saccharomyces cerevisiae with accession number ACCC 21399 instead of Saccharomyces cerevisiae with accession number CGMCC 21353 in Example 1. The method of Saccharomyces cerevisiae seed propagation in Comparative Example 1 is the same as the method of Saccharomyces cerevisiae seed propagation in Example 1.

[0123] Comparative Example 2 is the same as Example 1, except that Comparative Example 3 uses Saccharomyces cerevisiae with accession number ACCC 21278 instead of Saccharomyces cerevisiae with accession number CGMCC 21353 in Example 1. The method of Saccharomyces cerevisiae seed propagation in Comparative Example 3 is the same as the method of Saccharomyces cerevisiae seed propagation in Example 1.

[0124] Comparative Example 3 is the same as Example 1, except that Comparative Example 3 uses ACCC 21280 instead of the Saccharomyces cerevisiae with accession number CGMCC 21353 in Example 1. The method of Saccharomyces cerevisiae seed propagation in Comparative Example 3 is the same as the method of Saccharomyces cerevisiae seed propagation in Example 1.

[0125] Comparative Example 4 is the same as Example 1, except that Comparative Example 4 uses Candida pruriens with accession number ACCC 20060 instead of Saccharomyces cerevisiae with accession number CGMCC 21353 in Example 1. The method of Candida pruriens seed propagation in Comparative Example 4 is the same as the method of Saccharomyces cerevisiae seed propagation in Example 1.

[0126] Comparative Example 5 is the same as Example 1, except that Comparative Example 5 uses Lactobacillus plantarum with accession number ACCC 11095 instead of Lactobacillus plantarum with accession number CGMCC 24691 in Example 1. The method of Lactobacillus plantarum seed propagation in Comparative Example 5 is the same as the method of Lactobacillus plantarum seed propagation in Example 1.

[0127] Comparative Example 6 is the same as Example 1, except that Comparative Example 6 uses Lactobacillus reuteri with accession number ACCC 03960 instead of Lactobacillus plantarum with accession number CGMCC 24691 in Example 1. The method of seed propagation of Lactobacillus reuteri in Comparative Example 6 is the same as the method of seed propagation of Lactobacillus plantarum in Example 1.

[0128] Comparative Example 7 is the same as Example 1, except that Bacillus subtilis with accession number ACCC 19742 is used in Comparative Example 7 instead of Bacillus licheniformis with accession number CGMCC 13109 in Example 1. The method of seed propagation of Bacillus subtilis in Comparative Example 7 is the same as the method of seed propagation of Bacillus licheniformis in Example 1.

[0129] Comparative Example 8 is the same as Example 1, except that Bacillus subtilis with accession number ACCC 04396 is used in Comparative Example 8 instead of Bacillus licheniformis with accession number CGMCC 13109 in Example 1. The method of seed propagation of Bacillus subtilis in Comparative Example 8 is the same as the method of seed propagation of Bacillus licheniformis in Example 1.

[0130] Comparative Example 9 is the same as Example 1, except that Bacillus subtilis with accession number ACCC 04401 is used in Comparative Example 9 instead of Bacillus licheniformis with accession number CGMCC 13109 in Example 1. The method of seed propagation of Bacillus subtilis in Comparative Example 9 is the same as the method of seed propagation of Bacillus licheniformis in Example 1.

[0131] Comparative Example 10 is the same as Example 1, except that Bacillus licheniformis with accession number ACCC 19372 is used in Comparative Example 10 instead of Bacillus licheniformis with accession number CGMCC 13109 in Example 1. The method of seed propagation of Bacillus licheniformis in Comparative Example 10 is the same as the method of seed propagation of Bacillus licheniformis in Example 1.

[0132] Comparative Example 11 is the same as Example 1, except that no Lactobacillus plantarum and Saccharomyces cerevisiae were added in Comparative Example 11, and only Bacillus licheniformis (accession number CGMCC 13109) was used as the fermentation strain.

[0133] Comparative Example 12 is the same as Example 1, except that Bacillus licheniformis and Lactobacillus plantarum were not added in Comparative Example 12, and only Saccharomyces cerevisiae with accession number CGMCC 21353 was used as the fermentation strain.

[0134] Comparative Example 13 is the same as Example 1, except that Bacillus licheniformis was not added in Comparative Example 13, and only Saccharomyces cerevisiae and Lactobacillus plantarum were used as a compound strain. The fermentation seed liquid in Comparative Example 13 was obtained by thoroughly mixing Saccharomyces cerevisiae seed liquid and Lactobacillus plantarum seed liquid at a volume ratio of 2:1.

[0135] Comparative Example 14 is the same as Example 1, except that Lactobacillus plantarum was not added in Comparative Example 14. Only two strains, Saccharomyces cerevisiae (accession number CGMCC 21353) and Bacillus licheniformis (accession number CGMCC 13109), were used as the compound strain. The fermentation seed liquid in Comparative Example 14 was obtained by thoroughly mixing Saccharomyces cerevisiae seed liquid and Bacillus licheniformis seed liquid at a volume ratio of 2:1.

[0136] Comparative Example 15 is the same as Example 1, except that Comparative Example 15 uses Lactobacillus rhamnosus (ACCC 05450) instead of Lactobacillus plantarum with accession number CGMCC 24691 in Example 1. The method of Lactobacillus rhamnosus seed propagation in Comparative Example 15 is the same as the method of Lactobacillus plantarum seed propagation in Example 1.

[0137] Comparative Example 16 is the same as Example 1, except that Comparative Example 16 does not contain Bacillus licheniformis, Lactobacillus plantarum, Saccharomyces cerevisiae and phytase, but only uses acidic protease for fermentation.

[0138] Comparative Example 17 is the same as Example 1, except that Comparative Example 17 uses a neutral protease instead of the acidic protease in Example 1.

[0139] Comparative Example 18 is the same as Example 1, except that Comparative Example 18 does not contain Bacillus licheniformis, Lactobacillus plantarum, Saccharomyces cerevisiae and acid protease, but only uses phytase fermentation.

[0140] Performance testing

[0141] The components of the fermented feeds obtained in Examples 1-3 and Comparative Examples 1-18 of this invention were analyzed, and the specific test results are shown in Table 1.

[0142] The specific testing method is as follows:

[0143] Crude protein: Tested according to GB / T 24318-2009.

[0144] Crude fat: Tested according to GB / T 6433-2006.

[0145] Crude fiber: Tested according to GB / T 6434-2022.

[0146] Ash content: Tested according to GB / T 6438-2007.

[0147] Soluble proteins: tested according to the Folin-phenol method.

[0148] Polypeptides: Tested according to the trichloroacetic acid (TCA) precipitation method.

[0149] Viable bacteria count: Tested according to GB4789.2-2022.

[0150] Phosphorus content: Tested in accordance with GB / T 6437-2018.

[0151] Table 1

[0152]

[0153] Note: Inorganic phosphorus is a product of the complete hydrolysis of phytic acid (salt). The increased phosphorus and soluble protein content in the culture medium after fermentation indicates a high degree of degradation of phytic acid (salt) and hydrophobic proteins in the raw materials. Phytic acid is an anti-nutritional factor in feed, while protein is a nutrient. The high degradation rate of both indicates that fermentation reduces the content of anti-nutritional factors and improves the availability and digestibility of nutrients in the feed. Polypeptides are protein degradation products with potential antioxidant and immune-enhancing functions. A high polypeptide content in fermented feed suggests that the feed may possess these potential antioxidant and immune-enhancing functions.

[0154] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A fermented feed mainly composed of rice bran, characterized in that, It consists of fermentation raw materials, fermentation seed liquid, phytase, and acidic protease. The fermentation seed liquid is composed of Bacillus licheniformis seed liquid, Lactobacillus plantarum seed liquid, and Saccharomyces cerevisiae seed liquid. The Bacillus licheniformis seed culture, Lactobacillus plantarum seed culture, and Saccharomyces cerevisiae seed culture were obtained by seed propagation of Bacillus licheniformis YY-2 (accession number CGMCC 13109), Lactobacillus plantarum YY-7 (accession number CGMCC 24691), and Saccharomyces cerevisiae YY-4 (accession number CGMCC 21353), respectively. The volume ratio of Bacillus licheniformis seed liquid to Lactobacillus plantarum seed liquid and Saccharomyces cerevisiae seed liquid in the fermentation seed liquid is (6-10):(2-6):(1-3); The fermentation raw materials include rice bran, corn gluten meal, wheat bran, and molasses. The mass ratio of rice bran to corn gluten meal and wheat bran is 6:3:1, and the amount of molasses added is 1% of the total mass of the fermentation raw materials. The fermentation seed liquid includes a compound microbial seed liquid and sterile water. The inoculation amount of the compound microbial seed liquid is 8-12% of the total weight of the fermentation raw materials, and the moisture content of the fermented feed is 35-45%. The amount of phytase added to the fermented feed is 30-50 U / g; The acidic protease is added to the fermented feed at a rate of 70-90 U / g.

2. The fermented feed with rice bran as the main ingredient according to claim 1, characterized in that, The preparation method includes the following steps: (1) Bacillus licheniformis, Lactobacillus plantarum and Saccharomyces cerevisiae were cultured separately to obtain seed culture liquids, and then mixed thoroughly according to the formula volume ratio to prepare compound seed culture liquids; (2) According to the formula amount, mix the compound bacterial seed liquid obtained in step (1) with sterile water thoroughly to obtain fermentation seed liquid; (3) According to the formula, add rice bran, corn gluten powder, wheat bran and molasses into a mixer and mix thoroughly to obtain raw material premix; (4) According to the formula, add phytase, acidic protease and the fermented seed liquid obtained in step (2) to the raw material premix obtained in step (3), mix thoroughly, and then seal and anaerobic ferment to obtain fermented feed.

3. The fermented feed with rice bran as the main ingredient according to claim 2, characterized in that, The method for propagating brewer's yeast seed includes the following steps: (1) Slant culture of strain Single strains of Saccharomyces cerevisiae were inoculated onto slant culture medium and cultured at 28-30℃ for 1-3 days to obtain primary seed culture. The slant culture medium was YPD solid medium. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 28-30℃ to obtain a brewer's yeast seed liquid with an OD value of 0.55-0.

60. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10 g of yeast extract, 20 g of tryptone and 20 g of glucose in water and making the volume up to 1000 mL, and sterilizing at 121℃ for 30 min to obtain the brewer's yeast seed culture medium.

4. The fermented feed with rice bran as the main ingredient according to claim 2, characterized in that, The method for propagating Lactobacillus plantarum seeds includes the following steps: (1) Slant culture of strain Single strains of Lactobacillus plantarum were inoculated onto slant agar and cultured at 30-35℃ for 1-3 days to obtain primary seed culture. The slant agar was MRS solid medium. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 30-35℃ to obtain Lactobacillus plantarum seed liquid with an OD value of 0.55-0.

60. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making up to 1000 mL. The solution is then sterilized at 121℃ for 30 min to obtain Lactobacillus plantarum seed culture medium with pH=6.2-6.

6.

5. A fermented feed mainly composed of rice bran according to claim 2, characterized in that, The method for propagating Bacillus licheniformis seeds includes the following steps: (1) Slant culture of strain Single strains of Bacillus licheniformis were inoculated onto slant agar and cultured at 30-35℃ for 1-3 days to obtain primary seed culture. The slant agar was LB solid medium. (2) The primary seeds obtained in step (1) are inoculated into the seed culture medium and cultured at 30-35℃ to obtain Bacillus licheniformis seed liquid with an OD value of 0.55-0.

60. The inoculation amount is one loopful of bacteria. The seed culture medium is made by dissolving 10g tryptone, 5g yeast powder, 10g sodium chloride, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making the volume up to 1000 mL. The solution is then sterilized at 121℃ for 30 min to obtain Bacillus licheniformis seed culture medium with pH=6.8-7.

0.

6. The fermented feed with rice bran as the main ingredient according to claim 3, characterized in that, The YPD solid culture medium is obtained by dissolving 10 g yeast extract, 20 g tryptone, 20 g glucose, and 20 g agar powder in water to a final volume of 1000 mL, and then sterilizing at 121°C for 30 min.

7. The fermented feed with rice bran as the main ingredient according to claim 4, characterized in that, The MRS solid culture medium is prepared by dissolving 10g peptone, 5g yeast extract, 20g glucose, 5g sodium acetate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 2g dipotassium hydrogen phosphate, 1mL Tween 80, 2g diammonium hydrogen citrate, 10g beef extract, 20g agar, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water to a final volume of 1000mL, and then sterilizing at 121℃ for 30min to obtain a culture medium with a pH of 6.2-6.

6.

8. A fermented feed mainly composed of rice bran according to claim 5, characterized in that, The LB solid medium is a medium with a pH of 6.8-7.0 obtained by dissolving 10g tryptone, 5g yeast extract, 10g sodium chloride, 20g agar powder, and an appropriate amount of 0.5mol / L sodium hydroxide aqueous solution in water and making up to 1000mL, and then sterilizing at 121℃ for 30min.

9. A fermented feed mainly composed of rice bran according to any one of claims 2-8, characterized in that, The fermentation method is bag fermentation, the fermentation temperature is 25-37℃, and the fermentation time is 3-7 days.

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