Functional rapeseed meal as well as preparation method and application thereof

Abstract

The invention relates to the technical field of feed, and provides functional rapeseed meal as well as a preparation method and application thereof. The preparation method of the functional rapeseed meal provided by the invention comprises the following steps: sterilizing rapeseed meal to obtain a rapeseed meal solid culture medium; the method comprises the following steps: inoculating a rapeseed meal solid culture medium with a bacterial liquid containing serratia marcescens, and carrying out aerobic fermentation at 20-40 DEG C to obtain the functional rapeseed meal. Serratia marcescens is used for fermenting the rapeseed dregs, degradation of anti-nutritional factors such as thioglycoside is promoted, meanwhile, the content of tannin in the rapeseed dregs is increased, the advantages of strains can be fully utilized, the yield of nutrient substances in the rapeseed dregs is maximized, the nutrition and health care effects are diversified, and the cost is low.

Description

Technical Field

[0001] The present application belongs to the field of feed technology, and in particular relates to a functional rapeseed meal and a preparation method and application thereof. Background Art

[0002] With the improvement of living standards, dietary structure has changed greatly, people's consumption of meat, eggs and milk has increased year by year, which has brought about an increase in the demand for protein feed in the animal husbandry industry. At present, the shortage of feed protein resources has become a common problem in the world, which has seriously restricted the development of animal husbandry.

[0003] Rapeseed meal is a byproduct of rapeseed processing, with an annual output of more than 8 million tons. Rapeseed meal has a protein content of 35-42%, a crude fiber content of 12-13%, a balanced amino acid composition, high lysine and sulfur-containing amino acids, and is rich in a variety of mineral elements. It has a high feeding value and is a stable and relatively cheap protein feed resource with a wide source. It can effectively replace soybean meal and improve the current shortage of protein feed in my country. However, due to the presence of more anti-nutritional factors such as glucosinolates and tannins, especially glucosinolates, animals may cause goiter, restrict animal growth, and even poison and die after eating. Therefore, these substances have seriously restricted the application of rapeseed meal. However, with people's new understanding of tannins, it is found that an appropriate amount of tannins can improve animal growth efficiency and intestinal health, and improve antioxidant and anti-diarrhea capabilities. Therefore, tannins are widely used in livestock and poultry production as feed additives, but this has increased feed costs to a certain extent. Summary of the invention

[0004] The purpose of the present application is to provide a functional rapeseed meal and a preparation method thereof, aiming to solve the problem that the nutritional value of rapeseed meal is not ideal and the feeding cost is high.

[0005] Another object of the present application is to provide an animal feed, aiming to solve the problems of high cost and unbalanced nutrition of existing animal feed.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In a first aspect, the present application provides a method for preparing a functional rapeseed meal, comprising the following steps:

[0008] Step S10, sterilizing the rapeseed meal to obtain a rapeseed meal solid culture medium;

[0009] Step S20, inoculating the bacterial liquid containing Serratia marcescens into the rapeseed meal solid culture medium, and performing aerobic fermentation at 20-40° C. to obtain functional rapeseed meal.

[0010] In a second aspect, the present application provides a functional rapeseed meal prepared using the preparation method of the functional rapeseed meal of the present application.

[0011] In a third aspect, the present application provides an animal feed, comprising the functional rapeseed meal prepared by the preparation method of the functional rapeseed meal of the present application and / or the functional rapeseed meal of the present application.

[0012] The preparation method of the functional rapeseed meal provided in the first aspect of the present application utilizes Serratia marcescens to ferment the rapeseed meal, thereby promoting the degradation of anti-nutritional factors such as glucosinolates and increasing the tannin content in the rapeseed meal. The advantages of the bacterial strain can be fully utilized to maximize the yield of nutrients in the rapeseed meal, and the nutritional and health effects are diverse and the cost is low.

[0013] The functional rapeseed meal provided in the second aspect of the present application uses rapeseed meal as raw material, and after microbial fermentation, it forms a functional rapeseed meal with low glucosinolate content and high tannin content, giving it higher nutritional value and lower economic cost.

[0014] The animal feed provided in the third aspect of the present application includes functional rapeseed meal with low glucosinolate content and high tannin content. This characteristic can reduce the risk of slow growth of livestock and poultry and damage to their health caused by the high glucosinolate content when rapeseed meal is used as a feed raw material. In addition, its raw material resources are abundant and the cost is low, which is very suitable for the modern feed industry. It has a sour and fragrant taste, good palatability for livestock and poultry, and a high nutrient degradation rate. It can replace soybean meal in livestock and poultry feed, and can reduce the import volume of soybean meal by at least 10%. It can be widely used and promoted in animal husbandry production. DETAILED DESCRIPTION

[0015] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0016] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0017] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0018] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0019] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0020] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0021] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0022] The first aspect of the embodiment of the present application provides a method for preparing a functional rapeseed meal, comprising the following steps:

[0023] Step S10, sterilizing the rapeseed meal to obtain a rapeseed meal solid culture medium;

[0024] Step S20, inoculating the bacterial liquid containing Serratia marcescens into the rapeseed meal solid culture medium, and performing aerobic fermentation at 20-40° C. to obtain functional rapeseed meal.

[0025] The preparation method of the functional rapeseed meal provided in the first aspect of the embodiment of the present application utilizes Serratia marcescens to ferment the rapeseed meal, thereby promoting the degradation of anti-nutritional factors such as glucosinolates and increasing the tannin content in the rapeseed meal. The advantages of the bacterial strain can be fully utilized to maximize the yield of nutrients in the rapeseed meal, and the nutritional and health effects are diverse and the cost is low.

[0026] The preparation method provided in the embodiment of the present application is simple, can effectively prepare functional rapeseed meal with stable performance, and has the characteristics of being cheap and safe, and is suitable for wide application and promotion in animal husbandry production.

[0027] In some embodiments, in step S10, the step of sterilizing rapeseed meal comprises:

[0028] The rapeseed meal is mixed with water, sealed, and sterilized at 121-126°C.

[0029] Before inoculating the strains, the rapeseed meal is sterilized at high temperature. On the one hand, high temperature killing can eliminate the miscellaneous bacteria and harmful bacteria contained in the rapeseed meal, improving the cleanliness and safety of the fermentation process. On the other hand, the protein is moderately denatured under the action of high temperature, which is more conducive to the degradation of anti-nutritional factors while increasing the degradation rate of nutrients.

[0030] In some specific embodiments, in step S10, the material-water ratio of rapeseed meal to water is 1 to 3:1. Specifically, the material-water ratio of rapeseed meal to water may include but is not limited to 1:1, 2:1, and 3:1.

[0031] In this application, the feed-water ratio refers to the mass-to-volume ratio of rapeseed meal to water, where the unit of mass is g and the unit of volume is mL.

[0032] In some specific embodiments, in step S10, the sterilization treatment time is 10 to 20 minutes. The specific sterilization treatment time can be 10 minutes, 12 minutes, 15 minutes, 18 minutes, 20 minutes or any range thereof.

[0033] Exemplarily, rapeseed meal is mixed with water, and charged into a 250 mL beaker at a material-water ratio of 1:1 (w / v). After stirring evenly, the beaker is sealed with a sealing film, and the natural pH is set and autoclaved at 121° C. for 20 min. After being removed from the pot, the mixture is cooled and broken up for later use.

[0034] In some embodiments, in step S20, Serratia marcescens was deposited in the China Center for Type Culture Collection on March 2, 2023, with the deposit number being CCTCC NO: M 2023232.

[0035] In the embodiment of the present application, in the process of screening, isolating and purifying microorganisms in sunny, non-waterlogged soils where rapeseed is grown, a strain capable of degrading glucosinolates and increasing tannin content was found. By comparing its sequence number in the NCBI database, the strain was identified as a Gram-negative bacterium, Serratia marcescens, so it was named Serratia marcescens X-78, and was deposited in the China Center for Type Culture Collection (CCTCC), the deposit time was March 2, 2023, the deposit address was Wuhan University, China, and the strain deposit number was CCTCCNO: M 2023232.

[0036] In some embodiments, the bacterial solution further includes myrosinase and Bacillus subtilis.

[0037] In the examples of the present application, myrosinase promotes the degradation of glucosinolates without destroying other nutrients; Bacillus subtilis has a good degradation effect on glucosinolates in rapeseed meal, and can also increase the content of small peptides, and has good degradation of phytic acid and crude cellulose. When Serratia marcescens, myrosinase and Bacillus subtilis are used to ferment rapeseed meal in the form of bacterial enzyme combined fermentation, the synergistic effect of glucosinolate degradation can be fully exerted, and anti-nutritional substances such as glucosinolates in rapeseed meal can be degraded to the greatest extent, and tannins can be converted into hydrolyzed tannins with the effect of replacing antibiotics, and their content is increased, and a variety of growth-promoting factors can also be produced.

[0038] In some embodiments, the bacterial solution includes a first activation solution containing Serratia marcescens, a second activation solution containing myrosinase, and a third activation solution containing Bacillus subtilis, and the mass ratio of the first activation solution, the second activation solution and the third activation solution is (85-95): (3-7): (3-7).

[0039] The bacterial species or enzymes in the bacterial liquid are mixed in the form of an activation liquid before inoculation. At the same time, there is no antagonistic effect between strains in the fermentation of a single strain, which is conducive to the mass production of highly active enzymes. On the one hand, various highly active and high-level enzymes produced in the fermentation process can be fully utilized. On the other hand, the content of tannins with health benefits such as anti-oxidation and improving intestinal health can be greatly increased, thereby improving the safety and palatability of the functional rapeseed meal, which has a wide range of practical value. Specifically, the mass ratio of the first activation liquid, the second activation liquid and the third activation liquid may include but is not limited to 90:5:5, 86:7:7, and 94:3:3.

[0040] In some embodiments, the preparation process of the first activation solution containing Serratia marcescens comprises the following steps:

[0041] Add water to rapeseed meal and boil for 10-15 minutes, filter, adjust the filtrate to 1L, set the pH to natural, and sterilize at 121℃ for 15 minutes; then inoculate Serratia marcescens X-78 and culture at 37℃ for 48 hours.

[0042] In some embodiments, the preparation process of the second activation solution containing myrosinase comprises the following steps:

[0043] Add water to rapeseed meal and boil for 10-15 minutes, filter, adjust the volume of the filtrate to 1L, set the pH to natural, and sterilize at 121℃ for 15 minutes; then inoculate myrosinase and culture at 37℃ for 48 hours.

[0044] In some embodiments, the preparation process of the third activation solution containing Bacillus subtilis comprises the following steps:

[0045] Add water to rapeseed meal and boil for 10-15 minutes, filter, adjust the filtrate to 1L, set the pH to natural, and sterilize at 121℃ for 15 minutes; then inoculate Bacillus subtilis and culture at 37℃ for 48 hours.

[0046] In some embodiments, the number of viable bacteria of Serratia marcescens in the first activation solution is ≥ 9.5×10 8 cfu / g.

[0047] In some embodiments, the viable count of myrosinase in the second activation solution is ≥ 0.05 U / g.

[0048] In some embodiments, the number of viable Bacillus subtilis in the third activation solution is ≥ 1.5×10 7 cfu / g.

[0049] In some embodiments, in step S20, the inoculation amount of the bacterial solution in the rapeseed meal solid medium is 0.5-1.5%. In the present application, the inoculation amount refers to the volume of the bacterial solution inoculated in the rapeseed meal solid medium per unit mass. Specifically, the inoculation amount can be 0.5%, 0.8%, 1.0%, 1.2%, 1.5% or within the range of any of the above values.

[0050] In some embodiments, in step S20, the aerobic fermentation time is 60 to 72 hours. Specifically, the aerobic fermentation time may include but is not limited to 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, and 72 hours.

[0051] In some embodiments, in step S20, after the aerobic fermentation is completed, drying and crushing are also included.

[0052] In some embodiments, the temperature of the drying process is 60-65° C. Specifically, the temperature of the drying process may include but is not limited to 60° C., 61° C., 62° C., 63° C., 64° C., and 65° C.

[0053] In some embodiments, the particle size after the crushing process is 0.3-0.4 mm.

[0054] A second aspect of an embodiment of the present application provides a functional rapeseed meal prepared by a method for preparing a functional rapeseed meal.

[0055] The functional rapeseed meal provided in the second aspect of the embodiment of the present application is made of rapeseed meal as raw material, and after microbial fermentation, it forms a functional rapeseed meal with low glucosinolate content and high tannin content, giving it high nutritional value and low economic cost. The functional rapeseed meal not only has the effect of detoxification, but also can make it have health care effects such as anti-oxidation, which is very advantageous for the growth and development of animals.

[0056] In some embodiments, the glucosinolate content in the functional rapeseed meal is ≤7.5 μmol / L, and the tannin content is ≥160 mg / kg.

[0057] In some embodiments, the glucosinolate content in the functional rapeseed meal is ≤6.5 μmol / L, and the tannin content is ≥180 mg / kg.

[0058] In some embodiments, the glucosinolate content in the functional rapeseed meal is ≤5.6 μmol / L, and the tannin content is ≥200 mg / kg.

[0059] In some embodiments, the glucosinolate content in the functional rapeseed meal is ≤4.1 μmol / L, and the tannin content is ≥220 mg / kg.

[0060] A third aspect of the embodiments of the present application provides an animal feed, comprising the functional rapeseed meal prepared by the preparation method of the functional rapeseed meal of the present application and / or the functional rapeseed meal of the present application.

[0061] The animal feed provided in the third aspect of the embodiment of the present application includes functional rapeseed meal with low glucosinolate content and high tannin content. This characteristic can reduce the risk of slow growth of livestock and poultry and damage to their health caused by the high glucosinolate content when rapeseed meal is used as a feed raw material. In addition, its raw material resources are abundant and the cost is low, which is very suitable for the modern feed industry. It has a sour and fragrant taste, good palatability for livestock and poultry, and a high nutrient degradation rate. It can replace soybean meal in livestock and poultry feed, and can reduce the import volume of soybean meal by at least 10%. It can be widely used and promoted in animal husbandry production.

[0062] The following describes the invention in conjunction with specific embodiments.

[0063] Example 1 Screening of strains

[0064] In this embodiment, a strain capable of degrading glucosinolates and increasing tannin content was obtained by screening, isolating and purifying microorganisms from sunny, water-free soil where rapeseed was grown for many years.

[0065] The specific process is as follows:

[0066] Weigh 5 bags of rapeseed meal, 300g / bag, respectively, wrap them with a single layer of gauze and bury them in the soil at a depth of 15-20cm. Then, take out the fermented rapeseed meal (fermented silage) at the 2nd, 3rd, 4th and 5th week after burying in the soil (7d interval between two samplings). Scrape about 2g of the outer layer of the fermented silage and soil mixture, mark them with a marker, put the soil samples into a sterile plastic sample bag, mix them evenly, and then separate the strains.

[0067] Under sterile conditions, 1 g of fermented silage and soil mixture was placed in a triangular flask containing 99 mL of sterile deionized water, placed in a shaker at 37 °C at 200 r / min and fully shaken for 30 min, then taken out and allowed to stand for 10 min. After the silage soil sample was reprecipitated, a small amount of supernatant (1 mL) was taken from the clean bench and diluted with sterile deionized water at 1 × 10 -3 , 1×10 -4 , 1×10 -5 , 1×10 -6 , 1×10 -7 , 1×10 -8 Take 1×10 -6 , 1×10 -7 , 1×10 -8 0.2 mL of each was evenly spread on beef extract peptone agar medium, yeast extract peptone glucose agar medium and potato glucose agar medium, and cultured in a constant temperature incubator at 30°C. The culture time of beef extract peptone agar medium was 24 h, and the culture time of yeast extract peptone glucose agar medium and potato glucose agar medium was 48-96 h.

[0068] Before screening, the preserved strains were streaked on the corresponding flat agar medium. Bacteria were streaked on beef extract peptone agar medium and cultured and activated at 30°C for 24 hours; yeasts were streaked on yeast extract peptone glucose agar medium and cultured and activated at 30°C for 48 hours; molds were streaked on potato agar medium and cultured and activated at 30°C for 96 hours.

[0069] Under sterile conditions, activated bacteria, yeast colonies and mold spores were picked and inoculated into beef extract peptone liquid medium, yeast extract peptone glucose liquid medium and potato glucose liquid medium respectively. Bacteria and yeast were cultured at 30°C and 150rpm for 24h until the concentration of bacteria and yeast in the culture medium reached about 1×10 8 / mL (counted under a hemacytometer microscope).

[0070] The mold was cultured at 30°C for 96 h until spores grew. The spores were washed off with 5 mL of 0.9% sterile saline and shaken thoroughly with a vortex shaker for 5 seconds to dilute the bacterial solution to a spore concentration of about 1 × 10 8 / mL. Before spotting, shake thoroughly with a vortex oscillator for 5s, use an inoculation loop to dip a small amount of bacterial liquid and spot it on the glucosinolate screening medium and tannin screening medium respectively, spot 3 spots on each plate, and keep a certain approximately equal distance between the 3 spots. After spotting, incubate at 30℃ for 72h, and use a vernier caliper to measure the diameter of the colony and the hydrolysis transparent circle. The ability to produce a transparent hydrolysis circle around the colony indicates that the strain has the ability to degrade glucosinolates and tannins. The screening standard is the ratio (H / C) of the transparent circle diameter (H) / colony diameter (C), and the larger ratio is taken. The strain with the ability to degrade glucosinolates and increase the tannin content was screened.

[0071] in:

[0072] The beef extract peptone agar medium includes the following raw material components: peptone 10g / L, beef powder 3g / L, sodium chloride 5g / L, agar 15g / L, and pH 7.3±0.1. Weigh each component in proportion, weigh each raw material component (33.0g in total) and add it to 1L of distilled water, heat and stir until completely dissolved, and then sterilize at 121℃ for 15min.

[0073] Yeast extract powder peptone glucose agar medium includes the following raw material components: peptone 10g / L, yeast extract powder 5g / L, glucose 20g / L, agar 14g / L, natural pH. Weigh each component in proportion, weigh each raw material component (49g in total) and add it to 1L of distilled water, stir, heat and boil until completely dissolved, and then sterilize at 121℃ for 20min.

[0074] Potato glucose agar medium includes the following raw material components: 300 g / L potato (extracted powder), 20 g / L glucose, 15 g / L agar, 0.1 g / L chloramphenicol, natural pH. Weigh each component in proportion, weigh each raw material component (40.1 g in total) and add it to 1 L of distilled water, stir, heat and boil until completely dissolved, and then sterilize at 121° C. for 15 minutes.

[0075] The beef extract peptone liquid culture medium includes the following raw material components: peptone 10g / L, beef extract powder 3g / L, sodium chloride 5g / L, pH 7.4±0.2. Weigh each component in proportion, weigh each raw material component (a total of 18g) and add it to 1L of distilled water, stir and heat to boil until completely dissolved, and then sterilize it under high pressure at 121℃ for 15min.

[0076] Yeast extract peptone glucose liquid medium includes the following raw material components: peptone 20g / L, glucose 20g / L, yeast extract powder 10g / L, pH 6.5±0.2. Weigh each component in proportion, weigh each raw material component (50g in total) and add it to 1L of distilled water, stir, heat and boil until completely dissolved, and then sterilize at 121℃ for 15min.

[0077] Potato glucose liquid culture medium includes the following raw material components: 300 g / L potato (extracted powder), 20 g / L glucose, natural pH. Weigh each component in proportion, weigh each raw material component (24 g in total) and add it to 1 L of distilled water, stir, heat and boil until completely dissolved, and then sterilize at 121° C. for 15 minutes.

[0078] Glucosinolate screening medium (g / L): 10 g glucosinolate, 3 g NaNO3, 1 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g KCl, 0.01 FeSO4, 18 g agar, natural pH. After autoclaving at 121°C for 15 min, add glucosinolates sterilized by filtration with a 0.22 μm filter membrane to the ultra-close workbench.

[0079] Tannin screening medium (g / L): 20g tannic acid, 3g NaNO3, 1g K2HPO4, 0.5g MgSO4·7H2O, 0.5g KCl, 0.01 FeSO4, 18g agar, natural pH. After sterilization at 121℃ for 15min, add tannins sterilized by filtration with a 0.22μm pore size filter membrane to the ultra-close workbench.

[0080] The specific process of obtaining the genomic nucleotide sequence of the strain is as follows:

[0081] (1) Extraction of total bacterial DNA

[0082] The total bacterial DNA was extracted using the TSINGKE Plant DNA Extraction Kit (Universal).

[0083] (2) PCR amplification of 16S rDNA fragment

[0084] The primers used in the PCR reaction are 16S rDNA universal primers, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0085] SEQ ID NO. 1: 27F: 5'-AGTTTGATCMTGGCTCAG-3'.

[0086] SEQ ID NO. 2: 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0087] The PCR reaction system is shown in Table 1 below.

[0088] Table 1

[0089]

[0090] The reaction program was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 60°C for 30 s; extension at 72°C for 1 min, 30 cycles; and extension at 72°C for 10 min.

[0091] Prepare agarose gel: weigh 1.2g agarose and dissolve it in 100mL 1×TAE buffer, heat it in a microwave oven until it is completely dissolved, add 8μL nucleic acid dye, shake well, and pour it into a gel mold to prepare agarose gel. Take 5μL PCR product and 1.2% agarose gel, perform electrophoresis at 120V for 30min, and observe the bands under UV light after the electrophoresis.

[0092] (3) PCR product sequencing

[0093] After the PCR product is qualified, the target band is cut and purified and recovered, and the recovered product is used for Sanger sequencing.

[0094] (4) Comparison and analysis of sequencing results

[0095] The sequencing results were spliced ​​using the software ContigExpress, and the inaccurate parts at both ends were removed. After Blast alignment of the spliced ​​sequences in the NCBI database, the strain was identified as a Gram-negative bacterium, Serratia marcescens, so it was named Serratia marcescens X-78 and deposited in the China Center for Type Culture Collection (CCTCC) on March 2, 2023. The deposit address is Wuhan University, China, and the strain deposit number is CCTCC NO: M 2023232.

[0096] Example 2

[0097] This embodiment provides a functional rapeseed meal and a preparation method thereof.

[0098] A method for preparing functional rapeseed meal comprises the following steps:

[0099] Step S1, mixing rapeseed meal with water, according to the material-water ratio of 1:1 (w / v), mixing and loading into a 250 mL beaker, stirring evenly and sealing with a sealing film, natural pH, high pressure sterilization at 121° C. for 20 min, cooling and breaking up after being taken out of the pot, to obtain a rapeseed meal solid culture medium.

[0100] Step S2: 100 g of rapeseed meal was added with 100 g of water and boiled for 10 min, filtered through eight layers of gauze, the filtrate was fixed to 1 L, natural pH, sterilized at 121° C. for 15 min, and then inoculated with Serratia marcescens X-78 and cultured at 37° C. for 48 h to obtain an activated Serratia marcescens solution (the number of viable bacteria of Serratia marcescens was ≥ 9.5×10 8 cfu / g).

[0101] Step S3, inoculating the activated Serratia marcescens liquid into the rapeseed meal solid medium at an inoculum size of 0.5% (the inoculum size is calculated according to V / W, the unit of the activated liquid is mL, and the unit of the rapeseed meal solid medium is g), aerobically fermenting at 30° C. for 72 h (stirring once every 12 h during the fermentation process with a sterile glass rod), drying at 65° C. after the fermentation is completed, and crushing through a 40-mesh sieve to obtain functional rapeseed meal.

[0102] Example 3

[0103] This embodiment provides a functional rapeseed meal and a preparation method thereof.

[0104] A method for preparing functional rapeseed meal comprises the following steps:

[0105] Step S1 is the same as step S1 in embodiment 2.

[0106] Step S2: the same as step S2 in embodiment 2.

[0107] Step S3 is the same as step S3 in Example 2, except that the inoculation amount is 1.0%.

[0108] Example 4

[0109] This embodiment provides a functional rapeseed meal and a preparation method thereof.

[0110] A method for preparing functional rapeseed meal comprises the following steps:

[0111] Step S1 is the same as step S1 in embodiment 2.

[0112] Step S2: the same as step S2 in embodiment 2.

[0113] Step S3 is the same as step S3 in Example 2, except that the inoculation amount is 1.5%.

[0114] Example 5

[0115] This embodiment provides a functional rapeseed meal and a preparation method thereof.

[0116] A method for preparing functional rapeseed meal comprises the following steps:

[0117] Step S1 is the same as step S1 in embodiment 2.

[0118] Step S2: 100 g of rapeseed meal was added with 100 g of water and boiled for 10 min, filtered through eight layers of gauze, the filtrate was fixed to 1 L, natural pH, sterilized at 121° C. for 15 min, and then inoculated with Serratia marcescens X-78 and cultured at 37° C. for 48 h to obtain an activated Serratia marcescens solution (the number of viable bacteria of Serratia marcescens was ≥ 9.5×10 8 cfu / g);

[0119] 100 g of rapeseed meal was added with 100 g of water and boiled for 10 min, filtered through eight layers of gauze, the filtrate was fixed to 1 L, natural pH, sterilized at 121° C. for 15 min, and then inoculated with myrosinase and cultured at 37° C. for 48 h to obtain myrosinase activated solution (myrosinase viable bacteria count ≥ 0.05 U / g);

[0120] 100g rapeseed meal was added with 100g water and boiled for 10min, filtered through eight layers of gauze, and the filtrate was fixed to 1L, natural pH, sterilized at 121℃ for 15min, and then inoculated with Bacillus subtilis and cultured at 37℃ for 48h to obtain Bacillus subtilis activated liquid (the number of viable Bacillus subtilis was ≥1.5×10 7 cfu / g).

[0121] Step S3, the activated liquid of Serratia marcescens, the activated liquid of myrosinase and the activated liquid of Bacillus subtilis are mixed uniformly at a mass ratio of 90:5:5 to obtain a bacterial solution, the bacterial solution is inoculated into a rapeseed meal solid culture medium at an inoculum amount of 0.5% (the inoculum amount is calculated according to V / W, the unit of the bacterial solution is mL, and the unit of the rapeseed meal solid culture medium is g), and aerobic fermentation is carried out at 30° C. for 72 hours (stirring once every 12 hours with a sterile glass rod during the fermentation process), and after the fermentation is completed, drying is carried out at 65° C., and crushing is carried out through a 40-mesh sieve to obtain a functional rapeseed meal.

[0122] Example 6

[0123] This embodiment provides a functional rapeseed meal and a preparation method thereof.

[0124] A method for preparing functional rapeseed meal comprises the following steps:

[0125] Step S1 is the same as step S1 in embodiment 2.

[0126] Step S2: the same as step S2 in embodiment 5.

[0127] Step S3 is the same as step S3 in Example 5, except that the inoculation amount is 1.0%.

[0128] Example 7

[0129] This embodiment provides a functional rapeseed meal and a preparation method thereof.

[0130] A method for preparing functional rapeseed meal comprises the following steps:

[0131] Step S1 is the same as step S1 in embodiment 2.

[0132] Step S2: the same as step S2 in embodiment 5.

[0133] Step S3 is the same as step S3 in Example 5, except that the inoculation amount is 1.5%.

[0134] Comparative Example 1

[0135] This comparative example provides a functional rapeseed meal and a preparation method thereof.

[0136] A method for preparing functional rapeseed meal comprises the following steps:

[0137] Step S1 is the same as step S1 in embodiment 2.

[0138] Step S2, adding 100g of rapeseed meal to 100g of water and boiling for 10min, filtering through eight layers of gauze, fixing the filtrate to 1L, setting the pH to natural, sterilizing at 121°C for 15min, then inoculating myrosinase and culturing at 37°C for 48h to obtain myrosinase activated solution (the number of viable myrosinase bacteria is ≥0.05U / g).

[0139] Step S3, inoculating the myrosinase activation solution into the rapeseed meal solid medium at an inoculum size of 0.5% (the inoculum size is calculated according to V / W, the unit of the activation solution is mL, and the unit of the rapeseed meal solid medium is g), aerobically fermenting at 30° C. for 72 h (stirring once every 12 h during the fermentation process with a sterile glass rod), drying at 65° C. after the fermentation, and crushing through a 40-mesh sieve to obtain functional rapeseed meal.

[0140] Comparative Example 2

[0141] This comparative example provides a functional rapeseed meal and a preparation method thereof.

[0142] A method for preparing functional rapeseed meal comprises the following steps:

[0143] Step S1 is the same as step S1 in embodiment 2.

[0144] Step S2 is the same as step S2 in Comparative Example 1.

[0145] Step S3 is the same as step S3 in Comparative Example 1, except that the inoculation amount is 1.0%.

[0146] Comparative Example 3

[0147] This comparative example provides a functional rapeseed meal and a preparation method thereof.

[0148] A method for preparing functional rapeseed meal comprises the following steps:

[0149] Step S1 is the same as step S1 in embodiment 2.

[0150] Step S2 is the same as step S2 in Comparative Example 1.

[0151] Step S3 is the same as step S3 in Comparative Example 1, except that the inoculation amount is 1.5%.

[0152] Comparative Example 4

[0153] This comparative example provides a functional rapeseed meal and a preparation method thereof.

[0154] A method for preparing functional rapeseed meal comprises the following steps:

[0155] Step S1 is the same as step S1 in embodiment 2.

[0156] Step S2: 100 g of rapeseed meal was added with 100 g of water and boiled for 10 min, filtered through eight layers of gauze, the filtrate was fixed to 1 L, natural pH, sterilized at 121 ° C for 15 min, and then inoculated with Bacillus subtilis and cultured at 37 ° C for 48 h to obtain Bacillus subtilis activated liquid (the number of viable Bacillus subtilis was ≥ 1.5 × 10 7 cfu / g).

[0157] Step S3, inoculating the Bacillus subtilis activated liquid into the rapeseed meal solid culture medium at an inoculum size of 0.5% (the inoculum size is calculated according to V / W, the unit of the activated liquid is mL, and the unit of the rapeseed meal solid culture medium is g), aerobically fermenting at 30° C. for 72 h (stirring once every 12 h during the fermentation process with a sterile glass rod), drying at 65° C. after the fermentation, and crushing through a 40-mesh sieve to obtain functional rapeseed meal.

[0158] Comparative Example 5

[0159] This comparative example provides a functional rapeseed meal and a preparation method thereof.

[0160] A method for preparing functional rapeseed meal comprises the following steps:

[0161] Step S1 is the same as step S1 in embodiment 2.

[0162] Step S2 is the same as step S2 in Comparative Example 4.

[0163] Step S3 is the same as step S3 in Comparative Example 4, except that the inoculation amount is 1.0%.

[0164] Comparative Example 6

[0165] This comparative example provides a functional rapeseed meal and a preparation method thereof.

[0166] A method for preparing functional rapeseed meal comprises the following steps:

[0167] Step S1 is the same as step S1 in embodiment 2.

[0168] Step S2 is the same as step S2 in Comparative Example 4.

[0169] Step S3 is the same as step S3 in comparative example 4, except that the inoculation amount is 1.5%.

[0170] Comparative Example 7

[0171] This comparative example provides a blank control group.

[0172] Blank control group: The rapeseed meal solid culture medium without inoculation was used as the blank control group.

[0173] Mix rapeseed meal with water, put into a 250 mL beaker at a material-water ratio of 1:1 (w / v), stir evenly and seal with a sealing film, set to natural pH, autoclave at 121°C for 20 min, cool and break up after taking out of the pot, to obtain a rapeseed meal solid culture medium.

[0174] The functional rapeseed meal prepared in Examples 2 to 7 and Comparative Examples 1 to 6, and the blank control group provided in Comparative Example 7 were tested, and the degradation rate of glucosinolate and the increase rate of tannin were calculated respectively. To ensure the accuracy of the data, Examples 2 to 7 and Comparative Examples 1 to 6 were repeated 3 times, and the average value obtained was the final result. The results are shown in Table 2.

[0175] The content of glucosinolates in functional rapeseed meal was determined by referring to the test method for glucosinolates in rapeseed meal DB32 / T 384-2007. The content of tannins in functional rapeseed meal was determined by referring to the test method for tannins in feed GB / T 27985-2011.

[0176] Table 2

[0177]

[0178]

[0179] According to Table 2, the degradation rate of glucosinolates and the increase rate of tannins are positively correlated with the inoculation amount of bacterial solution. Specifically, X78 screened in the embodiment can not only degrade glucosinolates in feed, but also increase the tannin content in feed; although myrosinase and Bacillus subtilis have a certain effect on the degradation of glucosinolates, the increase in tannin content is negligible, and after the synergistic fermentation of Serratia marcescens, myrosinase and Bacillus subtilis by bacterial enzymes, the degradation rate of glucosinolates and the increase rate of tannins in rapeseed meal are greatly improved, the degradation rate of glucosinolates reaches more than 60%, and the increase rate of tannin content reaches more than 50%.

[0180] Example 8 Evaluation of the nutritional value of functional rapeseed cake using in vitro methods

[0181] The fistula animals were 3 healthy Xiangxi yellow cattle (steers) with permanent fistulas, 30 months old and weighing 360±20kg. The experimental animals had recovered to normal physiological state more than 2 months after the rumen fistula operation. The functional rapeseed meal prepared by fermentation in Example 7, the rapeseed meal solid medium provided in Comparative Example 7, and soybean meal were compared by nylon bag method for the degradation rates of dry matter, crude protein, neutral detergent fiber and acid detergent fiber within 48 hours, respectively. The results are shown in Table 3.

[0182] Table 3 Degradation rate of nutrients in protein feed raw materials (48h)

[0183] project Soybean meal Rapeseed meal solid medium Functional rapeseed meal Dry matter 45.50％ 45.20％ 48.10％ Crude Protein 58.30％ 55.60％ 60.30％ Neutral detergent fiber 55.30％ 54.90％ 57.20％ Acid detergent fiber 50.50％ 50.30％ 54.40％

[0184] As can be seen from Table 3, the functional rapeseed meal obtained after the synergistic fermentation of bacterial enzymes including Serratia marcescens, myrosinase and Bacillus subtilis is more conducive to the degradation of nutrients such as dry matter, crude protein, neutral detergent fiber and acid detergent fiber contained therein.

[0185] According to Tables 2 and 3, the use of Serratia marcescens to ferment rapeseed meal alone, or the use of a bacterial enzyme complex including Serratia marcescens to ferment rapeseed meal together, can ensure that the anti-nutritional factor glucosinolate is fully degraded while increasing the content of tannin, reducing the risk of slow growth of livestock and poultry and damage to body health caused by the high glucosinolate content when rapeseed meal is used as a feed raw material, and the fermented product is also conducive to the degradation of nutrients such as dry matter, crude protein, neutral detergent fiber and acid detergent fiber, so that the rapeseed meal not only has a higher nutritional value, but also has a lower economic cost (saving direct feed costs of 1.5 yuan / ton), thereby improving the feeding value of rapeseed meal, and is more conducive to its wide application and promotion in animal husbandry production.

[0186] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing functional rapeseed meal, characterized in that: The following steps are involved: sterilizing the rapeseed meal to obtain a rapeseed meal solid culture medium; The bacterial liquid containing Serratia marcescens is inoculated into the rapeseed meal solid culture medium, and aerobic fermentation is carried out at 20-40° C. to obtain the functional rapeseed meal.

2. The method for preparing the functional rapeseed meal according to claim 1, characterized in that: The Serratia marcescens was deposited in the China Center for Type Culture Collection on March 2, 2023, with the deposit number being CCTCC NO: M 2023232.

3. The method for preparing the functional rapeseed meal according to claim 1, characterized in that: The bacterial solution also includes myrosinase and Bacillus subtilis.

4. The method for preparing the functional rapeseed meal according to claim 3, characterized in that: The bacterial liquid includes: a first activation liquid containing the Serratia marcescens, a second activation liquid containing the myrosinase, and a third activation liquid containing the Bacillus subtilis, and the mass ratio of the first activation liquid, the second activation liquid and the third activation liquid is (85-95): (3-5): (3-5).

5. The method for preparing the functional rapeseed meal according to claim 4, characterized in that: The number of viable bacteria of Serratia marcescens in the first activation solution is ≥ 9.5×10 8 cfu / g; The viable bacterial count of the myrosinase in the second activation solution is ≥0.05U / g; The number of viable bacteria of the Bacillus subtilis in the third activation solution is ≥ 1.5×10 7 cfu / g.

6. The method for preparing the functional rapeseed meal according to claim 1, characterized in that: The inoculation amount of the bacterial liquid in the rapeseed meal solid culture medium is 0.5-1.5%; and / or The aerobic fermentation time is 60 to 72 hours.

7. The method for preparing the functional rapeseed meal according to claim 1, characterized in that: The steps of the rapeseed meal sterilization treatment include: The rapeseed meal is mixed with water, sealed, and sterilized at 121-126°C.

8. A functional rapeseed meal prepared by the method for preparing the functional rapeseed meal according to any one of claims 1 to 7.

9. The functional rapeseed meal according to claim 8, characterized in that The glucosinolate content in the functional rapeseed meal is ≤7.5 μmol / L, and the tannin content is ≥160 mg / kg.

10. An animal feed, characterized in that: The functional rapeseed meal comprises the functional rapeseed meal prepared by the preparation method of the functional rapeseed meal as claimed in any one of claims 1 to 7 and / or the functional rapeseed meal as claimed in any one of claims 8 to 9.

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