CN113229399B - Method for biologically degrading rapeseed meal toxin and improving nutritive value of rapeseed meal toxin - Google Patents
Method for biologically degrading rapeseed meal toxin and improving nutritive value of rapeseed meal toxin Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 28
- 239000003053 toxin Substances 0.000 title claims abstract description 26
- 231100000765 toxin Toxicity 0.000 title claims abstract description 26
- 230000000050 nutritive effect Effects 0.000 title abstract description 10
- 230000000593 degrading effect Effects 0.000 title abstract description 5
- 238000000855 fermentation Methods 0.000 claims abstract description 169
- 230000004151 fermentation Effects 0.000 claims abstract description 159
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 56
- 239000007787 solid Substances 0.000 claims abstract description 56
- 240000006439 Aspergillus oryzae Species 0.000 claims abstract description 54
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- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 53
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 9
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 3
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- 230000002378 acidificating effect Effects 0.000 description 1
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/26—Compounds containing phosphorus
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
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Abstract
The invention discloses a method for biologically degrading rapeseed meal toxin and improving the nutritive value of the rapeseed meal toxin, which comprises the following steps: firstly, respectively preparing a solid culture medium and a rapeseed meal fermentation culture medium; the rapeseed dreg fermentation culture medium is divided into two parts, and one part is inoculated with aspergillus oryzae strain and fermented to obtain a aspergillus oryzae fermented product; inoculating Aspergillus niger strain on the other part, and fermenting to obtain a Aspergillus niger fermented product; inoculating bacillus strain to the solid culture medium and fermenting to obtain bacillus fermentation product; and finally, uniformly mixing the rice starter ferment, the black starter ferment and the bacillus ferment, inoculating lactobacillus casei liquid strain, fermenting, and drying the ferment until the moisture content is 10-12wt% after the fermentation is finished to obtain the fermented rapeseed meal. The invention can reduce the toxin content in the rapeseed meal and improve the nutritive value of the rapeseed meal, so that the rapeseed meal can be better applied to animal feeds.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for biologically degrading rapeseed meal toxins and improving the nutritive value of the rapeseed meal toxins.
Background
Protein is an important component in animal diet and is generally provided by soybean meal, but the self-sufficiency rate of soybean in China is less than 20%, and the method mainly depends on import. In recent years, with the rapid development of the Chinese livestock breeding industry and the further expansion of the breeding body, the supply of conventional feed raw materials, especially protein raw materials, is short, and the healthy development of the Chinese livestock breeding industry is limited. According to statistics, 8851 ten thousand tons of soybean are imported in China in 2019, the same ratio is increased by 48 ten thousand tons, the imported quantity accounts for about 85% of the domestic consumption, and the imported proportions of Brazil, the United states and Argentina are 65%, 19% and 10% respectively. The imported soybeans in China in 2020 do not fall and rise under the influence of epidemic situation, and the quantity of the imported soybeans in China reaches 9280 ten thousand tons by the month 11 in 2020, and exceeds the synchronization level in 2019. Under the international background of the current increasingly vigorous competition of protein feed raw materials, the structure of the traditional corn-bean pulp type animal feed in China is changed, the development of novel protein feed resources is urgent, and the diversified feed formula for introducing mixed pulp such as rapeseed pulp is a new normal state of the livestock raising industry in China.
The rapeseed meal is a byproduct obtained after oil extraction of the rapeseeds, the crude protein content of the rapeseed meal is 35% -45%, the amino acid content of the rapeseed meal is rich, and the rapeseed meal is an important vegetable protein resource. However, the rapeseed meal contains more toxic and harmful substances such as isothiocyanate, thiocyanate, sinapine, tannin, phytic acid and the like, so that the palatability of the daily ration is influenced, the utilization of other nutrient substances is influenced, the goiter of animals can be caused, the growth of the animals is inhibited, and the application of the rapeseed meal in the daily ration of the animals is greatly limited. The solid state fermentation of the microorganism can effectively reduce the content of anti-nutritional factors in the rapeseed meal, and has higher development value in the aspect of replacing the soybean meal. However, the current fermentation of rapeseed meal also has the problems of different fermentation conditions, large difference of fermentation effects and the like. Meanwhile, the problems of degradation of crude protein of the rapeseed meal and detoxification are difficult to solve by utilizing a single microorganism strain. Therefore, a method is needed at present, which can not only reduce the toxin content in the rapeseed meal, but also improve the nutritive value of the rapeseed meal, so that the rapeseed meal can be better applied to animal feeds.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a method for biologically degrading rapeseed meal toxin and improving the nutritive value of the rapeseed meal toxin, and by the method, dominant enzyme systems in different microbial fermentation processes can be fully utilized to degrade toxic and harmful substances such as isothiocyanate, thiocyanate, sinapine, tannin, phytic acid and the like in the rapeseed meal, hydrolyze cell wall polysaccharide, crude protein and the like, and improve the nutritive value of the rapeseed meal. The method adopts a double-stage solid state fermentation mode of aerobic and anaerobic fermentation, utilizes the environment with relatively low water content and high ventilation rate in the aerobic fermentation stage, can ensure the enzyme activity of microorganisms, and can promote enzymolysis in the high-temperature and high-humidity environment in the anaerobic fermentation stage. Meanwhile, lactobacillus casei is utilized for anaerobic fermentation, products such as L-lactic acid with biological activity can be produced by metabolism, and the materials can avoid the growth of mixed bacteria under the acidic condition.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for biodegrading rapeseed meal toxins and increasing their nutritional value, comprising the steps of:
(1) Mixing the solid raw material with water until the water content is 45-50wt%, and sterilizing to obtain a bacillus solid fermentation medium; mixing rapeseed meal, whey powder and water until the water content is 42-46wt%, and sterilizing to obtain a rapeseed meal fermentation medium;
(2) Dividing the rapeseed dreg fermentation culture medium into two parts, inoculating aspergillus oryzae strain into one part, and fermenting to obtain a aspergillus oryzae fermented product; inoculating Aspergillus niger strain on the other part, and fermenting to obtain a Aspergillus niger fermented product; inoculating bacillus strain to the bacillus solid fermentation medium and fermenting to obtain bacillus fermentation product;
(3) Uniformly mixing a rice starter fermentation product, a black starter fermentation product and a bacillus fermentation product, inoculating lactobacillus casei liquid strain, and fermenting to obtain an anaerobic fermentation product after fermentation;
(4) And (3) drying the fermented product obtained in the step (3) until the moisture content is 10-12wt% to obtain the fermented rapeseed meal.
Preferably, in step (1), the solid raw materials comprise the following raw materials in weight percent:
80% of bran, 17% of rice husk, 2% of soybean meal, 0.15% of monopotassium phosphate, 0.2% of magnesium sulfate and 0.65% of light calcium carbonate.
Preferably, in the step (1), the addition amount of the whey powder is 0.1% of the total mass of the rapeseed meal; the sterilization temperature is 105-121 ℃ and the sterilization time is 15-30min. The bacillus solid fermentation medium and the rapeseed dreg fermentation medium are obtained through the same sterilization treatment conditions, and the sterilization temperature and the sterilization time are the same.
Preferably, in the step (2), the aspergillus oryzae strain is obtained by solid state culture until the number of spores of the aspergillus oryzae reaches 30 hundred million/g and the activity of neutral protease reaches 2000U/g;
the inoculum size of the Aspergillus oryzae strain is 0.5-1.0% of the mass of the rapeseed meal fermentation medium, and the Aspergillus oryzae strain is cultured for 30-32h at 28-30 ℃ to obtain a Aspergillus oryzae fermentation product; the aspergillus oryzae is purchased from China center for type culture collection of industrial microorganisms, and the culture collection number is: CICC 2013.
Preferably, in the step (2), the Aspergillus niger strain is obtained by solid state culture until the number of spores of the Aspergillus niger reaches 10 hundred million/g, the xylanase activity reaches 1000U/g, and the cellulase activity reaches 100U/g;
the inoculum size of the Aspergillus niger strain accounts for 1.0-1.5% of the mass of the rapeseed dreg fermentation medium, and the Aspergillus niger strain is cultured for 36-40h at 28-30 ℃ to obtain a Aspergillus niger fermentation product; the Aspergillus niger is purchased from China center for type culture collection of Industrial microorganisms, and the culture collection number is: CICC2041.
Preferably, in the step (2), the bacillus strain is obtained by culturing bacillus licheniformis or bacillus subtilis by shaking flask, and the number of the cultured bacillus strain is 1 multiplied by 10 10 cfu/mL; the mass ratio of the inoculum size of the bacillus strain to the bacillus solid state fermentation medium is (2-3) mL:100g, fermenting at 37-39deg.C for 44-48 hr to make the bacillus count reach 2×10 10 cfu/g, wherein the activity of neutral protease reaches 3000U/g, and the activity of alkaline protease reaches 5000U/g, so as to obtain bacillus fermented product; bacillus licheniformis is purchased from China industry microbiological culture Collection center, and has a culture collection number: CICC21886; bacillus subtilis is purchased from Chinese industrial microorganism strainPreservation management center, strain preservation number: CICC20872.
Preferably, in the step (3), the rice starter fermentate, the black starter fermentate and the bacillus fermentate are respectively according to (55-65): (25-30): (10-15) by mass ratio.
Preferably, in the step (3), the lactobacillus casei liquid strain is obtained by culturing lactobacillus casei in shake flask, and the lactobacillus casei bacterial count is cultured to 6×10 9 cfu/mL; the ratio of the inoculation amount of lactobacillus casei fermentation liquor to the total mass of the rice starter fermentation product, the black starter fermentation product and the bacillus fermentation product is (3-5) mL:100g; the fermentation temperature is not lower than 30deg.C and not higher than 40deg.C, and the fermentation time is 3-4d; lactobacillus casei was purchased from the China center for type culture Collection of microorganisms, accession number: CICC6117.
In a second aspect of the invention, fermented rapeseed meal prepared by the method is provided.
In a third aspect of the invention, there is provided the use of fermented rapeseed meal in the preparation of livestock feed.
The invention has the beneficial effects that:
(1) The multi-strain step fermentation adopted by the invention can utilize the complex enzyme system of microorganisms to degrade toxic and harmful substances such as thioglycoside, tannin, phytic acid and the like in the rapeseed meal in the fermentation process, and can hydrolyze cell wall polysaccharide, crude protein and the like, so that the organism is easier to absorb, and the nutritive value of the rapeseed meal is improved.
(2) The crude protein content of the fermented rapeseed meal obtained by the invention reaches more than 44%, is equivalent to the protein content of soybean meal, can be used as a protein feed raw material to replace soybean meal, and reduces the cultivation cost. Meanwhile, the fermented rapeseed meal is added into animal feed, so that the production performance of animals can be improved, the total bore-free rate, the pectoral muscle rate and the leg muscle rate are improved, and the economic benefit is increased.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background art, the rapeseed meal contains more toxic and harmful substances, such as isothiocyanate, thiocyanate, sinapine, tannin, phytic acid and the like, so that the palatability of the daily ration is influenced, the utilization of other nutrient substances is influenced, the goiter of animals can be caused, the growth of the animals is inhibited, and the application of the rapeseed meal in the daily ration of the animals is greatly limited.
Based on this, the object of the present invention is to provide a method for biodegradation of rapeseed meal toxins and for improving their nutritional value. The method finally obtains the method of the invention through screening strains and screening fermentation culture modes (see test 1): firstly, respectively preparing a solid culture medium and a rapeseed meal fermentation culture medium; the rapeseed dreg fermentation culture medium is divided into two parts, and one part is inoculated with aspergillus oryzae strain and fermented to obtain a aspergillus oryzae fermented product; inoculating Aspergillus niger strain on the other part, and fermenting to obtain a Aspergillus niger fermented product; inoculating bacillus licheniformis strain or bacillus subtilis strain to the solid culture medium and fermenting to obtain bacillus fermentation product; and finally, uniformly mixing the rice starter ferment, the black starter ferment and the bacillus ferment, inoculating lactobacillus casei fermentation liquor, and fermenting to obtain a ferment after the fermentation is finished, and drying until the moisture content is 10-12wt% to obtain the fermented rapeseed meal.
Test 1
The rapeseed meal fermentation medium is obtained by uniformly mixing the rapeseed meal, whey powder and water according to the mass percentage of the invention. Inoculating the strains in table 1 into a rapeseed dreg fermentation medium (the inoculum size is 1 wt%) for aerobic fermentation, fermenting for 34 hours at 28-30 ℃, and calculating the toxin removal rate according to the toxin content in the rapeseed dreg before and after fermentation, wherein the toxin removal rate is = (the content before treatment-the content after treatment)/the content before treatment is multiplied by 100%; the results obtained are shown in Table 1.
TABLE 1
According to Table 1, aspergillus oryzae and Aspergillus niger which have optimal rapeseed meal toxin removal rate are selected to be fermented respectively with equal quality rapeseed meal to obtain Aspergillus oryzae fermented product and Aspergillus niger fermented product; aspergillus oryzae fermented product and Aspergillus niger fermented product are mixed according to a ratio of 1:1, and then inoculating the strains in the table 2 for anaerobic fermentation, wherein the inoculation amount is 3mL of strains inoculated to each 100g of the mixture, and the fermentation starting temperature is controlled to be 30-32 ℃ and the fermentation is carried out for 1d. Calculating the removal rate of toxins according to the toxin content in the rapeseed meal before and after fermentation, wherein the removal rate is = (content before treatment-content after treatment)/content before treatment multiplied by 100%; the results obtained are shown in Table 2.
TABLE 2
As can be seen from Table 2, the Aspergillus oryzae fermented product and the Aspergillus niger fermented product were obtained according to the following ratio 1: the mass ratio of 1 is mixed, and the effect of fermenting by adding lactobacillus casei is optimal for removing toxins from the rapeseed meal.
The bran, the rice husk, the bean pulp powder, the monopotassium phosphate, the magnesium sulfate, the light calcium carbonate and the water are mixed according to the mass ratio of the invention to obtain a solid culture medium, the solid culture medium is inoculated with the strain in the table 3, and aerobic fermentation is carried out to obtain a solid fermentation product (the inoculation amount is 3 percent according to the mass volume percentage, and the fermentation is carried out for 46 hours at 37-39 ℃). The solid fermentation product, the aspergillus oryzae fermentation product and the aspergillus niger fermentation product are prepared according to the following steps of 1:1:1, and then inoculating the strains in the table 2 for anaerobic fermentation, wherein the inoculation amount is that 3mL of strains are inoculated to each 100g of the mixture, and the fermentation starting temperature is controlled to be 30-32 ℃ and the fermentation is carried out for 1d. Calculating the removal rate of toxins according to the toxin content in the rapeseed meal before and after fermentation, wherein the removal rate is = (content before treatment-content after treatment)/content before treatment multiplied by 100%; the results obtained are shown in Table 3.
TABLE 3 Table 3
As can be seen from Table 3, the solid fermentation obtained by inoculating the seed in Table 3 with the solid fermentation product was mixed with Aspergillus oryzae fermentation product and Aspergillus niger fermentation product according to 1:1:1, and then inoculating the strains in the table 2 for anaerobic fermentation, wherein the best effect is that the solid fermentation base is inoculated with bacillus licheniformis or bacillus subtilis to obtain bacillus fermentation product, and the bacillus fermentation product is mixed with aspergillus oryzae fermentation product and aspergillus niger fermentation product to be inoculated with lactobacillus caseii for anaerobic fermentation to obtain fermented rapeseed meal. Its average toxicity is most reduced.
The inventor evenly mixes the rice starter ferment, the black starter ferment and the bacillus ferment according to different mass ratios respectively, inoculates 4% (volume percent) of lactobacillus casei fermentation liquid, controls the fermentation initiation temperature to be 30-32 ℃ for 3d, and obtains the ferment after the fermentation is finished; the removal rate of toxin is calculated, and the mass percentage of the rice starter ferment, the black starter ferment and the bacillus ferment is 65:27.5:12.5, the removal rate of toxin is highest. The removal rates of isothiocyanate, thiocyanate, sinapine, tannin and phytic acid are 80.01%, 76.28%, 81.10%, 86.47% and 96.03%, respectively.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and are commercially available.
Description: aspergillus oryzae was purchased from China center for type culture Collection, accession number: CICC 2013;
aspergillus niger was purchased from China center for type culture Collection, accession number: CICC2041;
bacillus licheniformis is purchased from China industry microbiological culture Collection center, and has a culture collection number: CICC21886;
bacillus subtilis is purchased from the China center for type culture Collection of industrial microorganisms, and has a culture collection number: CICC20872;
lactobacillus casei was purchased from the China center for type culture Collection of microorganisms, accession number: CICC6117.
The seed liquid is inoculated with Bacillus licheniformis, bacillus subtilis and Lactobacillus casei in the examples in the amount of volume and mass percent. For example: inoculating 4% lactobacillus casei broth means inoculating 4mL of lactobacillus casei broth per 100g of substrate.
Example 1:
(1) Culturing Bacillus licheniformis and Lactobacillus casei fermentation seed liquid by shake flask culture method to obtain Bacillus licheniformis bacterial count of 1×10 10 cfu/mL, lactobacillus casei count of 6×10 9 cfu/mL; aspergillus oryzae and Aspergillus niger strains are respectively cultured by adopting a solid state culture method, so that the number of spores of Aspergillus oryzae reaches 30 hundred million/g, the activity of neutral protease reaches 2000U/g, the number of spores of Aspergillus niger reaches 10 hundred million/g, the activity of xylanase reaches 1000U/g, and the activity of cellulase reaches 100U/g.
(2) 80g of bran, 17g of rice husk, 2g of soybean meal powder, 0.15g of monopotassium phosphate, 0.2g of magnesium sulfate and 0.65g of light calcium carbonate are uniformly mixed to obtain a solid raw material. Mixing the solid raw materials with water until the water content is 47%, mixing 100g of rapeseed meal, 0.1g of whey powder with water until the water content is 44%, and sterilizing at 121 ℃ for 30min to obtain a solid fermentation medium and a rapeseed meal fermentation medium.
(3) The rapeseed dreg fermentation medium is divided into two parts by mass equally, one part is inoculated with 0.75wt% aspergillus oryzae strain, and the rice koji fermentation product is obtained after culturing for 31 hours at 29 ℃. The other part is inoculated with 1.25 weight percent of Aspergillus niger strain and cultured for 38 hours at 29 ℃ to obtain a Aspergillus niger fermentation product. Inoculating 2.5% Bacillus licheniformis strain into the solid fermentation culture medium, and culturing at 38deg.C for 46 hr to make the bacillus bacterial count reach 2×10 10 cfu/g, wherein the activity of neutral protease reaches 3000U/g, and the activity of alkaline protease reaches 5000U/g, so as to obtain the bacillus fermented product.
(4) Respectively fermenting rice starter ferment, black starter ferment and bacillus starter ferment according to the following proportion of 60:27.5: and (3) uniformly mixing the materials according to the mass ratio of 12.5, inoculating 4% lactobacillus casei fermentation liquor, controlling the initial fermentation temperature to be 32-38 ℃ and culturing for 3d, and obtaining a fermentation product after fermentation is finished.
(5) And (3) drying the fermented product obtained in the step (4) at 45-50 ℃ until the moisture is 11%, so as to obtain the fermented rapeseed meal.
Example 2
(1) Culturing Bacillus subtilis and Lactobacillus casei fermentation seed liquid by shake flask culture method to 1×10 10 cfu/mL, lactobacillus casei count of 6×10 9 cfu/mL; aspergillus oryzae and Aspergillus niger strains are respectively cultured by adopting a solid state culture method, so that the number of spores of Aspergillus oryzae reaches 30 hundred million/g, the activity of neutral protease reaches 2000U/g, the number of spores of Aspergillus niger reaches 10 hundred million/g, the activity of xylanase reaches 1000U/g, and the activity of cellulase reaches 100U/g.
(2) 80g of bran, 17g of rice husk, 2g of soybean meal powder, 0.15g of monopotassium phosphate, 0.2g of magnesium sulfate and 0.65g of light calcium carbonate are uniformly mixed to obtain a solid raw material. Mixing the solid raw materials with water until the water content is 45%, mixing 100g of rapeseed meal, 0.1g of whey powder with water until the water content is 46%, and sterilizing at 121 ℃ for 30min to obtain a solid fermentation medium and a rapeseed meal fermentation medium.
(3) The rapeseed dreg fermentation medium is divided into two parts by mass equally, one part is inoculated with 0.5 weight percent of Aspergillus oryzae strain, and the culture is carried out for 32 hours at 30 ℃ to obtain the Aspergillus oryzae fermentation product. The other part is inoculated with 1.5 weight percent of Aspergillus niger strain and cultured for 40 hours at 28 ℃ to obtain a Aspergillus niger fermentation product. Inoculating 2% bacillus subtilis strain into the solid fermentation culture medium, and culturing at 39deg.C for 48 hr to make bacillus count reach 2×10 10 cfu/g, wherein the activity of neutral protease reaches 3000U/g, and the activity of alkaline protease reaches 5000U/g, so as to obtain the bacillus fermented product.
(4) Respectively fermenting rice starter ferment, black starter ferment and bacillus ferment according to 55:30:15 mass ratio, inoculating 5% lactobacillus casei fermentation liquor, controlling the initial fermentation temperature to 31-34 ℃ and culturing for 4d, and obtaining the fermentation product after fermentation.
(5) And (3) drying the fermented product obtained in the step (4) at 45-50 ℃ until the moisture is 12%, so as to obtain the fermented rapeseed meal.
Example 3
(1) Respectively culturing lichen buds by shake flask culture methodFermenting seed liquid with bacillus and lactobacillus casei, and culturing bacillus licheniformis to 1×10 10 cfu/mL, lactobacillus casei count of 6×10 9 cfu/mL; aspergillus oryzae and Aspergillus niger strains are respectively cultured by adopting a solid state culture method, so that the number of spores of Aspergillus oryzae reaches 30 hundred million/g, the activity of neutral protease reaches 2000U/g, the number of spores of Aspergillus niger reaches 10 hundred million/g, the activity of xylanase reaches 1000U/g, and the activity of cellulase reaches 100U/g.
(2) 80g of bran, 17g of rice husk, 2g of soybean meal powder, 0.15g of monopotassium phosphate, 0.2g of magnesium sulfate and 0.65g of light calcium carbonate are uniformly mixed to obtain a solid raw material. Mixing the solid raw materials with water until the water content is 50%, mixing 100g of rapeseed meal, 0.1g of whey powder with water until the water content is 42%, and sterilizing at 121 ℃ for 30min to obtain a solid fermentation medium and a rapeseed meal fermentation medium.
(3) The rapeseed dreg fermentation culture medium is divided into two parts by mass averagely, one part is inoculated with 1.0 weight percent of Aspergillus oryzae strain, and the rice koji fermentation product is obtained after culturing for 30 hours at 28 ℃. Inoculating 1.0wt% Aspergillus niger strain on the other part, and culturing at 30deg.C for 36 hr to obtain Aspergillus niger fermentation product. Inoculating 3% Bacillus licheniformis strain into the solid fermentation culture medium, and culturing at 37deg.C for 48 hr to make the bacillus bacterial count reach 2×10 10 cfu/g, wherein the activity of neutral protease reaches 3000U/g, and the activity of alkaline protease reaches 5000U/g, so as to obtain the bacillus fermented product.
(4) Respectively fermenting rice starter ferment, black starter ferment and bacillus according to the following steps: 25:10, inoculating 3% lactobacillus casei fermentation liquid, controlling the initial fermentation temperature to be 32-38 ℃, culturing for 4d, and obtaining a fermentation product after fermentation.
(5) And (3) drying the fermented product obtained in the step (4) at 45-50 ℃ until the moisture is 10%, so as to obtain the fermented rapeseed meal.
Comparative example 1
Preparing rapeseed meal by using a physical detoxification method: putting the rapeseed meal into an autoclave, adding water to wet until the water content is 30%, introducing steam, treating for 2 hours at 121 ℃, taking out and air-drying to obtain the rapeseed meal by a physical detoxification method.
Comparative example 2
Preparing rapeseed meal by using a chemical detoxification method: taking rapeseed dregs, weighing ferrous sulfate according to 0.5% -1% of the weight of the rapeseed dregs, dissolving in 1/2 of the weight of water of the rapeseed dregs, pouring the ferrous sulfate into the rapeseed dregs after the ferrous sulfate is fully dissolved, uniformly mixing, and storing for 1h at room temperature. Steaming at 105 ℃ for 30min, taking out, and air drying to obtain the rapeseed meal by the chemical detoxification method.
Comparative example 3
(1) Culturing Bacillus licheniformis and Lactobacillus casei fermentation seed liquid by shake flask culture method to obtain Bacillus licheniformis bacterial count of 1×10 10 cfu/mL, lactobacillus casei count of 6×10 9 cfu/mL; aspergillus oryzae and Aspergillus niger strains are respectively cultured by adopting a solid state culture method, so that the number of spores of Aspergillus oryzae reaches 30 hundred million/g, and the number of spores of Aspergillus niger reaches 10 hundred million/g.
(2) 80g of bran, 17g of rice husk, 2g of soybean meal powder, 0.15g of monopotassium phosphate, 0.2g of magnesium sulfate and 0.65g of light calcium carbonate are uniformly mixed to obtain a solid raw material. Mixing the solid raw materials with water until the water content is 47%, mixing 100g of rapeseed meal, 0.1g of whey powder with water until the water content is 44%, and sterilizing at 121 ℃ for 30min to obtain a solid fermentation medium and a rapeseed meal fermentation medium.
(3) The rapeseed dreg fermentation medium is divided into two parts by mass equally, one part is inoculated with 0.75wt% aspergillus oryzae strain, and the rice koji fermentation product is obtained after culturing for 72 hours at 29 ℃. The other part is inoculated with 1.25 weight percent of Aspergillus niger strain and cultured for 72 hours at 29 ℃ to obtain a Aspergillus niger fermentation product. Inoculating 2.5% of bacillus licheniformis strain into the solid fermentation medium, and culturing at 38 ℃ for 72 hours to obtain bacillus fermentation product.
(4) Respectively fermenting rice starter ferment, black starter ferment and bacillus starter ferment according to the following proportion of 60:27.5: and (3) uniformly mixing the materials according to the mass ratio of 12.5, inoculating 4% lactobacillus casei fermentation liquor, controlling the initial fermentation temperature to be 32-38 ℃ and culturing for 6d, and obtaining a fermentation product after fermentation is finished.
(5) And (3) drying the fermented product obtained in the step (4) at 45-50 ℃ until the moisture is 11%, so as to obtain the fermented rapeseed meal.
Comparative example 4
(1) Culturing Bacillus subtilis and Lactobacillus casei fermentation seed liquid by shake flask culture method to 1×10 10 cfu/mL，Lactobacillus casei bacteria count to 6 x 10 9 cfu/mL; aspergillus oryzae and Aspergillus niger strains are respectively cultured by adopting a solid state culture method, so that the number of spores of Aspergillus oryzae reaches 30 hundred million/g, and the number of spores of Aspergillus niger reaches 10 hundred million/g.
(2) 80g of bran, 17g of rice husk, 2g of soybean meal powder, 0.15g of monopotassium phosphate, 0.2g of magnesium sulfate and 0.65g of light calcium carbonate are uniformly mixed to obtain a solid raw material. Mixing the solid raw materials with water until the water content is 47%, mixing 100g of rapeseed meal, 0.1g of whey powder with water until the water content is 44%, and sterilizing at 121 ℃ for 30min to obtain a solid fermentation medium and a rapeseed meal fermentation medium.
(3) The rapeseed dreg fermentation culture medium is divided into two parts by mass averagely, one part is inoculated with 0.75wt% of Aspergillus oryzae strain, and the culture is carried out for 12 hours at 29 ℃ to obtain the Aspergillus oryzae fermentation product. The other part is inoculated with 1.25 weight percent of Aspergillus niger strain and cultured for 12 hours at 29 ℃ to obtain a Aspergillus niger fermentation product. Inoculating 2.5% of bacillus subtilis strain into the solid fermentation medium, and culturing at 38 ℃ for 6 hours to obtain bacillus fermentation product.
(4) Respectively fermenting rice starter ferment, black starter ferment and bacillus starter ferment according to the following proportion of 60:27.5: and (3) uniformly mixing the materials according to the mass ratio of 12.5, inoculating 4% lactobacillus casei fermentation liquor, and controlling the initial fermentation temperature to be 32-38 ℃ for 1d, thus obtaining a fermentation product after fermentation.
(5) And (3) drying the fermented product obtained in the step (4) at 45-50 ℃ until the moisture is 11%, so as to obtain the fermented rapeseed meal.
Comparative example 5
(1) Culturing lactobacillus casei fermentation seed liquid by shake flask culture method to obtain lactobacillus casei bacterial count of 6×10 9 cfu/mL; aspergillus oryzae and Aspergillus niger strains are respectively cultured by adopting a solid state culture method, so that the number of spores of Aspergillus oryzae reaches 30 hundred million/g, the activity of neutral protease reaches 2000U/g, the number of spores of Aspergillus niger reaches 10 hundred million/g, the activity of xylanase reaches 1000U/g, and the activity of cellulase reaches 100U/g.
(2) 100g of rapeseed meal, 0.1g of whey powder and water are mixed until the water content is 44%, and sterilized for 30min at 121 ℃ to obtain the rapeseed meal fermentation medium. Inoculating 0.75wt% of Aspergillus oryzae strain and 1.25wt% of Aspergillus niger strain into the rapeseed dreg fermentation medium, and culturing at 29 ℃ for 36h to obtain a fermented product.
(3) Inoculating 4% lactobacillus casei fermentation liquor to the fermented product, controlling the initial fermentation temperature to be 32-38 ℃, and culturing for 3d to obtain the fermented product after the fermentation is finished.
(4) And (3) drying the fermented product obtained in the step (3) at 45-50 ℃ until the moisture is 11%, thus obtaining the fermented rapeseed meal.
Comparative example 6
(1) Culturing lactobacillus casei fermentation seed liquid by shake flask culture method to obtain lactobacillus casei bacterial count of 6×10 9 cfu/mL; aspergillus oryzae and Aspergillus niger strains are respectively cultured by adopting a solid state culture method, so that the number of spores of Aspergillus oryzae reaches 30 hundred million/g, the activity of neutral protease reaches 2000U/g, the number of spores of Aspergillus niger reaches 10 hundred million/g, the activity of xylanase reaches 1000U/g, and the activity of cellulase reaches 100U/g.
(2) 100g of rapeseed meal, 0.1g of whey powder and water are mixed until the water content is 44%, and sterilized for 30min at 121 ℃ to obtain the rapeseed meal fermentation medium. Inoculating 0.75wt% of Aspergillus oryzae strain and 1.25wt% of Aspergillus niger strain to the rapeseed dreg fermentation medium, inoculating 4% of Lactobacillus casei fermentation broth, controlling the initial fermentation temperature at 32-38deg.C, and culturing for 30 hr to obtain fermented product.
(3) And (3) drying the fermented product obtained in the step (2) at 45-50 ℃ until the moisture is 11%, so as to obtain the fermented rapeseed meal.
Comparative example 7
(1) Culturing Bacillus licheniformis and Lactobacillus casei fermentation seed liquid by shake flask culture method to obtain Bacillus licheniformis bacterial count of 1×10 10 cfu/mL, lactobacillus casei count of 6×10 9 cfu/mL; aspergillus oryzae and Aspergillus niger strains are respectively cultured by adopting a solid state culture method, so that the number of spores of Aspergillus oryzae reaches 30 hundred million/g, the activity of neutral protease reaches 2000U/g, the number of spores of Aspergillus niger reaches 10 hundred million/g, the activity of xylanase reaches 1000U/g, and the activity of cellulase reaches 100U/g.
(2) 80g of bran, 17g of rice husk, 2g of soybean meal powder, 0.15g of monopotassium phosphate, 0.2g of magnesium sulfate and 0.65g of light calcium carbonate are uniformly mixed to obtain a solid raw material. Mixing the solid raw materials with water until the water content is 47%, mixing 100g of rapeseed meal, 0.1g of whey powder with water until the water content is 44%, and sterilizing at 121 ℃ for 30min to obtain a solid fermentation medium and a rapeseed meal fermentation medium.
(3) Inoculating 0.75wt% of Aspergillus oryzae strain and 1.25wt% of Aspergillus niger strain into the rapeseed dreg fermentation medium, and culturing at 29 ℃ for 38 hours to obtain a mixed fermentation product. Inoculating 2.5% Bacillus licheniformis strain into the solid fermentation culture medium, and culturing at 38deg.C for 46 hr to make the bacillus bacterial count reach 2×10 10 cfu/g, wherein the activity of neutral protease reaches 3000U/g, and the activity of alkaline protease reaches 5000U/g, so as to obtain the bacillus fermented product.
(4) Mixing the mixed fermentation obtained in the step (3) with bacillus fermentation products, inoculating 4% lactobacillus casei fermentation liquor, controlling the initial fermentation temperature to be 32-38 ℃ and culturing for 3d, and obtaining fermentation products after fermentation.
(5) And (3) drying the fermented product obtained in the step (4) at 45-50 ℃ until the moisture is 11%, so as to obtain the fermented rapeseed meal.
Test example 1
Taking the rapeseed meal of examples 1-3 and comparative examples 1-7, and measuring the content of thioglycoside before and after fermentation of the rapeseed meal by adopting a palladium chloride colorimetric method; measuring the crude protein content of the sample by adopting a full-automatic azotometer; determining the acid soluble protein content by referring to a determination method of GB/T22492-2008 soybean peptide powder; the total acid content (as a result of organic acid) before and after fermentation was determined by titration. The measurement results are shown in Table 4.
Table 4 (absolute dry foundation)
As can be seen from the data in Table 4, the fermented rapeseed meal prepared in examples 1-3 has a lower thioglycoside content than that in comparative examples, and the crude protein, acid soluble protein and organic acid contents are higher than those in comparative examples 1-7, indicating that the nutritive value of the rapeseed meal after fermentation is improved.
Test example 2
360 healthy 1-day-old Ailaojia (AA) mixed chicks were selected and randomly divided into 3 treatments, 6 replicates per treatment, and 20 chickens per replicate. The test adopts a single-factor completely random test design, a control group is fed with basic diet, the basic diet is corn-bean pulp diet, and the nutrition level is prepared according to NRC (1994) broiler chicken standard. The test groups were recorded as 5% example 1, 10% example 1, substituting 5% and 10% of the fermented rapeseed meal prepared in example 1 for equivalent amounts of soybean meal, respectively. The test period is 42d, and the feed is carried out in two stages: early 1-21d and late 22-42d.
The test results are shown in tables 5 to 6.
TABLE 5 influence of fermented rapeseed meal on growth performance of broilers
| Project | Control group | 5% example 1 | 10% example 1 |
| Body weight (g) | 2234.0 | 2236.8 | 2238.0 |
| Average daily gain ADG/(g/d) | 52.41 | 52.46 | 52.47 |
| Average daily feed intake ADFI/(g/d) | 94.91 | 92.37 | 92.66 |
| Ratio of weight to weight F/G | 1.81 | 1.76 | 1.77 |
As shown in table 5, compared with the control group, the weight of broiler chickens can be increased and the feed/meat ratio can be reduced by adding fermented rapeseed meal to the broiler chickens feed instead of the equivalent amount of soybean meal.
TABLE 6 influence of fermented rapeseed meal on slaughtering Performance of broilers
As can be seen from table 6, the fermented rapeseed meal can improve slaughter rate, total evisceration rate, pectoral muscle rate and leg muscle rate of broiler chickens compared with the control group. Compared with bean pulp, the rapeseed meal has lower market price, can reduce the feed cost and increase the benefit.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (3)
1. A method for biodegrading rapeseed meal toxins and improving their nutritional value, comprising the steps of:
(1) Mixing the solid raw material with water until the water content is 45-50wt%, and sterilizing to obtain a bacillus solid fermentation medium; mixing rapeseed meal, whey powder and water until the water content is 42-46wt%, and sterilizing to obtain a rapeseed meal fermentation medium; the solid raw materials comprise the following raw materials in percentage by weight: 80% of bran, 17% of rice husk, 2% of soybean meal, 0.15% of monopotassium phosphate, 0.2% of magnesium sulfate and 0.65% of light calcium carbonate; the addition amount of the whey powder is 0.1% of the total mass of the rapeseed meal; the temperature of the sterilization treatment is 105-121 ℃, and the time of the sterilization treatment is 15-30 min;
(2) Dividing the rapeseed dreg fermentation culture medium into two parts, inoculating aspergillus oryzae strain into one part, and fermenting to obtain a aspergillus oryzae fermented product; inoculating Aspergillus niger strain on the other part, and fermenting to obtain a Aspergillus niger fermented product; inoculating bacillus strain to the bacillus solid fermentation medium and fermenting to obtain bacillus fermentation product; the Aspergillus oryzae strain is obtained by solid state culture until the number of spores of Aspergillus oryzae reaches 30 hundred million/g and the activity of neutral protease reaches 2000U/g; the inoculum size of the Aspergillus oryzae strain is 0.5-1.0wt% of half mass of the rapeseed dreg fermentation medium, and the Aspergillus oryzae strain is cultured at 28-30deg.C for 30-32h to obtain Aspergillus oryzae fermented product; the aspergillus oryzae is purchased from China center for type culture collection of industrial microorganisms, and the culture collection number is: CICC 2013;
the Aspergillus niger strain is obtained by solid state culture until the number of spores of the Aspergillus niger reaches 10 hundred million/g, the xylanase activity reaches 1000U/g, and the cellulase activity reaches 100U/g; the inoculum size of the Aspergillus niger strain accounts for 1.0-1.5wt% of the half mass of the rapeseed dreg fermentation medium, and is cultured at 28-30deg.C for 36-40h to obtain a Aspergillus niger fermented product; the Aspergillus niger is purchased from China center for type culture collection of Industrial microorganisms, and the culture collection number is: CICC2041; the bacillus strain is obtained by culturing bacillus licheniformis or bacillus subtilis by shaking bottles, and the number of the cultured bacillus strain is 1 multiplied by 10 10 cfu/mL; the mass ratio of the inoculum size of the bacillus strain to the bacillus solid state fermentation medium is (2-3) mL:100g, fermenting at 37-39deg.C for 44-48h to make the bacillus count reach 2×10 10 cfu/g, wherein the activity of neutral protease reaches 3000U/g, and the activity of alkaline protease reaches 5000U/g, so as to obtain bacillus fermented product; bacillus licheniformis is purchased from China industry microbiological culture collection center and strainDeposit number: CICC21886; bacillus subtilis is purchased from the China center for type culture Collection of industrial microorganisms, and has a culture collection number: CICC20872;
(3) Uniformly mixing a rice starter fermentation product, a black starter fermentation product and a bacillus fermentation product, inoculating lactobacillus casei liquid strain, and fermenting to obtain an anaerobic fermentation product after fermentation; the rice starter ferment, the black starter ferment and the bacillus fermentum are respectively prepared according to the following steps of (55-65): (25-30): mixing (10-15) by mass ratio; the Lactobacillus casei liquid strain is obtained by culturing Lactobacillus casei with shake flask, and the number of Lactobacillus casei is 6×10 9 cfu/mL; the ratio of the inoculation amount of the lactobacillus casei liquid strain to the total mass of the rice starter ferment, the black starter ferment and the bacillus ferment is (3-5) mL:100g, the initial temperature of fermentation is 30-40 ℃, and the total fermentation time is 3-4d; lactobacillus casei was purchased from the China center for type culture Collection of microorganisms, accession number: CICC6117;
(4) And (3) drying the anaerobic fermentation product obtained in the step (3) until the moisture content is 10-12wt% to obtain the fermented rapeseed meal.
2. The fermented rapeseed meal prepared by the method of claim 1.
3. Use of the fermented rapeseed meal of claim 2 in the preparation of livestock feed.
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| CN115851465B (en) * | 2022-12-14 | 2024-01-30 | 湖北省生物农药工程研究中心 | Composite fermentation inoculant, method for preparing fermented rapeseed meal for feeding and application of composite fermentation inoculant |
| CN115736092B (en) * | 2022-12-14 | 2024-07-12 | 山东泰山生力源集团股份有限公司 | Method for improving glutamine content in feed through solid state fermentation |
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