CN117223815A - Method for reducing free phenol of cottonseed meal by enzyme and bacterium cooperation - Google Patents
Method for reducing free phenol of cottonseed meal by enzyme and bacterium cooperation Download PDFInfo
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- 238000012258 culturing Methods 0.000 claims description 20
- 239000000463 material Substances 0.000 claims description 17
- 238000003756 stirring Methods 0.000 claims description 13
- 102100032487 Beta-mannosidase Human genes 0.000 claims description 11
- 108010059892 Cellulase Proteins 0.000 claims description 11
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 11
- 108091005804 Peptidases Proteins 0.000 claims description 11
- 239000004365 Protease Substances 0.000 claims description 11
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 11
- 108010055059 beta-Mannosidase Proteins 0.000 claims description 11
- 229940106157 cellulase Drugs 0.000 claims description 11
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- 150000003839 salts Chemical class 0.000 claims description 8
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- 240000007594 Oryza sativa Species 0.000 claims 1
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- QBKSWRVVCFFDOT-UHFFFAOYSA-N gossypol Chemical compound CC(C)C1=C(O)C(O)=C(C=O)C2=C(O)C(C=3C(O)=C4C(C=O)=C(O)C(O)=C(C4=CC=3C)C(C)C)=C(C)C=C21 QBKSWRVVCFFDOT-UHFFFAOYSA-N 0.000 abstract description 24
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Abstract
The invention discloses a method for cooperatively reducing free phenol in cottonseed meal by enzyme bacteria, which comprises the following steps: enzymolysis of cotton seed meal: and (3) uniformly mixing the fermentation raw materials and the compound enzyme preparation, adding water for enzymolysis, and sterilizing after the enzymolysis is finished. Packaging, encapsulating, and placing in a shake flask machine for inoculation and fermentation; primary fermentation of cotton seed meal: respectively inoculating the cultured bacillus subtilis strain liquid and saccharomyces cerevisiae strain liquid into the above enzymolysis-well suction filtration bottle, uniformly mixing and vibrating, and placing into a bottle shaking machine for fermentation; secondary fermentation of cotton seed meal: then the cultured lactobacillus plantarum strain liquid is equally inoculated into the suction filtration bottle after primary fermentation, and fermentation is continued after uniform shaking; drying cotton seed meal: and after the secondary fermentation is finished, drying to obtain the dephenolized cotton pulp. The invention adopts two steps of enzymolysis and microbial fermentation to remove the free gossypol in the cotton cake, and has obvious effect.
Description
Technical Field
The invention belongs to the technical field of cotton dreg dephenolization, and particularly relates to a method for reducing cotton dreg free phenol by enzyme bacteria.
Background
In order to alleviate the lack of conventional protein materials, the protein content must be treated by bioengineering technology and is not applicable to a large amount of cultivated non-conventional protein-containing agricultural or food processing byproducts.
In order to improve the utilization rate of cotton cake in feed cultivation, free gossypol in cotton cake must be removed greatly, and anti-nutritional factors are eliminated.
Based on the above, the invention aims to cooperatively remove free gossypol in cotton cake by adopting an enzymolysis and microbial fermentation two-step method so as to obtain a cotton cake biological fermentation feed raw material which is low in free phenol, rich in probiotics, protein, small molecular peptide, lactic acid, biological enzyme and the like and beneficial to animal digestion and absorption, so that the cotton cake biological fermentation feed raw material is widely used in the breeding industry, and one of effective ways for relieving serious shortage of feed protein raw materials is provided.
Disclosure of Invention
In order to solve the problems, the invention provides a method for reducing the free phenol of cotton pulp by enzyme bacteria. The method can effectively remove the free phenol in the cotton seed meal, and is suitable for industrial production.
The aim and the technical problems of the invention are realized by adopting the following technical proposal.
In one aspect, the invention provides a method for reducing free phenol in cottonseed meal by enzymatic synergy, which comprises the following steps:
enzymolysis of cotton seed meal: uniformly mixing a fermentation raw material and a compound enzyme preparation, adding hot water at 40-50 ℃ and uniformly stirring to control the water content of a system to be 38-40%, standing for 1-3 hours at a constant temperature of 35-38 ℃ for enzymolysis, sterilizing an enzymolysis material at 121 ℃ after the enzymolysis is finished, preserving heat for 40 minutes, finally, averagely split charging the enzymolysis material into 4 suction filter bottles of 5000 milliliters, sealing and fastening the suction filter bottles by flannelette and kraft paper, and placing the suction filter bottles in a bottle shaking machine for waiting for inoculation fermentation;
primary fermentation of cotton seed meal: respectively inoculating 5% (v/w) of the cultured bacillus subtilis strain liquid and saccharomyces cerevisiae strain liquid into 4 5000 ml suction filter bottles which are subjected to enzymolysis according to the ratio of 1:2, uniformly mixing and vibrating, placing the mixture into a shaking machine for fermentation, controlling the rotation speed of the shaking machine to be 200-400rpm at 30-35 ℃, and fermenting and culturing for 14-18h;
secondary fermentation of cotton seed meal: respectively inoculating the cultured lactobacillus plantarum strain liquid into 4 5000 ml suction filtration bottles after primary fermentation according to 10% (V/W) inoculum size of mixed raw materials before enzymolysis, continuously fermenting at 35-37 ℃ in a shaker machine at the temperature of 35-37 ℃ for 5min after shaking, starting the shaker machine every 4h, detecting pH change of a fermentation substrate in the process, controlling the pH at 4.2-5.0 and the fermentation time at 22-26h when the fermentation is finished, and performing fermentation on the lactobacillus plantarum strain liquid;
drying cotton seed meal: and after the secondary fermentation is finished, taking out a fermentation product, and drying the fermentation product by adopting an air flow dryer at the drying temperature of 40-45 ℃ to obtain the dephenolized cotton pulp.
Further, the fermentation raw material consists of 80-90% of cottonseed meal, 5-15% of corn meal and 4-6% of molasses in percentage by weight.
Further, the fermentation raw material and the complex enzyme preparation are added according to the mass ratio of 1:0.001-0.003.
Further, the composite enzyme preparation comprises the following components in percentage by weight: 15-18% of cellulase, 5-10% of xylanase, 5-10% of mannanase, 10-15% of protease, 15-35% of edible salt and 20-32% of rice hull powder.
Further, the cellulase activity is 2×10 4 u/g xylanase activity 1X 10 5 u/g, protease activity 1X 10 5 u/g, mannanase activity 5X 10 4 u/g。
Further, the bacillus subtilis strain liquid and the saccharomyces cerevisiae strain liquid are mixed according to the proportion of 1:2; the inoculation amount is 5% (v/w) of the mixed raw materials before enzymolysis.
Further, the inoculation amount of the lactobacillus plantarum strain liquid is 10% (V/W) of the mixed raw materials before enzymolysis.
Further, the colony number of the bacillus subtilis is 3.2-4.5X10 9 Hundred million cfu/g, the colony number of the saccharomyces cerevisiae is 2.8-3.5X10 9 Hundred million cfu/g, the colony number of the plant lactobacilli acid is 3.1-5.0X10 8 cfu/g。
By means of the technical scheme, the invention has at least the following advantages: in one aspect, the invention removes free gossypol in cotton cake by adopting an enzymolysis and microbial fermentation two-step method, thereby obtaining the cotton cake which is low in free phenol, rich in probiotics, protein, small molecular peptide, lactic acid, biological enzyme and the like and beneficial to animal digestion and absorption. On the other hand, the invention selects a compound enzyme preparation consisting of cellulase, xylanase, mannase and protease, can decompose macromolecular substances such as cellulose, sugar, protein and the like in cotton meal, and can provide enough energy sources for subsequent strain fermentation by additionally adding corn flour and molasses into fermentation raw materials, thereby improving fermentation efficiency; the strain for fermentation is Saccharomyces cerevisiae, bacillus subtilis and lactobacillus plantarum, and the three bacterial agents are synergistic to ferment, so that the free phenol in the cottonseed meal can be fully utilized as a capability source, the content of the free phenol in the cottonseed meal is reduced, and the content of the micromolecular peptide and crude protein is improved.
The foregoing description is only an overview of the present invention, and is intended to provide a more thorough understanding of the present invention, and is to be accorded the full scope of the present invention.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purposes and the effects of the present invention easy to understand, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1:
1 cultivation and preparation of microorganisms
1.1 Saccharomyces cerevisiae expansion culture
Taking 3g of yeast extract, 6g of peptone and 6g of glucose, dissolving in 300ml of water, split charging into 250ml triangular flasks, sealing and fastening 50ml of each flask by flannelette and kraft paper, sterilizing in a sterilizing pot at 115 ℃, preserving heat for 25min, cooling, inoculating yeast inclined plane strain, placing in a shaking table for culturing at 29-31 ℃, culturing at 180rpm for 24h, and reserving.
1.2 Bacillus subtilis expansion culture
Dissolving 15g of soybean meal, 10g of corn starch, 2.5g of glucose and 0.1g of manganese sulfate into 500ml of water, subpackaging into 500ml triangular bottles, sterilizing each bottle with 100ml of water, placing in a sterilizing pot, sterilizing at 121 ℃, preserving heat for 25min, cooling, and inoculating into inclined plane strains. The culture temperature of the shaking table is 35+/-1 ℃, the rotating speed is 180rpm, and the culture is carried out for 12 hours for standby.
1.3 Lactobacillus plantarum expanded culture
Taking 17.5g of glucose, 20g of soybean meal, 15g of corn steep liquor dry powder and 0.025g of manganese sulfate, dissolving in 500ml of water, subpackaging into 500ml triangular bottles, sealing and fastening 100ml of each bottle with flannelette and kraft paper, sterilizing in a sterilizing pot at the sterilizing temperature of 121 ℃, preserving heat for 25min, culturing in a shaking table at the temperature of 36+/-1 ℃ at the rotating speed of 50rpm for 12h, and standing for later use.
2 enzymolysis of cotton seed meal mixture
2.1 ratio of Complex enzyme preparation
The compound enzyme preparation comprises the following components: 18% cellulase (enzyme activity 2X 10) 4 u/g) +5% xylanase (enzyme activity 1×10) 5 u/g) +5% mannanase (enzyme activity 5×10) 4 u/g) +10% protease (enzyme activity 1×10) 5 u/g) +30% edible salt +32% rice hull powder.
2.2 enzymatic hydrolysis of cottonseed meal
The raw material requirements are as follows: the 10-mesh sieving rate of cotton seed meal and corn meal is more than 90%. After the detection raw materials are mixed, the crude protein is 38.28% (dry basis);
1700 g of cotton meal, 200 g of corn meal, 100 g of molasses and 2 g of compound enzyme preparation are respectively weighed and placed in a 10-liter plastic bucket to be uniformly mixed. Adding 950 ml of hot water at 45 ℃ which subtracts the water contained in the raw materials, stirring and mixing uniformly, and standing for 120 minutes at the constant temperature of 37 ℃ to carry out enzymolysis. After enzymolysis, sterilizing the enzymolysis materials at 121 ℃ and preserving heat for 40min, finally, split charging the enzymolysis materials into 4 suction filtration bottles of 5000 milliliters on average, sealing and fastening the suction filtration bottles by flannelette and kraft paper, and placing the suction filtration bottles in a shaking machine for waiting for inoculation and fermentation.
3 fermentation process of cotton seed meal
Taking cultured Bacillus subtilis strain liquid (colony number is 3.2-4.5X10) 9 Hundred million cfu/g) 35 ml of Saccharomyces cerevisiae seed solution (colony count of 2.8-3.5X10) 9 Hundred million cfu/g) 65 ml of the mixed raw materials before enzymolysis are respectively connected into 4 5000 ml of filter flasks after enzymolysis from the filter flask mouth on average according to 5% (v/w) of the mixed raw materials before enzymolysis, are uniformly mixed and vibrated, are placed into a shake flask machine for fermentation, the temperature is 34-35 ℃, the rotation speed of the shake flask machine is controlled to be 200rpm, and the fermentation culture time is 16h (about 50% of spores are produced).
Then the cultured lactobacillus plantarum seed solution (the colony number is 3.1-5.0X10) 8 cfu/g) 200 ml of the mixed raw materials before enzymolysis are respectively and equally inoculated into 4 5000 ml suction filtration bottles after primary fermentation according to the inoculation amount of 10% (V/W), after uniform shaking, the mixed raw materials are placed into a shaker machine for continuous fermentation at the temperature of 35-37 ℃ for 5min (the rotation speed of the shaker is 200 rpm) after starting the shaker machine every 4h, the pH change of a fermentation substrate is detected in the process, the pH is controlled to be 4.3 after the fermentation is finished, and the fermentation time is 24h.
Drying is carried out by adopting an air dryer, and the drying temperature is 43 ℃.
The quality of the obtained product is 1.92kg through detection, the appearance is light-yellow brown powder, the taste is soft, and the product is mellow and slightly sour.
Other index detection results of the product: 8.2% of water, pH4.3, and 21mg/kg of free gossypol (the national standard is 300 mg/kg) which is 7.0% of the national standard value; crude protein content 40.1%; the content of the small molecule peptide is 31.0%.
Example 2:
1 cultivation and preparation of microorganisms
1.1 Saccharomyces cerevisiae expansion culture
Taking 3g of yeast extract, 6g of peptone and 6g of glucose, dissolving in 300ml of water, split charging into 250ml triangular flasks, sealing and fastening 50ml of each flask by flannelette and kraft paper, sterilizing in a sterilizing pot at 115 ℃, preserving heat for 25min, cooling, inoculating yeast inclined plane strain, placing in a shaking table for culturing at 29-31 ℃, culturing at 180rpm for 24h, and reserving.
1.2 Bacillus subtilis expansion culture
Dissolving 15g of soybean meal, 10g of corn starch, 2.5g of glucose and 0.1g of manganese sulfate into 500ml of water, subpackaging into 500ml triangular bottles, sterilizing each bottle with 100ml of water, placing in a sterilizing pot, sterilizing at 121 ℃, preserving heat for 25min, cooling, and inoculating into inclined plane strains. The culture temperature of the shaking table is 35+/-1 ℃, the rotating speed is 180rpm, and the culture is carried out for 12 hours for standby.
1.3 Lactobacillus plantarum expanded culture
Taking 17.5g of glucose, 20g of soybean meal, 15g of corn steep liquor dry powder and 0.025g of manganese sulfate, dissolving in 500ml of water, subpackaging into 500ml triangular bottles, sealing and fastening 100ml of each bottle with flannelette and kraft paper, sterilizing in a sterilizing pot at the sterilizing temperature of 121 ℃, preserving heat for 25min, culturing in a shaking table at the temperature of 36+/-1 ℃ at the rotating speed of 50rpm for 12h, and standing for later use.
2 enzymolysis of cotton seed meal mixture
2.1 ratio of Complex enzyme preparation
The compound enzyme preparation comprises the following components: 16% cellulase (enzyme activity 2X 10) 4 u/g) +7% xylanase (enzyme activity 1×10) 5 u/g) +8% mannanase (enzyme activity 5×10) 4 u/g) +12% protease (enzyme activity 1×10) 5 u/g) +27% edible salt+30% rice hull powder.
2.2 enzymatic hydrolysis of cottonseed meal
The raw material requirements are as follows: the 10-mesh sieving rate of cotton seed meal and corn meal is more than 90%. After the detection raw materials are mixed, the crude protein is 38.28% (dry basis);
1600 g of cotton meal, 250 g of corn meal, 150 g of molasses and 1g of compound enzyme preparation are respectively weighed and placed in a 10 liter plastic bucket to be uniformly mixed. Adding 950 ml of hot water at 40 ℃ which is used for subtracting the water content of the raw materials, stirring and mixing uniformly, and standing for 60 minutes at the constant temperature of 38 ℃ for enzymolysis. After enzymolysis, sterilizing the enzymolysis materials at 121 ℃ and preserving heat for 40min, finally, split charging the enzymolysis materials into 4 suction filtration bottles of 5000 milliliters on average, sealing and fastening the suction filtration bottles by flannelette and kraft paper, and placing the suction filtration bottles in a shaking machine for waiting for inoculation and fermentation.
3 fermentation process of cotton seed meal
Taking cultured Bacillus subtilis strain liquid (colony number is 3.2-4.5X10) 9 Hundred million cfu/g) 35 ml of Saccharomyces cerevisiae seed solution (colony count of 2.8-3.5X10) 9 Hundred million cfu/g) 65 ml of the mixed raw materials before enzymolysis are respectively connected into 4 5000 ml of filter flasks after enzymolysis from the filter flask mouth on average according to 5% (v/w) of the mixed raw materials before enzymolysis, are uniformly mixed and vibrated, are placed into a shake flask machine for fermentation, the temperature is 34-35 ℃, the rotation speed of the shake flask machine is controlled to be 200rpm, and the fermentation culture time is 18h (about 50% of spores are produced).
Then the cultured lactobacillus plantarum seed solution (the colony number is 3.1-5.0X10) 8 cfu/g) 200 ml of the mixed raw materials before enzymolysis are respectively and equally inoculated into 4 5000 ml suction filtration bottles after primary fermentation according to the inoculation amount of 10% (V/W), after uniform shaking, the mixed raw materials are placed into a shaker machine for continuous fermentation at the temperature of 35-37 ℃ for 5min (the rotation speed of the shaker is 200 rpm) after starting the shaker machine every 4h, the pH change of a fermentation substrate is detected in the process, the pH is 5.0 after the fermentation is finished, and the fermentation time is 22h.
Drying is carried out by adopting an air dryer, and the drying temperature is 45 ℃.
The quality of the obtained product is 1.89kg through detection, the appearance is light-yellow brown powder, the taste is soft, and the product is mellow and slightly sour.
Other index detection results of the product: the water content is 7.9%, the pH is 4.3, the free gossypol is 24mg/kg (the national standard is 300 mg/kg), and the free gossypol is 8.0% of the national standard; crude protein content 37.9%; the content of the small molecule peptide is 28.2%.
Example 3:
1 cultivation and preparation of microorganisms
1.1 Saccharomyces cerevisiae expansion culture
Taking 3g of yeast extract, 6g of peptone and 6g of glucose, dissolving in 300ml of water, split charging into 250ml triangular flasks, sealing and fastening 50ml of each flask by flannelette and kraft paper, sterilizing in a sterilizing pot at 115 ℃, preserving heat for 25min, cooling, inoculating yeast inclined plane strain, placing in a shaking table for culturing at 29-31 ℃, culturing at 180rpm for 24h, and reserving.
1.2 Bacillus subtilis expansion culture
Dissolving 15g of soybean meal, 10g of corn starch, 2.5g of glucose and 0.1g of manganese sulfate into 500ml of water, subpackaging into 500ml triangular bottles, sterilizing each bottle with 100ml of water, placing in a sterilizing pot, sterilizing at 121 ℃, preserving heat for 25min, cooling, and inoculating into inclined plane strains. The culture temperature of the shaking table is 35+/-1 ℃, the rotating speed is 180rpm, and the culture is carried out for 12 hours for standby.
1.3 Lactobacillus plantarum expanded culture
Taking 17.5g of glucose, 20g of soybean meal, 15g of corn steep liquor dry powder and 0.025g of manganese sulfate, dissolving in 500ml of water, subpackaging into 500ml triangular bottles, sealing and fastening 100ml of each bottle with flannelette and kraft paper, sterilizing in a sterilizing pot at the sterilizing temperature of 121 ℃, preserving heat for 25min, culturing in a shaking table at the temperature of 36+/-1 ℃ at the rotating speed of 50rpm for 12h, and standing for later use.
2 enzymolysis of cotton seed meal mixture
2.1 ratio of Complex enzyme preparation
The compound enzyme preparation comprises the following components: 15% cellulase (enzyme activity 2X 10) 4 u/g) +10% xylanase (enzyme activity 1×10) 5 u/g) +10% mannanase (enzyme activity 5×10) 4 u/g) +15% protease (enzyme activity 1×10) 5 u/g) +30% edible salt+20% rice hull powder.
2.2 enzymatic hydrolysis of cottonseed meal
The raw material requirements are as follows: the 10-mesh sieving rate of cotton seed meal and corn meal is more than 90%. After the detection raw materials are mixed, the crude protein is 38.28% (dry basis);
respectively weighing 1800 g of cotton meal, 150 g of corn meal, 50 g of molasses and 3g of compound enzyme preparation, placing into a 10-liter plastic bucket, and uniformly mixing. Adding 950 ml of hot water (minus the water content of the raw materials) at 50 ℃, stirring and mixing uniformly, and standing for 180 minutes at the constant temperature of 35 ℃ for enzymolysis. After enzymolysis, sterilizing the enzymolysis materials at 121 ℃ and preserving heat for 40min, finally, split charging the enzymolysis materials into 4 suction filtration bottles of 5000 milliliters on average, sealing and fastening the suction filtration bottles by flannelette and kraft paper, and placing the suction filtration bottles in a shaking machine for waiting for inoculation and fermentation.
3 fermentation process of cotton seed meal
Taking cultured Bacillus subtilis strain liquid (colony number is 3.2-4.5X10) 9 Hundred million cfu/g) 35 ml of Saccharomyces cerevisiae seed solution (colony count of 2.8-3.5X10) 9 Hundred million cfu/g) 65 ml of the mixed raw materials before enzymolysis are respectively connected into 4 5000 ml of filter flasks after enzymolysis from the filter flask mouth on average according to 5% (v/w) of the mixed raw materials before enzymolysis, are uniformly mixed and vibrated, are placed into a shake flask machine for fermentation, the temperature is 34-35 ℃, the rotation speed of the shake flask machine is controlled to be 200rpm, and the fermentation culture time is 18h (about 50% of spores are produced).
Then the cultured lactobacillus plantarum seed solution (the colony number is 3.1-5.0X10) 8 cfu/g) 200 ml of the mixed raw materials before enzymolysis are respectively and equally inoculated into 4 5000 ml suction filtration bottles after primary fermentation according to the inoculation amount of 10% (V/W), after uniform shaking, the mixed raw materials are placed into a shaker machine for continuous fermentation at the temperature of 35-37 ℃ for 5min (the rotation speed of the shaker is 200 rpm) after starting the shaker machine every 4h, the pH change of a fermentation substrate is detected in the process, the pH is 4.6 after the fermentation is finished, and the fermentation time is 22h.
Drying is carried out by adopting an air dryer, and the drying temperature is 40 ℃.
The quality of the obtained product is 1.91kg through detection, the appearance is light-yellow brown powder, the taste is soft, and the product is mellow and slightly sour.
Other index detection results of the product: 8.0% of water, pH4.6, and free gossypol of 21mg/kg (300 mg/kg of national standard) of 7.0% of national standard value; crude protein content 39.2%; the content of the small molecule peptide is 30.7%.
Example 4:
1 cultivation and preparation of microorganisms
1.1 Saccharomyces cerevisiae expansion culture
Taking 3g of yeast extract, 6g of peptone and 6g of glucose, dissolving in 300ml of water, split charging into 250ml triangular flasks, sealing and fastening 50ml of each flask by flannelette and kraft paper, sterilizing in a sterilizing pot at 115 ℃, preserving heat for 25min, cooling, inoculating yeast inclined plane strain, placing in a shaking table for culturing at 29-31 ℃, culturing at 180rpm for 24h, and reserving.
1.2 Bacillus subtilis expansion culture
Dissolving 15g of soybean meal, 10g of corn starch, 2.5g of glucose and 0.1g of manganese sulfate into 500ml of water, subpackaging into 500ml triangular bottles, sterilizing each bottle with 100ml of water, placing in a sterilizing pot, sterilizing at 121 ℃, preserving heat for 25min, cooling, and inoculating into inclined plane strains. The culture temperature of the shaking table is 35+/-1 ℃, the rotating speed is 180rpm, and the culture is carried out for 12 hours for standby.
1.3 Lactobacillus plantarum expanded culture
Taking 17.5g of glucose, 20g of soybean meal, 15g of corn steep liquor dry powder and 0.025g of manganese sulfate, dissolving in 500ml of water, subpackaging into 500ml triangular bottles, sealing and fastening 100ml of each bottle with flannelette and kraft paper, sterilizing in a sterilizing pot at the sterilizing temperature of 121 ℃, preserving heat for 25min, culturing in a shaking table at the temperature of 36+/-1 ℃ at the rotating speed of 50rpm for 12h, and standing for later use.
2 enzymolysis of cotton seed meal mixture
2.1 ratio of Complex enzyme preparation
The compound enzyme preparation comprises the following components: 18% cellulase (enzyme activity 2X 10) 4 u/g) +5% xylanase (enzyme activity 1×10) 5 u/g) +5% mannanase (enzyme activity 5×10) 4 u/g) +10% protease (enzyme activity 1×10) 5 u/g) +30% edible salt +32% rice hull powder.
2.2 enzymatic hydrolysis of cottonseed meal
The raw material requirements are as follows: the 10-mesh sieving rate of cotton seed meal and corn meal is more than 90%. After the detection raw materials are mixed, the crude protein is 38.28% (dry basis);
pretreatment of fermentation raw materials: 1700 g of cotton meal is added into 6L of tryptophan alkali solution (tryptophan is dissolved by sodium hydroxide solution to prepare tryptophan alkali solution with pH of 7.5-8.2), the solution is heated to 90 ℃, the temperature is kept constant, 200 g of corn flour and 100 g of molasses are sequentially added after continuous stirring for 1h, and stirring is continued until the solution is uniform, so that a pretreated fermentation raw material is obtained.
2 g of the pretreated fermentation raw material and 2 g of the compound enzyme preparation are placed in a 10 liter plastic bucket and uniformly mixed. Adding 950 ml of hot water at 45 ℃ which subtracts the water contained in the raw materials, stirring and mixing uniformly, and standing for 120 minutes at the constant temperature of 37 ℃ to carry out enzymolysis. After enzymolysis, sterilizing the enzymolysis materials at 121 ℃ and preserving heat for 40min, finally, split charging the enzymolysis materials into 4 suction filtration bottles of 5000 milliliters on average, sealing and fastening the suction filtration bottles by flannelette and kraft paper, and placing the suction filtration bottles in a shaking machine for waiting for inoculation and fermentation.
3 fermentation process of cotton seed meal
Taking cultured Bacillus subtilis strain liquid (colony number is 3.2-4.5X10) 9 Hundred million cfu/g) 35 ml of Saccharomyces cerevisiae seed solution (colony count of 2.8-3.5X10) 9 Hundred million cfu/g) 65 ml of the mixed raw materials before enzymolysis are respectively connected into 4 5000 ml of filter flasks after enzymolysis from the filter flask mouth on average according to 5% (v/w) of the mixed raw materials before enzymolysis, are uniformly mixed and vibrated, are placed into a shake flask machine for fermentation, the temperature is 34-35 ℃, the rotation speed of the shake flask machine is controlled to be 200rpm, and the fermentation culture time is 16h (about 50% of spores are produced).
Then the cultured lactobacillus plantarum seed solution (the colony number is 3.1-5.0X10) 8 cfu/g) 200 ml of the mixed raw materials before enzymolysis are respectively and equally inoculated into 4 5000 ml suction filtration bottles after primary fermentation according to the inoculation amount of 10% (V/W), after uniform shaking, the mixed raw materials are placed into a shaker machine for continuous fermentation at the temperature of 35-37 ℃ for 5min (the rotation speed of the shaker is 200 rpm) after starting the shaker machine every 4h, the pH change of a fermentation substrate is detected in the process, the pH is controlled to be 4.3 after the fermentation is finished, and the fermentation time is 24h.
Drying is carried out by adopting an air dryer, and the drying temperature is 43 ℃.
The quality of the obtained product is 1.99kg through detection, the appearance is light-yellow brown powder, the taste is soft, and the product is mellow and slightly sour.
Other index detection results of the product: moisture 7.8%, pH4.3, free gossypol below detection limit 20mg/kg (national standard 300 mg/kg); crude protein content 49.6%; the content of the small molecule peptide is 40.2%.
Example 5:
1 cultivation and preparation of microorganisms
1.1 Saccharomyces cerevisiae expansion culture
Taking 3g of yeast extract, 6g of peptone and 6g of glucose, dissolving in 300ml of water, split charging into 250ml triangular flasks, sealing and fastening 50ml of each flask by flannelette and kraft paper, sterilizing in a sterilizing pot at 115 ℃, preserving heat for 25min, cooling, inoculating yeast inclined plane strain, placing in a shaking table for culturing at 29-31 ℃, culturing at 180rpm for 24h, and reserving.
1.2 Bacillus subtilis expansion culture
Dissolving 15g of soybean meal, 10g of corn starch, 2.5g of glucose and 0.1g of manganese sulfate into 500ml of water, subpackaging into 500ml triangular bottles, sterilizing each bottle with 100ml of water, placing in a sterilizing pot, sterilizing at 121 ℃, preserving heat for 25min, cooling, and inoculating into inclined plane strains. The culture temperature of the shaking table is 35+/-1 ℃, the rotating speed is 180rpm, and the culture is carried out for 12 hours for standby.
1.3 Lactobacillus plantarum expanded culture
Taking 17.5g of glucose, 20g of soybean meal, 15g of corn steep liquor dry powder and 0.025g of manganese sulfate, dissolving in 500ml of water, subpackaging into 500ml triangular bottles, sealing and fastening 100ml of each bottle with flannelette and kraft paper, sterilizing in a sterilizing pot at the sterilizing temperature of 121 ℃, preserving heat for 25min, culturing in a shaking table at the temperature of 36+/-1 ℃ at the rotating speed of 50rpm for 12h, and standing for later use.
2 enzymolysis of cotton seed meal mixture
2.1 ratio of Complex enzyme preparation
The compound enzyme preparation comprises the following components: 18% cellulase (enzyme activity 2X 10) 4 u/g) +5% xylanase (enzyme activity 1×10) 5 u/g) +5% mannanase (enzyme activity 5×10) 4 u/g) +10% protease (enzyme activity 1×10) 5 u/g) +30% edible salt +32% rice hull powder.
2.2 enzymatic hydrolysis of cottonseed meal
The raw material requirements are as follows: the 10-mesh sieving rate of cotton seed meal and corn meal is more than 90%. After the detection raw materials are mixed, the crude protein is 38.28% (dry basis);
pretreatment of fermentation raw materials: 1700 g of cotton meal is added into 5L of tryptophan alkali solution (tryptophan is dissolved by sodium hydroxide solution to prepare tryptophan alkali solution with pH of 7.5-8.2), the solution is heated to 90 ℃, the temperature is kept constant, 200 g of corn flour and 100 g of molasses are sequentially added after continuous stirring for 1h, and stirring is continued until the solution is uniform, so that a pretreated fermentation raw material is obtained.
2 g of the pretreated fermentation raw material and 2 g of the compound enzyme preparation are placed in a 10 liter plastic bucket and uniformly mixed. Adding 950 ml of hot water at 45 ℃ which subtracts the water contained in the raw materials, stirring and mixing uniformly, and standing for 120 minutes at the constant temperature of 37 ℃ to carry out enzymolysis. After enzymolysis, sterilizing the enzymolysis materials at 121 ℃ and preserving heat for 40min, finally, split charging the enzymolysis materials into 4 suction filtration bottles of 5000 milliliters on average, sealing and fastening the suction filtration bottles by flannelette and kraft paper, and placing the suction filtration bottles in a shaking machine for waiting for inoculation and fermentation.
3 fermentation process of cotton seed meal
Taking cultured Bacillus subtilis strain liquid (colony number is 3.2-4.5X10) 9 Hundred million cfu/g) 35 ml of Saccharomyces cerevisiae seed solution (colony count of 2.8-3.5X10) 9 Hundred million cfu/g) 65 ml of the mixed raw materials before enzymolysis are respectively connected into 4 5000 ml of filter flasks after enzymolysis from the filter flask mouth on average according to 5% (v/w) of the mixed raw materials before enzymolysis, are uniformly mixed and vibrated, are placed into a shake flask machine for fermentation, the temperature is 34-35 ℃, the rotation speed of the shake flask machine is controlled to be 200rpm, and the fermentation culture time is 16h (about 50% of spores are produced).
Then the cultured lactobacillus plantarum seed solution (the colony number is 3.1-5.0X10) 8 cfu/g) 200 ml of the mixed raw materials before enzymolysis are respectively and equally inoculated into 4 5000 ml suction filtration bottles after primary fermentation according to the inoculation amount of 10% (V/W), after uniform shaking, the mixed raw materials are placed into a shaker machine for continuous fermentation at the temperature of 35-37 ℃ for 5min (the rotation speed of the shaker is 200 rpm) after starting the shaker machine every 4h, the pH change of a fermentation substrate is detected in the process, the pH is controlled to be 4.3 after the fermentation is finished, and the fermentation time is 24h.
Drying is carried out by adopting an air dryer, and the drying temperature is 43 ℃.
The quality of the obtained product is 1.98kg through detection, the appearance is light-yellow brown powder, the taste is soft, and the product is mellow and slightly sour.
Other index detection results of the product: 8.0% of water, pH4.3, and free gossypol below a detection limit of 20mg/kg (the national standard is 300 mg/kg); crude protein content 48.9%; the content of the small molecule peptide is 39.7%.
Comparative example 1:
1 cultivation and preparation of microorganisms
1.1 Saccharomyces cerevisiae expansion culture
Taking 3g of yeast extract, 6g of peptone and 6g of glucose, dissolving in 300ml of water, split charging into 250ml triangular flasks, sealing and fastening 50ml of each flask by flannelette and kraft paper, sterilizing in a sterilizing pot at 115 ℃, preserving heat for 25min, cooling, inoculating yeast inclined plane strain, placing in a shaking table for culturing at 29-31 ℃, culturing at 180rpm for 24h, and reserving.
1.2 Bacillus subtilis expansion culture
Dissolving 15g of soybean meal, 10g of corn starch, 2.5g of glucose and 0.1g of manganese sulfate into 500ml of water, subpackaging into 500ml triangular bottles, sterilizing each bottle with 100ml of water, placing in a sterilizing pot, sterilizing at 121 ℃, preserving heat for 25min, cooling, and inoculating into inclined plane strains. The culture temperature of the shaking table is 35+/-1 ℃, the rotating speed is 180rpm, and the culture is carried out for 12 hours for standby.
1.3 Lactobacillus plantarum expanded culture
Taking 17.5g of glucose, 20g of soybean meal, 15g of corn steep liquor dry powder and 0.025g of manganese sulfate, dissolving in 500ml of water, subpackaging into 500ml triangular bottles, sealing and fastening 100ml of each bottle with flannelette and kraft paper, sterilizing in a sterilizing pot at the sterilizing temperature of 121 ℃, preserving heat for 25min, culturing in a shaking table at the temperature of 36+/-1 ℃ at the rotating speed of 50rpm for 12h, and standing for later use.
2 hydrolysis of cottonseed meal mixture
2.2 hydrolysis of cottonseed meal
The raw material requirements are as follows: the 10-mesh sieving rate of cotton seed meal and corn meal is more than 90%. After the detection raw materials are mixed, the crude protein is 38.28% (dry basis);
1700 g of cotton meal, 200 g of corn meal and 100 g of molasses are respectively weighed and placed in a 10 liter plastic bucket to be uniformly mixed. Adding 950 ml of hot water at 45 ℃ with water content of the raw materials subtracted, stirring and mixing uniformly, sterilizing at 121 ℃, preserving heat for 40min, finally, averagely split charging into 4 suction filtration bottles of 5000 ml, sealing and fastening by flannelette and kraft paper, and placing in a shaking machine for inoculation and fermentation.
3 fermentation process of cotton seed meal
Taking cultured bacillus subtilis strain liquid(colony count is 3.2-4.5X10) 9 Hundred million cfu/g) 35 ml of Saccharomyces cerevisiae seed solution (colony count of 2.8-3.5X10) 9 Hundred million cfu/g) 65 ml are respectively connected into the 4 5000 ml suction filtration bottles from the suction filtration bottle mouth according to 5% (v/w) of the mixed raw materials, evenly mixed and vibrated, and placed into a bottle shaking machine for fermentation at 34-35 ℃, the rotation speed of the bottle shaking machine is controlled to be 200rpm, and the fermentation culture time is 16h (about 50% of spores are produced).
Then the cultured lactobacillus plantarum seed solution (the colony number is 3.1-5.0X10) 8 cfu/g) 200 ml of the mixed raw materials are respectively and equivalently connected into 4 5000 ml suction filtration bottles after primary fermentation according to 10% (V/W) inoculum size of the mixed raw materials, and after uniform shaking, the mixed raw materials are placed into a shaker for continuous fermentation at the temperature of 35-37 ℃ for 5min (the rotational speed of the shaker is 200 rpm) after starting the shaker for every 4h, the pH change of a fermentation substrate is detected in the process, the pH is controlled to be 4.3 after the fermentation is finished, and the fermentation time is 24h.
Drying is carried out by adopting an air dryer, and the drying temperature is 43 ℃.
The quality of the obtained product is 0.99kg through detection, the appearance is light brown powder, and the sour taste is heavy.
Other index detection results of the product: 12.5% of water, pH4.3, 413mg/kg of free gossypol (the national standard is 300 mg/kg) and 137.7% of the national standard value; crude protein content 20.1%; the content of the small molecule peptide is 9.2%.
Comparative example 2:
1 enzymolysis of the cottonseed meal mixture
1.1 ratio of Complex enzyme preparation
The compound enzyme preparation comprises the following components: 18% cellulase (enzyme activity 2X 10) 4 u/g) +5% xylanase (enzyme activity 1×10) 5 u/g) +5% mannanase (enzyme activity 5×10) 4 u/g) +10% protease (enzyme activity 1×10) 5 u/g) +30% edible salt +32% rice hull powder.
1.2 enzymatic hydrolysis of cottonseed meal
The raw material requirements are as follows: the 10-mesh sieving rate of cotton seed meal and corn meal is more than 90%. After the detection raw materials are mixed, the crude protein is 38.28% (dry basis);
1700 g of cotton meal, 200 g of corn meal, 100 g of molasses and 2 g of compound enzyme preparation are respectively weighed and placed in a 10-liter plastic bucket to be uniformly mixed. Adding 950 ml of hot water at 45 ℃ which subtracts the water contained in the raw materials, stirring and mixing uniformly, and standing for 120 minutes at the constant temperature of 37 ℃ to carry out enzymolysis. After enzymolysis, sterilizing the enzymolysis materials at 121 ℃ and preserving heat for 40min, finally, split charging the enzymolysis materials into 4 suction filtration bottles of 5000 milliliters on average, sealing and fastening the suction filtration bottles by flannelette and kraft paper, and placing the suction filtration bottles in a shaking machine for waiting for inoculation and fermentation.
Drying is carried out by adopting an air dryer, and the drying temperature is 43 ℃.
The quality of the obtained product is 0.76kg, the appearance is white powder, and the taste is close to tasteless.
Other index detection results of the product: 11.3% of water, pH4.3, 521mg/kg of free gossypol (300 mg/kg of national standard) and 173.7% of national standard value; crude protein content 17.5%; the content of the small molecule peptide is 7.3 percent.
While the invention has been described with respect to preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and that any such changes and modifications as described in the above embodiments are intended to be within the scope of the invention.
Claims (8)
1. A method for reducing free phenol in cottonseed meal by enzymatic synergy, which is characterized by comprising the following steps:
enzymolysis of cotton seed meal: uniformly mixing a fermentation raw material and a compound enzyme preparation, adding hot water at 40-50 ℃ and uniformly stirring to control the water content of a system to be 38-40%, standing for 1-3 hours at a constant temperature of 35-38 ℃ for enzymolysis, sterilizing an enzymolysis material at 121 ℃ after the enzymolysis is finished, preserving heat for 40 minutes, finally, averagely split charging the enzymolysis material into 4 suction filter bottles of 5000 milliliters, sealing and fastening the suction filter bottles by flannelette and kraft paper, and placing the suction filter bottles in a bottle shaking machine for waiting for inoculation fermentation;
primary fermentation of cotton seed meal: respectively inoculating 5% (v/w) of the cultured bacillus subtilis strain liquid and saccharomyces cerevisiae strain liquid into 4 5000 ml suction filter bottles which are subjected to enzymolysis according to the ratio of 1:2, uniformly mixing and vibrating, placing the mixture into a shaking machine for fermentation, controlling the rotation speed of the shaking machine to be 200-400rpm at 30-35 ℃, and fermenting and culturing for 14-18h;
secondary fermentation of cotton seed meal: respectively inoculating the cultured lactobacillus plantarum strain liquid into 4 5000 ml suction filtration bottles after primary fermentation according to 10% (V/W) inoculum size of mixed raw materials before enzymolysis, continuously fermenting at 35-37 ℃ in a shaker machine at the temperature of 35-37 ℃ for 5min after shaking, starting the shaker machine every 4h, detecting pH change of a fermentation substrate in the process, controlling the pH at 4.2-5.0 and the fermentation time at 22-26h when the fermentation is finished, and performing fermentation on the lactobacillus plantarum strain liquid;
drying cotton seed meal: and after the secondary fermentation is finished, taking out a fermentation product, and drying the fermentation product by adopting an air flow dryer at the drying temperature of 40-45 ℃ to obtain the dephenolized cotton pulp.
2. The method according to claim 1, wherein the fermentation raw material consists of cotton seed meal, corn meal and molasses, and the content of each substance is 80-90% of cotton seed meal, 5-15% of corn meal and 4-6% of molasses in percentage by weight.
3. The method according to claim 1 or 2, characterized in that the fermentation feedstock and the complex enzyme preparation are added in a mass ratio of 1:0.001-0.003.
4. The method according to claim 1, wherein the composition of the complex enzyme preparation, in weight percent, is: 15-18% of cellulase, 5-10% of xylanase, 5-10% of mannanase, 10-15% of protease, 15-35% of edible salt and 20-32% of rice hull powder.
5. According to claim 1Characterized in that the cellulase activity is 2X 10 4 u/g xylanase activity 1X 10 5 u/g, protease activity 1X 10 5 u/g, mannanase activity 5X 10 4 u/g。
6. The method of claim 1, wherein the bacillus subtilis strain solution and the saccharomyces cerevisiae strain solution are mixed in a ratio of 1:2; the inoculation amount is 5% (v/w) of the mixed raw materials before enzymolysis.
7. The method according to claim 1, wherein the lactobacillus plantarum strain solution is inoculated in an amount of 10% (V/W) of the mixed raw material before enzymolysis.
8. The method of claim 1, wherein the number of bacillus subtilis colonies is 3.2-4.5x10 9 Hundred million cfu/g, the colony number of the saccharomyces cerevisiae is 2.8-3.5X10 9 Hundred million cfu/g, the colony number of the plant lactobacilli acid is 3.1-5.0X10 8 cfu/g。
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