CN113416673A - Complex microbial inoculant for detoxifying cottonseed meal as well as preparation method and application thereof - Google Patents
Complex microbial inoculant for detoxifying cottonseed meal as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of complex microbial inoculum, and provides a complex microbial inoculum for detoxifying cottonseed meal as well as a preparation method and application thereof. The composite microbial inoculum comprises Bacillus, Saccharomyces cerevisiae and Lactobacillus plantarum. When the compound microbial inoculum prepared by the invention is applied to the fermentation process of cottonseed meal, the content of free gossypol in the cottonseed meal can be effectively reduced, the content of protein and free amino acid in the cottonseed meal can be improved, when a certain amount of soybean meal in daily ration is replaced for feeding a mouse, the growth performance of the mouse can not be reduced, and the compound microbial inoculum has no obvious influence on the biochemical indexes of the liver. The invention has the characteristics of simple preparation process, stable and safe product quality, low cost and the like.
Description
Technical Field
The invention relates to the technical field of compound microbial inoculum, in particular to a compound microbial inoculum for detoxifying cottonseed meal.
Background
The protein feed resources, particularly high-quality protein feed raw materials, in China are seriously in short supply, the annual grain and oil feed resource demand amount reaches 3.5 hundred million tons, wherein the imported amount accounts for about 1/3, and the imported amount of soybean and fish meal is the first in the world. The cotton planting area in 2018 nationwide is 3352.3 kilo hectares, and the yield reaches 609.6 ten thousand tons. The seeds of cotton are called cottonseed, which is an important oil source, and 12% -18% of cottonseed oil and 45% of cottonseed cake meal by-products can be produced from the cottonseed by means of mechanical pressing or pre-pressing leaching and the like. The content of crude protein in the cottonseed meal is more than 35 percent, the price is relatively low, but the cottonseed meal contains endogenous toxin-Free Gossypol (FG), and the FG has certain toxic action on the growth and the reproduction of livestock and poultry, thereby limiting the application of the cottonseed meal in livestock and poultry feed. In recent years, scholars at home and abroad are dedicated to the detoxification research of the cottonseed meal, and the detoxified cottonseed meal not only has obviously reduced harmful components, but also has obviously improved feed quality.
According to the research progress of the cottonseed meal at home and abroad, the domestic research mainly aims at breeding the high-degradation gossypol strain, the obtained strain has better detoxification, but the single-strain fermentation effect is single, the cottonseed meal is fermented by mixing the composite strains, the nutrition quality of the cottonseed meal can be effectively improved while the detoxification rate is ensured, in addition, the microbial fermentation can generate various enzymes, the content of free gossypol can be effectively reduced in the growth and metabolism process, and the utilization rate of the cottonseed protein can be improved. At present, the research report of improving the nutrition quality of the cottonseed meal by adopting enzyme-producing strain composite fermentation is rare.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides the composite microbial inoculum with the cottonseed meal detoxification effect and the preparation method and the application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a composite microbial inoculum for detoxifying cottonseed meal, which comprises Bacillus, Saccharomyces cerevisiae and Lactobacillus plantarum.
The invention also provides a preparation method of the composite microbial inoculum for detoxifying the cottonseed meal, which comprises the following steps:
(1) adding bacillus into an LB broth culture medium, and performing shaking table shaking culture to obtain a bacillus seed solution for final fermentation;
(2) adding saccharomyces cerevisiae into the YPD liquid culture medium, and performing shaking table shaking culture to obtain a final saccharomyces cerevisiae seed liquid for fermentation;
(3) adding lactobacillus plantarum into an MRS liquid culture medium, and performing static culture to obtain lactobacillus plantarum seed liquid for final fermentation;
(4) and compounding the bacillus seed liquid, the saccharomyces cerevisiae seed liquid and the lactobacillus plantarum seed liquid for final fermentation to obtain the composite microbial inoculum.
Preferably, in the step (1), the LB broth culture medium comprises peptone 0.5-1.5 g, yeast powder 0.4-0.6 g, sodium chloride 0.4-0.6 g, pH 7.2-7.4, and sterilization is performed at 120-125 ℃ for 15-25 min;
in the step (2), the YPD liquid culture medium comprises 0.5-1.5 g of yeast extract powder, 1.5-2.5 g of peptone and 1.5-2.5 g of glucose, the pH value is 7.2-7.4, and the YPD liquid culture medium is sterilized at 120-125 ℃ for 15-25 min;
in the step (3), the MRS liquid culture medium comprises 0.5-1.5 g of peptone, 0.5-1.5 g of beef extract, 0.4-0.6 g of yeast extract powder, 1-3 g of glucose, 0.4-0.6 g of anhydrous sodium acetate, 0.1-0.3 g of diammonium citrate, 800.1-0.2 mL of Tween-2, 0.05-0.06 g of magnesium sulfate and 0.02-0.03 g of manganese sulfate, the pH value is 6.2-6.4, and the MRS liquid culture medium is sterilized at 120-125 ℃ for 15-25 min.
Preferably, in the step (1), the specific preparation method of the bacillus seed solution comprises: selecting 1-3 cyclosporins, inoculating the 1-3 cyclosporins into 4-6 mL of LB broth culture medium, carrying out shake culture for 18-22 h under the conditions of 24-26 ℃ and 180-220 r/min to obtain a first-stage seed solution, pouring the first-stage seed solution into 40-60 mL of LB broth culture medium, carrying out shake culture for 18-22 h under the conditions of 24-26 ℃ and 180-220 r/min to obtain a second-stage seed solution serving as a bacillus seed solution for final fermentation;
in the step (2), the specific preparation method of the saccharomyces cerevisiae seed liquid comprises the following steps: selecting 1-3 rings of saccharomyces cerevisiae, inoculating the saccharomyces cerevisiae into 4-6 mL of YPD liquid culture medium, carrying out shake culture for 22-26 h under the conditions of 26-30 ℃ and 160-200 r/min to obtain a first-stage seed solution, pouring the first-stage seed solution into 40-60 mL of YPD liquid culture medium, carrying out shake culture for 22-26 h under the conditions of 26-30 ℃ and 160-200 r/min to obtain a second-stage seed solution as a saccharomyces cerevisiae seed solution for final fermentation;
in the step (3), the specific preparation method of the lactobacillus plantarum seed liquid comprises the following steps: selecting 1-3 rings of lactobacillus plantarum, inoculating the lactobacillus plantarum into 4-6 mL of MRS liquid culture medium, performing static culture for 18-22 h at 35-40 ℃ to obtain first-stage seed liquid, pouring the first-stage seed liquid into 40-60 mLMRS liquid culture medium, performing static culture for 18-22 h at 35-40 ℃, and taking the obtained second-stage seed liquid as lactobacillus plantarum seed liquid for final fermentation.
Preferably, in the step (4), the compounding volume ratio of the bacillus seed liquid, the saccharomyces cerevisiae seed liquid and the lactobacillus plantarum seed liquid is 1-2: 1-3: 2-3.
The invention also provides the application of the compound microbial inoculum for detoxifying the cottonseed meal in the process of detoxifying the cottonseed meal.
Preferably, the application process includes: inoculating composite microbial inoculum for detoxifying cottonseed meal on a cottonseed meal fermentation culture medium, and then sequentially carrying out fermentation and enzymolysis.
Preferably, the cottonseed meal fermentation medium comprises 80-85 g of cottonseed meal, 9-10 g of bran, 5-10 g of brown sugar and 66-106 mL of distilled water, the cottonseed meal fermentation medium is sterilized for 15-25 min under the sterilization condition of 120-125 ℃, and the particle size of the cottonseed meal is 55-65 meshes.
Preferably, in the fermentation process, the inoculation amount of the composite microbial inoculum accounts for 3-9% of the total volume of the composite microbial inoculum, the material-water ratio is 48-52 g: 40-45 mL, the fermentation temperature is 30-40 ℃, and the fermentation time is 70-75 h.
Preferably, the enzymolysis condition is natural enzymolysis for 11-13 h.
The invention has the following beneficial effects:
(1) the bacillus adopted by the method has the characteristics of strong stress resistance, easy storage and the like, produces protease, can improve the protein quality of cottonseed meal, and improves the nutritional value of the cottonseed meal.
(2) The method of the invention selects three microorganisms of bacillus, saccharomyces cerevisiae and lactobacillus plantarum, and utilizes the comprehensive effect of the three microorganisms to degrade the content of free gossypol in the cottonseed meal, eliminate antinutritional factors in the cottonseed meal, improve the utilization rate of protein, and simultaneously has the function of a microecological preparation, and has the functions of improving the microecological balance of animal intestinal tracts, improving the immunity of animals and the like.
(3) The invention has the characteristics of simple production process, low energy consumption, no three wastes, small investment, easy large-scale production and the like.
Drawings
FIG. 1 shows the results of the neutral protease activity assay of Bacillus strain S77;
FIG. 2 shows the result of the activity of Saccharomyces cerevisiae ZY.003 in producing cellulase;
FIG. 3 shows the effect of the compound bacterial agent obtained in example 2 after fermentation for 12 h;
FIG. 4 is a response surface analysis of inoculum versus water-to-material ratio;
FIG. 5 is a graph of response surface analysis of inoculum size versus reaction time;
FIG. 6 is a graph of response surface analysis of reaction temperature versus reaction time;
FIG. 7 is the effect of different substitution ratios of detoxified cottonseed meal on the serum ALT activity in mice (day 21);
FIG. 8 is a graph showing the effect of various substitution ratios of detoxified cottonseed meal on the serum AST activity of mice (day 21).
Deposit description
The Bacillus S77 is named as Bacillus S77, the name of a Latin is China center for type culture Collection, the address is Wuhan university in China, the preservation date is 2021, 5 and 13 days, and the preservation number is CCTCC NO: M2021528;
lactobacillus plantarum LR002, Latin, named Lactobacillus plantarum LR002, and the preservation unit, named Chinese type culture Collection, with the address of Wuhan university, Wuhan, China, the preservation date of 2021, 5 and 13 days, and the preservation number of M2021527.
Detailed Description
The invention provides a composite microbial inoculum for detoxifying cottonseed meal, which comprises Bacillus, Saccharomyces cerevisiae and Lactobacillus plantarum.
The invention also provides a preparation method of the composite microbial inoculum for detoxifying the cottonseed meal, which comprises the following steps:
(1) adding bacillus into an LB broth culture medium, and performing shaking table shaking culture to obtain a bacillus seed solution for final fermentation;
(2) adding saccharomyces cerevisiae into the YPD liquid culture medium, and performing shaking table shaking culture to obtain a final saccharomyces cerevisiae seed liquid for fermentation;
(3) adding lactobacillus plantarum into an MRS liquid culture medium, and performing static culture to obtain lactobacillus plantarum seed liquid for final fermentation;
(4) and compounding the bacillus seed liquid, the saccharomyces cerevisiae seed liquid and the lactobacillus plantarum seed liquid for final fermentation to obtain the composite microbial inoculum.
In the invention, in the step (1), the LB broth culture medium preferably comprises 0.5-1.5 g of peptone, 0.4-0.6 g of yeast powder, 0.4-0.6 g of sodium chloride, more preferably 1g of peptone, 0.5g of yeast powder, 0.5g of sodium chloride, and preferably has a pH value of 7.2-7.4, more preferably 7.3, and the sterilization conditions preferably comprise 120-125 ℃ sterilization for 15-25 min, and more preferably 121 ℃ sterilization for 20 min;
in the step (2), the YPD liquid medium preferably comprises 0.5-1.5 g of yeast extract powder, 1.5-2.5 g of peptone and 1.5-2.5 g of glucose, more preferably comprises 1g of yeast extract powder, 2g of peptone and 2g of glucose, the pH value is preferably 7.2-7.4, more preferably 7.3, the sterilization condition is preferably sterilization at 120-125 ℃ for 15-25 min, and more preferably sterilization at 121 ℃ for 20 min;
in the step (3), the MRS liquid culture medium preferably comprises 0.5-1.5 g of peptone, 0.5-1.5 g of beef extract, 0.4-0.6 g of yeast extract powder, 1-3 g of glucose, 0.4-0.6 g of anhydrous sodium acetate, 0.1-0.3 g of diammonium citrate, 800.1-0.2 mL of tween-800, 0.05-0.06 g of magnesium sulfate and 0.02-0.03 g of manganese sulfate, more preferably 1g of peptone, 1g of beef extract, 0.5g of yeast extract powder, 2g of glucose, 0.5g of anhydrous sodium acetate, 0.2g of diammonium citrate, 0.3532 mL of tween-800.15, 0.058g of magnesium sulfate and 0.025g of manganese sulfate, the pH value is preferably 6.2-6.4, more preferably 6.3, the sterilization condition is preferably sterilization at 120-125 ℃ for 15-25 min, and the sterilization condition is more preferably sterilization at 121 ℃ for 20 min.
In the present invention, in the step (1), the bacillus seed solution is preferably prepared by a specific method comprising: selecting 1-3 cyclosporins to be inoculated into 4-6 mL of LB broth culture medium, carrying out shake culture for 18-22 h under the conditions of 24-26 ℃ and 180-220 r/min to obtain a first-stage seed solution, pouring the first-stage seed solution into 40-60 mL of LB broth culture medium, carrying out shake culture for 18-22 h under the conditions of 24-26 ℃ and 180-220 r/min to obtain a second-stage seed solution serving as the bacillus seed solution for final fermentation, further preferably selecting 2 cyclosporins to be inoculated into 5mL of LB broth culture medium, carrying out shake culture for 20h under the conditions of 25 ℃ and 200r/min to obtain a first-stage seed solution, pouring the first-stage seed solution into 50mL of LB broth culture medium, carrying out shake culture for 20h under the conditions of 25 ℃ and 200r/min to obtain a second-stage seed solution serving as the bacillus seed solution for final fermentation;
in the step (2), the specific preparation method of the saccharomyces cerevisiae seed liquid is preferably as follows: selecting 1-3 rings of saccharomyces cerevisiae, inoculating the selected 1-3 rings of saccharomyces cerevisiae into 4-6 mL of YPD liquid culture medium, performing shaking culture for 22-26 h under the conditions of 26-30 ℃ and 160-200 r/min to obtain a first-stage seed solution, pouring the first-stage seed solution into 40-60 mL of YPD liquid culture medium, performing shaking culture for 22-26 h under the conditions of 26-30 ℃ and 160-200 r/min to obtain a second-stage seed solution serving as saccharomyces cerevisiae seed solution for final fermentation, further preferably selecting 2 rings of saccharomyces cerevisiae, inoculating the selected 2 rings of saccharomyces cerevisiae into 5mLYPD liquid culture medium, performing shaking culture for 24h under the conditions of 28 ℃ and 180r/min to obtain a first-stage seed solution, pouring the first-stage seed solution into 50mL of YPD liquid culture medium, performing shaking culture for 24h under the conditions of 28 ℃ and 180r/min to obtain a second-stage seed solution serving as saccharomyces cerevisiae seed solution for final fermentation;
in the step (3), the specific preparation method of the lactobacillus plantarum seed liquid is preferably as follows: selecting 1-3 rings of lactobacillus plantarum, inoculating the lactobacillus plantarum into 4-6 mL of MRS liquid culture medium, performing static culture at 35-40 ℃ for 18-22 h to obtain a first-stage seed solution, pouring the first-stage seed solution into 40-60 mL of MRS liquid culture medium, performing static culture at 35-40 ℃ for 18-22 h to obtain a second-stage seed solution serving as lactobacillus plantarum seed solution for final fermentation, further preferably selecting 2 rings of lactobacillus plantarum, inoculating the selected lactobacillus plantarum into 5mL of MRS liquid culture medium, performing static culture at 37 ℃ for 20h to obtain a first-stage seed solution, pouring the first-stage seed solution into 50mL of MRS liquid culture medium, and performing static culture at 37 ℃ for 20h to obtain a second-stage seed solution serving as lactobacillus plantarum seed solution for final fermentation.
In the invention, in the step (4), the compounding volume ratio of the bacillus seed liquid, the saccharomyces cerevisiae seed liquid and the lactobacillus plantarum seed liquid is preferably 1-2: 1-3: 2-3, and more preferably 1:2: 2.
The invention also provides the application of the compound microbial inoculum for detoxifying the cottonseed meal in the process of detoxifying the cottonseed meal.
In the present invention, the application process preferably includes: inoculating composite microbial inoculum for detoxifying cottonseed meal on a cottonseed meal fermentation culture medium, and then sequentially carrying out fermentation and enzymolysis.
In the invention, the components of the cottonseed meal fermentation medium are preferably 80-85 g of cottonseed meal, 9-10 g of bran, 5-10 g of brown sugar, 66-106 mL of distilled water, more preferably 84.1g of cottonseed meal, 9.4g of bran, 6.5g of brown sugar and 86mL of distilled water, the sterilization condition of the cottonseed meal fermentation medium is preferably sterilization at 120-125 ℃ for 15-25 min, more preferably sterilization at 121 ℃ for 20min, and the particle size of the cottonseed meal is preferably 55-65 meshes, and more preferably 60 meshes.
In the invention, in the fermentation process, the inoculation amount of the complex microbial inoculum is preferably 3-9% of the total volume of the complex microbial inoculum, more preferably 4-7% of the total volume of the complex microbial inoculum, more preferably 5% of the total volume of the complex microbial inoculum, the feed-water ratio is preferably 48-52 g: 40-45 mL, more preferably 50g:43mL, the fermentation temperature is preferably 30-40 ℃, more preferably 35 ℃, the fermentation time is preferably 70-75 h, and more preferably 72 h.
In the invention, the feed-water ratio refers to the weight ratio of the dry matter weight of the cottonseed meal fermentation medium to the added water.
In the invention, the enzymolysis condition is preferably natural enzymolysis for 11-13 h, and is further preferably natural enzymolysis for 12 h.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
The bacillus in the embodiments of the invention is bacillus S77 with the preservation number of CCTCC NO: M2021528, and is preserved in the China center for type culture preservation in 2021, 5 months and 13 days; the saccharomyces cerevisiae is saccharomyces cerevisiae ZY.003 which is purchased from China industrial microorganism strain preservation management center with the preservation number of CICC 1027; the lactobacillus plantarum is lactobacillus plantarum LR002 with the preservation number of CCTCC NO: M2021527, and is preserved in the China center for type culture preservation in 2021, 5 months and 13 days.
Example 1
(1) Preparation of bacillus seed liquid:
selecting 1-ring bacillus from a slant for storing strains, inoculating the 1-ring bacillus into a 50mL triangular flask containing 4mL of LB broth culture medium (the culture medium comprises 0.5g of peptone, 0.4g of yeast powder, 0.4g of sodium chloride, the pH value of 7.2 and the sterilization condition of 120 ℃ for 15min), carrying out shaking culture in a constant-temperature shaking table at 24 ℃ and 180r/min for 18h to obtain a primary seed solution, pouring the primary seed solution into a 250mL triangular flask containing 40mL of LB broth culture medium, carrying out shaking culture in the constant-temperature shaking table at 24 ℃ and 180r/min for 18h, and obtaining a secondary seed solution as the bacillus seed solution for final fermentation.
(2) Preparing a saccharomyces cerevisiae seed solution:
picking 1-ring saccharomyces cerevisiae from a slant for storing strains, inoculating the 1-ring saccharomyces cerevisiae into a 50mL triangular flask containing a 4mL LYPD liquid culture medium (the culture medium comprises 0.5g of yeast extract powder, 1.5g of peptone, 1.5g of glucose, the pH value of 7.2, and the sterilization condition is sterilization for 15min at 120 ℃), carrying out shake culture for 22h in a constant-temperature shaking table under the conditions of 26 ℃ and 160r/min to obtain a primary seed solution, pouring the primary seed solution into a 250mL triangular flask containing 40mL YPD liquid culture medium, carrying out shake culture for 22h in the constant-temperature shaking table under the conditions of 26 ℃ and 160r/min to obtain a secondary seed solution as the saccharomyces cerevisiae seed solution for final fermentation.
(3) Preparing lactobacillus plantarum seed liquid:
selecting 1-ring lactobacillus plantarum from a slant for strain preservation, inoculating the lactobacillus plantarum into a 50mL triangular flask containing 4mL of MRS liquid culture medium (the culture medium comprises 0.5g of peptone, 0.5g of beef extract, 0.4g of yeast extract powder, 1g of glucose, 0.4g of anhydrous sodium acetate, 0.1g of diammonium citrate, 800.1mL of tween-800, 0.05g of magnesium sulfate, 0.02g of manganese sulfate, the pH value of 6.2 and the sterilization condition of 120 ℃ for 15min), performing standing culture in a 35 ℃ incubator for 18h to obtain a first-stage seed solution, pouring the first-stage seed solution into a 250mL triangular flask containing 40mL of MRS liquid culture medium, and performing standing culture in a 35 ℃ incubator for 18h to obtain a second-stage seed solution serving as the lactobacillus plantarum seed solution for final fermentation.
(4) Preparing a complex microbial inoculum: and (3) compounding the obtained bacillus seed liquid for final fermentation, the saccharomyces cerevisiae seed liquid and the lactobacillus plantarum seed liquid according to the volume ratio of 1:1:2 to obtain the composite microbial inoculum.
(5) Preparing a cottonseed meal fermentation liquid culture medium:
80g of cottonseed meal, 9g of bran and 5g of brown sugar are filled in a 500mL fermentation bag, 66mL of distilled water is added, and sterilization is carried out for 15min at 120 ℃, wherein the cottonseed meal is sieved by a 55-mesh sieve.
Example 2
(1) Preparation of bacillus seed liquid:
the method comprises the steps of selecting 2-ring bacillus from a slant for storing strains, inoculating the 2-ring bacillus into a 50mL triangular flask containing 5mL of LB broth culture medium (the culture medium comprises 1g of peptone, 0.5g of yeast powder, 0.5g of sodium chloride, 7.3 of pH value and 20min of sterilization at 121 ℃), carrying out shake culture in a constant-temperature shaking table at 25 ℃ and 200r/min for 20h to obtain a primary seed solution, pouring the primary seed solution into a 250mL triangular flask containing 50mL of LB broth culture medium, carrying out shake culture in the constant-temperature shaking table at 25 ℃ and 200r/min for 20h, and taking the obtained secondary seed solution as the bacillus seed solution for final fermentation.
(2) Preparing a saccharomyces cerevisiae seed solution:
2 rings of saccharomyces cerevisiae is picked from a slant for storing strains and is inoculated into a 50mL triangular flask containing a 5mL LYPD liquid culture medium (the culture medium comprises 1g of yeast extract powder, 2g of peptone and 2g of glucose, the pH value is 7.3, the sterilization condition is sterilization for 20min at 121 ℃), the shaking culture is carried out in a constant temperature shaking table for 24h under the conditions of 28 ℃ and 180r/min to obtain a primary seed solution, the primary seed solution is poured into a 250mL triangular flask containing the 50mL LYPD liquid culture medium, the shaking culture is carried out in the constant temperature shaking table for 24h under the conditions of 28 ℃ and 180r/min, and the obtained secondary seed solution is used as the saccharomyces cerevisiae seed solution for final fermentation.
(3) Preparing lactobacillus plantarum seed liquid:
picking 2-ring lactobacillus plantarum from a slant for strain preservation, inoculating the 2-ring lactobacillus plantarum into a 50mL triangular flask containing an MRS liquid culture medium (the culture medium comprises 1g of peptone, 1g of beef extract, 0.5g of yeast extract powder, 2g of glucose, 0.5g of anhydrous sodium acetate, 0.2g of diammonium citrate, Tween-800.15 mL, 0.058g of magnesium sulfate, 0.025g of manganese sulfate, 6.3 of pH value and sterilization conditions of 121 ℃ for 20min), standing and culturing for 20h in a 37 ℃ constant temperature box to obtain a primary seed solution, pouring the primary seed solution into a 250mL triangular flask containing 50mL of MRS liquid culture medium, and standing and culturing for 20h in a 37 ℃ constant temperature box to obtain a secondary seed solution as the lactobacillus plantarum seed solution for final fermentation.
(4) Preparing a complex microbial inoculum: and (3) compounding the obtained bacillus seed liquid for final fermentation, the saccharomyces cerevisiae seed liquid and the lactobacillus plantarum seed liquid according to the volume ratio of 1:2:2 to obtain the composite microbial inoculum.
(5) Preparing a cottonseed meal fermentation liquid culture medium:
putting 84.1g of cottonseed meal, 9.4g of bran and 6.5g of brown sugar into a 500mL fermentation bag, adding 86mL of distilled water, and sterilizing at 121 ℃ for 20min, wherein the cottonseed meal is sieved by a 60-mesh sieve.
Example 3
(1) Preparation of bacillus seed liquid:
picking 3-ring bacillus from a slant for storing strains, inoculating the 3-ring bacillus into a 50mL triangular flask containing 6mL of LB broth culture medium (the culture medium comprises 1.5g of peptone, 0.6g of yeast powder, 0.6g of sodium chloride, the pH value of 7.4 and the sterilization condition of 125 ℃ for 25min), carrying out shaking culture in a constant-temperature shaking table at 26 ℃ and 220r/min for 22h to obtain a primary seed solution, pouring the primary seed solution into a 250mL triangular flask containing 60mL of LB broth culture medium, carrying out shaking culture in the constant-temperature shaking table at 26 ℃ and 220r/min for 22h, and taking the obtained secondary seed solution as the bacillus seed solution for final fermentation.
(2) Preparing a saccharomyces cerevisiae seed solution:
picking 3-ring saccharomyces cerevisiae from a slant for storing strains, inoculating the 3-ring saccharomyces cerevisiae into a 50mL triangular flask containing 6mL LYPD liquid culture medium (the culture medium comprises 1.5g of yeast extract powder, 2.5g of peptone, 2.5g of glucose, 7.4 of pH value and 25min of sterilization at 125 ℃), carrying out shake culture in a constant temperature shaking table at 30 ℃ and 200r/min for 26h to obtain a primary seed solution, pouring the primary seed solution into a 250mL triangular flask containing 60mL YPD liquid culture medium, carrying out shake culture in the constant temperature shaking table at 30 ℃ and 200r/min for 26h to obtain a secondary seed solution as the saccharomyces cerevisiae seed solution for final fermentation.
(3) Preparing lactobacillus plantarum seed liquid:
picking 3-ring lactobacillus plantarum from a slant for strain preservation, inoculating the lactobacillus plantarum into a 50mL triangular flask containing 6mL of MRS liquid culture medium (the culture medium comprises 1.5g of peptone, 1.5g of beef extract, 0.6g of yeast extract powder, 3g of glucose, 0.6g of anhydrous sodium acetate, 0.3g of diammonium citrate, tween-800.2 mL, 0.06g of magnesium sulfate, 0.03g of manganese sulfate, the pH value of 6.4 and the sterilization condition of 25min at 125 ℃), performing standing culture for 22h in a constant temperature box at 40 ℃ to obtain a primary seed solution, pouring the primary seed solution into a 250mL triangular flask containing 60mL of MRS liquid culture medium, and performing standing culture for 22h in the constant temperature box at 40 ℃ to obtain a secondary seed solution as the lactobacillus plantarum seed solution for final fermentation.
(4) Preparing a complex microbial inoculum: and (3) compounding the obtained bacillus seed liquid for final fermentation, the saccharomyces cerevisiae seed liquid and the lactobacillus plantarum seed liquid according to the volume ratio of 2:3:3 to obtain the composite microbial inoculum.
(5) Preparing a cottonseed meal fermentation solid culture medium:
85g of cottonseed meal, 10g of bran and 10g of brown sugar are filled in a 500mL fermentation bag, 106mL of distilled water is added, and the mixture is sterilized at 125 ℃ for 25min, wherein the cottonseed meal is sieved by a 65-mesh sieve.
The complex microbial inoculum and the cottonseed meal fermentation liquid culture medium obtained in example 2 are taken as examples for experiments, and the results are shown in table 1:
TABLE 1 general rotational experiment design and test results
As can be seen from Table 1, compared with the comparative example, the highest detoxification rate of gossypol can be achieved by fermenting under the fermentation conditions of 5% inoculation amount (V/W) in the experimental example 1, the feed-water ratio of 50:43, the fermentation temperature of 35 ℃ and the fermentation period of 72h, and the detoxification rate of gossypol can be up to 92.12%.
Experimental example 5
Setting the inoculation amount (V/W) to be 5%, the material-water ratio to be 50:43, the fermentation temperature to be 35 ℃, the fermentation period to be 72h and the enzymolysis time to be 12h, researching various nutrition indexes of the fermented and enzymolyzed cottonseed meal, and obtaining the results shown in Table 2:
TABLE 2 detection of various nutritional indexes of cottonseed meal fermented and enzymolyzed by composite bacteria
As can be seen from Table 2, by adopting a fermentation condition that the inoculation amount is 5 percent (V/W), the ratio of material to water is 50:43, the fermentation temperature is 35 ℃, the fermentation period is 72 hours, after 12 hours of enzymolysis, the crude protein in the cottonseed meal is increased from 44.35 percent to 54.65 percent and is increased by 23.22 percent, the acid soluble protein is increased from 3.08 percent to 4.83 percent and is increased by 56.82 percent, the crude fiber is degraded from 11.4 percent to 8.1 percent and is reduced by 28.95 percent, the restrictive amino acid lysine is increased from 1.86 percent to 2.01 percent and is increased by 8 percent, the methionine is increased from 0.5 to 0.7 and is increased by 40 percent, the total amino acid amount is integrally increased by 22.42 percent, in addition, the contents of vitamin B1, B2 and B6 are respectively increased by 66.97 percent, 329.73 percent and 321.67 percent, and the nutritional quality of the cottonseed meal is greatly improved.
Experimental example 6
Taking the composite microbial inoculum and the cottonseed meal fermentation liquid culture medium obtained in the example 2 as an example, a feeding test is carried out on mice by adopting the detoxified cottonseed meal obtained under the fermentation conditions of 5 percent of inoculation amount, 72 hours of period, 50:43 of material-water ratio and 35 ℃ of fermentation temperature.
Cleaning grade 21 days old Kunming mouse 90 (female and male half), body mass (20 +/-2) g, license number: no.11400700309252, purchased from the laboratory animal research center, Lanzhou city.
After 3 days of acclimation, the mice were randomly divided into 6 groups, each group had 15 mice, and the mice were bred in cages, namely a control group D1 (commercial grain group), a control group D2 (non-detoxified cottonseed meal group), a test group S1 (25% detoxified cottonseed meal group), a test group S2 (50% detoxified cottonseed meal group), a test group S3 (75% detoxified cottonseed meal group) and a test group S4 (100% detoxified cottonseed meal group). The composition of the experimental group and the control group of the rat food is shown in table 3.
TABLE 3 mouse diet composition
Animal feeding conditions: the temperature is 22 +/-2) DEG C, the relative humidity is 40-60%, and the illumination is controlled for 12h/12h day-night circulation. Mice were housed for 3 weeks with free access to food and water during the experiment. The experimental animals were replaced with fresh food every 1d, the general behaviour of the mice was observed daily and diarrhea and death of the mice were recorded.
The test results show that the substitution of 25% and 50% did not have significant effect on the change in body weight of the mice as the test time increased, and the average daily gain of the mice was significantly reduced by 75%, 100% and non-detoxified groups, and the results are shown in table 4.
TABLE 4 measurement of average body weight of mouse (unit: g)
Note that the same row data does not use shoulder mark letters to indicate significant difference (P < 0.05), and the same or no shoulder mark indicates insignificant difference (P > 0.05).
The effect of detoxified cottonseed meal on the weight of the organs of the mice was studied and the results are shown in table 5:
TABLE 5 Effect of detoxified cottonseed meal on weight of organs in mice
As can be seen from Table 5, there was no significant difference in liver index, spleen index and kidney index (P >0.05) among the groups of mice, indicating that the detoxified cottonseed meal had no toxic or side effects on the mice.
The research results comprehensively show that the gossypol poisoning symptoms do not appear in the addition levels of 25%, 50% and 75% of fermented detoxified cottonseed meal, wherein the growth performance of a mouse is remarkably improved, the ALT and ALT activities are reduced and a certain positive influence on improvement of the liver function of the mouse is achieved compared with a control group D2 (non-detoxified group) by an S1 group (25% of addition) and an S2 group (50% of addition), in addition, the obvious difference is avoided compared with a control group D1 (commercial grain), and the AST activity in serum of the mouse is remarkably reduced when the mouse is fed for 21 days, which shows that the composite microbial inoculum has a good effect on the gossypol detoxified cottonseed meal, and when a certain amount of the gossypol in the daily ration is replaced by a certain amount, the growth performance of the mouse and the liver transaminase index are not remarkably affected, so that the gossypol poisoning symptoms can be better replaced by the compound microbial inoculum fermented cottonseed meal as a feed protein raw material.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The composite microbial inoculum for detoxifying the cottonseed meal is characterized by comprising Bacillus, Saccharomyces cerevisiae and Lactobacillus plantarum.
2. The method for preparing the complex microbial inoculum for detoxifying the cottonseed meal as claimed in claim 1, which is characterized by comprising the following steps:
(1) adding bacillus into an LB broth culture medium, and performing shaking table shaking culture to obtain a bacillus seed solution for final fermentation;
(2) adding saccharomyces cerevisiae into the YPD liquid culture medium, and performing shaking table shaking culture to obtain a final saccharomyces cerevisiae seed liquid for fermentation;
(3) adding lactobacillus plantarum into an MRS liquid culture medium, and performing static culture to obtain lactobacillus plantarum seed liquid for final fermentation;
(4) and compounding the bacillus seed liquid, the saccharomyces cerevisiae seed liquid and the lactobacillus plantarum seed liquid for final fermentation to obtain the composite microbial inoculum.
3. The preparation method of the complex microbial inoculum for detoxifying the cottonseed meal as claimed in claim 2, wherein in the step (1), the components of the LB broth culture medium comprise peptone 0.5-1.5 g, yeast powder 0.4-0.6 g, sodium chloride 0.4-0.6 g, pH value 7.2-7.4, and sterilization condition is 120-125 ℃ for 15-25 min;
in the step (2), the YPD liquid culture medium comprises 0.5-1.5 g of yeast extract powder, 1.5-2.5 g of peptone and 1.5-2.5 g of glucose, the pH value is 7.2-7.4, and the YPD liquid culture medium is sterilized at 120-125 ℃ for 15-25 min;
in the step (3), the MRS liquid culture medium comprises 0.5-1.5 g of peptone, 0.5-1.5 g of beef extract, 0.4-0.6 g of yeast extract powder, 1-3 g of glucose, 0.4-0.6 g of anhydrous sodium acetate, 0.1-0.3 g of diammonium citrate, 800.1-0.2 mL of Tween-2, 0.05-0.06 g of magnesium sulfate and 0.02-0.03 g of manganese sulfate, the pH value is 6.2-6.4, and the MRS liquid culture medium is sterilized at 120-125 ℃ for 15-25 min.
4. The preparation method of the complex microbial inoculum for detoxication of cottonseed meal according to claim 3, wherein in the step (1), the specific preparation method of the bacillus seed solution is as follows: selecting 1-3 cyclosporins, inoculating the 1-3 cyclosporins into 4-6 mL of LB broth culture medium, carrying out shake culture for 18-22 h under the conditions of 24-26 ℃ and 180-220 r/min to obtain a first-stage seed solution, pouring the first-stage seed solution into 40-60 mL of LB broth culture medium, carrying out shake culture for 18-22 h under the conditions of 24-26 ℃ and 180-220 r/min to obtain a second-stage seed solution serving as a bacillus seed solution for final fermentation;
in the step (2), the specific preparation method of the saccharomyces cerevisiae seed liquid comprises the following steps: selecting 1-3 rings of saccharomyces cerevisiae, inoculating the saccharomyces cerevisiae into 4-6 mL of YPD liquid culture medium, carrying out shake culture for 22-26 h under the conditions of 26-30 ℃ and 160-200 r/min to obtain a first-stage seed solution, pouring the first-stage seed solution into 40-60 mL of YPD liquid culture medium, carrying out shake culture for 22-26 h under the conditions of 26-30 ℃ and 160-200 r/min to obtain a second-stage seed solution as a saccharomyces cerevisiae seed solution for final fermentation;
in the step (3), the specific preparation method of the lactobacillus plantarum seed liquid comprises the following steps: selecting 1-3 rings of lactobacillus plantarum, inoculating the lactobacillus plantarum into 4-6 mL of MRS liquid culture medium, performing static culture for 18-22 h at 35-40 ℃ to obtain first-stage seed liquid, pouring the first-stage seed liquid into 40-60 mLMRS liquid culture medium, performing static culture for 18-22 h at 35-40 ℃, and taking the obtained second-stage seed liquid as lactobacillus plantarum seed liquid for final fermentation.
5. The preparation method of the composite microbial inoculum for detoxifying cottonseed meal according to claim 4, wherein in the step (4), the compounded volume ratio of the bacillus seed solution, the saccharomyces cerevisiae seed solution and the lactobacillus plantarum seed solution is 1-2: 1-3: 2-3.
6. The use of the complex microbial inoculum of claim 1 in the process of detoxifying cottonseed meal.
7. The application of the complex microbial inoculum in the process of detoxifying cottonseed meal as claimed in claim 6, wherein the application process comprises the following steps: the composite microbial inoculum of claim 1 is inoculated on a cottonseed meal fermentation medium, and then fermentation and enzymolysis are sequentially carried out.
8. The application of the composite microbial inoculum in a cottonseed meal detoxification process according to claim 7, wherein the cottonseed meal fermentation medium comprises 80-85 g of cottonseed meal, 9-10 g of bran, 5-10 g of brown sugar and 66-106 mL of distilled water, the cottonseed meal fermentation medium is sterilized at 120-125 ℃ for 15-25 min, and the particle size of the cottonseed meal is 55-65 meshes.
9. The application of the composite microbial inoculum in the process of detoxifying cottonseed meal according to claim 8, wherein in the fermentation process, the inoculation amount of the composite microbial inoculum accounts for 3-9% of the total volume of the composite microbial inoculum, the feed-water ratio is 48-52 g: 40-45 mL, the fermentation temperature is 30-40 ℃, and the fermentation time is 70-75 h.
10. The application of the composite microbial inoculum in the detoxication process of the cottonseed meal according to claim 9, wherein the enzymolysis condition is natural enzymolysis for 11-13 h.
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| CN113234641A (en) * | 2021-06-21 | 2021-08-10 | 西北民族大学 | Bacillus S77 capable of resisting low temperature and producing protease and application thereof |
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| CN101810244A (en) * | 2010-04-29 | 2010-08-25 | 湖南农业大学 | Biological detoxification method of cottonseed meal |
| CN103355472A (en) * | 2013-01-21 | 2013-10-23 | 上海创博生态工程有限公司 | Microbial fermentation agent for cottonseed meal detoxification and preparation method and application thereof |
| CN103525724A (en) * | 2013-09-23 | 2014-01-22 | 上海创博现代自然农业（集团）有限公司 | Microbial leavening agent of cottonseed meal as well as preparation method thereof |
| CN105255793A (en) * | 2015-11-20 | 2016-01-20 | 江南大学 | Lactobacillus plantarum capable of degrading gossypol and application of lactobacillus plantarum |
| CN105543131A (en) * | 2015-12-30 | 2016-05-04 | 中粮饲料有限公司 | Compound bacteria/cottonseed meal fermented feed and preparation method thereof |
| CN106615933A (en) * | 2016-12-15 | 2017-05-10 | 郑州禾丰牧业有限公司 | Premix mate for laying hens in egg producing period and preparation and application of premix mate |
| CN111184128A (en) * | 2018-11-14 | 2020-05-22 | 宁夏健力肽生物科技有限公司 | Method for producing multienzyme feed by solid fermentation |
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| CN113186139A (en) * | 2021-06-21 | 2021-07-30 | 西北民族大学 | Lactobacillus plantarum LR002 and application thereof |
| CN113234641A (en) * | 2021-06-21 | 2021-08-10 | 西北民族大学 | Bacillus S77 capable of resisting low temperature and producing protease and application thereof |
| CN113186139B (en) * | 2021-06-21 | 2022-06-07 | 西北民族大学 | Lactobacillus plantarum LR002 and application thereof |
| CN113234641B (en) * | 2021-06-21 | 2022-07-19 | 西北民族大学 | Bacillus S77 capable of resisting low temperature and producing protease and application thereof |
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