CN103719537A - Nonreactive biological fermented feed and preparation method thereof - Google Patents
Nonreactive biological fermented feed and preparation method thereof Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fodder In General (AREA)
Abstract
The invention discloses a preparation method of a nonreactive biological fermented feed, which is characterized by comprising the following steps: 1, preparing mixed seed liquid, i.e. after mixing seed culture solution of brevibacterium lactofermentum-QF-1 and seed culture solution of candida utilis QF-2, inoculating mixed solution into a mixed seed culture medium and culturing to obtain the mixed seed liquid; 2, preparing a mixed fermentation medium, i.e. sterilizing a feed raw material for later use; 3, carrying out segmental fermentation, i.e. firstly carrying out aerobic fermentation and then carrying out anaerobic fermentation. The preparation method has the benefits that two microorganisms of brevibacterium lactofermentum-QF-1 and candida utilis QF-2 are adopted to carry out mixed fermentation to process the raw material, so that not only is the total protein and amino acid content in the feed increased, but also non-starch substances such as celluloses and the like are decomposed and the biological value of the nonreactive biological fermented feed is greatly improved; various strains are adopted to carry out mixed fermentation so as to achieve the aims of treating bacteria by bacteria, treating diseases by bacteria, and promoting growth and disease resistance of livestocks, and effectively solve the problems of antibiotic residues and drug resistance of pathogenic bacterial strains.
Description
Technical Field
The invention relates to a feed and a preparation method thereof, in particular to a method for treating a mixture of various protein raw materials by adopting mixed fermentation of lactic acid bacteria and saccharomycetes, and an antibiotic-free biological fermented feed directly obtained by the method.
Background
In the production of the traditional feed, bran, corn, wheat, bean pulp, cottonseed meal, rapeseed meal and the like are used as raw materials, and because the raw materials cannot provide antibiotics, the antibiotics are usually required to be added into the feed in order to improve the disease prevention and disease resistance of livestock and poultry; it is well known that antibiotics also have growth promoting effects on animals, and therefore, antibiotics are widely used in the production of traditional feeds.
The application of antibiotics in feed will bring great disasters to human health due to the problems of residues and drug resistance of pathogenic strains. Therefore, the research of antibiotic-free fermented feed at home and abroad is continuously carried out.
At present, the microorganisms mainly used for producing mycoprotein at home are reported to be aspergillus, rhizopus, candida, lactobacillus, streptococcus lactis, bacillus subtilis, lysine producing bacteria, endoplasmic reticulum, geotrichum candidum and the like. The strains bred at present comprise:
(1) yeast: the yeast has strong acid resistance, large size, and easy recovery, and the commonly used yeasts include Candida tropicalis, Candida utilis, Trichosporon dermatomyces, and dendritic yeast.
(2) Actinomycetes: the actinomycetes have strong capability of decomposing cellulose, can directly utilize fiber raw materials to produce single-cell protein, and commonly used actinomycetes comprise nocardia and high-temperature actinomycetes.
(3) And (3) mould: the mould can generate high-activity cellulase to degrade cellulose in the feed raw materials, and the common mould is geotrichum candidum, aspergillus niger, trichoderma koningii and trichoderma viride.
(4) Basidiomycete, and the like. The method is mainly used for producing edible fungi and the like.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the antibiotic-free fermented feed which does not contain antibiotics and has the effects of treating bacteria by bacteria, treating diseases by bacteria, promoting the growth of livestock and poultry and resisting diseases, and the preparation method of the antibiotic-free fermented feed.
In order to achieve the above object, the present invention adopts the following technical solutions:
the preparation method of the antibiotic-free fermented feed is characterized by comprising the following steps of:
firstly, preparing mixed liquid:
(1) the preservation number of Brevibacterium lactofermentum-QF-1 is CCTCC NO: M2013453, the preservation date is 2013, 9 and 28 months, the preservation unit is: china center for type culture Collection, Collection Unit Address: the university of Wuhan, China is cultured in lactobacillus liquid culture medium at 37 deg.C to obtain cell concentration>109CFU/mL to obtain a third-level seed culture solution of the fermentation Brevibacterium lactofermentum;
(2) the preservation number of the Candida utilis (Candida utilis QF-2) is CCTCC NO: M2013454, the preservation date is 2013, 9 and 28 days, and the preservation unit is as follows: china center for type culture Collection, Collection Unit Address: the Wuhan university, Wuhan, China is cultured in malt juice liquid culture medium at 28 deg.C to obtain cell concentration>109CFU/mL to obtain a third-level seed culture solution of the candida utilis;
(3) and mixing the three-level seed culture solution of the fermented lactobacillus brevis and the three-level seed culture solution of the candida utilis according to the volume ratio of 1: mixing the materials in a ratio of 0.01-1 to obtain a mixed seed culture solution;
(4) and mixing the mixed seed culture solution according to the volume ratio of 1:10 in the mixed seed culture medium, culturing at 28-30 ℃ until the cell concentration is reached>109CFU/mL to obtain mixed liquid;
secondly, preparing a mixed fermentation medium:
(1) drying or squeezing the feed raw materials until the water content is 25-35% to obtain a mixed fermentation culture medium;
the feed raw materials comprise: bran, corn, wheat, rice bran, bean pulp, cottonseed meal, rapeseed meal, bean curd residue and the like, which can be used for fermenting a single raw material or fermenting a mixture of a plurality of raw materials according to specific conditions;
(2) adding CaCO to the mixed fermentation medium3；
(3) Adding water to adjust the humidity of the mixed fermentation medium to 50-60%;
(4) natural pH value, sterilizing for later use;
thirdly, a step of sectional fermentation:
(1) inoculating the mixed liquid into a mixed fermentation culture medium according to the volume ratio of 1: 10;
(2) firstly carrying out aerobic fermentation for 12h at the temperature of 30-32 ℃, then carrying out anaerobic fermentation for 48-72 h at the temperature of 35-37 ℃, and when the pH of the mixture is reduced to a stable state of 3.5-4.0, and the number of thalli reaches 107～108Stopping fermentation when the concentration of the carbon fiber is CFU/g;
(3) and drying the fermentation product at 40-62 ℃, and packaging to obtain a finished product.
The preparation method of the antibiotic-free fermented feed is characterized in that the preparation of the lactic acid bacteria liquid culture medium is as follows:
sterilizing under 666.4KPa pressure for 30 min.
The preparation method of the antibiotic-free fermented feed is characterized in that the wort liquid culture medium is prepared as follows:
diluting the raw material of fermented beer without hops to 12 Brix, and sterilizing at 666.4KPa pressure for 30 min.
The preparation method of the antibiotic-free biological fermentation feed is characterized in that the mixed seed culture medium is prepared as follows:
corn starch 5g urea 1g
CaHPO40.5g of distilled water was diluted to 100mL
Sterilizing at natural pH value of 666.4KPa for 30 min.
The preparation method of the antibiotic-free fermented feed is characterized in that the three-stage seed culture step comprises the following steps:
(1) inoculating the fermented lactobacillus brevis/candida utilis in 5mL of a lactobacillus liquid culture medium/malt wort liquid culture medium, and culturing for 16h to obtain a primary seed culture solution;
(2) inoculating 5mL of the primary seed culture solution into 100mL of a lactic acid bacteria liquid medium/wort liquid medium, culturing for 16h, and culturing to a cell concentration>109CFU/mL to obtain secondary seed culture solution;
(3) inoculating 100mL of secondary seed culture solution into 10L of lactobacillus liquid culture medium/malt juice liquid culture medium, culturing for 16h, and culturing to cell concentration>109CFU/mL to obtain a third-level seed culture solution of the fermented Brevibacterium lactofermentum/Candida utilis.
The preparation method of the antibiotic-free fermented feed is characterized in that the volume ratio of the three-stage seed culture solution of the fermented lactobacillus brevis to the three-stage seed culture solution of the candida utilis is 1: mixing at a ratio of 0.1.
The preparation method of the antibiotic-free fermented feed is characterized in that the mixed fermentation medium is also added with CaHPO4Any one of the mineral additive for feeding and the vitamin for feeding, or a mixture of any two of the mineral additive for feeding and the vitamin for feeding, or the three of the mineral additive for feeding and the vitamin for feeding are added simultaneously.
The preparation method of the antibiotic-free biological fermentation feed is characterized in that the feed mineral additive comprises the following raw materials in parts by weight:
the preparation method of the antibiotic-free fermented feed is characterized in that the sterilization method of the mixed fermentation medium comprises the following steps: and (3) intermittently sterilizing 2-3 times at 105 ℃ by steam, 1 hour each time, and 3-4 hours each time intermittently.
An antibiotic-free biological fermentation feed, which is characterized by being prepared by the method.
The invention has the advantages that:
1. the invention adopts two kinds of microorganisms of fermented Brevibacterium lactofermentum and Candida utilis to carry out mixed fermentation treatment on bran, corn, wheat, rice bran, bean pulp, cottonseed meal, rapeseed meal, bean curd residue and the like, thereby not only increasing the total protein content in the feed, increasing the crude protein content to more than 24% from 13% before fermentation, but also increasing the amino acid content to more than 16%, simultaneously decomposing non-starch substances such as cellulose and the like, reducing the cellulose content of the finished feed product to about 2.4%, and greatly improving the biological value of the antibiotic-free fermented feed;
2. the multi-strain mixed fermentation not only gives full play to the inherent nutritive value of the feed, but also realizes the aims of treating bacteria by bacteria, treating diseases by bacteria, promoting the growth and disease resistance of livestock and poultry, and effectively solves the problems of antibiotic residue and the drug resistance of pathogenic strains in the feed;
3. in the fermentation process, a sectional fermentation mode of aerobic fermentation and anaerobic fermentation is adopted, so that glucosinolates in protein raw materials such as rapeseed meal, cottonseed meal and the like are effectively removed, and in the finally obtained feed finished product, the content of the glucosinolates is lower than 0.01%, the content of isothiocyanates is lower than 0.045%, the content of oxazolidinethione is lower than 0.025%, and the feed safety requirement is met;
4. the gossypol in protein raw materials such as cottonseed meal and the like is effectively removed through the combined action of aerobic fermentation and anaerobic fermentation, and the content of the gossypol in the finally obtained feed finished product is lower than 0.01 percent and meets the feed safety requirement: namely, the content of gossypol is less than 0.02 percent;
5. the production conditions are simple, so that the pollution can be prevented, and the resources can be utilized to the maximum extent;
6. the feed prepared by the method has good palatability, no antibiotics and toxic substances, good feeding effect and safe eating.
Detailed Description
The present invention will be described in detail with reference to the following embodiments.
Example 1
Firstly, preparing a culture medium:
1. lactic acid bacteria liquid culture medium: yeast extract 7.5g, peptone 7.5g, glucose 10g, KH2PO42g, tomato juice 100mL, tween-800.5 mL and distilled water 900mL, adjusting the pH value to 7.0, and sterilizing for 30min under 666.4KPa pressure.
2. Wort liquid culture medium: diluting the raw material of fermented beer without hops to 12 Brix, and sterilizing at 666.4KPa for 30 min.
3. Mixing seed culture media: corn starch 5g, urea 1g, CaHPO4Diluting 0.5g and distilled water to 100mL, naturally adjusting pH, and sterilizing under 666.4KPa pressure for 30 min.
4. Mixing fermentation culture media: drying bran, semen Maydis, semen Tritici Aestivi, testa oryzae, bean cake, cotton cake, vegetable cake, and bean curd residue at 45 deg.C until the water content is 30%, mixing, and adding 0.1% (by weight) CaCO3、0.5%CaHPO4Adding water to adjust the humidity of the mixed fermentation medium to 50%, naturally sterilizing at 105 deg.C for 2 times with steam interval of 1 hr each time and 4 hr in the middle.
II, obtaining liquid:
1. fermenting a culture solution of a third-level seed of the Brevibacterium lactofermentum: placing fermented Brevibacterium lactofermentum in lactobacillus liquid culture medium, performing three-stage seed culture at 37 deg.C until cell concentration is reached>109CFU/mL. In particular, the method comprises the following steps of,
(1) selecting 2-3 inoculating loops of the thalli, inoculating the thalli into 5mL of lactobacillus liquid culture medium, and culturing at 220rpm and 37 ℃ for 16 hours to obtain a primary seed culture solution;
(2) inoculating 5mL of overnight-cultured primary seed culture medium into 100mL of lactic acid bacteria liquid medium, culturing at 37 ℃ for 16 hours at 220rpm, and culturing to cell concentration>109CFU/mL to obtain secondary seed culture solution;
(3) inoculating 100mL of secondary seed culture solution into 10L of lactobacillus liquid culture medium, culturing at 220rpm and 37 ℃ for 16 hours until the cell concentration is reached>109CFU/mL to obtain a third-level seed culture solution of the fermentation Brevibacterium lactofermentum.
2. Candida utilis third-level seed culture solution: placing Candida utilis in malt wort liquid culture medium, performing three-stage seed culture at 28 deg.C, and culturing to cell concentration>109CFU/mL to obtain a third-level seed culture solution of the candida utilis. In particular, the method comprises the following steps of,
(1) selecting 2-3 inoculating loops of thalli, inoculating the thalli into 5mL of malt wort liquid culture medium, and culturing at 220rpm and 28 ℃ for 16 hours to obtain a primary seed culture solution;
(2) inoculating 5mL of overnight-cultured primary seed culture medium into 100mL of malt wort liquid medium, culturing at 28 ℃ for 16 hours at 220rpm, and culturing to cell concentration>109CFU/mL to obtain secondary seed culture solution;
(3) inoculating 100mL of the secondary seed culture medium into 10L of malt wort liquid culture medium, culturing at 28 ℃ for 16 hours at 220rpm, and culturing to a cell concentration>109CFU/mL to obtain a third-level seed culture solution of the candida utilis.
3. Mixing seed culture solution: mixing the three-level seed culture solution of the fermentation Brevibacterium lactofermentum and the three-level seed culture solution of the Candida utilis according to the volume ratio of 1: 0.5.
4. Mixing liquid: mixing the mixed seed culture solution according to a volume ratio of 1:10 is inoculated into a mixed seed culture medium, the culture temperature is 30 ℃, the stirring speed is 220rpm, the culture is carried out for 48 hours, and the culture is carried out until the cell concentration is reached>109CFU/mL。
Thirdly, segmented fermentation:
1. inoculating the mixed liquid into a mixed fermentation culture medium according to the volume ratio of 1: 10.
2. Aerobic fermentation is carried out for 12 hours at the temperature of 30 ℃.
3. Then anaerobic fermenting at 35 deg.C for 60 hr, when pH of the mixture is reduced to 4.0 stable state, and the thallus number reaches 107～108And (5) stopping fermentation at CFU/g.
4. Drying the fermented product at 52 deg.C, and packaging to obtain the final product.
The main technical indexes of the product are as follows:
microorganism content 107～108CFU/g，pH<4.0, more than 24 percent of protein, more than 16 percent of amino acid, about 2.4 percent of cellulose and less than 0.01 percent of glucosinolate.
Example 2
Firstly, preparing a culture medium:
1. lactic acid bacteria liquid culture medium: yeast extract 7.5g, peptone 7.5g, glucose 10g, KH2PO42g, tomato juice 100mL, tween-800.5 mL and distilled water 900mL, adjusting the pH value to 7.0, and sterilizing for 30min under 666.4KPa pressure.
2. Wort liquid culture medium: diluting the raw material of fermented beer without hops to 12 Brix, and sterilizing at 666.4KPa for 30 min.
3. Mixing seed culture media: corn starch 5g, urea 1g, CaHPO4Diluting 0.5g and distilled water to 100mL, naturally adjusting pH, and sterilizing under 666.4KPa pressure for 30 min.
4. Mixing fermentation culture media: drying bran, semen Maydis, semen Tritici Aestivi, testa oryzae, bean cake, cotton cake, vegetable cake, and bean curd residue at 45 deg.C to water content of 35%, mixing, and adding 0.1% (by weight) CaCO3Adding 0.1% feeding mineral additive, adding water to adjust the humidity of the mixed fermentation medium to 60%, naturally adjusting pH, and intermittently sterilizing with 105 deg.C steam for 3 times, 1 hr each time, and 4 hr in the middle. Wherein,
the feed mineral additive comprises the following raw materials in parts by weight:
CuSO4·5H2o24 parts, FeSO4·7H2O250 parts, ZnSO4·H2O200 parts and MnSO4200 parts of KI5 parts with the mass percent concentration of 5 percent and Na with the mass percent concentration of 1 percent2SeO318 parts of zeolite powder (carrier) 303 parts;
the preparation method comprises the following steps: first 212.1 partsAdding zeolite powder into a mixer, and then adding CuSO4·5H2O、FeSO4·7H2O、ZnSO4·H2O、MnSO4、KI、Na2SeO3Finally, the rest zeolite powder is added and mixed evenly to obtain the catalyst.
II, obtaining liquid:
1. fermenting a culture solution of a third-level seed of the Brevibacterium lactofermentum: placing fermented Brevibacterium lactofermentum in lactobacillus liquid culture medium, performing three-stage seed culture at 37 deg.C until cell concentration is reached>109CFU/mL. In particular, the method comprises the following steps of,
(1) selecting 2-3 inoculating loops of the thalli, inoculating the thalli into 5mL of lactobacillus liquid culture medium, and culturing at 220rpm and 37 ℃ for 16 hours to obtain a primary seed culture solution;
(2) inoculating 5mL of overnight-cultured primary seed culture medium into 100mL of lactic acid bacteria liquid medium, culturing at 37 ℃ for 16 hours at 220rpm, and culturing to cell concentration>109CFU/mL to obtain secondary seed culture solution;
(3) inoculating 100mL of secondary seed culture solution into 10L of lactobacillus liquid culture medium, culturing at 220rpm and 37 ℃ for 16 hours until the cell concentration is reached>109CFU/mL to obtain a third-level seed culture solution of the fermentation Brevibacterium lactofermentum.
2. Candida utilis third-level seed culture solution: placing Candida utilis in malt wort liquid culture medium, performing three-stage seed culture at 28 deg.C, and culturing to cell concentration>109CFU/mL to obtain a third-level seed culture solution of the candida utilis. In particular, the method comprises the following steps of,
(1) selecting 2-3 inoculating loops of thalli, inoculating the thalli into 5mL of malt wort liquid culture medium, and culturing at 220rpm and 28 ℃ for 16 hours to obtain a primary seed culture solution;
(2) 5mL of overnight-cultured primary seed culture medium was inoculated into 100mL of wort liquid medium and cultured at 28 ℃ at 220rpmCulturing for 16 hours until the cell concentration is reached>109CFU/mL to obtain secondary seed culture solution;
(3) inoculating 100mL of the secondary seed culture medium into 10L of malt wort liquid culture medium, culturing at 28 ℃ for 16 hours at 220rpm, and culturing to a cell concentration>109CFU/mL to obtain a third-level seed culture solution of the candida utilis.
3. Mixing seed culture solution: mixing the three-level seed culture solution of the fermentation Brevibacterium lactofermentum and the three-level seed culture solution of the Candida utilis according to the volume ratio of 1: 1.
4. Mixing liquid: mixing the mixed seed culture solution according to a volume ratio of 1:10 is inoculated into a mixed seed culture medium, the culture temperature is 28 ℃, the stirring speed is 220rpm, the culture is carried out for 50h, and the culture is carried out until the cell concentration is reached>109CFU/mL。
Thirdly, segmented fermentation:
1. inoculating the mixed liquid into a mixed fermentation culture medium according to the volume ratio of 1: 10.
2. Aerobic fermentation is carried out for 12 hours at the temperature of 30 ℃.
3. Then anaerobic fermenting at 35 deg.C for 60 hr, when pH of the mixture is reduced to 4.0, and the thallus number reaches 107～108And (5) stopping fermentation at CFU/g.
4. Drying the fermented product at 40 deg.C, and packaging to obtain the final product.
The main technical indexes of the product are as follows:
microorganism content 107～108CFU/g，pH<4.0, more than 24 percent of protein, more than 16 percent of amino acid, about 2.4 percent of cellulose and less than 0.01 percent of glucosinolate.
Example 3
Firstly, preparing a culture medium:
1. lactic acid bacteria liquid culture medium: yeast extract 7.5g, peptone 7.5g, glucose 10g, KH2PO42g, tomato juice 100mL, tween-800.5 mL and distilled water 900mL, adjusting the pH value to 7.0, and sterilizing for 30min under 666.4KPa pressure.
2. Wort liquid culture medium: diluting the raw material of fermented beer without hops to 12 Brix, and sterilizing at 666.4KPa for 30 min.
3. Mixing seed culture media: corn starch 5g, urea 1g, CaHPO4Diluting 0.5g and distilled water to 100mL, naturally adjusting pH, and sterilizing under 666.4KPa pressure for 30 min.
4. Mixing fermentation culture media: drying bran, semen Maydis, semen Tritici Aestivi, testa oryzae, bean cake, cotton cake, vegetable cake, and bean curd residue at 55 deg.C to water content of 25%, mixing, and adding 0.1% (by weight) CaCO30.1% of feeding vitamin (purchased from Beijing Junde co-creation company, trade name is universal multivitamin for pigs), adding water to adjust the humidity of the mixed fermentation medium to 55%, naturally adjusting pH value, sterilizing 3 times with 105 deg.C steam intermittently, 1h each time, and 4h intermittently.
II, obtaining liquid:
1. fermenting a culture solution of a third-level seed of the Brevibacterium lactofermentum: placing fermented Brevibacterium lactofermentum in lactobacillus liquid culture medium, performing three-stage seed culture at 37 deg.C until cell concentration is reached>109CFU/mL. In particular, the method comprises the following steps of,
(1) selecting 2-3 inoculating loops of the thalli, inoculating the thalli into 5mL of lactobacillus liquid culture medium, and culturing at 220rpm and 37 ℃ for 16 hours to obtain a primary seed culture solution;
(2) inoculating 5mL of overnight-cultured primary seed culture medium into 100mL of lactic acid bacteria liquid medium, culturing at 37 ℃ for 16 hours at 220rpm, and culturing to cell concentration>109CFU/mL to obtain secondary seed culture solution;
(3) then, 100mL of the secondary seed culture medium was inoculated into 10L of lactic acidCulturing in liquid medium at 220rpm and 37 deg.C for 16 hr until cell concentration>109CFU/mL to obtain the third-level seed culture solution of Brevibacterium fermentum.
2. Candida utilis third-level seed culture solution: placing Candida utilis in malt wort liquid culture medium, performing three-stage seed culture at 28 deg.C, and culturing to cell concentration>109CFU/mL to obtain a third-level seed culture solution of the candida utilis. In particular, the method comprises the following steps of,
(1) selecting 2-3 inoculating loops of thalli, inoculating the thalli into 5mL of malt wort liquid culture medium, and culturing at 220rpm and 28 ℃ for 16 hours to obtain a primary seed culture solution;
(2) inoculating 5mL of overnight-cultured primary seed culture medium into 100mL of malt wort liquid medium, culturing at 28 ℃ for 16 hours at 220rpm, and culturing to cell concentration>109CFU/mL to obtain secondary seed culture solution;
(3) inoculating 100mL of the secondary seed culture medium into 10L of malt wort liquid culture medium, culturing at 28 ℃ for 16 hours at 220rpm, and culturing to a cell concentration>109CFU/mL to obtain a third-level seed culture solution of the candida utilis.
3. Mixing seed culture solution: mixing the three-level seed culture solution of the fermentation Brevibacterium lactofermentum and the three-level seed culture solution of the Candida utilis according to the volume ratio of 1: 0.01.
4. Mixing liquid: mixing the mixed seed culture solution according to a volume ratio of 1:10 is inoculated into a mixed seed culture medium, the culture temperature is 30 ℃, the stirring speed is 300rpm, the culture is carried out for 72 hours, and the culture is carried out until the cell concentration is reached>109CFU/mL。
Thirdly, segmented fermentation:
1. inoculating the mixed liquid into a mixed fermentation culture medium according to the volume ratio of 1: 10.
2. Aerobic fermentation is carried out for 12 hours at the temperature of 30 ℃.
3. Then anaerobic fermenting at 35 deg.C for 60 hr, when pH of the mixture is reduced to 4.0, and the thallus number reaches 107～108And (5) stopping fermentation at CFU/g.
4. Drying the fermented product at 60 deg.C, and packaging to obtain the final product.
The main technical indexes of the product are as follows:
microorganism content 107～108CFU/g，pH<4.0, more than 24 percent of protein, more than 16 percent of amino acid, about 2.4 percent of cellulose and less than 0.01 percent of glucosinolate.
Example 4
Firstly, preparing a culture medium:
1. lactic acid bacteria liquid culture medium: yeast extract 7.5g, peptone 7.5g, glucose 10g, KH2PO42g, tomato juice 100mL, tween-800.5 mL and distilled water 900mL, adjusting the pH value to 7.0, and sterilizing for 30min under 666.4KPa pressure.
2. Wort liquid culture medium: diluting the raw material of fermented beer without hops to 12 Brix, and sterilizing at 666.4KPa for 30 min.
3. Mixing seed culture media: corn starch 5g, urea 1g, CaHPO4Diluting 0.5g and distilled water to 100mL, naturally adjusting pH, and sterilizing under 666.4KPa pressure for 30 min.
4. Mixing fermentation culture media: drying bran, semen Maydis, semen Tritici Aestivi, testa oryzae, bean cake, cotton cake, vegetable cake, and bean curd residue at 45 deg.C to water content of 32%, mixing, and adding 0.1% (by weight) CaCO30.1% of feeding vitamin (purchased from Beijing Junde co-creation company, trade name is universal multivitamin for pigs), adding water to adjust the humidity of the mixed fermentation medium to 50%, naturally adjusting pH value, sterilizing intermittently with 105 deg.C steam for 2 times, 1h each time, and 3h in the middle.
II, obtaining liquid:
1. fermenting a culture solution of a third-level seed of the Brevibacterium lactofermentum: placing fermented Brevibacterium lactofermentum in lactobacillus liquid culture medium, performing three-stage seed culture at 37 deg.C until cell concentration is reached>109CFU/mL. In particular, the method comprises the following steps of,
(1) selecting 2-3 inoculating loops of the thalli, inoculating the thalli into 5mL of lactobacillus liquid culture medium, and culturing at 220rpm and 37 ℃ for 16 hours to obtain a primary seed culture solution;
(2) inoculating 5mL of overnight-cultured primary seed culture medium into 100mL of lactic acid bacteria liquid medium, culturing at 37 ℃ for 16 hours at 220rpm, and culturing to cell concentration>109CFU/mL to obtain secondary seed culture solution;
(3) inoculating 100mL of secondary seed culture solution into 10L of lactobacillus liquid culture medium, culturing at 220rpm and 37 ℃ for 16 hours until the cell concentration is reached>109CFU/mL to obtain a third-level seed culture solution of the fermentation Brevibacterium lactofermentum.
2. Candida utilis third-level seed culture solution: placing Candida utilis in malt wort liquid culture medium, performing three-stage seed culture at 28 deg.C, and culturing to cell concentration>109CFU/mL to obtain a third-level seed culture solution of the candida utilis. In particular, the method comprises the following steps of,
(1) selecting 2-3 inoculating loops of thalli, inoculating the thalli into 5mL of malt wort liquid culture medium, and culturing at 220rpm and 28 ℃ for 16 hours to obtain a primary seed culture solution;
(2) inoculating 5mL of overnight-cultured primary seed culture medium into 100mL of malt wort liquid medium, culturing at 28 ℃ for 16 hours at 220rpm, and culturing to cell concentration>109CFU/mL to obtain secondary seed culture solution;
(3) inoculating 100mL of the secondary seed culture medium into 10L of malt wort liquid culture medium, culturing at 28 ℃ for 16 hours at 220rpm, and culturing to a cell concentration>109CFU/mL to obtain a third-level seed culture solution of the candida utilis.
3. Mixing seed culture solution: mixing the three-level seed culture solution of the fermentation Brevibacterium lactofermentum and the three-level seed culture solution of the Candida utilis according to the volume ratio of 1: 0.1.
4. Mixing liquid: mixing the mixed seed culture solution according to a volume ratio of 1:10 is inoculated into a mixed seed culture medium, the culture temperature is 30 ℃, the stirring speed is 220rpm, the culture is carried out for 55 hours, and the culture is carried out until the cell concentration is reached>109CFU/mL。
Thirdly, segmented fermentation:
1. inoculating the mixed liquid into a mixed fermentation culture medium according to the volume ratio of 1: 10.
2. Aerobic fermentation is carried out for 12 hours at the temperature of 30 ℃.
3. Then anaerobic fermenting at 35 deg.C for 60 hr, when pH of the mixture is reduced to 4.0, and the thallus number reaches 107～108And (5) stopping fermentation at CFU/g.
4. Drying the fermented product at 40-50 deg.C, and packaging to obtain the final product.
The main technical indexes of the product are as follows:
microorganism content 107～108CFU/g，pH<4.0, more than 24 percent of protein, more than 16 percent of amino acid, about 2.4 percent of cellulose and less than 0.01 percent of glucosinolate.
In summary, the method of the present invention has the following advantages:
1. the production conditions are simple, so that the pollution can be prevented, and the resources can be utilized to the maximum extent;
2. the fermented Brevibacterium lactofermentum and Candida utilis are mixed and fermented to treat bran, corn, wheat, rice bran, bean pulp, cottonseed meal, rapeseed meal, bean curd residue and the like, so that the total protein content in the feed is increased, the content of crude protein is increased to more than 24% from 13% before fermentation, the content of amino acid is increased to more than 16%, non-starch substances such as cellulose are decomposed, the cellulose content of a feed finished product is reduced to about 2.4%, and the biological value of the antibiotic-free fermented feed is greatly improved;
3. the feed prepared by the method has good palatability, no antibiotics and toxic substances, good feeding effect and safe eating.
It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.
Claims (10)
1. The preparation method of the antibiotic-free biological fermentation feed is characterized by comprising the following steps:
firstly, preparing mixed liquid:
(1) placing the fermented lactobacillus Brevibacterium with the preservation number of CCTCC NO: M2013453 in a lactobacillus liquid culture medium, carrying out three-stage seed culture at 37 ℃, and culturing until the cell concentration is reached>109CFU/mL to obtain a third-level seed culture solution of the fermentation Brevibacterium lactofermentum;
(2) the preservation number of the candida utilis is CCTCC NO: M2013454, placing in malt liquid culture medium, performing three-stage seed culture at 28 deg.C, and culturing to cell concentration>109CFU/mL to obtain a third-level seed culture solution of the candida utilis;
(3) and mixing the three-level seed culture solution of the fermented lactobacillus brevis and the three-level seed culture solution of the candida utilis according to the volume ratio of 1: mixing the materials in a ratio of 0.01-1 to obtain a mixed seed culture solution;
(4) and mixing the mixed seed culture solution according to the volume ratio of 1:10 in the mixed seed culture medium, culturing at 28-30 ℃ until the cell concentration is reached>109CFU/mL to obtain mixed liquid;
secondly, preparing a mixed fermentation medium:
(1) drying or squeezing the feed raw materials until the water content is 25-35% to obtain a mixed fermentation culture medium;
(2) adding CaCO to the mixed fermentation medium3；
(3) Adding water to adjust the humidity of the mixed fermentation medium to 50-60%;
(4) natural pH value, sterilizing for later use;
thirdly, a step of sectional fermentation:
(1) inoculating the mixed liquid into a mixed fermentation culture medium according to the volume ratio of 1: 10;
(2) firstly carrying out aerobic fermentation for 12h at the temperature of 30-32 ℃, then carrying out anaerobic fermentation for 48-72 h at the temperature of 35-37 ℃, and when the pH of the mixture is reduced to a stable state of 3.5-4.0, and the number of thalli reaches 107～108Stopping fermentation when the concentration of the carbon fiber is CFU/g;
(3) and drying the fermentation product at 40-62 ℃, and packaging to obtain a finished product.
2. The method for preparing a non-antibiotic fermented feed according to claim 1, wherein the liquid culture medium of lactic acid bacteria is prepared as follows:
3. the method for preparing a non-antibiotic fermented feed according to claim 1, wherein the wort liquid culture medium is prepared as follows:
diluting the raw material of fermented beer without hops to 12 Brix, and sterilizing at 666.4KPa for 30 min.
4. The method for preparing a non-antibiotic fermented feed according to claim 1, wherein the mixed seed culture medium is prepared by:
corn starch 5g urea 1g
CaHPO40.5g of distilled water was diluted to 100mL
Sterilizing at natural pH value of 666.4KPa for 30 min.
5. The method for preparing a fermented feed without antibiotic according to claim 1, wherein the step of tertiary seed cultivation is:
(1) inoculating the fermented lactobacillus brevis/candida utilis in 5mL of a lactobacillus liquid culture medium/malt wort liquid culture medium, and culturing for 16h to obtain a primary seed culture solution;
(2) inoculating 5mL of the primary seed culture solution into 100mL of a lactic acid bacteria liquid medium/wort liquid medium, culturing for 16h, and culturing to a cell concentration>109CFU/mL to obtain secondary seed culture solution;
(3) inoculating 100mL of secondary seed culture solution into 10L of lactobacillus liquid culture medium/malt wort liquid culture medium, culturing for 16h, and culturing to cell concentration>109CFU/mL to obtain a third-level seed culture solution of the fermented Brevibacterium lactofermentum/Candida utilis.
6. The method for preparing a fermented feed without antibiotic according to claim 1, wherein the volume ratio of the tertiary seed culture solution of Brevibacterium lactofermentum to the tertiary seed culture solution of Candida utilis is 1: mixing at a ratio of 0.1.
7. The method for preparing a non-antibiotic fermented feed according to claim 1, wherein CaHPO is further added to the mixed fermentation medium4Any one of the mineral additive for feeding and the vitamin for feeding, or a mixture of any two of the mineral additive for feeding and the vitamin for feeding, or the three of the mineral additive for feeding and the vitamin for feeding are added simultaneously.
8. The preparation method of the antibiotic-free fermented feed according to claim 7, wherein the feed mineral additive comprises the following raw materials in parts by weight:
9. the method for preparing the antibiotic-free fermented feed according to claim 1, wherein the sterilization method of the mixed fermentation medium is: and (3) intermittently sterilizing 2-3 times at 105 ℃ by steam, 1 hour each time, and 3-4 hours each time intermittently.
10. A non-antibiotic fermented feed, prepared by the method of any one of claims 1 to 9.
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| CN104824363A (en) * | 2015-05-28 | 2015-08-12 | 河南双成生物科技有限公司 | Production method of high protein feed rich in threonine |
| CN104824364A (en) * | 2015-05-28 | 2015-08-12 | 河南双成生物科技有限公司 | Production method of functional protein feed rich in five essential amino-acids |
| CN104920789A (en) * | 2015-05-28 | 2015-09-23 | 河南双成生物科技有限公司 | Production method of high-protein feed containing arginine |
| CN106262955A (en) * | 2016-09-24 | 2017-01-04 | 青岛嘉瑞生物技术有限公司 | A kind of method preparing Herba suadeae glaucae dietary fiber |
| CN106387925A (en) * | 2016-09-16 | 2017-02-15 | 青岛嘉瑞生物技术有限公司 | Method for preparing dietary fibers from salicornia stalks by biological method |
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