CN108157673B - Preparation method of fermented prawn compound feed - Google Patents
Preparation method of fermented prawn compound feed Download PDFInfo
- Publication number
- CN108157673B CN108157673B CN201711326369.1A CN201711326369A CN108157673B CN 108157673 B CN108157673 B CN 108157673B CN 201711326369 A CN201711326369 A CN 201711326369A CN 108157673 B CN108157673 B CN 108157673B
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- fermentation
- liquid
- culture medium
- bacterial liquid
- fermented
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
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Abstract
The invention discloses a preparation method of a fermented prawn compound feed. The invention adopts a solid and liquid mixed fermentation process, preferably selects Aspergillus niger, Bacillus subtilis and Bacillus licheniformis which are rich in enzyme and can jointly grow, performs combined fermentation, and fully exerts the advantage of rich enzyme production of Aspergillus niger and Bacillus subtilis through aerobic and anaerobic two-step fermentation, so that basic fermentation raw materials are comprehensively decomposed, macromolecular proteins are decomposed into peptides and amino acids, cellulose is degraded into fermentable sugars, starch is decomposed into fermentable glucose, xylan is decomposed into xylose, glucan and the like, and the growth and the propagation of the Lactobacillus reuteri in an anaerobic stage are supported by a large amount of nutrient substances. The indexes of the content of the biological peptide, the content of the L-lactic acid, the colony number and the like can reflect that the growth and metabolism of the microorganisms in the aerobic stage and the anaerobic stage in the preparation method provided by the invention are very vigorous.
Description
The technical field is as follows:
the invention belongs to the technical field of aquatic feeds and bioengineering, and particularly relates to a preparation method of a fermented prawn compound feed.
Background art:
the prawn breeding of China forms a complete industrial system, various diseases frequently occur along with the continuous expansion of breeding density and scale, bacterial diseases caused by vibrio are fast in transmission and high in infection rate, huge economic losses are caused to the prawn breeding industry, and the development of prawn breeding in China and all over the world is severely restricted at present. Culturists usually use a large amount of antibiotic drugs, and although the effect is achieved in a short period of time, the problems of enhanced drug resistance of pathogenic microorganisms, reduced diversity of microbial communities, drug residues, worsening of the cultivation environment and the like are caused. The preparation method comprises the steps of carrying out treatment such as puffing or crushing on feed raw materials, degrading part of macromolecular substances such as polysaccharide, protein and fat by microbial fermentation, generating micromolecular substances such as organic acid and soluble polypeptide, accumulating beneficial metabolites, eliminating anti-nutritional factors, improving the digestibility of the prawn on the feed, promoting the proliferation of a series of functional strains in the intestinal tract and a culture water body of the prawn, stabilizing the advantageous microbial population structure of the intestinal tract and the water body of the prawn, and constructing a long-term stable and effective culture mode for efficient and healthy culture of the prawn.
The invention content is as follows:
compared with common prawn compound feeds or conventionally fermented prawn compound feeds, the fermented prawn compound feed obtained by the preparation method provided by the invention has very obvious improvements on indexes such as biological peptide content, protein, L-lactic acid content, probiotic bacterial colony number and the like on one hand, and the improvements can greatly promote the digestion, absorption and utilization of the feed by the prawns, improve the intestinal health of the prawns, improve the immunity of the prawns, improve the survival rate, reduce the use of antibiotics in prawn culture and reduce the environmental pollution; on the other hand, the fermented compound feed for prawns can promote the proliferation of series functional strains in intestinal tracts of prawns and aquaculture water, stabilize the beneficial dominant microbial population structure of microorganisms in the intestinal tracts and the water of prawns, and construct a long-term stable and effective culture mode for efficient and healthy culture of prawns; and finally, the fermented aspergillus niger can produce a plurality of enzymes by one bacterium, enzymolysis is carried out on the fermented raw material bean pulp anti-nutritional factors, the fermented raw material bean pulp is degraded into digestible nutrient substances, the fermented raw material is pretreated, granulation is not needed after fermentation, and the fermented soybean pulp is directly fed, so that the production process is simple.
The fermented prawn compound feed is prepared by the following method:
(1) solid fermentation medium raw materials: the solid fermentation medium comprises, by 100% of total mass fraction, 10-30% of corn grit, 30-55% of soybean meal, 10-30% of fish meal, 0.5-1.5% of prawn compound premix feed and the balance of bran, wherein the raw materials are uniformly mixed to obtain a mixture, and water is added until the water content of the mixture is 25-35% by weight to obtain a solid fermentation medium;
(2) inoculating Aspergillus niger liquid, Bacillus subtilis liquid and Bacillus licheniformis liquid into a solid fermentation culture medium, uniformly mixing, and performing aerobic fermentation to obtain an aerobically fermented solid fermentation culture medium;
(3) adding sterile water to keep the water content of the solid fermentation culture medium after aerobic fermentation at 25-35%, inoculating lactobacillus reuteri liquid, lactobacillus plantarum liquid and saccharomyces cerevisiae liquid into the solid fermentation culture medium after aerobic fermentation, uniformly mixing, performing anaerobic fermentation, and obtaining the fermented prawn compound feed after fermentation.
The preparation method comprises the steps of inoculating an Aspergillus niger liquid, a Bacillus subtilis liquid and a Bacillus licheniformis liquid into a solid fermentation culture medium, uniformly mixing, and then carrying out aerobic fermentation, preferably inoculating the Aspergillus niger liquid, the Bacillus subtilis liquid and the Bacillus licheniformis liquid into the solid fermentation culture medium in a volume ratio of 6-8: 1-2, uniformly mixing, and then carrying out aerobic fermentation until white hyphae are fully distributed on the material, namely the material is regarded as an aerobic fermentation end point, and fermenting for 24-72 hours at the temperature of 28-32 ℃. Further preferred inoculation amounts are in terms of the total mass of the three bacteria: the solid fermentation medium was inoculated in an amount of 1:10 by mass.
Inoculating lactobacillus reuteri liquid, lactobacillus plantarum liquid and saccharomyces cerevisiae liquid into a solid fermentation culture medium after aerobic fermentation, uniformly mixing, and then carrying out anaerobic fermentation, preferably inoculating the mixed liquid of lactobacillus reuteri liquid, lactobacillus plantarum liquid and saccharomyces cerevisiae liquid into the solid fermentation culture medium after aerobic fermentation according to the volume ratio of 5-7: 1, uniformly mixing, and then carrying out anaerobic fermentation. Further preferred inoculation amounts are in terms of the total mass of the three bacteria: the solid fermentation medium was inoculated in an amount of 1:10 by mass. The anaerobic fermentation conditions are as follows: the temperature is 28-35 ℃, the time is 36-72 hours, and after the fermentation is finished, the fermented prawn compound feed is obtained.
1000kg of the prawn composite premixed feed is prepared from the following raw materials in parts by mass: 1.0kg of vitamin A, 30.22kg of vitamin D, 32.0kg of vitamin K, 8.08kg of dl-alpha-tocopheryl acetate, 11.06kg of vitamin B, 21.52kg of vitamin B, 62.03kg of vitamin B, 120.20kg of vitamin B, 7.12kg of nicotinamide, 3.16kg of D-calcium pantothenate, 0.73kg of folic acid, 0.92kg of D-biotin, 15.29kg of L-ascorbic acid-2-phosphate, 8.29kg of inositol, 0.52kg of ethoxyquinoline, 45.50kg of magnesium sulfate monohydrate, 18.0kg of ferrous sulfate monohydrate, 12.49kg of zinc sulfate monohydrate, 3.0kg of pentahydrate, 2.72kg of manganese sulfate monohydrate, 1% of cobalt sulfate, 1.0kg of 1% of calcium iodate, 1% of sodium selenite, and the balance of zeolite powder and half shell powder of rice.
The lowest concentration of each bacterial liquid is as follows: aspergillus niger liquid 2.0X 107cfu/mL, 1.0X 10 Bacillus subtilis liquid9cfu/mL, Bacillus licheniformis liquid 8.0 × 108cfu/mL, Lactobacillus plantarum bacterial liquid 0.8 × 109cfu/mL, Lactobacillus reuteri bacterial liquid 1.0 × 108cfu/mL, Saccharomyces cerevisiae bacterial liquid 7.0 × 108cfu/mL。
The invention provides a fermented prawn compound feed, which mainly takes corn grit, soybean meal, fish meal, bran, trace element additives and vitamin additives as fermentation raw materials, and adopts two-step fermentation of mixed bacteria, wherein Aspergillus niger, Bacillus subtilis and Bacillus licheniformis are subjected to aerobic fermentation to generate a large amount of enzymes such as cellulase, xylanase, protease, amylase, glucoamylase, lipase, glucanase and the like, so that starch, crude fiber and xylan of the fermentation raw materials are decomposed into fermentable sugar and protein is decomposed into biological peptide and the like, then Lactobacillus reuteri, Lactobacillus plantarum, Saccharomyces cerevisiae and the like are added for anaerobic fermentation to continuously decompose macromolecular substances of the fermentation raw materials, and L-lactic acid, Roy's hormone and other antibacterial substances and aromatic odor are generated, and in addition, the pH value of the lactic acid is reduced, so that the growth of harmful bacteria is effectively inhibited.
According to the invention, Aspergillus niger, Bacillus subtilis and Bacillus licheniformis are used for producing abundant cellulase, xylanase, protease, amylase, glucoamylase, lipase, glucanase and the like, so that basic fermentation raw materials are comprehensively and synergistically decomposed, macromolecular proteins are decomposed into peptides and amino acids, cellulose is degraded into fermentable sugars, starch is decomposed into fermentable glucose, and xylan is decomposed into xylose, glucan and the like. Through aerobic fermentation, a large amount of nutrient substances of the prawn compound feed are released, and necessary nutrients can be fully provided for other microorganisms with weak decomposition capacity, such as lactobacillus, saccharomycetes and the like. After the aerobic fermentation is finished, inoculating a mixed strain consisting of lactobacillus reuteri, lactobacillus plantarum and saccharomyces cerevisiae immediately according to a certain proportion, carrying out anaerobic fermentation, and after the aerobic and anaerobic fermentation, the content of crude protein in the fermented prawn compound feed is 45-51%, the content of biological peptide is 15-17%, the content of L-lactic acid is 60-80 mg/g, the pH is 4.1-4.5, and the total number of bacillus is more than 1 multiplied by 109cfu/g, total number of lactic acid bacteria > 1 × 1010cfu/g; in addition, the fermentation metabolite contains a large amount of digestive enzymes, bacteriostatic substances and immune enhancement factors, after the fermentation of the probiotics, the anti-nutritional factors contained in the vegetable protein are basically degraded, the fermented product has good acid aroma and wine aroma, the pH is 4.0-4.5, and the fermented product is favorable for storage.
The preparation method adopts a solid and liquid mixed fermentation process, preferably selects Aspergillus niger, Bacillus subtilis and Bacillus licheniformis which are rich in enzyme production and can jointly grow, performs combined fermentation, and fully exerts the advantage of rich enzyme production of Aspergillus niger and Bacillus subtilis through aerobic and anaerobic two-step fermentation, so that basic fermentation raw materials are comprehensively decomposed, macromolecular proteins are decomposed into peptides and amino acids, cellulose is degraded into fermentable sugars, starch is decomposed into fermentable glucose, xylan is decomposed into xylose, glucan and the like, and the growth and the propagation of the Lactobacillus reuteri in an anaerobic stage are supported by a large amount of nutrient substances. The indexes of the content of the biological peptide, the content of the L-lactic acid, the colony number and the like can reflect that the growth and metabolism of the microorganisms in the aerobic stage and the anaerobic stage in the preparation method provided by the invention are very vigorous.
The Aspergillus niger, Bacillus subtilis, Bacillus licheniformis, Lactobacillus reuteri, Lactobacillus plantarum and Saccharomyces cerevisiae are all conventional strains in the field and can be purchased from various large collections or strain sales organizations.
Unless otherwise defined, the present invention is directed to the definitions of terms having the same meaning as commonly understood by one of ordinary skill in the art.
Compared with the prior art, the invention has the following advantages:
(1) the fermented prawn compound feed provided by the invention has high content of biological peptide, the peptide can be directly absorbed by animal intestinal tracts, the digestibility is higher than that of amino acid, the absorption of the peptide does not compete with the absorption of the amino acid, the combination of the small peptide with biological activity and trace elements can greatly promote the absorption and utilization of the trace elements by aquatic animals, thereby promoting the growth of the aquatic animals and reducing the cost and environmental pollution.
(2) The fermented compound feed for prawns provided by the invention produces a large amount of digestive enzymes, L-lactic acid, bacteriostatic substances and the like through fermentation and metabolism, can improve the nutrition level and the utilization rate of the feed, inhibit the propagation of harmful bacteria in intestinal tracts of prawns, and enhance the disease resistance of prawns; on the other hand, the lactobacillus reuteri and the metabolite thereof have a certain repairing effect on prawn intestinal tracts; the fermented prawn compound feed has unique effects of improving prawn intestinal environment, inhibiting harmful bacteria growth, reducing antibiotic usage, increasing aquatic animal feed conversion rate, and the like.
(3) The fermented prawn compound feed provided by the invention has high content of bacillus subtilis and bacillus licheniformis, can quickly degrade residual bait, excrement and other organic debris in water, reduces ammonia nitrogen, hydrogen sulfide and the like in water environment, avoids organic waste accumulation in a water pool, and maintains water ecological balance.
(4) The preparation method of the fermented prawn compound feed adopts a solid and liquid mixed fermentation process, preferably selects Aspergillus niger, Bacillus subtilis and Bacillus licheniformis which are rich in enzyme production and can jointly grow, performs combined fermentation, and fully exerts the advantage of rich enzyme production of the Aspergillus niger and the Bacillus subtilis through aerobic and anaerobic two-step fermentation, so that basic fermentation raw materials are comprehensively decomposed, macromolecular proteins are decomposed into peptides and amino acids, cellulose is degraded into fermentable sugars, starch is decomposed into fermentable glucose, and xylan is decomposed into xylose, glucan and the like, thereby supporting the growth and the propagation of the Lactobacillus reuteri in an anaerobic stage to obtain a large amount of nutrient substances.
The specific implementation mode is as follows:
in order to describe the invention, examples are set forth below. It is to be understood that the invention is not limited to these embodiments, but is provided as a means of practicing the invention.
Example 1:
(1) preparation of liquid fermentation strain
a. Preparation of Aspergillus niger (purchased from Guangdong province culture Collection of microorganisms with a collection number of GIM 3.576) fermentation liquor: placing the slant strains of the Aspergillus niger in an incubator, activating for 24 hours at 28 ℃, selecting bacterial colonies on a slant culture medium, inoculating the bacterial colonies to a 250mL triangular flask filled with 100mL PDA liquid culture medium, and carrying out fermentation culture under the culture conditions: the temperature is 28 ℃ and the time is 24 hours, the Aspergillus niger fermentation liquor is obtained, and the Aspergillus niger is fermentedThe content of Aspergillus niger in the liquid is (2.0-4.0) x 107cfu/mL。
Wherein, the PDA liquid culture medium formula is as follows: peeling 200g of potato, cutting, boiling, filtering, removing residues, adding 20g of sucrose, 10g of peptone and 18g of agar, adding water to a constant volume of 1L, and adjusting the pH value to 7.0.
b. Preparation of a bacillus subtilis (purchased from Guangdong province culture Collection of microorganisms with a collection number of GIM1.372) fermentation liquid: selecting bacillus subtilis, inoculating the bacillus subtilis into an LB culture medium, and activating for 16 hours in a shaking table at 37 ℃ with the rotation speed of the shaking table of 180 r/min; then inoculating the activated bacterial liquid into a first-stage seed culture medium according to the inoculation amount of 3% of the volume ratio for fermentation culture, wherein the culture conditions are as follows: the temperature is 37 ℃ and the time is 24 hours, so that the bacillus subtilis fermentation liquor is obtained, and the content of the bacillus subtilis in the bacillus subtilis fermentation liquor is (1.0-1.8) × 109cfu/mL。
Wherein the LB culture medium (Luria-Bertani culture medium) formula is as follows: 10g of tryptone, 5g of yeast powder and 10g of sodium chloride were added to 1L of distilled water, and the pH was adjusted to 7.0.
The first-order seed culture medium is as follows: adding 5g of glucose, 15g of corn starch, 20g of soybean meal, 5g of yeast powder, 2g of magnesium sulfate, 2g of dipotassium phosphate, 0.3g of manganese sulfate and 2.4g of calcium carbonate into distilled water, then adding the distilled water to a constant volume of 1L, and adjusting the pH value to 7.0.
c. Preparation of a bacillus licheniformis (purchased from Guangdong province culture Collection of microorganisms with a collection number of GIM1.11) fermentation liquid: selecting Bacillus licheniformis to inoculate into LB culture medium, activating for 24h in a shaking table at 37 ℃, wherein the rotation speed of the shaking table is 180 r/min; then inoculating the activated bacterial liquid into a first-stage seed culture medium according to the inoculation amount of 3% of the volume ratio for fermentation culture, wherein the culture conditions are as follows: the temperature is 37 ℃ and the time is 24 hours, thus obtaining the bacillus licheniformis fermentation liquor, wherein the content of the bacillus licheniformis in the bacillus licheniformis fermentation liquor is (8.0-8.8) multiplied by 108cfu/mL。
Wherein the LB culture medium (Luria-Bertani culture medium) formula is as follows: 10g of tryptone, 5g of yeast powder and 10g of sodium chloride were added to 1L of distilled water, and the pH was adjusted to 7.0.
The first-order seed culture medium is as follows: adding 5g of glucose, 10g of sucrose, 20g of peptone, 3g of yeast powder, 2g of magnesium sulfate, 2.5g of dipotassium hydrogen phosphate and 5g of ammonium chloride into distilled water, then adding distilled water to a constant volume of 1L, and adjusting the pH value to 7.0.
d. Preparation of lactobacillus plantarum (purchased from Guangdong province culture Collection of microorganisms with accession number GIM1.191) fermentation broth: inoculating lactobacillus plantarum into an MRS culture medium, standing and activating at 37 ℃ for 24 hours, then inoculating activated bacterium liquid into a first-level seed culture medium according to the inoculation amount of 5% of the volume ratio for fermentation culture, wherein the culture conditions are as follows: standing at 37 ℃ for 18-20 h to obtain lactobacillus plantarum fermentation liquor, wherein the content of lactobacillus plantarum in the lactobacillus plantarum fermentation liquor is (0.8-1.5) multiplied by 109cfu/mL。
The MRS culture medium comprises the following components in percentage by weight: adding 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium hydrogen citrate, 20g of glucose, 801 mL of tween, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate and 0.25g of manganese sulfate into water, then adding distilled water to a constant volume of 1000mL, and adjusting the pH value to 6.2.
The first-order seed culture medium is as follows: adding 5g of glucose, 5g of molasses, 10g of peptone, 5g of yeast extract, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate and 0.5g of manganese sulfate into distilled water, then adding distilled water to a constant volume of 1.0L, and adjusting the pH value to 6.8.
e. Preparation of lactobacillus reuteri (purchased from Guangdong province microorganism culture collection center, with the preservation number of CICC6118) fermentation liquor: inoculating lactobacillus reuteri into an MRS culture medium, standing and activating for 24h at 37 ℃, then inoculating the activated bacterium liquid into a first-level seed culture medium according to the inoculation amount of 5% of the volume ratio for fermentation culture, wherein the culture conditions are as follows: standing at 37 ℃ for 20-22 h to obtain lactobacillus reuteri fermentation liquor, wherein the content of lactobacillus reuteri in the lactobacillus reuteri fermentation liquor is (1.0-1.4) multiplied by 108cfu/mL。
The MRS culture medium comprises the following components in percentage by weight: adding 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium hydrogen citrate, 20g of glucose, 801 mL of Tween, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.5g of magnesium sulfate and 0.25g of manganese sulfate into distilled water, then diluting to 1000mL with distilled water, and adjusting the pH to 6.2.
The first-order seed culture medium is as follows: adding 5g of glucose, 5g of molasses, 10g of peptone, 5g of yeast extract, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate and 0.5g of manganese sulfate into distilled water, then adding distilled water to a constant volume of 1L, and adjusting the pH value to 6.8.
f. Preparation of a saccharomyces cerevisiae (purchased from Guangdong province culture Collection of microorganisms with a collection number of GIM 2.167): selecting a saccharomyces cerevisiae slant strain to be inoculated into a PDA liquid culture medium, activating for 24 hours by a shaking table at 28 ℃, wherein the rotating speed of the shaking table is 180 r/min; then inoculating the activated bacterial liquid into a first-stage seed culture medium according to the inoculation amount of 2% of the volume ratio for fermentation culture, wherein the culture conditions are as follows: the temperature is 28 ℃, the time is 24 hours, and the saccharomyces cerevisiae fermentation liquor is obtained, wherein the content of the saccharomyces cerevisiae in the fermentation liquor is (7.0-8.0) multiplied by 108cfu/mL。
The formula of the PDA liquid culture medium is the same as that of the PDA liquid culture medium.
The first-order seed culture medium is as follows: adding 20g of glucose, 20g of sucrose, 30g of soybean peptone, 10g of yeast powder, 5g of dipotassium hydrogen phosphate and 5g of urea into distilled water, and then adding the distilled water to a constant volume of 1.0L and a pH value of 5.8-6.2.
(2) Raw materials of the solid fermentation medium: 10% of corn residue, 55% of soybean meal, 10% of fish meal, 0.5% of prawn compound premix feed and 24.5% of bran, uniformly mixing the components according to the content of the components to obtain a mixture, and then adding water into the mixture until the water content is 25% by weight to obtain the solid fermentation culture medium.
The compound premixed feed for the prawns (the same as the embodiments 2 and 3) is prepared from the following raw materials in parts by mass per 1000 kg: 1.0kg of vitamin A, 30.22kg of vitamin D, 32.0kg of vitamin K, 8.08kg of dl-alpha-tocopheryl acetate, 11.06kg of vitamin B, 21.52kg of vitamin B, 62.03kg of vitamin B, 120.20kg of vitamin B, 7.12kg of nicotinamide, 3.16kg of D-calcium pantothenate, 0.73kg of folic acid, 0.92kg of D-biotin, 15.29kg of L-ascorbic acid-2-phosphate, 8.29kg of inositol, 0.52kg of ethoxyquinoline, 45.50kg of magnesium sulfate monohydrate, 18.0kg of ferrous sulfate monohydrate, 12.49kg of zinc sulfate monohydrate, 3.0kg of copper sulfate pentahydrate, 2.72kg of manganese sulfate monohydrate, 1% of cobalt sulfate, 1.0kg of 1% of calcium iodate, 1% of sodium selenite, the balance of zeolite powder and half shell meal of rice, and uniformly mixing the raw materials to obtain the compound feed. Wherein the 1% cobalt sulfate, the 1% calcium iodate and the 1% sodium selenite are all purchased from Guangzhou Zhi special fodder Co.
(3) An aerobic fermentation stage: inoculating the liquid fermentation strain prepared in the step (1) into the solid fermentation culture medium obtained in the step (2) according to the volume ratio of Aspergillus niger fermentation liquid to Bacillus subtilis fermentation liquid to Bacillus licheniformis fermentation liquid of 6:1:1, wherein the inoculation amount is 10% by mass (namely the total mass of the Aspergillus niger fermentation liquid, the total mass of the Bacillus subtilis fermentation liquid and the Bacillus licheniformis fermentation liquid is 1g, the total mass of the three bacteria is inoculated into 10g of the solid fermentation culture medium), and placing the two bacteria in an open container for aerobic fermentation, wherein the fermentation conditions are as follows: the temperature is 28 ℃, the time is 24 hours, a solid fermentation culture medium after aerobic fermentation is obtained, white hypha is fully distributed on the material, the aerobic fermentation end point is regarded as the aerobic fermentation end point, and then the fermentation of the next stage is carried out.
(4) An anaerobic fermentation stage: adding sterile water to keep the water content of the solid fermentation medium after aerobic fermentation to be 25%, inoculating the prepared liquid fermentation strain into the solid fermentation medium after aerobic fermentation according to the volume ratio of lactobacillus reuteri fermentation liquor to saccharomyces cerevisiae fermentation liquor of 5:5:1, wherein the inoculation amount is 10% (namely the total mass of lactobacillus reuteri fermentation liquor, lactobacillus plantarum fermentation liquor and saccharomyces cerevisiae fermentation liquor is 1g, the lactobacillus reuteri fermentation liquor and the saccharomyces cerevisiae fermentation liquor are inoculated into 10g of the solid fermentation medium after aerobic fermentation), placing the solid fermentation medium in a sealed airtight container for anaerobic fermentation, and the fermentation conditions are as follows: the temperature is 28 ℃, the time is 36h, and after the fermentation is finished, the fermented prawn compound feed is obtained.
And (3) determining indexes of the feed crude protein content, the biological peptide content, the L-lactic acid content, the total number of bacillus, the total number of lactic acid bacteria and the like of the unfermented prawn compound feed (namely the solid fermentation culture medium in the step (2)) and the fermented prawn compound feed, and the indexes are shown in table 1.
TABLE 1
| Item | Unfermented compound feed for prawn | Example 1 the fermented prawn Compound feed |
| Crude protein (%) | 36 | 45 |
| Biological peptide (%) | 0.4 | 15 |
| L-lactic acid (mg/g) | 0.5 | 60 |
| Total number of Bacillus | 1.2×103 | 1.0×109 |
| Total number of lactic acid bacteria | 0 | 1.4×1010 |
Example 2:
(1) liquid fermentation strains (Aspergillus niger, Bacillus subtilis, Bacillus licheniformis, Lactobacillus reuteri, Lactobacillus plantarum, Saccharomyces cerevisiae) were prepared according to the method of example 1.
(2) Weighing the following raw materials in parts by mass: 20% of corn residue, 45% of soybean meal, 20% of fish meal, 0.8% of prawn compound premix feed and 14.2% of bran, uniformly mixing the components to obtain a mixture, and adding water into the mixture until the water content is 30% by weight to obtain the solid fermentation culture medium.
(3) An aerobic fermentation stage: and (2) mixing the liquid fermentation strain prepared in the step (1) with Aspergillus niger fermentation liquor, Bacillus subtilis fermentation liquor and Bacillus licheniformis fermentation liquor according to a volume ratio of 7: 2: inoculating the solid fermentation culture medium prepared in the step (2) in a ratio of 1, wherein the inoculation amount is 10% by mass (the total mass of the three bacteria: the mass of the solid fermentation culture medium is 1:10), placing the solid fermentation culture medium in an open container for aerobic fermentation, and the fermentation conditions are as follows: the temperature is 30 ℃, the time is 48 hours, a solid fermentation culture medium after aerobic fermentation is obtained, white hypha is fully distributed on the material, the aerobic fermentation end point is regarded as the aerobic fermentation end point, and then the fermentation of the next stage is carried out.
(4) An anaerobic fermentation stage: adding sterile water to keep the water content of the solid fermentation culture medium after aerobic fermentation at 30%, and mixing the prepared liquid fermentation strain with lactobacillus reuteri fermentation liquor according to the volume ratio of lactobacillus plantarum fermentation liquor to saccharomyces cerevisiae fermentation liquor of 6: 6: inoculating the solid fermentation culture medium obtained after the aerobic fermentation in the step (3) in a ratio of 1, wherein the inoculation amount is 10% (the total mass of the three bacteria: the mass of the solid fermentation culture medium obtained after the aerobic fermentation is 1:10), and placing the solid fermentation culture medium in a sealed airtight container for anaerobic fermentation under the fermentation conditions that: the temperature is 32 ℃, the time is 54h, and after the fermentation is finished, the fermented prawn compound feed is obtained.
And (3) determining indexes of the feed crude protein content, the biological peptide content, the L-lactic acid content, the total number of bacillus, the total number of lactic acid bacteria and the like of the unfermented prawn compound feed (namely the solid fermentation culture medium in the step (2)) and the fermented prawn compound feed, as shown in the table 2.
TABLE 2
| Item | Unfermented compound feed for prawn | Example 2 the fermented prawn Compound feed |
| Crude protein (%) | 37.6 | 48.5 |
| Biological peptide (%) | 0.6 | 16 |
| L-lactic acid (mg/g) | 0.62 | 67 |
| Total number of Bacillus | 1.1×103 | 2.4×109 |
| Total number of lactic acid bacteria | 0 | 1.8×1010 |
Example 3:
(1) liquid fermentation strains (Aspergillus niger, Bacillus subtilis, Bacillus licheniformis, Lactobacillus reuteri, Lactobacillus plantarum, Saccharomyces cerevisiae) were prepared according to the method of example 1.
(2) Weighing the following raw materials in parts by mass: 30% of corn residue, 30% of soybean meal, 30% of fish meal, 1.5% of prawn compound premix feed and 8.5% of bran, uniformly mixing the components according to the content to obtain a mixture, and adding water into the mixture until the water content is 35% by weight to obtain the solid fermentation culture medium.
(3) An aerobic fermentation stage: and (2) mixing the liquid fermentation strain prepared in the step (1) according to the volume ratio of Aspergillus niger fermentation liquor to Bacillus subtilis fermentation liquor to Bacillus licheniformis fermentation liquor of 8: 1: inoculating the solid fermentation culture medium prepared in the step (2) in a ratio of 10% by mass (the total mass of the three bacteria: the mass of the solid fermentation culture medium is 1:10), and placing the mixture in an open container for aerobic fermentation under the fermentation conditions that: the temperature is 32 ℃, the time is 72 hours, a solid fermentation culture medium after aerobic fermentation is obtained, white hypha is fully distributed on the material, the aerobic fermentation end point is regarded as the aerobic fermentation end point, and then the fermentation of the next stage is carried out.
(4) An anaerobic fermentation stage: adding sterile water to keep the water content of the aerobically fermented solid fermentation medium at 35%, and mixing the prepared liquid fermentation strain with the fermentation liquid of lactobacillus reuteri in a volume ratio of 7: 7: inoculating the solid fermentation culture medium obtained after the aerobic fermentation in the step (3) in a ratio of 1, wherein the inoculation amount is 10% (the total mass of the three bacteria: the mass of the solid fermentation culture medium obtained after the aerobic fermentation is 1:10), and placing the solid fermentation culture medium in a sealed airtight container for anaerobic fermentation under the fermentation conditions that: the temperature is 35 ℃, the time is 72 hours, and after the fermentation is finished, the fermented prawn compound feed is obtained.
And (3) determining indexes of the feed crude protein content, the biological peptide content, the L-lactic acid content, the total number of bacillus, the total number of lactic acid bacteria and the like of the unfermented prawn compound feed (namely the solid fermentation culture medium in the step (2)) and the fermented prawn compound feed, as shown in the table 3.
TABLE 3
| Item | Unfermented compound feed for prawn | Example 3 the fermented prawn Compound feed |
| Crude protein (%) | 38 | 51 |
| Biological peptide (%) | 0.8 | 17 |
| L-lactic acid (mg/g) | 1.0 | 80 |
| Total number of Bacillus | 1.1×103 | 1.5×109 |
| Total number of lactic acid bacteria | 0 | 2.0×1010 |
As can be seen from tables 1 to 3, in the fermented prawn compound feeds prepared in examples 1 to 3, compared with the unfermented prawn compound feed ((i.e., the solid fermentation medium in each step (2)), the content of crude protein is increased from 36% to 38% to 45% to 51%, the content of biological peptide is increased from 0.4% to 0.8% to 15% to 17%, the content of L-lactic acid is increased from 0.5 to 1mg/g to 60 to 80mg/g, the pH value is decreased from 6.3 to 6.5 to 4.1 to 4.5, and the total number of bacillus is more than 1 × 109cfu/g, total number of lactic acid bacteria > 1 × 1010 cfu/g.
Claims (2)
1. A preparation method of a fermented prawn compound feed is characterized by comprising the following steps:
(1) preparing a solid fermentation culture medium raw material: the raw material components comprise 10-30% of corn grit, 30-55% of soybean meal, 10-30% of fish meal, 0.5-1.5% of prawn composite premixed feed and the balance of bran, the raw material components are uniformly mixed to obtain a mixture, water is added until the water content of the mixture is 25-35%, and a solid fermentation culture medium with the pH value of 6.3-6.5 is obtained;
(2) inoculating Aspergillus niger liquid, Bacillus subtilis liquid and Bacillus licheniformis liquid into a solid fermentation culture medium in a volume ratio of 6-8: 1-2, uniformly mixing, and performing aerobic fermentation, wherein the mass ratio of the total mass of the Aspergillus niger liquid, the Bacillus subtilis liquid and the Bacillus licheniformis liquid to the solid fermentation culture medium is 1:10, and the aerobic fermentation conditions are as follows: fermenting for 24-72 h at the temperature of 28-32 ℃;
(3) adding sterile water to keep the water content of the solid fermentation culture medium after aerobic fermentation to be 25-35%, inoculating the mixed bacterial liquid of the lactobacillus reuteri bacterial liquid, the lactobacillus plantarum bacterial liquid and the saccharomyces cerevisiae bacterial liquid into the solid fermentation culture medium after the aerobic fermentation in a volume ratio of 5-7: 1, uniformly mixing, and then carrying out anaerobic fermentation, thus obtaining the fermented prawn compound feed with the pH value of 4.1-4.5 after the fermentation is finished, wherein the mass ratio of the total mass of the lactobacillus reuteri bacterial liquid, the lactobacillus plantarum bacterial liquid and the saccharomyces cerevisiae bacterial liquid to the solid fermentation culture medium is 1:10, and the anaerobic fermentation conditions are as follows: the temperature is 28-3 ℃, and the time is 36-72 h;
the concentration of each bacterial liquid of the aspergillus niger bacterial liquid, the bacillus subtilis bacterial liquid, the bacillus licheniformis bacterial liquid, the lactobacillus reuteri bacterial liquid, the lactobacillus plantarum bacterial liquid and the saccharomyces cerevisiae bacterial liquid is the lowest: aspergillus niger liquid 2.0X 107cfu/mL, 1.0X 10 Bacillus subtilis liquid9cfu/mL, Bacillus licheniformis liquid 8.0 × 108cfu/mL, Lactobacillus plantarum bacterial liquid 0.8 × 109cfu/mL, Lactobacillus reuteri bacterial liquid 1.0 × 108cfu/mL, Saccharomyces cerevisiae bacterial liquid 7.0 × 108cfu/mL; the compound premixed feed for prawns comprises, per 1000 kg: 1.0kg of vitamin A, 30.22 kg of vitamin D, 32.0 kg of vitamin K, 8.08kg of dl-a-tocopheryl acetate, 11.06kg of vitamin B, 21.52kg of vitamin B, 62.03 kg of vitamin B, 120.20 kg of vitamin B, 7.12kg of nicotinamide, 3.16kg of D-calcium pantothenate, 0.73kg of folic acid, 0.92kg of D-biotin, 15.29kg of L-ascorbic acid-2-phosphate, 8.29kg of inositol, 0.52kg of ethoxyquinoline, 0.52kg of magnesium sulfate monohydrate45.50kg, 18.0kg of ferrous sulfate monohydrate, 12.49kg of zinc sulfate monohydrate, 3.0kg of copper sulfate pentahydrate, 2.72kg of manganese sulfate monohydrate, 1.0kg of 1% cobalt sulfate, 1% of calcium iodate, 1.5kg of 1% sodium selenite, and the balance of zeolite powder and half of rice hull powder in mass.
2. A fermented prawn compound feed prepared according to the preparation method of claim 1.
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| CN111066945A (en) * | 2019-12-19 | 2020-04-28 | 广东省微生物研究所（广东省微生物分析检测中心） | Pelletization-free prawn fermented feed |
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| CN116076631B (en) * | 2022-10-21 | 2024-04-02 | 广州朴成生物科技有限公司 | Functional fermented feed for regulating and controlling glycolipid metabolism of micropterus salmoides and preparation method thereof |
| CN115708547B (en) * | 2022-10-21 | 2024-04-05 | 华中农业大学 | Application of functional fermented feed beneficial to improvement of glycolipid metabolism of micropterus salmoides |
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