CN115708547A - Application of functional fermented feed beneficial to improving micropterus salmoides glycolipid metabolism - Google Patents
Application of functional fermented feed beneficial to improving micropterus salmoides glycolipid metabolism Download PDFInfo
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- CN115708547A CN115708547A CN202211297013.0A CN202211297013A CN115708547A CN 115708547 A CN115708547 A CN 115708547A CN 202211297013 A CN202211297013 A CN 202211297013A CN 115708547 A CN115708547 A CN 115708547A
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- liquid
- culture medium
- feed
- fermentation
- micropterus salmoides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention discloses application of a functional fermented feed beneficial to improving micropterus salmoides glycolipid metabolism. The application of the fermented feed in preparing the feed for improving the glycolipid metabolism capability of micropterus salmoides. Mixing bacillus subtilis, saccharomyces cerevisiae and a reuteri bacterium solution for producing reuterin to obtain a composite microbial inoculum, inoculating the composite microbial inoculum into a solid culture medium for anaerobic fermentation, adding micropterus salmoides premixed feed and konjac powder after the anaerobic fermentation, and granulating to obtain the fermented feed. The invention promotes the utilization rate and nutrient digestion and absorption of micropterus salmoides to the glycolipid in the feed, improves the feed conversion rate of animals, can effectively improve the utilization of micropterus salmoides to the glycolipid metabolism of the feed, reduces fat accumulation in abdominal cavity, reduces the occurrence of fatty liver, enhances the immunity of organisms, reduces the occurrence of diseases, and improves the economic and market values of commercial fish.
Description
The technical field is as follows:
the invention belongs to the field of animal feed, and particularly relates to application of a functional fermented feed beneficial to improving micropterus salmoides glycolipid metabolism.
Background art:
micropterus salmoides (Micropterus salmoides) belongs to the family Sunglyudae of Perciformes (Cerutrachidae) and is also called Micropterus salmoides, is carnivorous warm water fishes, is an important fresh water culture famous and excellent variety in China, and has delicious meat and high nutritional value. According to the '2022 Chinese fishery statistics yearbook', the culture yield of the micropterus salmoides in China reaches 70.21 ten thousand tons, the problem of culture from ice fresh fish to compound feed is solved basically at present, but because the micropterus salmoides per se has poor utilization rate on glycolipids, ingestion of artificial feed easily causes glycolipid metabolic disturbance of the micropterus salmoides, abdominal fat accumulation and liver swelling, and the quality and the commodity value of the micropterus salmoides are seriously influenced. The intestinal flora is an important member of the intestinal barrier and influences the nutrient absorption, energy metabolism and immune response of the host. The nutrition metabolic disturbance of the organism and the imbalance of the intestinal flora are closely related, and the regulation of the intestinal microorganisms is an important way for regulating and controlling the nutrition metabolic disturbance of the organism.
The feed is fermented by functional microorganisms to feed bred animals, and the targeting regulation of intestinal flora and metabolites by the functional microorganisms and secondary metabolites is a very effective method for preventing or treating the diseases of nutrition metabolism disorder of organisms.
At present, no patent related to a preparation method of functional fermented feed for promoting the metabolism of micropterus salmoides glycolipid is found.
The invention content is as follows:
the invention aims to provide application of fermented feed in preparing functional fermented feed for improving glycolipid metabolism of micropterus salmoides.
The fermented feed of the invention, namely the functional fermented feed for improving the glycolipid metabolism of micropterus salmoides, has the preparation method that:
mixing bacillus subtilis, saccharomyces cerevisiae and lactobacillus reuteri liquid for producing reuterin to obtain a composite microbial inoculum, inoculating the composite microbial inoculum into a solid culture medium for anaerobic fermentation, adding micropterus salmoides premixed feed and konjac fine powder after the anaerobic fermentation, and granulating to obtain fermented feed;
the solid culture medium is as follows: comprises 10-20 percent of corn flour, 35-55 percent of bean pulp, 25-45 percent of fish meal and 5-10 percent of wheat bran by mass; mixing, adding water until the water content is 25-40%, and mixing to obtain solid culture medium.
Preferably, bacillus subtilis, saccharomyces cerevisiae and lactobacillus reuteri liquid for producing reuterin are mixed according to the volume ratio of 1-3.
Preferably, the viable count of the bacteria liquid of bacillus subtilis, saccharomyces cerevisiae and lactobacillus reuteri for producing reuterin is 1.0 multiplied by 10 9 cfu/mL or more.
Preferably, the preparation method of the bacillus subtilis liquid comprises the following steps: selecting Bacillus subtilis, inoculating to LB culture medium, and activating with shaker at 37 + -1 deg.C for 16-24 hr; then inoculating the activated bacterial liquid into a seed liquid culture medium according to the inoculation amount of 3-6% of the volume ratio, and culturing at 37 +/-1 ℃ for 16-24h to obtain a bacillus subtilis seed liquid;
the seed liquid culture medium comprises: the compound premix comprises, by mass, 0.5% of glucose, 1.5% of soluble starch, 2% of peptone, 0.5% of yeast powder, 0.2% of magnesium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.03% of manganese sulfate, 0.24% of calcium carbonate and pH7.0 +/-0.2.
Preferably, the saccharomyces cerevisiae bacterial liquid is prepared by selecting saccharomyces cerevisiae slant strains to be inoculated into a PDA liquid culture medium and activating the strains for 20 to 24 hours in a shaking table at the temperature of 28 +/-1 ℃; then inoculating the activated bacterial liquid into a seed liquid culture medium according to the inoculation amount of 2-5% of the volume ratio, and culturing at 28 +/-1 ℃ for 18-24h to obtain a saccharomyces cerevisiae seed liquid;
the seed liquid culture medium comprises: the nutrient solution comprises, by mass, 2% of glucose, 2% of sucrose, 3% of soybean peptone, 1% of yeast powder, 0.5% of dipotassium hydrogen phosphate, 0.5% of urea and pH6.0 +/-0.2.
Preferably, the lactobacillus reuteri liquid for producing the reuterin is prepared by selecting lactobacillus reuteri strains for producing the reuterin, inoculating the lactobacillus reuteri strains into an MRS liquid culture medium, carrying out static culture at 37 +/-1 ℃ for 18-26h, then transferring the activated liquid strains into a fermentation tank of the liquid seed culture medium according to the inoculation amount of 3-6% of the activated liquid strains by volume ratio, wherein the liquid filling amount of the fermentation tank is 60-80%, the fermentation temperature is 37 +/-1 ℃, carrying out sealed static culture for 18-26h, and preparing a seed liquid;
transferring the seed liquid into a fermentation medium containing glycerol-producing dehydratase for fermentation by an inoculum size of 3-6% in volume ratio, controlling the liquid loading amount of a fermentation tank to be 50-65%, the fermentation temperature to be 28-32 ℃, carrying out micro-aerobic culture, rotating speed to be 80-100rpm, controlling the pH value of the whole fermentation process to be 5.5-7.0, and after the fermentation time is 18-25h, feeding sterilized and cooled glycerol into a bacterial liquid in a variable-speed feeding manner, wherein the concentration of the glycerol is 150-450mmol/L, and the volume ratio of the glycerol solution to the bacterial liquid is as follows: 1-5: 99-95, enzyme conversion temperature: preparing a bacterium liquid of the lactobacillus reuteri containing the reuterin at the temperature of 28-32 ℃ for 1-5 h;
the MRS liquid culture medium and the liquid seed culture medium are prepared by the following steps of: adding water into 10g of peptone, 5g of yeast powder, 10g of beef extract, 5g of sodium acetate, 2g of diammonium hydrogen citrate, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 1mL of tween 80 and 10g of calcium carbonate to 1000mL, and adjusting the pH value to 6.8;
the preparation method of each liter of the glycerol-producing dehydratase fermentation culture medium comprises the following steps: adding 25g of molasses, 4g of beef extract and 4g of yeast powder into water, adding water to 1000ml, and adjusting the pH value to 6.0.
The content of reuterin is 120-200mmol/L and the number of viable bacteria of the reuterin is 1.0 × 10 9 cfu/mL～5.0×10 9 cfu/mL。
Preferably, the improvement of the micropterus salmoides glycolipid metabolism is to improve the utilization of the micropterus salmoides in the feed and the digestion and absorption of nutrition, and improve the feed conversion rate of animals.
The invention can regulate and control the intestinal microbial community structure, improve the intestinal flora distribution, antagonize the harmful bacteria colonization, simultaneously can target and adjust the intestinal flora and metabolites of the micropterus salmoides, promote the utilization rate and nutrient digestion and absorption of the micropterus salmoides on the glycolipid in the feed, improve the feed conversion rate of animals, effectively improve the utilization of the micropterus salmoides on the glycolipid metabolism of the feed, reduce the fat accumulation in the abdominal cavity, reduce the occurrence of fatty liver, enhance the immunity of organisms, reduce the occurrence of diseases and improve the economic and market values of commercial fish.
Description of the drawings:
FIG. 1 is the experimental results of example 4, each set of histograms is Ctr0, test groups I and II from left to right, respectively;
FIG. 2 is the content of the intestinal metabolite biogenic amine;
FIG. 3 is the fatty acid content of the intestinal metabolite exercise, with Ctr0, and test group I on each group of bar graphs from left to right, respectively;
FIG. 4 is the lipid metabolism content, with Ctr0, test groups I and II from left to right for each group of histograms;
FIG. 5 is a graph of enzyme activity, where each set of bar graphs is Ctr0, test set I and II from left to right.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Every 1000kg of the micropterus salmoides premixed feed used in the following steps is prepared from the following raw materials in parts by mass: 0.8kg of vitamin A, 0.24kg of vitamin D, 2.16kg of vitamin K, 8.40kg of dl-alpha-tocopheryl acetate, 0.86kg of vitamin B, 1.20kg of vitamin B, 0.73kg of vitamin B, 0.30kg of vitamin B, 6.19kg of nicotinamide, 2.45kg of D-calcium pantothenate, 0.49kg of folic acid, 0.60kg of D-biotin, 20.57kg of L-ascorbic acid-2-phosphate, 3.67kg of inositol, 2.40kg of ethoxyquinoline, 20.63kg of magnesium sulfate monohydrate, 12.83kg of ferrous sulfate monohydrate, 5.74kg of zinc sulfate monohydrate, 3.52kg of copper sulfate pentahydrate, 3.46kg of manganese sulfate monohydrate, 13.2kg of 1% cobalt sulfate, 9.9kg of calcium iodate, 3.9 kg of 1% sodium selenite, zeolite powder and half shell powder of rice are added to obtain a premixed feed. Wherein the 1% cobalt sulfate, the 1% calcium iodate and the 1% sodium selenite are all purchased from Guangzhou Zhi special feed Co.
Konjac refined powder obtained from Kyosen Konjac science and technology Limited
Lactobacillus reuteri, strain number, GDMCC 1.614, deposited at the guangdong province collection of microorganisms (GDMCC), used below is lactobacillus reuteri: GDMCC 1.614.
The following bacillus subtilis used is bacillus subtilis GDMCC 1.372, which is deposited in the Guangdong province culture collection (GDMCC), strain number: GDMCC 1.372.
The saccharomyces cerevisiae used below was saccharomyces cerevisiae GDMCC 2.167, deposited at the guangdong province collection of microorganisms (GDMCC), strain number: GDMCC 2.167.
Example 1
1, preparing a fermentation strain: preparing Bacillus subtilis, saccharomyces cerevisiae seed solution, and Roy's bacillus solution for producing Roy's bacterin with viable count of 1.0 × 10 9 cfu/mL or more;
1.1 preparation of Bacillus subtilis seed solution
Selecting bacillus subtilis, inoculating the bacillus subtilis into an LB culture medium, and activating for 16 hours in a shaking table at the temperature of 37 +/-1 ℃; then inoculating the activated bacterial liquid into a seed liquid culture medium according to the inoculation amount of 3 percent of the volume ratio, and culturing for 16h at the temperature of 37 +/-1 ℃ to obtain the bacillus subtilis seed liquid.
The LB culture medium (Luria-Bertani culture medium) formula is as follows: adding 10g of tryptone, 5g of yeast powder and 10g of sodium chloride into 1L of distilled water, adjusting the pH value to 7.0, and sterilizing for later use.
The seed liquid culture medium comprises: the nutrient solution comprises, by mass, 0.5% of glucose, 1.5% of soluble starch, 2% of peptone, 0.5% of yeast powder, 0.2% of magnesium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.03% of manganese sulfate, 0.24% of calcium carbonate and water as a solvent, wherein the pH value is 7.0 +/-0.2, and the preparation method comprises the steps of uniformly mixing the components and sterilizing for later use.
2.2 preparation of Roisella reuteri seed solutions for Roisella production
2.2.1 selecting a reuterin-producing reuterin strain, inoculating the strain into an MRS liquid seed culture medium, standing and culturing for 18 hours at 37 +/-1 ℃, then transferring the activated liquid strain into a fermentation tank of the MRS liquid fermentation culture medium according to the inoculum size of 3 percent in volume ratio, wherein the liquid filling amount of the fermentation tank is 60 percent, the fermentation temperature is 37 +/-1 ℃, and sealing and standing and culturing for 18 hours to prepare a seed solution;
transferring the seed liquid into a fermentation medium containing glycerol-producing dehydratase for fermentation by an inoculation amount of 3% in volume ratio, wherein the liquid loading amount of a fermentation tank is 50%, the fermentation temperature is 28 ℃, the micro-aerobic culture is carried out, the rotating speed is 80rpm, the pH is adjusted by 6mol/L NaOH, the pH of the whole fermentation enzyme is controlled to be 5.5, after the fermentation time is 18h, glycerol (the glycerol concentration is 150 mmol/L) which is sterilized and cooled is fed into a bacterial liquid in a variable-speed feeding mode, and the volume ratio of the glycerol solution to the bacterial liquid is as follows: 1: 99, enzyme conversion temperature: and (3) preparing the bacterium liquid of the lactobacillus reuteri containing the reuterin at the temperature of 28 ℃ for 1 h.
The MRS culture medium and the seed culture medium are all as follows: in a 1L system, 10g of peptone, 5g of yeast powder, 10g of beef extract, 5g of sodium acetate, 2g of diammonium hydrogen citrate, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 80 mL of tween, 10g of calcium carbonate and 1000mL of water are added to prepare a mixture with a pH value of 6.8; sterilizing at 115 deg.C for 30min.
The glycerol-producing dehydratase fermentation medium comprises: adding 25g of molasses, 4g of beef extract and 4g of yeast powder into 1000mL of water in a 1L system, and sterilizing at 115 ℃ for 30min.
2.3 preparation of Saccharomyces cerevisiae seed liquid
Selecting saccharomyces cerevisiae slant strains to be inoculated into a PDA liquid culture medium, and activating for 20 hours by a shaking table at the temperature of 28 +/-1 ℃; then inoculating the activated bacterial liquid into a seed liquid culture medium according to the inoculation amount of 2 percent of the volume ratio, and culturing at the temperature of 28 +/-1 ℃ for 18 hours to obtain the saccharomyces cerevisiae seed liquid.
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.
The seed liquid culture medium comprises: the composition comprises, by mass, 2% of glucose, 2% of sucrose, 3% of soybean peptone, 1% of yeast powder, 0.5% of dipotassium hydrogen phosphate, 0.5% of urea and water as a solvent, wherein the pH value is 6.0 +/-0.2.
3, crushing and sieving feed raw materials: the feed raw materials are crushed and sieved by a 40-mesh sieve, and are weighed according to the following mass ratio: 10% of corn flour, 55% of soybean meal, 25% of fish meal and 10% of wheat bran, uniformly mixing, adding water until the water content is 35%, and uniformly mixing by using a mixer to obtain the solid culture medium.
4 inoculating and fermenting
Mixing the strain bacillus subtilis seed liquid to be fermented, the saccharomyces cerevisiae seed liquid and the reuteri bacterium liquid for producing reuterin according to the volume ratio of 1.
Taking Lactobacillus reuteri bacterial liquid as a reference, uniformly inoculating the lactobacillus reuteri bacterial liquid into the solid culture medium by 10% of the wet weight of the solid culture medium, and carrying out anaerobic fermentation at a constant temperature of 28 ℃ for 72h.
5, after the anaerobic fermentation is finished, adding 0.5 percent by mass of anaerobic fermentation material premixed feed and 0.3 percent by mass of konjac refined powder into the anaerobic fermentation material, and uniformly mixing the soft pellet feed with the particle size of 2.0 prepared at normal temperature, namely the micropterus salmoides fermented compound feed (namely the functional fermentation feed for improving the glycolipid metabolism of micropterus salmoides).
The unfermented micropterus salmoides compound feed is prepared by adding 0.5 mass percent of solid culture medium premixed feed and 0.3 mass percent of konjac fine powder into a solid culture medium, and uniformly mixing soft pellet feed with the particle size of 2.0 prepared at normal temperature, namely the unfermented micropterus salmoides compound feed.
6 test results
The contents of crude protein, acid soluble protein, bioactive peptide, free amino acid, L-lactic acid and reuterin in the compound feed fermented by Micropterus salmoides before and after fermentation, and the viable count indexes of bacillus, lactic acid bacteria and yeast are respectively measured (Table 1).
TABLE 1
Example 2
1, preparing a fermentation strain: preparing Bacillus subtilis, saccharomyces cerevisiae seed solution, and Roy's bacillus solution for producing Roy's bacterin with viable count of 1.0 × 10 9 More than cfu/mL;
1.1 preparation of Bacillus subtilis seed solution
Selecting bacillus subtilis, inoculating the bacillus subtilis into an LB culture medium, and activating for 20 hours in a shaking table at the temperature of 37 +/-1 ℃; then inoculating the activated bacterial liquid into a seed liquid culture medium according to the inoculation amount of 4% of the volume ratio, and culturing at 37 +/-1 ℃ for 20 hours to obtain the bacillus subtilis seed liquid.
The LB culture medium (Luria-Bertani culture medium) formula is as follows: adding tryptone 10g, yeast powder 5g, and sodium chloride 10g into 1L distilled water, adjusting pH to 7.0, and sterilizing.
The seed liquid culture medium comprises: the nutrient solution comprises, by mass, 0.5% of glucose, 1.5% of soluble starch, 2% of peptone, 0.5% of yeast powder, 0.2% of magnesium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.03% of manganese sulfate, 0.24% of calcium carbonate and pH7.0 +/-0.2.
2.2 preparation of Roisella reuteri seed solutions for Roisella production
2.2.1 selecting a reuterin-producing reuterin strain, inoculating the strain into an MRS liquid seed culture medium, standing and culturing for 22 hours at 37 +/-1 ℃, then transferring the activated liquid strain into a fermentation tank of the MRS liquid fermentation culture medium according to the inoculum size of 4 percent in volume ratio, wherein the liquid filling amount of the fermentation tank is 70 percent, the fermentation temperature is 37 +/-1 ℃, and sealing and standing and culturing for 22 hours to prepare a seed liquid;
inoculating the seed solution into a fermentation medium containing glycerol-producing dehydratase for fermentation by an inoculum size of 4 percent in volume ratio, wherein the liquid loading amount of a fermentation tank is 58 percent, the fermentation temperature is 30 ℃, the micro-aerobic culture is carried out, the rotating speed is 90rpm, the pH is adjusted by 6mol/L NaOH, the pH of the whole fermentation enzyme is controlled to be 6.0, after the fermentation time is 21 hours, the sterilized and cooled glycerol (the glycerol concentration is 300 mmol/L) is fed into a bacterial solution in a variable speed feeding manner, and the volume ratio of the glycerol solution to the bacterial solution is as follows: 2: 98, enzyme conversion temperature: the time is 2.5h at 30 ℃, thus preparing the lactobacillus reuteri bacterial liquid containing the reuterin.
The MRS liquid fermentation culture medium comprises the following seed culture media: in a 1L system, adding 10g of peptone, 5g of yeast powder, 10g of beef extract, 5g of sodium acetate, 2g of diammonium hydrogen citrate, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 80 mL of tween and 10g of calcium carbonate into 1000mL of water, and adjusting the pH value to 6.8; sterilizing at 115 deg.C for 30min.
The glycerol producing dehydratase fermentation medium comprises: in a 1L system, 25g of molasses, 4g of beef extract and 4g of yeast powder are added with water to 1000mL at the pH value of 6.0, and sterilized for 30min at 115 ℃.
2.3 preparation of Saccharomyces cerevisiae seed liquid
Selecting a saccharomyces cerevisiae slant strain, inoculating the saccharomyces cerevisiae slant strain into a PDA liquid culture medium, and activating the strain for 22 hours in a shaking table at the temperature of 28 +/-1 ℃; then inoculating the activated bacterial liquid into a seed liquid culture medium according to the inoculation amount of 3 percent of the volume ratio, and culturing for 22 hours at the temperature of 28 +/-1 ℃ to obtain the saccharomyces cerevisiae seed liquid.
The PDA liquid culture medium formula is as follows: peeling 200g of potato, cutting, boiling, filtering to remove residues, adding 20g of sucrose, 10g of peptone and 18g of agar, adding water to a constant volume of 1L, adjusting the pH value to 7.0, and sterilizing for later use.
The seed liquid culture medium comprises: comprises 2 percent of glucose, 2 percent of sucrose, 3 percent of soybean peptone, 1 percent of yeast powder, 0.5 percent of dipotassium hydrogen phosphate, 0.5 percent of urea and pH6.0 +/-0.2 by mass fraction, and the preparation method comprises the steps of uniformly mixing the components and sterilizing for later use.
3, crushing and sieving feed raw materials: crushing the feed raw materials, sieving the crushed feed raw materials with a 40-mesh sieve, and weighing the crushed feed raw materials according to the following mass ratio: corn flour 20%, soybean meal 30%, fish meal 45%, wheat bran 5%, water content 35%, and mixing in a mixer to obtain solid culture medium.
4 inoculating and fermenting
Uniformly inoculating the strains of bacillus subtilis, saccharomyces cerevisiae seed liquid and the reuteri liquid for producing reuteri into the solid culture medium according to the volume ratio of 2 to 6, wherein the mass is 15 percent of the wet weight of the solid culture medium, and carrying out anaerobic fermentation at the constant temperature of 32 ℃ for 84 hours.
5 after the anaerobic fermentation is finished, adding 0.75 percent of micropterus salmoides premix feed and 0.1 percent of konjac fine powder by weight of anaerobic fermentation material into the anaerobic fermentation material, uniformly mixing and preparing the soft pellet feed (micropterus salmoides fermentation compound feed, and micropterus salmoides metabolism functional fermentation feed) with the particle size of 2.0 at normal temperature.
The unfermented micropterus salmoides compound feed is prepared by adding 0.75 mass percent of solid culture medium premixed feed and 0.1 mass percent of konjac fine powder into a solid culture medium, uniformly mixing soft pellet feed with the particle size of 2.0 prepared at normal temperature, and is the unfermented micropterus salmoides compound feed.
6 test results
The contents of crude protein, acid soluble protein, bioactive peptide, free amino acid, L-lactic acid and reuterin in the compound feed fermented by micropterus salmoides and the indexes of viable count of bacillus, lactic acid bacteria and saccharomycetes (table 2) are respectively measured before and after fermentation.
TABLE 2
Example 3
1, preparing a fermentation strain: preparing Bacillus subtilis, saccharomyces cerevisiae seed solution, and Roy's bacillus solution for producing Roy's bacterin with viable count of 1.0 × 10 9 cfu/mL or more;
1.1 preparation of Bacillus subtilis seed solution
Selecting bacillus subtilis, inoculating the bacillus subtilis into an LB culture medium, and activating for 24 hours in a shaking table at the temperature of 37 +/-1 ℃; then inoculating the activated bacterial liquid into a seed liquid culture medium according to the inoculation amount of 6 percent of the volume ratio, and culturing at 37 +/-1 ℃ for 24 hours to obtain the bacillus subtilis seed liquid.
The LB culture medium (Luria-Bertani culture medium) formula is as follows: adding tryptone 10g, yeast powder 5g, and sodium chloride 10g into 1L distilled water, adjusting pH to 7.0, and sterilizing.
The seed liquid culture medium comprises: the nutrient solution comprises, by mass, 0.5% of glucose, 1.5% of soluble starch, 2% of peptone, 0.5% of yeast powder, 0.2% of magnesium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.03% of manganese sulfate, 0.24% of calcium carbonate and pH7.0 +/-0.2.
2.2 preparation of Roisella reuteri seed solutions for Roisella production
2.2.1 selecting a lactobacillus reuteri strain producing reuterin, inoculating the lactobacillus reuteri strain into an MRS liquid seed culture medium, carrying out static culture at 37 +/-1 ℃ for 26 hours, then transferring the activated liquid strain into a fermentation tank of the MRS liquid fermentation culture medium according to the inoculation amount of 6% by volume, wherein the liquid filling amount of the fermentation tank is 80%, the fermentation temperature is 37 +/-1 ℃, carrying out closed static culture, and the fermentation time is 26 hours to prepare a seed solution;
transferring the seed liquid into a fermentation medium containing glycerol-producing dehydratase for fermentation by an inoculation amount of 6% in volume ratio, wherein the liquid loading amount of a fermentation tank is 65%, the fermentation temperature is 32 ℃, the micro-aerobic culture is carried out, the rotating speed is 100rpm, the pH is adjusted by 6mol/L NaOH, the pH of the whole fermentation enzyme is controlled to be 7.0, after the fermentation time is 25 hours, the sterilized and cooled glycerol (the glycerol concentration is 450 mmol/L) is fed into a bacterial liquid in a variable-speed feeding mode, and the volume ratio of the glycerol solution to the bacterial liquid is as follows: 5: 95, enzyme conversion temperature: and (3) preparing the lactobacillus reuteri liquid containing the reuterin at the temperature of 32 ℃ for 5 hours.
The MRS liquid fermentation culture medium comprises the following seed culture media: adding 10g of peptone, 5g of yeast powder, 10g of beef extract, 5g of sodium acetate, 2g of diammonium hydrogen citrate, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 80 mL of tween and 10g of calcium carbonate into a 1L system, adding water to 1000mL, and adjusting the pH value to 6.8; sterilizing at 115 deg.C for 30min.
The glycerol producing dehydratase fermentation medium comprises: in a 1L system, 25g of molasses, 4g of beef extract and 4g of yeast powder are added with water to 1000mL at the pH value of 6.0, and the mixture is sterilized for 30min at the temperature of 115 ℃.
2.3 preparation of Saccharomyces cerevisiae seed liquid
Selecting saccharomyces cerevisiae slant strains to be inoculated into a PDA liquid culture medium, and activating for 24 hours in a shaking table at the temperature of 28 +/-1 ℃; then inoculating the activated bacterial liquid into a seed liquid culture medium according to the inoculation amount of 5 percent of the volume ratio, and culturing at the temperature of 28 +/-1 ℃ for 24 hours to obtain the saccharomyces cerevisiae seed liquid.
The PDA liquid culture medium formula is as follows: peeling 200g of potato, cutting, boiling, filtering to remove residues, adding 20g of sucrose, 10g of peptone and 18g of agar, adding water to a constant volume of 1L, adjusting the pH value to 7.0, and sterilizing for later use.
The seed culture medium is as follows: comprises 2 percent of glucose, 2 percent of sucrose, 3 percent of soybean peptone, 1 percent of yeast powder, 0.5 percent of dipotassium hydrogen phosphate, 0.5 percent of urea and pH6.0 +/-0.2 by mass fraction, and the preparation method comprises the steps of uniformly mixing the components and sterilizing for later use.
3, crushing and sieving feed raw materials: crushing the feed raw materials, sieving the crushed feed raw materials with a 40-mesh sieve, and weighing the crushed feed raw materials according to the following mass ratio: 15% of corn flour, 43% of soybean meal, 35% of fish meal and 7% of wheat bran, adding water until the water content is 35%, and uniformly mixing by using a mixer to obtain the solid culture medium.
4 inoculating and fermenting
Uniformly inoculating the strains of bacillus subtilis, saccharomyces cerevisiae seed liquid and the reuteri bacterium liquid for producing reuterin into the solid culture medium according to the volume ratio of 3 to 4, wherein the mass ratio of the strains of bacillus subtilis, saccharomyces cerevisiae seed liquid and the reuteri bacterium liquid is 20 percent of the wet weight of the solid culture medium, and carrying out anaerobic fermentation at the constant temperature of 35 ℃ for 96 hours.
Taking Lactobacillus reuteri bacterial liquid as a reference, uniformly inoculating the lactobacillus reuteri bacterial liquid into the solid culture medium by 20% of the wet weight of the solid culture medium, and performing anaerobic fermentation at the constant temperature of 35 ℃ for 96 hours.
5 after the anaerobic fermentation is finished, 1.0 percent of micropterus salmoides premix feed and 0.5 percent of konjak flavor powder by weight of the anaerobic fermentation material are added into the anaerobic fermentation material, and the soft pellet feed (micropterus salmoides fermentation compound feed, namely the functional fermentation feed for improving micropterus salmoides glycolipid metabolism) with the particle size of 2.0 which is prepared at normal temperature is mixed uniformly.
The unfermented micropterus salmoides compound feed is prepared by adding 1% of solid culture medium mass of a premixed feed of micropterus salmoides and 0.5% of konjac fine powder of solid culture medium mass into a solid culture medium, and uniformly mixing soft pellet feed with the particle size of 2.0 prepared at normal temperature, namely the unfermented micropterus salmoides compound feed.
6 test results
The contents of crude protein, acid soluble protein, bioactive peptide, free amino acid, L-lactic acid and reuterin in the compound feed fermented by Micropterus salmoides before and after fermentation, and the viable count indexes of bacillus, lactic acid bacteria and yeast are respectively measured (Table 3).
TABLE 3
Example 4
The method comprises the steps of feeding the micropterus salmoides fermented compound feed prepared by fermenting in the embodiment 1 by about 8g to obtain a test group II, recording the test group II, simultaneously taking the unfermented micropterus salmoides fermented compound feed as a blank control group and recording the blank control group as Ctr0, taking the single lactobacillus reuteri fermented compound feed of the micropterus salmoides fermented compound feed as a test group I, recording the blank control group and recording the test group I, feeding the unpermus salmoides fermented compound feed of the single lactobacillus reuteri fermented compound feed for 8 weeks, and repeating the test group I by 3, wherein each test group is repeated by 30 fish. The test result is shown in figure 1, and the apparent digestibility of the fat and the total sugar of the micropterus salmoides feed in the test group I is respectively improved by 7.93 percent and 14.97 percent compared with the control group. The apparent digestibility of fat and total sugar of the Micropterus salmoides feed in the test group II is respectively improved by 26.80 percent and 39.19 percent compared with that of a control group, is obviously higher than Ctr0 (P is less than 0.05), and is improved by 17.48 percent and 21.07 percent compared with that of the test group I (P is less than 0.05). The result shows that the micropterus salmoides fermented compound feed prepared by the invention can obviously improve the utilization rate of the glycolipid of the feed, and the effect of the micropterus salmoides fermented compound feed is superior to that of the unfermented micropterus salmoides fermented compound feed and the single lactobacillus reuteri fermented micropterus salmoides fermented compound feed.
Example 5
The micropterus salmoides fermented compound feed prepared by fermenting in the example 2 is fed to about 25g of micropterus salmoides as a test group, which is marked as I, meanwhile, the unfermented micropterus salmoides compound feed is used as a blank control group, which is marked as Ctr0, the micropterus salmoides is fed for 8 weeks, 3 times are set for each test, and 20 fish times are repeated for each test. After the test is finished and after the food is fast-frozen for 24 hours, 10 fishes are randomly taken from each jar, the surfaces of the fish bodies are wiped dry by using clean gauze, intestinal tracts are dissected from ice trays, and the fish bodies are placed into a freezing tube, and are quickly frozen by using liquid nitrogen and then stored in a refrigerator at the ultralow temperature of-80 ℃. After freeze-drying intestinal tract samples of Micropterus salmoides, the content of short-chain fatty acids such as acetic acid and the like is determined by adopting a gas chromatography, and the content of biogenic amine is detected by adopting a high performance liquid chromatography.
The test results are shown in fig. 2 and 3: compared with the control group, the content of the intestinal metabolite group amine, cadaverine and putrescine in the test group I is reduced by 67.84%, 89.36% and 84.53%, and is obviously lower than that in the control group (P < 0.05). The content of the intestinal metabolites acetic acid, propionic acid and butyric acid in the test group I is respectively improved by 119.26 percent, 340.21 percent and 426.90 percent compared with the control group. The results show that: by feeding the micropterus salmoides fermented compound feed, the content of intestinal metabolite histamine, cadaverine and putrescine biogenic amine of micropterus salmoides is obviously reduced, and the content of intestinal metabolite acetic acid, propionic acid and butyric acid short-chain fatty acid of micropterus salmoides is increased. The intestinal metabolite short-chain fatty acid is regulated and controlled by feeding the micropterus salmoides fermented compound feed disclosed by the invention, so that the digestion and absorption of intestinal nutrient substances of micropterus salmoides are promoted and improved. The micropterus salmoides fermented compound feed provided by the invention is fed to regulate and control the biogenic amine level of intestinal metabolites, and can protect intestinal tracts from being damaged by glycolipid metabolic disturbance to a certain extent.
Example 6
The micropterus salmoides fermented compound feed prepared by fermenting in the embodiment 3 is fed to about 40g of micropterus salmoides test group II, which is recorded as II, meanwhile, the unfermented micropterus salmoides fermented compound feed is used as a blank control group and is recorded as Ctr0, the single lactobacillus reuteri fermented compound feed for micropterus salmoides is used as test group I, which is recorded as I, the micropterus salmoides fermented compound feed is fed for 8 weeks, each test is set to be 3, and each test is 20 repeated. After the test is finished and after the fasting for 24 hours, randomly taking 5 fishes per jar, wiping the surfaces of the fish bodies by using clean gauze, collecting blood from tail veins, centrifuging at 3000rpm and 4 ℃ for 10min, separating serum, quickly freezing by using liquid nitrogen, and storing in an ultra-low temperature refrigerator at-80 ℃ for serum glycolipid metabolism related index analysis.
The test results are shown in fig. 4 and 5. The content of triglyceride, total cholesterol, free fatty acid and low-density lipoprotein cholesterol in the micropterus salmoides serum of the test group I is respectively reduced by 9.19 percent, 10.60 percent, 8.49 percent and 10.63 percent compared with the content of the cholesterol in the control group. The serum pyruvate kinase and glucokinase of the weever in the test group I are respectively improved by 8.85 percent and 9.33 percent compared with the control group. The serum triglyceride, total cholesterol and free fatty acid contents of the weever II in the test group are respectively reduced by 41.84%, 40.63%, 36.39% and 31.50% compared with the control group, are obviously lower than those of the control group (P is less than 0.05), and are respectively reduced by 35.95%, 33.58%, 30.49% and 23.35% compared with the test group I in the test group I (P is less than 0.05). The serum pyruvate kinase and glucokinase of the weever in the test group II are respectively improved by 34.07 percent and 39.39 percent compared with those of the control group (P is less than 0.05), and the serum pyruvate kinase and the glucokinase are respectively improved by 23.17 percent and 27.49 percent compared with those of the test group I (P is less than 0.05). The result shows that the micropterus salmoides fermented compound feed can obviously improve the glycolipid metabolic capability of micropterus salmoides, and the effect of the micropterus salmoides fermented compound feed is superior to that of the conventional micropterus salmoides fermented compound feed and the single lactobacillus reuteri fermented compound feed.
Claims (7)
1. The application of the fermented feed in preparing the functional fermented feed for improving the glycolipid metabolism of the micropterus salmoides comprises the following steps:
mixing bacillus subtilis, saccharomyces cerevisiae and lactobacillus reuteri liquid for producing reuterin to obtain a composite microbial inoculum, inoculating the composite microbial inoculum into a solid culture medium for anaerobic fermentation, adding micropterus salmoides premixed feed and konjac fine powder after the anaerobic fermentation, and granulating to obtain fermented feed;
the solid culture medium is as follows: comprises 10-20% of corn flour, 35-55% of bean pulp, 25-45% of fish meal and 5-10% of wheat bran by mass; mixing, adding water until the water content is 25-40%, and mixing to obtain solid culture medium.
2. The application of the compound microbial inoculum according to claim 1 is characterized in that bacillus subtilis, saccharomyces cerevisiae and lactobacillus reuteri liquid for producing reuterin are mixed according to the volume ratio of 1-3 to 4-8, so as to obtain the compound microbial inoculum, the compound microbial inoculum is inoculated into a solid culture medium according to 10-20% of the weight of the solid culture medium, the constant-temperature anaerobic fermentation is carried out at 28-35 ℃, the fermentation time is 72-96h, after the anaerobic fermentation is finished, 0.5-1% of micropterus salmoides premixed feed and 0.1-0.5% of konjac refined powder by weight of anaerobic fermentation materials are added into the anaerobic fermentation materials, and the micropterus salmoides feed is prepared at normal temperature according to the requirements of the particle sizes of the micropterus salmoides feed in different growth stages.
3. The use of claim 1, wherein the viable count of the bacteria liquid of Bacillus subtilis, saccharomyces cerevisiae, and Roy I's bacterin producing Roy I's bacterin is 1.0 x 10 9 More than cfu/mL.
4. The application of claim 1, wherein the preparation method of the bacillus subtilis liquid comprises the following steps: selecting Bacillus subtilis, inoculating to LB culture medium, and activating with shaker at 37 + -1 deg.C for 16-24 hr; then inoculating the activated bacterial liquid into a seed liquid culture medium according to the inoculation amount of 3-6% of the volume ratio, and culturing at 37 +/-1 ℃ for 16-24h to obtain a bacillus subtilis seed liquid;
the seed liquid culture medium comprises: the compound premix comprises, by mass, 0.5% of glucose, 1.5% of soluble starch, 2% of peptone, 0.5% of yeast powder, 0.2% of magnesium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.03% of manganese sulfate, 0.24% of calcium carbonate and pH7.0 +/-0.2.
5. The application of claim 1, wherein the saccharomyces cerevisiae bacterial liquid is prepared by selecting saccharomyces cerevisiae slant strains, inoculating the strains into a PDA liquid culture medium, and performing table activation at 28 +/-1 ℃ for 20-24h; then inoculating the activated bacterial liquid into a seed liquid culture medium according to the inoculation amount of 2-5% of the volume ratio, and culturing at 28 +/-1 ℃ for 18-24h to obtain a saccharomyces cerevisiae seed liquid;
the seed liquid culture medium comprises: the nutrient solution comprises, by mass, 2% of glucose, 2% of sucrose, 3% of soybean peptone, 1% of yeast powder, 0.5% of dipotassium hydrogen phosphate, 0.5% of urea and pH6.0 +/-0.2.
6. The use of claim 1, wherein the reuterin-producing bacterium solution of lactobacillus reuteri is prepared by selecting a reuterin-producing lactobacillus reuteri strain, inoculating the reuterin-producing lactobacillus reuteri strain into an MRS liquid culture medium, performing static culture at 37 +/-1 ℃ for 18-26h, transferring the activated liquid strain into a fermentation tank of the liquid seed culture medium in an inoculation amount of 3-6% by volume, wherein the liquid filling amount of the fermentation tank is 60-80%, the fermentation temperature is 37 +/-1 ℃, performing closed static culture, and the fermentation time is 18-26h to prepare a seed solution;
transferring the seed liquid into a fermentation medium containing glycerol-producing dehydratase for fermentation by an inoculum size of 3-6% in volume ratio, controlling the liquid loading amount of a fermentation tank to be 50-65%, the fermentation temperature to be 28-32 ℃, carrying out micro-aerobic culture, rotating speed to be 80-100rpm, controlling the pH value of the whole fermentation process to be 5.5-7.0, and after the fermentation time is 18-25h, feeding sterilized and cooled glycerol into a bacterial liquid in a variable-speed feeding manner, wherein the concentration of the glycerol is 150-450mmol/L, and the volume ratio of the glycerol solution to the bacterial liquid is as follows: 1-5: 99-95, enzyme conversion temperature: preparing lactobacillus reuteri bacterial liquid containing the reuterin at the temperature of 28-32 ℃ for 1-5 hours;
the preparation method of each liter of MRS liquid culture medium and liquid seed culture medium is as follows: adding water into 10g of peptone, 5g of yeast powder, 10g of beef extract, 5g of sodium acetate, 2g of diammonium hydrogen citrate, 2g of dipotassium hydrogen phosphate, 0.2g of magnesium sulfate, 0.05g of manganese sulfate, 1mL of tween 80 and 10g of calcium carbonate to 1000mL, and adjusting the pH value to 6.8;
the preparation method of each liter of the glycerol-producing dehydratase fermentation culture medium comprises the following steps: adding 25g of molasses, 4g of beef extract and 4g of yeast powder into water, adding water to 1000ml, and adjusting the pH value to 6.0.
7. The use of claim 1, wherein said improving the metabolism of micropterus salmoides is improving the utilization of glycolipids and the digestion and absorption of nutrients in the feed by micropterus salmoides, and improving the feed conversion rate of animals.
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