CN115708547A - Application of functional fermented feed beneficial to improving micropterus salmoides glycolipid metabolism - Google Patents

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

Application of a functional fermented feed beneficial to improve glucose and lipid metabolism in California perch

Technical field:

The invention belongs to the field of animal feed, and in particular relates to the application of a functional fermented feed that is beneficial to improving the glucose and lipid metabolism of California perch.

Background technique:

Micropterus salmoides belongs to the family Ceutrarchidae of the order Perciformes, also known as largemouth bass. It is a warm-water carnivorous fish. According to the "2022 China Fishery Statistical Yearbook", the aquaculture output of California perch in my country has reached 702,100 tons. At present, California perch has basically solved the problem of farming from chilled fish to compound feed. Artificial feed can easily lead to glucose and lipid metabolism disorder, abdominal fat accumulation, and hepatomegaly, which seriously affect the meat quality and commodity value of California sea bass. Gut flora is an important member of the intestinal barrier, affecting the host's nutrient absorption, energy metabolism and immune response. There is a close relationship between the body's nutrient metabolism disorder and the intestinal flora imbalance, and the regulation of intestinal microbes is an important way to regulate the body's nutrient metabolism disorder.

The feed is fermented by functional microorganisms to feed farmed animals, and the targeted regulation of intestinal flora and metabolites through functional microorganisms and secondary metabolites is a very effective method for preventing or treating nutritional metabolism disorders in the body.

At present, there is no relevant patent related to the preparation method of functional fermented feed that promotes glucose and lipid metabolism in California sea bass.

Invention content:

The purpose of the invention is to provide the application of fermented feed in the preparation of functional fermented feed for improving the glucose and lipid metabolism of California perch.

Fermented feed of the present invention, promptly improves the functional fermented feed of California perch glucose and lipid metabolism, and its preparation method is:

Mix Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus reuteri producing reuterin to obtain a composite bacterial agent, inoculate the composite bacterial agent into a solid medium for anaerobic fermentation, and then add it after anaerobic fermentation California perch premixed feed and konjac powder, pelleted to obtain fermented feed;

The solid culture medium is: in terms of mass fraction, it includes 10%-20% of corn flour, 35-55% of soybean meal, 25-45% of fish meal, and 5-10% of wheat bran; after mixing, add water until the water content is 25- 40%, mixed evenly to obtain a solid medium.

Preferably, Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus reuteri bacteria liquid producing reuterin are mixed according to the volume ratio of 1-3:1-3:4-8 to obtain a composite bacterial agent, and the composite bacterial agent Inoculate 10-20% of the weight of the solid medium into the solid medium, perform anaerobic fermentation at a constant temperature of 28-35°C, and the fermentation time is 72-96h. After the anaerobic fermentation is completed, add the weight of the anaerobic fermentation material to the anaerobic fermentation material 0.5%-1% California perch premixed feed and 0.1-0.5% konjac fine powder, according to the particle size requirements of California perch feed at different growth stages, soft pellet feed is made at room temperature.

Preferably, the number of live bacteria of Bacillus subtilis, Saccharomyces cerevisiae, and reuterin-producing Lactobacillus reuteri are all above 1.0×10 ⁹ cfu/mL.

Preferably, the preparation method of the Bacillus subtilis bacterial liquid is as follows: pick the Bacillus subtilis and inoculate it into LB medium, activate it on a shaker at 37±1°C for 16-24h; then mix the activated bacterial liquid with a volume ratio of 3 Inoculate -6% of the inoculation amount into the seed liquid medium, and cultivate at 37±1°C for 16-24 hours to obtain the Bacillus subtilis seed liquid;

The seed liquid culture medium is: by mass fraction, including glucose 0.5%, soluble starch 1.5%, peptone 2%, yeast powder 0.5%, magnesium sulfate 0.2%, dipotassium hydrogen phosphate 0.2%, manganese sulfate, 0.03% , calcium carbonate 0.24%, pH7.0±0.2.

Preferably, the Saccharomyces cerevisiae liquid is inoculated into PDA liquid culture medium by picking Saccharomyces cerevisiae slant strains and activating it on a shaker at 28±1°C for 20-24h; then the activated bacterium liquid is 2-5% by volume The inoculation amount was inoculated into the seed liquid medium, and cultivated at 28±1°C for 18-24 hours to obtain the Saccharomyces cerevisiae seed liquid;

The seed liquid culture medium is: by mass fraction, including 2% of glucose, 2% of sucrose, 3% of soybean peptone, 1% of yeast powder, 0.5% of dipotassium hydrogen phosphate, 0.5% of urea, pH 6.0±0.2.

Preferably, the reuterin-producing Lactobacillus reuteri bacteria liquid is selected to inoculate the reuterin-producing Lactobacillus reuteri species into the MRS liquid medium, and cultured statically at 37±1°C for 18 -26h, then transfer the activated liquid strain to the fermenter of the liquid seed culture medium with an inoculation amount of 3-6% by volume, the liquid volume of the fermenter is 60-80%, and the fermentation temperature is 37±1°C. Sealed static culture, fermentation time is 18-26h, made into seed liquid;

The seed liquid is transferred to the fermentation medium containing glycerol dehydratase with a volume ratio of 3-6% for fermentation, the liquid volume of the fermentation tank is 50-65%, the fermentation temperature is 28-32 °C, micro-aerobic culture, The rotation speed is 80-100rpm, and the pH of the whole fermentation process is controlled to be 5.5-7.0. After the fermentation time is 18-25h, the sterilized and cooled glycerin is fed into the bacterial liquid by feeding at a variable speed. The glycerol concentration is 150-450mmol/L Volume ratio of solution and bacterial liquid: 1-5:99-95, enzyme conversion temperature: 28-32°C, time 1-5h, thus preparing Lactobacillus reuteri bacterial liquid containing reuterin;

Described MRS liquid culture medium, liquid seed culture medium, its preparation method per liter is: peptone 10g, yeast powder 5g, beef extract 10g, sodium acetate 5g, diamine hydrogen citrate 2g, dipotassium hydrogen phosphate 2g, Magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 80 1mL, calcium carbonate 10g, add water to 1000mL, pH6.8;

The preparation method per liter of the fermentation medium for producing glycerol dehydratase is as follows: add 25g of molasses, 4g of beef extract, and 4g of yeast powder into water, add water to 1000ml, and pH 6.0.

The content of reuterin and the number of live bacteria of Lactobacillus reuteri were detected, the content of reuterin was 120-200mmol/L, and the number of live bacteria of Lactobacillus reuteri was 1.0×10 ⁹ cfu/mL～5.0×10 ⁹ cfu/mL.

Preferably, the improvement of glycolipid metabolism in California perch is to improve the utilization of glycolipids in feed and the digestion and absorption of nutrients by California perch, and improve the feed conversion rate of animals.

The invention can regulate the structure of intestinal microbial flora, improve the distribution of intestinal flora, antagonize the colonization of harmful bacteria, and at the same time, it can target and regulate the intestinal flora and metabolites of California perch, and promote the utilization rate of glycolipids in feed and nutrition of California perch Digestion and absorption, improve the feed conversion rate of animals, and can effectively improve the utilization of California perch on the metabolism of feed glucose and lipids, reduce the accumulation of abdominal fat, reduce the occurrence of fatty liver, enhance the body's immunity, reduce the occurrence of diseases, and improve the economy and market of commercial fish value.

Description of drawings:

Fig. 1 is the experimental result of embodiment 4, and each group of histograms is respectively Ctr0, test group I and II from left to right;

Figure 2 is the content of intestinal metabolite biogenic amine;

Figure 3 shows the fatty acid content of intestinal metabolite exercise, and the bar graphs of each group are Ctr0 and test group I from left to right;

Fig. 4 is lipid metabolism content, and each group of histograms is respectively Ctr0, test group I and II from left to right;

Fig. 5 is a graph of enzyme activity, and each group of histograms is respectively Ctr0, test groups I and II from left to right.

Detailed ways:

The following examples are to further illustrate the present invention, rather than limit the present invention.

The premixed feed for California perch used below is prepared from the following raw materials in mass ratio per 1000kg: vitamin A 0.8kg, vitamin D3 0.24kg, vitamin K3 2.16kg, dl-α-tocopheryl acetate 8.40kg, vitamin B10 .86kg, vitamin B2 1.20kg, vitamin B6 0.73kg, vitamin B12 0.30kg, niacinamide 6.19kg, D-calcium pantothenate 2.45kg, folic acid 0.49kg, D-biotin 0.60kg, L-ascorbic acid-2-phosphate 20.57 kg, inositol 3.67kg, ethoxyquinoline 2.40kg, magnesium sulfate monohydrate 20.63kg, ferrous sulfate monohydrate 12.83kg, zinc sulfate monohydrate 5.74kg, copper sulfate pentahydrate 3.52kg, manganese sulfate monohydrate 3.46kg , 1% cobalt sulfate 13.20kg, 1% calcium iodate 9.9kg, 1% sodium selenite 3.3kg, all the other are zeolite powder and rice husk powder quality half and half, above-mentioned raw material is mixed, obtains California perch premixed feed. Wherein, the 1% cobalt sulfate, 1% calcium iodate and 1% sodium selenite were purchased from Guangzhou Zhizhuan Feed Co., Ltd.

Konjac fine powder was purchased from Hubei Qiangsen Konjac Technology Co., Ltd.

Lactobacillus reuteri used below is Lactobacillus reuteri GDMCC 1.614, which is preserved in Guangdong Microbial Culture Collection Center (GDMCC), strain number: GDMCC 1.614.

The Bacillus subtilis used below is Bacillus subtilis GDMCC 1.372, which is preserved in Guangdong Microbial Culture Collection Center (GDMCC), strain number: GDMCC 1.372.

The Saccharomyces cerevisiae used below is Saccharomyces cerevisiae GDMCC 2.167, which is preserved in Guangdong Microbial Culture Collection Center (GDMCC), strain number: GDMCC 2.167.

Example 1

1 Preparation of fermentation strains: preparation of Bacillus subtilis, Saccharomyces cerevisiae seed liquid, and reuterin-producing Lactobacillus reuteri bacterial liquid, the number of viable bacteria of which is above 1.0×10 ⁹ cfu/mL;

1.1 Preparation of Bacillus subtilis seed solution

Pick Bacillus subtilis and inoculate it into LB medium, and activate it on a shaking table at 37±1°C for 16 hours; then inoculate the activated bacterial liquid into the seed liquid medium according to the volume ratio of 3% inoculum, and culture it at 37±1°C for 16 hours to obtain Bacillus subtilis seed liquid.

The formula of LB medium (Luria-Bertani medium) is: add 10 g of tryptone, 5 g of yeast powder, and 10 g of sodium chloride into 1 L of distilled water, adjust the pH to 7.0, and sterilize for later use.

The seed liquid culture medium is: by mass fraction, including glucose 0.5%, soluble starch 1.5%, peptone 2%, yeast powder 0.5%, magnesium sulfate 0.2%, dipotassium hydrogen phosphate 0.2%, manganese sulfate, 0.03% , calcium carbonate 0.24%, solvent is water, pH 7.0 ± 0.2, its preparation method is to mix each component uniformly, sterilize for later use.

2.2 Preparation of Lactobacillus reuteri seed solution producing reuterin

2.2.1 Select the reuterin-producing Lactobacillus reuteri species and inoculate them into the MRS liquid seed medium, culture them statically at 37±1°C for 18 hours, and then inoculate the activated liquid strains with a volume ratio of 3%. Transfer to the fermenter of the MRS liquid fermentation medium, the liquid volume of the fermenter is 60%, the fermentation temperature is 37±1°C, the airtight static culture, the fermentation time is 18h, and the seed liquid is made;

The seed liquid is transferred to the fermentation medium containing glycerol dehydratase with a volume ratio of 3% for fermentation. The liquid volume of the fermentation tank is 50%. Adjust the pH with LNaOH to control the enzyme pH5.5 in the entire fermentation. After 18 hours of fermentation time, add sterilized and cooled glycerin (glycerin concentration 150mmol/L) to the bacterial liquid by feeding at a variable speed. The glycerin solution and the bacterial liquid volume Ratio: 1:99, enzyme conversion temperature: 28° C., and time of 1 hour, thereby preparing Lactobacillus reuteri bacteria liquid containing reuterin.

The MRS medium and seed medium are: in a 1L system, 10g of peptone, 5g of yeast powder, 10g of beef extract, 5g of sodium acetate, 2g of diamine hydrogen citrate, 2g of dipotassium hydrogen phosphate, and magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 801mL, calcium carbonate 10g, add water to 1000mL, pH6.8; sterilize at 115°C for 30min.

The fermentation medium for producing glycerol dehydratase is as follows: in a 1L system, add 25g of molasses, 4g of beef extract, and 4g of yeast powder, add water to 1000mL, pH 6.0, and sterilize at 115°C for 30min.

2.3 Preparation of Saccharomyces cerevisiae seed solution

Pick the Saccharomyces cerevisiae slant and inoculate it into the PDA liquid medium, and activate it on a shaking table at 28±1°C for 20 hours; then inoculate the activated bacterial liquid into the seed liquid medium at 28±1°C Cultivate for 18 hours to obtain Saccharomyces cerevisiae seed liquid.

The formula of PDA liquid medium is: 200g of peeled potatoes, chopped boiled juice, filtered to remove slag, added 20g of sucrose, 10g of peptone, 18g of agar, added water to adjust the volume to 1L, and adjusted the pH to 7.0.

The seed liquid culture medium is: by mass fraction, including glucose 2%, sucrose 2%, soybean peptone 3%, yeast powder 1%, dipotassium hydrogen phosphate 0.5%, urea 0.5%, solvent is water, pH6. 0±0.2, the preparation method is to mix the ingredients evenly and sterilize for later use.

3 Feed raw materials crushing and sieving: Feed raw materials are crushed through 40 meshes, weighed according to the following mass ratio: 10% corn flour, 55% soybean meal, 25% fish meal, 10% wheat bran, mix evenly, and then add water until the moisture content is 35% %, mixed evenly with a mixer to obtain a solid medium.

4 inoculation fermentation

Mix the Bacillus subtilis seed liquid, the Saccharomyces cerevisiae seed liquid, and the reuterin-producing Lactobacillus reuteri bacterial liquid in a ratio of 1:1:8 by volume, and use a solid medium with a wet weight of 10 % uniformly inoculated into the above-mentioned solid medium, 28 ℃ constant temperature anaerobic fermentation, the fermentation time is 72h.

Using the Lactobacillus reuteri bacteria liquid as a control, 10% of the wet weight of the solid medium was evenly inoculated into the above solid medium, and the constant temperature anaerobic fermentation was carried out at 28° C., and the fermentation time was 72 hours.

5 After the anaerobic fermentation is over, add the anaerobic fermentation material to the anaerobic fermentation material, which accounts for 0.5% of the California perch premix feed and the anaerobic fermentation material, which accounts for 0.3% of the konjac powder, and mix the 2.0 particle size prepared at room temperature The soft pellet feed is the California perch fermented compound feed (that is, the functional fermented feed for improving the glucose and lipid metabolism of California perch).

The unfermented California perch compound feed is made by adding 0.5% California perch premix feed and 0.3% konjac powder to the solid medium, and mixing it at room temperature to make a 2.0-grain-size soft pellet feed , which is the unfermented California perch compound feed.

6 test results

The content of crude protein, acid-soluble protein, biologically active peptide, free amino acid, L-lactic acid, reuterin and the number of viable bacteria of Bacillus, lactic acid bacteria and yeast were measured before and after fermentation (Table 1).

Table 1

Example 2

1 Preparation of fermentation strains: preparation of Bacillus subtilis, Saccharomyces cerevisiae seed liquid, and reuterin-producing Lactobacillus reuteri bacterial liquid, the number of viable bacteria of which is above 1.0×10 ⁹ cfu/mL;

1.1 Preparation of Bacillus subtilis seed solution

Pick Bacillus subtilis and inoculate it into LB medium, and activate it on a shaking table at 37±1°C for 20 hours; then inoculate the activated bacterial liquid into the seed liquid medium according to the volume ratio of 4% inoculum, and cultivate it at 37±1°C for 20 hours to obtain Bacillus subtilis seed liquid.

The formula of LB medium (Luria-Bertani medium) is: add 10 g of tryptone, 5 g of yeast powder, and 10 g of sodium chloride into 1 L of distilled water, adjust the pH to 7.0, and sterilize for later use.

The seed liquid culture medium is: by mass fraction, including glucose 0.5%, soluble starch 1.5%, peptone 2%, yeast powder 0.5%, magnesium sulfate 0.2%, dipotassium hydrogen phosphate 0.2%, manganese sulfate, 0.03% , calcium carbonate 0.24%, pH7.0±0.2, its preparation method is to mix each component uniformly, and sterilize for later use.

2.2 Preparation of Lactobacillus reuteri seed solution producing reuterin

2.2.1 Select the reuterin-producing Lactobacillus reuteri species and inoculate them into the MRS liquid seed medium, culture them statically at 37±1°C for 22 hours, and then inoculate the activated liquid strains with a volume ratio of 4%. Transfer to the fermenter of the MRS liquid fermentation medium, the liquid volume of the fermenter is 70%, the fermentation temperature is 37±1°C, the airtight static culture, the fermentation time is 22h, and the seed liquid is made;

The seed liquid is transferred to the fermentation medium containing glycerol dehydratase with a volume ratio of 4% for fermentation. The liquid volume of the fermentation tank is 58%. Adjust the pH with LNaOH to control the pH of the entire fermentation to produce enzymes at pH 6.0. After 21 hours of fermentation time, add sterilized and cooled glycerin (glycerin concentration 300mmol/L) into the bacterial solution by feeding at a variable speed. The glycerol solution and bacterial solution volume Ratio: 2:98, enzyme conversion temperature: 30° C., time 2.5 hours, thus preparing Lactobacillus reuteri bacteria liquid containing reuterin.

The MRS liquid fermentation medium and the seed medium are: in a 1L system, 10g of peptone, 5g of yeast powder, 10g of beef extract, 5g of sodium acetate, 2g of diamine hydrogen citrate, 2g of dipotassium hydrogen phosphate, Magnesium sulfate 0.2g, manganese sulfate 0.05g, Tween 80 1mL, calcium carbonate 10g, add water to 1000mL, pH6.8; sterilize at 115°C for 30min.

The fermentation medium for producing glycerol dehydratase is as follows: in a 1L system, add molasses 25g, beef extract 4g, yeast powder 4g, pH 6.0, add water to 1000mL, and sterilize at 115°C for 30min.

2.3 Preparation of Saccharomyces cerevisiae seed solution

Pick the Saccharomyces cerevisiae slant and inoculate it into the PDA liquid medium, and activate it on a shaking table at 28±1°C for 22 hours; then inoculate the activated bacterial liquid into the seed liquid medium at 28±1°C Cultivate for 22 hours to obtain Saccharomyces cerevisiae seed liquid.

The formula of PDA liquid medium is: 200g of peeled potatoes, chopped boiled juice, filtered to remove slag, added 20g of sucrose, 10g of peptone, 18g of agar, added water to make the volume to 1L, adjusted to pH 7.0, and sterilized for later use.

The seed liquid medium is: by mass fraction, including glucose 2%, sucrose 2%, soybean peptone 3%, yeast powder 1%, dipotassium hydrogen phosphate 0.5%, urea 0.5%, pH6.0±0.2, The preparation method is to mix the ingredients uniformly and sterilize for standby use.

3 Feed raw material crushing and sieving: Feed raw material is crushed through 40 meshes, weighed according to the following mass ratio: 20% corn flour, 30% soybean meal, 45% fish meal, 5% wheat bran, add water until the water content is 35%, and use The mixer mixes evenly to obtain a solid medium.

4 inoculation fermentation

Inoculate Bacillus subtilis, Saccharomyces cerevisiae seed liquid, and reuterin-producing Lactobacillus reuteri bacterial liquid in a volume ratio of 2:2:6, and uniformly inoculate them with a solid medium with a wet weight of 15%. Into the above solid medium, 32 ° C constant temperature anaerobic fermentation, the fermentation time is 84h.

5. After the anaerobic fermentation, add anaerobic fermentation material weight 0.75% California perch premixed feed and anaerobic fermentation material weight 0.1% konjac fine powder in the anaerobic fermentation material, and mix it at room temperature to make a soft pellet feed with a particle size of 2.0 (California perch fermented compound feed, functional fermented feed for improving glucose and lipid metabolism of California perch).

The unfermented California perch compound feed is made by adding 0.75% California perch premix feed and 0.1% konjac fine powder to the solid medium, and mixing it at room temperature to make soft pellet feed with a particle size of 2.0 , which is the unfermented California perch compound feed.

6 test results

The content of crude protein, acid-soluble protein, biologically active peptide, free amino acid, L-lactic acid, reuterin and the number of live bacteria of Bacillus, lactic acid bacteria and yeast were measured before and after fermentation (Table 2).

Table 2

Example 3

1 Preparation of fermentation strains: preparation of Bacillus subtilis, Saccharomyces cerevisiae seed liquid, and reuterin-producing Lactobacillus reuteri bacterial liquid, the number of viable bacteria of which is above 1.0×10 ⁹ cfu/mL;

1.1 Preparation of Bacillus subtilis seed solution

Pick Bacillus subtilis and inoculate it into LB medium, and activate it on a shaking table at 37±1°C for 24 hours; then inoculate the activated bacterial liquid into the seed liquid medium according to the volume ratio of 6% inoculum, and cultivate it at 37±1°C for 24 hours to obtain Bacillus subtilis seed liquid.

The formula of LB medium (Luria-Bertani medium) is: add 10 g of tryptone, 5 g of yeast powder, and 10 g of sodium chloride into 1 L of distilled water, adjust the pH to 7.0, and sterilize for later use.

The seed liquid culture medium is: by mass fraction, including glucose 0.5%, soluble starch 1.5%, peptone 2%, yeast powder 0.5%, magnesium sulfate 0.2%, dipotassium hydrogen phosphate 0.2%, manganese sulfate, 0.03% , calcium carbonate 0.24%, pH7.0±0.2, its preparation method is to mix each component uniformly, and sterilize for later use.

2.2 Preparation of Lactobacillus reuteri seed solution producing reuterin

2.2.1 Select the reuterin-producing Lactobacillus reuteri species and inoculate them into the MRS liquid seed medium, culture them statically at 37±1°C for 26 hours, and then inoculate the activated liquid strains with a volume ratio of 6%. Transfer to the fermenter of the MRS liquid fermentation medium, the liquid volume of the fermenter is 80%, the fermentation temperature is 37±1°C, the airtight static culture, the fermentation time is 26h, and the seed liquid is made;

The seed liquid is transferred to the fermentation medium containing glycerol dehydratase with a volume ratio of 6% for fermentation. The liquid volume of the fermentation tank is 65%. Adjust the pH with LNaOH to control the pH of the entire fermentation enzyme production to 7.0. After 25 hours of fermentation time, add sterilized and cooled glycerin (glycerin concentration 450mmol/L) into the bacterial solution by feeding at a variable speed. The glycerin solution and the bacterial solution volume Ratio: 5:95, enzyme conversion temperature: 32° C., time 5 h, thus preparing Lactobacillus reuteri bacteria liquid containing reuterin.

The MRS liquid fermentation medium and the seed medium are: 10 g of peptone, 5 g of yeast powder, 10 g of beef extract, 5 g of sodium acetate, 2 g of diamine hydrogen citrate, 2 g of dipotassium hydrogen phosphate, and 2 g of sulfuric acid in a 1 L system. Magnesium 0.2g manganese sulfate 0.05g, Tween 80 1mL, calcium carbonate 10g, add water to 1000mL, pH6.8; sterilize at 115°C for 30min.

The fermentation medium for producing glycerol dehydratase is as follows: in a 1L system, add molasses 25g, beef extract 4g, yeast powder 4g, pH 6.0, add water to 1000mL, and sterilize at 115°C for 30min.

2.3 Preparation of Saccharomyces cerevisiae seed solution

Pick the Saccharomyces cerevisiae slant and inoculate it into the PDA liquid medium, and activate it on a shaking table at 28±1°C for 24 hours; then inoculate the activated bacterial liquid into the seed liquid medium at 28±1°C Cultivate for 24 hours to obtain Saccharomyces cerevisiae seed liquid.

The formula of PDA liquid medium is: 200g of peeled potatoes, chopped boiled juice, filtered to remove slag, added 20g of sucrose, 10g of peptone, 18g of agar, added water to make the volume to 1L, adjusted to pH 7.0, and sterilized for later use.

The seed medium is: by mass fraction, including 2% glucose, 2% sucrose, 3% soybean peptone, 1% yeast powder, 0.5% dipotassium hydrogen phosphate, 0.5% urea, pH6.0±0.2, and The preparation method is to mix all components uniformly, and sterilize for standby use.

3 Feed raw material crushing and sieving: Feed raw material is crushed through 40 meshes, weighed according to the following mass ratio: 15% corn flour, 43% soybean meal, 35% fish meal, 7% wheat bran, add water until the water content is 35%, use The mixer mixes evenly to obtain a solid medium.

4 inoculation fermentation

Inoculate Bacillus subtilis, Saccharomyces cerevisiae seed liquid, and reuterin-producing Lactobacillus reuteri bacterial liquid in a volume ratio of 3:3:4, and uniformly inoculate the solid medium with a wet weight of 20% Into the above solid medium, 35 ° C constant temperature anaerobic fermentation, the fermentation time is 96h.

Lactobacillus reuteri bacteria liquid was used as a control, and 20% of the wet weight of the solid medium was evenly inoculated into the above-mentioned solid medium, and the anaerobic fermentation was carried out at a constant temperature of 35° C., and the fermentation time was 96 hours.

5. After the anaerobic fermentation finishes, add anaerobic fermentation material weight 1.0% California perch premixed feed and 0.5% konjac powder in the anaerobic fermentation material, mix the soft pellet feed of 2.0 grain diameters (California perch fermentation compound feed , that is, functional fermented feed for improving glucose and lipid metabolism in California perch).

The unfermented California perch compound feed is a 2.0-grain-size soft pellet feed prepared by adding 1% California perch premix feed and 0.5% solid medium mass to the solid medium and mixing them at room temperature. , which is the unfermented California perch compound feed.

6 test results

The content of crude protein, acid-soluble protein, biologically active peptide, free amino acid, L-lactic acid, reuterin and the number of viable bacteria of Bacillus, lactic acid bacteria and yeast were measured before and after fermentation (Table 3).

table 3

Example 4

About 8g of California perch fed with the California perch fermented compound feed obtained by the fermentation of the above-mentioned Example 1 was regarded as test group II, denoted as II, and at the same time, the unfermented California perch compound feed was used as a blank control group, denoted as Ctr0, and single Roy's milk The California perch fermented compound feed fermented by Bacillus was the experimental group Ⅰ, denoted as Ⅰ, fed for 8 weeks, and each experiment was repeated 3 times, with 30 fish in each repetition. The test results are shown in Figure 1. The apparent digestibility of dietary fat and total sugar in test group I increased by 7.93% and 14.97% respectively compared with the control group. Compared with the control group, the apparent digestibility of fat and total sugar in the experimental group II increased by 26.80% and 39.19%, significantly higher than Ctr0 (P<0.05), and increased by 17.48% and 21.07% compared with the experimental group I, significantly higher than Test group I (P<0.05). The results show that the California perch fermented compound feed prepared by the invention can significantly improve the glycolipid utilization rate of the California perch, and its effect is better than that of the unfermented California perch compound feed and the California perch fermented compound feed fermented by a single Lactobacillus reuteri.

Example 5

The California perch fermented compound feed obtained by the above-mentioned embodiment 2 was fed with about 25 g of California perch as the test group, which was designated as I, while the unfermented California perch compound feed was used as the blank control group, which was recorded as Ctr0, and fed for 8 weeks. Three replicates were set up for each experiment, with 20 fish in each replicate. After the test, after fasting for 24 hours, randomly select 10 fish from each tank, dry the surface of the fish with clean gauze, dissect the intestines on an ice tray and put them into a freezing tube. save. Intestinal samples of California perch were freeze-dried, and the content of short-chain fatty acids such as acetic acid was determined by gas chromatography, and the content of biogenic amines was detected by high-performance liquid chromatography.

The test results are shown in Figure 2 and Figure 3: compared with the control group, the contents of the intestinal metabolites histamine, cadaverine and putrescine in the test group I decreased by 67.84%, 89.36% and 84.53%, which were significantly lower than the control group (P< 0.05). The contents of intestinal metabolites acetic acid, propionic acid and butyric acid in the test group Ⅰ were increased by 119.26%, 340.21% and 426.90% respectively compared with the control group. The results show: feeding the California perch fermented compound feed of the present invention significantly reduces the content of the intestinal metabolites histamine, cadaverine and putrescine biogenic amine in the California perch, and improves the intestinal metabolites acetic acid, propionic acid and butyric short-chain fatty acids in the California perch. content. Feeding the California perch fermented compound feed of the present invention regulates short-chain fatty acids of intestinal metabolites, and promotes and improves the digestion and absorption of intestinal nutrients of California perch. Feeding the California perch fermented compound feed of the present invention regulates the level of intestinal metabolite biogenic amine, and can protect the intestinal tract from intestinal damage caused by glucose and lipid metabolism disorders to a certain extent.

Example 6

About 40g of California perch test group II was fed with the California perch fermented compound feed obtained by the fermentation in Example 3 above, which was designated as II. At the same time, the unfermented California perch compound feed was used as a blank control group, which was designated as Ctr0. A single Lactobacillus reuteri The fermented California perch fermented compound feed was the test group Ⅰ, denoted as Ⅰ, fed for 8 weeks, and each experiment was repeated 3 times, with 20 fish in each repetition. After the test, after fasting for 24 hours, randomly select 5 fish from each tank, dry the surface of the fish with clean gauze, collect blood from the tail vein, centrifuge at 3000rpm at 4°C for 10min, separate the serum, freeze it in liquid nitrogen, and store it in a -80°C ultra-low temperature refrigerator Used for the analysis of related indicators of serum glucose and lipid metabolism.

The test results are shown in Figure 4 and Figure 5. The content of serum triglyceride, total cholesterol, free fatty acid and low-density lipoprotein cholesterol in test group Ⅰ California perch decreased by 9.19%, 10.60%, 8.49% and 10.63% respectively compared with the control group. Serum pyruvate kinase and glucokinase of California perch in test group Ⅰ increased by 8.85% and 9.33% respectively compared with the control group. The content of serum triglyceride, total cholesterol and free fatty acid in California perch in test group Ⅱ was 41.84%, 40.63%, 36.39% and 31.50% lower than that of control group, significantly lower than that of control group (P<0.05), and lower than that of test group Ⅰ. 35.95%, 33.58%, 30.49% and 23.35%, significantly lower than the test group I (P <0.05). Compared with the control group, the serum pyruvate kinase and glucokinase of California perch in the test group II increased by 34.07% and 39.39%, which were significantly higher than the control group (P<0.05). Compared with the test group I, they increased by 23.17% and 27.49% respectively , significantly higher than that of the experimental group I (P<0.05). The results show that feeding the California perch fermented compound feed of the present invention can significantly improve the California perch's glycolipid metabolism, and its effect is better than that of the conventional California perch compound feed and the California perch fermented compound feed fermented by a single Lactobacillus reuteri.

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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