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CN111700157A - Probiotic feed additive for improving immunity of aquatic animals - Google Patents

Probiotic feed additive for improving immunity of aquatic animals Download PDF

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Publication number
CN111700157A
CN111700157A CN202010706369.XA CN202010706369A CN111700157A CN 111700157 A CN111700157 A CN 111700157A CN 202010706369 A CN202010706369 A CN 202010706369A CN 111700157 A CN111700157 A CN 111700157A
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parts
fermentation
feed additive
probiotic
bacillus
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张凯
王浩宇
万雪
李晓晓
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Qingdao Beibao Ocean Technology Co ltd
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Qingdao Beibao Ocean Technology Co ltd
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract

The invention discloses a probiotic feed additive for improving immunity of aquatic animals, which comprises the following raw materials in parts by mass: 20-30 parts of probiotic fermentation bacteria powder, 30-50 parts of oyster powder, 5-10 parts of cyclodextrin, 2-5 parts of vitamin, 1-2 parts of brown algae polysaccharide and 0-10 parts of complex enzyme preparation. The invention adopts the aerobic and anaerobic mixed bacteria synergistic fermentation technology to prepare the probiotic fermented feed additive through co-fermentation, obviously improves the immunity enhancing effect of the feed additive through the synergistic effect between the fermentation product and the microorganism, and has important effects on inhibiting the infection and growth of pathogenic microorganisms, recovering the balance of intestinal flora and enhancing the intestinal immune function and physiological function. The experimental result shows that the product can obviously improve the immunity of the feed additive to the organism of the aquaculture animals such as the penaeus vannamei boone and the like, improve the growth performance of the animals, reduce the feed coefficient, reduce the culture risk and improve the culture economic benefit.

Description

Probiotic feed additive for improving immunity of aquatic animals
Technical Field
The invention relates to a probiotic feed additive for improving the immunity of aquatic animals, in particular to a probiotic feed additive which is prepared by mixed fermentation of probiotics and has the function of obviously improving the immunity of aquatic animals such as penaeus vannamei boone and the like, belonging to the technical field of feed additives.
Background
The method aims to meet the increasing demand of aquatic products and solve the key problems that physiological dysfunction and diseases frequently occur to the aquaculture due to the continuous expansion of the aquaculture scale and the reduction of the growth cycle, and is also the key for the development of the aquaculture feed industry. Under the background, the antibiotic feed additive is hot-held, is widely applied to the feed industry, promotes the development of the aquaculture industry to a certain extent, but has more serious problems of high residue, high pollution, drug resistance and the like caused by antibiotics. At present, environment-friendly feed additives such as microecologics, Chinese herbal medicine feed additives and the like are developed successively, but the defects of uncertain action and efficacy, large influence of external environment and the like exist, particularly for marine cultured animals, the defects of low germination rate of probiotics, short action duration and the like exist in the existing feed additives, so that the development of novel probiotic feed additives is favorable for further promoting the development of the aquatic ecological breeding industry.
The application of the probiotic feed additive in aquaculture is increasingly wide, and the probiotic feed additive is generally used for improving the aquaculture ecological environment, improving the immunity of cultured animals and inhibiting pathogenic microorganisms, so that the occurrence of diseases is reduced. In recent years, people begin to try to use beneficial microorganisms to regulate water quality in aquaculture production, but in view of the fact that the existing probiotic feed additive for aquaculture mainly directly adds microbial agents, for example, patent CN201810145012.1 discloses a preparation method of probiotic feed for aquaculture, which takes leftovers from processing agricultural products and/or leftovers from processing marine products as raw materials, and prepares probiotic feed rich in three different habits of probiotics by performing primary fermentation curing and decontamination on the leftovers and then performing secondary fermentation by using a biological fermentation technology; patent CN201811363435.7 discloses a bacillus coagulans feed additive containing germinants for aquaculture and a preparation method thereof; the bacillus coagulans feed additive containing the germinant for aquaculture comprises the following components: bacillus coagulans powder, glucose, trehalose and corn steep liquor dry powder; the content of spores in the bacillus coagulans powder is not less than 100 hundred million CFU/g, and the problem of poor product effect of the bacillus coagulans feed additive after application due to slow germination and low germination rate of spores is solved. Although the probiotic feed additive has remarkable technical effects, a great technical bottleneck still exists in improving the comprehensive immunity of aquaculture animals and further playing the nutritional role of probiotics, and the requirement of modern high-density intensive aquaculture is difficult to meet.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the probiotic feed additive for improving the immunity of aquatic animals, and the staged aerobic and anaerobic combined mixed bacteria fermentation technology is adopted, so that the immunity of the feed additive to the organisms of the aquatic animals such as penaeus vannamei boone and the like is obviously improved, the growth performance of the animals is improved, the feed coefficient is reduced, the pollution of the aquaculture industry caused by the large-scale use of antibiotics is reduced, the aquaculture risk is reduced, and the aquaculture economic benefit is improved; in addition, the production process of the probiotic feed additive has strong operability and is convenient for large-scale popularization and application.
The invention realizes the technical effects through the following technical scheme:
a probiotic feed additive for improving the immunity of aquatic animals comprises the following raw materials in parts by mass: 20-30 parts of probiotic fermentation bacteria powder, 30-50 parts of oyster powder, 5-10 parts of cyclodextrin, 2-5 parts of vitamin, 1-2 parts of brown algae polysaccharide and 0-10 parts of complex enzyme preparation.
Preferably, the probiotic feed additive for improving the immunity of aquatic animals comprises the following raw materials in parts by mass: 28 parts of probiotic zymocyte powder, 45 parts of oyster powder, 6 parts of cyclodextrin, 3 parts of vitamin, 1.8 parts of brown alga polysaccharide and 8 parts of complex enzyme preparation.
Further, the probiotics comprise a mixed microbial inoculum of saccharomyces cerevisiae, bacillus and lactobacillus.
Further, the saccharomyces cerevisiae is purchased from Guangdong province microorganism culture collection center and is numbered as GDMCC 2.167.
Further, the bacillus is one or a mixed microbial inoculum of a plurality of bacillus subtilis, bacillus licheniformis and bacillus pumilus.
Preferably, the bacillus subtilis, the bacillus licheniformis and the bacillus pumilus can all be obtained from the China general microbiological culture Collection center; the serial numbers are respectively CGMCC1.2163, CGMCC1.0809 and CGMCC 1.3533.
Furthermore, the lactobacillus is lactobacillus casei, which can be obtained from the common microorganism center of China Committee for culture Collection of microorganisms; the serial numbers are CGMCC1.159 respectively.
Further, the preparation process of the probiotic fermentation powder specifically comprises the following steps:
(1) preparing fermentation liquor: preparing a liquid fermentation liquid according to the mass percentage of 2% of bran, 2% of soybean meal, 1% of peptone, 1% of corn steep liquor, 0.02% of potassium dihydrogen phosphate, 0.01% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate, 0.01% of calcium chloride and the balance of water, and adjusting the pH to 6.5-7.0;
(2) aerobic fermentation: inoculating saccharomyces cerevisiae and bacillus seed liquid, wherein the inoculation amount is 1-2% and 2-4% respectively, controlling the fermentation temperature to be 28-37 ℃ and the rotation speed to be 150-300 rpm; fermenting for 12-24 h under the fermentation condition;
(3) anaerobic fermentation: after the aerobic fermentation is finished, inoculating lactobacillus powder, wherein the inoculation amount is 2-4%, the fermentation temperature is 28-37 ℃, and controlling the temperature for anaerobic fermentation for 12-24 hours;
(4) low-temperature concentration: concentrating the fermentation liquor at low temperature after the anaerobic fermentation is finished;
(5) adsorption drying: and (3) adsorbing the concentrated mixture by using bran with the mass ratio of 0.5-1.0 time, and drying the mixture in a blast dryer at the temperature of 40-60 ℃ until the water content is controlled to be 8-10% to obtain the probiotic fermentation bacteria powder.
The above-mentioned BacillusThe preparation process of the seed liquid comprises the following steps: inoculating the activated bacillus to a seed culture medium, wherein the seed culture medium comprises the following components: 2% of glucose, 2% of peptone, 1% of yeast extract, 0.01% of magnesium sulfate, 0.003% of manganese sulfate, and 6.5-7.0 of pH, and culturing for 16-24 h at the temperature of 28-37 ℃ and the rotating speed of 150-3000 rpm, wherein the effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL。
The process for activating the bacillus comprises the following steps: the method adopts a culture medium LB solid culture medium commonly used in the field, and cultures the strain for 16-24 hours at the temperature of 28-37 ℃.
The preparation method of the yeast seed liquid comprises the following steps: under the aseptic operation condition, two-ring strains are picked from the inclined plane of the yeast YDP by using an inoculating ring, inoculated into a 500mL triangular flask filled with 300mL PDA liquid culture medium, subjected to shaking culture at the temperature of between 28 and 32 ℃ at 140r/min for 24 hours, and then inoculated into a seed tank for expanded culture according to the process to prepare the yeast seed liquid, wherein the effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL。
Most preferably, the bacillus is a mixed microbial inoculum of bacillus subtilis, bacillus licheniformis and bacillus pumilus, and the inoculation ratio of the bacillus is 1:2: 1.
Further, the compound enzyme preparation is one or a mixture of more of amylase, mannanase and cellulase.
Optimally, the compound enzyme preparation is a mixture of amylase, mannase and cellulase, and the mass ratio of the compound enzyme preparation to the cellulase is 3:1: 2.
The invention also provides a preparation method of the probiotic feed additive for improving the immunity of aquatic animals, which specifically comprises the following steps:
(1) preparing oyster powder particles, namely performing ultrasonic oscillation on oyster shell powder to respectively form particles of 40-80 nm;
(2) adding oyster powder into deionized water with the mass ratio of 10 times, then adding cyclodextrin, and uniformly stirring and dispersing to obtain a mixed solution of the oyster powder and the cyclodextrin;
(3) adding probiotic zymocyte powder, vitamins, brown algae polysaccharide and a complex enzyme preparation into a mixed solution of oyster powder and cyclodextrin according to a proportion;
(4) and after mixing, heating at the temperature of 60-65 ℃, ultrasonically oscillating for 1-2 hours, cooling, filtering, and vacuum drying at low temperature to obtain the probiotic feed additive for improving the immunity of aquatic animals.
The invention provides a probiotic feed additive for improving the immunity of aquatic animals, which has multiple effects of prevention, health care, treatment, immunity regulation and the like, and is an ideal, green, environment-friendly, safe and novel feed additive for replacing antibiotic medicines. The feed additive can effectively condition the intestinal health of aquatic animals such as penaeus vannamei boone and the like, treat both symptoms and root causes, consolidate the constitution and cultivate the vitality, keep the intestinal microecological balance and the optimal immune state, improve the digestibility of nutrient substances and improve the immunity of animal organisms; secondly, the occurrence of pathogenic coliform bacteria, clostridium, salmonella and other harmful bacteria can be inhibited and prevented, intestinal probiotics are increased, and healthy intestinal flora is maintained; thirdly, the immunity is enhanced, and the formation of local antibodies and the activity of macrophages are increased by stimulating immune system cells in intestinal tracts; fourthly, the feed additive enters into intestines to resist harmful bacteria, reduce the formation of stink and harmful ingredients, eliminate the stink of the discharged excrement, protect the breeding environment and simultaneously improve the meat quality of aquaculture animals such as the penaeus vannamei boone and the like.
The invention provides a probiotic feed additive for improving immunity of aquatic animals, wherein the mass ratio of a probiotic feed additive product used in an aquatic feed complete feed is 0.1-0.5%: or the feed additive is added into premix and concentrated feed according to the proportion required by complete feed for use.
The invention provides a probiotic feed additive for improving the immunity of aquatic animals, which has the following remarkable advantages compared with the prior art:
(1) the probiotic fermented feed additive is prepared by co-fermenting aerobic bacteria (saccharomyces cerevisiae and bacillus) and anaerobic bacteria (lactobacillus casei), the immunity enhancing effect of the feed additive is remarkably improved through the synergistic effect among microorganisms, and the probiotic fermented feed additive has important effects on promoting the growth of intestinal probiotics such as lactobacillus and the like, inhibiting the growth of pathogenic bacteria such as vibrio parahaemolyticus and the like, recovering the balance of intestinal flora and enhancing the intestinal immunity and physiological functions after entering animal intestinal tracts; the product efficacy is obviously higher than that of a single fermentation strain feed additive;
(2) oyster shell powder is applied to the feed additive, the oyster shell contains a large number of 2-10 mu m microporous structures, contains a plurality of biological activity amino polysaccharides and characteristic proteins, has strong adsorption capacity and can contain molecules with a certain size, and the oyster shell powder is crosslinked with beta-cyclodextrin, so that probiotics, vitamins, brown algae polysaccharide and a complex enzyme preparation can be well adsorbed and coated, a stable feed additive reaction system is constructed, and the action time of the feed additive is prolonged;
(3) according to the invention, the brown algae polysaccharide is added into the feed, so that the function of promoting the germination of probiotics disclosed in the patent CN201811363435.7 can be exerted, the physiological activity of the brown algae polysaccharide can be exerted, and the immune function of the product is further enhanced; meanwhile, experimental data show that the exertion of the functions of the algal polysaccharides is closely related to the composition of the complex enzyme preparation;
(4) the probiotic feed additive is simple in production process and convenient to popularize and apply in large scale.
Detailed Description
In the following examples, the preparation process of the bacillus seed liquid was: inoculating the activated bacillus to a seed culture medium, wherein the seed culture medium comprises the following components: 2% of glucose, 2% of peptone, 1% of yeast extract, 0.01% of magnesium sulfate, 0.003% of manganese sulfate, and 6.5-7.0 of pH, and culturing for 16-24 h at the temperature of 28-37 ℃ and the rotating speed of 150-3000 rpm, wherein the effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL; the process for activating the bacillus comprises the following steps: the method adopts a culture medium LB solid culture medium commonly used in the field, and cultures the strain for 16-24 hours at the temperature of 28-37 ℃.
The preparation method of the yeast seed liquid comprises the following steps: under the aseptic operation condition, two rings of strains are picked from the inclined plane of the yeast YDP by using an inoculating ring, inoculated into a 500mL triangular flask containing 300mLPDA liquid culture medium, subjected to shaking culture at the temperature of between 28 and 32 ℃ and at the speed of 140r/min for 24 hours, and then inoculated into a seed tank for expanded culture according to the process to prepare the yeast seed liquidThe effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL。
The saccharomyces cerevisiae is purchased from Guangdong province microorganism strain preservation center and is numbered as GDMCC 2.167; the bacillus subtilis, the bacillus licheniformis and the bacillus pumilus can all be obtained from the common microorganism center of China Committee for culture Collection of microorganisms; the serial numbers are respectively CGMCC1.2163, CGMCC1.0809 and CGMCC 1.3533; the lactobacillus is lactobacillus casei, and can be obtained from the common microorganism center of China Committee for culture Collection of microorganisms; the serial numbers are CGMCC1.159 respectively.
Example 1
A probiotic feed additive for improving the immunity of aquatic animals comprises the following specific preparation process steps:
(1) preparing fermentation liquor: preparing a liquid fermentation liquid according to the mass percentage of 2% of bran, 2% of soybean meal, 1% of peptone, 1% of corn steep liquor, 0.02% of potassium dihydrogen phosphate, 0.01% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate, 0.01% of calcium chloride and the balance of water, and adjusting the pH to 6.5-7.0;
(2) aerobic fermentation: inoculating 2% and 3% of saccharomyces cerevisiae and bacillus seed liquid respectively, controlling the fermentation temperature to be 28-37 ℃ and the rotation speed to be 150-300 rpm; fermenting for 24h under the fermentation condition; the bacillus is a mixed microbial inoculum of bacillus subtilis, bacillus licheniformis and bacillus pumilus, and the inoculation ratio of the bacillus is 1:2: 1;
(3) anaerobic fermentation: after the aerobic fermentation is finished, inoculating lactobacillus powder, wherein the inoculation amount is 3%, the fermentation temperature is 28-37 ℃, and controlling the temperature for anaerobic fermentation for 18 h;
(4) low-temperature concentration: concentrating the fermentation liquor at low temperature after the anaerobic fermentation is finished;
(5) adsorption drying: after concentration, wheat bran with the mass ratio of 0.8 times is used for adsorption, and the mixture is dried in a blast drier at the temperature of 40-60 ℃, and the moisture is controlled to be 8-10%, so that the probiotic fermentation bacteria powder is obtained;
(6) preparing oyster powder particles, namely performing ultrasonic oscillation on 45 parts of oyster shell powder to respectively form particles of 40-80 nm;
(7) adding oyster powder into deionized water with the mass ratio of 10 times, then adding 6 parts of cyclodextrin, and uniformly stirring and dispersing to obtain a mixed solution of the oyster powder and the cyclodextrin;
(8) adding 28 parts of probiotic zymocyte powder, 3 parts of vitamin, 1.8 parts of brown alga polysaccharide and 8 parts of complex enzyme preparation into a mixed solution of oyster powder and cyclodextrin according to the proportion; the compound enzyme preparation is a mixture of amylase, mannase and cellulase, and the mass ratio of the compound enzyme preparation to the cellulase is 3:1: 2;
(9) and after mixing, heating at 60-65 ℃, ultrasonically oscillating for 2h, cooling, filtering, and vacuum drying at low temperature to obtain the probiotic feed additive for improving the immunity of aquatic animals.
Example 2
(1) Preparing fermentation liquor: preparing a liquid fermentation liquid according to the mass percentage of 2% of bran, 2% of soybean meal, 1% of peptone, 1% of corn steep liquor, 0.02% of potassium dihydrogen phosphate, 0.01% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate, 0.01% of calcium chloride and the balance of water, and adjusting the pH to 6.5-7.0;
(2) aerobic fermentation: inoculating 2% and 3% of saccharomyces cerevisiae and bacillus seed liquid respectively, controlling the fermentation temperature to be 28-37 ℃ and the rotation speed to be 150-300 rpm; fermenting for 24h under the fermentation condition; the bacillus is a mixed microbial inoculum of bacillus subtilis, bacillus licheniformis and bacillus pumilus, and the inoculation ratio of the bacillus is 1:2: 1;
(3) anaerobic fermentation: after the aerobic fermentation is finished, inoculating lactobacillus powder, wherein the inoculation amount is 3%, the fermentation temperature is 28-37 ℃, and controlling the temperature for anaerobic fermentation for 18 h;
(4) low-temperature concentration: concentrating the fermentation liquor at low temperature after the anaerobic fermentation is finished;
(5) adsorption drying: after concentration, wheat bran with the mass ratio of 0.8 times is used for adsorption, and the mixture is dried in a blast drier at the temperature of 40-60 ℃, and the moisture is controlled to be 8-10%, so that the probiotic fermentation bacteria powder is obtained;
(6) preparing oyster powder particles, namely performing ultrasonic oscillation on 50 parts of oyster shell powder to respectively form particles of 40-80 nm;
(7) adding oyster powder into deionized water with the mass ratio of 10 times, then adding 5 parts of cyclodextrin, and uniformly stirring and dispersing to obtain a mixed solution of the oyster powder and the cyclodextrin;
(8) adding 30 parts of probiotic zymocyte powder, 2 parts of vitamin and 2 parts of brown alga polysaccharide into a mixed solution of oyster powder and cyclodextrin according to the proportion;
(9) and after mixing, heating at the temperature of 60-65 ℃, ultrasonically oscillating for 1-2 hours, cooling, filtering, and vacuum drying at low temperature to obtain the probiotic feed additive for improving the immunity of aquatic animals.
Example 3
A probiotic feed additive for improving the immunity of aquatic animals comprises the following specific preparation process steps:
(1) preparing fermentation liquor: preparing a liquid fermentation liquid according to the mass percentage of 2% of bran, 2% of soybean meal, 1% of peptone, 1% of corn steep liquor, 0.02% of potassium dihydrogen phosphate, 0.01% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate, 0.01% of calcium chloride and the balance of water, and adjusting the pH to 6.5-7.0;
(2) aerobic fermentation: inoculating 2% and 3% of saccharomyces cerevisiae and bacillus seed liquid respectively, controlling the fermentation temperature to be 28-37 ℃ and the rotation speed to be 150-300 rpm; fermenting for 24h under the fermentation condition; the bacillus is a mixed microbial inoculum of bacillus subtilis, bacillus licheniformis and bacillus pumilus, and the inoculation ratio of the bacillus is 1:2: 1;
(3) anaerobic fermentation: after the aerobic fermentation is finished, inoculating lactobacillus powder, wherein the inoculation amount is 3%, the fermentation temperature is 28-37 ℃, and controlling the temperature for anaerobic fermentation for 18 h;
(4) low-temperature concentration: concentrating the fermentation liquor at low temperature after the anaerobic fermentation is finished;
(5) adsorption drying: after concentration, wheat bran with the mass ratio of 0.8 times is used for adsorption, and the mixture is dried in a blast drier at the temperature of 40-60 ℃, and the moisture is controlled to be 8-10%, so that the probiotic fermentation bacteria powder is obtained;
(6) preparing oyster powder particles, namely performing ultrasonic oscillation on 30 parts of oyster shell powder to respectively form particles of 40-80 nm;
(7) adding oyster powder into deionized water with the mass ratio of 10 times, then adding 10 parts of cyclodextrin, and uniformly stirring and dispersing to obtain a mixed solution of the oyster powder and the cyclodextrin;
(8) adding 20 parts of probiotic zymocyte powder, 5 parts of vitamin, 1 part of brown alga polysaccharide and 10 parts of complex enzyme preparation into a mixed solution of oyster powder and cyclodextrin according to the proportion; the compound enzyme preparation is a mixture of amylase, mannase and cellulase, and the mass ratio of the compound enzyme preparation to the cellulase is 3:1: 2;
(9) and after mixing, heating at the temperature of 60-65 ℃, ultrasonically oscillating for 1-2 hours, cooling, filtering, and vacuum drying at low temperature to obtain the probiotic feed additive for improving the immunity of aquatic animals.
Example 4
A probiotic feed additive for improving the immunity of aquatic animals comprises the following specific preparation process steps:
(1) preparing fermentation liquor: preparing a liquid fermentation liquid according to the mass percentage of 2% of bran, 2% of soybean meal, 1% of peptone, 1% of corn steep liquor, 0.02% of potassium dihydrogen phosphate, 0.01% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate, 0.01% of calcium chloride and the balance of water, and adjusting the pH to 6.5-7.0;
(2) aerobic fermentation: inoculating 2% and 3% of saccharomyces cerevisiae and bacillus seed liquid respectively, controlling the fermentation temperature to be 28-37 ℃ and the rotation speed to be 150-300 rpm; fermenting for 24h under the fermentation condition; the bacillus is bacillus subtilis;
(3) anaerobic fermentation: after the aerobic fermentation is finished, inoculating lactobacillus powder, wherein the inoculation amount is 3%, the fermentation temperature is 28-37 ℃, and controlling the temperature for anaerobic fermentation for 18 h;
(4) low-temperature concentration: concentrating the fermentation liquor at low temperature after the anaerobic fermentation is finished;
(5) adsorption drying: after concentration, wheat bran with the mass ratio of 0.8 times is used for adsorption, and the mixture is dried in a blast drier at the temperature of 40-60 ℃, and the moisture is controlled to be 8-10%, so that the probiotic fermentation bacteria powder is obtained;
(6) preparing oyster powder particles, namely performing ultrasonic oscillation on 45 parts of oyster shell powder to respectively form particles of 40-80 nm;
(7) adding oyster powder into deionized water with the mass ratio of 10 times, then adding 6 parts of cyclodextrin, and uniformly stirring and dispersing to obtain a mixed solution of the oyster powder and the cyclodextrin;
(8) adding 28 parts of probiotic zymocyte powder, 3 parts of vitamin, 1.8 parts of brown alga polysaccharide and 8 parts of complex enzyme preparation into a mixed solution of oyster powder and cyclodextrin according to the proportion; the compound enzyme preparation is a mixture of amylase, mannase and cellulase, and the mass ratio of the compound enzyme preparation to the cellulase is 3:1: 2;
(9) and after mixing, heating at 60-65 ℃, ultrasonically oscillating for 2h, cooling, filtering, and vacuum drying at low temperature to obtain the probiotic feed additive for improving the immunity of aquatic animals.
Example 5
A probiotic feed additive for improving the immunity of aquatic animals comprises the following specific preparation process steps:
(1) preparing fermentation liquor: preparing a liquid fermentation liquid according to the mass percentage of 2% of bran, 2% of soybean meal, 1% of peptone, 1% of corn steep liquor, 0.02% of potassium dihydrogen phosphate, 0.01% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate, 0.01% of calcium chloride and the balance of water, and adjusting the pH to 6.5-7.0;
(2) aerobic fermentation: inoculating 2% and 3% of saccharomyces cerevisiae and bacillus seed liquid respectively, controlling the fermentation temperature to be 28-37 ℃ and the rotation speed to be 150-300 rpm; fermenting for 24h under the fermentation condition; the bacillus is bacillus licheniformis;
(3) anaerobic fermentation: after the aerobic fermentation is finished, inoculating lactobacillus powder, wherein the inoculation amount is 3%, the fermentation temperature is 28-37 ℃, and controlling the temperature for anaerobic fermentation for 18 h;
(4) low-temperature concentration: concentrating the fermentation liquor at low temperature after the anaerobic fermentation is finished;
(5) adsorption drying: after concentration, wheat bran with the mass ratio of 0.8 times is used for adsorption, and the mixture is dried in a blast drier at the temperature of 40-60 ℃, and the moisture is controlled to be 8-10%, so that the probiotic fermentation bacteria powder is obtained;
(6) preparing oyster powder particles, namely performing ultrasonic oscillation on 45 parts of oyster shell powder to respectively form particles of 40-80 nm;
(7) adding oyster powder into deionized water with the mass ratio of 10 times, then adding 6 parts of cyclodextrin, and uniformly stirring and dispersing to obtain a mixed solution of the oyster powder and the cyclodextrin;
(8) adding 28 parts of probiotic zymocyte powder, 3 parts of vitamin, 1.8 parts of brown alga polysaccharide and 8 parts of complex enzyme preparation into a mixed solution of oyster powder and cyclodextrin according to the proportion; the compound enzyme preparation is a mixture of amylase, mannase and cellulase, and the mass ratio of the compound enzyme preparation to the cellulase is 3:1: 2;
(9) and after mixing, heating at 60-65 ℃, ultrasonically oscillating for 2h, cooling, filtering, and vacuum drying at low temperature to obtain the probiotic feed additive for improving the immunity of aquatic animals.
Example 6
A probiotic feed additive for improving the immunity of aquatic animals comprises the following specific preparation process steps:
(1) preparing fermentation liquor: preparing a liquid fermentation liquid according to the mass percentage of 2% of bran, 2% of soybean meal, 1% of peptone, 1% of corn steep liquor, 0.02% of potassium dihydrogen phosphate, 0.01% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate, 0.01% of calcium chloride and the balance of water, and adjusting the pH to 6.5-7.0;
(2) aerobic fermentation: inoculating 2% and 3% of saccharomyces cerevisiae and bacillus seed liquid respectively, controlling the fermentation temperature to be 28-37 ℃ and the rotation speed to be 150-300 rpm; fermenting for 24h under the fermentation condition; the bacillus is bacillus pumilus;
(3) anaerobic fermentation: after the aerobic fermentation is finished, inoculating lactobacillus powder, wherein the inoculation amount is 3%, the fermentation temperature is 28-37 ℃, and controlling the temperature for anaerobic fermentation for 18 h;
(4) low-temperature concentration: concentrating the fermentation liquor at low temperature after the anaerobic fermentation is finished;
(5) adsorption drying: after concentration, wheat bran with the mass ratio of 0.8 times is used for adsorption, and the mixture is dried in a blast drier at the temperature of 40-60 ℃, and the moisture is controlled to be 8-10%, so that the probiotic fermentation bacteria powder is obtained;
(6) preparing oyster powder particles, namely performing ultrasonic oscillation on 45 parts of oyster shell powder to respectively form particles of 40-80 nm;
(7) adding oyster powder into deionized water with the mass ratio of 10 times, then adding 6 parts of cyclodextrin, and uniformly stirring and dispersing to obtain a mixed solution of the oyster powder and the cyclodextrin;
(8) adding 28 parts of probiotic zymocyte powder, 3 parts of vitamin, 1.8 parts of brown alga polysaccharide and 8 parts of complex enzyme preparation into a mixed solution of oyster powder and cyclodextrin according to the proportion; the compound enzyme preparation is a mixture of amylase, mannase and cellulase, and the mass ratio of the compound enzyme preparation to the cellulase is 3:1: 2;
(9) and after mixing, heating at 60-65 ℃, ultrasonically oscillating for 2h, cooling, filtering, and vacuum drying at low temperature to obtain the probiotic feed additive for improving the immunity of aquatic animals.
Example 7
A probiotic feed additive for improving the immunity of aquatic animals comprises the following specific preparation process steps:
(1) preparing fermentation liquor: preparing a liquid fermentation liquid according to the mass percentage of 2% of bran, 2% of soybean meal, 1% of peptone, 1% of corn steep liquor, 0.02% of potassium dihydrogen phosphate, 0.01% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate, 0.01% of calcium chloride and the balance of water, and adjusting the pH to 6.5-7.0;
(2) aerobic fermentation: inoculating 2% and 3% of saccharomyces cerevisiae and bacillus seed liquid respectively, controlling the fermentation temperature to be 28-37 ℃ and the rotation speed to be 150-300 rpm; fermenting for 24h under the fermentation condition; the bacillus is a mixed microbial inoculum of bacillus subtilis, bacillus licheniformis and bacillus pumilus, and the inoculation ratio of the bacillus is 1:2: 1;
(3) anaerobic fermentation: after the aerobic fermentation is finished, inoculating lactobacillus powder, wherein the inoculation amount is 3%, the fermentation temperature is 28-37 ℃, and controlling the temperature for anaerobic fermentation for 18 h;
(4) low-temperature concentration: concentrating the fermentation liquor at low temperature after the anaerobic fermentation is finished;
(5) adsorption drying: after concentration, wheat bran with the mass ratio of 0.8 times is used for adsorption, and the mixture is dried in a blast drier at the temperature of 40-60 ℃, and the moisture is controlled to be 8-10%, so that the probiotic fermentation bacteria powder is obtained;
(6) preparing oyster powder particles, namely performing ultrasonic oscillation on 45 parts of oyster shell powder to respectively form particles of 40-80 nm;
(7) adding oyster powder into deionized water with the mass ratio of 10 times, then adding 6 parts of cyclodextrin, and uniformly stirring and dispersing to obtain a mixed solution of the oyster powder and the cyclodextrin;
(8) adding 28 parts of probiotic zymocyte powder, 3 parts of vitamin, 1.8 parts of brown alga polysaccharide and 8 parts of complex enzyme preparation into a mixed solution of oyster powder and cyclodextrin according to the proportion; the compound enzyme preparation is a mixture of amylase and cellulase, and the mass ratio of the compound enzyme preparation to the cellulase is 3: 2;
(9) and after mixing, heating at 60-65 ℃, ultrasonically oscillating for 2h, cooling, filtering, and vacuum drying at low temperature to obtain the probiotic feed additive for improving the immunity of aquatic animals.
Example 8
A probiotic feed additive for improving the immunity of aquatic animals comprises the following specific preparation process steps:
(1) preparing fermentation liquor: preparing a liquid fermentation liquid according to the mass percentage of 2% of bran, 2% of soybean meal, 1% of peptone, 1% of corn steep liquor, 0.02% of potassium dihydrogen phosphate, 0.01% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate, 0.01% of calcium chloride and the balance of water, and adjusting the pH to 6.5-7.0;
(2) aerobic fermentation: inoculating 2% and 3% of saccharomyces cerevisiae and bacillus seed liquid respectively, controlling the fermentation temperature to be 28-37 ℃ and the rotation speed to be 150-300 rpm; fermenting for 24h under the fermentation condition; the bacillus is a mixed microbial inoculum of bacillus subtilis, bacillus licheniformis and bacillus pumilus, and the inoculation ratio of the bacillus is 1:2: 1;
(3) anaerobic fermentation: after the aerobic fermentation is finished, inoculating lactobacillus powder, wherein the inoculation amount is 3%, the fermentation temperature is 28-37 ℃, and controlling the temperature for anaerobic fermentation for 18 h;
(4) low-temperature concentration: concentrating the fermentation liquor at low temperature after the anaerobic fermentation is finished;
(5) adsorption drying: after concentration, wheat bran with the mass ratio of 0.8 times is used for adsorption, and the mixture is dried in a blast drier at the temperature of 40-60 ℃, and the moisture is controlled to be 8-10%, so that the probiotic fermentation bacteria powder is obtained;
(6) preparing oyster powder particles, namely performing ultrasonic oscillation on 45 parts of oyster shell powder to respectively form particles of 40-80 nm;
(7) adding oyster powder into deionized water with the mass ratio of 10 times, then adding 6 parts of cyclodextrin, and uniformly stirring and dispersing to obtain a mixed solution of the oyster powder and the cyclodextrin;
(8) adding 28 parts of probiotic zymocyte powder, 3 parts of vitamin, 1.8 parts of brown alga polysaccharide and 8 parts of complex enzyme preparation into a mixed solution of oyster powder and cyclodextrin according to the proportion; the compound enzyme preparation is mannanase;
(9) and after mixing, heating at 60-65 ℃, ultrasonically oscillating for 2h, cooling, filtering, and vacuum drying at low temperature to obtain the probiotic feed additive for improving the immunity of aquatic animals.
Example 9
A probiotic feed additive for improving the immunity of aquatic animals comprises the following specific preparation process steps:
(1) preparing fermentation liquor: preparing a liquid fermentation liquid according to the mass percentage of 2% of bran, 2% of soybean meal, 1% of peptone, 1% of corn steep liquor, 0.02% of potassium dihydrogen phosphate, 0.01% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate, 0.01% of calcium chloride and the balance of water, and adjusting the pH to 6.5-7.0;
(2) aerobic fermentation: inoculating 2% and 3% of saccharomyces cerevisiae and bacillus seed liquid respectively, controlling the fermentation temperature to be 28-37 ℃ and the rotation speed to be 150-300 rpm; fermenting for 24h under the fermentation condition; the bacillus is a mixed microbial inoculum of bacillus subtilis, bacillus licheniformis and bacillus pumilus, and the inoculation ratio of the bacillus is 1:2: 1;
(3) anaerobic fermentation: after the aerobic fermentation is finished, inoculating lactobacillus powder, wherein the inoculation amount is 3%, the fermentation temperature is 28-37 ℃, and controlling the temperature for anaerobic fermentation for 18 h;
(4) low-temperature concentration: concentrating the fermentation liquor at low temperature after the anaerobic fermentation is finished;
(5) adsorption drying: after concentration, wheat bran with the mass ratio of 0.8 times is used for adsorption, and the mixture is dried in a blast drier at the temperature of 40-60 ℃, and the moisture is controlled to be 8-10%, so that the probiotic fermentation bacteria powder is obtained;
(6) preparing oyster powder particles, namely performing ultrasonic oscillation on 45 parts of oyster shell powder to respectively form particles of 40-80 nm;
(7) adding oyster powder into deionized water with the mass ratio of 10 times, then adding 6 parts of cyclodextrin, and uniformly stirring and dispersing to obtain a mixed solution of the oyster powder and the cyclodextrin;
(8) adding 28 parts of probiotic zymocyte powder, 3 parts of vitamin, 1.8 parts of brown algae polysaccharide and a complex enzyme preparation in proportion into a mixed solution of oyster powder and cyclodextrin;
(9) and after mixing, heating at 60-65 ℃, ultrasonically oscillating for 2h, cooling, filtering, and vacuum drying at low temperature to obtain the probiotic feed additive for improving the immunity of aquatic animals.
Example 10
Experimental animals: penaeus vannamei Boone;
address: an Lankangbao ocean science and technology Limited Langmuir cultivation base;
experimental groups: respectively adding the feed additives of examples 1-9 and comparative examples 1-2 into feed according to the proportion of 0.5%, uniformly mixing, and feeding, wherein the comparative example 1 is a blank control group, no feed additive is added into the feed, and no bacillus is added into the feed additive added in the comparative example 2 in aerobic fermentation;
3 parallel experiments are set in each group, the size of the pool is 4.2 multiplied by 5.4 multiplied by 1.2m, 20000 shrimp larvae are thrown in each pool, and the feeding time is 3 months. The enzyme activity index is as follows: randomly selecting 10 shrimps from each pond, and detecting the content of superoxide dismutase (SOD) and lysozyme (detected by Shanghai Tong Ulmus laboratory Co., Ltd.);
the results of the experiments are shown in table 1:
TABLE 1 cultivation experimental results
Figure DEST_PATH_IMAGE001
The experimental results show that the feed additive in the example 1 can obviously promote the growth of the penaeus vannamei boone and improve the contents of superoxide dismutase and lysozyme, and compared with a blank control group (comparative example 1), the difference is very obvious; the experimental results of examples 2-3 show that the effect of enhancing immunity of the feed additive can be obviously reduced when the composition of the raw materials of the feed additive is changed, and particularly, the experimental results of example 2 show that when the raw materials of the additive do not contain the compound enzyme preparation, the effect is obviously different from that of example 1 although brown algae polysaccharide is added in the raw materials; the experimental results of examples 4-6 show that when the bacillus is a single bacillus subtilis, bacillus licheniformis or bacillus pumilus, the immune enhancing efficacy of the additive is severely affected, and the difference is not significant compared with the control group without bacillus (comparative example 2), especially the experimental group with bacillus pumilus (example 6), and a negative effect is generated; the experimental results of examples 7-9 show that the composition of the complex enzyme preparation has a significant effect on the immune enhancing effect of the additive, the amylase, the cellulase and the mannase have a significant synergistic effect, and the analysis reason may be the reason that the mannase can degrade brown algae polysaccharides into brown algae oligosaccharides with stronger activity. For comparison, example 1 is the best mode of carrying out the invention.
The above embodiments are only used for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; such modifications and substitutions do not depart from the spirit and scope of the present invention as set forth in the appended claims.

Claims (10)

1. The probiotic feed additive for improving the immunity of aquatic animals is characterized by comprising the following raw materials in parts by mass: 20-30 parts of probiotic fermentation bacteria powder, 30-50 parts of oyster powder, 5-10 parts of cyclodextrin, 2-5 parts of vitamin, 1-2 parts of brown algae polysaccharide and 0-10 parts of complex enzyme preparation.
2. The probiotic feed additive for improving the immunity of aquatic animals according to claim 1, wherein the raw materials of the additive comprise, by mass: 28 parts of probiotic zymocyte powder, 45 parts of oyster powder, 6 parts of cyclodextrin, 3 parts of vitamin, 1.8 parts of brown alga polysaccharide and 8 parts of complex enzyme preparation.
3. The probiotic feed additive for improving the immunity of aquatic animals according to claim 1, wherein the probiotic comprises a mixed microbial inoculum of saccharomyces cerevisiae, bacillus and lactobacillus; the saccharomyces cerevisiae is purchased from Guangdong province microorganism strain preservation center and is numbered as GDMCC 2.167.
4. The probiotic feed additive for improving the immunity of aquatic animals according to claim 3, wherein the bacillus is one or more of bacillus subtilis, bacillus licheniformis and bacillus pumilus; the bacillus subtilis, the bacillus licheniformis and the bacillus pumilus can all be obtained from the common microorganism center of China Committee for culture Collection of microorganisms; the serial numbers are respectively CGMCC1.2163, CGMCC1.0809 and CGMCC 1.3533.
5. The probiotic feed additive for improving the immunity of aquatic animals according to claim 3, wherein the lactobacillus is lactobacillus casei, which is obtained from the China general microbiological culture Collection center; the serial numbers are CGMCC1.159 respectively.
6. The probiotic feed additive for improving the immunity of aquatic animals according to claim 1, wherein the preparation process of the probiotic fermented bacteria powder specifically comprises the following steps:
(1) preparing fermentation liquor: preparing a liquid fermentation liquid according to the mass percentage of 2% of bran, 2% of soybean meal, 1% of peptone, 1% of corn steep liquor, 0.02% of potassium dihydrogen phosphate, 0.01% of dipotassium hydrogen phosphate, 0.02% of magnesium sulfate, 0.01% of calcium chloride and the balance of water, and adjusting the pH to 6.5-7.0;
(2) aerobic fermentation: inoculating saccharomyces cerevisiae and bacillus seed liquid, wherein the inoculation amount is 1-2% and 2-4% respectively, controlling the fermentation temperature to be 28-37 ℃ and the rotation speed to be 150-300 rpm; fermenting for 12-24 h under the fermentation condition;
(3) anaerobic fermentation: after the aerobic fermentation is finished, inoculating lactobacillus powder, wherein the inoculation amount is 2-4%, the fermentation temperature is 28-37 ℃, and controlling the temperature for anaerobic fermentation for 12-24 hours;
(4) low-temperature concentration: concentrating the fermentation liquor at low temperature after the anaerobic fermentation is finished;
(5) adsorption drying: and (3) adsorbing the concentrated mixture by using bran with the mass ratio of 0.5-1.0 time, and drying the mixture in a blast dryer at the temperature of 40-60 ℃ until the water content is controlled to be 8-10% to obtain the probiotic fermentation bacteria powder.
7. The probiotic feed additive for improving the immunity of aquatic animals according to claim 6, wherein the preparation process of the bacillus seed solution comprises the following steps: inoculating the activated bacillus to a seed culture medium, wherein the seed culture medium comprises the following components: 2% of glucose, 2% of peptone, 1% of yeast extract, 0.01% of magnesium sulfate, 0.003% of manganese sulfate, and 6.5-7.0 of pH, and culturing for 16-24 h at the temperature of 28-37 ℃ and the rotating speed of 150-3000 rpm, wherein the effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL; the process for activating the bacillus comprises the following steps: culturing for 16-24 h at 28-37 ℃ by adopting a LB solid culture medium commonly used in the field; the preparation method of the yeast seed liquid comprises the following steps: under the aseptic operation condition, two rings of strains are picked from the inclined plane of the yeast YDP by using an inoculating ring, inoculated into a 500mL triangular flask containing 300mLPDA liquid culture medium, subjected to shaking culture at the temperature of between 28 and 32 ℃ and at the speed of 140r/min for 24 hours, and then inoculated into a seed tank for expanded culture according to the process to prepare the yeast seed liquid, wherein the effective viable count of the seed liquid is required to be more than or equal to 109cfu/mL。
8. The probiotic feed additive for improving the immunity of aquatic animals according to claim 6, wherein the bacillus is a mixed microbial inoculum of bacillus subtilis, bacillus licheniformis and bacillus pumilus, and the inoculation ratio of the bacillus subtilis, the bacillus licheniformis and the bacillus pumilus is 1:2: 1.
9. The probiotic feed additive for improving the immunity of aquatic animals according to claim 1, wherein the complex enzyme preparation is one or a mixture of amylase, mannanase and cellulase; optimally, the compound enzyme preparation is a mixture of amylase, mannase and cellulase, and the mass ratio of the compound enzyme preparation to the cellulase is 3:1: 2.
10. The probiotic feed additive for improving the immunity of aquatic animals according to claim 1, wherein the preparation method of the probiotic feed additive specifically comprises the following steps:
(1) preparing oyster powder particles, namely performing ultrasonic oscillation on oyster shell powder to respectively form particles of 40-80 nm;
(2) adding oyster powder into deionized water with the mass ratio of 10 times, then adding cyclodextrin, and uniformly stirring and dispersing to obtain a mixed solution of the oyster powder and the cyclodextrin;
(3) adding probiotic zymocyte powder, vitamins, brown algae polysaccharide and a complex enzyme preparation into a mixed solution of oyster powder and cyclodextrin according to a proportion;
(4) and after mixing, heating at the temperature of 60-65 ℃, ultrasonically oscillating for 1-2 hours, cooling, filtering, and vacuum drying at low temperature to obtain the probiotic feed additive for improving the immunity of aquatic animals.
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Application publication date: 20200925