CN116058430A - Feed-modified microbial agent for fish and shrimp culture and preparation method thereof - Google Patents
Feed-modified microbial agent for fish and shrimp culture and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a feed-modified microbial agent for fish and shrimp culture and a preparation method thereof, belonging to the technical field of microbial agents, and comprising the following components in parts by weight: 50-85 parts of microorganism composite bacterial powder, 10-15 parts of water flower extract, 5-10 parts of red algae extract, 5-10 parts of cercis chinensis extract, 3-8 parts of achyranthes bidentata polysaccharide, 1-2 parts of cordyceps militaris powder, 0.5-3 parts of citric acid, 0.5-1 part of zinc methionine, 7-10 parts of oyster shell powder and 1-2 parts of sodium laurate glutamate; the microbial agent prepared by the invention realizes the double technical effects of accelerating the growth and development of fishes and shrimps and improving the immunity of the fishes and shrimps by compounding the microbial agent and the plant extract, and realizes the efficient immune response of the fishes and shrimps to pathogenic microorganisms by regulating and controlling the expression of immune related genes, thereby solving the problems of drug resistance and residue caused by abuse of antibiotics which are difficult to solve in the prior art, being capable of being used as an alternative of antibiotics, being used for producing high-quality fishes and shrimps products and having important application prospects in aquaculture.
Description
Technical Field
The invention belongs to the technical field of microbial agents, and particularly relates to an improved microbial agent for fish and shrimp culture and a preparation method thereof.
Background
Along with the increase of the demand of human beings on fishes and shrimps, the fish and shrimp farming industry is developed, a large amount of antibiotics are used for preventing and treating fish and shrimp diseases in the traditional fish and shrimp farming process, the survival rate of the fishes and shrimps is improved, however, along with the development and progress of the times, green healthy farming is becoming a development trend, the supervision of the fishery industry on the fishery medicine is also more and more strict, antibiotics drugs have caused great side effects on the shrimp farming industry, on one hand, bacterial drug resistance is increased when the antibiotics are excessively used, so that excessive bacterial propagation with strong drug resistance causes ecological imbalance of microorganisms, on the other hand, antibiotics can remain in the shrimps, people can accumulate in the bodies for a long time to affect health, and therefore, how to avoid abuse of the fishery medicine and develop healthy green ecological high-quality farming of the shrimps are hot problems of current research.
The microbial feed additive is prepared by applying the basic technical principle of modern microecology, screening out needed target microorganisms or screening out beneficial microorganisms with promotion effect on microbial growth through a series of screening, and can be used as a microbial additive for direct feeding, for improving organism immunity, preventing diseases and improving production performance, for example, the microbial agent for aquatic product feed is proposed in Chinese patent publication No. CN105918620A, and the microbial agent comprises the following components in parts by weight: 8-15 parts of photosynthetic bacteria, 13-25 parts of nitrifying bacteria, 9-17 parts of lactic acid bacteria, 3-8 parts of lactobacillus acidophilus, 9-14 parts of saccharomycetes, 15-35 parts of bacillus, 5-9 parts of white rot fungi, 4-13 parts of streptococcus thermophilus, 1-8 parts of enterococcus faecium, 2-5 parts of enterococcus faecalis, 1-4 parts of stenotrophomonas maltophilia, 0-3 parts of pseudomonas cepacia, 0-0.5 part of yersinia ruckeri, and 15-23 parts of medical stone powder in the microbial agent, so that the survival rate can be improved, the ammonia nitrogen content and the nitrite content in the aquaculture environment can be reduced, however, the microbial agent has complex components, high production cost and general efficacy, and therefore, the microbial agent for fish and shrimp culture still needs to be further developed to improve the production level and the production quality.
Disclosure of Invention
Aiming at the situation, the invention provides the feed improved microbial agent for fish and shrimp culture and the preparation method thereof, and aims to solve the problem of poor effect of the microbial agent in the prior art.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the invention provides an improved microbial agent for fish and shrimp culture feed, which comprises the following components in parts by weight: 50-85 parts of microorganism composite bacterial powder, 10-15 parts of water flower extract, 5-10 parts of red ball algae extract, 5-10 parts of cercis extract, 3-8 parts of achyranthes polysaccharide, 1-2 parts of cordyceps militaris powder, 0.5-3 parts of citric acid, 0.5-1 part of zinc methionine, 7-10 parts of oyster shell powder and 1-2 parts of sodium laurate glutamate.
Preferably, the improved microbial agent comprises the following components in parts by weight: 65-80 parts of microorganism composite bacterial powder, 10-12 parts of water flower extract, 5-7 parts of red algae extract, 5-7 parts of cercis extract, 3-5 parts of achyranthes polysaccharide, 1-2 parts of cordyceps militaris powder, 0.5-3 parts of citric acid, 0.5-1 part of zinc methionine, 7-10 parts of oyster shell powder and 1-2 parts of sodium laurate glutamate.
Further, the microbial composite bacterial powder comprises the following components in parts by weight:
12-20 parts of candida utilis powder;
5-15 parts of trichoderma longibrachiatum powder;
5-10 parts of lactobacillus rhamnosus bacterial powder;
10-20 parts of bacillus subtilis powder.
Preferably, the microbial composite bacterial powder comprises the following components in parts by weight:
15-18 parts of candida utilis powder;
8-12 parts of trichoderma longibrachiatum powder;
8-10 parts of lactobacillus rhamnosus bacterial powder;
12-18 parts of bacillus subtilis powder.
Wherein the spore concentration of the candida utilis powder is 3-8.5X10 8 CFU/g, the spore concentration of the trichoderma longibrachiatum powder is 2-7.5X10 8 CFU/g, the spore concentration of the lactobacillus rhamnosus powder is 1-8 multiplied by 10 8 CFU/g, the spore concentration of the bacillus subtilis powder is 1-9 multiplied by 10 8 CFU/g。
In addition, the invention also provides a preparation method of the feed-modified microbial agent for fish and shrimp culture, which specifically comprises the following steps:
(1) Preparing a water flower extract, a red algae extract, a cercis chinensis extract and a microorganism composite bacterial powder;
(2) Mixing the microorganism composite bacterial powder with conventional feed for fishes and shrimps, and adding the water-lily flower extract, the haematococcus extract, the cercis extract, the achyranthes polysaccharide, the cordyceps militaris powder, the citric acid, the zinc methionine, the oyster shell powder and the sodium laurate glutamate according to a proportion to obtain the feed for fishes and shrimps;
(3) And (3) carrying out primary fermentation on the fish and shrimp feed, and preparing the fish and shrimp feed into granules or directly spraying water in a pond for feeding after fermentation.
Preferably, one or more of fruit shells, cotton seed hulls, buckwheat hulls, sugar residues, vinegar residues, bagasse, oil residues, corn stover and edible fungus residues are added into the conventional feed for fish and shrimp to serve as a microbial fermentation carrier.
Preferably, the primary fermentation temperature is 30-32 ℃, and the primary fermentation time is 2-3 days.
The preparation method of the microbial composite bacterial powder comprises the following steps:
(1) Activating bacterial liquid: diluting candida utilis, trichoderma longibrachiatum, lactobacillus rhamnosus and bacillus subtilis 5-10 times, respectively coating on a solid growth culture medium, and culturing at 28-32 ℃ for 12-24 hours to obtain activated strains respectively;
(2) Expansion and activation culture of strains: inoculating the activated strain into a liquid growth medium, and culturing for 12-24 hours at 28-32 ℃ to obtain an enlarged culture strain;
(3) Enrichment culture of bacterial liquid: sucking 5mL expanded culture strain into liquid growth medium, and culturing at 28-32deg.C for 12-24 hr to obtain enriched thallus;
(4) Powder preparation: centrifuging the enriched thalli for 1 minute at 150rpm, removing supernatant, and then placing in a temperature of 30-37 ℃ for drying for 30-60 minutes to respectively obtain candida utilis powder, trichoderma longibrachiatum powder, lactobacillus rhamnosus powder and bacillus subtilis powder.
Preferably, the preparation method of the water flower extract, the haematococcus extract and the cercis chinensis extract comprises the following steps: pulverizing flos Pulsatillae, haematococcus and Cercis chinensis, respectively adding into ethanol solution, reflux extracting for 3-5 times for 2-3 hr each time, filtering, concentrating, and drying to obtain dry powders of flos Pulsatillae extract, haematococcus extract and Cercis chinensis extract.
By adopting the scheme, the beneficial effects obtained by the invention are as follows: the invention provides a feed improvement microbial agent for fish and shrimp culture and a preparation method thereof, and aims to solve the problem of poor effect of the microbial agent in the prior art;
by regulating the up-regulated expression of immune related genes (interleukin 1 beta and C-type lysozyme), the high-efficiency immune response of fishes and shrimps to pathogenic microorganisms is realized, the contents of immune related enzymes (alkaline phosphatase) and antioxidase (superoxide dismutase and catalase) in the fishes and shrimps are improved, the resistance and antioxygenic property of the fishes and shrimps to the pathogenic microorganisms are enhanced, and the problems of drug resistance and residues caused by abuse of antibiotics which are difficult to solve in the prior art are solved;
the microbial agent prepared by the invention has the advantages of realizing multiple technical effects of accelerating the growth and development of fishes and shrimps, improving the immunity of the fishes and shrimps, improving the oxidation resistance and the like, can be used as an antibiotic substitute, is used for producing high-quality fish and shrimp products, and has important application prospects in aquaculture.
Drawings
FIG. 1 is a graph showing the results of measuring the content of superoxide dismutase (SOD) after being treated by the microbial agent prepared by the invention;
FIG. 2 is a graph showing the measurement result of the content of Catalase (CAT) after the microbial agent prepared by the invention is treated;
FIG. 3 is a graph showing the results of measurement of alkaline phosphatase (AKP) content after treatment with the microbial agent prepared in the present invention;
FIG. 4 is mortality of an invasive challenge test using Streptococcus agalactiae bacteria solution;
FIG. 5 is a thermal diagram of the expression content of the immune related genes of the paralichthys olivaceus by the microbial agent prepared by the invention.
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are illustrative only and should not be construed as limiting the scope of the present application.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials and test strains used in the examples described below, unless otherwise specified, were commercially available.
Example 1
Feed improved microbial agent for fish and shrimp culture
The improved microbial agent comprises the following components in parts by weight: 65 parts of microorganism composite bacterial powder, 10 parts of water flower extract, 5 parts of haematococcus extract, 5 parts of cercis chinensis extract, 3 parts of achyranthes bidentata polysaccharide, 1 part of cordyceps militaris powder, 0.5 part of citric acid, 0.5 part of zinc methionine, 7 parts of oyster shell powder and 1 part of lauric acid sodium glutamate.
The microbial composite bacterial powder comprises the following components in parts by weight:
15 parts of candida utilis powder;
8 parts of trichoderma longibrachiatum powder;
8 parts of lactobacillus rhamnosus bacterial powder;
12 parts of bacillus subtilis powder.
Wherein, candida utilis is purchased from China center for industry microbiological culture collection center, and the strain preservation number is as follows: CICC13145; trichoderma longibrachiatum was purchased from China center for type culture Collection, strain accession number: CICC40202; lactobacillus rhamnosus was purchased from the industrial microorganism strain collection management center, sichuan province, strain collection number: SICC1.342; bacillus subtilis was purchased from the Sichuan province industrial microorganism strain collection management center, strain collection number: SICC1.561
The spore concentration of the candida utilis powder is 3 multiplied by 10 8 CFU/g, the spore concentration of the trichoderma longibrachiatum powder is 2 multiplied by 10 8 CFU/g, the spore concentration of the lactobacillus rhamnosus powder is 1 multiplied by 10 8 CFU/g, the spore concentration of the bacillus subtilis powder is 1 multiplied by 10 8 CFU/g。
In addition, the invention also provides a preparation method of the feed-modified microbial agent for fish and shrimp culture, which specifically comprises the following steps: preparing a water flower extract, a red algae extract, a cercis chinensis extract and a microorganism composite bacterial powder; mixing the microorganism composite bacterial powder with conventional feed for fishes and shrimps, and adding the water-lily flower extract, the haematococcus extract, the cercis extract, the achyranthes polysaccharide, the cordyceps militaris powder, the citric acid, the zinc methionine, the oyster shell powder and the sodium laurate glutamate according to a proportion to obtain the feed for fishes and shrimps; and (3) carrying out primary fermentation on the fish and shrimp feed, and preparing the fish and shrimp feed into granules or directly spraying water in a pond for feeding after fermentation.
One or more of fruit shells, cotton seed hulls, buckwheat hulls, sugar residues, vinegar residues, bagasse, oil residues, corn stover and edible fungus residues are added into the conventional feed for fish and shrimp to serve as a microbial fermentation carrier, the primary fermentation temperature is 30-32 ℃, and the primary fermentation time is 3 days.
The preparation method of the microbial composite bacterial powder comprises the following steps:
(1) Activating bacterial liquid: diluting candida utilis, trichoderma longibrachiatum, lactobacillus rhamnosus and bacillus subtilis 5-10 times, respectively coating on a solid growth culture medium, and culturing at 28-32 ℃ for 24 hours to obtain activated strains respectively;
(2) Expansion and activation culture of strains: inoculating the activated strain into a liquid growth medium, and culturing at 28-32 ℃ for 24 hours to obtain an enlarged culture strain;
(3) Enrichment culture of bacterial liquid: sucking 5mL of the expanded culture strain into a liquid growth medium, and culturing at 28-32 ℃ for 24 hours to obtain enriched thalli;
(4) Powder preparation: centrifuging the enriched thalli for 1 minute at 150rpm, removing supernatant, and then placing in a temperature of 30-37 ℃ for drying for 30 minutes to respectively obtain candida utilis powder, trichoderma longibrachiatum powder, lactobacillus rhamnosus powder and bacillus subtilis powder.
The preparation method of the water flower extract, the haematococcus extract and the cercis chinensis extract comprises the following steps: pulverizing flos Pulsatillae, haematococcus and Cercis chinensis, respectively adding into ethanol solution, reflux extracting for 3 times and 3 hr each time, filtering, concentrating, and drying to obtain dry powders of flos Pulsatillae extract, haematococcus extract and Cercis chinensis extract.
The inhibition effect of the microbial agent prepared by the invention on common pathogenic bacteria of fish and shrimp is measured.
The aeromonas hydrophila mainly has pathogenicity to cold blood animals such as fishes and frogs and warm-blooded animals such as mice, guinea pigs and rabbits through enterotoxins, streptococcus agalactiae becomes one of main pathogens of fish streptococcosis, common clinical symptoms of fish infection streptococcus agalactiae comprise protrusion of eyeballs, belly swelling, spinal curvature, basal bleeding of fin and the like, the test is carried out by an agar plate oxforum test method, the microbial inoculum prepared by the invention is added into an LB agar plate culture dish added with streptococcus agalactiae (ATCC 13813) or aeromonas hydrophila (ATCC 7966), the conventional feed for fishes and shrimps is added as a blank control, the conventional antibiotic-penicillin is added as a positive control, and the antibacterial circle is measured after the common culture is carried out for 15 hours at 37 ℃.
TABLE 1 inhibition effect of the microbial agent prepared by the present invention on Streptococcus agalactiae
TABLE 2 inhibition effect of microbial inoculants prepared in accordance with the present invention on Aeromonas hydrophila
As shown in tables 1 and 2, compared with a blank control, the microbial agent prepared by the invention can inhibit common streptococcus agalactiae or aeromonas hydrophila which are pathogenic bacteria in fish and shrimp intestinal tracts, so that the microbial agent has the effect of preventing diseases, has obvious effect although the antibacterial effect is deficient, can be used as a substitute material of antibiotics, and reduces adverse effects caused by mass use of the antibiotics.
Example 2
Feed improved microbial agent for fish and shrimp culture
The improved microbial agent comprises the following components in parts by weight: 80 parts of microorganism composite bacterial powder, 12 parts of water flower extract, 7 parts of haematococcus extract, 7 parts of cercis chinensis extract, 5 parts of achyranthes bidentata polysaccharide, 1 part of cordyceps militaris powder, 3 parts of citric acid, 1 part of zinc methionine, 10 parts of oyster shell powder and 2 parts of sodium laurate glutamate.
The microbial composite bacterial powder comprises the following components in parts by weight:
18 parts of candida utilis powder;
12 parts of trichoderma longibrachiatum powder;
10 parts of lactobacillus rhamnosus bacterial powder;
18 parts of bacillus subtilis powder.
Wherein, candida utilis is purchased from China center for industry microbiological culture collection center, and the strain preservation number is as follows: CICC13145; trichoderma longibrachiatum was purchased from China center for type culture Collection, strain accession number: CICC40202; lactobacillus rhamnosus was purchased from the industrial microorganism strain collection management center, sichuan province, strain collection number: SICC1.342; bacillus subtilis was purchased from the Sichuan province industrial microorganism strain collection management center, strain collection number: SICC1.561
The spore concentration of the candida utilis powder is 5.5x10 8 CFU/g, the spore concentration of the trichoderma longibrachiatum powder is 7.5X10 8 CFU/g, the spore concentration of the lactobacillus rhamnosus powder is 3 multiplied by 10 8 CFU/g, the spore concentration of the bacillus subtilis powder is 7.5X10 8 CFU/g。
In addition, the invention also provides a preparation method of the feed-modified microbial agent for fish and shrimp culture, which specifically comprises the following steps: preparing a water flower extract, a red algae extract, a cercis chinensis extract and a microorganism composite bacterial powder; mixing the microorganism composite bacterial powder with conventional feed for fishes and shrimps, and adding the water-lily flower extract, the haematococcus extract, the cercis extract, the achyranthes polysaccharide, the cordyceps militaris powder, the citric acid, the zinc methionine, the oyster shell powder and the sodium laurate glutamate according to a proportion to obtain the feed for fishes and shrimps; and (3) carrying out primary fermentation on the fish and shrimp feed, and preparing the fish and shrimp feed into granules or directly spraying water in a pond for feeding after fermentation.
One or more of fruit shells, cotton seed hulls, buckwheat hulls, sugar residues, vinegar residues, bagasse, oil residues, corn stover and edible fungus residues are added into the conventional feed for fish and shrimp to serve as a microbial fermentation carrier, the primary fermentation temperature is 30-32 ℃, and the primary fermentation time is 3 days.
The preparation method of the microbial composite bacterial powder comprises the following steps:
(1) Activating bacterial liquid: diluting candida utilis, trichoderma longibrachiatum, lactobacillus rhamnosus and bacillus subtilis 5-10 times, respectively coating on a solid growth culture medium, and culturing at 28-32 ℃ for 24 hours to obtain activated strains respectively;
(2) Expansion and activation culture of strains: inoculating the activated strain into a liquid growth medium, and culturing at 28-32 ℃ for 24 hours to obtain an enlarged culture strain;
(3) Enrichment culture of bacterial liquid: sucking 5mL of the expanded culture strain into a liquid growth medium, and culturing at 28-32 ℃ for 24 hours to obtain enriched thalli;
(4) Powder preparation: centrifuging the enriched thalli for 1 minute at 150rpm, removing supernatant, and then placing in a temperature of 30-37 ℃ for drying for 30 minutes to respectively obtain candida utilis powder, trichoderma longibrachiatum powder, lactobacillus rhamnosus powder and bacillus subtilis powder.
The preparation method of the water flower extract, the haematococcus extract and the cercis chinensis extract comprises the following steps: pulverizing flos Pulsatillae, haematococcus and Cercis chinensis, respectively adding into ethanol solution, reflux extracting for 3 times and 3 hr each time, filtering, concentrating, and drying to obtain dry powders of flos Pulsatillae extract, haematococcus extract and Cercis chinensis extract.
Example 3
Feed improved microbial agent for fish and shrimp culture
The improved microbial agent comprises the following components in parts by weight: 75 parts of microorganism composite bacterial powder, 12 parts of water flower extract, 8 parts of haematococcus extract, 8 parts of cercis extract, 5 parts of achyranthes bidentata polysaccharide, 1 part of cordyceps militaris powder, 0.5 part of citric acid, 1 part of zinc methionine, 10 parts of oyster shell powder and 1 part of lauric acid sodium glutamate.
The microbial composite bacterial powder comprises the following components in parts by weight:
15 parts of candida utilis powder;
12 parts of trichoderma longibrachiatum powder;
10 parts of lactobacillus rhamnosus bacterial powder;
12 parts of bacillus subtilis powder.
Wherein, candida utilis is purchased from China center for industry microbiological culture collection center, and the strain preservation number is as follows: CICC13145; trichoderma longibrachiatum was purchased from China center for type culture Collection, strain accession number: CICC40202; lactobacillus rhamnosus was purchased from the industrial microorganism strain collection management center, sichuan province, strain collection number: SICC1.342; bacillus subtilis was purchased from the Sichuan province industrial microorganism strain collection management center, strain collection number: SICC1.561
The spore concentration of the candida utilis powder is 3 multiplied by 10 8 CFU/g, the spore concentration of the trichoderma longibrachiatum powder is 7.5X10 8 CFU/g, the spore concentration of the lactobacillus rhamnosus powder is 1 multiplied by 10 8 CFU/g, the spore concentration of the bacillus subtilis powder is 9 multiplied by 10 8 CFU/g。
In addition, the invention also provides a preparation method of the feed-modified microbial agent for fish and shrimp culture, which specifically comprises the following steps: preparing a water flower extract, a red algae extract, a cercis chinensis extract and a microorganism composite bacterial powder; mixing the microorganism composite bacterial powder with conventional feed for fishes and shrimps, and adding the water-lily flower extract, the haematococcus extract, the cercis extract, the achyranthes polysaccharide, the cordyceps militaris powder, the citric acid, the zinc methionine, the oyster shell powder and the sodium laurate glutamate according to a proportion to obtain the feed for fishes and shrimps; and (3) carrying out primary fermentation on the fish and shrimp feed, and preparing the fish and shrimp feed into granules or directly spraying water in a pond for feeding after fermentation.
One or more of fruit shells, cotton seed hulls, buckwheat hulls, sugar residues, vinegar residues, bagasse, oil residues, corn stover and edible fungus residues are added into the conventional feed for fish and shrimp to serve as a microbial fermentation carrier, the primary fermentation temperature is 30-32 ℃, and the primary fermentation time is 3 days.
The preparation method of the microbial composite bacterial powder comprises the following steps:
(1) Activating bacterial liquid: diluting candida utilis, trichoderma longibrachiatum, lactobacillus rhamnosus and bacillus subtilis 5-10 times, respectively coating on a solid growth culture medium, and culturing at 28-32 ℃ for 24 hours to obtain activated strains respectively;
(2) Expansion and activation culture of strains: inoculating the activated strain into a liquid growth medium, and culturing at 28-32 ℃ for 24 hours to obtain an enlarged culture strain;
(3) Enrichment culture of bacterial liquid: sucking 5mL of the expanded culture strain into a liquid growth medium, and culturing at 28-32 ℃ for 24 hours to obtain enriched thalli;
(4) Powder preparation: centrifuging the enriched thalli for 1 minute at 150rpm, removing supernatant, and then placing in a temperature of 30-37 ℃ for drying for 30 minutes to respectively obtain candida utilis powder, trichoderma longibrachiatum powder, lactobacillus rhamnosus powder and bacillus subtilis powder.
The preparation method of the water flower extract, the haematococcus extract and the cercis chinensis extract comprises the following steps: pulverizing flos Pulsatillae, haematococcus and Cercis chinensis, respectively adding into ethanol solution, reflux extracting for 3 times and 3 hr each time, filtering, concentrating, and drying to obtain dry powders of flos Pulsatillae extract, haematococcus extract and Cercis chinensis extract.
Comparative example 1
The comparative example provides a feed-improving microbial agent for fish and shrimp culture, which is different from example 1 only in that the water flower extract and the cercis extract are not added to all the components, and the contents of the rest components and components are the same as those in example 1.
Comparative example 2
This comparative example provides a feed-improving microbial agent for fish and shrimp culture, which differs from example 1 only in that candida utilis, trichoderma longibrachiatum powder, lactobacillus rhamnosus powder and bacillus subtilis are not added to all components, and the rest components and the content of the components are the same as those in example 1.
Performance measurement test
Commercial flounder seedlings are purchased, sterilized by using a salt solution with the concentration of 2%, the fish and shrimp are fed with the feed conventionally for 15 days or so to adapt to the environment, 360 flounder seedlings with good states and equivalent initial weights are selected and distributed to a circular water tank for experiment, 6 treatments are set, 3 treatments are set, each treatment is repeated for 20 times, the daily feeding amount is 3% -5% of the weight of the fish body (9:00 in the morning and 18:00 in the evening), the test fish is weighed for 1 time every 2 weeks, the feeding amount is adjusted according to the weight increment condition of the test fish, wherein 5 treatments are respectively fed with the microbial inoculum prepared in example 1, example 3 and comparative example 1 and comparative example 2, 1 treatment is fed with the feed conventionally used for the fish and shrimp as a blank control group, other environments and management methods are kept consistent, and the test period is 35 days.
Analysis of results
1.1 influence of the microbial inoculant prepared by the invention on the growth of Paralichthys olivaceus
The weight of the test fish at the end of the test was weighed and the weight gain (%) was calculated as the percentage of the difference between the weight of the test fish at the end of the test and the weight of the fish before the test.
As shown in Table 3, compared with a blank control, the weight gain rate of the paralichthys olivaceus is improved after the microbial agent is used, which shows that the microbial agent can promote the growth of fish and shrimp and further improve the cultivation yield of the fish and shrimp, and the results according to comparative examples and comparative example 2 show that the common use of candida utilis, trichoderma longibrachiatum powder, lactobacillus rhamnosus powder, bacillus subtilis, a water flower extract, a rhodococcus rhodochrous extract and a cercis chinensis extract is key for playing the efficacy, and the synergistic efficacy exists among the components.
TABLE 3 influence of microbial inoculants on growth of Paralichthys olivaceus
1.2 influence of the microbial inoculant prepared by the invention on the immunity activity of the paralichthys olivaceus
(1) Serum non-specific immune index
After the cultivation test is finished, sampling is carried out rapidly, blood is taken from the tail vein by a 1mL syringe, standing is carried out for 24 hours at 4 ℃, and then supernatant is taken by centrifugation at 3000r/min for 20 minutes, serum is collected and placed at-80 ℃ for temporary storage.
Superoxide dismutase (SOD), catalase (CAT) and alkaline phosphatase (AKP) were measured in serum using the kit (purchased from institute of bioengineering, built in south kyo):
wherein, superoxide dismutase (SOD): the measurement by the WST-1 method is defined as follows: in each 1mL of serum, the corresponding enzyme amount is an enzyme activity unit when the SOD inhibition rate reaches 50%, and the result is expressed as U/mL;
catalase (CAT): the determination by the ammonium molybdate method is defined as: the amount of HO2 decomposed 1. Mu. Mol per second in 1mL of serum is one enzyme activity unit, and the result is expressed as U/mL;
alkaline phosphatase (AKP): the trace enzyme-labeled method is adopted for measurement, and definition is that: the disodium phosphate was used as a matrix, and 1mg phenol was produced as 1 gold unit per 100mL of serum by reacting with the matrix at 37℃for 15 minutes, and the result was expressed as gold unit per 100 mL.
As shown in figures 1, 2 and 3, superoxide dismutase (SOD), catalase (CAT) and alkaline phosphatase (AKP) in the fish body are all improved after the microbial agent prepared by the invention is used for raising, and the microbial agent can improve the immunity and the antioxidant capacity of the fish.
(2) Streptococcus agalactiae bacterial liquid infection toxicity attack test
After the sample collection is finished, the remaining 15 fish in each jar are fished out, and after the anesthesia by MS222, 0.3mL of the fish with the concentration of 1.5x10 is injected into each fish in the abdominal cavity 6 CFU/mL streptococcus agalactiae bacterial liquid, returning the injected fish to the original culture tank, calculating the accumulated dead fish mantissa after 14 days, and calculating the death rate (%), wherein the death rate is the ratio percentage of the dead fish number to the total number (10).
As shown in fig. 4, after the paralichthys olivaceus is infected again by the streptococcus agalactiae bacterial liquid, the mortality rate of the treatment of feeding the microbial inoculum is obviously lower than that of the blank control, which indicates that the microbial inoculum feeding can improve the resistance of the fish to pathogenic bacteria and can possibly play a role in inhibiting harmful flora.
1.3 Effect of the microbial inoculant prepared by the invention on the expression of the immune-related genes of Paralichthys olivaceus
In the streptococcus agalactiae bacterial liquid infection toxicity attack test, 0.3mL of the streptococcus agalactiae bacterial liquid with the concentration of 1.5x10 is injected into each tail fish abdominal cavity 6 CFU/mL streptococcus agalactiae bacterial liquid, the injected fish returns to the original culture tank, and after 5 days of culture, the fish is cultured along withSelecting 5 pieces, anesthetizing with MS-222, separating hindgut in ice tray, removing content, collecting fish blocks, extracting RNA with TRIzol, and using Prime Script TM Reverse transcription of the RT Reagent kit into cDNA, and real-time fluorescence quantitative PCR (polymerase chain reaction) is carried out by taking the cDNA as a template, wherein the reaction conditions are as follows: 95 ℃,10 minutes, 1 cycle; 95 ℃,30 seconds, 60 ℃,30 seconds, 72 ℃,30 seconds, 40 cycles; the expression levels of interleukin 1 beta (primer: 5'-CACATCAGAGCAAGA-3' and 5'-TGGTAGCACCGGGCATTCT-3') and C-type lysozyme (primer: 5'-CATTGTGGCGATCAAATG-3' and 5'-GCTCCGATCCCGTTT-3') were measured by using the beta-actin reference gene (primer: 5'-TCCACGAAACCACCTACAACA-3' and 5'-CCAGACGGAGTATTTACGCTCA-3') at 72℃for 5 minutes in 1 cycle, and the result of gene expression was 2 -△△CT The method was used for analysis.
As shown in figure 5, wherein A represents interleukin 1 beta, B represents C-type lysozyme, and 1-6 are blank control groups, example 1, example 2, example 3, comparative example 1 and comparative examples 2,5-15 in sequence, which represent relative expression amounts of genes (lighter colors represent higher gene expression levels), after the streptococcus agalactiae bacterial liquid infects the paralichthys olivaceus again, immune-related genes in the paralichthys olivaceus are obviously regulated and improved, and the mechanism of improving the resistance of the paralichthys olivaceus to pathogenic bacteria is probably improved, so that the microbial agent prepared by the invention can improve the immunity of resisting fishes and shrimps and simultaneously accelerate the growth and development speed, and has application prospect.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
The invention and its embodiments have been described above with no limitation, and the invention is illustrated in the figures of the accompanying drawings as one of its embodiments, without limitation in practice. In summary, those skilled in the art, having benefit of this disclosure, will appreciate that the invention can be practiced without the specific details disclosed herein.
Claims (10)
1. A feed-improving microbial agent for fish and shrimp culture is characterized in that: the improved microbial agent comprises the following components in parts by weight: 50-85 parts of microorganism composite bacterial powder, 10-15 parts of water flower extract, 5-10 parts of red ball algae extract, 5-10 parts of cercis extract, 3-8 parts of achyranthes polysaccharide, 1-2 parts of cordyceps militaris powder, 0.5-3 parts of citric acid, 0.5-1 part of zinc methionine, 7-10 parts of oyster shell powder and 1-2 parts of sodium laurate glutamate.
2. The microbial agent for improving fish and shrimp culture feed according to claim 1, which is characterized in that: the improved microbial agent comprises the following components in parts by weight: 65-80 parts of microorganism composite bacterial powder, 10-12 parts of water flower extract, 5-7 parts of red algae extract, 5-7 parts of cercis extract, 3-5 parts of achyranthes polysaccharide, 1-2 parts of cordyceps militaris powder, 0.5-3 parts of citric acid, 0.5-1 part of zinc methionine, 7-10 parts of oyster shell powder and 1-2 parts of sodium laurate glutamate.
3. The microbial agent for improving fish and shrimp culture feed according to claim 2, which is characterized in that: the microbial composite bacterial powder comprises the following components in parts by weight:
12-20 parts of candida utilis powder;
5-15 parts of trichoderma longibrachiatum powder;
5-10 parts of lactobacillus rhamnosus bacterial powder;
10-20 parts of bacillus subtilis powder.
4. A feed-modifying microbial agent for fish and shrimp farming as defined in claim 3, wherein: the microbial composite bacterial powder comprises the following components in parts by weight:
15-18 parts of candida utilis powder;
8-12 parts of trichoderma longibrachiatum powder;
8-10 parts of lactobacillus rhamnosus bacterial powder;
12-18 parts of bacillus subtilis powder.
5. The microbial agent for improving fish and shrimp farming feed according to claim 4, wherein: the spore concentration of the candida utilis powder is 3-8.5X10 8 CFU/g, the spore concentration of the trichoderma longibrachiatum powder is 2-7.5X10 8 CFU/g, the spore concentration of the lactobacillus rhamnosus powder is 1-8 multiplied by 10 8 CFU/g, the spore concentration of the bacillus subtilis powder is 1-9 multiplied by 10 8 CFU/g。
6. A method for preparing the feed-modified microbial agent for fish and shrimp culture according to claim 5, which is characterized in that: the method specifically comprises the following steps:
(1) Preparing a water flower extract, a red algae extract, a cercis chinensis extract and a microorganism composite bacterial powder;
(2) Mixing the microorganism composite bacterial powder with conventional feed for fishes and shrimps, and adding the water-lily flower extract, the haematococcus extract, the cercis extract, the achyranthes polysaccharide, the cordyceps militaris powder, the citric acid, the zinc methionine, the oyster shell powder and the sodium laurate glutamate according to a proportion to obtain the feed for fishes and shrimps;
(3) And (3) carrying out primary fermentation on the fish and shrimp feed, and preparing the fish and shrimp feed into granules or directly spraying water in a pond for feeding after fermentation.
7. The method for preparing the feed-modified microbial agent for fish and shrimp farming according to claim 6, which is characterized by comprising the following steps: one or more of fruit shells, cotton seed hulls, buckwheat hulls, sugar residues, vinegar residues, bagasse, oil residues, corn stover and edible fungus residues are added into the conventional feed for fish and shrimp to serve as a microbial fermentation carrier.
8. The method for preparing the feed-modified microbial agent for fish and shrimp farming according to claim 7, which is characterized in that: the primary fermentation temperature is 30-32 ℃, and the primary fermentation time is 2-3 days.
9. The method for preparing the feed-modified microbial agent for fish and shrimp culture of claim 8, which is characterized by comprising the following steps: the preparation method of the microbial composite bacterial powder comprises the following steps:
(1) Activating bacterial liquid: diluting candida utilis, trichoderma longibrachiatum, lactobacillus rhamnosus and bacillus subtilis 5-10 times, respectively coating on a solid growth culture medium, and culturing at 28-32 ℃ for 12-24 hours to obtain activated strains respectively;
(2) Expansion and activation culture of strains: inoculating the activated strain into a liquid growth medium, and culturing for 12-24 hours at 28-32 ℃ to obtain an enlarged culture strain;
(3) Enrichment culture of bacterial liquid: sucking 5mL expanded culture strain into liquid growth medium, and culturing at 28-32deg.C for 12-24 hr to obtain enriched thallus;
(4) Powder preparation: centrifuging the enriched thalli for 1 minute at 150rpm, removing supernatant, and then placing in a temperature of 30-37 ℃ for drying for 30-60 minutes to respectively obtain candida utilis powder, trichoderma longibrachiatum powder, lactobacillus rhamnosus powder and bacillus subtilis powder.
10. The method for preparing the feed-modified microbial agent for fish and shrimp culture according to claim 9, which is characterized by comprising the following steps: the preparation method of the water flower extract, the haematococcus extract and the cercis chinensis extract comprises the following steps: pulverizing flos Pulsatillae, haematococcus and Cercis chinensis, respectively adding into ethanol solution, reflux extracting for 3-5 times for 2-3 hr each time, filtering, concentrating, and drying to obtain dry powders of flos Pulsatillae extract, haematococcus extract and Cercis chinensis extract.
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