CN113678962B - Micro-ecological feed additive for aquaculture and use method thereof - Google Patents
Micro-ecological feed additive for aquaculture and use method thereof Download PDFInfo
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- CN113678962B CN113678962B CN202110946729.8A CN202110946729A CN113678962B CN 113678962 B CN113678962 B CN 113678962B CN 202110946729 A CN202110946729 A CN 202110946729A CN 113678962 B CN113678962 B CN 113678962B
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- microbacterium oxydans
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- WWUZIQQURGPMPG-UHFFFAOYSA-N (-)-D-erythro-Sphingosine Natural products CCCCCCCCCCCCCC=CC(O)C(N)CO WWUZIQQURGPMPG-UHFFFAOYSA-N 0.000 description 1
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- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
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- PMVSDNDAUGGCCE-TYYBGVCCSA-L Ferrous fumarate Chemical compound [Fe+2].[O-]C(=O)\C=C\C([O-])=O PMVSDNDAUGGCCE-TYYBGVCCSA-L 0.000 description 1
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- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
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- 235000007079 manganese sulphate Nutrition 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 230000001546 nitrifying effect Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
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- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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- YZRQUTZNTDAYPJ-UHFFFAOYSA-N sanguinarine pseudobase Natural products C1=C2OCOC2=CC2=C3N(C)C(O)C4=C(OCO5)C5=CC=C4C3=CC=C21 YZRQUTZNTDAYPJ-UHFFFAOYSA-N 0.000 description 1
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- 239000002904 solvent Substances 0.000 description 1
- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 description 1
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- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
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-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biochemistry (AREA)
- Physiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Health & Medical Sciences (AREA)
- Insects & Arthropods (AREA)
- Marine Sciences & Fisheries (AREA)
- Birds (AREA)
- Fodder In General (AREA)
- Feed For Specific Animals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to an aquatic product cultureThe micro-ecological feed additive for reproduction comprises rhizobium and microbacterium oxydans, wherein the rhizobium is rhizobium (A), (B)Rhizobium rosettiformans) The preservation number is CGMCC No.22739, and the microbacterium oxydans is microbacterium oxydans (A.Microbacterium oxydans) The preservation number is CGMCC No.22738. The invention can promote the growth of the Australia crayfish, improve the immunity and disease resistance of the organism, greatly improve the survival rate and the growth rate of the culture, and has the effect obviously superior to the well-known immunopotentiation microbial inoculum such as lactobacillus and bacillus subtilis; but also can effectively stabilize the level of ammonia nitrogen and nitrite nitrogen in the water body. The used bacterial strain can be cultured by using an inorganic nitrogen source and a cheap organic carbon source, so that the culture cost is obviously reduced.
Description
Technical Field
The invention relates to a micro-ecological feed additive for aquaculture and a using method thereof, in particular to a micro-ecological feed additive for Australia crayfish, belonging to the technical field of aquaculture microorganisms.
Background
With the rapid development of aquaculture industry and the great increase of aquaculture density, in order to reduce aquaculture diseases, on one hand, the aquaculture ecological environment needs to be improved, and on the other hand, the immunity of cultured animals needs to be improved, and pathogenic microorganisms are inhibited. At present, in the aspect of improving the culture environment, various chemical preparation products for bottom modification and disinfection are available, and photosynthetic bacteria, autotrophic nitrifying bacteria and anoxic denitrifying bacteria are also available; in the aspect of improving the immunity of the cultured animals, allicin, complex enzyme, traditional Chinese medicine components (such as lemongrass) and microbial preparations of lactobacillus plantarum and bacillus subtilis are used as substitute additives of antibiotics, so that a series of adverse consequences of drug residue, increase of drug-resistant microorganisms, imbalance of normal microbial population in water, functional damage of internal organs of the cultured animals and the like are avoided. However, the prior art still has the following problems:
1. the existing product for improving the immunity of the aquaculture animals is not outstanding in the aspect of improving the culture environment, and the continuous addition of the feed and the bait can easily cause the nitrite nitrogen and the ammonia nitrogen in the water body to exceed the standards, so that the aquaculture animals are poisoned and killed;
2. the product for improving the immunity of the aquaculture animals has higher application cost;
3. the current product for improving the breeding environment has a weak effect of improving the immunity of aquaculture animals, and although the product is continuously added, the breeding environment is still easily interfered, and the bacterial phase and the algal phase are changed greatly, so that the disease death of the aquaculture animals is aggravated.
Chinese patent with publication number CN109043225A found by retrieval discloses a feed for increasing the individual weight of crayfish, which comprises wheat bran, soybean cake, ma Youzha, lotus powder, monochoria vaginalis fermentation material, silkworm chrysalis meal, alfalfa meal, bacillus subtilis, bone meal and a regulator; wherein the regulator comprises D-glucuronic acid, L-pyroglutamic acid calcium, ferrous fumarate, sanguinarine, and betaine; the preparation method of the herba monochoriae vaginalis fermented material comprises adding molasses, EDTA chelated iron and rhizomatous nitrogen-fixing rhizobium into the collected herba monochoriae vaginalis whole plant, mixing, and pulping to obtain a pulping material; mixing the beating slurry and the vinegar residue biogas slurry, and fermenting. The fermentation material of the crayfish feed contains nodule nitrogen fixing rhizobia which is mixed in the pulping material of the monochoria vaginalis and the like, and the effect of the fermentation material is to decompose high-content cellulose and other substances which are difficult to decompose in the monochoria vaginalis and then ferment together with acetic acid fermentation bacteria in the vinegar residue biogas slurry to promote the generation of acetic acid. Wherein the rhizomatous nitrogen-fixing rhizobia can be inhibited by the low pH value of acetic acid, and the pH value needs to be adjusted to about 5.6 for fermentation; meanwhile, the rhizoma nitrogen-fixing rhizobia is a self-growing nitrogen-fixing bacterium, and can still fix and convert nitrogen into ammonium of organisms under the condition of no symbiosis of leguminous plants.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: aiming at the problems in the prior art, a microecological feed additive for aquaculture and a use method thereof are provided.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a microecological feed additive for aquaculture comprises rhizobium and microbacterium oxydans, wherein the rhizobium is rhizobium: (A), (B), (C) and (C)Rhizobium rosettiformans) The preservation number is CGMCC No.22739, and the microbacterium oxydans is microbacterium oxydans (A.Microbacterium oxydans) The preservation number is CGMCC No.22738.
The invention adopts rhizobia (Rhizobium rosettiformansThe preservation number is CGMCC No. 22739) as a feed additive. Extracellular polysaccharide secreted by rhizobium can effectively improve the immunity of Australia freshwater lobster; the rhizobia left on the feed can grow and propagate by taking the ammonia nitrogen, the nitrite nitrogen or the nitrate nitrogen and other inorganic nitrogen in the water body as a unique nitrogen source, and the control of the concentrations of the ammonia nitrogen and the nitrite nitrogen in the water body is facilitated; the carbon source is more widely utilized, and the rhizobia can take cheap wastes such as plant straws as the carbon source, so that the culture cost is obviously reduced.
The invention adopts microbacterium oxydans (Microbacterium oxydansThe preservation number is CGMCC No. 22738) as the feed additive. The microbacterium oxydans can enrich trace elements in a water body, the absorption amount of the microbacterium oxydans to phosphorus reaches more than 3 times of that of common bacteria, the high requirements of growth of the Australian crayfish on the trace elements and the phosphorus can be effectively supplemented, and the body immunity of the Australian crayfish is further ensured to play a remarkable role; the microbacterium oxydans on the residual feed is beneficial to reducing the concentration of phosphate in the water body, so that the occurrence risk of cyanobacterial bloom is reduced; the microbacterium oxydans also has antagonistic action on pathogenic bacteria of Australia crayfish, and is beneficial to reducing the occurrence of breeding diseases; oxygen (O)The microbacterium warpouns has antagonistic action on bare algae in the pond, and is beneficial to reducing the risk of bare algae bloom; the carbon source of the microbacterium oxydans is widely utilized, and cheap wastes such as plant straws can be used as the carbon source, so that the culture cost is obviously reduced.
In a word, the special microbial inoculum is added into the feed or the plant bait, so that the growth of the Australia crayfish can be promoted, the immunity and the disease resistance of the organism can be improved, and the survival rate of the culture can be greatly improved; secondly, the water quality deterioration caused by the accumulation of a large amount of feed residues and animal wastes can be effectively reduced, and ammonia nitrogen and nitrite nitrogen in the water body are stabilized; furthermore, the used bacterial strain can utilize inorganic nitrogen source and cheap organic carbon source, so that the culture cost is obviously reduced.
In the technical scheme, the feed additive is formed by mixing a liquid microbial inoculum obtained by enlarged culture of rhizobia and a liquid microbial inoculum obtained by enlarged culture of microbacterium oxydans.
Further, the number of viable rhizobia in the feed additive is 1 × 10 8 CFU/ml～1×10 9 CFU/ml, viable count of Microbacterium oxydans 1X 10 8 CFU/ml～1×10 9 CFU/ml。
The invention further provides the feed additive which is prepared by mixing the rhizobium freeze-dried powder and the microbacterium oxydans freeze-dried powder according to the mass ratio of the rhizobium freeze-dried powder: microbacterium oxydans = 1-2:3-1.
The invention also provides a use method of the microecological feed additive, wherein the liquid feed additive is directly sprayed on the surface of feed or plant bait and is used after being dried in the air.
The additive disclosed by the invention is directly applied to the feed, the rhizobium in the feed can improve the intestinal health of the shrimps and promote the digestion and absorption of nutrient substances, and extracellular polysaccharide secreted by the rhizobium is favorable for improving the immunity of the Australia crayfish; the rhizobia on the residual feed can take the ammonia nitrogen, the nitrite nitrogen or the nitrate nitrogen in the water body as the only nitrogen source, thereby being beneficial to controlling the concentration of the ammonia nitrogen and the nitrite nitrogen in the water body and maintaining the healthy water body environment. Meanwhile, the rhizobia used by the method is not an autogenous azotobacter, does not have the symbiosis of leguminous plants, does not have the independent azotobacter capacity, can use ammonia nitrogen, nitrite nitrogen or inorganic nitrogen such as nitrate nitrogen in the water body as the only nitrogen source, has the aerobic denitrification denitrogenation capacity, and is beneficial to improving the survival rate of the cultured Australian crayfish from the two aspects of water quality and immunity. The microbacterium oxydans used in the invention can enrich trace elements in water, and the absorption capacity of thalli to phosphorus is obviously higher than that of other bacteria, and the microbacterium oxydans also has antagonism to pathogenic bacteria and antagonism to bare algae in water bloom. These advantages are all beneficial to the application of the microbacterium oxydans in aquaculture.
Further, the content of rhizobia in the feed is 1 x 10 6 CFU/g～2×10 7 CFU/g, content of Microbacterium oxydans 1 × 10 6 CFU/g～2×10 7 CFU/g。
Further, the feed additive is a liquid composite microbial inoculum formed by mixing a liquid microbial inoculum obtained by enlarged culture of rhizobia and a liquid microbial inoculum obtained by enlarged culture of microbacterium oxydans, wherein the number of viable bacteria of the rhizobia is 1 multiplied by 10 8 CFU/ml～1×10 9 CFU/ml, viable count of Microbacterium oxydans 1X 10 8 CFU/ml～1×10 9 CFU/ml。
Further, the feed additive is a solid-state composite microbial agent formed by mixing rhizobium freeze-dried powder and microbacterium oxydans freeze-dried powder, and the mass ratio of the rhizobium freeze-dried powder is as follows: the microbacterium oxydans = 1-2:3-1, and the solid complex microbial inoculum is dissolved by water to be a liquid microbial inoculum for use.
Furthermore, the number of viable rhizobia in the liquid microbial inoculum is 1 multiplied by 10 8 CFU/ml～1×10 9 CFU/ml, viable count of Microbacterium oxydans 1X 10 8 CFU/ml～1×10 9 CFU/ml。
Furthermore, the dosage form of the feed additive is powder or liquid.
When the feed additive is a liquid, respectively placing the rhizobia and the microbacterium oxydans in an LB culture medium for culture to obtain a rhizobia enlarged culture solution and a microbacterium oxydans enlarged culture solution;
the rhizobium enlarged culture solution and the microbacterium oxydans enlarged culture solution are mixed to form a liquid composite microbial inoculum which is used as a feed additive.
When the feed additive is powder, the rhizobium enlarged culture solution and the microbacterium oxydans enlarged culture solution are respectively subjected to centrifugal collection and low-temperature freeze drying to obtain rhizobium freeze-dried powder and microbacterium oxydans freeze-dried powder, and the two are mixed to obtain the solid compound microbial inoculum serving as the feed additive.
The invention has the advantages that:
firstly, the growth of the Australia crayfish can be promoted, the immunity and disease resistance of the organism can be improved, the survival rate and the growth rate of the culture can be greatly improved, and the effect is obviously superior to the well-known immunopotentiation microbial inoculum such as lactobacillus and bacillus subtilis;
secondly, the levels of ammonia nitrogen and nitrite nitrogen in the water body can be effectively stabilized;
finally, the strain used can be cultured by using an inorganic nitrogen source and a cheap organic carbon source, so that the culture cost is obviously reduced.
Drawings
The invention will be further described with reference to the accompanying drawings.
FIG. 1 shows NO in water after feeding different feeds in example 1 of the present invention 2 Graph of concentration profile.
FIG. 2 shows NH in water after feeding different feeds in example 1 of the present invention 4 Graph of concentration profile.
Fig. 3 is a schematic diagram of the survival rate of the Australian crayfish fed with different feeds in the embodiment 1 of the invention.
FIG. 4 is a diagram showing the survival rate and specific growth rate of the Australian crayfish after addition of Aeromonas hydrophila in example 1 of the present invention.
Fig. 5 is a schematic diagram of the survival rate of the Australian crayfish fed with different feeds in the embodiment 2 of the invention.
Fig. 6 is a schematic diagram of the survival rate of the Australian crayfish fed with different feeds in example 3 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples. The invention is not limited to the examples given.
The invention provides a micro-ecological feed additive for aquaculture, which comprises rhizobia and microbacterium oxydans. Wherein the rhizobia is rhizobiumRhizobium rosettiformansThe preservation number is CGMCC No.22739; the microbacterium oxydans is microbacterium oxydansMicrobacterium oxydansThe preservation number is CGMCC No.22738. The preservation unit of the strains is the common microorganism center of China Committee for culture Collection of microorganisms, the address is No. 3 Xilu No. 1 of Beijing, chaoyang, and the preservation date is 21 days at 6 months in 2021.
The micro-ecological feed additive can be liquid or powder. When the micro-ecological feed additive is a liquid agent, the micro-ecological feed additive is a composite liquid agent formed by mixing a liquid agent obtained by enlarged culture of rhizobia and a liquid agent obtained by enlarged culture of microbacterium oxydans; when the micro-ecological feed additive is powder, the micro-ecological feed additive is prepared by mixing the rhizobium freeze-dried powder and the microbacterium oxydans freeze-dried powder according to the mass ratio of the rhizobium freeze-dried powder to the microbacterium oxydans freeze-dried powder: the compound solid microbial inoculum is formed by mixing microbacterium oxydans = 1-2:3-1, and can be used after being dissolved into a compound liquid microbial inoculum by adopting solvents such as water and the like when in use. In the above two composite liquid microbial inoculum, the viable count of Rhizobium is 1 × 10 8 CFU/ml～1×10 9 CFU/ml, viable count of Microbacterium oxydans 1X 10 8 CFU/ml～1×10 9 CFU/ml。
The compound liquid microbial inoculum is directly sprayed on the surfaces of various formula feeds for aquaculture, and can be directly used after being dried in the air. After the feed is fed with the Australia crayfish, a small amount of feed can be left at the bottom of a water area, the microbial inoculum on the residual feed can have good influence on water quality, the feed eaten by the Australia crayfish can have good influence on the Australia crayfish, and the immunity of the Australia crayfish is improved.
The chemical reagents and materials used in the invention are all commercially available.
Example 1
RhizobiumRhizobium rosettiformansMicrobacterium oxydansMicrobacterium oxydansRespectively inoculating into LB culture medium for amplification culture. Basic formula of LB culture mediumThe following method is adopted: 5g/L yeast powder, 10g/L peptone and 10g/L sodium chloride. The culture medium has high cost, and in large-scale use, corn starch can be added into a conventional inorganic salt culture medium as a carbon source to reduce the cost, and inorganic ammonia contained in the culture medium is used as a nitrogen source. The basal medium formulation may be MgSO 4 ·7H 2 O 1g/L, KH 2 PO 4 0.5g/L, K 2 HPO 4 4.0g/L, NaCl 1.0 g/L,CaCl 2 20 mg/L，FeSO 4 5 mg/L,ZnCl 2 5 mg/L, MnCl 2 ·4H 2 O 5mg/L,CuCl 2 0.5mg/L。
The lactic acid bacteria used for the comparative treatment were inoculated in an MRS medium for the scale-up culture, and the bacillus was inoculated in an LB medium for the scale-up culture. The formula of the MRS culture medium is as follows: 10g/L of protein orange, 5g/L of beef powder, 4g/L of yeast powder, 1ml/L of Tween 80, 2g/L of dipotassium phosphate, 5g/L of sodium acetate, 2g/L of triammonium citrate, 0.2g/L of magnesium sulfate and 0.05g/L of manganese sulfate.
Respectively concentrating the bacterial liquid after the rhizobium enlarged culture and the bacterial liquid after the microbacterium oxydans enlarged culture, and mixing to prepare mixed bacterial liquid, wherein the viable bacteria number of the rhizobium in the mixed bacterial liquid is 6 multiplied by 10 8 CFU/ml, viable count of Microbacterium oxydans 6X 10 8 CFU/ml. Meanwhile, the lactic acid bacteria liquid and the bacillus liquid which are used for comparison treatment and subjected to expanded culture are respectively concentrated to prepare a mixed bacterial liquid, and the number of viable bacteria of the two strains in the mixed bacterial liquid is the same as that of rhizobium and microbacterium oxydans.
In addition, the rhizobium enlarged culture solution and the microbacterium oxydans enlarged culture solution can be respectively centrifugally collected and frozen and dried at low temperature to prepare the freeze-dried rhizobium powder and the freeze-dried microbacterium oxydans powder, the freeze-dried rhizobium powder and the freeze-dried microbacterium oxydans powder are mixed according to the mass ratio of 1-2:3-1 to obtain the solid composite microbial agent, and the solid composite microbial agent is dissolved and diluted by water when in use.
And respectively spraying the mixed bacterial liquid containing rhizobium and microbacterium oxydans and the mixed bacterial liquid containing lactobacillus and bacillus on the surface of the feed of the Australian crayfish, and air-drying. Wherein, in the feed sprayed with the bacterial liquid containing rhizobium and microbacterium oxydans, the contents of both rhizobium and microbacterium oxydans are 6 multiplied by 10 6 CFU/g; the spraying containsThe contents of lactobacillus and bacillus in the feed of lactobacillus and bacillus liquid are both 6 × 10 6 CFU/g. Meanwhile, the following test was performed using a feed sprayed with an equal volume of deionized water as a blank control group.
(1) The influence of feeding feed containing rhizobium and microbacterium oxydans on the maintenance of water quality and survival rate of Australian freshwater lobster in the rough period
The test shrimp was young shrimp with a weight of about 0.02 g/shrimp and a feeding density of 185/m 2 The method is characterized in that a shrimp nest is constructed by tiles, the daily feeding amount is 10% of the weight of shrimps, 1/3 of water is changed every day, test water is aerated tap water, liquid ionized calcium is supplemented every 2 days, the test period is 20 days, and the water temperature is 25 ℃. In the test, formula feed without any microbial inoculum is added as a blank control group, and formula feed added with lactobacillus and bacillus which are recognized to improve immunity at present is used as comparison treatment. The concentration of single bacterium agent contained in the feed is 6 multiplied by 10 6 CFU/g. As shown in figures 1 to 3, the feed containing rhizobium and microbacterium oxydans has good maintenance effect on the water quality of the Australian freshwater lobster in the standard rough period, can keep ammonia ions and nitrite ions in the water body below a danger threshold value under the conditions of no aquatic plant dimension, no photosynthetic bacteria and denitrifying bacteria regulation, and improves the survival rate by 16.4 percent compared with the common formula feed and by 8.7 percent compared with the formula feed of lactic acid bacteria and bacillus which is currently recognized to improve the immunity.
(2) Feeding feed containing rhizobia and microbacterium oxydans to resist pathogenic bacteria invasion of Australian freshwater lobster under the condition that water quality is cooperatively regulated by microbial inoculum and eel grass
The test shrimp was adult shrimp with a weight of about 17 g/adult shrimp and a feeding density of 37 shrimp/m 2 The daily feeding amount is 5 percent of the weight of the shrimps, the eel coverage is 75 percent, the test water is pond water which is treated by eel, photosynthetic bacteria and denitrifying bacteria in advance, the shrimps are put after the water quality is stable, and liquid ionized calcium is supplemented every 5 days. Supplementing brown sugar carbon source, and maintaining CODc r About 20mg/L. The water changing treatment is carried out according to the water quality condition. The test period is 30 days, and the water temperature is 30 ℃. The adding of the medicine can cause the crustacean disease and the enteritis,Aeromonas hydrophila of edematosis, tail rot and necrobiosis: (Aeromonas hydrophila) Initial concentration of 3X 10 4 Adding into Aeromonas hydrophila every 5 days, wherein the concentration is 3 × 10 4 One per ml. The experiment uses a formula feed without any microbial inoculum as a blank control, and uses a formula feed added with lactobacillus and bacillus which are recognized to improve immunity at present as comparison treatment. The concentration of single bacterium agent contained in the feed is 6 multiplied by 10 6 CFU/g. As shown in figure 4, the feed containing rhizobium and microbacterium oxydans is beneficial to the Australian crayfish to resist the invasion of aeromonas hydrophila, the survival rate is improved by 124.2 percent compared with the blank, and is improved by 15.6 percent compared with the formula feed of 'lactobacillus + bacillus' which is currently recognized to improve immunity, and the feed containing the rhizobium and microbacterium oxydans is equivalent to the feed containing the 'lactobacillus + bacillus' in terms of specific growth rate.
(3) Phosphorus enrichment capacity of microbacterium oxydans
The experiment was carried out using 0.1 XHoaglands medium (to which 0.125 g of yeast powder and 0.25 g of peptone were added) and having a TP content of 5.79mg/L. Different strains were inoculated into 0.1 × hoagland media according to table 1, with an inoculum size of 2% for each strain. The culture was carried out at 28 ℃ and 120rpm for 48 hours. Test results show that the absorption removal rate of the microbacterium oxydans to phosphorus in a water body is the highest and reaches 69.48%.
TABLE 1
| Bacteria | Absorption removal rate of phosphorus |
| Brevundimonas sp | 4.98% |
| Sphingosine bacteria | 8.62% |
| Photosynthetic rhodospirillum | 5.32% |
| Arthrobacter | 11.64% |
| Pseudomonas sp | 11.65% |
| Bacillus sp | 14.63% |
| Microbacterium oxydans | 69.48% |
| Agrobacterium tumefaciens | 23.06% |
(4) Enrichment capacity of microbacterium oxydans on trace elements
LB culture medium is adopted for the experiment, feSO is added 4 The concentration of Fe ions was 100mg/L and the inoculum size of Microbacterium oxydans was 2%, and the cells were cultured at 28 ℃ and 120rpm for 48 hours. The results showed that the iron-enriching capacity of the cells reached 8.7mg/g dry weight, which is comparable to the iron content of iron-rich yeast.
LB culture medium is adopted for the experiment, znSO is added 4 The Zn ion concentration was 100mg/L and the inoculum size of the Microbacterium oxydans was 2%, and the cells were cultured at 28 ℃ and 120rpm for 48 hours. The result shows that the zinc enrichment capacity of the bacteria reaches 14.6mg/g dry weight, which is much higher than the zinc enrichment capacity of common probiotics by 0.55mg/L.
(5) Antagonistic ability of microbacterium oxydans against some pathogenic bacteria and bare algae
The test uses LB culture medium to amplify micro-bacillus oxydans and PD culture medium to amplify black pythium aphanidermatum (pythium aphanidermatum is causing shrimp pest disease)The fungal pathogen of (1) respectively adding the Saccharomycopsis nigricans and the microbacterium oxydans into a co-culture medium according to certain concentrations for culturing, wherein the co-culture medium is obtained by mixing an LB culture medium and a PDA culture medium according to a weight ratio of 1:1. The addition amount of black Humicola is 5 × 10 3 The addition amount of Microbacterium oxydans is 5 × 10 4 Each/ml and 5X 10 5 One cell/ml, blank without adding Microbacterium oxydans. The number of colonies of the fungus Saccharum niveum was counted using PDA medium after three days of culture, and the results are shown in Table 2.
TABLE 2
| Inhibition of mold after 3 | |
| Blank space | |
| 0% | |
| Microbacterium oxydans (5X 10) 4 One/ml) | 49.09% |
| Microbacterium oxydans (5X 10) 5 One/ml) | 73.47% |
The result shows that the microbacterium oxydans has good inhibition effect on the black pythium aphanidermatum.
The test adopts LB culture medium to expand microbacterium oxydans, HUT culture medium to expand euglena flos-aquae (euglena flos-aquae can make ecological system collapse and pathogenic bacteria mass-propagate), respectively adding euglena flos-aquae and microbacterium oxydans into co-culture medium according to a certain concentration, the co-culture medium is HUT culture medium. The addition amount of Euglena sanguinea is 5 × 10 6 The addition amount of Microbacterium oxydans is 5 × 10 7 Each/ml and 5X 10 8 One cell/ml, blank without adding Microbacterium oxydans. The colony count of euglena flos-aquae was counted using HUT medium after three days of culture, and the results are shown in Table 3.
TABLE 3
| Inhibition rate of Euglena haemolytica after 3 | |
| Blank space | |
| 0% | |
| Microbacterium oxydans (5X 10) 7 One/ml) | 68.95% |
| Microbacterium oxydans (5X 10) 8 One/ml) | 87.01% |
The result shows that the microbacterium oxydans has good inhibition effect on the euglena haemoglobin.
Example 2
The rhizobia concentration was 1X 10 as prepared in example 1 6 CFU/g, concentration of oxidizing micro-bacteria is 3 x 10 6 CFU/g feed.
The test shrimp was adult shrimp with a weight of about 17 g/adult shrimp and a feeding density of 37 shrimp/m 2 The daily feeding amount is 5 percent of the weight of the shrimps, the picrasm coverage is 75 percent, the test water is pond water which is treated by the picrasm, the photosynthetic bacteria and the nitrification and denitrification bacteria in advance, the shrimps are put into the pond water after the water quality is stable, and the liquid ionized calcium is supplemented every 5 days.Supplementing brown sugar carbon source, and maintaining CODC r About 20mg/L. The water changing treatment is carried out according to the water quality condition. The test period is 30 days, and the water temperature is 30 ℃. Aeromonas hydrophila (capable of causing crustacean disease, enteritis, edema, tail rot and necrobiosis) is addedAeromonas hydrophila) Initial concentration of 3X 10 4 The cells per ml were then supplemented every 5 days with Aeromonas hydrophila at a concentration of 3X 10 4 Each/ml. The test takes the formula feed without any microbial inoculum as a blank control. The feed contains rhizobia at a concentration of 1 × 10 6 CFU/g, microbacterium oxydans 3X 10 6 CFU/g. As shown in FIG. 5, feeding of the feed containing rhizobium and microbacterium oxydans is beneficial to the resistance of the Australian crayfish to the invasion of aeromonas hydrophila, and the survival rate is improved by 118.3 percent compared with the blank.
Example 3
Rhizobia concentrations of 2X 10 were made up as in example 1 7 CFU/g, concentration of oxidizing micro-bacteria is 1 x 10 7 CFU/g of feed.
The test shrimp was adult shrimp with a weight of about 17 g/adult shrimp and a feeding density of 37 shrimp/m 2 The daily feeding amount is 5 percent of the weight of the shrimps, the picrasm coverage is 75 percent, the test water is pond water which is treated by the picrasm, the photosynthetic bacteria and the nitrification and denitrification bacteria in advance, the shrimps are put into the pond water after the water quality is stable, and the liquid ionized calcium is supplemented every 5 days. Supplementing brown sugar carbon source, and maintaining CODC r About 20mg/L. The water changing treatment is carried out according to the water quality condition. The test period is 30 days, and the water temperature is 30 ℃. Aeromonas hydrophila (capable of causing crustacean disease, enteritis, edema, tail rot and necrobiosis) is addedAeromonas hydrophila) Initial concentration of 3X 10 4 Adding into Aeromonas hydrophila every 5 days, wherein the concentration is 3 × 10 4 One per ml. The test takes the formula feed without any microbial inoculum as a blank control. The feed contains rhizobia at a concentration of 2 × 10 7 CFU/g, microbacterium oxydans 1X 10 7 CFU/g. As shown in figure 6, the feedstuff containing rhizobium and microbacterium oxydans is beneficial to the Australian crayfish to resist the invasion of aeromonas hydrophila, and the survival rate is improved by 130.3 percent compared with the blank.
In addition to the above embodiments, the present invention may have other embodiments. All technical solutions formed by adopting equivalent substitutions or equivalent transformations fall within the protection scope of the claims of the present invention.
Claims (10)
1. A micro-ecological feed additive for aquaculture is characterized in that: comprises rhizobium and microbacterium oxydans, wherein the rhizobium is rhizobium (A), (B)Rhizobium rosettiformans) The preservation number is CGMCC No.22739, and the microbacterium oxydans is microbacterium oxydans (A.Microbacterium oxydans) The preservation number is CGMCC No.22738.
2. The micro-ecological feed additive for aquaculture according to claim 1, characterized in that: the feed additive is formed by mixing a liquid microbial inoculum obtained by enlarged culture of rhizobia and a liquid microbial inoculum obtained by enlarged culture of microbacterium oxydans.
3. The micro-ecological feed additive for aquaculture according to claim 2, characterized in that: the number of viable rhizobia in the feed additive is 1 × 10 8 CFU/ml～1×10 9 CFU/ml, viable count of Microbacterium oxydans 1X 10 8 CFU/ml～1×10 9 CFU/ml。
4. The micro-ecological feed additive for aquaculture according to claim 1, characterized in that: the feed additive is formed by mixing rhizobium freeze-dried powder and microbacterium oxydans freeze-dried powder, and the mass ratio of the rhizobium freeze-dried powder is as follows: microbacterium oxydans = 1-2:3-1.
5. The use of the micro-ecological feed additive for aquaculture according to any one of claims 1 to 4, characterized in that: the liquid feed additive is directly sprayed on the surface of feed or plant bait, and is used after being dried in the air.
6. The use method of the micro-ecological feed additive for aquaculture according to claim 5, characterized in that: the feed contains rhizobium of1×10 6 CFU/g～2×10 7 CFU/g, content of Microbacterium oxydans 1X 10 6 CFU/g～2×10 7 CFU/g。
7. The use method of the micro-ecological feed additive for aquaculture according to claim 5, characterized in that: the feed additive is a liquid composite microbial inoculum formed by mixing a liquid microbial inoculum obtained by enlarged culture of rhizobia and a liquid microbial inoculum obtained by enlarged culture of microbacterium oxydans, wherein the number of viable bacteria of the rhizobia is 1 multiplied by 10 8 CFU/ml～1×10 9 CFU/ml, viable count of Microbacterium oxydans 1X 10 8 CFU/ml～1×10 9 CFU/ml。
8. The use method of the micro-ecological feed additive for aquaculture according to claim 5, characterized in that: the feed additive is a solid-state composite microbial agent formed by mixing rhizobium freeze-dried powder and microbacterium oxydans freeze-dried powder, and the mass ratio of the rhizobium freeze-dried powder is as follows: the microbacterium oxydans = 1-2:3-1, and the solid complex microbial inoculum is dissolved by water to be a liquid microbial inoculum for use.
9. The use method of the micro-ecological feed additive for aquaculture as claimed in claim 8, wherein the viable count of rhizobia in the liquid microbial inoculum is 1 x 10 8 CFU/ml～1×10 9 CFU/ml, viable count of Microbacterium oxydans 1X 10 8 CFU/ml～1×10 9 CFU/ml。
10. The use method of the micro-ecological feed additive for aquaculture as claimed in claim 7 or 8, wherein rhizobia and microbacterium oxydans are cultured in LB medium to obtain rhizobia amplification culture solution and microbacterium oxydans amplification culture solution;
mixing the rhizobium enlarged culture solution and the microbacterium oxydans enlarged culture solution to form a liquid composite microbial agent serving as a feed additive;
or centrifugally collecting the rhizobium enlarged culture solution and the microbacterium oxydans enlarged culture solution respectively, and freeze-drying at low temperature to obtain rhizobium freeze-dried powder and microbacterium oxydans freeze-dried powder, and mixing the two to obtain the solid compound microbial inoculum serving as a feed additive.
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| WO2014169046A1 (en) * | 2013-04-09 | 2014-10-16 | Novozymes A/S | Compositions and methods for improving the health of aquatic animals |
| CN104017757B (en) * | 2014-06-03 | 2016-05-18 | 江南大学 | The one plant height effect end, changed bacillus and the composite base made changes microbial inoculum and application |
| CN106754549A (en) * | 2017-01-20 | 2017-05-31 | 国家海洋局第三海洋研究所 | Compound Bacillus acidi lactici powder used for aquiculture with long preservation period and preparation method thereof |
| CN109043225A (en) * | 2018-07-12 | 2018-12-21 | 铜陵市五华生态农业发展有限公司 | A kind of feed improving cray individual weight |
| CN109498744A (en) * | 2018-08-24 | 2019-03-22 | 四川长重宝生物工程有限责任公司 | A kind of preparation method that composite bacteria fermentation compound eucommia bark leaf is scattered |
| CN110402856B (en) * | 2019-08-30 | 2022-05-13 | 三亚福联水产发展有限公司 | High-yield culture method for groupers |
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Effective date of registration: 20240617 Address after: No. 8-1 Yiyu Road, Changfu Street, Suzhou City, Jiangsu Province, 215000 Patentee after: SUZHOU HENGYANG AOLONG AGRICULTURAL TECHNOLOGY CO.,LTD. Country or region after: China Address before: Zhongshan outside the Nanjing city of Jiangsu province 210014 Zhongling Street No. 50 Patentee before: JIANGSU ACADEMY OF AGRICULTURAL SCIENCES Country or region before: China |