CN108277174B - Complex microbial inoculant for aquaculture and application thereof - Google Patents
Complex microbial inoculant for aquaculture and application thereof Download PDFInfo
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Abstract
The invention discloses a novel composite microbial inoculum fermentation method, a propagation method and application thereof for aquaculture, and belongs to the technical field of feed additives, aquaculture water quality improvement and microorganisms. The compound microbial inoculum for aquaculture is prepared from lactobacillus plantarum, leuconostoc mesenteroides, pediococcus acidilactici and gibberella barnacii. The compound microbial inoculum is mixed with fish, shrimp and crab feed, can promote the balance of intestinal flora of the fish, shrimp and crab and the disease resistance of the fish, shrimp and crab, can enhance the appetite of the fish, shrimp and crab and improve the utilization rate of the feed; is sprinkled on the aquaculture water body according to a certain proportion, and has good effects on the prevention and inhibition of blue algae, the degradation of pH and nitrite and the promotion of the ecological balance of the aquaculture water body. The application of the complex microbial inoculum in aquaculture has the advantages of short fermentation period, wide suitable temperature range, stable activity, simple process, less equipment investment and easy implementation, and is suitable for self-production and self-use of farmers.
Description
Technical Field
The invention discloses a novel method for fermenting and expanding culture of a complex microbial inoculant for aquaculture and application of the complex microbial inoculant, and belongs to the technical field of feed additives, water quality improvement of aquaculture water and microorganisms.
Background
China is the largest aquatic product breeding country in the world at present and is the only country in the world with the breeding yield exceeding the fishing yield. In order to avoid polluting the water area environment and ensure the healthy and sustainable development of aquaculture industry, the aquaculture industry in China is changed from a quantitative type to a quality benefit type, the large-scale and intensive culture mode gradually replaces the original extensive culture mode, and bait, space competition and accumulation of residual bait and excrement directly cause the deterioration of the water quality of the aquaculture water body, serious eutrophication, overproof ammonia nitrogen and nitrite in the aquaculture water body and the like in the high-density culture process, so that the aquaculture water body has toxic action on aquatic animals. Secondly, harmful microorganisms in the aquaculture environment are bred in a large quantity, the immunity of cultured animals is reduced, and finally, aquaculture diseases are caused to frequently burst, so that the quality of aquatic products is reduced, and the like, wherein the disease problem seriously puzzles the majority of culturists and hinders the healthy and sustainable development of the aquaculture industry. Meanwhile, the environment quality of the aquaculture water area is gradually reduced due to the discharge of industrial wastewater and domestic sewage, the over-high content of ammonia nitrogen is one of the main factors of water pollution and eutrophication, and the improvement of aquaculture water quality becomes one of the key problems of success and failure of aquaculture.
Practice proves that the bioremediation technology for decomposing toxic and harmful substances by using microorganisms is a low-price and effective method for treating large-area polluted areas, and the steps of inoculating exogenous high-efficiency microorganisms, adding nutrient salts of the microorganisms, providing electron acceptors, providing co-metabolic substrates and improving the bioavailability are measures for promoting bioremediation. The microbial preparation can improve the environment of a culture water area, has the denitrification performance with high efficiency, low cost and no secondary pollution, improves the organism immunity of a culture object, reduces the occurrence of diseases and makes culture activities develop towards a virtuous circle direction. In recent years, probiotics are concerned by people with the advantages of no toxicity, no residue and the like, and are widely applied to aquaculture, including application of feed additives and pond water quality purifiers.
Lactic acid bacteria are commonly used probiotics, and nitrite reductase and organic acid can be generated in the fermentation process. The compound feed additive is mainly used for feed additives in aquaculture, and can degrade carbohydrates to generate lactic acid and other organic acids, so that the pH value in intestinal tracts of cultured animals is reduced, the propagation of harmful bacteria is inhibited, the growth performance and the digestive enzyme activity of fishes and shrimps are improved, and the non-specific immunity of the fishes and the shrimps is improved. The lactobacillus can also promote mass propagation and growth of bait organisms in the aquaculture water, decompose residual bait, excrement and organic matters in water, improve water quality and inhibit propagation and growth of harmful bacteria in the water. However, if the viable count of lactic acid bacteria is low, it is not easy to become a dominant bacterial group in a water environment, and the efficacy of lactic acid bacteria cannot be exerted. The yeast is unicellular fungus with high content of nutrient components, is widely used for feed additives, can be propagated in a large amount in the alimentary canal, becomes a dominant population in competition with the survival of harmful bacteria, and effectively inhibits the growth of harmful pathogenic bacteria. Therefore, the yeast is combined with various lactic acid bacteria, and the application of the yeast and the lactic acid bacteria in aquaculture has good prospects. However, microbial preparations are greatly influenced by temperature and external conditions, the existing lactobacillus series microbial agents are mostly fermented at a single temperature, the types and the number of metabolites are limited, the quality guarantee period is short, the activity is continuously reduced in the storage process, and the product quality is difficult to guarantee.
Disclosure of Invention
The invention aims to provide a novel compound microbial inoculum for aquaculture, which has the advantages of short fermentation period, wide suitable temperature range, stable product activity and simple process, and is suitable for farmers to produce and use by themselves. The invention also aims to disclose the application of the complex microbial inoculum provided by the invention in improving the water quality of aquaculture water.
The strain used in the invention is compounded by 4 strains separated and screened by the inventor. The composite microbial inoculum is prepared from Lactobacillus plantarum, Leuconostoc mesenteroides, Pediococcus acidilactici, Saccharomyces pastorianus and liquid culture medium, the pH of the microbial inoculum is less than or equal to 4.0, and the number of viable bacteria is 6 multiplied by 10 respectively8-3×1010CFU/mL. Four kinds of bacteria are preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number of the lactobacillus plantarum is CGMCC No.15024, gram-positive bacteria and no spores are obtained by separating pickle samples, and the lactobacillus plantarum is identified as lactobacillus plantarum by 16S rDNA analysis and identification, has 99 percent of homology with other published lactobacillus plantarum 16S rDNA sequences; leuconostoc mesenteroides with the preservation number of CGMCC number 15023 is separated from a pickle sample, gram-positive coccus, no motion, no spore and facultative anaerobe, the growth temperature range is 2-37 ℃, the acid resistance is strong, and the homology with other published Leuconostoc mesenteroides 16S rDNA sequences is 99 percent through 16S rDNA analysis and identification; pediococcus acidilactici with preservation number of CGMCC No.5959, applied to the invention patent No. 201210110496.9 and in the shape of a sphereThe cells are divided alternately in two planes at right angles to form a tetrad, the general cells are paired, and the single-born cells are rare and are not arranged in a chain. Gram positive, no movement, acid production and facultative anaerobic. Colonies were small on MRS medium and white. The growth along the agar puncture line is filamentous. Catalase negative, no cytochrome. The Babbitt gibberella with the preservation number of CGMCC No.5960 is applied to the invention patent No. 201210110496.9, has high-temperature fermentation capacity, can rapidly decompose substrates such as lactic acid, amino acid, organic matters, protein and the like at the temperature of 60 ℃, has stronger reproductive capacity compared with other yeasts, can coexist with lactic acid bacteria, and has important significance for promoting the proliferation of the lactic acid bacteria.
The identification of the 4 microorganisms is described in the Bergey Manual of bacteria identification (eighth edition), according to their morphological and physiological and biochemical characteristics, and according to their 16S rDNA gene sequences, which are searched in Genbank.
1. The complex microbial inoculum of claim 1, which is characterized by comprising the following components in parts by weight:
3-4 parts of lactobacillus plantarum microbial inoculum;
3-4 parts of leuconostoc mesenteroides microbial inoculum;
3-4 parts of pediococcus acidilactici microbial inoculum;
2-3 parts of Babbitt gibberella;
wherein the pH of the microbial inoculum is not more than 4.0, and the viable count is not less than 6 × 108-3×1010CFU/mL。
2. The method of claim 1, wherein the preparation method comprises the following steps
Now: (1) respectively inoculating the four bacteria as described in claim 2 on a solid culture medium for activation; (2) selecting single colonies of the four activated strains, respectively inoculating the single colonies on respective liquid culture media, and culturing for 48 hours at the temperature of 37 ℃; (3) inoculating the cultured strain in (2) into a fermentation tank according to the proportion in claim 2, fermenting at 37 deg.C for 24 hr to obtain thallus suspensionThe pH of the bacterial liquid is less than or equal to 4.0, and the viable count is not less than 6 multiplied by 108-3×1010And (5) CFU/mL, namely preparing a finished compound bacterium agent product, performing quality inspection on the bacterium agent, filling after the bacterium agent is qualified, and storing in a cool and dry place. Wherein in the step (1), the solid culture medium comprises 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract and diammonium hydrogen citrate [ (NH) per 1000 mL4)2HC6H5O7]2.0 g, glucose (C)6H12O6·H2O) 20.0 g, Tween 801.0 mL, sodium acetate (CH)3COONa·3H2O) 5.0 g, dipotassium hydrogen phosphate (K)2HPO4·3H2O) 2.0 g, magnesium sulfate (MgSO)4·7H2O) 0.58 g, manganese sulfate (MnSO)4·H2O) 0.25 g, agar 18.0 g and 1000 mL of distilled water. In the step (2), every 1000 mL of liquid culture medium comprises 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract and diammonium hydrogen citrate [ (NH)4)2HC6H5O7]2.0 g, glucose (C)6H12O6·H2O) 20.0 g, Tween 801.0 mL, sodium acetate (CH)3COONa·3H2O) 5.0 g, dipotassium hydrogen phosphate (K)2HPO4·3H2O) 2.0 g, magnesium sulfate (MgSO)4·7H2O) 0.58 g, manganese sulfate (MnSO)4·H2O) 0.25 g and 1000 mL of distilled water.
3. The method for preparing the composite propagation inoculant according to claim 1, wherein the preparation method of the inoculant in fresh water is realized according to the following steps (taking propagation of 100L as an example):
(1) moving a 100L plastic white barrel to an outdoor direct sunlight place (the temperature in summer is more than 25 ℃, indoor expanding culture can be carried out in winter, heating is needed to keep the expanding culture temperature between 10 ℃ and 30 ℃), and adding about 50L of clean water (which can be well water or tap water, and the tap water needs to be aired for a period of time to prevent the residual chlorine in the tap water from damaging the growth of bacteria); (2) adding weighed culture medium, 2kg brown sugar and 5L of the composite probiotic preparation product in claim 1 into a 100L white barrel, and uniformly stirring by using clean rollersAnd then, injecting water into the plastic white barrel until the plastic white barrel is full, sealing and fermenting for 3 days until the pH value of the fermentation liquor is less than or equal to 5.0, and properly prolonging the culture expanding time of one to two days if the pH value does not reach the expected effect in rainy days or at the temperature of less than 20 ℃. Wherein in the step (2), every 1000 mL of the liquid culture medium comprises 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract and diammonium hydrogen citrate [ (NH)4)2HC6H5O7]2.0 g, glucose (C)6H12O6·H2O) 20.0 g, Tween 801.0 mL, sodium acetate (CH)3COONa·3H2O) 5.0 g, dipotassium hydrogen phosphate (K)2HPO4·3H2O) 2.0 g, magnesium sulfate (MgSO)4·7H2O) 0.58 g, manganese sulfate (MnSO)4·H2O) 0.25 g and 1000 mL of distilled water.
4. The propagation of the complex microbial inoculum of claim 1 in seawater is characterized in that: the method for preparing the composite propagation inoculant according to claim 1, which is characterized by comprising the following steps (taking propagation 100L as an example):
(1) moving a 100L plastic white barrel to an outdoor direct sunlight place (the temperature in summer is more than 25 ℃, indoor expanding culture can be carried out in winter, the expanding culture temperature needs to be kept between 10 ℃ and 30 ℃ by heating), and adding about 50L seawater into the barrel; (2) adding weighed culture medium, 2kg brown sugar and 5L of the compound microbial inoculum product in claim 1 into a 100L white barrel, uniformly stirring by using a clean roller, then injecting seawater into the plastic white barrel until the plastic white barrel is full, sealing and fermenting for 3 days until the pH of fermentation liquor is less than or equal to 5.0, and properly prolonging the culture time by one to two days if the pH does not reach the expected effect in rainy days or at the temperature of less than 20 ℃. Wherein in the step (2), every 1000 mL of the liquid culture medium comprises 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast extract and diammonium hydrogen citrate [ (NH)4)2HC6H5O7]2.0 g, glucose (C)6H12O6·H2O) 20.0 g, Tween 801.0 mL, sodium acetate (CH)3COONa·3H2O) 5.0 g and 1000 mL of distilled water.
5. The use method of the complex microbial inoculum of claim 1 is characterized in that: the microbial inoculum can be mixed for feeding or directly sprinkled into culture water.
The specific use method comprises the following steps: (1) the compound microbial inoculum of claim 1 is added into feed (the compound microbial inoculum can be diluted by clean water and then uniformly mixed with the feed) according to 1-3% of the weight of the feed, the compound microbial inoculum is added for 2 times every day, the compound microbial inoculum and the feed are premixed once in the morning and evening, the microbial inoculum and the feed are required to be premixed in advance for 1-2 hours, the microbial inoculum is completely immersed into the feed to be fed, and the uniformly mixed feed is directly fed when the feed is fed; (2) the compound microbial inoculum of claim 1 is directly sprinkled into the culture pond according to 5-10L/mu, and the sprinkling amount can be properly increased when the water quality is poor, such as severe blue algae.
Drawings
FIG. 1 shows the influence of the composite microbial inoculum on the pH value of a water body of a traditional soil pond.
FIG. 2 shows the influence of the complex microbial inoculum on nitrite in the water body of the traditional soil pond.
FIG. 3 shows the influence of the composite microbial inoculum on the pH value of the water body of the cement pond.
FIG. 4 shows the effect of the composite microbial inoculum on nitrite in water body of a cement pond.
Detailed Description
Example 1: method for producing aquaculture composite microbial inoculum
1. The method of the aquaculture complex microbial inoculum comprises the following steps: (1) respectively inoculating the four bacteria as described in claim 2 on a solid culture medium for activation; (2) selecting single colonies of the four activated strains, respectively inoculating the single colonies on respective liquid culture media, and culturing for 48 hours at the temperature of 37 ℃; (3) inoculating the cultured strain in (2) into a fermentation tank according to the proportion in claim 2, fermenting at 37 deg.C for 24 hr to obtain a strain suspension, wherein the viable count in the strain suspension is 6 × 109CFU/mL, pH3.8, and the preparation of the complex microbial inoculum is completed.
2. The production method of the compound propagation microbial inoculum product (taking propagation 100L as an example) comprises the following steps: (1) moving a 100L plastic white barrel to an outdoor direct sunlight place, and adding about 50L of clean water (which can be well water, tap water or seawater, wherein the tap water needs to be aired for a period of time to prevent the residual chlorine in the tap water from damaging the growth of bacteria); (2) adding weighed culture medium, 2kg of brown sugar and 5L of the compound microbial inoculum product into a 100L white barrel, uniformly stirring by using a clean roller, then injecting water into the plastic white barrel until the plastic white barrel is full, and sealing and fermenting for 3 days until the pH value of fermentation liquor is 4.04, thus completing fermentation.
Example 2: influence of aquaculture composite microbial inoculum on water quality of artificially constructed cement culture pond
1. The basic situation before the application of the pond microbial inoculum of an aquaculture base is as follows:
the depth of water in the selected No.5 pond and No. 7 pond is 1.5-2m, the water surface size is about 2 mu/pond, the bottom of the pond is a cement surface, the cultured variety is weever, and the fry feeding amount is 3000 tails. When the test is started by 8 months and 1 day, the average size of 2 pond fishes is 350 g-400 g, the feed feeding amount is 20 kg/day/pond, the feed protein content is about 45%, the measured original nitrite values are 1.16mg/L and 1.06mg/L respectively, the pH values are 8.79 and 8.56 respectively, and the blue algae outbreak is serious. Selecting a pond with No. 10 as a blank, not putting the fry in the pond, not spraying the bactericide, not putting the feed, and having an original pH value of 8.80 at 8 months and 1 day and nitrite less than 0.04 mg/L. And normally feeding the feed in the process of sprinkling the compound fungicide.
2. The application scheme of the microbial inoculum is as follows:
the fungicide expanded in the embodiment 1 is sprayed by 10L/mu. The sprinkling period is that the water is sprinkled according to the weather and the water quality. Monitoring the change of the pH value and nitrite of the aquaculture water, wherein the observation time is 8 months, 1 day to 18 days, and the total splashing frequency is 4 times. The nitrite begins to decline when the No.5 pond and the No. 7 pond are splashed on the next day, the nitrite respectively declines from 1.16mg/L and 1.06mg/L to 0.159mg/L and 0.24 mg/L when the nitrite is splashed on the 18 th day, the blue algae begins to float like flocculent from the 3 rd day when the bactericide is splashed on the blue algae, and the blue algae completely disappears when the blue algae disappears on the 8 th day. As No. 10 pond without any treatment, the pond water shows a trend of continuously increasing pH, and at the 18 th day, the pH of the pond water is 9.63, and large-area blue algae float.
3. Application effects
The result shows that the composite microbial inoculum has obvious effect of inhibiting blue algae in the water body of the artificially constructed cement culture pond
It has strong resistance to external environment change, and has obvious effect on regulating pH and nitrite of water (see figure 1 and figure 2). In addition, in the process of sprinkling the composite microbial inoculum, the pond fishes grow normally without adverse reaction, the quantity of the mixed bacteria in the water body is also obviously inhibited, and a safe, healthy, nontoxic and harmless culture environment is created for pond culture.
Example 3: influence of aquaculture composite microbial inoculum on water quality of traditional soil pond culture pond
1. The basic situation before the application of the pond microbial inoculum of an aquaculture base is as follows:
three ponds, namely a north No. 8 pond, a north No. 10 pond and a south No. 2 pond are respectively selected as test ponds, and the pond conditions are approximately as follows: the area of the pond No. 8 north is 10 mu, the water depth is 2m, the average size of the fish is about 200-; the No. 10 north pond has the water area of 12 mu and the water depth of 2m, the fish size is about 300g, and the number is 16000; south 2 good ponds, the water area is 6 mu, the water depth is 2 meters, the number is 13000 tails, and the size is about 300 g. The fish species are all white fish, the feed is added in an amount of 2% per day of the total weight of the fish, and the protein content in the feed is about 40%. The original nitrite values measured in the No. 8 pond, the No. 10 pond and the No. 2 pond are respectively 0.243 mg/L, 0.59 mg/L and 2.42 mg/L, the pH values are respectively 8.87, 8.28 and 7.84, and the blue algae is seriously exploded.
2. Application scheme of microbial inoculum
The fungicide expanded in the embodiment 1 is sprayed by 5L/mu. The sprinkling period is that the water is sprinkled according to the weather and the water quality. Monitoring the change of the pH and nitrite of the aquaculture water body, and testing for 2017.7.21-2017.8.18.
3. Application effects
The result shows that the composite microbial inoculum has obvious effect of inhibiting blue algae in the traditional soil pond culture water body and can be used for environment protection
The environmental change resistance is strong, and the effect of adjusting pH and nitrite is obvious (see figure 3 and figure 4). In addition, the compound microbial inoculum is used in a splashing way, the pond fishes grow normally without adverse reaction, and a safe, healthy, nontoxic and harmless culture environment is created for pond culture.
Claims (7)
1. The compound microbial inoculum for aquaculture is characterized by comprising lactobacillus plantarum (Lactobacillus)Lactobacillus plantarum) Leuconostoc mesenteroides (A), (B), (C)Leuconostocmesenteroides) Pediococcus acidilactici (Pediococcusacidilactici) Pichia pastoris (A), (B), (C)Pichiapastoris) Composition is carried out; the preservation number of the lactobacillus plantarum is CGMCC No. 15024; the preservation number of the Leuconostoc mesenteroides is CGMCC number 15023; the preservation number of the pediococcus acidilactici is CGMCC No. 5959; the preservation number of the pichia pastoris is CGMCC No. 5960.
2. The complex microbial inoculant according to claim 1, which is prepared from the following components in parts by weight:
3-4 parts of plant lactobacillus agent;
3-4 parts of leuconostoc mesenteroides agent;
3-4 parts of lactic acid pellet bacterium agent;
2-3 parts of a pichia pastoris agent;
wherein the viable count of each strain is 6 × 108-3×1010CFU/mL, and the pH value of the complex microbial inoculum is less than or equal to 4.0.
3. The preparation method of the aquaculture complex microbial inoculum according to claim 2, which is realized by the following steps: (1) respectively inoculating four bacteria in the aquaculture composite bacterial agent on a solid culture medium for activation; (2) selecting single colonies of the four activated strains, respectively inoculating the single colonies on respective liquid culture media, and culturing for 48 hours at the temperature of 37 ℃; (3) inoculating the cultured strain in (2) into a fermentation tank, fermenting at 37 deg.C for 24 hr to obtain a suspension with pH of 4.0 or less, and viable count of each strain in the suspension is 6 × 108-3×1010And (5) CFU/mL, namely preparing the compound bacterial agent, and storing the compound bacterial agent in a cool and dry place.
4. The method for expanding the culture of the complex microbial inoculum for aquaculture in fresh water according to claim 1, which is characterized by comprising the following steps:
(1) when the temperature in summer is higher than 25 ℃, moving a 100L plastic white barrel to an outdoor direct sunlight place; or heating a 100L plastic white barrel in winter to keep the propagation temperature between 10 ℃ and 30 ℃ for indoor propagation; adding 50L of clean water into the barrel; (2) adding weighed culture medium, 2 kilograms of brown sugar and 5 liters of the compound microbial inoculum for aquaculture according to claim 1 into a 100L white barrel, uniformly stirring by using a clean roller, then injecting water into the plastic white barrel till the plastic white barrel is full, sealing and fermenting for 3 days until the pH of fermentation liquor is less than or equal to 5.0, and prolonging the culture time by one to two days if the temperature is lower than 20 ℃ or the pH does not reach the expected effect in rainy days.
5. The method for expanding the culture of the complex microbial inoculum for aquaculture in seawater according to claim 1, which is realized by the following steps:
(1) when the temperature in summer is higher than 25 ℃, moving a 100L plastic white barrel to an outdoor direct sunlight place; or heating a 100L plastic white barrel in winter to keep the propagation temperature between 10 ℃ and 30 ℃ for indoor propagation; adding 50L of seawater into the barrel; (2) adding weighed culture medium, 2kg brown sugar and 5L of the compound microbial inoculum for aquaculture according to claim 1 into a 100L white barrel, uniformly stirring by using a clean roller, then injecting seawater into the plastic white barrel until the plastic white barrel is full, sealing and fermenting for 3 days until the pH of fermentation liquor is less than or equal to 5.0, and prolonging the culture expanding time for one to two days if the temperature is lower than 20 ℃ or the pH does not reach the expected effect in rainy days.
6. A complex microbial inoculum for aquaculture prepared by the propagation method of claim 4 or 5.
7. The use of the complex microbial inoculum for aquaculture in the preparation of aquaculture feed according to claim 1, wherein the complex microbial inoculum is added into the feed according to 1-3% of the weight of the feed for mixing and feeding.
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| CN108850729A (en) * | 2018-08-30 | 2018-11-23 | 安徽金牧饲料有限公司 | One kind containing compound probiotic and Chinese herbal medicine feed for river crab |
| CN109456914A (en) * | 2018-11-19 | 2019-03-12 | 江苏渔多多生物科技有限公司 | One kind low temperature resistant lactic acid bacteria used for aquiculture and its application |
| CN109897803B (en) * | 2019-03-22 | 2021-02-02 | 北京好实沃生物技术有限公司 | Aquatic probiotic and preparation method and application thereof |
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Address after: 100089 No. 27 West Third Ring Road North, Beijing, Haidian District Patentee after: Institute of resources and environment, Beijing Academy of science and technology Address before: 100089 No. 27 West Third Ring Road North, Beijing, Haidian District Patentee before: ENVIRONMENTAL PROTECTION Research Institute OF LIGHT INDUSTRY |