CN102559545B - Composite micro-ecological preparation for aquatic product and premix thereof - Google Patents
Composite micro-ecological preparation for aquatic product and premix thereof Download PDFInfo
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- CN102559545B CN102559545B CN201110419406.XA CN201110419406A CN102559545B CN 102559545 B CN102559545 B CN 102559545B CN 201110419406 A CN201110419406 A CN 201110419406A CN 102559545 B CN102559545 B CN 102559545B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of application biology and relates to a composite micro-ecological preparation for an aquatic product and a premix thereof. The efficient and multifunctional composite micro-ecological preparation is formed by combining multiple beneficial strains having different functions in a certain proportion; the preparation can obviously promote individual weight increment of an aquatic animal and improve yield per unit area and total yield, thereby increasing the income of aquaculture and improving benefits; and the preparation provided by the invention has the effects in multiple aspects of improving water quality, increasing algae in water, inhibiting harmful flora, improving the environment in the intestinal tract of an aquaculture animal, increasing feed and the like.
Description
Technical field
The invention belongs to Applied Biotechnology field, relate to a kind of aquatic products compound micro-ecological preparation and Preblend thereof.
Background technology
Along with aquaculture intensive degree improves constantly, physicochemical environment and the ecotope of aquaculture water worsen increasingly; Meanwhile, the excess discharges such as human lives's sewage, various trade effluent, agricultural effluent, have all aggravated the decline of aquaculture water quality; These problems cause the farming disease harms to be on the rise; Meanwhile, people's living standard improves, the raising of Consciousness of food security, and Post-WTO is to more and more standard of the requirement of Animal by-product, in the time producing and use fishing medicine, essential ground need to consider whether this fishing medicine meets food safety requirements, otherwise can not produce or use, and this is to produce and the maximum difference using with the fishing medicine in past.People only paid attention to the effect of fishing medicine from the past, and do not take the residual impact on human health of destruction, medicine on environment into account, till now, the countries and regions such as the U.S. and European Union have in succession been put into effect relevant law and have been limited some fishing medicines as the application in aquaculture such as Victoria Green WPB and microbiotic, and therefore the fishery products of the nuisanceless green of safety, high-quality must be provided is trends of the times in aquaculture.A lot of results of study show that probiotics not only can reduce the organic contamination of water body, and purifying water body, can also suppress killing microorganism, thereby and can be used as the use of appropriate additive part substitute antibiotics.Compound micro-ecological preparation, owing to having the intestinal environment of improvement, increase feed, suppress pernicious bacteria, improving many-sided effects such as water quality simultaneously, is more and more applied in culture fishery by people.
On market, existed multiple can at water quality improvement and preventing disease be a certain or aspect some, obtain the compound micro-ecological preparation of positive effect, but still there is the problems such as bacterial classification unstable properties, function singleness and environmental adaptability be poor in a lot of preparations, make it can not adapt to the environment that water body is complicated and changeable, after imposing on water body, effect is unstable, thereby has limited probiotics applying in aquaculture.
Summary of the invention:
The object of this invention is to provide a kind of aquatic products compound micro-ecological preparation, said preparation can be used as aquatic products fodder additives, also can be used for water body purification of aquaculture and conditioning, the pollutents such as said preparation environmental compatibility is strong, function-stable and ammonia nitrogen, nitric nitrogen, nitrite nitrogen and the COD that can effectively degrade in water body.
The object of the invention is to be achieved through the following technical solutions:
The invention provides a kind of aquatic products compound micro-ecological preparation, the main component of said preparation is subtilis CGMCC No. 4628, Bacillus licheniformis CGMCC No.5094 and enterococcus faecalis CGMCC No.5092.
Wherein, in preparation of the present invention, the proportioning of subtilis CGMCC No.4628, Bacillus licheniformis CGMCC No.5094 and enterococcus faecalis CGMCC No.5092 is 1-5: 1-5: 2-7.
Further, in preparation of the present invention, the proportioning of subtilis CGMCC No.4628, Bacillus licheniformis CGMCC No.5094 and enterococcus faecalis CGMCC No.5092 is 1: 1: 2.
Compound micro-ecological preparation of the present invention can be prepared into liquid formulation or granule; Described liquid formulation is the mixed solution of fermented liquid, the fermented liquid of Bacillus licheniformis CGMCC No.5094 and the fermented liquid of enterococcus faecalis CGMCC No.5092 of subtilis CGMCC No.4628; Described granule is that bacterium mud, the bacterium mud of Bacillus licheniformis CGMCC No.5094 and the bacterium mud of enterococcus faecalis CGMCC No.5092 of subtilis CGMCC No.4628 mixes with carrier respectively, granulation agent.
Wherein, described liquid formulation pH is 6.4-7.2, and viable count is 1-2 × 10
10cfu/ml.
Described carrier is one or both and the mixing of stone flour in W-Gum, maltodextrin, corn cob meal, wheat bran powder, and the viable count in granule is 3-4 × 10
10cfu/g.
The present invention also provides a kind of Preblend that comprises this aquatic products compound micro-ecological preparation, it is characterized in that the weight percent that in Preblend, compound micro-ecological preparation adds is 1-10 ‰.
Bacillus licheniformis of the present invention (Bacillus licheniformis) separates from Roll road for applicant, obtains through directed primary dcreening operation and multiple sieve.Its vegetative cell is shaft-like, and its gemma is oval or long tubular, is middle life or the life of inferior end, and sporangiocyst slightly expands.Isolated or be short chain, rod end semicircle.In 1 2～16h, can form bacterium colony, bacterium colony is circular, or irregular, and edge is hair-like, and bacteria colony white is opaque, corrugationless.Gram-positive.Applicant has carried out the experiments such as acid resistance, bile tolerance, high thermal resistance, fermentation character and bacteriostatic activity to this bacillus licheniformis, this bacillus licheniformis has the features such as strong to contrary environmental resistance, adhesivity strong, growth fast, biomass is large, bacteriostatic activity is good.Bacillus licheniformis of the present invention (Bacillus licheniformis) applicant has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 26th, 2011, be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.5094.Enterococcus faecalis of the present invention (Enterococcus faecalis) obtains for applicant separates from Radix Polygalae Crotalarioidis, and its biological property is as follows: bacterium colony rounding, and smooth surface, opaque, oyster white, the smooth of the edge, gram-positive microorganism; Single thalline is spherical or ellipticity.Enterococcus faecalis of the present invention is through simulated gastric fluid, bile fluid and stable on heating screening; Again through enzymatic productivity screening and bacteriostasis screening, can tolerate pH2.0,1% pepsic simulated gastric fluid, can tolerate 0.3% artificial bile fluid, can tolerate the pelleting temperature of 85 ℃, also can suppress pathogenic colon bacillus K88, K99 and streptococcus aureus, have stronger product acid and the ability that suppresses mould in dregs of beans, cotton dregs, maize straw.Enterococcus faecalis of the present invention (Enterococcus faecalis) applicant has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 26th, 2011, be called for short CGMCC, address is the same, and deposit number is CGMCC No.5092.The present invention subtilis (Bacillus subtilis) used obtains for applicant separates from traditional zymotic fermented soya bean, and its biological property is as follows: bacterium colony surface irregularity is opaque, dirty white, bacterium colony circle, edge indentation, gram-positive microorganism; Gemma form is oval to column, is positioned at thalline central authorities or slightly inclined to one side, and after sporulation, thalline does not expand.Subtilis of the present invention is significantly different from existing subtilis, can tolerate pH2.0,1% pepsic simulated gastric fluid, can tolerate 0.3% artificial cholate, can tolerate the pelleting temperature of 80 ℃, can suppress pathogenic colon bacillus K88, K99 and streptococcus aureus, there is the ability of stronger cellulase-producing, can degraded cellulose.Subtilis of the present invention (Bacillus subtilis) has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 2nd, 2011, be called for short CGMCC, and address is the same, and deposit number is CGMCC No.4628.
Beneficial effect:
1, preparation of the present invention is containing three kinds of high-quality probiotic bacteriums, its bacterial classification in strict accordance with microecology rule choose seeds, seed selection, guarantee the good of bacterial classification, viable bacteria content is high, and wherein the liquid probiotics quality guaranteed period reaches 6 months, and the solid-state probiotics quality guaranteed period can reach 24 months, all there is the growth of good micro-Ecological regulation, optimizing water quality, promotion aquatic animal, the effect of strengthening immunity, and steady quality, safe and reliable, environmental protection, pollution-free.
2, preparation of the present invention can significantly promote the individual weight gain of aquatic animal, increase the yield per unit area and total amount, thereby the income of increase aquaculture is increased the benefit.
3, high, the long quality-guarantee period of viable bacteria content of the present invention, sphere of action is large, production cost is low, be to improve specially water quality, increase the multiple microbial inoculum of ideal that aquatic animal immunizing power and control aquatic product disease occur.
4, the independent use of the micro-ecology of liquid aquatic products of the present invention can be spread rapidly in water, and has good fecundity, better improves water quality and suppresses pathogenic microorganism in water.
5, the independent use of the micro-ecological product of particle of the present invention can sink under water rapidly, improves the organic substance at the bottom of water-bed environment and degradation water, optimizes culture environment of aquatic products.
, it should be understood that described embodiment is only for the present invention is described, rather than limit the scope of the invention in any form more specific description the present invention by the following example.
Embodiment:
The high density fermentation liquid of 1 three kinds of bacterium of embodiment and the preparation of liquid compound micro-ecological preparation
1, the preparation of enterococcus faecalis CGMCC No.5092 high density fermentation liquid
1) dull and stereotyped cultivation rejuvenation: enterococcus faecalis bacterial classification is inoculated on MRS plate culture medium, in 37 ℃ of cultivation 18h, makes enterococcus faecalis rejuvenation, and form single bacterium colony, picking list bacterium colony, on inoculation medium, is cultivated 24h for 37 ℃;
2) preparation of first order seed: by step 1) cultivate enterococcus faecalis strain transfer be equipped with in 300m1MRS liquid culture medium 2L shaking flask, 37 ℃ leave standstill cultivate 24h, to the logarithm later stage, obtain first order seed;
3) preparation of secondary seed: by step 2) first order seed prepared is transferred in the 100L seeding tank that 80LMRS seed culture medium is housed, 37 ℃ of temperature, rotating speed 60rpm, cultivates 12h, obtains secondary seed solution.
4) preparation of Enterococcus faecalis fermentation liquid: by step 3) secondary seed solution prepared is inoculated into 0.5-0.8m according to 1% inoculum size
3in the fermentor tank of fermention medium, 37 ℃ of temperature, rotating speed 60rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 0.6, cultivate 12h, can obtain bacterium number is 7.72 × 10
9the Enterococcus faecalis fermentation liquid of cfu/ml.
The fermention medium of using when fermentation enterococcus faecalis consists of the following composition: brown sugar 18g/L, and peptone 4g/L, extractum carnis 3g/L, yeast soaks powder, 0.8g/L, bitter salt 0.4g/L, manganous sulfate 0.005g/L, pH is 7.2.
2, the preparation of Bacillus licheniformis CGMCC No.5094 high density fermentation liquid
1) dull and stereotyped cultivation rejuvenation: Bacillus licheniformis strain is inoculated on BPY plate culture medium, in 32 ℃ of cultivation 24h, makes Bacillus licheniformis rejuvenation, and form single bacterium colony, picking list bacterium colony, on inoculation medium, is cultivated 24h for 32 ℃;
2) preparation of first order seed: by step 1) on the Bacillus licheniformis strain switching eggplant bottle BPY slant medium cultivated, cultivate 16h, make, in the logarithm later stage, to obtain first order seed for 32 ℃;
3) preparation of secondary seed: by step 2) the first order seed sterilized water prepared makes bacteria suspension, is inoculated in the 100L seeding tank that 60L BPY seed culture medium is housed 32 ℃ of temperature, rotating speed 250rpm, tank pressure 0.05MPA, ventilating ratio: 1: 0.8, cultivate 14h, obtain secondary seed solution.
4) preparation of the lichen bacillus ferments liquid: by step 3) secondary seed solution prepared is inoculated into 6m according to 5% inoculum size
3in the fermentor tank of fermention medium, 32 ℃ of temperature, rotating speed 300rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 1, cultivate 16h, obtain gemma production rate more than 90%, viable count is 1.32 × 10
10the lichen bacillus ferments liquid of cfu/ml;
Described fermention medium is (mass percent): wheat bran 1.5%, Semen Maydis powder 1.0%, dregs of beans 1.2%, ammonium sulfate 0.6%, magnesium sulfate 0.04%; Ammonium citrate 0.8%, defoamer 0.06%, pH is 6.8.
3, the preparation of subtilis CGMCC No.4628 high density fermentation liquid
1) dull and stereotyped cultivation rejuvenation: Bacillus subtilis strain is inoculated on BPY plate culture medium, in 30 ℃ of cultivation 24h, makes subtilis rejuvenation, and form single bacterium colony, picking list bacterium colony, on inoculation medium, is cultivated 24h for 30 ℃;
2) preparation of first order seed: by step 1) on the Bacillus subtilis strain switching eggplant bottle BPY slant medium cultivated, cultivate 24h, make, in the logarithm later stage, to obtain first order seed for 37 ℃;
3) preparation of secondary seed: by step 2) the first order seed sterilized water prepared makes bacteria suspension, is inoculated in the 100L seeding tank that 60L BPY seed culture medium is housed 30 ℃ of temperature, rotating speed 300rpm, tank pressure 0.05MPA, ventilating ratio: cultivate 14h, obtain secondary seed solution at 1: 1.
4) preparation of fermentation of bacillus subtilis liquid: by step 3) secondary seed solution of preparing is inoculated into the 1m of 600L fermention medium by 10% inoculum size
3in fermentor tank, 30 ℃ of temperature, rotating speed 300rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 1, cultivate 16h, obtain gemma production rate more than 90%, viable count is 1.09 × 10
10the fermentation of bacillus subtilis liquid of cfu/ml;
Described fermention medium is (mass percent): sucrose 1.0%, and peptone 1.0%, yeast soak powder 0.5%, ammonium sulfate 0.2%, magnesium sulfate 0.04%; Manganous sulfate 0.08%, defoamer 0.06%, pH is 6.8.
4, the preparation of probiotics of the present invention
1,2,3 fermented liquids that obtain are mixed to rear adjusting pH to 7.0 left and right in the ratio of 2: 1: 1, carry out bag distribution packaging, every bag of 1000mL, in preparation, viable count is 1 × 10
10cfu/mL.
The preparation of embodiment 2 probiotics granule of the present invention
Getting respectively by volume in embodiment 11,2,3 fermented liquid carries out centrifugal, its ratio is: enterococcus faecalis: Bacillus licheniformis: subtilis=2: 1: 1, centrifugal rotational speed is 15000rpm, the bacterium mud obtaining after centrifugal is mixed, then add 25% carrier of centrifugal front 3 kinds of fermented liquid cumulative volumes to mix with bacterium mud, adopt rotary squeezing comminution granulation to carry out granulation, and be dried by vibrations fluidized-bed, 70 ℃ of air intakes, 35 ℃ of air-out, input speed is 120kg/h; Obtaining moisture content is 8.5% probiotics granule, and viable count is 3.5 × 10
10cfu/g.
The impact of embodiment 3 probiotics of the present invention on flora in crucian digestive enzyme activity and enteron aisle
180 tail fingerlings are divided into 3 groups at random, every group of 3 repetitions, the 3 kinds of feeds of throwing something and feeding respectively: basal diet (I group), in basal diet (moiety and trophic level are as shown in table 1), adding mass percent is the compound microecological granular preparation (II group) in 0.02% embodiment 2, adds mass percent and be 0.02% compound microecological liquid state (III group) in basal diet.Test aquarium volume is 0.85m × 0.45m × 0.55m, and every case is thrown 20 tail fishes.
Composition and the trophic level of table 1 crucian basal diet
Note: additive is containing trace element and VITAMIN etc.
The preparation of test group feed: by above-mentioned each raw material pulverizing, after crossing 60 mesh sieves, fully mix, in embodiment 2, compound micro-ecological preparation granule is fine ground with grinding alms bowl, first mix with monocalcium phosphate, doping again, rod soil, zeolite powder etc., finally add suitable quantity of water, making diameter with small meat mincer is the wet granular material about 2.5mm, and the making method of liquid compound micro-ecological preparation group is to replace water with liquid compound microecological, add, then also make the wet granular material of diameter 2.5mm size, then Three feed all at 40 ℃, dry 12h (to quality moisture in feed be 10% left and right).
Feeding method: crucian domestication was weighed after 15 days, by 2% one 4% of the fish body weight test daily ration of throwing something and feeding, throw something and feed every day 3 times (8: 30,13: 30,18: 30), the unnecessary material of sucking-off, throw according to the situation adjustment of feeding the amount of raising, make it be satiated with food and remain without feed as far as possible.Between feeding period, 24.4 ± 3 ℃ of water temperature average out to.Sooner or later each soil pick-up once, adopts flowing water system.Raise 75d post analysis digestive enzyme activity, intestinal microflora etc.Adopt forint phenol reagent process to measure protease activity, starch enzyme reagent kit is measured amylase activity, the results are shown in Table 2.
The impact of table 2 compound micro-ecological preparation on crucian digestive ferment
As seen from the results in Table 2, compared with I group, protein, diastatic activity in the enteron aisle chyme of II group have improved respectively 24.3% and 58.56%, significant difference (p < 0.05), not remarkable (p > 0.05) of proteolytic enzyme and amylase activity difference in liver, but the latter has improved 16.20%; Compared with I group, the protein amylase activity in the enteron aisle chyme of III group is also greatly improved, as can be seen here, the secretion of adding compound micro-ecological preparation of the present invention in fish meal and can improving the digestive ferment of crucian, thus the utilization ratio of feed improved.
By the analysis of the flora in three groups of enteron aisle chymes, obtain a result as shown in table 3.Be the known method of scientific research personnel to the analytical procedure of flora.
The impact of table 3 compound micro-ecological preparation on crucian intestinal microflora
From table 3 result, compared with I group, genus bacillus in the enteron aisle chyme of II and III group, the quantity of milk-acid bacteria has improved respectively 9.6%, 6.9% and 23.6%, 19.6%, intestinal bacteria reduce 9.45% and 9.67%, as can be seen here, in feed, add compound micro-ecological preparation of the present invention, can promote microbial population of animal intestinal tract to increase, produce the voltaile fatty acid that some can suppress harmful bacteria, thereby promote the growth of milk-acid bacteria, thereby the probiotics in enteron aisle is increased, pathogenic bacteria reduces, form the dominant microflora of probiotics, maintain and adjust intestinal microecology balance.
Embodiment 4 fishpond tests detect compound micro-ecological preparation result of use of the present invention
Test is divided into three ponds, processes a and b for two, a contrast c, 2 hectares, each pond.Fishpond initial stage water source is identical, and treating pond is identical with fry amount with the shrimp seedling that throw in contrast pond.Within the whole culture-cycle, add two treating pond a and the b of compound micro-ecological preparation, do not use any microbiotic and sterilant, average 15d throws in once, injected volume is that liquid micro-ecological preparations 30L, particle probiotics are 3 kilograms, contrast sterilant for the average 15d in pond, service condition is as shown in table 4.
The fishpond result of use of table 4 compound micro-ecological preparation of the present invention
As seen from the results in Table 4, the nitrogenous source pollutant load in contrast pond presents the trend of rising, and two treating ponds all decrease adding bacterium after stain thing content, wherein the treatment effect for the treatment of pond b is more effective than processing a, its imido nitrogen, nitrate, nitrate down ratio are respectively 42.4%, 74.3% and 4.28%, also show that the micro-ecology of particle can sink to the bottom of water, start to play a role below water body, its action effect than upper strata effect obviously simultaneously.
After off-test, the aquatic animal in each pond is sampled, draw mean body weight and rate of increase, its result is as shown in table 5.
Table 5 fishes and shrimps Gain weight
As seen from the results in Table 5, the improvement that is not only conducive to water quality with compound micro-ecological preparation of the present invention improves fishes and shrimps surviving rate, also help individual weightening finish, thereby significantly improve output and the ultimate production of unit surface, wherein obvious to living in the individual weight gain of crucian of middle lower floor of water.
The results show, probiotics product of the present invention not only safety, regulating water quality, improve the growing environment of aquatic animal, and can significantly promote the individual weight gain of aquatic animal, increase the yield per unit area and total amount, thereby increase the income of aquaculture, increase the benefit.
Claims (2)
1. an aquatic products compound micro-ecological preparation, the main component that it is characterized in that said preparation is subtilis (Bacillus subtilis) CGMCC No.4628, Bacillus licheniformis (Bacillus licheniformis) CGMCC No.5094 and enterococcus faecalis (Enterococcus faccalis) CGMCC No.5092, and in preparation, the proportioning of subtilis CGMCC No.4628, Bacillus licheniformis CGMCC No.5094 and enterococcus faecalis CGMCC No.5092 is 1: 1: 2.
2. aquatic products compound micro-ecological preparation as claimed in claim 1, is characterized in that described preparation is liquid formulation or granule; Described liquid formulation is the mixed solution of fermented liquid, the fermented liquid of Bacillus licheniformis CGMCC No.5094 and the fermented liquid of enterococcus faecalis CGMCC No.5092 of subtilis CGMCC No.4628; Described granule is that bacterium mud, the bacterium mud of Bacillus licheniformis CGMCC No.5094 and the bacterium mud of enterococcus faecalis CGMCC No.5092 of subtilis CGMCC No.4628 mixes with carrier respectively, granulation agent.
3. aquatic products compound micro-ecological preparation as claimed in claim 2, is characterized in that described liquid formulation pH is 6.4-7.2, and viable count is 1-2 × 10
10cfu/ml.
4. aquatic products compound micro-ecological preparation as claimed in claim 2, is characterized in that described carrier is one or both and the mixing of stone flour in W-Gum, maltodextrin, corn cob meal, wheat bran powder, and the viable count in granule is 3-4 × 10
10cfu/g.
5. comprise a Preblend for aquatic products compound micro-ecological preparation described in claim 1, it is characterized in that the weight percent that in Preblend, compound micro-ecological preparation adds is 0.1-1%.
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Effective date of registration: 20180903 Address after: 223300 Jiangsu province Huaian Huaiyin District Xu Yao town industrial concentration area Co-patentee after: Beijing Dabeinong Technology Group Co., Ltd. Patentee after: Jiangsu Da Bei Nong Aquatic Technology Co., Ltd. Address before: 100080 Beijing Haidian District Zhongguancun Street 27 Zhongguancun building 14 level big north agriculture group Co-patentee before: Beijing DBN Agricultural Feed Co., Ltd. Patentee before: Beijing Dabeinong Technology Group Co., Ltd. Co-patentee before: Harbin Dabei Farming and Animal Husbandry Technology Co., Ltd. |
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