CN102559545A - Composite micro-ecological preparation for aquatic product and premix thereof - Google Patents

Composite micro-ecological preparation for aquatic product and premix thereof Download PDF

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CN102559545A
CN102559545A CN201110419406XA CN201110419406A CN102559545A CN 102559545 A CN102559545 A CN 102559545A CN 201110419406X A CN201110419406X A CN 201110419406XA CN 201110419406 A CN201110419406 A CN 201110419406A CN 102559545 A CN102559545 A CN 102559545A
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cgmcc
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ecological preparation
compound micro
subtilis
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CN102559545B (en
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杜建涛
付维来
吴雅琨
李晓清
王安如
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Jiangsu Da Bei Nong Aquatic Technology Co., Ltd.
Beijing Dabeinong Technology Group Co Ltd
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BEIJING DBN AGRICULTURAL FEED Co Ltd
HARBIN DABEI FARMING AND ANIMAL HUSBANDRY TECHNOLOGY Co Ltd
Beijing Dabeinong Technology Group Co Ltd
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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

A kind of aquatic products is with compound micro-ecological preparation and Preblend thereof
Technical field
The invention belongs to the Applied Biotechnology field, relate to a kind of aquatic products with compound micro-ecological preparation and Preblend thereof.
Background technology
Along with aquaculture intensification degree improves constantly, the physicochemical environment and the ecotope of aquaculture water worsen day by day; Simultaneously, the discharging of excess such as human lives's sewage, various trade effluent, agricultural effluent has all aggravated the decline of aquaculture water quality; These problems cause the breed disease to be on the rise; Meanwhile; People's living standard improves, the raising of food safety consciousness, and the accession to WTO back is to animal source requirement of products standard more and more; When producing and use the fishing medicine; Need essentially to consider whether this fishing medicine meets food safety requirements, otherwise can not produce or use that this is to produce and the maximum difference of using with the fishing medicine in past.People only paid attention to the effect of fishing medicine from the past; And do not take into account environment damage, the residual influence to human health of medicine; Till now; Countries and regions such as the U.S. and European Union have been put into effect relevant law in succession and have been limited some fishing medicines such as the application in aquaculture such as Victoria Green WPB and microbiotic, thus aquaculture the fishery products of safety, fine nuisanceless green must be provided is trends of the times.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 an amount of additive part and substitute antibiotic use.Compound micro-ecological preparation is owing to have the intestinal environment of improvement, increase feed, suppress pernicious bacteria, improve many-sided effect such as water quality simultaneously, increasingly is applied in the culture fishery by people.
Existed on the market multiple can at water quality improvement and preventing disease be a certain or obtain the compound micro-ecological preparation of positive effect aspect some; But still there are problems such as bacterial classification unstable properties, function singleness and environmental adaptability difference in a lot of preparations; Make it can not adapt to water body environment complicated and changeable; Effect is unstable after imposing on water body, thereby has limited probiotics applying in aquaculture.
Summary of the invention:
The purpose of this invention is to provide a kind of aquatic products and use compound micro-ecological preparation; Said preparation can be used as aquatic products and uses fodder additives; Also can be used for water body purification of aquaculture and conditioning, the said preparation environmental compatibility is strong, the pollutents such as ammonia nitrogen, nitric nitrogen, nitrite nitrogen and COD in function-stable and the water body of can effectively degrading.
The objective of the invention is to realize through following technical scheme:
The present invention provides a kind of aquatic products to use compound micro-ecological preparation, and the staple of said preparation is subtilis CGMCC No. 4628, Bacillus licheniformis CGMCC No.5094 and enterococcus faecalis CGMCC No.5092.
Wherein, 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 in the preparation of the present invention.
Further, the proportioning of subtilis CGMCC No.4628, Bacillus licheniformis CGMCC No.5094 and enterococcus faecalis CGMCC No.5092 is 1: 1: 2 in the preparation of the present invention.
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 of fermented liquid and enterococcus faecalis CGMCC No.5092 of fermented liquid, the Bacillus licheniformis CGMCC No.5094 of subtilis CGMCC No.4628; Described granule is that the bacterium mud of subtilis CGMCC No.4628, the bacterium mud of Bacillus licheniformis CGMCC No.5094 and the bacterium mud of enterococcus faecalis CGMCC No.5092 mix with carrier respectively, processes granule.
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, the wheat bran powder, and the viable count in the granule is 3-4 * 10 10Cfu/g.
The present invention also provides a kind of Preblend that comprises this aquatic products with compound micro-ecological preparation, it is characterized in that the weight percent that compound micro-ecological preparation adds in the Preblend is 1-10 ‰.
Bacillus licheniformis of the present invention (Bacillus licheniformis) separates from the Roll road for the applicant, obtains through directed primary dcreening operation and multiple sieve.Its vegetative cell is shaft-like, and its gemma is oval or grows tubular, is middle life or the life of inferior end, and sporangiocyst expands slightly.Isolated or be short chain, rod end is semicircle.In 1 2~16h, can form bacterium colony, bacterium colony is circular, or irregular, and the edge is hair-like, and bacteria colony white is opaque, corrugationless.Gram-positive.The applicant has carried out experiments such as acid resistance, bile tolerance, high thermal resistance, fermentation character and bacteriostatic activity to this bacillus licheniformis, this bacillus licheniformis has characteristics such as strong to contrary environmental resistance, that adhesivity strong, the growth fast, living weight is big, bacteriostatic activity is good.Bacillus licheniformis of the present invention (Bacillus licheniformis) applicant has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 26th, 2011; Be called for short CGMCC; The address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC No.5094.Enterococcus faecalis of the present invention (Enterococcus faecalis) obtains for the applicant separates from Radix Polygalae Crotalarioidis, and its biological property is following: the bacterium colony rounding, and smooth surface, opaque, oyster white, the edge is smooth, 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 85 ℃ pelleting temperature; 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, the corn straw.Enterococcus faecalis of the present invention (Enterococcus faecalis) applicant has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 26th, 2011, be called for short CGMCC, and the address is the same, and deposit number is CGMCC No.5092.The used subtilis (Bacillus subtilis) of the present invention obtains for the applicant separates from the traditional zymotic fermented soya bean, and its biological property is following: the bacterium colony surface irregularity, and opaque, dirty white, bacterium colony is circular, edge indentation, gram-positive microorganism; The gemma form is oval to column, is positioned at thalline central authorities or inclined to one side slightly, and gemma forms the back thalline and do not expand.Subtilis of the present invention significantly is different from existing subtilis, can tolerate pH2.0,1% pepsic simulated gastric fluid; Can tolerate 0.3% artificial cholate; Can tolerate 80 ℃ pelleting temperature, can suppress pathogenic colon bacillus K88, K99 and streptococcus aureus; Ability with stronger cellulase-producing can degraded cellulose.Subtilis of the present invention (Bacillus subtilis) has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on March 2nd, 2011, be called for short CGMCC, and the address is the same, and deposit number is CGMCC No.4628.
Beneficial effect:
1, preparation of the present invention contains three kinds of high-quality probiotic bacteriums, its bacterial classification in strict accordance with the microecology rule choose seeds, seed selection, guarantee the good of bacterial classification; Viable bacteria content is high, and the wherein liquid probiotics quality guaranteed period reaches 6 months, and the solid-state probiotics quality guaranteed period can reach 24 months; All have good little ecological adjusting, optimize water quality, the growth that promotes 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 body weight gains of aquatic animal, increase the yield per unit area and total amount, thereby the income of increase aquaculture is increased the benefit.
3, viable bacteria content height of the present invention, long quality-guarantee period, reach is big, production cost is low, be to improve water quality specially, increase the multiple microbial inoculum of ideal that aquatic animal immunizing power and control aquatic product disease take place.
4, the independent use of the little ecology of liquid aquatic products of the present invention can be spread rapidly in water, and good prolificacy is arranged, and better improves water quality and suppresses pathogenic microorganism in the water.
5, the independent use of the little ecological product of particle of the present invention can sink under water rapidly, improves water-bed environment and the organic substance at the bottom of the degradation water, optimizes culture environment of aquatic products.
Through the following example the present invention will be described more specifically, and it should be understood that said embodiment only is for the present invention is described, rather than limit scope of the present invention in any form.
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: the enterococcus faecalis bacterial classification inoculation on the MRS plate culture medium, in 37 ℃ of cultivation 18h, is made the enterococcus faecalis rejuvenation, and forms single bacterium colony, and picking list bacterium colony is cultivated 24h for 37 ℃ on inoculation medium;
2) preparation of first order seed: the liquid substratum 2L of 300m1MRS is equipped with in the enterococcus faecalis bacterial classification switching of step 1) cultivation shakes in the bottle, 37 ℃ leave standstill cultivation 24h, to the logarithm later stage, get first order seed;
3) preparation of secondary seed: with step 2) first order seed of preparation is transferred in the 100L seeding tank that the 80LMRS seed culture medium is housed, 37 ℃ of temperature, and rotating speed 60rpm cultivates 12h, gets secondary seed solution.
4) enterococcus faecalis preparation of fermentation liquid: the secondary seed solution of step 3) preparation 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 get the bacterium number is 7.72 * 10 9The enterococcus faecalis fermented liquid of cfu/ml.
The fermention medium of using during the fermentation enterococcus faecalis is grouped into by following one-tenth: brown sugar 18g/L, and peptone 4g/L, Carnis Bovis seu Bubali cream 3g/L, yeast soaks powder, 0.8g/L, bitter salt 0.4g/L, manganous sulfate 0.005g/L, pH are 7.2.
2, the preparation of Bacillus licheniformis CGMCC No.5094 high density fermentation liquid
1) dull and stereotyped cultivation rejuvenation: the Bacillus licheniformis bacterial classification inoculation on the BPY plate culture medium, in 32 ℃ of cultivation 24h, is made the Bacillus licheniformis rejuvenation, and forms single bacterium colony, and picking list bacterium colony is cultivated 24h for 32 ℃ on inoculation medium;
2) preparation of first order seed: on the Bacillus licheniformis bacterial classification switching eggplant bottle BPY slant medium with the step 1) cultivation, cultivate 16h for 32 ℃, make to be in the logarithm later stage, get first order seed;
3) preparation of secondary seed: with step 2) first order seed of preparation is processed bacteria suspension with sterilized water, 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, get secondary seed solution.
4) preparation of the lichen bacillus ferments liquid: the secondary seed solution of step 3) preparation 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, get the 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%, sal epsom 0.04%; Ammonium citrate 0.8%, skimmer 0.06%, pH are 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 the BPY plate culture medium, in 30 ℃ of cultivation 24h, makes the subtilis rejuvenation, and form single bacterium colony, picking list bacterium colony is cultivated 24h for 30 ℃ on inoculation medium;
2) preparation of first order seed: on the Bacillus subtilis strain switching eggplant bottle BPY slant medium with the step 1) cultivation, cultivate 24h for 37 ℃, make to be in the logarithm later stage, get first order seed;
3) preparation of secondary seed: with step 2) first order seed of preparation is processed bacteria suspension with sterilized water, 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, get secondary seed solution at 1: 1.
4) preparation of fermentation of bacillus subtilis liquid: the 1m that the secondary seed solution of step 3) preparation is inoculated into the 600L fermention medium by 10% inoculum size 3In the fermentor tank, 30 ℃ of temperature, rotating speed 300rpm, tank pressure 0.05Mpa, ventilating ratio: 1: 1, cultivate 16h, get the 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%, sal epsom 0.04%; Manganous sulfate 0.08%, skimmer 0.06%, pH are 6.8.
4, the preparation of probiotics of the present invention
1,2,3 fermented liquids that obtain are evenly regulated about pH to 7.0 back by 2: 1: 1 mixed, carried out bag distribution packaging, every bag of 1000mL, viable count is 1 * 10 in the preparation 10Cfu/mL.
The preparation of embodiment 2 probiotics granules of the present invention
Get among the embodiment 11,2,3 fermented liquid by volume respectively and carry out centrifugally, its ratio is: enterococcus faecalis: Bacillus licheniformis: subtilis=2: 1: 1, centrifugal rotational speed are 15000rpm; The bacterium mud that obtains after centrifugal is mixed, and 25% the carrier that adds centrifugal preceding 3 kinds of fermented liquid TVs then mixes with bacterium mud, adopts the rotation extrusion comminution granulation to carry out granulation; And through vibrations fluidized-bed carry out drying; 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.
Embodiment 3 probioticses of the present invention are to the influence of flora in crucian digestive enzyme activity and the 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 (moity and trophic level are as shown in table 1), add mass percent and be the compound microecological granular preparation (II group) among 0.02% the embodiment 2, in basal diet, add mass percent and be 0.02% compound microecological liquid state (III group).Test Shui nationality casing is long-pending to be 0.85m * 0.45m * 0.55m, and every case is thrown 20 tail fishes.
The composition and the trophic level of table 1 crucian basal diet
Figure BDA00001204195000051
Figure BDA00001204195000061
Annotate: additive contains trace element and VITAMINs etc.
The preparation of test group feed: with above-mentioned each raw material pulverizing, thorough mixing behind mistake 60 mesh sieves, the compound micro-ecological preparation granule is fine ground with grinding alms bowl among the embodiment 2; Mix with monocalcium phosphate earlier; Doping again, rod soil, zeolite powder etc. add suitable quantity of water at last, use small meat mincer to process the wet granular material of diameter about as 2.5mm; And the making method of liquid compound micro-ecological preparation group is to replace water with the liquid compound microecological; Add, also process the wet granular material of diameter 2.5mm size then, three kinds of feeds are all at 40 ℃ of oven dry 12h (quality moisture is about 10% to the feed) down then.
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 is thrown the amount of raising according to the situation adjustment of feeding, and it is satiated with food and does not have the feed residue.Between feeding period, 24.4 ± 3 ℃ of water temperature average out to.Sooner or later each soil pick-up once adopts the flowing water system.Raise 75d post analysis digestive enzyme activity, intestinal microflora etc.Adopt the forint phenol reagent process to measure protease activity, starch enzyme reagent kit is measured amylase activity, and the result sees table 2.
Table 2 compound micro-ecological preparation is to the influence of crucian digestive ferment
Figure BDA00001204195000062
Can know by table 2 result; Compare with the I group, protein, diastatic activity in the enteron aisle chyme of II group have improved 24.3% and 58.56% respectively, significant difference (p<0.05); Proteolytic enzyme and amylase activity difference not significantly (p>0.05) in the liver, but the latter has improved 16.20%; Compare with the I group, the protein amylase activity in the enteron aisle chyme of III group also is greatly improved, and this shows, adds the secretion that compound micro-ecological preparation of the present invention can improve the digestive ferment of crucian in the fish meal, thereby improves the utilization ratio of feed.
Through the analysis to the flora in three groups of enteron aisle chymes, it is as shown in table 3 to obtain a result.Analytical procedure to flora is the method that the scientific research personnel knew.
Table 3 compound micro-ecological preparation is to the influence of crucian intestinal microflora
Figure BDA00001204195000063
Visible by table 3 result, to compare with the I group, the genus bacillus in the enteron aisle chyme of II and III group, the quantity of milk-acid bacteria have improved 9.6%, 6.9% and 23.6%, 19.6% respectively; Intestinal bacteria then reduce 9.45% and 9.67%, this shows, add compound micro-ecological preparation of the present invention in the feed; Can promote microbial population of animal intestinal tract to increase, produce the voltaile fatty acid that some can suppress harmful bacterium, thereby promote the growth of milk-acid bacteria; Thereby make the probiotics in the enteron aisle increase; Pathogenic bacteria reduces, and forms the dominant microflora of probiotics, keeps and adjust the intestinal microecology balance.
The test of embodiment 4 fishponds detects compound micro-ecological preparation result of use of the present invention
Test is divided into three ponds, handles a and b for two, a contrast c, 2 hectares in each pond.Fishpond initial stage water source is identical, and treating pond is identical with the fry amount with the shrimp seedling that throw in the contrast pond.In the whole culture-cycle; Add two the treating pond a and the b of compound micro-ecological preparation; Do not use any microbiotic and sterilant, average 15d throws in once, and injected volume is that liquid micro-ecological preparations 30L, particle probiotics are 3 kilograms; The average 15d in pond is with a sterilant in contrast, and service condition is as shown in table 4.
The fishpond result of use of table 4 compound micro-ecological preparation of the present invention
Figure BDA00001204195000071
Can know by table 4 result; The nitrogenous source pollutant load in contrast pond presents trend of rising, and two treating ponds all decrease adding bacterium after stain thing content, and wherein the treatment effect of treating pond b is more effective than handling a; Its imido nitrogen, nitrate salt, nitrate salt decline ratio are respectively 42.4%, 74.3% and 4.28%; Simultaneously show that also the little ecology of particle can sink to the bottom of water, below water body, begin to play a role that its action effect than the upper strata effect obviously.
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 weightening finish situation
Figure BDA00001204195000081
Can know by table 5 result; The improvement that not only helps water quality with compound micro-ecological preparation of the present invention improves the fishes and shrimps surviving rate; Also help individual weightening finish, thereby significantly improve the output and the ultimate production of unit surface, wherein obvious to the individual body weight gains of the crucian of the middle lower floor that lives in water.
Experimental result proves; Probiotics product of the present invention not only safely, regulate water quality, improve the growing environment of aquatic animal, and can significantly promote the individual body weight gains of aquatic animal, increase the yield per unit area and total amount; Thereby increase the income of aquaculture, increase the benefit.

Claims (7)

1. an aquatic products is used compound micro-ecological preparation, and the staple 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.
2. weigh 1 described aquatic products and use compound micro-ecological preparation, it is characterized in that the proportioning of subtilis CGMCC No.4628 in the preparation, Bacillus licheniformis CGMCC No.5094 and enterococcus faecalis CGMCC No.5092 is 1-5: 1-5: 2-7.
3. weigh 2 described aquatic products and use compound micro-ecological preparation, it is characterized in that the proportioning of subtilis CGMCC No.4628 in the preparation, Bacillus licheniformis CGMCC No.5094 and enterococcus faecalis CGMCC No.5092 is 1: 1: 2.
4. weigh 1 described aquatic products and use compound micro-ecological preparation, it is characterized in that described preparation is liquid formulation or granule; Described liquid formulation is the mixed solution of fermented liquid of fermented liquid and enterococcus faecalis CGMCC No.5092 of fermented liquid, the Bacillus licheniformis CGMCC No.5094 of subtilis CGMCC No.4628; Described granule is that the bacterium mud of subtilis CGMCC No.4628, the bacterium mud of Bacillus licheniformis CGMCC No.5094 and the bacterium mud of enterococcus faecalis CGMCC No.5092 mix with carrier respectively, processes granule.
5. weigh 4 described aquatic products and use compound micro-ecological preparation, it is characterized in that described liquid formulation pH is 6.4-7.2, viable count is 1-2 * 10 10Cfu/ml.
6. weigh 4 described aquatic products and use compound micro-ecological preparation, it is characterized in that described carrier is one or both and the mixing of stone flour in W-Gum, maltodextrin, corn cob meal, the wheat bran powder, the viable count in the granule is 3-4 * 10 10Cfu/g.
7. a Preblend that comprises power 1 said aquatic products with compound micro-ecological preparation is characterized in that the weight percent that compound micro-ecological preparation adds in the Preblend is 1-10 ‰.
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