CN101595943B - Production method of additive of bait for photosynthetic bacteria aquaculture - Google Patents
Production method of additive of bait for photosynthetic bacteria aquaculture Download PDFInfo
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- CN101595943B CN101595943B CN2009100395240A CN200910039524A CN101595943B CN 101595943 B CN101595943 B CN 101595943B CN 2009100395240 A CN2009100395240 A CN 2009100395240A CN 200910039524 A CN200910039524 A CN 200910039524A CN 101595943 B CN101595943 B CN 101595943B
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Abstract
The invention relates to a production method of an additive of a bait for photosynthetic bacteria aquaculture, adopting the following steps: 1. strain amplification culture: adjusting the pH value of a strain amplification medium from 6.0 to 8.5; placing the medium into a glass bottle and carrying out heat disinfection; inoculating a mixed culture of two photosynthetic bacteria including Rhodopeudomonas palustris and Rhodobacter sphaeroides based on 5-40% of the volume of a strain medium; anaerobically culturing the mixed culture for 3-4 days under the conditions that the temperature is 15-35DEG C and the illumination intensity is 1000-3000lux, thus obtaining strain culture fluid; 2. fermentation production: placing a fermentation medium into a fermentation cylinder and carrying out heatdisinfection; inoculating the strain culture fluid based on the inoculation amount, namely 5-40% of the volume of the fermentation production medium; the strain culture fluid is fermented for 6 days to obtain the additive of the bait for photosynthetic bacteria aquaculture under the conditions that the temperature is 28-30 DEG C, the stirring rate is 150-300r/m, the cylinder pressure is kept between 0.02MPa and 0.05MPa and the illumination intensity is 2000-3000lux. Compared with the prior art, the additive has the advantages of stable product quality and diversified functions.
Description
Technical field
The present invention relates to microorganism field, relate in particular to a kind of production method of additive of bait for photosynthetic bacteria aquaculture.
Background technology
Along with the high speed development of China worker, agricultural, the raising of living standards of the people, the direct discharging of a large amount of undressed workers, agricultural production waste water and sanitary sewage; Add the raising of China's aquaculture intensive degree, China's cultivation water pollution is on the rise.The cultivation aquatic animal disease that causes thus rolls up, and frequent occurrence.And blindly frequently use in a large number fishing (beast) medicines, antibiotic, hormones etc., make the quality safety of aquatic products be subject to serious threat.In order to protect fishery biologic environment, improve aquatic product quality and output, promote sustainable fisheries industry development, application photosynthetic bacteria probiotics control fishery biologic environment pollutes and destroys, and control aquatic products disease is significant undoubtedly.Chinese invention patent numbers 94106402.6 discloses the technology that a kind of name is called " animal feed additive and the production method thereof of utilizing micro-organisms ".This animal feed additive is a kind of liquid preparation of producing with photosynthetic bacteria, also use in aquaculture, but because its raw materials for production adopt the amidin of 10-40%, nutrient is not comprehensive, be difficult to satisfy photosynthetic bacterium growth to the needs of a great number of elements such as nitrogenous source, phosphorus source, sulphur source, thereby undergrowth.Simultaneously, the amidin that this patent is used adopts the mode of self-souring to obtain acid fermentation liquid after saccharification, because acidifying bacterial classification manual control in addition not, thereby the difficult quality guarantee of acid fermentation liquid.And sterilized is processed before inoculation photosynthetic bacteria bacterial classification, so contain a large amount of heterotrophs in the acid fermentation liquid, these heterotrophs can suppress the growth of photosynthetic bacteria, thereby cause the severe contamination of product, pollution rate can reach more than 80%, thereby causes the unstable of application effect.
Summary of the invention
Goal of the invention of the present invention is in order to overcome the deficiency of prior art, the production method of the additive of bait for photosynthetic bacteria aquaculture of a kind of steady quality and functional diversities to be provided.
Goal of the invention of the present invention is to realize like this; The production method of this additive of bait for photosynthetic bacteria aquaculture is taked following steps:
1, bacterial classification enlarges cultivation
Produce bacterial classification: adopting culture presevation number is CCTCCNO; The Rhodopseudomonas palustris of M209077 (H1) (Rhodopeudomonas palustris) and culture presevation number are CCTCCNO; 1: 1 the mixed culture of two kinds of photosynthetic bacterias of the Spherical red antibacterial of M209078 (H2) (Rhodobacter sphaeroides) is as producing bacterial classification.This two strains bacterial classification is deposited in " the Chinese Typical Representative culture collection center " of Wuhan University on April 27th, 2009.
Bacterial classification is enlarged the following composition of culture medium: natrium malicum 1.0 grams; Sodium acetate 1.0 grams; Ammonium chloride 1.0 grams; Potassium dihydrogen phosphate 1.0 grams; Calcium chloride 0.1 gram; Sodium acid carbonate 3.0 grams; Magnesium chloride 0.5 gram; Trace element solution 1.0ml; Vitamin solution 1.0ml; Yeast extract 0.5 gram is dissolved in respectively in 1.0 liters of distilled water.Yong Qing Yangization Na transfers in the pH6.0-8.5 scope, and preferably pH is 6.8-7.2, the vial of packing into.121 ℃ of sterilizations 30 minutes.When treating that culture medium is cooled to 30-35 ℃, press the 5%-40% of bacterium culture medium volume, preferably the inoculum concentration of 10%-20% accesses the mixed culture of above-mentioned two kinds of photosynthetic bacterias.In 15 ℃ of-35 ℃ of scopes of temperature, best 28 ℃-30 ℃, intensity of illumination is that anaerobism was cultivated 3-4 days under the 1000lux-3000lux condition, and bacteria containing amount reaches 5 * 10
8-10 * 10
8Cfu/ml, red bacteria culture fluid.
Trace element solution forms: four water frerrous chlorides, 1.8 grams; CoCL2 6H2O 0.25 gram; Six water nickel chlorides, 0.01 gram; Copper chloride dihydrate 0.01 gram; Tetrahydrate manganese chloride 0.7 gram; Zinc chloride 0.1 gram; Boric acid 0.5 gram; Sodium Molybdate Dihydrate 0.03 gram; Sodium selenite (Na2SeO3) pentahydrate 0.01 gram; 1.0 liters of distilled water.
Vitamin solution forms: biotin 0.1 gram; Nicotinic acid 0.35 gram; Thiamine hydrochloride 0.3 gram; Para-Aminobenzoic 0.2 gram; Hydrochloric acid pyridoxamine 0.1 gram; Calcium pantothenate 0.1 gram; Cobastab
120.05 gram; 1.0 liters of distilled water.
2, fermenting and producing
(1) fermentation medium forms: malic acid 1.0 grams; Sodium acetate 1.5 grams; Sodium propionate 0.5 gram; Yeast extract 1.0 grams; Ammonium sulfate 1.3 grams; Epsom salt 0.2 gram; Calcium chloride dihydrate 0.07 gram; Ironic citrate 0.01 gram; Ethylenediamine tetra-acetic acid (EDTA) 0.02 gram; Potassium dihydrogen phosphate 0.6 gram; Dipotassium hydrogen phosphate 0.9 gram; Trace element solution 1.0ml; Vitamin solution 7.5ml; 1.0 liters in running water; PH 6.8-7.2.
Wherein:
Trace element solution: ironic citrate 3 grams; Manganese sulfate monohydrate 0.02 gram; Boric acid 0.01 gram; Cupric sulfate pentahydrate 0.01 gram; Four water ammonium molybdates, 0.02 gram; Zinc sulfate 0.01 gram; Ethylenediamine tetra-acetic acid (EDTA) 0.5 gram; Calcium chloride dihydrate 0.2 gram; 1.0 liters of distilled water.
Vitamin solution: nicotinic acid 0.2 gram; Niacinamide 0.2 gram; Thiamine salt hydrochlorate 0.4 gram; Biotin 0.08 gram; 1.0 liters of distilled water.
(2) production process and condition control: in full-automatic photosynthesis microorganism fermentation tank, by above-mentioned composition preparation fermentation medium.Sterilized 60 minutes for 121 ℃.When treating that culture medium is cooled to 30 ℃-35 ℃, by the 5%-40% of fermenting and producing culture volume, be preferably the inoculum concentration access bacteria culture fluid of 10%-20%.At temperature 28-30 ℃; Mixing speed 150-300 rev/min; Keep tank pressure with filtrated air, tank pressure keeps 0.02-0.05MPa; Intensity of illumination initial stage 2000lux, mid-term 2500lux, later stage 3000lux condition bottom fermentation 6 days, the rufous additive of bait for photosynthetic bacteria aquaculture.Include living bacteria count 5 * 10
9-6 * 10
9Cfu/ml; PH7.0-8.5; The miscellaneous bacteria rate is less than 2%.
The present invention compared with prior art has the following advantages:
1, the additive of bait steady quality of producing with the present invention.
The present invention adopts pure chemical reagent to make raw materials for production, and quality and quantity are strictly controlled, nutritive element balance, comprehensive.Can satisfy the photosynthetic bacterium growth breeding to the demand of carbon source, nitrogenous source, phosphorus source, sulphur source and various trace elements.In addition, before inoculation photosynthetic bacteria mixed culture, adopt 121 ℃ of sterilizations 60 minutes, killed vegetative cell, brood cell and the mold spore of all miscellaneous bacterias in the fermenting and producing culture medium.Adopt again in process of production filtrated air to keep tank pressure, make photosynthetic bacteria growth and breeding under the condition of fully pure culture.Every batch of product includes living bacteria count 5 * 10
9-6 * 10
9Cfu/ml, the miscellaneous bacteria rate is less than 2%, thereby guaranteed quality and the application effect of additive of bait.
2, the additive of bait of producing with the present invention has following multiple efficacies:
This additive of bait for photosynthetic bacteria aquaculture is the bioactive additive that integrates nutrition and health care, and its effect is equivalent to feed addictive ten growth accelerators ten antiviral agents ten water purification agents.
1), provides nutrition, promotion growth, raising bait utilization, increasing both production and income
The main component photosynthetic bacteria cell of this additive of bait contains profuse protein, B family vitamin, carotenoids, trace element, also is rich in nucleic acid, ubiquinone
10With somatomedin etc.In eel culture, in bait, by behind 1% this additive of interpolation, on average increase by 8.19% than conventional cultivation output; Feed coefficient on average descends 12.6%; Growth cycle has shortened 15 days.In the Micropterus salmonoides cultivation, by behind 1% this additive of interpolation, than conventional cultivation, output increases by 7.78% in bait; Feed coefficient descends 5.56%; Growth cycle shortens 28 days.
2), purify water, safeguard the water ecology balance
In eel culture, every water quality index of measuring before not using this additive is: pH6.7; Dissolved oxygen 5.5mg/L; Ammonia nitrogen 1.132mg/L; Nitrite 0.413mg/L; Hydrogen sulfide 0.003mg/L.Then by 1kg/ mu * rice this additive of splashing, the water quality detection result is as follows after 5 days: pH7.6 (becoming alkalescent by faintly acid); Dissolved oxygen 7.8mg/L (increasing 41.8%); Ammonia nitrogen 0.465mg/L (reducing by 58.9%); Nitrite 0.101mg/L (reducing by 75.5%); Hydrogen sulfide 0mg/L (degraded 100%).Indices all is better than " fresh water fishery water quality cultivation standard ".In Micropterus salmonoides and culture of Penaeus vannamei process, adhere to per 15 days by 1kg/ mu * rice this additive of splashing, sampling and measuring water quality weekly, indices all is better than " fresh water fishery cultivation water standard ", and the useful zooplankter quantity such as wheel animalcule, branch angle worm obviously increases in the water body.
3), degraded medicament residue and water pollutant
(1), this aquaculture additive is very obvious to multiple aquaculture medicament residue degradation effect.Can reach 84.6% to 120 hours the degradation rate of water body that contains the 10mg/L malachite green; To containing the water body of 10mg/L furazolidone, 72 hours degradation rate reaches 100%.
(2), in outlet eel live body detects, find that the fish body includes nitrofuran metabolite 3.15mg/kg, add this additive by 3% subsequently and in bait, feed, after 10 days again testing result be: can't detect, the vivo degradation rate reaches 100%, reaches export standard.
(3), this additive contains enzyme and the plasmid of multiple degradation of contaminant, except each type organic of degradable, also material of the many difficult for biological degradation of degradable such as Halogen can reach 96.4% for aromatic compound, amine, alkanes etc. to the chlorobenzenes clearance that is widely used in the industrial and agricultural production.
4), fixing Heavy Metals in Waters, reduce heavy metal to the harm of aquatic animal; And reduce aquatic livestock heavy metal of body content, ensure food safety.This additive can be converted into water-soluble poisonous and harmful heavy metallic water-fast metal sulfide, thereby has avoided the harm of heavy metal to aquatic animal to grow; Also reduce the enrichment of heavy metal in the aquiculture animal body, reduced heavy metal of body content.Test shows: this additive is to Pb
2+, As
5+, As
3+, Cd
2 +, Cr
2+, Hg
2+, Cu
2+Etc. various heavy very strong resistance is arranged.Be the Cd of 10-25mg/L to initial concentration
2+, 36 hours clearance can be more than 85%.Be the Pb of 100mg/L to concentration
2+Clearance can reach 95% in 24 hours.
5), disease resistance, improve immunity and survival rate
This additive contains effectively passivation water body virus of antiviral ingredients, reduces the generation of virosis.The multiple pathogenic bacterias such as Aeromonas sobria, pseudomonas putida also there is better inhibitory action.This additive can promote the growth of animal immune organ in addition, the Effective Raise animal immunizing power.Eel culture experiment shows, use this additive after, the fish disease loss reduces 15%, survival rate improves 4.26%; The Micropterus salmonoides culture experiment shows, uses the diseases such as the common fin rot of California perch behind this additive, mashed body disease, fish sore not occur; Survival rate improves 3.19%; Penaeus Vannmei high-density breeding experiment shows, use this additive after, substantially do not re-use the fishing medicine, survival rate is up to more than 95%.
6), improve the quality of aquatic products
(1), in eel culture, use this additive after, the adult fish size is evenly neat, color crow profit gloss, meat is good, mouthfeel is good.The live body examining report shows that antibiotics, sulfamido, BHC class, DDT class, steroids, chrysanthemum ester class and all kinds of poisonous and harmful heavy metallics etc. are and do not detect.
(2), in Micropterus salmonoides cultivation, use this additive after, the fish body is neat, fish scale is glossy especially, and is fresh and alive vigorous, meat is abundant soft.The live body examining report shows that antibiotics, sulfamido, BHC class, DDT class, steroids, chrysanthemum ester class and all kinds of poisonous and harmful heavy metallics etc. are and do not detect.
The specific embodiment
The present invention is further illustrated below in conjunction with specific embodiment:
Embodiment 1
Step 1: put vial after the various compositions that above-mentioned bacterial classification is enlarged culture medium dissolve in distilled water, transfer pH6.0 with NaOH, 121 ℃ of sterilizations 30 minutes, when being cooled to 30 ℃, inoculation enters Rhodopseudomonas palustris and the Spherical red antibacterial mixed culture of bacterium culture medium volume 5%, is 15 ℃ in temperature, and intensity of illumination is that anaerobism was cultivated 3 days under the 1000lux condition, namely get the bacteria culture fluid of rufous, bacteria containing amount reaches 5 * 10
8Cfu/ml.
Step 2: put in the full-automatic photosynthesis microorganism fermentation tank after above-mentioned fermentation dissolved in running water with the various compositions of culture medium, 121 ℃ of sterilizations 60 minutes, when treating that culture medium is cooled to 30 ℃, by the inoculum concentration access bacteria culture fluid of fermenting and producing culture volume 5%.It is 30 ℃ in temperature; 150 rev/mins of mixing speeds; Use the filtrated air pressurize, tank pressure remains 0.02MPa; The intensity of illumination initial stage is 2000lux, and be 2500lux mid-term, and the later stage is 3000lux condition bottom fermentation 6 days, namely gets the rufous additive of bait for photosynthetic bacteria aquaculture, includes living bacteria count 6 * 10
9Cfu/ml, the miscellaneous bacteria rate is less than 2%.
Embodiment 2
Step 1: will put vial into after the dissolving in the various composition distilled water of above-mentioned bacterial classification expansion culture medium, transfer pH8.5 with NaOH, 121 ℃ of sterilizations 30 minutes, when being cooled to 35 ℃, inoculation enters Rhodopseudomonas palustris and the Spherical red antibacterial mixed culture of bacterium culture medium volume 20%, is 35 ℃ in temperature, and intensity of illumination is that anaerobism was cultivated 4 days under the 3000lux condition, namely get the bacteria culture fluid of rufous, bacteria containing amount reaches 8 * 10
8Cfu/ml.
Step 2: put in the full-automatic photosynthesis microorganism fermentation tank after above-mentioned fermentation dissolved in running water with the various compositions of culture medium, 121 ℃ of sterilizations 60 minutes, when treating that culture medium is cooled to 35 ℃, by the inoculum concentration access bacteria culture fluid of fermenting and producing culture volume 40%.It is 30 ℃ in temperature; 300 rev/mins of mixing speeds; Use the filtrated air pressurize, tank pressure remains 0.05MPa; The intensity of illumination initial stage is 2000lux, and be 2500lux mid-term, and the later stage is 3000lux condition bottom fermentation 6 days, namely gets the rufous additive of bait for photosynthetic bacteria aquaculture, includes living bacteria count 5.8 * 10
9Cfu/ml, the miscellaneous bacteria rate is less than 2%.
Embodiment 3
Step 1: put vial after the various compositions that above-mentioned bacterial classification is enlarged culture medium dissolve in distilled water, transfer pH7.0 with NaOH, 121 ℃ of sterilizations 30 minutes, when being cooled to 32 ℃, inoculation enters Rhodopseudomonas palustris and the Spherical red antibacterial mixed culture of bacterium culture medium volume 15%, is 25 ℃ in temperature, and intensity of illumination is that anaerobism was cultivated 4 days under the 2000lux condition, namely get the bacteria culture fluid of rufous, bacteria containing amount reaches 8 * 10
8Cfu/ml.
Step 2: put in the full-automatic photosynthesis microorganism fermentation tank after above-mentioned fermentation dissolved in running water with the various compositions of culture medium, 121 ℃ of sterilizations 60 minutes, when treating that culture medium is cooled to 32 ℃, by the inoculum concentration access bacteria culture fluid of fermenting and producing culture volume 15%.It is 29 ℃ in temperature; 250 rev/mins of mixing speeds; Use the filtrated air pressurize, tank pressure remains 0.04MPa; The intensity of illumination initial stage is 2000lux, and be 2500lux mid-term, and the later stage is 3000lux condition bottom fermentation 6 days, namely gets the rufous additive of bait for photosynthetic bacteria aquaculture, includes living bacteria count 6 * 10
9Cfu/ml, the miscellaneous bacteria rate is less than 2%.
Claims (5)
1. the production method of an additive of bait for photosynthetic bacteria aquaculture is characterized in that taking following steps:
1) bacterial classification enlarges cultivation
Bacterial classification enlarges culture medium and forms: natrium malicum 1.0 grams; Sodium acetate 1.0 grams; Ammonium chloride 1.0 grams; Potassium dihydrogen phosphate 1.0 grams; Calcium chloride 0.1 gram; Sodium acid carbonate 3.0 grams; Magnesium chloride 0.5 gram; Trace element solution 1.0ml; Vitamin solution 1.0ml; Yeast extract 0.5 gram; 1.0 liters of distilled water.
With the vial of packing into behind the culture medium Yong Qing Yangization Na accent pH6.0-8.5,121 ℃ of sterilizations 30 minutes, to be cooledly press the 5-40% access Rhodopseudomonas palustris (H1) that bacterial classification enlarges culture volume during to 30-35 ℃ (Rhodopeudomonas palustris culture presevation number is CCTCCNO; M209077) (Rhodobacter sphaeroides culture presevation number is CCTCCNO with Spherical red antibacterial (H2); M209078) 1: 1 mixed culture of two kinds of photosynthetic bacterias, 15 ℃-35 ℃ of temperature, intensity of illumination is that anaerobism was cultivated 3-4 days under the 1000lux-3000lux condition, red bacteria culture fluid;
2) fermenting and producing
The fermenting and producing culture medium forms: malic acid 1 gram; Sodium acetate 1.5 grams; Sodium propionate 0.5 gram; Yeast extract 1.0 grams; Ammonium sulfate 1.3 grams; Epsom salt 0.2 gram; Calcium chloride dihydrate 0.07 gram; Ironic citrate 0.01 gram; Ethylenediamine tetra-acetic acid (EDTA) 0.02 gram; Potassium dihydrogen phosphate 0.6 gram; Dipotassium hydrogen phosphate 0.9 gram; Trace element solution 1.0ml; Vitamin solution 7.5ml; 1.0 liters in running water; PH 6.8-7.2.
Various compositions until the fermenting and producing culture medium are added in the fermentation tank after the dissolving in water fully, sterilized 60 minutes for 121 ℃, when treating that culture medium is cooled to 30-35 ℃, press the inoculum concentration access bacteria culture fluid of the 5%-40% of fermenting and producing culture volume, 15 °-35 ℃ of temperature; Mixing speed 150-300 rev/min; Use the filtrated air pressurize, tank pressure keeps 0.02-0.05MPa; Intensity of illumination initial stage 2000lux, mid-term 2500lux, later stage 3000lux condition bottom fermentation 6 days, the photosynthetic bacteria aquatic feed additive of rufous.
2. the production method of additive of bait for photosynthetic bacteria aquaculture according to claim 1 is characterized in that described bacterial classification enlarges culture medium and transfers pH6.8-7.2 with NaOH.
3. the production method of additive of bait for photosynthetic bacteria aquaculture according to claim 1 is characterized in that it is the 10%-20% that bacterial classification enlarges culture volume that described bacterial classification enlarges the inoculum concentration of cultivating.
4. the production method of additive of bait for photosynthetic bacteria aquaculture according to claim 1 is characterized in that it is 28 ℃-30 ℃ that described bacterial classification enlarges the temperature of cultivating.
5. the production method of additive of bait for photosynthetic bacteria aquaculture according to claim 1, the inoculum concentration that it is characterized in that described fermenting and producing is the 10%-20% of fermenting and producing culture volume.
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| WO2017007830A1 (en) * | 2015-07-06 | 2017-01-12 | Parabel Ltd. | Methods and systems for extracting a polysaccharide product from a microcrop and compositions thereof |
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Address after: 528322 Guangdong province Foshan City Leliu Shunde Zhongyu road Yuyong Industrial Zone Applicant after: GUANGDONG VITAFOOD Co.,Ltd. Address before: 528322 Guangdong province Foshan City Leliu Shunde Zhongyu road Yuyong Industrial Zone Applicant before: SHUNDE HONGLONG BIOLOGY SCIENCE AND TECHNOLOGY Co.,Ltd. |
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Effective date of registration: 20230915 Address after: Room 102, Building 3, Weihe New Science Park, No. 2, Keji 2nd Road, Xingtan Industrial Zone, Qixing Community, Xingtan Town, Shunde District, Foshan City, Guangdong Province, 528311 Patentee after: Guangdong Fengshu Industrial Co.,Ltd. Address before: 528322 Guangdong province Foshan City Leliu Shunde Zhongyu road Yuyong Industrial Zone Patentee before: GUANGDONG VITAFOOD Co.,Ltd. |