CN101595943B - Production method of additive of bait for photosynthetic bacteria aquaculture - Google Patents

Abstract

The invention relates to a production method of a photosynthetic bacteria aquaculture bait additive. The method takes the following steps: 1. Expanded culture of strains: adjust the pH of the expanded culture medium to 6.0-8.5, put it into a glass bottle and carry out high-temperature disinfection, and insert marsh red by 5%-40% of the volume of the culture medium A mixed culture of two photosynthetic bacteria, Pseudomonas and Rhodobacter sphaericus. Under the condition of temperature 15°C-35°C and light intensity of 1000lux-3000lux, anaerobic culture is carried out for 3-4 days to form the culture medium of the strain; 2. Fermentation production: put the fermentation medium into the fermenter and perform high-temperature disinfection. The inoculation amount of 5%-40% of the volume of the fermentation production medium is inserted into the strain culture solution. Ferment for 6 days under the conditions of temperature 28-30°C; stirring speed 150-300 rev/min; tank pressure 0.02-0.05MPa; light intensity 2000-3000lux, and it becomes a bait additive for photosynthetic bacteria aquaculture. Compared with the prior art, the invention has the advantages of stable product quality and diversified functions.

Description

A kind of production method of photosynthetic bacteria aquaculture bait additive

technical field

The invention relates to the field of microorganisms, in particular to a production method of a photosynthetic bacteria aquaculture bait additive.

Background technique

With the rapid development of industry and agriculture in our country and the improvement of people's living standards, a large amount of untreated industrial and agricultural production wastewater and domestic sewage are directly discharged; coupled with the improvement of the degree of intensification of aquaculture in our country, the pollution of aquaculture waters in our country is becoming more and more serious. serious. The resulting diseases of farmed aquatic animals have increased in a large number and occur frequently. The blind and frequent use of fish (veterinary) drugs, antibiotics, hormones, etc. has seriously threatened the quality and safety of aquatic products. In order to protect the fishery ecological environment, improve the quality and output of aquatic products, and promote the sustainable development of the aquaculture industry, it is undoubtedly of great significance to apply photosynthetic bacteria microecological preparations to prevent and control the pollution and destruction of the fishery ecological environment and aquatic product diseases. Chinese invention patent No. 94106402.6 discloses a technology called "animal feed additive produced by microorganisms and its production method". This animal feed additive is a liquid preparation produced by photosynthetic bacteria, which is also used in aquaculture, but because its production raw materials use 10-40% starch aqueous solution, the nutrients are not comprehensive, and it is difficult to meet the nitrogen source required by the growth of photosynthetic bacteria , phosphorus source, sulfur source and other large elements, so the growth is poor. Simultaneously, after the aqueous starch solution used in this patent is saccharified, the acidic fermented liquid is obtained by natural acidification. Since the acidified strains are not artificially controlled, the quality of the acidic fermented liquid is difficult to guarantee. Moreover, there is no sterilization treatment before inoculation of photosynthetic bacteria strains, so the acidic fermentation liquid contains a large number of heterotrophic bacteria, which will inhibit the growth of photosynthetic bacteria, thereby causing serious pollution of the product, and the pollution rate can reach 80%. Above, resulting in the instability of the application effect.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art and provide a production method of a photosynthetic bacteria aquaculture bait additive with stable quality and diversified functions.

The purpose of the invention of the present invention is achieved like this; The production method of this photosynthetic bacteria aquaculture bait additive takes the following steps:

1. Expansion of strains

Production of strains: Two species of photosynthetic strains were used: Rhodopseudomonas palustris (H1) (Rhodopeudomonas palustris) with strain preservation number CCTCCNO; M209077 and CCTCCNO; M209078 Rhodobacter sphaeroides A 1:1 mixed culture of bacteria was used as the production strain. These two strains were preserved on April 27, 2009 in the "Chinese Type Culture Collection Center" of Wuhan University.

The following ingredients of the strain expansion medium: 1.0 grams of sodium malate; 1.0 grams of sodium acetate; 1.0 grams of ammonium chloride; 1.0 grams of potassium dihydrogen phosphate; 0.1 grams of calcium chloride; 3.0 grams of sodium bicarbonate; 0.5 grams of magnesium chloride; 1.0ml of trace element solution; 1.0ml of vitamin solution; 0.5g of yeast extract were dissolved in 1.0 liter of distilled water respectively. Use sodium hydroxide to adjust the pH within the range of 6.0-8.5, preferably 6.8-7.2, and put it into a glass bottle. Sterilize at 121°C for 30 minutes. When the culture medium is cooled to 30-35 DEG C, insert the mixed culture of the above two photosynthetic bacteria according to 5%-40% of the volume of the culture medium, preferably 10%-20%. In the temperature range of 15°C-35°C, preferably 28°C-30°C, under the condition of light intensity of 1000lux-3000lux, anaerobic culture for 3-4 days, the bacterial content reaches 5×10 ⁸ -10×10 ⁸ cfu/ml , Serve as a red culture medium.

Trace element solution composition: 1.8 grams of ferrous chloride tetrahydrate; 0.25 grams of cobalt chloride hexahydrate; 0.01 grams of nickel chloride hexahydrate; 0.01 grams of copper chloride dihydrate; 0.7 grams of manganese chloride tetrahydrate; 0.1 grams of zinc chloride gram; 0.5 gram of boric acid; 0.03 gram of sodium molybdate dihydrate; 0.01 gram of sodium selenite pentahydrate; 1.0 liter of distilled water.

Vitamin solution composition: 0.1 g of biotin; 0.35 g of nicotinic acid; 0.3 g of thiamine hydrochloride; 0.2 g of p-aminobenzoic acid; 0.1 g of pyridoxamine hydrochloride; 0.1 g of calcium pantothenate; 0.05 g of vitamin _B12 ; .

2. Fermentation production

(1) Composition of fermentation medium: 1.0 grams of malic acid; 1.5 grams of sodium acetate; 0.5 grams of sodium propionate; 1.0 grams of yeast extract; 1.3 grams of ammonium sulfate; 0.2 grams of magnesium sulfate heptahydrate; 0.07 grams of calcium chloride dihydrate; 0.01 g of ferric citrate; 0.02 g of ethylenediaminetetraacetic acid (EDTA); 0.6 g of potassium dihydrogen phosphate; 0.9 g of dipotassium hydrogen phosphate; 1.0 ml of trace element solution; 7.5 ml of vitamin solution; 1.0 liter of tap water; pH 6.8-7.2 .

in:

Trace element solution: 3 grams of iron citrate; 0.02 grams of manganese sulfate monohydrate; 0.01 grams of boric acid; 0.01 grams of copper sulfate pentahydrate; 0.02 grams of ammonium molybdate tetrahydrate; 0.01 grams of zinc sulfate; grams; 0.2 grams of calcium chloride dihydrate; 1.0 liters of distilled water.

Vitamin solution: niacin 0.2 g; nicotinamide 0.2 g; thiamine hydrochloride 0.4 g; biotin 0.08 g; distilled water 1.0 liter.

(2) Production process and condition control: In the fully automatic photosynthetic microorganism fermentation tank, the fermentation medium is prepared according to the above composition. Sterilize at 121°C for 60 minutes. When the culture medium is cooled to 30°C-35°C, insert the strain culture solution with an inoculum amount of 5%-40%, preferably 10%-20%, of the volume of the fermentation production medium. The temperature is 28-30°C; the stirring speed is 150-300 rpm; the tank pressure is kept at 0.02-0.05MPa with sterile air; the light intensity is 2000lux in the early stage, 2500lux in the middle stage, and 3000lux in the later stage. Ferment for 6 days and serve. Red-brown photosynthetic bacteria aquaculture bait additive. The number of effective viable bacteria in it is 5×10 ⁹ -6×10 ⁹ cfu/ml; pH7.0-8.5; the miscellaneous bacteria rate is less than 2%.

Compared with the prior art, the present invention has the following advantages:

1. The quality of the bait additive produced by the invention is stable.

The invention adopts pure chemical reagents as raw materials for production, the quality and quantity are strictly controlled, and the nutritional elements are balanced and comprehensive. It can meet the needs of photosynthetic bacteria for carbon source, nitrogen source, phosphorus source, sulfur source and various trace elements for the growth and reproduction of photosynthetic bacteria. In addition, before inoculating the mixed culture of photosynthetic bacteria, it was sterilized at 121°C for 60 minutes to kill the vegetative cells, spores and mold spores of all the miscellaneous bacteria in the fermentation production medium. In the production process, sterile air is used to maintain the tank pressure, so that photosynthetic bacteria can grow and reproduce under completely pure culture conditions. Each batch of products contains 5×10 ⁹ -6×10 ⁹ cfu/ml of effective viable bacteria, and the miscellaneous bacteria rate is less than 2%, thus ensuring the quality and application effect of the bait additive.

2, the bait additive produced with the present invention has following multiple effects:

The photosynthetic bacteria aquaculture bait additive is a biologically active additive integrating nutrition and health care, and its functions are equivalent to feed additive ten growth promoters ten disease resistance agents ten water purifiers.

1) Provide nutrition, promote growth, improve bait utilization, increase production and income

The main component of this bait additive is photosynthetic bacteria cells, which are extremely rich in protein, B vitamins, carotenoids, trace elements, and also rich in nucleic acids, coenzyme Q ₁₀ and growth-promoting factors. In eel culture, after adding this additive in 1% of bait, the output of eel culture will increase by 8.19% on average; the coefficient of bait will decrease by 12.6% on average; and the growth cycle will be shortened by 15 days. In California sea bass culture, after adding this additive to the bait at a rate of 1%, the yield increased by 7.78% compared with conventional culture; the feed coefficient decreased by 5.56%; the growth period was shortened by 28 days.

2) Purify water quality and maintain ecological balance of water body

In eel farming, the water quality indicators measured before using this additive are: pH6.7; dissolved oxygen 5.5mg/L; ammonia nitrogen 1.132mg/L; nitrite 0.413mg/L; hydrogen sulfide 0.003mg/L . Sprinkle this additive by 1kg/mu* rice then, water quality testing result is as follows after 5 days: pH7.6 (becoming weakly alkaline by weak acidity); Dissolved oxygen 7.8mg/L (increase 41.8%); Ammonia nitrogen 0.465mg/L ( 58.9% reduction); Nitrite 0.101mg/L (75.5% reduction); Hydrogen sulfide 0mg/L (100% degradation). All indicators are better than the "Freshwater Fishery Aquaculture Standards". During the breeding process of California perch and vannamei, the additive was sprayed every 15 days at a rate of 1kg/mu*meter, and the water quality was measured by sampling every week. The number of beneficial zooplankton such as Cladocera increased significantly.

3) Degradation of drug residues and water pollutants

(1) The aquaculture additive has a very obvious effect on the degradation of various aquaculture drug residues. For the water body containing 10mg/L malachite green, the degradation rate can reach 84.6% in 120 hours; for the water body containing 10mg/L furazolidone, the degradation rate can reach 100% in 72 hours.

(2) In the living test of eels for export, it was found that the fish contained 3.15 mg/kg of nitrofuran metabolites, and then this additive was added to the bait at 3% to feed, and the test result again after 10 days was: Unable to detect, The degradation rate in the body reaches 100%, reaching the export standard.

(3) This additive contains a variety of enzymes and plasmids for degrading pollutants. In addition to degrading various organic substances, it can also degrade many difficult-to-biodegrade substances, such as halogenated aromatic compounds, amines, alkanes, etc. The removal rate of chlorobenzene compounds widely used in industrial and agricultural production can reach 96.4%.

4) Fix heavy metals in water, reduce the harm of heavy metals to aquatic animals; and reduce the content of heavy metals in aquatic animals to ensure food safety. This additive can convert toxic and harmful heavy metals soluble in water into metal sulfides insoluble in water, thereby avoiding the harm of heavy metals to the growth of aquatic animals; it also reduces the accumulation of heavy metals in aquaculture animals and reduces the content of heavy metals in the body. Tests show that this additive has strong resistance to Pb ²⁺ , As ^{5+ ,} As ³⁺ , Cd ²⁺ , Cr ²⁺ , Hg ²⁺ , Cu ²⁺ and other heavy metals. For Cd ²⁺ with an initial concentration of 10-25mg/L, the removal rate can be over 85% within 36 hours. The removal rate of Pb ²⁺ with a concentration of 100mg/L can reach 95% in 24 hours.

5), disease prevention and disease resistance, improve immunity and survival rate

The additive contains antiviral ingredients, which can effectively passivate water body viruses and reduce the occurrence of viral diseases. It also has a good inhibitory effect on a variety of aquaculture pathogens such as Aeromonas sobria and Pseudomonas putida. In addition, the additive can promote the development of animal immune organs and effectively improve animal immunity. The eel breeding experiment shows that after using this additive, the fish disease loss is reduced by 15%, and the survival rate is increased by 4.26%; the California perch breeding experiment shows that after using this additive, common diseases such as gill rot, body rot, and fish sores in California perch have not occurred The survival rate increased by 3.19%. The high-density culture experiment of Penaeus vannamei showed that after using this additive, no fishing medicine was basically used anymore, and the survival rate was as high as 95%.

6) Improve the quality of aquatic products

(1) After using this additive in eel breeding, the adult fish will be uniform in size, dark in color and lustrous, with good meat quality and good taste. The biopsy report showed that antibiotics, sulfonamides, H66, DDT, hormones, pyrethroids and various toxic and harmful heavy metals were not detected.

(2) After using this additive in California perch breeding, the fish body is neat, the fish scales are particularly shiny, fresh and vigorous, and the meat is rich and tender. The biopsy report showed that antibiotics, sulfonamides, H66, DDT, hormones, pyrethroids and various toxic and harmful heavy metals were not detected.

Detailed ways

The present invention will be further described below in conjunction with specific embodiment:

Example 1

Step 1: Dissolve the various components of the above strain expansion medium in distilled water and put them into a glass bottle, adjust the pH to 6.0 with sodium hydroxide, sterilize at 121°C for 30 minutes, cool to 30°C, inoculate into the bacteria The mixed culture of Rhodopseudomonas palustris and Rhodobacter sphaericus with 5% seed medium volume was anaerobically cultured for 3 days at a temperature of 15°C and a light intensity of 1000lux to obtain a reddish-brown strain culture solution containing The amount of bacteria reached 5×10 ⁸ cfu/ml.

Step 2: Dissolve the various components of the above-mentioned fermentation medium in tap water and put them into a fully automatic photosynthetic microorganism fermenter, sterilize at 121°C for 60 minutes, and when the medium is cooled to 30°C, produce the medium by fermentation 5% of the volume of the inoculum was inserted into the culture medium of the bacteria. The temperature is 30°C; the stirring speed is 150 rpm; the tank pressure is maintained at 0.02 MPa with sterile air; the light intensity is 2000 lux in the early stage, 2500 lux in the middle stage, and 3000 lux in the later stage. Ferment for 6 days to obtain reddish brown The bait additive for photosynthetic bacteria aquaculture contains 6×10 ⁹ cfu/ml of effective viable bacteria, and the miscellaneous bacteria rate is less than 2%.

Example 2

Step 1: Dissolve the various components of the above strain expansion medium in distilled water and put them into a glass bottle, adjust the pH to 8.5 with sodium hydroxide, sterilize at 121°C for 30 minutes, cool to 35°C, and inoculate the strains The mixed culture of Rhodopseudomonas palustris and Rhodobacter sphaericus with a medium volume of 20% was anaerobically cultured at a temperature of 35°C and a light intensity of 3000lux for 4 days to obtain a reddish-brown strain culture solution containing bacteria The amount reached 8×10 ⁸ cfu/ml.

Step 2: Dissolve the various components of the above-mentioned fermentation medium in tap water and put them into a fully automatic photosynthetic microbial fermentation tank, sterilize at 121°C for 60 minutes, and when the medium is cooled to 35°C, press the fermentation medium 40% of the volume of the inoculum into the culture medium. The temperature is 30°C; the stirring speed is 300 rpm; the tank pressure is maintained at 0.05 MPa with sterile air; the light intensity is 2000 lux in the early stage, 2500 lux in the middle stage, and 3000 lux in the later stage. Ferment for 6 days to obtain reddish brown Photosynthetic bacteria aquaculture bait additive, containing 5.8×10 ⁹ cfu/ml of effective viable bacteria, and the rate of miscellaneous bacteria is less than 2%.

Example 3

Step 1: Dissolve the various components of the above strain expansion medium in distilled water and put them into a glass bottle, adjust the pH to 7.0 with sodium hydroxide, sterilize at 121°C for 30 minutes, cool to 32°C, and inoculate into the bacteria The mixed culture of Rhodopseudomonas palustris and Rhodobacter sphaericus with a volume of 15% of the seed medium was anaerobically cultured for 4 days at a temperature of 25°C and a light intensity of 2000lux to obtain a reddish-brown strain culture solution containing The amount of bacteria reached 8×10 ⁸ cfu/ml.

Step 2: Dissolve the various components of the above-mentioned fermentation medium in tap water and put them into a fully automatic photosynthetic microbial fermentation tank, sterilize at 121°C for 60 minutes, and when the medium is cooled to 32°C, produce the medium according to the fermentation method 15% of the volume of the inoculum into the culture medium. The temperature is 29°C; the stirring speed is 250 rpm; the tank pressure is maintained at 0.04MPa with sterile air; the light intensity is 2000lux in the early stage, 2500lux in the middle stage, and 3000lux in the later stage. Ferment for 6 days to obtain reddish brown The bait additive for photosynthetic bacteria aquaculture contains 6×10 ⁹ cfu/ml of effective viable bacteria, and 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.