CN101353636B - Production method of lactobacillus micro-ecological preparation for cultivation water regulation - Google Patents
Production method of lactobacillus micro-ecological preparation for cultivation water regulation Download PDFInfo
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- CN101353636B CN101353636B CN2008101984746A CN200810198474A CN101353636B CN 101353636 B CN101353636 B CN 101353636B CN 2008101984746 A CN2008101984746 A CN 2008101984746A CN 200810198474 A CN200810198474 A CN 200810198474A CN 101353636 B CN101353636 B CN 101353636B
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Abstract
The invention discloses a production method of lactobacillus microecologics which is used for regulating and controlling culture water quality, comprising the following steps of: (1) tube culture; (2) shaking culture; (3) seed tank culture; and (4) fermentor culture: culture solution in the seed tank is inoculated into the fermentor filled with culture solution, and placed for culturing of 24 to 36 hours at the temperature of 25 to 35 DEG C; when the final pH of the fermenting liquid is 3.0 to 4.0 and the density of the lactobacillus is 5 billion/ml, the fermentation is stopped; the pH of theculture solution of the step (3) and the step (4) is 5.5 to 6.5; the culture solution comprises the following ingredients in 1L of water by weight portion: 3 to 5 portions of molasses, 3 to 5 portions of yeast extract, 0.8 to 1 portion of corn steep liquor, 0.1 to 0.3 portion of KH2PO4, 0.3 to 0.5 portion of MgSO4, 3 to 5 portions of NaCl, 8 to 10 portions of glucose and 0.8 to 1 portion of peptone; the method adopts a totally open culture mode to carry out the production of lactobacillus microecologics which is used for regulating and controlling the culture water quality, and is characterized by little equipment investment, little energy consumption, simple technique and low production cost.
Description
Technical field
The present invention relates to the production method of a kind of milk-acid bacteria, relate in particular to a kind of production method that is used for the lactobacillus micro-ecological preparation of cultivation water regulation.
Background technology
Milk-acid bacteria is in close relations with the mankind's, and it has wide range of applications, and in the clinical medicine aspect, milk-acid bacteria can be treated the intestinal function disorder as probiotics, keeps enteron aisle thalline balance etc.Be applied in the feed, can promote to grow, disease preventing and treating and the quality of improving poultry product; Can be used as foodstuff additive, improve acidity and improve local flavor, increase nutrient and improve nutritive value, prolong effective period of food quality, prevent putrid effect.Use in a large number in intensification prawn culturing process, its regulation and control algae phase, the effect that purifies water are obvious, have improved the success ratio of prawn culturing.
The technology of existing cultivation lactic acid bacteria formulation generally is to stir mixing after the substratum batching, is placed in the fermentation unit then and adds bacterial classification and cultivate, and commonly used now is that the interior standard fermentor tank of microbial fermentation industry ferments.There is interlayer standard fermentor tank inside, mainly utilizes water vapor or cold water to heat or lowers the temperature, and this culture tank requires high to envrionment conditions, and sterilization must thoroughly cultivate the cost height, and facility investment be big.
On the other hand, the prawn culturing pond microecological preparation of lactobacillus usage quantity of using of splashing is big, just can reduce the aquaculture input cost greatly if can reduce production cost of products.
Summary of the invention
The object of the invention provides a kind of production method that is used for the lactobacillus micro-ecological preparation of cultivation water regulation, this method adopts the training method that standard-sized sheet is put to carry out the production of lactobacillus micro-ecological microbial inoculum, it is few to have facility investment, the characteristics that energy consumption is little, technology is simple, production cost is low.
The objective of the invention is to be achieved through the following technical solutions: a kind of production method that is used for the lactobacillus micro-ecological preparation of cultivation water regulation may further comprise the steps:
(1) test tube is cultivated: lactic acid bacteria culturers is inoculated in the test tube that substratum is housed, cultivates 28～30 ℃ of temperature-activated;
(2) shake-flask culture: the activation culture liquid of bacterial classification is inoculated in the triangular flask that substratum is housed, and 28～30 ℃ of temperature leave standstill cultivation;
(3) seed tank culture: the nutrient solution in the triangular flask is inoculated in the seeding tank that substratum is housed, and temperature leaves standstill cultivation for 25～35 ℃;
(4) fermentor cultivation: the nutrient solution in the seeding tank is inoculated in the fermentor tank that substratum is housed, and temperature leaves standstill for 25～35 ℃ cultivated 24～36 hours; When the fermented liquid final pH is 3.0～4.0, the nectar degree stops fermentation when being 5,000,000,000/ml;
The pH of the substratum in described step (3) and the step (4) is 5.5～6.5; Described substratum is the component that comprises following weight part in 1000 weight parts waters: molasses 3～5, yeast extract paste 3～5, corn steep liquor 0.8～1, KH
2PO
40.1～0.3, MgSO
40.3～0.5, NaCl3～5, glucose 8～10, peptone 0.8～1.
The inoculum size of the nutrient solution in the activation culture liquid of described step (2), step (3) and the step (4) is 3～5% of a substratum gross weight.
Substratum in described step (1) and the step (2) is improved MRS substratum, and its pH is 5.5～6.5; Described MRS substratum is the component that comprises following weight part in 1000 weight parts waters: peptone 8～10, yeast extract paste 3～5, glucose 13～15, KH
2PO
41.5～2, MgSO
40.3～0.5, MnSO
40.20～0.25, CH
3COONa3～5.
Described MRS substratum was 121 ℃ of following autoclavings 20 minutes.
The described seed culture of step (3) is to carry out first order seed earlier to cultivate, and then carries out secondary seed and cultivate.Described first order seed is cultivated and is cultivated at the seeding tank of smaller size smaller for lactic acid bacteria culturers; Described secondary seed lactic acid bacteria culturers is cultivated at the seeding tank of comparatively large vol.
Nutrient solution in described step (4) seeding tank stirs insert in the fermentor tank that substratum is housed with 3～5% inoculum size after, is divided in the plastics pot of narrow-mouth bottle and leaves standstill cultivation.
The present invention compared with prior art has the following advantages: test tube is cultivated, the two stage medium sterilization of shake-flask culture, and seeding tank, fermentor tank production phase substratum are unsterilised, adopt and leave standstill training method, thereby cut down the consumption of energy greatly; Can adopt plastics pot to replace the formal fermentor tank of microorganism culturing, thus the input that reduces production costs significantly.As the main raw material of producing fermention medium, to reduce production costs, the realization resource that turns waste into wealth is utilized once more with molasses, corn steep liquor.
Embodiment
Set forth below in conjunction with embodiment with to comparison the present invention, yet protection scope of the present invention is not only to be confined to following examples.The person of an ordinary skill in the technical field all can realize purpose of the present invention according to content disclosed by the invention.
Embodiment one
A kind of production method that is used for the lactobacillus micro-ecological preparation of cultivation water regulation may further comprise the steps:
(1) test tube is cultivated: the two strains of lactic acid bacteria bacterial classification is inoculated in respectively in two test tubes that the MRS substratum is housed, cultivates 28 ℃ of temperature-activated; Merge the gained nutrient solution then;
(2) shake-flask culture: the activation culture liquid of bacterial classification 5% inoculum size with the substratum gross weight is inoculated in the erlenmeyer flask that the MRS substratum is housed, and 28 ℃ of temperature leave standstill cultivation;
(3) seed tank culture: 5% the inoculum size of the nutrient solution in the triangular flask with the substratum gross weight is inoculated in the polyethylene bottle of the substratum that 4500ml is housed, leaves standstill 35 ℃ of temperature and carried out first order seed in 24 hours and cultivate, fill bottle; And then 5% the inoculum size of first order seed nutrient solution by the substratum gross weight be inoculated in the plastics pot of 500L, carry out secondary seed by the first order seed culture condition and cultivate;
(4) fermentor cultivation: the nutrient solution in the seeding tank inserted with 5% inoculum size of substratum gross weight be equipped with in the 1000L fermentor tank of substratum, temperature leaves standstill for 35 ℃ cultivated 24 hours; When the fermented liquid final pH is 4.0, the nectar degree stops fermentation when being 5,000,000,000/ml.
The pH of the substratum in described step (3) and the step (4) is 6.5; Described substratum is the component that comprises following weight part in every premium on currency: molasses 5g, yeast extract paste 5g, corn steep liquor 1g, KH
2PO
40.3g, MgSO
40.5g, NaCl5g, glucose 10g, peptone 1g.
Substratum in described step (1) and the step (2) is improved MRS substratum, and its pH is 6.5; Described MRS substratum is the component that comprises following weight part in every premium on currency: peptone 10g, yeast extract paste 5g, glucose 15g, KH
2PO
42g, MgSO
40.5g, MnSO
40.25g, CH
3COONa5g.Described MRS substratum was 121 ℃ of following autoclavings 20 minutes.
Embodiment two
A kind of production method that is used for the lactobacillus micro-ecological preparation of cultivation water regulation may further comprise the steps:
(1) test tube is cultivated: the two strains of lactic acid bacteria bacterial classification is inoculated in respectively in two test tubes that the MRS substratum is housed, cultivates 29 ℃ of temperature-activated; Merge the gained nutrient solution then;
(2) shake-flask culture: the activation culture liquid of bacterial classification 4% inoculum size with the substratum gross weight is inoculated in the triangular flask that the MRS substratum is housed, and 29 ℃ of temperature leave standstill cultivation;
(3) seed tank culture: 4% the inoculum size of the nutrient solution in the triangular flask with the substratum gross weight is inoculated in the polyethylene bottle of the substratum that 4500ml is housed, leaves standstill 30 ℃ of temperature and carried out first order seed in 30 hours and cultivate, fill bottle; And then 4% the inoculum size of first order seed nutrient solution by the substratum gross weight be inoculated in the plastics pot of 500L, carry out secondary seed by the first order seed culture condition and cultivate;
(4) fermentor cultivation: with the nutrient solution in the seeding tank with 4% inoculum size of substratum gross weight insert the 1000L fermentor tank of substratum is housed after, stir, be divided in the plastics pot of 5 narrow-mouth bottles, leave standstill 30 ℃ of temperature and cultivated 30 hours; When the fermented liquid final pH is 3.5, the nectar degree stops fermentation when being 5,000,000,000/ml.
The pH of the substratum in described step (3) and the step (4) is 5.5; Described substratum is the component that comprises following weight part in every premium on currency: molasses 3g, yeast extract paste 3g, corn steep liquor 0.8g, KH
2PO
40.1g, MgSO
40.3g, NaCl3g, glucose 8g, peptone 0.8g.
Substratum in described step (1) and the step (2) is improved MRS substratum, and its pH is 5.5; Described MRS substratum is the component that comprises following weight part in every premium on currency: peptone 8g, yeast extract paste 3g, glucose 13g, KH
2PO
41.5g, MgSO
40.3g, MnSO
40.20g, CH
3COONa3g.Described MRS substratum was 121 ℃ of following autoclavings 20 minutes.
Embodiment three
A kind of production method that is used for the lactobacillus micro-ecological preparation of cultivation water regulation may further comprise the steps:
(1) test tube is cultivated: the two strains of lactic acid bacteria bacterial classification is inoculated in respectively in two test tubes that the MRS substratum is housed, cultivates 30 ℃ of temperature-activated; Merge the gained nutrient solution then;
(2) shake-flask culture: the activation culture liquid of bacterial classification 3% inoculum size with the substratum gross weight is inoculated in the triangular flask that the MRS substratum is housed, and 30 ℃ of temperature leave standstill cultivation;
(3) seed tank culture: 3% the inoculum size of the nutrient solution in the triangular flask with the substratum gross weight is inoculated in the polyethylene bottle of the substratum that 4500ml is housed, leaves standstill 25 ℃ of temperature and carried out first order seed in 36 hours and cultivate, fill bottle; And then 3% the inoculum size of first order seed nutrient solution by the substratum gross weight be inoculated in the plastics pot of 500L, carry out secondary seed by the first order seed culture condition and cultivate;
(4) fermentor cultivation: the nutrient solution in the seeding tank inserted with 3% inoculum size of substratum gross weight be equipped with in the 1000L fermentor tank of substratum, temperature leaves standstill for 25 ℃ cultivated 36 hours; When the fermented liquid final pH is 3.0, the nectar degree stops fermentation when being 5,000,000,000/ml.
The pH of the substratum in described step (3) and the step (4) is 6; Described substratum is the component that comprises following weight part in every premium on currency: molasses 4g, yeast extract paste 4g, corn steep liquor 0.9g, KH
2PO
40.2g, MgSO
40.4g, NaCl4g, glucose 9g, peptone 0.9g.
Substratum in described step (1) and the step (2) is improved MRS substratum, and its pH is 6; Described MRS substratum is the component that comprises following weight part in every premium on currency: peptone 9g, yeast extract paste 4g, glucose 14g, KH
2PO
41.8g, MgSO
40.4g, MnSO
40.2g, CH
3COONa4g.Described MRS substratum was 121 ℃ of following autoclavings 20 minutes.
Product lactobacillus micro-ecological preparation provided by the invention is applied to aquaculture water, and every 1000ml microbial inoculum is converted 10 kilograms in the pool water during use, stirs, and splashing in the full pool, according to the water quality situation, added microbial inoculum once in per 10～15 days.Using lactobacillus micro-ecological preparation can significance improve aquaculture water water quality, promotes useful algae grows, regulation and control algae phase, and water colour is fresh and alive, and is not dim, improves the aquaculture water dissolved oxygen, and degradable organic pollutant reduces objectionable impurities NO
2-N, NH
4The amount of-N improves and cultures success ratio.
Claims (4)
1. production method that is used for the lactobacillus micro-ecological preparation of cultivation water regulation may further comprise the steps:
(1) test tube is cultivated: lactic acid bacteria culturers is inoculated in the test tube that substratum is housed, cultivates 28～30 ℃ of temperature-activated;
(2) shake-flask culture: the activation culture liquid of bacterial classification is inoculated in the triangular flask that substratum is housed, and 28～30 ℃ of temperature leave standstill cultivation;
(3) seed tank culture: the nutrient solution in the triangular flask is inoculated in the seeding tank that substratum is housed, and temperature leaves standstill cultivation for 25～35 ℃;
(4) fermentor cultivation: the nutrient solution in the seeding tank is inoculated in the fermentor tank that substratum is housed, and temperature leaves standstill for 25～35 ℃ cultivated 24～36 hours; When the fermented liquid final pH is 3.0～4.0, the nectar degree stops fermentation when being 5,000,000,000/ml;
Substratum in described step (1) and the step (2) is improved MRS substratum, and its pH is 5.5～6.5; Described improved MRS substratum is the component that comprises following weight part in 1000 weight parts waters: peptone 8～10, yeast extract paste 3～5, glucose 13～15, KH
2PO
41.5～2, MgSO
40.3～0.5, MnSO
40.20～0.25, CH
3COONa 3～5; Described MRS substratum was 121 ℃ of following autoclavings 20 minutes;
The pH of the substratum in described step (3) and the step (4) is 5.5～6.5; Described substratum is the component that comprises following weight part in 1000 weight parts waters: molasses 3～5, yeast extract paste 3～5, corn steep liquor 0.8～1, KH
2PO
40.1～0.3, MgSO
40.3～0.5, NaCl 3～5, glucose 8～10, peptone 0.8～1.
2. the production method that is used for the lactobacillus micro-ecological preparation of cultivation water regulation according to claim 1, it is characterized in that the inoculum size of the nutrient solution in the activation culture liquid of described step (2), step (3) and the step (4) is 3～5% of a substratum gross weight.
3. the production method that is used for the lactobacillus micro-ecological preparation of cultivation water regulation according to claim 1 is characterized in that, the described seed culture of step (3) is to carry out first order seed earlier to cultivate, and then carries out secondary seed and cultivate.
4. the production method that is used for the lactobacillus micro-ecological preparation of cultivation water regulation according to claim 1, it is characterized in that, nutrient solution in described step (4) seeding tank inserts in the fermentor tank that substratum is housed with 3～5% inoculum size after, leave standstill cultivation after stirring, or be divided in the plastics pot of narrow-mouth bottle and leave standstill cultivation.
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| CN101353636B true CN101353636B (en) | 2010-09-01 |
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Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101940167A (en) * | 2009-06-15 | 2011-01-12 | 江声 | Aquaculture method by using symbiotic bacteria |
| US8398856B2 (en) | 2011-06-10 | 2013-03-19 | West Bengal University Of Technology | Self-sustained microbial detoxification of soluble sulfate from environmental effluent |
| JP5793617B2 (en) * | 2011-06-10 | 2015-10-14 | ウエスト ベンガル ユニバーシティ オブ テクノロジー | Microbial self-sustained detoxification of soluble sulfate from environmental effluents |
| CN102524117B (en) * | 2011-12-12 | 2013-07-03 | 中国水产科学研究院黑龙江水产研究所 | Method for improving water quality of fresh water culture pond and promoting fish growth |
| CN102992493B (en) * | 2012-10-22 | 2018-08-21 | 北京盛拓达生物技术有限公司 | A kind of improved combination microorganism renovation agent for cultivation water environment regulation and control |
| CN103146611A (en) * | 2013-03-12 | 2013-06-12 | 通威股份有限公司 | Preparation method for composite-type lactobacillus microecological preparation |
| CN103405479B (en) * | 2013-06-26 | 2016-02-24 | 浙江省农业科学院 | A kind of Lemonal for prevention and control cultivation later stage shrimp pond vibrios |
| CN107777780A (en) * | 2016-08-30 | 2018-03-09 | 张继红 | Water process preparation, preparation method and applications |
| CN106508761A (en) * | 2016-10-19 | 2017-03-22 | 南通维尔思水产科技有限公司 | Method for culturing penaeus vannamei in cement pool by means of microecological preparations |
| CN108048381A (en) * | 2018-02-28 | 2018-05-18 | 苏州昆蓝生物科技有限公司 | A kind of preparation method of high-activity lactic acid bacteria solid formulation |
| CN109652333A (en) * | 2019-01-04 | 2019-04-19 | 广州市欣海利生生物科技有限公司 | A kind of photosynthetic bacteria microbial inoculum cultural method and photosynthetic bacteria microbial inoculum |
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Inventor after: Yang Yingying Inventor after: Li Zhuojia Inventor after: Liang Xiaohua Inventor after: Hong Minna Inventor after: Chen Yongqing Inventor after: Wen Guoliang Inventor after: Cao Yucheng Inventor before: Yang Yingying Inventor before: Liang Xiaohua Inventor before: Hong Minna Inventor before: Chen Yongqing Inventor before: Li Jiazhuo Inventor before: Wen Guoliang Inventor before: Cao Yucheng |
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Free format text: CORRECT: INVENTOR; FROM: YANG YINGYING LIANG XIAOHUA HONG MINNUO CHEN YONGQING LI JIAZHUO WEN GUOGUO CAO YUCHENG TO: YANG YINGYING LI ZHUOJIA LIANG XIAOHUA HONG MINNUO CHEN YONGQING WEN GUOGUO CAO YUCHENG |
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Granted publication date: 20100901 Termination date: 20110911 |