CN103283950A - Pediococcus acidilactici viable preparation and preparation method thereof - Google Patents
Pediococcus acidilactici viable preparation and preparation method thereof Download PDFInfo
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- CN103283950A CN103283950A CN2013102344061A CN201310234406A CN103283950A CN 103283950 A CN103283950 A CN 103283950A CN 2013102344061 A CN2013102344061 A CN 2013102344061A CN 201310234406 A CN201310234406 A CN 201310234406A CN 103283950 A CN103283950 A CN 103283950A
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- pediococcus acidilactici
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Abstract
The invention relates to a production method of a Pediococcus acidilactici viable preparation, particularly a production method of a Pediococcus acidilactici viable preparation by liquid anaerobic fermentation, belonging to the technical field of microorganisms. The method comprises the following steps: inoculating Pediococcus acidilactici used as a zymophyte strain into an optimized liquid nutrient medium, culturing at 37-40 DEG C for 16-18 hours, putting in a tank, determining the viable count, adsorbing the fermentation liquid with plant starch, drying, pulverizing, and packaging to obtain the stable-activity Pediococcus acidilactici preparation. The invention can implement high-density fermentation of Pediococcus acidilactici in a common fermentation tank, is simple to operate, has the advantages of stable effect and low energy consumption, does not use any centrifugal machine, freeze drier or the like, lowers the production cost, enhances the production efficiency, and thus, is easy to popularize and use.
Description
Technical field
The present invention relates to the microbial fermentation engineering field, specifically a kind of fluid nutrient medium prepares the method for probiotic feed additive through the Pediococcus acidilactici anaerobic fermentation.
Background technology
Pediococcus acidilactici (
Pediococcusacidilactici) gram-positive bacteria, do not move amphimicrobian.The cell spherical shape, two planes alternately divide formation tetrad shape at the right angle, and general cell is given birth in pairs, and single survivor is rare, does not become catenation.Bacterium colony is little on the MRS culture medium, is white in color.Growth-gen along agar puncture line is thread.The catalase feminine gender is not produced cytochromes.
The physiological property following points of Pediococcus acidilactici: produce acid, regulate gastrointestinal bacterial flora, keep the intestinal microecology balance.In animal body pathogenic microorganism there is antagonism, can suppresses pathogenic microorganism competitively, strengthen immunity function of animal, produce useful metabolite, activate the activity of acid protease, participate in the metabolism of body, prevent that harmful substance from producing.
Pediococcus acidilactici is a kind of novel fodder additive, and its purposes is identical with the probiotics of China with classification, belongs to a kind of of probiotic, but again the function that possesses health-care agent than probiotic more, especially to people and the nontoxic no pathogenicity of animal, acid there is very strong resistance, can waltzs through stomach, and in enteron aisle, breed rapidly, reconcile gut flora, secretion class sterilization factors, stimulating immune system improves immunocompetence, promoting the full group of animal healthy water equality aspect, is the green additive in the feed industry.
Pediococcus acidilactici is fermented through high density liquid, obtain the fermentation culture of high cell density, after plant amylum absorption, dry, pulverize, the Pediococcus acidilactici probiotics of making is applied to livestock-raising, can regulate the microbial population of animal intestinal tract balance, strengthen immunity of organisms, improve resistance against diseases.
Mainly there is following problem and shortage in the fermentation mode of Pediococcus acidilactici in the past: (1) is adopted anaerobic jar or is kept its anaerobic environment by lasting feeding carbon dioxide or nitrogen and carry out, and raw material can not be reused, and operation is complicated, and cost is higher; (2) post processing of liquid fermentation production needs high speed freezing centrifuge to carry out centrifugal and frozen drying, and complex process is with high costs, yields poorly; (3) the viable bacteria density of tunning is not high enough.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of Pediococcus acidilactici active bacteria formulation is with above shortcomings on the preparation method who overcomes existing Pediococcus acidilactici active bacteria formulation.
The technical scheme that realizes the object of the invention is as follows:
A kind of preparation method of Pediococcus acidilactici active bacteria formulation may further comprise the steps:
(1) preparation culture medium;
(2) culture medium of jar in the dissolving is loaded onto the pH electrode and is adjusted the pH value, in the fermentation tank of then culture medium being packed into;
(3) to the sterilization of heating of the culture medium in the fermentation tank;
(4) inoculating lactic acid sheet coccus kind daughter bacteria: add inorganic salt solution in fermentation tank cooling back, add atoleine then and insert kind of a daughter bacteria;
(5) temperature controlled fermentation; Control pH value and temperature are at the state bottom fermentation of insulation;
(6) put jar and measure zymotic fluid bacterium number;
(7) with corn flour, wheat flour or soy meal and composition adsorbs zymotic fluid thereof, dry and pulverizing makes stable Pediococcus acidilactici active bacteria formulation with the mixture after the absorption.
Compared with prior art, technical advantage of the present invention:
(1) directly utilize the common fermentation jar to produce, simple to operate, steady quality, with low cost, be easy to industrialization production;
(2) product cost reduces with the fermentation pollution risk: the present invention is because the production cycle shortens, and production cost reduces, and simultaneously, the fermentation base-material produces in earlier stage that acid is very fast, pH value descends rapidly, can better suppress living contaminants, and the product risk of failure of fermenting reduces greatly;
(3) adopt anaerobic jar or keep its anaerobic environment and carry out by continuing to feed carbon dioxide or nitrogen, raw material can not be reused, and operation is complicated, and cost is higher; The atoleine that is used for secluding air can repeatedly use repeatedly, greatly reduces production cost;
(4) the present invention adopts the Pediococcus acidilactici liquid fermentation medium through optimizing, and the Pediococcus acidilactici bacterium is counted content and obviously improves, and tunning viable bacteria concentration can reach 1.0 * 10
9More than the CFU/mL;
(5) the present invention adopts plant amylum absorption zymotic fluid, and aftertreatment technology does not need to use high speed freezing centrifuge, and the present invention is adsorbed mixture convection drying and pulverizing with plant amylum, namely makes product after the packing, and aftertreatment technology is simplified greatly, and production cost is lower;
(6) both at home and abroad about the powder Study and manufacture on starter culture, adopt vacuum freeze-drying method mostly, the advantage of the method is somatic cells survival rate height, but complex process, the cost of manufacture height, and the production cost of absorption back seasoning is low, operating procedure is simple, be easy for large-scale industrial production and apply, be suitable for China's Feed Enterprise and under the existing equipment condition, produce Pediococcus acidilactici probiotics product.
The specific embodiment
The technological process of embodiment 1 preparation Pediococcus acidilactici active bacteria formulation may further comprise the steps:
(1) actication of culture:
The present invention adopts Pediococcus acidilactici as fermented bacterium, purchase the microorganism fungus kind preservation administrative center in Guangdong Province, deposit number: GIM1.263, with the Pediococcus acidilactici viable bacteria bacterial classification of-80 ℃ of preservations at MRS culture medium (peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween 80 1.0 mL, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, agar powder 15.0 g, distilled water 1 000 mL, pH 6.2 ~ 6.6, autoclave sterilization) solid rules on the inclined-plane, cultivated general 1 ~ 3 time of actication of culture about 16 hours for 37 ℃;
(2) preparation first order seed:
Pediococcus acidilactici with activation, be inoculated in 20mL MRS liquid (peptone 10.0 g with oese picking one ring, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween 80 1.0 mL, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, distilled water 1 000 mL, pH 6.2 ~ 6.6, autoclave sterilization) test tube in, 37 ℃ of deep layers leave standstill cultivated about 16 hours, bacterium liquid is inoculated in the triangular flask that proper volume MRS culture medium is housed with 3 ~ 5% inoculum concentration, 37 ℃ of deep layers leave standstill cultivate about 16 hours standby; If do not use immediately behind the medium sterilization, then need to use after heating 10min drives oxygen in boiling water bath;
(3) obtaining liq fermentation medium:
The raw material that culture medium comprises and parts by weight are: corn flour 70, wheat flour 20, soya-bean cake powder 10, glucose 15, yeast extract powder 2, peptone 4, raw material is worked as in gram, the corresponding umber in mL of distilled water is 800 ~ 1000, and dissolving adds in the fermentation tank and carries out 121 ℃, time is 20 minutes autoclave sterilization, when fermentation tank is cooled to 35 ~ 45 ℃, add inorganic salts 1.2g/1000mL base-material, inorganic salts: CaCl
20.02%, MgSO
47H
2O 0.04%, MnSO
4H
2O 0.04%,, for avoiding forming precipitation, in adition process, stir, adding atoleine to thickness then is 2cm;
(4) liquid fermentation:
To insert the first order seed bacterium according to 5% inoculum concentration through the liquid fermentation medium of sterilization treatment, stir, adding atoleine to thickness then is 2cm, keeping temperature is 37 ~ 40 ℃, pH 6.0-6.8, cultivate and put jar in 20 hours, Pediococcus acidilactici viable bacteria culture is carried out colony counting, reach 1.9 * 10 through the viable bacteria bulk concentration that ferments like this
9CFU/mL;
(5) adsorb zymotic fluid with plant amylum: be that the corn flour, wheat flour soya-bean cake powder of 7:2:1 are as the adsorbent of zymotic fluid with the mass ratio, and the zymotic fluid mixture that plant amylum is adsorbed carries out drying, heat under 65 ℃ and handle about 60min, through super-dry, pulverizing and packing, make the microbial forage additive for livestock and poultry cultivation.
What embodiment 2 and above-described embodiment 1 were different is:
(1) culture medium prescription: corn flour 60, wheat flour 30, soya-bean cake powder 10, glucose 10, yeast extract powder 1.5, peptone 3.0, raw material is worked as in gram, and the corresponding umber in mL of distilled water is 800 ~ 1000, dissolving is through 100 ℃, 30min autoclave sterilization, when fermentation tank is cooled to 35 ~ 45 ℃, add inorganic salts 1.2g/1000mL base-material, the composition of inorganic salts: MgSO
47H
2O 0.04%, MnSO
4H
2O 0.04%, for avoiding forming precipitation, stirs in adition process, and adding atoleine thickness then is about 1cm;
(2) temperature controlled fermentation: the inoculum concentration that inserts the Pediococcus acidilactici liquid seeds is 3%, stir, initial pH 6.6, control pH 6.0 ~ 6.8, fermentation temperature control was fermented 18 hours at 37 ~ 40 ℃, put jar, the Pediococcus acidilactici culture is carried out colony counting, is 1.5 * 10 through the viable bacteria bulk concentration that ferments like this
9CFU/mL;
(3) adsorb zymotic fluid with plant amylum: with etc. corn flour and the wheat flour of quality mix, as the adsorbent of zymotic fluid, the mixture of absorption through super-dry, pulverizing and packing, is made the microbial forage additive for livestock and poultry cultivation.
Claims (6)
1. the preparation method of a Pediococcus acidilactici active bacteria formulation may further comprise the steps:
(1) actication of culture:
The present invention adopts Pediococcus acidilactici as fermented bacterium, purchase the microorganism fungus kind preservation administrative center in Guangdong Province, deposit number: GIM1.263, with the Pediococcus acidilactici viable bacteria bacterial classification of-80 ℃ of preservations at MRS culture medium (peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween 80 1.0 mL, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, agar powder 15.0 g, distilled water 1 000 mL, pH 6.2 ~ 6.6, autoclave sterilization) solid rules on the inclined-plane, cultivated general 1 ~ 3 time of actication of culture about 16 hours for 37 ℃;
(2) preparation first order seed:
Pediococcus acidilactici with activation, be inoculated in 20mL MRS liquid (peptone 10.0 g with oese picking one ring, beef extract 10.0 g, yeast extract 5.0 g, diammonium hydrogen citrate 2.0 g, glucose 20.0 g, Tween 80 1.0 mL, sodium acetate 5.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.58 g, manganese sulfate 0.25 g, distilled water 1 000 mL, pH 6.2 ~ 6.6, autoclave sterilization) test tube in, 37 ℃ of deep layers leave standstill cultivated about 16 hours, bacterium liquid is inoculated in the triangular flask that proper volume MRS culture medium is housed with 3 ~ 5% inoculum concentration, 37 ℃ of deep layers leave standstill cultivate about 16 hours standby; If do not use immediately behind the medium sterilization, then need to use after heating 10min drives oxygen in boiling water bath;
(3) obtaining liq fermentation medium:
The raw material that culture medium comprises and parts by weight are: corn flour 40 ~ 70, wheat flour 15 ~ 30, soya-bean cake powder 0 ~ 15, glucose 10 ~ 15, yeast extract powder 1 ~ 3, peptone 2 ~ 4, raw material is worked as in gram, the corresponding umber in mL of distilled water is 800 ~ 1000, and dissolving adds in the fermentation tank and carries out 100 ~ 121 ℃, time is 10 ~ 30 minutes autoclave sterilization, when fermentation tank is cooled to 35 ~ 45 ℃, add inorganic salts 0.7 ~ 1.5g/1000mL base-material, inorganic salts are selected from CaCl
2, MgSO
47H
2O and MnSO
4H
2Among the O two kinds or three kinds for avoiding forming precipitation, stir in adition process, and adding atoleine to thickness then is 1 ~ 2cm;
(4) liquid fermentation:
To insert the first order seed bacterium according to 3 ~ 5% inoculum concentration through the liquid fermentation medium of sterilization treatment, stir, adding atoleine to thickness then is 1 ~ 2cm, keeping temperature is 37 ~ 40 ℃, pH 6.0-6.8, cultivate and put jar in 16 ~ 18 hours, Pediococcus acidilactici viable bacteria culture is carried out colony counting, can reach 1.0 * 10 through the viable bacteria bulk concentration that ferments like this
9More than the CFU/mL;
(5) adsorb zymotic fluid with plant amylum: with corn flour, soya-bean cake powder or wheat flour, perhaps its any combination as the adsorbent of zymotic fluid, and is carried out bake drying with the zymotic fluid mixture of plant amylum absorption, pulverize and packing, make the microbial forage additive for livestock and poultry cultivation.
2. Pediococcus acidilactici active bacteria formulation according to claim 1, it is characterized in that described liquid fermentation medium is: corn flour 40 ~ 70, wheat flour 15 ~ 30, soya-bean cake powder 0 ~ 15, glucose 10 ~ 15, yeast extract powder 1 ~ 3, peptone 2 ~ 4, raw material is worked as in gram, and the corresponding umber in mL of distilled water is 800 ~ 1000, dissolving, add in the fermentation tank and carry out 100 ~ 121 ℃, the time is 10 ~ 30 minutes autoclave sterilization, when fermentation tank is cooled to 35 ~ 45 ℃, add inorganic salts 0.7 ~ 1.5g/1000mL base-material, inorganic salts are selected from CaCl
2, MgSO
47H
2O and MnSO
4H
2Among the O two kinds or three kinds for avoiding forming precipitation, stir in adition process, and adding atoleine to thickness then is 1 ~ 2cm.
3. Pediococcus acidilactici active bacteria formulation according to claim 1, it is characterized in that: cultivation temperature is that temperature is 37 ~ 40 ℃, and pH 6.0 ~ 6.8.
4. Pediococcus acidilactici active bacteria formulation according to claim 1 is characterized in that: at different levels kinds of daughter bacteria inoculum concentrations are 3 ~ 5% inoculum concentration.
5. Pediococcus acidilactici active bacteria formulation according to claim 1, it is characterized in that: used bacterial classification is the Pediococcus acidilactici kind of Pediococcus.
6. Pediococcus acidilactici active bacteria formulation according to claim 1, it is characterized in that: the application as feed addictive is additive for farm animal feed.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450565A (en) * | 2014-11-14 | 2015-03-25 | 湖南农业大学 | Forage pediococcus acidilactici solid raw material fermentation medium and fermentation method |
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| CN1995330A (en) * | 2006-12-26 | 2007-07-11 | 华中农业大学 | Clostridium butyricum active bacteria agent production method |
| CN101050437A (en) * | 2007-03-21 | 2007-10-10 | 华东理工大学 | Method for producing bacterial strain of Pediococcus acidilactici, and bacterin of Pediococcus acidilactici |
| CN102115720A (en) * | 2010-11-30 | 2011-07-06 | 天津大学 | Pediococcus acidilactici strain and method for producing pediocin by using same |
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2013
- 2013-06-14 CN CN2013102344061A patent/CN103283950A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1995330A (en) * | 2006-12-26 | 2007-07-11 | 华中农业大学 | Clostridium butyricum active bacteria agent production method |
| CN101050437A (en) * | 2007-03-21 | 2007-10-10 | 华东理工大学 | Method for producing bacterial strain of Pediococcus acidilactici, and bacterin of Pediococcus acidilactici |
| CN102115720A (en) * | 2010-11-30 | 2011-07-06 | 天津大学 | Pediococcus acidilactici strain and method for producing pediocin by using same |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450565A (en) * | 2014-11-14 | 2015-03-25 | 湖南农业大学 | Forage pediococcus acidilactici solid raw material fermentation medium and fermentation method |
| CN104450565B (en) * | 2014-11-14 | 2016-08-24 | 湖南农业大学 | A kind of feeding addicted to acid micrococcus solid raw material fermentation culture medium and fermentation process |
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Application publication date: 20130911 |