CN101412983B - Bacillus coagulans, preparation of high-density cultivated spore preparation, and use thereof - Google Patents
Bacillus coagulans, preparation of high-density cultivated spore preparation, and use thereof Download PDFInfo
- Publication number
- CN101412983B CN101412983B CN2008102355120A CN200810235512A CN101412983B CN 101412983 B CN101412983 B CN 101412983B CN 2008102355120 A CN2008102355120 A CN 2008102355120A CN 200810235512 A CN200810235512 A CN 200810235512A CN 101412983 B CN101412983 B CN 101412983B
- Authority
- CN
- China
- Prior art keywords
- bacillus coagulans
- gemma
- culture
- preparation
- tank
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The present invention discloses a preparation method and application of Bacillus coagulans and a spore preparation cultured in high density, and belongs to the microbe technical field. A Bacillus coagulans strain is classified and named Bacillus coagulans JSSW-LA-07 and is preserved as CGMCC No.2602 in China General Microbiological Culture Collection Center. The Bacillus coagulans strain JSSW-LA-07 can produce spore under an aerobic condition and produce a small amount of L-lactic acid in the metabolism during a course of fermentation. The Bacillus coagulans strain can produce L-lactic acid with a higher purity under an anaerobic condition. The strain does not produce substances which can be harmful to human and cultured animals, has the characteristics of innocuity, on residue, no side effect and the like, has a great significance for production of green and safe livestock, fowls and marine products and improvement of cultivation ecological environment, and can be widely applied in the cultivation production of high yield, high quality, high efficiency, synusiologic and safe livestock, fowls and marine products, and the regulation and control of the ecological environment of cultivation.
Description
Technical field
The preparation of the cultivated spore preparation of a kind of Bacillus coagulans and high-density culture and application thereof belong to microbial technology field.
Background technology
China is a large agricultural country, aquaculture is occupied suitable proportion in whole agricultural, along with the fast development of China's herding, culture fishery, meat and bird output leap into the front ranks of the world, but, but ignored the edible safety problem of livestock and poultry, fishery products when aquaculture economy develops rapidly.The mode of propagating artificially with tuple amount lightweight of high-density intensification in recent years develops, and causes the breeding ecological environment to suffer broken ring, and the cultivated animals disease frequently breaks out; People are long-term a large amount of to use microbiotic and chemical synthetic drug to cause resistance, resistance more and more obvious, the cultivated animals resistance against diseases constantly descends, and drug residue brings secondary pollution directly to influence human immunity and healthy, thereby the edible safety of livestock and poultry, fishery products is threatened, have a strong impact on the Sustainable development of aquaculture.In recent years, a series of great food safety incidents such as Belgian Dioxins incident, Britain's mad cow disease incident, Japanese intestinal bacteria " 0-157 " food poisoning, French Listeria pollution incident, " clenbuterol hydrochloride " incident of China, how precious fish incident have taken place to comprise in the whole world in succession, make food-safety problem cause whole world concern rapidly.Drug residue problems such as paraxin, malachite green, nitrofuran make the fishery products of China meet with " Green Trade Barrier " in the international market repeatly.The alarm bell of food safety has been beaten in Sanlu milk powder trimeric cyanamide pollution incident in the recent period once more, and also eliminating food-safety problem to people's caution on the source of agricultural-food production simultaneously is the task of top priority.Therefore, under the prerequisite that does not influence animal products, output of aquatic products and quality, how to reduce the environmental pollution that livestock and poultry, culture fishery bring, the edible safety that improves livestock and poultry, fishery products has become the focus that people pay close attention to, also is simultaneously the focus of research.Aquaculture, livestock and poultry cultivation industry change to high yield, high-quality, efficient, ecological, safe modern breeding way and evolution in, all is badly in need of substituting or novel green, safe feed additive and water quality, the environment conditioning technology of alternative microbiotic of part and chemicals.
(Microbial ecological agent) carries out the control of livestock and poultry, aquatic animal disease with probiotics, and repair and culture contaminate environment, be a kind of novel method that developed recently gets up.Probiotics is based on the microecological balance theory, theoretical and the microniological proudcts that grow up of the theoretical and little bionomic control of little ecoalimental, it is controls of a kind of active form to the control of Animal diseases, probiotics helps to help probiotic bacterium growth in the body, the breeding of antagonism pathogenic bacterium, the enhancing body immunologic function, the generation of control and minimizing disease, improve the host health level, promote nutrient digestion to absorb, purify the animal body internal and external environment, suppress the growth of putrefactive bacterium, and then reduce ammonia, the generation of objectionable impuritiess such as amine reduces the pollution of foul smell in the environment; In addition, it can be directly used in the livestock and poultry contaminate environment, relies on microorganism to organic Degradation in the feces of livestock and poultry, reduces the pollution of feces of livestock and poultry to environment; Or be applied in the aquaculture water, by to the decomposition of water body objectionable impurities, the inhibition of pathogenic bacteria and the cultivation of water body profitable strain, improve water quality, keep the water ecology balance, thereby keep the coordinated development of livestock and poultry, aquaculture production and environment protection.Probiotics has toxicological harmless, has no side effect, noresidue, pollution-free, have no drug resistance, can improve breeding performonce fo animals, therefore many advantages such as improve food conversion ratio are subject to people's attention just day by day, its bright prospects are mathematical.
At present, probiotics has been widely used in livestock and poultry, the culture fishery, to the R and D of probiotics, has entered climax both at home and abroad, and has formed powerful industry, and annual earns quite a handsome income.There is the scholar to point out " epoch after the microbiotic will be the epoch of probiotics ".As microorganism fodder additive, the lactic acid bacteria class probiotic bacterium is present China Ministry of Agriculture, the quantity of government permission use both at home and abroad such as U.S. food and FAD is maximum, effect is a class safety microbial strains preferably, has good biological function, it has improves food conversion ratio, improve weightening finish, replenish, regulate, keep microbial balance in the animal intestinal, suppress pathogenic bacteria, enhancing body immunizing power, generation wards off disease, the treatment established condition, reduce mortality ratio, effects such as promoting digestion, also can improve livestock and poultry fishing class final product quality, avoid drug residue and the pollution of using chemicals and microbiotic to cause in a large number, therefore, it has nontoxic, no resistance, noresidue, characteristics such as have no side effect.In addition, milk-acid bacteria can utilize organism to grow, and reduces the biological oxygen demand (BOD) and the chemical oxygen demand of polluted-water, is the important sewage disposal microorganism of a class, can repair the aquaculture water environment.
Because the biological characteristics of bacterial strain self is the key factor that influences lactic acid bacteria class microorganism fodder additive effect, most lactic acid bacteria does not produce gemma, the nutrition thalline is poor to environment resistance, oxytolerant not, thermo-labile, be unfavorable for feed processing and preservation, have a strong impact on its quality and effect, the application in aquaculture and fodder industry is very restricted.Thus, based on above-mentioned background, have adjustment, the promotion growth of participating in microecological balance in the cultivated animals body directly concurrently if can develop, disease preventing and treating, can improve simultaneously the probiotics of breeding environment again, then to reducing the use of microbiotic and chemicals, production pollution-free green livestock and poultry, fishery products, it is significant to improve the culturing economic benefit.
Lactic acid is the organic acid that extensively is present in the organisms such as people, animal, plant and microorganism.Therefore contain a unsymmetrical carbon in its molecular structure, have opticity, can divide three kinds of D (-)-lactic acid, L (+)-lactic acid and DL-lactic acid by its configuration.Human body only contains the L-serum lactic dehydrogenase, therefore can only metabolism utilize L-lactic acid, and too much edible D-lactic acid or DL-lactic acid all can cause human metabolism function's disorder, are detrimental to health, and L-lactic acid is not then added restriction.Therefore, the mankind take the eating options that the probiotic bacterium that can produce L-lactic acid in vivo just becomes a kind of ten minutes green health, and it can reduce digestive tube pH, makes enteron aisle be in sour environment, suppress the pathogenic bacteria growth, and human intestinal health is played a positive role.Mammals all can only metabolism utilize L-lactic acid with human the same, therefore adds the milk-acid bacteria that can produce L-lactic acid in animal body and just have very vast market prospect in feed, and will become the effective way of development non-harmful product.
Summary of the invention
The object of the present invention is to provide a kind of high-density cultivation method and application thereof of Bacillus coagulans gemma, under aerobic conditions, can produce gemma, the small amount of L-lactic acid of metabolism output during the fermentation at Bacillus coagulans under this cultural method; Leave standstill in anaerobism that Bacillus coagulans can the higher L-lactic acid of output purity under the condition.
Technical scheme of the present invention: a bacillus coagulans bacterial strain, its called after of classifying: Bacillus coagulans (Bacillus coagulans) JSSW-LA-07, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center the sixth of the twelve Earthly Branches, preserving number is CGMCC No.2602; This bacterial strain JSSW-LA-07 can produce gemma under aerobic conditions, and the small amount of L-lactic acid of metabolism output during the fermentation; In that leave standstill can the higher L-lactic acid of output purity under the anaerobic condition.
The preparation method of the high-density cultivated spore preparation of described Bacillus coagulans is to finish by following steps:
(1) inclined-plane thalline activation
The freeze-drying preservation bacterial classification of aseptic unlatching bacterial strain JSSW-LA-07, streak inoculation are in wheat bran nutrient agar medium test tube slant, and slant medium (wheat bran nutrient agar) composition is counted with g/L: peptone 10, extractum carnis 3, NaCl5, wheat bran 10, agar 15-20, pH7.0-7.2; Cultivate 24-48h for 30-60 ℃; Line is transferred in wheat bran nutrient agar medium eggplant bottle inclined-plane then, cultivates 24-48h for 30-60 ℃, and microscopy when 90% above thalline forms gemma, is maturation, prepares culture transferring;
(2) shake bottle and seed tank culture
Prepare the aseptic triangular flask of an interior glaze pearl, with sterilized water the ripe bacterium mud on the eggplant bottle inclined-plane is scraped to wash, the triangular flask of packing into, vibrating dispersion bacterium mud, obtain uniform bacteria suspension, bacteria suspension was heated 10 minutes in 80 ℃ of water-baths, and with the inoculum size access 250mL triangular flask of volume ratio 1%-10%, liquid amount is 15mL; Or the 500mL triangular flask, liquid amount is 30mL; Shake-flask culture, or insert seed tank culture, the seeding tank volume is selected 10L, 20L, 50L or 100L for use, and the loading amount coefficient volume ratio of seeding tank is 60%-70%;
Substratum consists of in g/L: wheat bran 5-20, yeast extract paste 5.0-10, bean cake powder 5-10, K
2HPO
43, NaCl 5, MnSO
4H
2O 0.3, and pH 7.0;
Culture condition is: shake a bottle rotating speed 200r/min; Seeding tank mixing speed 〉=150r/min, air flow 1-2m
3/ h, culture temperature is 30-60 ℃, incubation time is 18-48h;
(3) fermentor cultivation
Seed culture fluid inserts fermentor tank with the inoculum size of volume ratio 1%-10%, and fermentor tank is selected the fermentor tank of 1T, 2T or 3T volume for use, and fermentor tank loading amount coefficient volume ratio is 60%-70%;
Fermentation tank culture medium is formed:
Fermention medium is made up of carbon source, nitrogenous source and inorganic salt, in g/L: carbon source 5-20, and nitrogenous source 5-20, K
2HPO
43, NaCl 5, MnSO
4H
2O 0.3, lime carbonate 2-10, pH6.0-8.0;
The fermentor cultivation condition is: mixing speed 〉=150r/min, air flow 1-2m
3/ h, culture temperature is 30-60 ℃, incubation time is 18-48h; In culturing process, add soya-bean oil or bubble enemy carries out froth breaking, prevent to cause living contaminants because of the foam fermentor tank of escaping out according to the surging situation of foamy; Get fermented liquid and carry out microscopy, when the thalline more than 90% has formed gemma, can put a jar end and cultivate; Total count and gemma number are surveyed in sampling;
Viable bacteria and gemma detect with tomato juice agar counting substratum in g/L: yeast extract paste 10, peptone 10, tomato juice 200mL, CaCO
35, agar 15-20, distilled water 800mL, pH7.0-7.2; Record fermented liquid gemma quantity and reach 1 * 10
9More than the cfu/mL;
(4) gemma is collected and the preparation of bacterium powder
It is that the continuous centrifugal machine of 15000r/min carries out centrifugal that nutrient solution is pumped into rotating speed, obtain gemma, gemma is mixed with weight ratio 1:1-5 with dry starch, at 40-50 ℃ of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, adds in batching stone flour, wheat bran or the corn cob meal one or more, make probiotics after the mixing, gemma content reaches 1 * 10
9More than the cfu/mL.
Described preparation method, the carbon source of the aerobic liquid fermenting of its Bacillus coagulans and the selection of nitrogenous source are very extensive, and carbon source is selected one or more in wheat bran, glucose, sucrose, lactose, maltose, fructose, pectinose, primverose, rice candy, Zulkovsky starch, W-Gum, tapioca (flour), wheat starch, sweet potato starch or the yam starch for use; Nitrogenous source is selected one or more in peptone, yeast extract paste, extractum carnis, corn starch, bean cake powder, soybean peptides, cottonseed protein, soybean cake powder or the groundnut meal for use.
Leave standstill anaerobic fermentation L-lactic acid: the Bacillus coagulans gemma is inserted in the following substratum, and the substratum composition is counted with g/L: peptone 5-10, yeast extract paste 5-10, glucose 10-20, extractum carnis 1-3, tween-80 0.5, pH7.0.Culture condition: culture temperature 30-60 ℃, leave standstill anaerobically fermenting and cultivate incubation time 24-72h.
L-lactate detection: fermented liquid is heated to 80-100 ℃ earlier, centrifugal 5 minutes then in 4000rpm/min, get supernatant liquor and survey the L-lactic acid content by the SBA-40C bio-sensing analyser of principle (biological study of Shandong academy of sciences is developed), record the L-lactic acid content and reach more than the 8.0g/L with L-lactic dehydrogenase enzyme process mensuration or with this method.
Beneficial effect of the present invention:
1. Bacillus coagulans belongs to facultative good (detesting) oxygen bacterium, and under aerobic conditions, can ferment forms high density, highly active gemma; Leaving standstill under the oxygen free condition, can ferment produces the L-lactic acid useful to humans and animals.
2. selectable C source in the fermention medium, N source are very extensive, so when applying to produce, can select for use economical and practical raw material as C, N source; And its temperature growth scope is wide, all can grow 30 ℃ of-60 ℃ of scopes, grows under comparatively high temps, has reduced the chance of microbiological contamination, cultivates acquisition gemma concentration for single jar and can reach 1 * 10
9More than the cfu/ml.
3. Bacillus coagulans is a kind of probiotic bacterium of lactic acid bacteria class, what it had a general milk-acid bacteria keeps the intestinal microecology balance, immune stimulatory, improve the body health level, outside the effects such as raising humans and animals digestive function, also have unique biological characteristics such as the not available environment strong stress resistance of ordinary lactic acid bacteria, anti-hydrochloric acid in gastric juice, resist drying, high temperature high voltage resistant, easy storage simultaneously.
4. Bacillus coagulans can be used as the animal probiotics, directly make an addition in fowl poultry kind feed, the aquatic feed, replenish, regulate, keep the balance of microorganism in the animal intestinal, improve the growth and breeding of animal body immunizing power, inhibition unwanted bacteria, and the L-lactic acid that Bacillus coagulans produces under the oxygen free condition in mammalian body can directly be absorbed by body, improve animal immunizing power and resistibility, thereby reduce the use of antibiotic medicine.It also can be applicable to the regulation and control of breeding ecological environment, and by the decomposition to organic pollutant, objectionable impurities in the breeding environment, the inhibition of pathogenic bacteria etc. are carried out little restoration of the ecosystem to breeding environment, thereby improved breeding environment, keep the environmental ecology balance.
5. this bacterium does not produce human body, the deleterious material of cultivated animals, characteristics such as have nontoxic, noresidue, have no side effect, and it is to the protection of environment and administer the positive effect of also having played simultaneously.Therefore, it is to producing livestock and poultry, the fishery products of green, safety, and it is all significant to improve the breeding ecological environment, is widely used in the regulation and control of high yield, high-quality, efficient, ecological, safe livestock and poultry, aquaculture production and breeding ecological environment.
The biological material specimens preservation
One bacillus coagulans bacterial strain, its called after of classifying: Bacillus coagulans (Bacillus coagulans) JSSW-LA-07, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center the sixth of the twelve Earthly Branches, preservation date on July 28th, 2008, preserving number is CGMCC No.2602.
Embodiment
Embodiment 1: the cultivation of high-density Bacillus coagulans gemma
1, the activation of thalline
The freeze-drying lactobacillus of aseptic unlatching Bacillus coagulans JSSW-LA-07, streak inoculation is in test tube wheat bran nutrient agar medium inclined-plane, and slant medium consists of: peptone 10g/L, extractum carnis 3g/L, NaCl 5g/L, wheat bran 10g/L, agar 15-20g/L, distilled water 1L, pH 7.0-7.2.Cultivated 20-24 hour for 40 ℃, line is transferred in wheat bran nutrient agar medium eggplant bottle inclined-plane then, cultivates 20-24 hour for 40 ℃, and microscopy when 90% above thalline forms gemma, is maturation, can prepare culture transferring.
2,100L seed tank culture
Prepare the aseptic triangular flask of an interior glaze pearl, with sterilized water the ripe bacterium mud on the eggplant bottle inclined-plane is scraped and washed, the triangular flask of packing into, vibrating dispersion bacterium mud obtains uniform bacteria suspension.Bacteria suspension was heated 10 minutes in 80 ℃ of water-baths, and with the inoculum size access 100L seeding tank of volume ratio 2%-10%, the loading amount coefficient of seeding tank is 60%-70% (v/v), i.e. the canned substratum 60-70L of 100L seed.
Seed culture medium consists of: wheat bran 20g/L, yeast extract paste 5g/L, bean cake powder 10g/L, K
2HPO
43g/L, NaCl 5g/L, MnSO
4H
2O 0.3g/L, pH7.0.
The seed tank culture condition is: mixing speed 200r/min, air flow 1-2m
3/ h, culture temperature is 40 ℃, incubation time is 20-24h.
3,1T fermentor cultivation
Seed culture fluid inserts the 1T fermentor tank with the inoculum size of volume ratio 5%-10%, and fermentor tank loading amount coefficient is 60%-70% (v/v), i.e. the 1T canned substratum 600-700L that ferments.
Fermention medium consists of: wheat bran 20g/L, yeast extract paste 5g/L, bean cake powder 10g/L, K
2HPO
43g/L, NaCl 5g/L, MnSO
4H
2O 0.3g/L, lime carbonate 5g/L, pH 7.0.
The fermentor cultivation condition is: mixing speed 200r/min, air flow 1.5m
3/ h, culture temperature is 45 ℃, incubation time is 48h.In culturing process, add soya-bean oil or bubble enemy carries out froth breaking, prevent to cause living contaminants because of the foam fermentor tank of escaping out according to the surging situation of foamy; Get fermented liquid and carry out microscopy, when the thalline more than 90% has formed gemma, can put a jar end and cultivate; Total count and gemma number are surveyed in sampling;
Tomato juice agar counting substratum: yeast extract paste 10g/L, peptone 10g/L, tomato juice 200mL, distilled water 800mL, agar 15-20g/L, CaCO
35g/L, pH7.0-7.2.
After the fermentation ends, recording the fermented liquid total count is 6.0 * 10
9CFU/mL, the gemma number reaches 5.8 * 10
9CFU/mL, gemma rate 96.7%.It is 3.0g/L that SBA bio-sensing analyser records the L-concentration of lactic acid.
Embodiment 2: Bacillus coagulans leaves standstill to cultivate and produces L-lactic acid
1, standing for fermentation is cultivated
The Bacillus coagulans gemma is inserted in the following substratum, and substratum consists of: peptone 5g/L, yeast extract 5g/L, glucose 10g/L, extractum carnis 1g/L, tween-80 0.5g/L, distilled water 1000mL, pH7.0.
Culture condition: culture temperature: 45 ℃, incubation time: 72h leaves standstill anaerobism and cultivates.
2, L-lactate detection
Fermented liquid is heated to 80-100 ℃ earlier, centrifugal 5 minutes then in 4000rpm/min, get supernatant liquor and survey the L-lactic acid content with SBA-40C bio-sensing analyser, recording the L-lactic acid content is 8.5g/L.
Embodiment 3: make probiotics
1, the cultural method of Bacillus coagulans gemma is described identical with embodiment 1
2, the preparation of Bacillus coagulans bacterium powder
Bacillus coagulans wet thallus and dry starch 1 with high-density pure culture collection: 1-5 mixes, dry 20-24h under 40-50 ℃, pulverizer is pulverized, and crosses 40 mesh sieves, with the gemma number in the dry starch adjustment bacterium powder, make gemma number 〉=5 * 10 of Bacillus coagulans in the bacterium powder at last
9Individual/gram.
3, finished product preparation
After getting the auxiliary material stone flour and crossing 40 mesh sieves, mix, mix thoroughly, make Bacillus coagulans gemma sum 〉=1 * 10 in the finished product with feed mixing machine with Bacillus coagulans bacterium powder
9Individual/gram.Finished product adopts aluminium foil packed, every packed amount 1kg, and 15 bags is 1 packed in cases, carton material adopts fluting board.
Embodiment 4: the application example 1 of Bacillus coagulans preparation
Get cultivating pool water, in breadboard pond, carry out the test that the Bacillus coagulans formulation products purifies water.The experimental water pond is a ceramic tile paster cement pit, water body volume 0.05m
325 ℃ of water temperatures, aeration are after 20 hours, and interpolation ammonium chloride, potassium nitrite are adjusted initial ammonia nitrogen and nitrite concentration.Initial ammonia nitrogen concentration is 1.20mg/L, nitrite concentration 0.50mg/L, chemical oxygen demand COD is 12.36mg/L, test is divided into control group and test group, every group 3 parallel, addition by 0.3ppm adds each test group with the Bacillus coagulans preparation, detects the change of water quality of test group and control group after 5 days respectively, and it is as follows that each index of water quality changes mean value:
Test group: ammonia-nitrogen content is 0.23mg/L, and nitrite content is 0mg/L, and COD is 6.92mg/L.
Control group: ammonia-nitrogen content 1.26mg/L, nitrite content 0.68mg/L, COD are 14.22mg/L.
Therefore, Bacillus coagulans JSSW-LA-07 can obviously reduce harmful substance contents such as ammonia nitrogen in the aquaculture water, nitrite, improves culture-pool water quality, can be used as water quality cleansing agent and is applied in the aquaculture.
Embodiment 5: the application example 2 of Bacillus coagulans preparation
300 of the near 1 age in days Bi Dexun chickens of test and Selection birth heavy phase, test is divided into control group and test group, and 3 every group are parallel, support respectively at same breed inner greenhouse ground face is flat.Control group, the basal diet of only feeding does not add the Bacillus coagulans preparation, and test group is added the Bacillus coagulans preparation of weight ratio 0.5% in basal diet.Each group all weighs up birth weight before opening food, and birth back 24h opens food, will feed behind Bacillus coagulans preparation and the abundant mixing of feed.Trial period is 49d, weighs weekly 1 time, and gets on an empty stomach intestinal contents, after its dilution, is seeded in coating process and carries out microbial culture on the SS nutrient agar, carries out enumeration after 37 ℃ of aerobics are cultivated 24h.During to 7 ages in week, test-results is as follows:
Control group: average weight gain 2058g, material anharmonic ratio 2.20, survival rate 94.67%, Salmonellas positive rate 29.60%
Test group: average weight gain 2385g, material anharmonic ratio 1.90, survival rate 97.33%, Salmonellas positive rate 23.50%
Test group and control group compare, and the test group average weight gain improves 15.89% than control group, and through one-way analysis of variance, difference is (P＜0.01) extremely significantly; Test group reduces extremely remarkable than control group material anharmonic ratio; From the survival rate situation, test group improves than control group survival rate; Test group reduces by 20.61% than control group Salmonellas positive rate.Above result shows, adds the Bacillus coagulans preparation in the chicken feed growing of chicken benefited, and it is improved food conversion ratio improving weightening finish, reduces the material anharmonic ratio, improves survival rate, and it is obvious to strengthen resistance against diseases and immunizing power aspect effect.Therefore, this Bacillus coagulans preparation can substitute microbiotic or antimicrobial drug uses as growth stimulant.
Claims (3)
1. a bacillus coagulans bacterial strain, its called after of classifying: Bacillus coagulans (Bacillus coagulans) JSSW-LA-07, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number is CGMCC No.2602; This bacterial strain JSSW-LA-07 can produce gemma under aerobic conditions, and the small amount of L-lactic acid of metabolism output during the fermentation; In that leave standstill can the higher L-lactic acid of output purity under the anaerobic condition.
2. the preparation method of the high-density cultivated spore preparation of the described Bacillus coagulans of claim 1 is characterized in that finishing by following steps:
(1) inclined-plane thalline activation
The freeze-drying preservation bacterial classification of aseptic unlatching bacterial strain JSSW-LA-07, streak inoculation are in wheat bran nutrient agar medium test tube slant, and the slant medium composition is counted with g/L: peptone 10, and extractum carnis 3, NaCl 5, wheat bran 10, agar 15-20, pH 7.0-7.2; Cultivate 24-48h for 30-60 ℃; Line is transferred in wheat bran nutrient agar medium eggplant bottle inclined-plane then, cultivates 24-48h for 30-60 ℃, and microscopy when 90% above thalline forms gemma, is maturation, prepares culture transferring;
(2) shake bottle and seed tank culture
Prepare the aseptic triangular flask of an interior glaze pearl, with sterilized water the ripe bacterium mud on the eggplant bottle inclined-plane is scraped to wash, the triangular flask of packing into, vibrating dispersion bacterium mud, obtain uniform bacteria suspension, bacteria suspension was heated 10 minutes in 80 ℃ of water-baths, and with the inoculum size access 250mL triangular flask of volume ratio 1%-10%, liquid amount is 15mL; Or the 500mL triangular flask, liquid amount is 30mL; Shake-flask culture, or insert seed tank culture, the seeding tank volume is selected 10L, 20L, 50L or 100L for use, and the loading amount coefficient volume ratio of seeding tank is 60%-70%;
Substratum consists of in g/L: wheat bran 5-20, yeast extract paste 5.0-10, bean cake powder 5-10, K
2HPO
43, NaCl5, MnSO
4H
2O 0.3, and pH 7.0;
Culture condition is: shake a bottle rotating speed 200r/min; Seeding tank mixing speed 〉=150r/min, air flow 1-2m
3/ h, culture temperature is 30-60 ℃, incubation time is 18-48h;
(3) fermentor cultivation
Seed culture fluid inserts fermentor tank with the inoculum size of volume ratio 1%-10%, and fermentor tank is selected the fermentor tank of 1T, 2T or 3T volume for use, and fermentor tank loading amount coefficient volume ratio is 60%-70%;
Fermentation tank culture medium is formed:
Fermention medium is made up of carbon source, nitrogenous source and inorganic salt, in g/L: carbon source 5-20, and nitrogenous source 5-20, K
2HPO
43, NaCl 5, MnSO
4H
2O 0.3, lime carbonate 2-10, pH 6.0-8.0;
The fermentor cultivation condition is: mixing speed 〉=150r/min, air flow 1-2m
3/ h, culture temperature is 30-60 ℃, incubation time is 18-48h; In culturing process, add soya-bean oil or bubble enemy carries out froth breaking, prevent to cause living contaminants because of the foam fermentor tank of escaping out according to the surging situation of foamy; Get fermented liquid and carry out microscopy, when the thalline more than 90% has formed gemma, can put a jar end and cultivate; Total count and gemma number are surveyed in sampling;
Viable bacteria and gemma detect with tomato juice agar counting substratum in g/L: yeast extract paste 10, peptone 10, tomato juice 200mL, CaCO
35, agar 15-20, distilled water 800mL, pH 7.0-7.2; Record fermented liquid gemma quantity and reach 1 * 10
9More than the cfu/mL;
(4) gemma is collected and the preparation of bacterium powder
It is that the continuous centrifugal machine of 15000r/min carries out centrifugal that nutrient solution is pumped into rotating speed, obtain gemma, gemma is mixed with weight ratio 1:1-5 with dry starch, at 40-50 ℃ of dry 20-24h, pulverizer is pulverized, and crosses 40 mesh sieves, adds in batching stone flour, wheat bran or the corn cob meal one or more, make probiotics after the mixing, gemma content reaches 1 * 10
9More than the cfu/mL.
3. preparation method according to claim 2, the selection that it is characterized in that the carbon source of Bacillus coagulans liquid fermenting and nitrogenous source is very extensive, and carbon source is selected one or more in wheat bran, glucose, sucrose, lactose, maltose, fructose, pectinose, primverose, rice candy, Zulkovsky starch, W-Gum, tapioca (flour), wheat starch, sweet potato starch or the yam starch for use; Nitrogenous source is selected one or more in peptone, yeast extract paste, extractum carnis, corn starch, bean cake powder, soybean peptides, cottonseed protein, soybean cake powder or the groundnut meal for use.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102355120A CN101412983B (en) | 2008-11-20 | 2008-11-20 | Bacillus coagulans, preparation of high-density cultivated spore preparation, and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102355120A CN101412983B (en) | 2008-11-20 | 2008-11-20 | Bacillus coagulans, preparation of high-density cultivated spore preparation, and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101412983A CN101412983A (en) | 2009-04-22 |
| CN101412983B true CN101412983B (en) | 2011-01-26 |
Family
ID=40593723
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008102355120A Active CN101412983B (en) | 2008-11-20 | 2008-11-20 | Bacillus coagulans, preparation of high-density cultivated spore preparation, and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101412983B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018163094A1 (en) * | 2017-03-08 | 2018-09-13 | Symbiosis International University | A method of inducing sporulation in bacillus coagulans |
Families Citing this family (23)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851594A (en) * | 2010-03-09 | 2010-10-06 | 成都趋元科技有限责任公司 | Liquid fermentation culturing method of bacillus strain |
| JP5892580B2 (en) * | 2011-04-14 | 2016-03-23 | 国立大学法人 琉球大学 | Method for producing novel lactic acid bacteria and L-lactic acid, and food and medicine containing lactic acid bacteria |
| CN102329005B (en) * | 2011-06-20 | 2013-04-24 | 大连金砣水产食品有限公司 | Preparation method of microbial preparation for aquaculture |
| CN102492641A (en) * | 2011-12-16 | 2012-06-13 | 上海绿乐生物科技有限公司 | Fermentation culture method of Bacillus vallismortis and production method of microbial fertilizer |
| CN103589654A (en) * | 2012-08-14 | 2014-02-19 | 湖南普菲克生物科技有限公司 | Bacillus coagulans strain and fast identification of anaerobic metabolism and lactic acid production of the strain in animal digestive tract hindgut segment simulator |
| CN103289912A (en) * | 2012-10-23 | 2013-09-11 | 广州格拉姆生物科技有限公司 | Solid fermentation method of bacillus coagulans |
| CN102943057B (en) * | 2012-11-16 | 2014-06-25 | 南京工业大学 | Bacillus coagulans and process for high-density fermentation of bacillus coagulans |
| CN103160455B (en) * | 2013-03-27 | 2014-12-10 | 杭州保安康生物技术有限公司 | Preparation method of spore preparation of bacillus coagulans |
| CN104059945B (en) * | 2014-07-10 | 2016-07-27 | 中国科学院亚热带农业生态研究所 | A kind of method of the feeding composite organic acid of sweet potato waste fermenting and producing |
| CN104480052A (en) * | 2015-01-05 | 2015-04-01 | 江苏省苏微微生物研究有限公司 | Composite bacillus preparation containing three strains, preparation method of composite bacillus preparation and application of composite bacillus preparation to ecological breeding |
| CN105524855B (en) * | 2015-02-04 | 2019-03-12 | 江苏省苏微微生物研究有限公司 | A kind of bacillus coagulans and its application with aquatic pathogenic bacterium antagonistic properties |
| CN104974966B (en) * | 2015-07-22 | 2017-12-12 | 江南大学 | A kind of bacillus coagulans and its fermentation process in high density and dry bacterium powder preparation method |
| CN105002123B (en) * | 2015-08-18 | 2018-08-03 | 北京农学院 | A kind of preparation method of the bacillus coagulans Liu-g1 active bacteria formulations of production neutral proteinase |
| CN105112326B (en) * | 2015-08-19 | 2019-08-20 | 华南理工大学 | A kind of bacillus and its high-density cultivation method |
| CN105176870B (en) * | 2015-09-16 | 2019-03-05 | 浙江省农业科学院 | A kind of method that solid fermentation produces feeding bacillus coagulans |
| CN105524866B (en) * | 2016-01-05 | 2019-02-15 | 中牧实业股份有限公司 | Improve the fermentation process of bacillus coagulans bud ratio and Pfansteihl yield |
| CN105695304B (en) * | 2016-03-31 | 2018-02-09 | 昆明三正生物科技（集团）有限公司 | A kind of bacillus coagulans fast culture tank |
| CN108102967A (en) * | 2018-01-11 | 2018-06-01 | 天津生机集团股份有限公司 | One breeder source coagulating bacillus strain and its production spore method |
| CN109207406A (en) * | 2018-10-24 | 2019-01-15 | 广东绿百多生物开发有限公司 | A kind of method and culture medium for improving bacillus coagulans cell and forming gemma |
| CN109593790B (en) * | 2018-11-22 | 2021-10-19 | 广东轻工职业技术学院 | Oil-soluble monascus pigment prepared from alcohol-soluble monascus pigment, and preparation method and application thereof |
| CN110804574A (en) * | 2019-12-06 | 2020-02-18 | 湖北华扬科技发展有限公司 | High-concentration bacillus coagulans liquid fermentation method |
| CN112624350A (en) * | 2020-12-25 | 2021-04-09 | 自然资源部第三海洋研究所 | Microecological preparation for freshwater aquaculture |
| CN114437984A (en) * | 2022-02-18 | 2022-05-06 | 鑫九通科技(武汉)有限公司 | Preparation method of lactic acid-producing bacillus coagulans microbial preparation |
-
2008
- 2008-11-20 CN CN2008102355120A patent/CN101412983B/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018163094A1 (en) * | 2017-03-08 | 2018-09-13 | Symbiosis International University | A method of inducing sporulation in bacillus coagulans |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101412983A (en) | 2009-04-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101412983B (en) | Bacillus coagulans, preparation of high-density cultivated spore preparation, and use thereof | |
| CN103981118B (en) | A kind of bacillus subtilis feed addictive and its preparation method and application | |
| CN100412187C (en) | Microecological preparation-tetragenous viable bacteria preparation and method for preparing same | |
| CN102488084B (en) | Method for preparing protein feed by fermentation on straw and cake using aerobic mixed bacteria | |
| CN101099543B (en) | Aquatic biological feed and preparation method thereof | |
| CN101914460B (en) | Method for fermenting phodotorula benthica culture | |
| CN106260504B (en) | Method for producing microbial fermentation wet feed by using beer yeast paste | |
| CN103184174B (en) | Production method of bacillus subtilis biological agent used for sodium humate-containing feed in medium | |
| CN102220262A (en) | Composite microbiological preparation and preparation method and use thereof | |
| CN101974463B (en) | Lactobacillus reuteri and composite viable bacteria preparation thereof | |
| CN104480052A (en) | Composite bacillus preparation containing three strains, preparation method of composite bacillus preparation and application of composite bacillus preparation to ecological breeding | |
| CN109897803A (en) | A kind of aquatic products probiotics and its preparation method and application | |
| CN102286376A (en) | Microbial inoculum for high-efficiency fermenting bed and preparation method thereof | |
| CN107279462A (en) | The method of chrysanthemum meal fermenting and producing aquatic products livestock and poultry active peptide powder | |
| CN110257292A (en) | A kind of microbial bacterial agent used for aquiculture and preparation method thereof | |
| CN105524855B (en) | A kind of bacillus coagulans and its application with aquatic pathogenic bacterium antagonistic properties | |
| CN107821789A (en) | A kind of biologic ferment for improving fishes and shrimps intestinal health degree and its preparation method and application | |
| CN111593010B (en) | Lactobacillus plantarum and method for producing fermented feed by using same | |
| CN111685228A (en) | Fungus enzyme synergistic fermented feed containing stevia rebaudiana residue and application thereof | |
| CN106508821A (en) | Method for breeding earthworms through combination of wastewater from cassava starch factories with municipal refuse | |
| CN107760612B (en) | Aspergillus niger yy07 strain and application thereof in solid fermentation production of acidic protease for feed | |
| CN102334602B (en) | Preparation method of bioactive composite high-efficiency feed nutrient solution used for livestock and poultry | |
| RU2546880C2 (en) | Method of probiotic feed preparation for farm animals | |
| CN106035988B (en) | Production method of arginine active peptide powder for livestock and poultry | |
| CN1322113C (en) | Sporolactobacillaceae and produced preparation of living fungus thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |