CN101503708A - Cultivation fermentation method of Bacillus coagulans antimycotics active substance - Google Patents
Cultivation fermentation method of Bacillus coagulans antimycotics active substance Download PDFInfo
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- CN101503708A CN101503708A CNA2009100680273A CN200910068027A CN101503708A CN 101503708 A CN101503708 A CN 101503708A CN A2009100680273 A CNA2009100680273 A CN A2009100680273A CN 200910068027 A CN200910068027 A CN 200910068027A CN 101503708 A CN101503708 A CN 101503708A
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Abstract
The invention relates to a culture fermentation method for a Bacillus coagulans antifungal active substance. The method can obtain a bacterium with high biomass and antifungal activity by taking the Bacillus coagulans (CGMCC 1.949) as material, and a fermentation process of the method can be used for preparing fungicides for preventing and controlling plant diseases. The method has the following three advantages: 1, the Bacillus coagulans is a type of probiotics beneficial to human bodies, and the biggest difference between biological fungicides prepared from metabolite of the Bacillus coagulans and the prior fungicides lies in natural property and environment-friendly property thereof; 2, a set of fermentation process established according to the bacterium substitutes soybean cake powder and corn starch for beef extract, peptone and the like greatly reduces production cost; and 3, after the bacterium is fermented, fermentation-product supernatant can be used for preparing the biological fungicides, and thalli obtained through centrifugation can continue to be utilized for producing probiotic products, so that economic benefit is greatly increased.
Description
Technical field
The invention belongs to technical field of bioengineering, relate in particular to the cultivation and fermentation method of a kind of Bacillus coagulans (Bacilluscoagulans) (CGMCC 1.0949) antifungus active substance.
Background technology
Worldwide, agriculture production every year, due to illness the Chinese caterpillar fungus evil brought about great losses, and wherein accounted for 80% by fungus-caused disease.For a long time, people mainly adopt chemical pesticide to control the disease pest and weed of farm crop to guarantee stable yields and the high yield of farm crop.But a large amount of uses of chemical pesticide have brought pollution not only for soil, water body and atmosphere; And chemical pesticide is residual in the agricultural byproducts, with the healthy and existence of direct harm humans.Simultaneously, because the use for a long time, in a large number and repeatedly of chemical pesticide, increasing plant pathogenic fungi has produced resistance to chemosynthesis anti-mycotic agent and microbiotic.Therefore, anti-mycotic agent development of new, natural is very necessary.
Bacillus coagulans (Bacillus coagulans) claims lactic acid bacillus in the past, is a kind ofly can carry out the lactic acid fermented genus bacillus of homotype.This bacterium also has following good characteristic except that the general advantage with ordinary lactic acid bacteria: (1) facultative aerobic microbiological is easy to cultivate; (2) owing to form gemma, the keeping quality of viable bacteria product is good, can overcome the existing keeping quality of ordinary lactic acid bacteria product poor, prolong the shortcoming that number of viable seriously descends degradation to be difficult to overcome with the shelf time; (3) high temperature resistant, stomach juice-resistant; (5) can in enteron aisle, breed.This bacterium is mainly as probiotic bacterium, to the remarkable effect that has of animal disease resistance and reduction animal dead rate at present.About the research of this bacterium resisting pathogenic microbes, reported that this bacterium is inhibited to enteron aisle putrefactive bacteriums such as intestinal bacteria, Shigellaes.But do not see that this bacterium is to the inhibited research of fungi report with utilize this bacterium to produce the report of the biological pesticide of anti-Plant diseases fungi.
In the substratum of milk-acid bacterias such as cultivation Bacillus coagulans, nitrogenous source commonly used is peptone, soy peptone, Tryptones, yeast extract/powder and extractum carnis, because these material costs are higher, therefore, if can adopt inexpensive nitrogenous source class material to replace above material, then can reduce the cost that milk-acid bacteria such as Bacillus coagulans produces probiotic composition and production desirable metabolites, especially concerning big production, its meaning is more important.
We find that Bacillus coagulans can produce multiple meta-bolites, these meta-bolitess not only have the number of chemical structure, and in agricultural and foodstuffs industry, have wide biological activity, as stronger antibacterial vigor, for the exploitation of natural biological agricultural chemicals provides new resource.At present, Bacillus coagulans is produced the correlation technique of application of the fermentation condition of antifungus active substance and fermented product and the document or the patent of method do not appeared in the newspapers.
Summary of the invention
The objective of the invention is with Bacillus coagulans CGMCC 1.0949 is material, provides a kind of Bacillus coagulans (Bacillus coagulans) to produce the cultivation and fermentation method of antifungus active substance.
Technical scheme of the present invention is:
A kind of cultivation and fermentation method of Bacillus coagulans antimycotics active substance, the step of cultivation and fermentation method is:
(1) inclined-plane seed culture: get ring bacterium mud streak inoculation on nutrition nutrient agar substratum, cultivate 20h down in 40 ℃;
(2) seed shake-flask culture: get a ring inclined-plane access and be equipped with in the 250ml triangular flask of 25ml MRS substratum, 40 ℃, 140r/min cultivates 20h, becomes primary seed solution;
(3) shake flask fermentation is cultivated: with 5% inoculum size primary seed solution is inserted and be equipped with in the 500ml triangular flask of 120ml fermention medium, 40 ℃, 180r/min cultivates 18h, becomes secondary seed solution;
(4) fermentor cultivation: secondary seed solution is equipped with in the automatic fermentor tank of fermention medium with the access of 3-6% inoculum size, and main control parameters is as follows between yeast phase: dissolved oxygen and rotating speed: mixing speed 100-400r/min, and the control dissolved oxygen is 30%～40%; Temperature: 40 ℃, cultivate 48h, can obtain fermented product, fermented product is filtered, remove cell and promptly obtain fermented liquid.
And described Bacillus coagulans (Bacillus coagulans) is preserved in Chinese common micro-organisms preservation administrative center CGMCC, deposit number 1.949.
And, described nutrition nutrient agar substratum: peptone 10.0g, extractum carnis 3.0g, NaCl5.0g, agar 15.0g, water 1.0L, pH7.0; Described MRS substratum: peptone 10g, extractum carnis 10g, yeast extract paste 5g, tween-80 1ml, Triammonium citrate 2g, K
2HPO
42g, glucose 20g, sodium acetate 5g, MgSO
47H
2O 0.1g, MnSO
40.05g, water 1.0L, pH7.0; Described fermention medium: soybean cake powder 10.0g, corn starch 10.0g, glucose 6.0g, MgSO
47H
2O 1.0g, K
2HPO
4, MnSO
450ppm, water 1.0L, pH7.0.
Advantage of the present invention and positively effect are:
1. Bacillus coagulans involved in the present invention is the probiotic bacterium of a class to the human body beneficial, and the biological bactericide that its meta-bolites is made is its natural sex and the feature of environmental protection with the maximum difference of sterilant in the past.
2. the present invention is directed in the cover zymotechnique that this bacterium sets up and utilize soybean cake powder and corn starch to replace extractum carnis, peptone etc., greatly reduce production cost.
3. product bacterium of the present invention by fermentation after, fermentation-product supernatant can be used for preparing biological mycocide, the thalline of centrifugal acquisition can continue utilize to produce probiotic composition, has improved economic interests greatly.
4. technology of the present invention can stably obtain the Bacillus coagulans fermented product, and resulting bacterium has higher biomass and anti-microbial activity, can be used for preparing the controlling plant diseases sterilant, and its production cost is relatively low, is suitable for producing in batches.
Embodiment
The present invention is further described below in conjunction with embodiment; Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
Bacillus coagulans involved in the present invention (Bacillus coagulans) is preserved in Chinese common micro-organisms preservation administrative center (CGMCC), and deposit number is 1.949.
Embodiment 1:
A kind of cultivation and fermentation method of Bacillus coagulans antimycotics active substance, step is:
(1) inclined-plane seed culture: get ring bacterium mud streak inoculation on nutrition nutrient agar substratum, cultivate 20h down in 40 ℃;
(2) seed shake-flask culture: get a ring inclined-plane access and be equipped with in the 250ml triangular flask of 25ml MRS substratum, 40 ℃, 140r/min cultivates 20h, becomes primary seed solution;
(3) shake flask fermentation is cultivated: with 5% inoculum size primary seed solution is inserted and be equipped with in the 500ml triangular flask of 120ml fermention medium, 40 ℃, 180r/min cultivates 18h, becomes secondary seed solution;
(4) 5L fermentor cultivation: secondary seed solution is equipped with in the automatic fermentor tank of 5L of 3.5L fermention medium with the access of 5% inoculum size, and setting liquid amount is 70%, and initial mixing speed is 100r/min, and air flow is 1:1, and decide dissolved oxygen this moment is 100%; Reduce to 30% when following when dissolved oxygen, improve rotating speed with the control dissolved oxygen more than 30%, temperature is cultivated 48h for 40 ℃, can obtain fermented product, and fermented product is filtered, and removes cell, can obtain fermented liquid.
The formation of each substratum is in the present embodiment:
Nutrition nutrient agar substratum: peptone 10.0g, extractum carnis 3.0g, NaCl 5.0g, agar 15.0g, water 1.0L, pH7.0;
MRS substratum: peptone 10g, extractum carnis 10g, yeast extract paste 5g, tween-80 1ml, Triammonium citrate 2g, K
2HPO
42g, glucose 20g, sodium acetate 5g, MgSO
47H
2O 0.1g, MnSO
40.05g, water 1.0L, pH7.0;
Fermention medium: soybean cake powder 10.0g, corn starch 10.0g, glucose 6.0g, MgSO
47H
2O1.0g, K
2HPO
4, MnSO
450ppm, water 1.0L, pH7.0.
Embodiment 2:
(1) inclined-plane seed culture: get ring bacterium mud streak inoculation on nutrition nutrient agar substratum, cultivate 20h down in 40 ℃;
(2) seed shake-flask culture: get a ring inclined-plane access and be equipped with in the 250ml triangular flask of 25mlMRS substratum, 40 ℃, 140r/min cultivates 20h, becomes primary seed solution;
(3) shake flask fermentation is cultivated: with 5% inoculum size primary seed solution is inserted and be equipped with in the 500ml triangular flask of 120ml fermention medium, 40 ℃, 180r/min cultivates 18h, becomes secondary seed solution;
(4) 30L fermentor cultivation: secondary seed solution is inserted in the automatic fermentor tank of 30L that the 21L fermention medium is housed (liquid amount is 70%) with 3.6% inoculum size, and initial mixing speed is 100r/min, and air flow is 1:0.3, and decide dissolved oxygen this moment is 100%.Reduce to 30% when following when dissolved oxygen, improve rotating speed with the control dissolved oxygen more than 30%, temperature is cultivated 48h for 40 ℃, can obtain fermented product, and fermented product is filtered, and removes cell and promptly obtains fermented liquid.
The formation of each substratum is identical with embodiment 1 in the present embodiment.
Narrate stripped bacteriostatic test of the present invention (growth velocity inhibition method) below, the application of Bacillus coagulans fermented liquid is to carry out on the basis of embodiment 1,2:
1, with the fermented liquid that obtains in the technology further at 14000r/min, 4 ℃ of centrifugal 10min, collect supernatant liquor.
2, adding the 1ml supernatant liquor in the 9ml PDA substratum that melts, the 10ml substratum that obtains is poured in the sterilization culture dish of diameter 9.0cm, make flat board, is contrast with the PDA that adds aseptic culture medium.From cultivating the plant pathogenic fungi colony edge of 3～6d, be cut into the bacterium piece of diameter 5mm with punch tool, place dull and stereotyped central authorities, cultivate 6d in 24 ℃ of incubators, measure the pathogenic bacteria colony diameter;
3, from following table, can see: Bacillus coagulans fermented product supernatant liquor 100～200ml (fermented liquid)/L to tomato early the mycelial growth of vaccine, tomato gray mould, cucumber phytophthora, muskmelon Fusarium oxysporum, apple anthrax-bacilus stronger restraining effect is arranged, inhibiting rate is 60～100%, and wherein fermented product is the highest to the restraining effect of tomato gray mould mycelial growth.
Claims (3)
1, a kind of cultivation and fermentation method of Bacillus coagulans antimycotics active substance, it is characterized in that: the step of cultivation and fermentation method is:
(1) inclined-plane seed culture: get ring bacterium mud streak inoculation on nutrition nutrient agar substratum, cultivate 20h down in 40 ℃;
(2) seed shake-flask culture: get a ring inclined-plane access and be equipped with in the 250ml triangular flask of 25ml MRS substratum, 40 ℃, 140r/min cultivates 20h, becomes primary seed solution;
(3) shake flask fermentation is cultivated: with 5% inoculum size primary seed solution is inserted and be equipped with in the 500ml triangular flask of 120ml fermention medium, 40 ℃, 180r/min cultivates 18h, becomes secondary seed solution;
(4) fermentor cultivation: secondary seed solution is equipped with in the automatic fermentor tank of fermention medium with the access of 3-6% inoculum size, and main control parameters is as follows between yeast phase: dissolved oxygen and rotating speed: mixing speed 100-400r/min, and the control dissolved oxygen is 30%～40%; Temperature: 40 ℃, cultivate 48h, can obtain fermented product, fermented product is filtered, remove cell and promptly obtain fermented liquid.
2, the cultivation and fermentation method of a kind of Bacillus coagulans antimycotics active substance according to claim 1, it is characterized in that: described Bacillus coagulans (Bacillus coagulans) is preserved in Chinese common micro-organisms preservation administrative center CGMCC, deposit number 1.949.
3, the cultivation and fermentation method of a kind of Bacillus coagulans antimycotics active substance according to claim 1 is characterized in that: described nutrition nutrient agar substratum: peptone 10.0g, extractum carnis 3.0g, NaCl 5.0g, agar 15.0g, water 1.0L, pH 7.0; Described MRS substratum: peptone 10g, extractum carnis 10g, yeast extract paste 5g, tween-80 1ml, Triammonium citrate 2g, K
2HPO
42g, glucose 20g, sodium acetate 5g, MgSO
47H
2O 0.1g, MnSO
40.05g, water 1.0L, pH7.0; Described fermention medium: soybean cake powder 10.0g, corn starch 10.0g, glucose 6.0g, MgSO
47H
2O 1.0g, K
2HPO
4, MnSO
450ppm, water 1.0L, pH7.0.
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| CN2009100680273A CN101503708B (en) | 2009-03-05 | 2009-03-05 | Culture fermentation method of bacillus coagulans antifungal active substance |
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| CN2009100680273A CN101503708B (en) | 2009-03-05 | 2009-03-05 | Culture fermentation method of bacillus coagulans antifungal active substance |
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| CN101503708B CN101503708B (en) | 2012-04-11 |
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| CN101948783A (en) * | 2010-08-31 | 2011-01-19 | 广州广牧丰生物技术有限公司 | Culture medium capable of improving viable count of lactic acid bacillus |
| CN101775360B (en) * | 2009-09-04 | 2012-02-01 | 武汉科诺生物科技股份有限公司 | Preparation method of Bacillus licheniformis and raw materials |
| CN103300126A (en) * | 2013-06-07 | 2013-09-18 | 浙江工商大学 | Application of bacillus coagulans serving as biological preservative in preservation and fresh keeping of pseudosciaena crocea |
| CN105189733A (en) * | 2013-12-24 | 2015-12-23 | 穆罕迈德.玛杰德 | Method for producing partially purified extracellular metabolites from bacillus coagulans and biological applications thereof |
| WO2016150152A1 (en) * | 2015-03-25 | 2016-09-29 | 南京工业大学 | Preparation method for bacillus coagulans bacterial suspension |
| CN106191178A (en) * | 2016-08-25 | 2016-12-07 | 江南大学 | Method for producing bacteriostatic active substance by using bacillus coagulans |
| 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 |
| CN108721336A (en) * | 2018-06-15 | 2018-11-02 | 江苏远山生物技术有限公司 | The preparation method and application of bacillus coagulans and bacillus licheniformis cocktail spray |
| CN110382686A (en) * | 2017-03-08 | 2019-10-25 | 共生国际大学 | The method that inducing spore is formed in bacillus coagulans |
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| CN1472327A (en) * | 2003-06-25 | 2004-02-04 | 中国科学院沈阳应用生态研究所 | Fermentation of antifungal antibiotic produced by oceanic bacillus |
| CN1952117A (en) * | 2006-04-18 | 2007-04-25 | 兰州大学 | Bacillus subtilis strain and application thereof |
| CN100473720C (en) * | 2007-10-18 | 2009-04-01 | 中国科学院微生物研究所 | Method for producing L-lactic acid and coagulate bacillus cereus special for the same |
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2009
- 2009-03-05 CN CN2009100680273A patent/CN101503708B/en not_active Expired - Fee Related
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| CN101775360B (en) * | 2009-09-04 | 2012-02-01 | 武汉科诺生物科技股份有限公司 | Preparation method of Bacillus licheniformis and raw materials |
| CN101948783A (en) * | 2010-08-31 | 2011-01-19 | 广州广牧丰生物技术有限公司 | Culture medium capable of improving viable count of lactic acid bacillus |
| CN101948783B (en) * | 2010-08-31 | 2012-03-21 | 广州广牧丰生物技术有限公司 | Culture medium capable of improving viable count of lactic acid bacillus |
| CN103300126A (en) * | 2013-06-07 | 2013-09-18 | 浙江工商大学 | Application of bacillus coagulans serving as biological preservative in preservation and fresh keeping of pseudosciaena crocea |
| CN103300126B (en) * | 2013-06-07 | 2014-11-26 | 浙江工商大学 | Application of bacillus coagulans serving as biological preservative in preservation and fresh keeping of pseudosciaena crocea |
| CN105189733B (en) * | 2013-12-24 | 2018-09-18 | 穆罕迈德.玛杰德 | Method for producing partially purified extracellular metabolites from bacillus coagulans and biological applications thereof |
| CN105189733A (en) * | 2013-12-24 | 2015-12-23 | 穆罕迈德.玛杰德 | Method for producing partially purified extracellular metabolites from bacillus coagulans and biological applications thereof |
| WO2016150152A1 (en) * | 2015-03-25 | 2016-09-29 | 南京工业大学 | Preparation method for bacillus coagulans bacterial suspension |
| CN106191178A (en) * | 2016-08-25 | 2016-12-07 | 江南大学 | Method for producing bacteriostatic active substance by using bacillus coagulans |
| CN106191178B (en) * | 2016-08-25 | 2019-05-10 | 江南大学 | Method for producing bacteriostatic active substance by using bacillus coagulans |
| CN110382686A (en) * | 2017-03-08 | 2019-10-25 | 共生国际大学 | The method that inducing spore is formed in bacillus coagulans |
| 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 |
| CN108721336A (en) * | 2018-06-15 | 2018-11-02 | 江苏远山生物技术有限公司 | The preparation method and application of bacillus coagulans and bacillus licheniformis cocktail spray |
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| CN101503708B (en) | 2012-04-11 |
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