CN104004679A - Feeding lactic acid bacteria fermentation culture medium, and preparation method and application thereof - Google Patents
Feeding lactic acid bacteria fermentation culture medium, and preparation method and application thereof Download PDFInfo
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- CN104004679A CN104004679A CN201410199313.4A CN201410199313A CN104004679A CN 104004679 A CN104004679 A CN 104004679A CN 201410199313 A CN201410199313 A CN 201410199313A CN 104004679 A CN104004679 A CN 104004679A
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Abstract
The invention provides a feeding lactic acid bacteria fermentation culture medium. The formula comprises 37-43 g/L of glucose, 47-55 g/L of a corn steep liquor dried powder, 42-48 g/L of fish meal, 0-2 g/L of K2HPO4.3H2O, 8-12 g/L of sodium acetate, 2-4 g/L of triammonium citrate, 0-0.5 g/L of MgSO4.7H2O, 0.25-0.5 g/L of MnSO4.H2O and 1-2 ml/L of Twain-80, wherein the pH value is 7.0-7.2, and water is used for preparation. The invention also provides a preparation method and an application of the fermentation culture medium. The cheap and easily available corn steep liquor dried powder and the fish meal are adopted to replace a nitrogen source ingredient in an MRS culture medium, the culture medium components are optimized, and the price of the obtained culture medium is only 40% of that of the MRS culture medium; through optimization of the fermentation initial pH value and the fermentation temperature, and simultaneously through adopting of a way of flowing addition of an NaOH solution to relieve feedback inhibition of lactic acid, the bacteria density is significantly improved compared with that of normal fermentation, the number of viable bacteria in a fermentation liquid is up to 10<10> CFU/ml or more, high density fermentation of feeding lactic acid bacteria is realized, and the specific production rate and the product quality are improved.
Description
Technical field
The present invention relates to microorganism fermentation field, specifically, relate to a kind of feeding lactobacillus fermention medium, its preparation method and application.
Background technology
For a long time, in feed, add microbiotic animal husbandry development has been played to huge pushing effect, but along with the widespread use of feeding antibiotic, cause the untoward reactions such as microbial population of animal intestinal tract imbalance, superinfection and bacterial drug resistance also to highlight gradually, bring serious threat to aquaculture and fodder industry.Therefore, exploitation has no side effect, the Substitutes For Antibiotic of pollution-free and noresidue, significant to livestock industry.Milk-acid bacteria, as dominant microflora in swine alimentary canal, has at home and abroad been subject to paying close attention to widely and studying.Research shows, at least has hundreds of milk-acid bacteria in chitling road, and the general character ratio of the interior normal microflora of its energy regulating intestinal canal, maintains the balance of intestinal ecosystem.
Because milk-acid bacteria can not be synthesized much amino acid, VITAMIN and the relative growth factor, therefore cause its nutritional requirement and culture condition complexity, nitrogenous source composition in MRS culture medium raw material costliness, particularly substratum at present used, its price accounts for the more than 80% of MRS substratum total price.
Milk-acid bacteria can produce the organic acid meta-bolites such as lactic acid in process of growth, makes the constantly decline of pH value of substratum, thereby the growth of lactic acid bacteria inhibiting causes the even thalline death that declines of lactic-acid bacteria cells activity.Therefore, adopt traditional production technique, strain quality can not be guaranteed, and production cost is high.
High density fermentation refers to the culture technique that application is certain or installs the fermentation density that improves thalline, cell density is significantly improved compared with cellar culture method.Utilize high-density culture technology to realize the high density fermentation of microorganism, can improve the concentration of thalline in unit volume nutrient solution, improve specific production rate, reduce production costs.
Summary of the invention
The object of this invention is to provide a kind of feeding lactobacillus fermention medium cheap and easy to get.
Another object of the present invention is to provide the preparation method of described fermention medium.
A further object of the present invention is to provide the application of described fermention medium in feeding lactobacillus high density fermentation.
In order to realize the object of the invention, a kind of feeding lactobacillus fermention medium of the present invention, the formula of described fermention medium is: glucose 37～43g/L, Dried Corn Steep Liquor Powder 47～55g/L, fish meal 42～48g/L, K
2hPO
43H
2o0～2g/L, sodium acetate 8～12g/L, Triammonium citrate 2～4g/L, MgSO
47H
2o0～0.5g/L, MnSO
4h
2o0.25～0.5g/L and tween-80 1～2ml/L, pH value 7.0～7.2, prepares with water.
Wherein, the preparation method of described Dried Corn Steep Liquor Powder is: by corn making beating, and the spray-dried Dried Corn Steep Liquor Powder that obtains of gained corn steep liquor, in dry powder, protein content is not less than 42%.
The preparation method of described fish meal is: the flesh of fish through deoiling, dehydration and grinding and processing obtain fish meal, in fish meal, protein content is not less than 55%.
In view of above-mentioned two kinds of cost of material are cheap, there are the potentiality of nitrogenous source composition in alternative MRS.
The present invention also provides the preparation method of described fermention medium, comprises the following steps:
1) take respectively the each component except Dried Corn Steep Liquor Powder and fish meal, dissolve with distilled water;
2) take Dried Corn Steep Liquor Powder and fish meal adds step 1) in the solution of preparation, supply distilled water, filtered through gauze;
(3) adjust pH to 7.0～7.2;
(4) 121 DEG C, sterilizing 15min, is cooled to room temperature, to obtain final product.
The present invention further provides the application of described fermention medium in feeding lactobacillus high density fermentation.
The feeding lactobacillus relating in the present invention includes but not limited to plant lactobacillus etc.For example, plant lactobacillus BM-1 can produce bacteriocin, and pathogenic bacterium Listeria monocytogenes is had to stronger restraining effect, has application potential in fodder industry.
Particularly, the fermentation process in high density of described plant lactobacillus comprises the following steps:
(1) plant lactobacillus of freezing is inoculated in MRS liquid nutrient medium, in 37 DEG C of cultivations 12 hours, activates continuously after three generations, as seed liquor;
(2) initial pH value of the above-mentioned fermention medium preparing is adjusted to 6.2～6.6;
(3) by the inoculum size of 2v/v%, the seed liquor of step (1) is inoculated in to shaking culture in the fermention medium of step (2);
(4) in the time that the pH of fermention medium value is down to 6.0, stream adds 6mol/L NaOH solution makes the pH value stabilization of substratum 5.8～6.0, and stuffiness in fermenting process, in 30～34 DEG C, rotating speed 100rpm condition bottom fermentation 14～16 hours, in the plant lactobacillus fermented liquid of acquisition, viable count reaches 10
10more than CFU/ml.
The present invention adopts Dried Corn Steep Liquor Powder cheap and easy to get and fish meal to substitute the nitrogenous source composition in MRS substratum, and the component of substratum is optimized, the price of gained substratum is only 40% of MRS substratum price, by the optimization to fermentation initial pH value and leavening temperature, the mode that simultaneously adopts stream to add NaOH solution is removed the feedback inhibition of lactic acid, cell density is significantly improved compared with common fermentation, make in fermented liquid viable count up to 10
10more than CFU/ml, realize the high density fermentation of feeding lactobacillus (for example plant lactobacillus), improved specific production rate and quality product.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, the raw materials used commercial goods that is.
The Dried Corn Steep Liquor Powder using in following examples is purchased from Shanghai Xi Wang β-amylose company limited, and its preparation method is: by corn making beating, and the spray-dried Dried Corn Steep Liquor Powder that obtains of gained corn steep liquor, in dry powder, protein content is not less than 42%.
The fish meal using in following examples is purchased from Rongcheng Li Tai fish meal company limited, and its preparation method is: the flesh of fish through deoiling, dehydration and grinding and processing obtain fish meal, in fish meal, protein content is not less than 55%.
The plant lactobacillus BM-1 relating in following examples separates from Fujian dried yellow croaker goods, is provided with metabolic engineering laboratory by China Agricultural University's Food science and the milk-acid bacteria heredity of nutrition engineering college.
High density fermentation culture medium and the fermentation process of embodiment 1 plant lactobacillus BM-1
1, the component of plant lactobacillus BM-1 fermention medium and content
Component and the content of high density fermentation culture medium are as follows: glucose 40g/L, Dried Corn Steep Liquor Powder 52g/L, fish meal 45g/L, K
2hPO
43H
2o1g/L, sodium acetate 10g/L, Triammonium citrate 4g/L, MgSO
47H
2o0.25g/L, MnSO
4h
2o0.375g/L and tween-80 1ml/L, pH value 7.0～7.2, prepares with water.
2, the compound method of substratum
(1) take respectively the each component except Dried Corn Steep Liquor Powder and fish meal by content, dissolve with distilled water;
(2) take Dried Corn Steep Liquor Powder and fish meal and add the solution in step (1), supply distilled water, filtered through gauze;
(3) regulate pH value to 7.0～7.2;
(4) 121 DEG C, sterilizing 15min.
3, fermentation process in high density
(1) plant lactobacillus of freezing is inoculated in MRS liquid nutrient medium, in 37 DEG C of cultivations 12 hours, activates continuously after three generations, as seed liquor;
(2) regulating the initial pH value of the fermention medium of preparation is 6.6;
(3) seed liquor of step (1) is inoculated in to shaking culture in the substratum of step (2) with the inoculum size of 2v/v%;
(4) in the time that the pH of nutrient solution value is down to 6.0, stream adds 6mol/L NaOH solution makes the pH value stabilization of nutrient solution 5.8～6.0, thereby removes the feedback inhibition of lactic acid.Stuffiness in fermenting process, in 30 DEG C, rotating speed 100rpm condition bottom fermentation 14～16 hours, obtains plant lactobacillus BM-1 viable count and reaches 1.32 × 10
10fermented liquid more than CFU/ml.
High density fermentation culture medium and the fermentation process of embodiment 2 plant lactobacillus BM-1
1, the component of plant lactobacillus BM-1 fermention medium and content
Component and the content thereof of high density fermentation culture medium are as follows: glucose 43g/L, Dried Corn Steep Liquor Powder 47g/L, fish meal 42g/L, sodium acetate 10g/L, Triammonium citrate 2g/L, MnSO
4h
2o0.375g/L and tween-80 1ml/L, pH value 7.0～7.2, prepares with water.
2, the compound method of substratum
(1) take respectively the each component except Dried Corn Steep Liquor Powder and fish meal by content, dissolve with distilled water;
(2) take Dried Corn Steep Liquor Powder and fish meal and add the solution in step (1), supply distilled water, filtered through gauze;
(3) regulate pH value to 7.0～7.2;
(4) 121 DEG C, sterilizing 15min.
3, fermentation process in high density
(1) plant lactobacillus of freezing is inoculated in MRS liquid nutrient medium, in 37 DEG C of cultivations 12 hours, activates continuously after three generations, as seed liquor;
(2) regulating the initial pH value of the fermention medium of preparation is 6.2;
(3) seed liquor of step (1) is inoculated in to shaking culture in the substratum of step (2) with the inoculum size of 2v/v%;
(4) in the time that the pH of nutrient solution value is down to 6.0, stream adds 6mol/L NaOH solution makes the pH value stabilization of nutrient solution 5.8～6.0, thereby removes the feedback inhibition of lactic acid.Stuffiness in fermenting process, in 34 DEG C, rotating speed 100rpm condition bottom fermentation 14～16 hours, obtains plant lactobacillus BM-1 viable count and reaches 1.05 × 10
10fermented liquid more than CFU/ml.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (7)
1. a feeding lactobacillus fermention medium, is characterized in that, the formula of described fermention medium is: glucose 37～43g/L, Dried Corn Steep Liquor Powder 47～55g/L, fish meal 42～48g/L, K
2hPO
43H
2o0～2g/L, sodium acetate 8～12g/L, Triammonium citrate 2～4g/L, MgSO
47H
2o0～0.5g/L, MnSO
4h
2o0.25～0.5g/L and tween-80 1～2ml/L, pH value 7.0～7.2, prepares with water.
2. fermention medium according to claim 1, is characterized in that, the preparation method of described Dried Corn Steep Liquor Powder is: by corn making beating, and the spray-dried Dried Corn Steep Liquor Powder that obtains of gained corn steep liquor, in dry powder, protein content is not less than 42%.
3. fermention medium according to claim 1, is characterized in that, the preparation method of described fish meal is: the flesh of fish through deoiling, dehydration and grinding and processing obtain fish meal, in fish meal, protein content is not less than 55%.
4. the preparation method of fermention medium described in claim 1-3 any one, is characterized in that, comprises the following steps:
1) take respectively the each component except Dried Corn Steep Liquor Powder and fish meal, dissolve with distilled water;
2) take Dried Corn Steep Liquor Powder and fish meal adds step 1) in the solution of preparation, supply distilled water, filtered through gauze;
(3) adjust pH to 7.0～7.2;
(4) 121 DEG C, sterilizing 15min, is cooled to room temperature, to obtain final product.
5. the application of fermention medium in feeding lactobacillus high density fermentation described in claim 1-3 any one.
6. application according to claim 5, is characterized in that, described feeding lactobacillus includes but not limited to plant lactobacillus.
7. application according to claim 6, is characterized in that, fermentation process comprises the following steps:
(1) plant lactobacillus of freezing is inoculated in MRS liquid nutrient medium, in 37 DEG C of cultivations 12 hours, activates continuously after three generations, as seed liquor;
(2) initial pH value of fermention medium described in claim 1-3 any one is adjusted to 6.2～6.6;
(3) by the inoculum size of 2v/v%, the seed liquor of step (1) is inoculated in to shaking culture in the fermention medium of step (2);
(4) in the time that the pH of fermention medium value is down to 6.0, stream adds 6mol/L NaOH solution makes the pH value stabilization of substratum 5.8～6.0, and stuffiness in fermenting process, in 30～34 DEG C, rotating speed 100rpm condition bottom fermentation 14～16 hours, in the plant lactobacillus fermented liquid of acquisition, viable count reaches 10
10more than CFU/ml.
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Cited By (7)
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| CN104450506A (en) * | 2014-11-20 | 2015-03-25 | 成都瑞琦科技实业股份有限公司 | Anaerobic blood culture medium, culture bottle and preparation method of culture bottle |
| CN104774795A (en) * | 2015-04-28 | 2015-07-15 | 深圳比利美英伟营养饲料有限公司 | Lactobacillus plantarum fluid nutrient medium and lactobacillus plantarum fermental cultivation method |
| CN105087420A (en) * | 2015-03-30 | 2015-11-25 | 北京伟嘉人生物技术有限公司 | High-density fermentation medium and fermentation technology for forage-use enterococcus faecium |
| CN105925515A (en) * | 2016-07-14 | 2016-09-07 | 贝嘉美（天津）生物技术研发有限公司 | Method for culturing lactic acid bacteria by using candied jujube wastewater as medium |
| CN107937322A (en) * | 2018-01-09 | 2018-04-20 | 倪同艳 | A kind of alkaline medium and its application |
| CN109082397A (en) * | 2018-09-26 | 2018-12-25 | 黄拥亮 | The cultural method of feeding lactobacillus |
| CN109837222A (en) * | 2017-11-28 | 2019-06-04 | 广州中国科学院先进技术研究所 | A kind of cultural method of Bacillus acidi lactici culture medium and preparation method thereof and Bacillus acidi lactici |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104450506A (en) * | 2014-11-20 | 2015-03-25 | 成都瑞琦科技实业股份有限公司 | Anaerobic blood culture medium, culture bottle and preparation method of culture bottle |
| CN105087420A (en) * | 2015-03-30 | 2015-11-25 | 北京伟嘉人生物技术有限公司 | High-density fermentation medium and fermentation technology for forage-use enterococcus faecium |
| CN104774795A (en) * | 2015-04-28 | 2015-07-15 | 深圳比利美英伟营养饲料有限公司 | Lactobacillus plantarum fluid nutrient medium and lactobacillus plantarum fermental cultivation method |
| CN105925515A (en) * | 2016-07-14 | 2016-09-07 | 贝嘉美（天津）生物技术研发有限公司 | Method for culturing lactic acid bacteria by using candied jujube wastewater as medium |
| CN109837222A (en) * | 2017-11-28 | 2019-06-04 | 广州中国科学院先进技术研究所 | A kind of cultural method of Bacillus acidi lactici culture medium and preparation method thereof and Bacillus acidi lactici |
| CN107937322A (en) * | 2018-01-09 | 2018-04-20 | 倪同艳 | A kind of alkaline medium and its application |
| CN107937322B (en) * | 2018-01-09 | 2021-01-26 | 倪同艳 | Alkaline culture medium and application thereof |
| CN109082397A (en) * | 2018-09-26 | 2018-12-25 | 黄拥亮 | The cultural method of feeding lactobacillus |
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