CN102334684B - Preparation method of microbial product with specific function - Google Patents
Preparation method of microbial product with specific function Download PDFInfo
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- CN102334684B CN102334684B CN 201010231320 CN201010231320A CN102334684B CN 102334684 B CN102334684 B CN 102334684B CN 201010231320 CN201010231320 CN 201010231320 CN 201010231320 A CN201010231320 A CN 201010231320A CN 102334684 B CN102334684 B CN 102334684B
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Abstract
The invention relates to a preparation method of a microbial product with specific function. According to the method provided by the invention, high quality Lactobacillus in lactic acid bacteria is used and cultured to obtain high activity lactobacillus fermentation, and a live bacteria additive of which the viable count being as high as 1011/gram is made by the use of vacuum freeze dryer with skim milk as a protective agent. The microbial additive with specific function has advantages of high and strong activity of lactic acid bacteria live bacteria additive obtained from vacuum freeze drying, stable biological function of fermentation product, low cost and convenient operation, and it is applicable to industrial continuous cultivation. The lactic acid bacteria additive can be directly added into food and drink or be used to ferment food and drink. Food or drink obtained by fermentation of the microbial additive with specific function can promote the growth of human-beings and animals, regulate gastrointestinal normal flora, maintain microecological balance, degrade endotoxin, inhibit the growth of intestinal spoilage bacteria, improve the body immunity and simultaneously has an antagonistic effect to pathogenic bacteria such as Shigella flexneri, Salmonella typhi, Bacillus paratyphosus, Campylobacter, staphylococcus and so on.
Description
Technical field: the preparation method who the present invention relates to a kind of microbial product with specific function.
Technical background
It is more and more higher to the requirement of quality of the life and quality of life to enter the 21 century people, and its requirement to nutritional quality is also more and more higher, improves its source of material life and is the food that uses and the quality of drink.Therefore the nutritional quality and the nutritional labeling that improve the food drink are very important for the mankind's health.At present, some food and drink production have realized commercialization, standardization, at present mainly with the food that contains vitamin and drink; The food and the drink that contain mineral matter, the food and the drink that contain Chinese herbal medicine are main, but ubiquitous one to ask questions be that the utilization rate of additive contained in these food and the drink is low, contain side effect, and these add the material great majority all is synthetic, its long-term use enters digestive system and hematological system, namely can harm humans health can work the mischief to environment again.
Summary of the invention: the purpose of this invention is to provide and a kind ofly can promote people and animal intestinal digestive function, reduce drug use, reduce production costs, improve the nutrient contents such as protein of food and drink, the preparation method of the lactic acid bacteria biological additive of vitamin and calcium content in raising food and the drink.Technical solution of the present invention is: a kind of preparation method of microbial product with specific function: 1) (1) under the extramalization environment separating lactic acid lactobacillus to obtain to such an extent that bacterial strain is taked the screening test of extreme environmental conditions: (1) is through 50 degrees centigrade of high temperature resistant tests; (2) meaning of the acid resistance test of PH3: PH3 is that the pH value of adjusting culture medium is 3 test; (3) anti-high salt test; (4) to the demonstration test of the antagonistic experiment antagonistic ability of Salmonella, shigella dysenteriae, typhoid bacillus, paratyphosum Bacterium, campylobacter, grape ball; The Lactobacillus delbrueckii bacterial strain that therefrom selects (delbrueckii subsp.Lactis), lactobacillus casei bacterial strain (paracasei) Lactobacillus brevis (Lactobacillus brevis) are as engineered strain, the picking engineered strain is inoculated into respectively on the mrs slant medium for subsequent use, the structural constituent of mrs culture medium:
Remarks: at 121 ℃ of sterilization 15min, regulate pH6.2～6.4 with pressure cooker;
2) culture medium of an activation culture of preparation, the prescription of its culture medium is as follows: soy peptone 50g, powdered beef 30g, dusty yeast 30g, glucose 200g, lactose 20g, tomato juice 50ml, deionized water 1000ml, above component is dissolved in the deionized water, in the triangular flask with its 2l that packs into, the refrigerator of behind high pressure steam sterilization 20min under 121 ℃ the condition, putting into-4 ℃ is stand-by;
3) activation culture: with the culture medium 20ml of an activation culture, divide and install in the 100ml blake bottle, with 5ml normal saline flushing Lactobacillus delbrueckii (delbrueckii subsp.Lactis), Lactobacillus casei (paracasei) Lactobacillus brevis (Lactobacillus brevis) strain inclined plane, in the culture medium with bacterium liquid transferred species to the time activation culture of washing, be under 36 ± 1 ℃ of conditions in temperature, the static cultivation 16～18h of anaerobism, ph value to be determined shows that an activation culture state reaches requirement when the 3.8-4.5 scope;
4) the preparation secondary enlarges the culture medium of cultivating: soy peptone 200g, raffinose 20g, tomato juice 100ml, sodium chloride 10g, deionized water 2000ml, in deionized water, the capacity of joining is in the small-sized fermentation tank of 4l with above each components dissolved, and high pressure steam sterilization 20min is stand-by under 121 ℃ of conditions;
5) secondary expansion cultivation is inoculated into the zymotic fluid of an activation culture of each bacterial strain in the nutrient solution of secondary expansion cultivation simultaneously, temperature is transferred to 40 ℃ of constant temperature, anaerobism, static cultivation 12-14h, when the ph that treats zymotic fluid reaches 3.8-4.5, show that secondary enlarges cultivation conditions and reaches requirement;
6) preparation freeze drying protectant: skimmed milk 720g, glycerine 30ml, lactose 200g, glucose 200g, deionized water 6000ml, above component is dissolved in the deionized water, installs to after stirring in the triangular flask of 10l, the refrigerator of putting into-4 ℃ behind high pressure steam sterilization 15min under 118 ℃ of conditions is stand-by;
7) collect zymophyte mud: secondary is enlarged cultivate the zymotic fluid that obtains and under aseptic condition, pack in the centrifuge tube of centrifuge, under the 10000rpm/min condition, behind the centrifugal 5min, in aseptic superclean bench, outwell supernatant, resulting dry is bacterium mud, collects in the triangular flask that joins immediately the 10l that freeze drying protectant is housed behind the bacterium mud stand-by;
8) with bacterium mud and the freeze drying protectant mixing collected, wherein the ratio of bacterium mud and freeze drying protectant is 1: 3 after the fermentation;
9) freeze-drying: the bacterium mud that mixes and freeze drying protectant are packed in the freeze-drying bottle, and the freeze-drying bottle changes pre-freeze 4h in-40 ℃ the refrigerator over to, then changes the freeze-drying bottle over to vacuum freeze drier, and freeze-drying 24h namely can obtain viable count and reach 10
11The lactic bacteria additive of/g.
The invention has the advantages that:
Utilize the lactic acid bacteria strains of independent separate to make the fermentation strain of microbe additive, its stable physical-chemical indexes, the biological function stability and safety, effect in enteron aisle is thorough, be easy in enteron aisle, adhere to and bring into play work, and stronger fertility and biologically active are arranged, be convenient to the quality strains that batch production is cultivated.And simple to operate aspect production technology, the low amount of cost can continued operation, and use this microbial product and can promote the people and the animal intestinal digestive function, reduce drug use, reduce production costs, improve the nutrient content of food and drink, the content of vitamin and calcium in raising food and the drink.The preparation method of above-mentioned directed microbial product, its characteristics are that three aspects one are the optimization that bacterial classification is selected; We break through the obtain manner of traditional lactic acid bacteria strains, and separating plant source lactic acid bacteria and advances strict match choosing test to bacterial strain as engineered strain from extreme environment, therefrom obtains to have the bacterial strain of certain resistance and certain biological function as engineered strain; The 2nd, the simple low cost of zymotechnique; Applied culture medium is conventional chemical articles for use and simple to operate during the fermentation, is convenient to batch production and uses; The 3rd, freeze-dry process efficient finally obtains to be fit to lactic acid bacteria strains through our experimental study for many years and can make viable count reach 10 at the freeze drying protectant of vacuum freeze drying
11/ g.
Characteristics of the present invention are to utilize the quality strains in the lactic acid bacteria: Lactobacillus delbrueckii strain (delbrueckii subsp.Lactis), lactobacterium casei strains (paracasei)) to cultivate through once enlarging behind Lactobacillus brevis (Lactobacillus brevis) purifying, secondary enlarges cultivates the viable count 10 that obtains as protective agent process vacuum freeze drying with skimmed milk glycerol-glucose lactose sucrose again
11The directed microbe additive goods of/g.
And lactic acid bacteria is the close relationship of having digested and assimilated in the normal beneficial microbe of people and animal intestinal and the enteron aisle.When keeping the interior beneficial flora of enteron aisle normal, namely can improve food digestion rate and biological value; The control endotoxin; Suppress corrupt bacteria growing in the enteron aisle.The biological rhzomorph that simultaneously probio metabolism produces has antagonism to do for pathogenic bacteria such as shigella dysenteriae, typhoid bacillus, paratyphosum Bacterium, campylobacter, grape ball Lin etc., improves immunity of organisms etc.
Description of drawings:
Fig. 1 is process chart of the present invention
The specific embodiment:
As shown in Figure 1, application and the preparation method of the multi-functional lactic bacteria additive of a kind of orientation: 1) (1) under the extramalization environment separating lactic acid lactobacillus to obtain to such an extent that bacterial strain is taked the screening test of extreme environmental conditions: (1) is through 50 degrees centigrade of high temperature resistant tests; (2) meaning of the acid resistance test of PH3: PH3 is that the pH value of adjusting culture medium is 3 test; (3) anti-high salt test; (4) to the demonstration test of the antagonistic experiment antagonistic ability of Salmonella, shigella dysenteriae, typhoid bacillus, paratyphosum Bacterium, campylobacter, grape ball; The Lactobacillus delbrueckii bacterial strain that therefrom selects (delbrueckii subsp.Lactis), lactobacillus casei bacterial strain (paracasei) Lactobacillus brevis (Lactobacillus brevis) are as engineered strain, the picking engineered strain is inoculated into respectively on the mrs slant medium for subsequent use, the structural constituent of mrs culture medium:
Remarks: at 121 ℃ of sterilization 15min, regulate pH6.2～6.4 with pressure cooker;
2) culture medium of an activation culture of preparation, the prescription of its culture medium is as follows: soy peptone 50g, powdered beef 30g, dusty yeast 30g, glucose 200g, lactose 20g, tomato juice 50ml, deionized water 1000ml, above component is dissolved in the deionized water, in its triangular flask of 21 of packing into, the refrigerator of behind high pressure steam sterilization 20min under 121 ℃ the condition, putting into-4 ℃ is stand-by;
3) activation culture: with the culture medium 20ml of an activation culture, divide and install in the 100ml blake bottle, with 5ml normal saline flushing Lactobacillus delbrueckii (delbrueckii subsp.Lactis), Lactobacillus casei (paracasei) Lactobacillus brevis (Lactobacillus brevis) strain inclined plane, in the culture medium with bacterium liquid transferred species to the time activation culture of washing, be under 36 ± 1 ℃ of conditions in temperature, the static cultivation 16～18h of anaerobism, ph value to be determined shows that an activation culture state reaches requirement when the 3.8-4.5 scope;
4) the preparation secondary enlarges the culture medium of cultivating: soy peptone 200g, raffinose 20g, tomato juice 100ml, sodium chloride 10g, deionized water 2000ml, in deionized water, the capacity of joining is that high pressure steam sterilization 20min is stand-by under 121 ℃ of conditions in 41 the small-sized fermentation tank with above each components dissolved;
5) secondary expansion cultivation is inoculated into the zymotic fluid of an activation culture of each bacterial strain in the nutrient solution of secondary expansion cultivation simultaneously, temperature is transferred to 40 ℃ of constant temperature, anaerobism, static cultivation 12-14h, when the ph that treats zymotic fluid reaches 3.8-4.5, show that secondary enlarges cultivation conditions and reaches requirement;
6) preparation freeze drying protectant: skimmed milk 720g, glycerine 30ml, lactose 200g, glucose 200g, deionized water 6000ml, above component is dissolved in the deionized water, installs to after stirring in the triangular flask of 10l, the refrigerator of putting into-4 ℃ behind high pressure steam sterilization 15min under 118 ℃ of conditions is stand-by;
7) collect zymophyte mud: secondary is enlarged cultivate the zymotic fluid that obtains and under aseptic condition, pack in the centrifuge tube of centrifuge, under the 10000rpm/min condition, behind the centrifugal 5min, in aseptic superclean bench, outwell supernatant, resulting dry is bacterium mud, collects in the triangular flask that joins immediately the 10l that freeze drying protectant is housed behind the bacterium mud stand-by;
8) with bacterium mud and the freeze drying protectant mixing collected, wherein the ratio of bacterium mud and freeze drying protectant is 1: 3 after the fermentation;
9) freeze-drying: the bacterium mud that mixes and freeze drying protectant are packed in the freeze-drying bottle, and the freeze-drying bottle changes pre-freeze 4h in-40 ℃ the refrigerator over to, then changes the freeze-drying bottle over to vacuum freeze drier, and freeze-drying 24h namely can obtain viable count and reach 10
11The lactic bacteria additive of/g.
Claims (1)
1. the preparation method of a microbial product with specific function is characterized in that:
1) under the extramalization environment separating lactic acid lactobacillus to obtain to such an extent that bacterial strain is taked the screening test of extreme environmental conditions: (1) is through 50 degrees centigrade of high temperature resistant tests; (2) meaning of the acid resistance test of PH3: PH3 is that the pH value of adjusting culture medium is 3 test; (3) anti-high salt test; (4) to the demonstration test of the antagonistic experiment antagonistic ability of Salmonella, shigella dysenteriae, typhoid bacillus, paratyphosum Bacterium, campylobacter, grape ball; The Lactobacillus delbrueckii bacterial strain that therefrom selects (delbrueckii subsp.Lactis), lactobacillus casei bacterial strain (paracasei) Lactobacillus brevis (Lactobacillus brevis) are as engineered strain, the picking engineered strain is inoculated into respectively on the mrs slant medium for subsequent use, the structural constituent of mrs culture medium:
Remarks: at 121 ℃ of sterilization 15min, regulate pH6.2～6.4 with pressure cooker;
2) culture medium of an activation culture of preparation, the prescription of its culture medium is as follows: soy peptone 50g, powdered beef 30g, dusty yeast 30g, glucose 200g, lactose 20g, tomato juice 50ml, deionized water 1000ml, above component is dissolved in the deionized water, in its triangular flask of 21 of packing into, the refrigerator of behind high pressure steam sterilization 20min under 121 ℃ the condition, putting into-4 ℃ is stand-by;
3) activation culture: with the culture medium 20ml of an activation culture, divide and install in the 100ml blake bottle, with 5ml normal saline flushing Lactobacillus delbrueckii (delbrueckii subsp.Lactis), Lactobacillus casei (paracasei) Lactobacillus brevis (Lactobacillus brevis) strain inclined plane, in the culture medium with bacterium liquid transferred species to the time activation culture of washing, be under 36 ± 1 ℃ of conditions in temperature, the static cultivation 16～18h of anaerobism, ph value to be determined shows that an activation culture state reaches requirement when the 3.8-4.5 scope;
4) the preparation secondary enlarges the culture medium of cultivating: soy peptone 200g, raffinose 20g, tomato juice 100ml, sodium chloride 10g, deionized water 2000ml, in deionized water, the capacity of joining is in the small-sized fermentation tank of 4l with above each components dissolved, and high pressure steam sterilization 20min is stand-by under 121 ℃ of conditions;
5) secondary expansion cultivation is inoculated into the zymotic fluid of an activation culture of each bacterial strain in the nutrient solution of secondary expansion cultivation simultaneously, temperature is transferred to 40 ℃ of constant temperature, anaerobism, static cultivation 12-14h, when the ph that treats zymotic fluid reaches 3.8-4.5, show that secondary enlarges cultivation conditions and reaches requirement;
6) preparation freeze drying protectant: skimmed milk 720g, glycerine 30ml, lactose 200g, glucose 200g, deionized water 6000ml, above component is dissolved in the deionized water, installs to after stirring in the triangular flask of 10l, the refrigerator of putting into-4 ℃ behind high pressure steam sterilization 15min under 118 ℃ of conditions is stand-by;
7) collect zymophyte mud: secondary is enlarged cultivate the zymotic fluid that obtains and under aseptic condition, pack in the centrifuge tube of centrifuge, under the 10000rpm/min condition, behind the centrifugal 5min, in aseptic superclean bench, outwell supernatant, resulting dry is bacterium mud, collects in the triangular flask that joins immediately the 10l that freeze drying protectant is housed behind the bacterium mud stand-by;
8) with bacterium mud and the freeze drying protectant mixing collected, wherein the ratio of bacterium mud and freeze drying protectant is 1: 3 after the fermentation;
9) freeze-drying: the bacterium mud that mixes and freeze drying protectant are packed in the freeze-drying bottle, and the freeze-drying bottle changes pre-freeze 4h in-40 ℃ the refrigerator over to, then changes the freeze-drying bottle over to vacuum freeze drier, and freeze-drying 24h namely can obtain viable count and reach 10
11The lactic bacteria additive of/g.
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| CN102742728B (en) * | 2012-06-15 | 2015-11-18 | 北京大北农科技集团股份有限公司 | Livestock and poultry composite probiotics feed additive and premix, concentrate feed and batch |
| CN102934735A (en) * | 2012-11-15 | 2013-02-20 | 北京大北农科技集团股份有限公司 | Probiotics leavening agent suitable for fermenting cake dreg type feed raw materials and preparation method of probiotics leavening agent |
| CN106916759A (en) * | 2015-12-24 | 2017-07-04 | 新疆康美瑞农业发展有限公司 | A kind of preparation method of high anti-oxidation lactic acid bacteria powder |
| CN106929421B (en) * | 2017-02-27 | 2020-12-01 | 中国计量科学研究院 | Method for preparing freeze-dried pellets for quantitatively preserving microorganisms |
| CN107190046A (en) * | 2017-05-24 | 2017-09-22 | 中国检验检疫科学研究院 | EHEC proficiency testing sample and preparation method thereof in medicine |
| CN109259210A (en) * | 2018-09-07 | 2019-01-25 | 浙江九田环保科技有限公司 | The preparation method of probiotic microorganisms fermented product |
| CN112852666A (en) * | 2021-01-19 | 2021-05-28 | 新疆河润水业有限责任公司 | Preparation method of microbial agent and microbial fertilizer prepared by adopting microbial agent |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101153315A (en) * | 2007-09-14 | 2008-04-02 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting lactobacillus acidophilus in probiotic bacteria milk product |
| CN101486986A (en) * | 2009-02-12 | 2009-07-22 | 上海谱莱生物技术有限公司 | Preparation of freeze-dried Lactobacillus acidophilus powder |
| CN101715876A (en) * | 2009-04-13 | 2010-06-02 | 四川渴望生物科技有限公司 | Preparation method of high-efficiency compound microorganism soybean meal leaven |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101153315A (en) * | 2007-09-14 | 2008-04-02 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting lactobacillus acidophilus in probiotic bacteria milk product |
| CN101486986A (en) * | 2009-02-12 | 2009-07-22 | 上海谱莱生物技术有限公司 | Preparation of freeze-dried Lactobacillus acidophilus powder |
| CN101715876A (en) * | 2009-04-13 | 2010-06-02 | 四川渴望生物科技有限公司 | Preparation method of high-efficiency compound microorganism soybean meal leaven |
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