CN112322531A - Production method and application of high-activity lactobacillus acidophilus freeze-dried powder - Google Patents
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Abstract
The invention relates to the technical field of lactobacillus acidophilus, in particular to a production method and application of high-activity lactobacillus acidophilus freeze-dried powder, wherein the process is simple, the obtained freeze-dried powder has large quantity of viable bacteria and high yield of thalli; the method comprises the following steps: s1, streaking and culturing lactobacillus acidophilus strains; s2, selecting dominant strains and culturing; s3, selecting dominant strains again and continuing culturing; s4, fermentation culture in a fermentation tank: selecting the single colony with a large calcium-dissolving ring on the plate in the step S3, inoculating the single colony into a selenium-rich culture medium, and culturing at 35-38 ℃ and pH of 5.5-6.0 for 48 h; s5, centrifugally concentrating the culture solution, and collecting bacterial sludge; s6, freeze-drying the centrifuged bacterial sludge, adding a protective agent, fully and uniformly mixing, carrying out vacuum freeze-drying, and taking out when the moisture content is extremely low; s7, crushing, sieving and packaging.
Description
Technical Field
The invention relates to the technical field of lactobacillus acidophilus, in particular to a production method and application of high-activity lactobacillus acidophilus freeze-dried powder.
Background
Lactobacillus acidophilus (Lactobacillus acidophilus, abbreviated as A bacteria) belongs to Lactobacillus in Lactobacillus family, is gram-positive bacteria, has slender rod shape, no flagellum, no motion, and is anaerobic or facultative anaerobic; it can utilize glucose, fructose, lactose and cane sugar to make homotype fermentation, and can produce DL-type lactic acid by means of fermentation.
The lactobacillus acidophilus regulates the micro-ecological environment from top to bottom in the whole gastrointestinal tract of a human body. "lower" means that they can also release substances that are beneficial for the growth of other probiotics such as bifidobacteria in the large intestine, increase the number of probiotics in the large intestine, and enhance their vitality.
Based on the fact that lactobacillus acidophilus has good nutrition and health care effects on human bodies, more and more enterprises take lactobacillus acidophilus freeze-dried powder as a food additive to prepare biological products and health food, and the number of living bacteria in a unit product directly influences the efficacy of the product. Therefore, it is important to increase the viable count during the production and preparation of the freeze-dried powder.
Selenium has various probiotic effects of resisting oxidation, improving the immunity of the organism and the like, certain probiotics can convert inorganic selenium into organic selenium, and because the probiotics can be conveniently added into food, sufficient selenium element can be supplemented for human bodies by taking a small amount of selenium-rich probiotics, the selenium-rich probiotic has good absorption effect and low toxicity, and simultaneously has double effects of organic selenium and probiotics.
Disclosure of Invention
In order to solve the technical problems, the invention provides a production method and application of high-activity lactobacillus acidophilus freeze-dried powder, the process is simple, the number of viable bacteria of the obtained freeze-dried powder is large, and the yield of thalli is high.
The invention relates to a production method of high-activity lactobacillus acidophilus freeze-dried powder, which comprises the following steps:
s1, streaking and culturing lactobacillus acidophilus strains: diluting lactobacillus acidophilus powder with sterile water, carrying out partition marking on an improved MC culture medium, inverting a plate after marking is finished, and carrying out constant-temperature culture in an incubator at 35 ℃ for 40-72 hours;
s2, selecting dominant strains for culture: selecting a single colony with a large calcium dissolving ring on a plate, inoculating the single colony on an improved MC culture medium, and carrying out partition scribing, inverting the plate after scribing is finished, and carrying out constant-temperature culture in a 35 ℃ incubator for 40-72 hours;
s3, selecting dominant strains again to culture continuously: selecting the single bacterial colony with the larger calcium dissolving ring on the plate in the step S2, inoculating the single bacterial colony on the improved MC culture medium, and carrying out partition scribing, wherein after the scribing is finished, the plate is inverted, and the single bacterial colony is cultured in a 35 ℃ incubator for 40-72 hours at constant temperature;
s4, fermentation culture in a fermentation tank: selecting the single colony with a large calcium-dissolving ring on the plate in the step S3, inoculating the single colony into a selenium-rich culture medium, and culturing at 35-38 ℃ and pH of 5.5-6.0 for 48 h;
s5, centrifugally concentrating the culture solution, and collecting bacterial sludge;
s6, freeze-drying the centrifuged bacterial sludge, adding a protective agent, fully and uniformly mixing, carrying out vacuum freeze-drying, and taking out when the moisture content is extremely low;
s7, crushing, sieving and packaging.
The invention relates to a production method of high-activity lactobacillus acidophilus freeze-dried powder, wherein in the steps S1, S2 and S3, the improved MC culture medium comprises 5.0g/L of soybean peptone, 3.0g/L of beef extract powder, 3.0g/L of yeast extract powder, 20.0g/L of lactose, 10.0g/L of calcium carbonate, 15.0g/L of agar, 10.0g/L of carrot extract, 10.0g/L of vitamin, 5g/L of casein hydrolysate and the balance of sterile water, the pH value is 5.0-7.0, and dilute hydrochloric acid or sodium hydroxide is used for adjusting the pH value.
The invention relates to a production method of high-activity lactobacillus acidophilus freeze-dried powder, wherein a selenium-rich culture medium in the step S4 comprises the following components: the selenium-rich culture medium in the step S4 comprises 5.0g/L of soybean meal, 3.0g/L of beef extract powder, 3.0g/L of yeast extract powder, 20.0g/L of pork kidney powder, 10.0g/L of broad bean powder, 10.0g/L of calcium carbonate, 10.0g/L of carrot extract, 10.0g/L of vitamin and 5g/L of casein hydrolysate, the balance of sterile water, the pH is 5.0-7.0, and the pH is adjusted by using dilute hydrochloric acid or sodium hydroxide.
In the steps S1, S2 and S3, sodium selenate is added after the culture is started for 10 hours in the culture process to ensure that the selenium concentration reaches 100 mug/ml.
The invention relates to a production method of high-activity lactobacillus acidophilus freeze-dried powder, and the protective agent in the step S6 comprises the following components: 70g/L of skimmed milk powder, 10g/L of cane sugar, 20g/L of stachyose, 10g/L of fructo-oligosaccharide, 10g/L of galacto-oligosaccharide, 5g/L of sodium ascorbate and the balance of sterile water.
According to the production method of the high-activity lactobacillus acidophilus freeze-dried powder, the inoculation amount in the step S4 is 6%.
The invention discloses application of high-activity lactobacillus acidophilus freeze-dried powder, and relates to application of high-activity lactobacillus acidophilus freeze-dried powder prepared by a production method in preparation of a medicine for treating diseases caused by intestinal dysbacteriosis.
Compared with the prior art, the invention has the beneficial effects that: firstly, through three times of culture, a strain with vigorous activity is selected, and through adding inorganic selenium for culture, the lactobacillus acidophilus converts the inorganic selenium into organic selenium in the body of the lactobacillus acidophilus, so that the activity of the lactobacillus acidophilus is enhanced, the immunity of the lactobacillus acidophilus is improved, and the activity of the lactobacillus acidophilus is higher; secondly, the selenium content in the lactobacillus acidophilus is further increased by culturing in a selenium-rich culture medium, so that the immunity of a user is improved, and the disease resistance is enhanced; thirdly, by adding stachyose, fructo-oligosaccharide and galacto-oligosaccharide, the sugar is a general term of linear chain component polymerized by 2-10 same or different monosaccharide units through glycosidic bonds or branched low-degree polymerized sugar, no enzyme metabolized in the human body is available in the sugar, so that the sugar and lactobacillus acidophilus directly enter the intestinal tract to be utilized by probiotics, become proliferation factors of the probiotics and promote the development of beneficial flora in the body.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1:
the invention relates to a production method of high-activity lactobacillus acidophilus freeze-dried powder, which comprises the following steps:
s1, streaking and culturing lactobacillus acidophilus strains: diluting lactobacillus acidophilus powder with sterile water, partitioning and scribing on an improved MC culture medium (5.0 g/L of soybean peptone, 3.0g/L of beef extract powder, 3.0g/L of yeast extract powder, 20.0g/L of lactose, 10.0g/L of calcium carbonate, 15.0g/L of agar, 10.0g/L of carrot extract, 10.0g/L of vitamin, 5g/L of casein hydrolysate and the balance of sterile water), inverting a plate after scribing is finished, culturing for 56 hours at the constant temperature of a 35 ℃ culture box, and adding sodium selenate after 10 hours of culture is started to enable the selenium concentration to reach 100 mu g/ml;
s2, selecting dominant strains for culture: selecting a single colony with a large calcium dissolving ring on a plate, inoculating the single colony to an improved MC culture medium (5.0 g/L of soybean peptone, 3.0g/L of beef extract powder, 3.0g/L of yeast extract powder, 20.0g/L of lactose, 10.0g/L of calcium carbonate, 15.0g/L of agar, 10.0g/L of carrot extract, 10.0g/L of vitamin, 5g/L of casein hydrolysate and the balance of sterile water), carrying out partition streaking, after finishing the streaking, inverting the plate, carrying out constant-temperature culture in a 35 ℃ incubator for 70 hours, adding sodium selenate after beginning to carry out the culture for 10 hours, and enabling the selenium concentration to reach 100 mu g/ml;
s3, selecting dominant strains again to culture continuously: selecting a single colony with a large calcium dissolving ring on the plate in the step S2, inoculating the single colony to an improved MC culture medium (5.0 g/L of soybean peptone, 3.0g/L of beef extract powder, 3.0g/L of yeast extract powder, 20.0g/L of lactose, 10.0g/L of calcium carbonate, 15.0g/L of agar, 10.0g/L of carrot extract, 10.0g/L of vitamin, 5g/L of casein hydrolysate and the balance of sterile water), carrying out partition streaking, inverting the plate after the streaking is finished, carrying out constant-temperature culture in a 35 ℃ incubator for 62 hours, adding sodium selenate after 10 hours of culture is started, and enabling the selenium concentration to reach 100 mu g/ml;
s4, fermentation culture in a fermentation tank: selecting a single colony with a large calcium-dissolving ring on the plate in the step S3, inoculating the single colony into a selenium-rich culture medium (5.0 g/L of soybean meal, 3.0g/L of beef extract powder, 3.0g/L of yeast extract powder, 20.0g/L of pork kidney powder, 10.0g/L of broad bean powder, 10.0g/L of calcium carbonate, 10.0g/L of carrot extract, 10.0g/L of vitamin, 5g/L of casein hydrolysate and the balance of sterile water), wherein the inoculation amount is 6%, the temperature is 37 ℃, the pH value is 5.5-6.0, and the culture time is 48 h;
s5, centrifugally concentrating the culture solution, and collecting bacterial sludge;
s6, freeze-drying the centrifuged bacterial sludge, adding protective agents (70 g/L of skimmed milk powder, 10g/L of cane sugar, 20g/L of stachyose, 10g/L of fructo-oligosaccharide, 10g/L of galacto-oligosaccharide, 5g/L of sodium ascorbate and the balance of sterile water), fully mixing uniformly, carrying out vacuum freeze-drying, and taking out when the water content is extremely low through freeze-drying;
s7, crushing, sieving and packaging.
Example 2:
the difference from example 1 is that: the culture time in S1 was 50h, the culture time in S2 was 60h, the culture time in S3 was 50h, and the temperature in step S4 was 36 ℃.
Comparative example 1:
the difference from example 1 is that: sodium selenate is not added in the steps S1, S2 and S3;
comparative example 2:
the difference from example 1 is that: in step S4, soybean meal, pork kidney meal and broad bean meal are not added;
comparative example 3:
the difference from example 1 is that: sodium selenate is not added in steps S1, S2 and S3, and soybean meal, pork kidney meal and broad bean meal are not added in step S4;
the viable count of the lactobacillus acidophilus lyophilized powder prepared in examples 1-2 and comparative examples 1-3 was measured to obtain the following data:
from the data, when the steps S1, S2 and S3 are carried out simultaneously, a higher viable count can be obtained, which mainly comes from three times of culture, a strain with stronger activity is selected, and inorganic selenium is added for culture, so that the lactobacillus acidophilus converts the inorganic selenium into organic selenium in the body of the lactobacillus acidophilus, the activity of the lactobacillus acidophilus is enhanced, the immunity of the lactobacillus acidophilus is improved, and the activity of the lactobacillus acidophilus is higher;
the main reason for performing step S4 is to make lactobacillus acidophilus further absorb selenium and increase the selenium content in lactobacillus acidophilus, thereby increasing the selenium content in user' S body and achieving the purpose of preventing and treating diseases.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Claims (7)
1. A production method of high-activity lactobacillus acidophilus freeze-dried powder is characterized by comprising the following steps:
s1, streaking and culturing lactobacillus acidophilus strains: diluting lactobacillus acidophilus powder with sterile water, carrying out partition marking on an improved MC culture medium, inverting a plate after marking is finished, and carrying out constant-temperature culture in an incubator at 35 ℃ for 40-72 hours;
s2, selecting dominant strains for culture: selecting a single colony with a large calcium dissolving ring on a plate, inoculating the single colony on an improved MC culture medium, and carrying out partition scribing, inverting the plate after scribing is finished, and carrying out constant-temperature culture in a 35 ℃ incubator for 40-72 hours;
s3, selecting dominant strains again to culture continuously: selecting the single bacterial colony with the larger calcium dissolving ring on the plate in the step S2, inoculating the single bacterial colony on the improved MC culture medium, and carrying out partition scribing, wherein after the scribing is finished, the plate is inverted, and the single bacterial colony is cultured in a 35 ℃ incubator for 40-72 hours at constant temperature;
s4, fermentation culture in a fermentation tank: selecting the single colony with a large calcium-dissolving ring on the plate in the step S3, inoculating the single colony into a selenium-rich culture medium, and culturing at 35-38 ℃ and pH of 5.5-6.0 for 48 h;
s5, centrifugally concentrating the culture solution, and collecting bacterial sludge;
s6, freeze-drying the centrifuged bacterial sludge, adding a protective agent, fully and uniformly mixing, carrying out vacuum freeze-drying, and taking out when the moisture content is extremely low;
s7, crushing, sieving and packaging.
2. The method of claim 1, wherein the modified MC medium of steps S1, S2, and S3 comprises soy peptone 5.0g/L, beef extract 3.0g/L, yeast extract 3.0g/L, lactose 20.0g/L, calcium carbonate 10.0g/L, agar 15.0g/L, carrot extract 10.0g/L, vitamins 10.0g/L, and casein hydrolysate 5g/L, and the balance is sterile water, pH is 5.0-7.0, and pH is adjusted with diluted hydrochloric acid or sodium hydroxide.
3. The method for producing highly active lactobacillus acidophilus lyophilized powder of claim 2, wherein the selenium-rich medium of step S4 comprises: the selenium-rich culture medium in the step S4 comprises 5.0g/L of soybean meal, 3.0g/L of beef extract powder, 3.0g/L of yeast extract powder, 20.0g/L of pork kidney powder, 10.0g/L of broad bean powder, 10.0g/L of calcium carbonate, 10.0g/L of carrot extract, 10.0g/L of vitamin and 5g/L of casein hydrolysate, the balance of sterile water, the pH is 5.0-7.0, and the pH is adjusted by using dilute hydrochloric acid or sodium hydroxide.
4. The method of claim 3, wherein during the culturing steps S1, S2 and S3, sodium selenate is added after 10 hours of culturing to make selenium concentration reach 100 μ g/ml.
5. The method for producing highly active lactobacillus acidophilus lyophilized powder of claim 4, wherein the protective agent in step S6 comprises: 70g/L of skimmed milk powder, 10g/L of cane sugar, 20g/L of stachyose, 10g/L of fructo-oligosaccharide, 10g/L of galacto-oligosaccharide, 5g/L of sodium ascorbate and the balance of sterile water.
6. The method of claim 5, wherein the inoculation amount in step S4 is 6%.
7. Use of highly active lactobacillus acidophilus lyophilized powder prepared by the method of any of claims 1 to 6 for the preparation of a medicament for the treatment of disorders caused by a disturbance of intestinal flora.
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| CN113956996A (en) * | 2021-08-03 | 2022-01-21 | 镇江市远胜生物工程有限公司 | Production method of freeze-dried lactobacillus acidophilus powder |
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Application publication date: 20210205 |