CN115261298A - Domestication method of high-temperature-resistant lactobacillus plantarum and preparation method of lactobacillus plantarum powder - Google Patents
Domestication method of high-temperature-resistant lactobacillus plantarum and preparation method of lactobacillus plantarum powder Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/36—Adaptation or attenuation of cells
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D1/00—Evaporating
- B01D1/16—Evaporating by spraying
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- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
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Abstract
The invention discloses a domestication method of high-temperature-resistant lactobacillus plantarum and a preparation method of lactobacillus plantarum powder. According to the invention, the heat resistance of the domesticated lactobacillus plantarum can be promoted to be improved by domesticating the lactobacillus plantarum, so that the survival rate of the spray-dried lactobacillus plantarum is obviously improved. Meanwhile, the method does not influence the bacterial quantity. The domestication method provided by the invention is simple and feasible, the strain is not easy to degenerate, the domestication method is suitable for production in most factories, and the domestication method has a great promoting effect on large-scale production and application of plant lactobacillus.
Description
Technical Field
The invention relates to the technical field of probiotic culture, in particular to a domestication method of high-temperature-resistant lactobacillus plantarum and a preparation method of lactobacillus plantarum powder.
Background
Lactobacillus plantarum is one of lactic acid bacteria, a gram-positive bacterium which cannot form spores and can produce lactic acid. Anaerobic or facultative anaerobic, the bacterial body is in a straight or bent rod shape, and is single, sometimes paired or chained. The microbial inoculum has strong reproductive capacity and high unit viable bacteria amount, can produce a large amount of acid, can play roles in maintaining intestinal flora and inhibiting pathogenic bacteria, and also can play roles in resisting oxidation, tumors and the like.
However, although lactobacillus plantarum is excellent in reproductive ability and acid-producing ability, it does not have high stress resistance of bacillus, and even as lactobacillus, it is inferior in heat resistance, acid resistance, and the like, as compared to lactobacillus such as enterococcus faecalis. At present, the stress resistance of the strains with poor acid resistance and heat resistance is generally improved by a coating mode.
The existing coating methods include: press curing in advance, formation of microcapsules in advance by chemical means, spray drying, and the like. Among them, the method of extruding and curing in advance and forming microcapsules in advance by a chemical method have the disadvantages of low efficiency, many equipment limitations and the like. The most convenient way to form the microcapsule powder directly is by spray drying. The survival rate of the lactobacillus plantarum is difficult to improve due to poor heat resistance and high spray drying temperature, and the spray effect is greatly influenced due to low spray drying temperature, so that how to improve the survival rate of the lactobacillus plantarum at reasonable spray drying temperature is particularly important.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a domestication method of high-temperature-resistant lactobacillus plantarum and a preparation method of lactobacillus plantarum powder, so that the survival rate of lactobacillus plantarum at a reasonable spray drying temperature is improved.
The invention is realized in the following way:
the invention provides a domestication method of high-temperature-resistant lactobacillus plantarum, which comprises the following steps:
first-stage domestication: inoculating lactobacillus plantarum to be domesticated into a culture medium, and culturing at 36-38 ℃ for 23-25h; then mixing the cultured bacterial liquid with a protective agent, placing the mixture in a water bath at 59-61 ℃, and calculating the survival rate of the lactobacillus plantarum after the water bath; inoculating the bacterial liquid after water bath into a new culture container containing a culture medium; repeating the first-stage domestication for at least 2 times until the survival rate is more than or equal to 90 percent, and finishing the first-stage domestication;
second-stage domestication: inoculating the bacterial liquid after the first-stage domestication into a new culture container containing a culture medium, and culturing at 36-38 ℃ for 23-25h; then mixing the protective agent with the cultured bacterial liquid, carrying out water bath at 64-66 ℃, and calculating the survival rate of the lactobacillus plantarum subjected to the water bath; inoculating the bacterial liquid after water bath into a new culture container containing a culture medium; repeating the second-stage domestication for at least 3 times until the survival rate is more than or equal to 85 percent, and finishing the second-stage domestication;
third-stage domestication: inoculating the bacterial liquid after the second-stage domestication into a new culture container containing a culture medium, and culturing at 36-38 ℃ for 23-25h; then mixing the protective agent with the cultured bacterial liquid, carrying out water bath at 69-71 ℃, and calculating the survival rate of the lactobacillus plantarum after the water bath; inoculating the bacterial liquid after water bath into a new culture container containing a culture medium; repeating the third stage acclimatization for at least 4 times until the survival rate is more than or equal to 80 percent, and finishing the third stage acclimatization;
in the bacterial liquid, the protective agent comprises the following raw materials in percentage by weight: 1.5-2% of sodium alginate, 1-1.2% of calcium chloride, 1.5-2% of chitosan, 2.5-3% of yeast cell wall, 0.2-0.4% of gluconic acid-delta-lactone, 3-4% of skim milk powder and 1-1.2% of sodium stearate;
survival = (bacterial count after water bath/bacterial count before water bath) 100%.
The inventor finds that the heat resistance of the domesticated lactobacillus plantarum can be improved by domesticating the lactobacillus plantarum, so that the survival rate of the spray-dried lactobacillus plantarum is obviously improved.
If the strain domestication is carried out by directly increasing the culture temperature, the problem that the culture temperature generally cannot exceed 55 ℃ exists, the air outlet temperature during actual spray drying is about 70 ℃, and the survival rate of the lactobacillus plantarum during spray drying still cannot be effectively increased at a temperature with a large difference. The inventor adopts gradient temperature rise, and adopts a water bath mode with the temperature of about 70 ℃ to simulate the spray drying process in the third stage, so that the conditions are more approximate to those in actual production, the survival rate after spray drying is obviously improved, and the genetic stability of the strain is maintained.
The inventor carries out acclimatization of the strain by adopting a gradient temperature rise mode for the following two aspects: on one hand, lactobacillus plantarum has extremely poor heat resistance, and complete death of thalli in the acclimatization process can occur due to direct adoption of 70 ℃ water bath, so that acclimatization failure is caused, and on the other hand, the high lethality rate caused by direct adoption of 70 ℃ water bath can possibly cause large mutation of the strain, so that the hereditary stability of the strain is reduced.
The initial (first stage acclimatization) bath temperature was set at 59-61 ℃ because the temperature setting was too low for lactobacillus plantarum to die substantially, and too high a bath temperature was likely to cause the entire strain to die. The temperature of the water bath in the third stage of acclimatization is 69-71 deg.C according to the conventional temperature of spray drying.
The inventor sets 3 stages of acclimatization because the temperature difference between every two stages is kept at 5 ℃ and has higher acclimatization effect, the temperature difference is too small to lose the acclimatization meaning, and the bacteria can die if the temperature difference is too high.
The inventor mixes the bacterial liquid with the protective agent before water bath, on one hand, the actual spray drying process can be simulated more truly, and the heat resistance of the mixed thallus and protective agent is acclimated in a targeted manner; on the other hand, some protective agents have an effect of protecting the bacterial cells during spray drying, but the protective agents themselves have a certain harm to the bacterial cells, and strains which are tolerant to harmful components in the protective agents can also be screened by mixing the protective agents and then carrying out acclimatization in a water bath. Thereby further improving the survival rate of the strain.
The inventors have also found that setting a certain number of repetitions at different stages helps to improve survival. Therefore, the lowest repetition times are set in different stages, and the current stage is ended when the survival rate reaches a certain value, on one hand, accidental situations in the actual domestication process are prevented, on the other hand, if the domestication repetition times are too low, or the survival rate does not reach the standard, the domestication is ended, the domesticated thalli are likely to be incompletely domesticated, strains with poor heat resistance and unstable genes are still remained in the domesticated thalli, the subsequent propagation of a large number of strains with poor heat resistance is caused, and the survival rate of the lactobacillus plantarum after spray drying is further low. The number of repetitions of the first stage is set to at least 2, such as 2-10, for example 2, 3, 4 or 5 repetitions. The number of repetitions of the second stage is set to be at least 3, such as 3-10, for example 3, 4, 5, 6 or 7. The number of repetitions of the third stage is set to at least 4, such as 4-10, for example 4, 5, 6, 7 or 8.
In addition, the inventor also improves the components of the protective agent, wherein sodium alginate and calcium chloride are used as main packing materials of the microcapsule structure, the sodium alginate structure has polyguluronic acid chain segments, and after contacting with calcium ions, the polyguluronic acid chain segments can generate gelation reaction to form a completely polymerized net structure, so that the hard shell of the microcapsule is formed.
Casein in the skim milk powder is used as a main component for forming a microcapsule structure, and under the action of gluconic acid-delta-lactone, cross linking occurs among proteins, so that the proteins in the skim milk powder are solidified to form a structural framework of the microcapsule, in addition, the gluconic acid-delta-lactone can effectively reduce the pH value and provide an acidic environment, and the hard shell of calcium alginate has a more stable structure under the condition of low pH.
Meanwhile, in an acidic environment, amino groups on chitosan molecular chains and carboxyl groups on calcium alginate molecular chains have electrostatic interaction to form a layer of polyelectrolyte film to cover the surface of the calcium alginate microcapsules, so that the embedding rate of the microcapsules is improved.
The main components of the yeast cell wall are beta-glucan and mannan, which can play the effect of a filling agent, and meanwhile, the yeast cell wall is rich in a large amount of bioactive substances and nutrient substances and can provide most of components necessary for the survival of thalli. Sodium stearate functions as an emulsifier and a dispersant, has a good affinity with calcium ions, can form a coagulated calcium soap, can compensate shrinkage when microcapsules are formed, prevents cracking by filling gaps generated when microcapsules are formed, has good long-term heat resistance, and is suitable for granulation, storage and transportation in the later period.
Therefore, the microcapsule structure outside the lactobacillus plantarum is more stable under the condition of low pH through the compatibility of the plurality of protective agent components, the embedding rate of the microcapsule is improved, the long-term heat resistance of the microcapsule is also improved through the arrangement of the sodium stearate, and the granulation, storage and transportation at the later stage are facilitated. The protective agent also provides necessary bioactive substances and nutrient substances for the survival of the thalli. If the components in the protective agent are deleted, for example, the survival rate is obviously reduced without adding yeast cell wall, glucono-delta-lactone and sodium stearate in the protective agent.
In conclusion, the domestication method of the high-temperature-resistant lactobacillus plantarum provided by the invention obviously improves the survival capability of the lactobacillus plantarum in spray drying, and does not influence the bacterial load of the lactobacillus plantarum. The domestication method provided by the invention is simple and feasible, the strain is not easy to degenerate, the domestication method is suitable for production in most factories, and the domestication method has a great promoting effect on large-scale production and application of plant lactobacillus.
In order to calculate the survival rate, viable bacteria count is performed after a protective agent is added before the water bath, and then viable bacteria count is performed after the water bath is finished, so that the survival rate is calculated.
In the preferred embodiment of the present invention, the time of the water bath in the first stage is 10-12min; the water bath time in the second stage is 10-12min; the water bath time in the third stage is 10-12min.
For example, the water bath time of the first to third stages is set to be 10min,11min or 12min. In the water bath time, the acclimatization of the lactobacillus plantarum can be quickly realized.
In a preferred embodiment of the invention, the first stage acclimatization is repeated at least 3 times, the second stage acclimatization is repeated at least 4 times, and the third stage acclimatization is repeated at least 6 times. The survival rate of the strain can exceed 90 percent and even exceed 95 percent under the repeated times. Reducing the number of repetition times of the acclimation stage affects the survival rate of the acclimated bacteria solution. For example, the first stage acclimatization is repeated at least 4 times, the second stage acclimatization is repeated at least 5 times, and the third stage acclimatization is repeated at least 7 times.
If the domestication is not carried out, the survival rate of the lactobacillus plantarum is greatly reduced; deletion of any stage of acclimation step, reduction of acclimation temperature or no addition of protective agent in the acclimation step can result in significant reduction of the survival rate of lactobacillus plantarum.
In a preferred embodiment of the application of the invention, the rejuvenation before the acclimation of the strain to be acclimated is carried out before the acclimation of the first stage, wherein the rejuvenation comprises inoculating lactobacillus plantarum into a culture medium, and carrying out standing culture for 23-25h at 36-38 ℃; the rejuvenated lactobacillus plantarum was then used for the first stage acclimatization. The bacterial liquid inoculation amount in the rejuvenation stage is 1-1.2%. For example, 1ml of the bacterial suspension is inoculated into 100ml of the culture medium.
In a preferred embodiment of the present invention, the culture medium comprises the following raw materials: 8-10g/L of peptone, 4-5g/L of yeast powder, 10-12g/L of glucose, 4-5g/L of sodium acetate, 2-2.2g/L of diammonium citrate, 2-2.2g/L of dipotassium phosphate and water. In other embodiments, the corresponding medium formula can also be set according to the type of lactobacillus plantarum, and is not limited to the above medium formula.
In a preferred embodiment of the invention, the bacterial liquid inoculum size of the first stage acclimatization, the second stage acclimatization and the third stage acclimatization is 1-1.2%.
In a preferred embodiment of the present invention, the first stage acclimatization, the second stage acclimatization, and the third stage acclimatization are all static cultures. For example, the culture is performed by static culture in an Erlenmeyer flask.
The invention also provides a preparation method of the lactobacillus plantarum powder, which comprises the following steps: inoculating the domesticated strain obtained by the domestication method of the high-temperature-resistant lactobacillus plantarum into a culture medium to prepare a seed solution, then inoculating the seed solution into a fermentation tank for culture, mixing the cultured bacterial solution with a protective agent, and performing spray drying to obtain bacterial powder.
The preparation method of the powder has the advantages of convenient and fast operation of the fermentation process, suitability for production in most factories and great promotion effect on large-scale production and application of lactobacillus plantarum.
In a preferred embodiment of the invention, the culture medium in the fermenter comprises the following raw materials: 10-12g/L of peptone, 10-12g/L of yeast powder, 10-12g/L of corn flour, 20-24g/L of glucose, 4-5g/L of sodium acetate, 2-2.2g/L of diammonium citrate, 2-2.2g/L of dipotassium hydrogen phosphate, 0.2-0.3g/L of magnesium sulfate monohydrate, 0.2-0.3g/L of manganese sulfate monohydrate and water. In other embodiments, the medium formulation in the fermentor may also be set according to the type of lactobacillus plantarum, and is not limited to the above-described medium formulation, for example, the type of raw materials is set according to the carbon source, the nitrogen source.
In the preferred embodiment of the present invention, the culture temperature in the fermentation tank is 36-38 deg.C, and the static culture is 30-32 hr.
The air outlet temperature during spray drying is 70 +/-3 ℃. The air outlet temperature corresponds to the water bath temperature of the third stage, so that the temperature of the domesticated strain can be ensured to be tolerant to the temperature of spray drying.
In a preferred embodiment of the present invention, the culture medium of the seed liquid comprises the following raw materials: 8-10g/L of peptone, 4-5g/L of yeast powder, 10-12g/L of glucose, 4-5g/L of sodium acetate, 2-2.2g/L of diammonium citrate, 2-2.2g/L of dipotassium phosphate and water.
The invention also provides the lactobacillus plantarum powder prepared by the preparation method of the lactobacillus plantarum powder. The powder prepared by the preparation method has high bacterial count and high survival rate. The bacterial amount can reach 6-8 × 10 10 CFU/g。
The invention has the following beneficial effects:
according to the domestication method of the high-temperature-resistant lactobacillus plantarum, provided by the invention, the domestication of the lactobacillus plantarum is carried out, so that the heat resistance of the domesticated lactobacillus plantarum can be promoted to be improved, and the survival rate of the spray-dried lactobacillus plantarum is obviously improved. Meanwhile, the method does not influence the bacterial quantity. The domestication method provided by the invention is simple and feasible, the strain is not easy to degenerate, the domestication method is suitable for production in most factories, and the domestication method has a great promoting effect on large-scale production and application of the plant lactobacillus. Compared with the prior art, the invention has the advantages that:
(1) The inventor adopts gradient temperature rise, and adopts a water bath mode with the temperature of about 70 ℃ to simulate the spray drying process in the third stage, so that the conditions are more approximate to those in actual production, the survival rate after spray drying is obviously improved, and the genetic stability of the strain is maintained.
(2) The inventor mixes the bacterial liquid with the protective agent before water bath, on one hand, the actual spray drying process can be simulated more truly, and the heat resistance of the mixed thallus and protective agent is acclimated in a targeted manner; on the other hand, some protective agents have an effect of protecting the bacterial cells during spray drying, but the protective agents themselves have a certain harm to the bacterial cells, and strains which are tolerant to harmful components in the protective agents can also be screened by mixing the protective agents and then carrying out acclimatization in a water bath. Thereby further improving the survival rate of the strain.
(3) And certain repetition times are set at different stages, so that the survival rate is improved. Therefore, the lowest repetition times are set in different stages, and the current stage is ended when the survival rate reaches a certain value, on one hand, accidental situations in the actual domestication process are prevented, on the other hand, if the domestication repetition times are too low, or the survival rate does not reach the standard, the domestication is ended, the domesticated thalli are likely to be incompletely domesticated, strains with poor heat resistance and unstable genes are still remained in the domesticated thalli, the subsequent propagation of a large number of strains with poor heat resistance is caused, and the survival rate of the lactobacillus plantarum after spray drying is further low.
(4) The inventor also improves the components of the protective agent, wherein sodium alginate and calcium chloride are used as main packing materials of the microcapsule structure, the sodium alginate structure has polyguluronic acid chain segments, and the polyguluronic acid chain segments can generate gelation reaction after being contacted with calcium ions to form a completely polymerized network structure, so as to form the hard shell of the microcapsule.
Casein in the skim milk powder is used as a main component for forming a microcapsule structure, and under the action of gluconic acid-delta-lactone, cross linking occurs among proteins, so that the proteins in the skim milk powder are solidified to form a structural framework of the microcapsule, in addition, the gluconic acid-delta-lactone can effectively reduce the pH value and provide an acidic environment, and the hard shell of calcium alginate has a more stable structure under the condition of low pH.
Meanwhile, in an acidic environment, amino groups on chitosan molecular chains and carboxyl groups on calcium alginate molecular chains have electrostatic interaction to form a layer of polyelectrolyte film to cover the surface of the calcium alginate microcapsules, so that the embedding rate of the microcapsules is improved.
The main components of the yeast cell wall are beta-glucan and mannan, which can play the effect of a filling agent, and meanwhile, the yeast cell wall is rich in a large amount of bioactive substances and nutrient substances and can provide most of components necessary for the survival of thalli. Sodium stearate functions as an emulsifier and a dispersant, has a good affinity with calcium ions, can form a coagulated calcium soap, can compensate shrinkage when microcapsules are formed, prevents cracking by filling gaps generated when microcapsules are formed, has good long-term heat resistance, and is suitable for granulation, storage and transportation in the later period.
Therefore, the microcapsule structure outside the lactobacillus plantarum is more stable under the condition of low pH through the compatibility of the plurality of protective agent components, the embedding rate of the microcapsule is improved, the long-term heat resistance of the microcapsule is also improved through the arrangement of the sodium stearate, and the granulation, storage and transportation at the later stage are facilitated. The protective agent also provides necessary bioactive substances and nutrient substances for the survival of the thalli. If the components in the protective agent are omitted, for example, yeast cell walls, glucono-delta-lactone and sodium stearate are not added into the protective agent, the survival rate is obviously reduced.
In addition, the preparation method of the lactobacillus plantarum powder is simple and easy to implement.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a process flow diagram of a acclimatization method of high temperature resistant Lactobacillus plantarum.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a method for domesticating high-temperature-resistant lactobacillus plantarum and a method for preparing lactobacillus plantarum powder, and the process flow is shown in fig. 1.
The domestication method comprises the following steps:
1. and (5) rejuvenating the strains.
The objective Lactobacillus plantarum stored on the slant was inoculated into a 150mL Erlenmeyer flask containing 100mL of liquid, and incubated at 37 ℃ for 24 hours.
The formula of the culture medium in the conical flask is as follows: 10g/L of peptone, 5g/L of yeast powder, 10g/L of glucose, 5g/L of sodium acetate, 2g/L of diammonium citrate and 2g/L of dipotassium phosphate.
2. The first stage acclimatization.
And (3) sucking 1mL of bacterial liquid from the cultured conical flask, transferring the bacterial liquid to a new 150mL conical flask with the liquid content of 100mL per bottle, performing static culture at 37 ℃ for 24 hours, adding a protective agent, uniformly mixing, quickly counting viable bacteria, performing water bath at 60 ℃ for 10 minutes, counting the viable bacteria of the bacterial liquid after the water bath, calculating the survival rate, sucking 1mL of bacterial liquid, and transferring the bacterial liquid to the new 150mL conical flask with the liquid content of 100mL per bottle.
Wherein the formula of the culture medium in the conical flask is the same as that in the step 1.
Wherein, the protective agent comprises the following raw materials in percentage by weight: 1.5% of sodium alginate, 1.2% of calcium chloride, 2% of chitosan, 3% of yeast cell wall, 0.2% of gluconic acid-delta-lactone, 4% of skimmed milk powder and 1% of sodium stearate.
Wherein, survival rate = bacterial count after water bath/bacterial count before water bath 100%.
3. Repeating the steps until the acclimatization is completed.
And (3) repeating the step (2) at least, and finishing the first-stage domestication when the survival rate is more than or equal to 90 percent.
4. And second stage domestication.
And (3) sucking 1mL of bacterial liquid from the cultured conical flask, transferring the bacterial liquid to a new 150mL conical flask with the liquid content of 100mL per bottle, performing static culture at 37 ℃ for 24 hours, adding a protective agent, uniformly mixing, quickly counting viable bacteria, performing water bath at 65 ℃ for 10 minutes, counting the viable bacteria of the bacterial liquid after the water bath, calculating the survival rate, sucking 1mL of bacterial liquid, and transferring the bacterial liquid to the new 150mL conical flask with the liquid content of 100mL per bottle.
Wherein the formula of the culture medium in the conical flask is the same as that in the step 1.
Wherein the formula of the protective agent is the same as that in the step 2.
Wherein survival = post-water bath/pre-water bath bacterial count 100%.
5. Repeating the steps until the acclimatization is completed.
And (4) repeating the step (4) at least 4 times, and finishing the second-stage domestication when the survival rate is more than or equal to 85 percent.
6. And (3) domestication in the third stage.
And (3) sucking 1mL of bacterial liquid from the cultured conical flask, transferring the bacterial liquid to a new 150mL conical flask with the liquid content of 100mL per bottle, performing static culture at 37 ℃ for 24 hours, adding a protective agent, uniformly mixing, quickly counting viable bacteria, performing water bath at 70 ℃ for 10 minutes, counting the viable bacteria of the bacterial liquid after the water bath, calculating the survival rate, sucking 1mL of bacterial liquid, and transferring the bacterial liquid to the new 150mL conical flask with the liquid content of 100mL per bottle.
Wherein the formula of the culture medium in the conical flask is the same as that in the step 1.
Wherein the formula of the protective agent is the same as that in the step 2.
Wherein survival = post-water bath/pre-water bath bacterial count 100%.
7. Repeating the steps until the domestication is completed
And (5) repeating the step (6) at least 6 times, and finishing the third-stage domestication when the survival rate is more than or equal to 80%.
The preparation method of the lactobacillus plantarum powder comprises the following steps:
inoculating the domesticated strain into a new 150mL conical flask with the liquid loading capacity of 100mL, standing and culturing at 37 ℃ for 24 hours to serve as seed liquid, then inoculating into a sterilized fermentation tank for culturing, uniformly mixing the cultured bacterial liquid with a protective agent, quickly sampling for viable bacteria counting, performing spray drying on the bacterial liquid mixed with the protective agent through a spray drying tower, performing viable bacteria counting on the spray-dried bacterial powder, and counting the weight of the spray-dried bacterial powder.
Wherein the formula of the culture medium in the conical flask is the same as that in the step 1.
Wherein the formula of the culture medium in the fermentation tank is as follows: 10g/L of peptone, 12g/L of yeast powder, 12g/L of corn flour, 20g/L of glucose, 5g/L of sodium acetate, 2g/L of diammonium citrate, 2g/L of dipotassium phosphate, 0.3g/L of magnesium sulfate monohydrate, 0.2g/L of manganese sulfate monohydrate, 300L of liquid loading capacity, 37 ℃ of culture temperature and 31 hours of static culture.
Wherein the formula of the protective agent is the same as that in the step 2.
Then, taking example 1 as an example, examples 2 to 4 and comparative examples 1 to 9 were set
Example 2
Example 2 differs from example 1 only in that: the formula of the culture medium adopted in the steps 1, 2, 4, 6 and 8 is as follows: 8g/L of peptone, 4g/L of yeast powder, 12g/L of glucose, 4g/L of sodium acetate, 2.2g/L of diammonium citrate and 2.2g/L of dipotassium phosphate.
Example 3
Example 3 differs from example 1 only in that the protectant formulations in steps 2, 4, 6, and 8 are: 2% of sodium alginate, 1% of calcium chloride, 1.5% of chitosan, 2.5% of yeast cell wall, 0.4% of gluconic acid-delta-lactone, 3% of skimmed milk powder and 1.2% of sodium stearate.
Example 4
Example 4 differs from example 1 only in that the formulation of the fermenter medium in step 8 is: 12g/L of peptone, 10g/L of yeast powder, 10g/L of corn flour, 24g/L of glucose, 4g/L of sodium acetate, 2.2g/L of diammonium citrate, 2.2g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate monohydrate, 0.3g/L of manganese sulfate monohydrate, 300L of liquid loading capacity, 37 ℃ of culture temperature and 31 hours of static culture.
Example 5
The only difference from example 1 is that the number of repetitions is reduced, where step 3 is: repeating the step 2 for 2 times, wherein the step 5 is as follows: repeating the step 4 for 3 times, wherein the step 7 is as follows: repeat step 64 times.
Comparative example 1
The difference between comparative example 1 and example 1 is that there are no steps 1-7 and the protectant formulation in step 8 is: 3-5% of skimmed milk powder and 6-8% of water-soluble starch.
Comparative example 2
Comparative example 2 differs from test example 1 in that there are no steps 1 to 7.
Comparative example 3
Comparative example 3 differs from example 1 in that steps 6-7 are absent.
Comparative example 4
Comparative example 4 differs from example 1 in that no protective agent was added in steps 2, 4, 6.
Comparative example 5
The difference between the comparative example 5 and the example 1 is that the protective agent formula in the steps 2, 4, 6 and 8 is as follows: 3-5% of skimmed milk powder and 6-8% of water-soluble starch.
Comparative example 6
The difference between comparative example 6 and example 1 is that the yeast cell walls were removed from the composition of the protective agent in steps 2, 4, 6, 8.
Comparative example 7
The difference between the comparative example 7 and the example 1 is that the glucono delta lactone is removed from the components of the protective agent in the steps 2, 4, 6 and 8.
Comparative example 8
Comparative example 8 differs from example 1 in that sodium stearate was removed from the composition of the protectant in steps 2, 4, 6, and 8.
Comparative example 9
Comparative example 9 differs from example 1 in that the yeast cell walls in the composition of the protective agent in steps 2, 4, 6, 8 are replaced with trehalose.
Comparative example 10
The difference between comparative example 10 and example 1 is that the glucono delta lactone in the composition of the protectant in steps 2, 4, 6, 8 is replaced with calcium chloride.
Comparative example 11
Comparative example 11 differs from example 1 in that the sodium stearate in the components of the protectant in steps 2, 4, 6, 8 is replaced with tween 80.
Experimental example 1
The unit bacterial count of the bacterial liquid, the unit bacterial count of the bacterial powder after spray drying, and the mass of the bacterial powder after spray drying in examples 1-5 and comparative examples 1-8 after mixing the protective agent were counted, and the spray survival rate was calculated, and the results are shown in table 1.
Wherein, the spray survival rate = bacterial powder unit bacterial count weight (kg)/(bacterial liquid unit bacterial count 300) 100%.
TABLE 1 bacterial load and survival rate after fermentation
As can be seen from examples 1 to 5 in Table 1, the change of the medium ratio, the protective agent ratio and the process in examples 1 to 5 did not significantly affect the acclimatization effect. And reducing the number of repetitions of the acclimatization phase results in a slight decrease in the survival rate of lactobacillus.
As can be seen from comparative examples 1, 2 and 5, the survival rate was very severely reduced without acclimatization or without the use of the protective agent of the present invention, and also with acclimatization alone without the use of the protective agent of the present invention. It can be seen from comparative examples 3 and 4 that varying the acclimation procedure resulted in varying degrees of decreased survival, especially the most significant decrease in survival with decreased acclimation temperature and no added protectant during acclimation.
As can be seen from comparative examples 6, 7 and 8, the survival rate is obviously reduced without adding the yeast cell wall, the glucono-delta-lactone and the sodium stearate into the protective agent.
It can be seen from comparative examples 9, 10 and 11 that replacing the yeast cell wall in the protective agent with trehalose which is also a bulking agent, replacing glucono-delta-lactone with calcium chloride which is also a coagulant, and replacing sodium stearate with tween 80 which is also an emulsifier all resulted in a certain decrease in survival rate.
As can be seen from the experimental example 1, the bacterial load of the lactobacillus plantarum powder prepared by the domestication method and the spray drying protective agent of the invention can reach 5.91-7.29 x 10 10 CFU/g, the survival rate reaches 83.73-98.89%, compared with a method which is not domesticated and uses a conventional protective agent, the survival rate is improved by 376%, the method is beneficial to subsequent granulation, storage and transportation, and the method is greatly suitable forPromoting the production activity of lactobacillus plantarum.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A domestication method of high temperature resistant lactobacillus plantarum is characterized by comprising the following steps:
first-stage domestication: inoculating lactobacillus plantarum to be domesticated into a culture medium, and culturing at 36-38 ℃ for 23-25h; then mixing the cultured bacterial liquid with a protective agent, placing the mixture in a water bath at 59-61 ℃, and calculating the survival rate of the lactobacillus plantarum after the water bath; inoculating the bacterial liquid after water bath into a new culture container containing a culture medium; repeating the first-stage domestication for at least 2 times until the survival rate is more than or equal to 90 percent, and finishing the first-stage domestication;
second-stage domestication: inoculating the bacterial liquid after the first-stage domestication into a new culture container containing a culture medium, and culturing at 36-38 ℃ for 23-25h; then mixing the protective agent with the cultured bacterial liquid, carrying out water bath at 64-66 ℃, and calculating the survival rate of the lactobacillus plantarum subjected to the water bath; inoculating the bacterial liquid after water bath into a new culture container containing a culture medium; repeating the second-stage domestication for at least 3 times until the survival rate is more than or equal to 85 percent, and finishing the second-stage domestication;
the third stage acclimatization: inoculating the bacterial liquid after the second-stage domestication into a new culture container containing a culture medium, and culturing at 36-38 ℃ for 23-25h; then mixing the protective agent with the cultured bacterial liquid, carrying out water bath at 69-71 ℃, and calculating the survival rate of the lactobacillus plantarum subjected to the water bath; inoculating the bacterial liquid after water bath into a new culture container containing a culture medium; repeating the third stage acclimatization for at least 4 times until the survival rate is more than or equal to 80 percent, and finishing the third stage acclimatization;
in the bacterial liquid, the protective agent comprises the following raw materials in percentage by weight: 1.5-2% of sodium alginate, 1-1.2% of calcium chloride, 1.5-2% of chitosan, 2.5-3% of yeast cell wall, 0.2-0.4% of gluconic acid-delta-lactone, 3-4% of skimmed milk powder and 1-1.2% of sodium stearate;
survival = (bacterial count after water bath/bacterial count before water bath) 100%.
2. The acclimatization method of high-temperature-resistant lactobacillus plantarum according to claim 1, wherein the time of water bath in the first stage is 10-12min; the water bath time in the second stage is 10-12min; the water bath time in the third stage is 10-12min;
preferably, the first stage acclimatization is repeated at least 3 times, the second stage acclimatization is repeated at least 4 times, and the third stage acclimatization is repeated at least 6 times.
3. The domestication method of the high temperature resistant lactobacillus plantarum according to claim 1, wherein the rejuvenation before domestication of the strain to be domesticated is further included before domestication in the first stage, and comprises inoculating lactobacillus plantarum into a culture medium, standing and culturing at 36-38 ℃ for 23-25h; the rejuvenated lactobacillus plantarum was then used for the first stage acclimatization.
4. The acclimatization method of high-temperature-resistant lactobacillus plantarum according to claim 3, wherein the culture medium comprises the following raw materials: 8-10g/L of peptone, 4-5g/L of yeast powder, 10-12g/L of glucose, 4-5g/L of sodium acetate, 2-2.2g/L of diammonium citrate, 2-2.2g/L of dipotassium phosphate and water.
5. The acclimatization method of high-temperature-resistant lactobacillus plantarum according to claim 1, wherein the bacterial liquid inoculum size of the first-stage acclimatization, the second-stage acclimatization and the third-stage acclimatization is 1-1.2%.
6. The acclimatization method of high-temperature-resistant lactobacillus plantarum according to claim 1, wherein the culture of the first-stage acclimatization, the second-stage acclimatization, and the third-stage acclimatization is static culture.
7. A preparation method of lactobacillus plantarum powder is characterized by comprising the following steps: inoculating the domesticated strain obtained by the domestication method of the high temperature resistant lactobacillus plantarum described in any one of claims 1 to 6 into a culture medium to prepare a seed solution, then inoculating the seed solution into a fermentation tank for culture, mixing the cultured bacterial solution with a protective agent, and spray-drying to obtain bacterial powder.
8. A method for preparing Lactobacillus plantarum powder according to claim 7, characterized in that the culture medium in the fermentor comprises the following raw materials: 10-12g/L of peptone, 10-12g/L of yeast powder, 10-12g/L of corn flour, 20-24g/L of glucose, 4-5g/L of sodium acetate, 2-2.2g/L of diammonium citrate, 2-2.2g/L of dipotassium hydrogen phosphate, 0.2-0.3g/L of magnesium sulfate monohydrate, 0.2-0.3g/L of manganese sulfate monohydrate and water;
preferably, the culture temperature in the fermentation tank is 36-38 ℃, and the standing culture is carried out for 30-32 hours;
preferably, the air outlet temperature during spray drying is 70 ℃ +/-3 ℃.
9. The method for preparing lactobacillus plantarum powder according to claim 7, wherein the culture medium of the seed liquid comprises the following raw materials: 8-10g/L of peptone, 4-5g/L of yeast powder, 10-12g/L of glucose, 4-5g/L of sodium acetate, 2-2.2g/L of diammonium citrate, 2-2.2g/L of dipotassium phosphate and water.
10. A lactobacillus plantarum powder obtained by the method for its preparation according to any one of claims 7-9.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616656A (en) * | 2003-11-10 | 2005-05-18 | 中国科学院大连化学物理研究所 | Method for preparing fixed yeast cell micro capsule |
| CN101869149A (en) * | 2010-05-18 | 2010-10-27 | 中国农业大学 | Method for producing bacterial powder for preparing sour liquid bean curd |
| CN101914518A (en) * | 2010-08-02 | 2010-12-15 | 天津商业大学 | Method for preparing Lactobacillus casei microcapsule bacterial powder by spray drying |
| CN108740013A (en) * | 2018-06-07 | 2018-11-06 | 宁夏亿美生物科技有限公司 | A kind of sour milk powder and preparation method thereof |
| CN109234205A (en) * | 2018-11-07 | 2019-01-18 | 福建傲农生物科技集团股份有限公司 | A kind of the preservation protective agent and freeze-drying store method of lactobacillus acidophilus |
| CN110623066A (en) * | 2019-08-21 | 2019-12-31 | 厦门益力康生物科技有限公司 | Composite probiotic product and preparation method thereof |
| CN111154689A (en) * | 2020-01-20 | 2020-05-15 | 内蒙古农业大学 | Method for improving heat-resistant survival rate of probiotics |
| CN111206006A (en) * | 2020-03-25 | 2020-05-29 | 福建傲农生物科技集团股份有限公司 | Screening and domesticating method of high-temperature-resistant enterococcus faecalis |
-
2022
- 2022-09-06 CN CN202211083770.8A patent/CN115261298A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616656A (en) * | 2003-11-10 | 2005-05-18 | 中国科学院大连化学物理研究所 | Method for preparing fixed yeast cell micro capsule |
| CN101869149A (en) * | 2010-05-18 | 2010-10-27 | 中国农业大学 | Method for producing bacterial powder for preparing sour liquid bean curd |
| CN101914518A (en) * | 2010-08-02 | 2010-12-15 | 天津商业大学 | Method for preparing Lactobacillus casei microcapsule bacterial powder by spray drying |
| CN108740013A (en) * | 2018-06-07 | 2018-11-06 | 宁夏亿美生物科技有限公司 | A kind of sour milk powder and preparation method thereof |
| CN109234205A (en) * | 2018-11-07 | 2019-01-18 | 福建傲农生物科技集团股份有限公司 | A kind of the preservation protective agent and freeze-drying store method of lactobacillus acidophilus |
| CN110623066A (en) * | 2019-08-21 | 2019-12-31 | 厦门益力康生物科技有限公司 | Composite probiotic product and preparation method thereof |
| CN111154689A (en) * | 2020-01-20 | 2020-05-15 | 内蒙古农业大学 | Method for improving heat-resistant survival rate of probiotics |
| CN111206006A (en) * | 2020-03-25 | 2020-05-29 | 福建傲农生物科技集团股份有限公司 | Screening and domesticating method of high-temperature-resistant enterococcus faecalis |
Non-Patent Citations (1)
| Title |
|---|
| 胡国才;黄静;吴晓玉;: "猪源肠道嗜酸乳杆菌的分离、鉴定与驯化", 动物营养学报, vol. 27, no. 04, pages 1 * |
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