CN114292788A - Preparation method of active probiotics by freeze drying - Google Patents
Preparation method of active probiotics by freeze drying Download PDFInfo
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- CN114292788A CN114292788A CN202111663525.XA CN202111663525A CN114292788A CN 114292788 A CN114292788 A CN 114292788A CN 202111663525 A CN202111663525 A CN 202111663525A CN 114292788 A CN114292788 A CN 114292788A
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application relates to the technical field of probiotics, and particularly discloses a preparation method of freeze drying of active probiotics. The preparation method of freeze-drying active probiotics comprises the following steps: preparing a culture medium, culturing probiotics, collecting thalli, preparing a protective agent and carrying out vacuum freeze drying. This application is through compound as the protective agent with soluble starch, trehalose, skim milk and inorganic salt, through four synergistic effect, has improved the survival rate of thallus when freeze-drying.
Description
Technical Field
The application relates to the technical field of probiotics, in particular to a preparation method for freeze drying of active probiotics.
Background
The freeze drying technology is a drying technology that freezes wet materials or solutions into solid state at a lower temperature, then directly sublimates the moisture in the solid state into gas state under vacuum, and finally dehydrates the materials. The drying of the material is completed in a frozen state, and compared with other drying methods, the material has the advantages of little change of physical structure and molecular structure, better preservation of organization structure and appearance form, excellent water-rich property and capability of recovering the state before drying in a short time. Because the drying process is carried out at a very low temperature and basically isolates air, the biological, chemical or physical changes of the heat-sensitive substances are effectively inhibited, and the active substances in the raw materials are well preserved. Therefore, the freeze drying technology is one of the best methods for preserving strains at present, and is widely applied to probiotic freeze-dried powder, probiotic preserved strains and the like.
However, in the freeze-drying process, too low a temperature may cause intracellular water crystallization, cell dehydration, or protein inactivation, which may result in cell damage or death, and may adversely affect the production and storage of the bacterial strain.
In view of the above-mentioned related technologies, the inventors of the present invention have considered that the freeze-drying technology of active probiotics has a problem of low survival rate of the bacterial cells, and thus have limited the use and development thereof.
Disclosure of Invention
In order to improve the survival rate of active probiotics in a freeze drying technology, the application provides a preparation method of freeze drying the active probiotics.
The application provides a preparation method of freeze drying of active probiotics, which adopts the following technical scheme:
a preparation method of freeze-drying active probiotics comprises the following steps:
preparation of a culture medium: the culture medium is prepared and sterilized by the following raw materials in parts by weight: 5-10 parts of soybean peptone, 1-3 parts of yeast extract powder, 2-4 parts of beef extract, 0.1-0.5 part of magnesium sulfate, 10-15 parts of agar and the balance of distilled water;
culturing probiotics: inoculating the preserved probiotic strains into the culture medium for culturing, and cooling and standing the culture medium when the culture medium is cultured to a logarithmic phase to obtain probiotic fermentation liquor;
and (3) collecting thalli: centrifuging the probiotic fermentation liquor, and removing supernatant to obtain probiotic mud;
preparing a protective agent: the protective agent is prepared and sterilized by the following raw materials in parts by weight: 1-3 parts of soluble starch, 1-5 parts of trehalose, 3-5 parts of skim milk, 0.5-1 part of inorganic salt and the balance of distilled water;
vacuum freeze drying: mixing the probiotic bacteria mud with a protective agent to prepare an emulsion; and (3) carrying out vacuum freeze drying on the emulsion to prepare freeze-dried fungus powder, and storing after vacuum packaging.
By adopting the technical scheme, the protective agent is added during vacuum freeze drying, so that the strain can be protected from being damaged by low temperature, and the survival rate of the strain is further improved; in the application, the soluble starch in the protective agent is a starch derivative obtained by processing starch by an oxidant, acid, glycerol, enzyme or other methods, and the soluble starch is used as an excipient for preparing probiotic freeze-dried powder, so that the defects of high viscosity and difficult granulation of trehalose and skim milk can be overcome, and the solution can be maintained in a supercooled state due to low permeability although the trehalose and the skim milk cannot enter cells, so that the concentration of the solution is reduced, and the protective effect is achieved; trehalose can permeate cell walls but cannot permeate cell membranes, and skim milk can permeate cell walls and cell membranes at the same time, and both belong to permeable substances, so that the viscosity of the solution is increased after the trehalose is combined with water in freeze drying, the crystallization process of the water is weakened, and the thallus is protected; the inorganic salt is used as a macromolecular substance, can achieve a buffering effect, reduces the damage of low-temperature action to thalli, can form a protective layer on the surface of cells, wraps the thalli, reduces the area of the cells exposed in a medium, and stimulates micromolecular substances to play a protective role, so that the protective effect is achieved; therefore, the protective agent with different permeabilities is compounded from different mechanisms of the protective agent for the permeability of cells, and under the combined action mechanism of micromolecules and macromolecules, the freeze-drying protective agent formed by compounding the soluble starch, the trehalose, the skim milk and the inorganic salt has an excellent protective effect on strains, so that the survival rate of the strains of the probiotics in the freeze-drying technology is improved.
In a specific embodiment, the soluble starch is prepared by the following method: reacting starch with dry hydrogen chloride under a vacuum condition to obtain acidic soluble starch, neutralizing the acidic soluble starch, and crushing to obtain soluble starch; the starch is one of corn starch, sweet potato starch or potato starch.
By adopting the technical scheme, the reaction efficiency in preparation is high, centrifugation and drying are not needed, the reaction is simple, no wastewater is generated, and the production is environment-friendly.
In a particular embodiment, the inorganic salt is one of sodium citrate, sodium acetate or phosphate.
In a specific embodiment, the mass ratio of the probiotic bacteria mud and the protective agent is 1: (1-3).
By adopting the technical scheme, the mass ratio of the probiotic bacteria mud to the protective agent is optimized, and the survival rate of the probiotic bacteria after freeze drying is favorably improved.
In a particular embodiment, the probiotic bacterial strain is one of bifidobacterium, lactobacillus bulgaricus and streptococcus thermophilus.
By adopting the technical scheme, the bifidobacteria is used as the most beneficial bacteria in the intestinal tract, can form a protective barrier on the surface of the intestinal tract mucosa, is lower than the invasion and damage of harmful bacteria on the intestinal tract mucosa, generates acetic acid and lactic acid to ensure that the intestinal tract is acidic, inhibits the growth and reproduction of the harmful bacteria, and maintains the normal flora balance of the intestinal tract; the functions of the lactobacillus bulgaricus, the streptococcus thermophilus and the bifidobacteria in intestinal tracts are similar, and only the existing parts are different, so that the lactobacillus bulgaricus, the streptococcus thermophilus and the bifidobacteria can be commonly used under the same preparation method, and the prepared probiotic freeze-dried powder has higher survival rate.
In a specific embodiment, the probiotic is cultured by activated culture and then expanded culture, specifically: inoculating the preserved probiotic bacteria into the culture medium according to the inoculation amount of 5-10%, culturing at 37-43 ℃ for 24-32h to obtain an activated culture solution, inoculating 5-10% of the activated culture solution into the culture medium again, culturing at 37-43 ℃ for 24-32h to obtain an expanded culture solution, and cooling and standing the expanded culture solution to obtain the probiotic fermentation broth.
By adopting the technical scheme, the collected probiotics are firstly activated and cultured and then expanded, so that the influence on strain production caused by insufficient living space or overlarge density and nutrition obstacle of strains is avoided.
In a specific embodiment, the cooling temperature of the expanding culture solution is 10-20 ℃, and the standing time is 2-6 h.
By adopting the technical scheme, the cooling temperature and the standing time of the culture solution are optimized and enlarged, so that the probiotic fermentation liquor can be generated better.
In a specific possible embodiment, the emulsion is freeze-dried using a vacuum freeze-dryer, in particular: placing the emulsifier in a vacuum freeze dryer, pre-freezing for 1h at-40 ℃ to-30 ℃, starting a vacuum pump, vacuumizing until the vacuum degree is 60-80Pa, and freeze-drying for 20-40h in vacuum.
By adopting the technical scheme, the pre-freezing is a process of freezing strains containing a large amount of water into solid, and the freeze-drying is to separate the water in the freeze-dried strains in a sublimation way, namely, the water is directly changed from ice into gas, so the pre-freezing is a freezing basis, and the speed of the drying process and the quality of the freeze-dried strains are determined to a greater extent in the pre-freezing process.
In a specific embodiment, the probiotic fermentation broth is centrifuged by pipeline centrifugation, wherein the temperature is 4-10 ℃, the rotation speed is 5000-7000r/min, and the time is 8-10 min.
By adopting the technical scheme, the high-density probiotic mud can be obtained, so that the survival rate of probiotics is improved.
In a specific embodiment, the probiotic fermentation broth further comprises a second centrifugation, specifically: washing the precipitate obtained by pipeline type centrifugation with sterile normal saline with the same volume, centrifuging at the rotating speed of 3000-5000r/min for 4-7min, and discarding the supernatant to obtain the probiotic bacterial mud.
Through adopting above-mentioned technical scheme, aseptic normal saline washs the impurity in the precipitate and separates out impurity to guarantee the purity of probiotic bacterial mud, reduce the negative effects that impurity produced in to the follow-up freeze-drying process of bacterial.
In summary, the present application has at least one of the following beneficial technical effects:
1. according to the method, the soluble starch, the trehalose, the skim milk and the inorganic salt are compounded, and the protective effect of the protective agent on the strain during low-temperature freeze drying is improved through the synergistic effect of the soluble starch, the trehalose, the skim milk and the inorganic salt, so that the strain has a higher survival rate after freeze drying;
2. the survival rate of the protective agent to the strains is further improved by optimizing the mass ratio of the protective agent to the bacterial sludge during mixing;
2. this application adopts the resistant degradation of dry hydrogen chloride mixed starch to prepare soluble starch, not only has reaction efficiency height, does not have waste water to produce moreover, has the environmental protection effect.
Detailed Description
The present application is described in further detail below with reference to preparation examples and examples.
Among the relevant raw materials used in the preparation examples and examples:
bifidobacterium, Lactobacillus bulgaricus and Streptococcus thermophilus were purchased from Jiangsu microbial research institute, Inc.; soy peptone, yeast extract and beef extract were purchased from beijing obozin biotechnology, llc; magnesium sulfate was purchased from Tianjin university Kewei; agar was purchased from Tianjin northern Tianyi chemical reagent factory; the trehalose brand is 6138-23-4; skim milk was purchased from Olympic Experimental facilities, Inc., Fuzhou; corn starch, sweet potato starch and potato starch are all available from open market all industries, ltd.
Preparation example
Preparation example 1
The preparation example prepares the soluble starch, and the specific method comprises the following steps:
adding 50kg corn starch into a vacuum drier, heating with 60 deg.C water in a jacket, vacuumizing to-0.09 Mpa, adding dry hydrogen chloride, stopping adding hydrogen chloride until the vacuum degree is-0.02 Mpa, and introducing 280g hydrogen chloride; heating at 60 deg.C, rotating for 3 hr, vacuumizing to-0.09 Mpa for 30min, introducing ammonia gas to neutralize to pH 6.5, vacuumizing for 30min, and pulverizing to obtain the final product.
Preparation example 2
This preparation is essentially the same as preparation 1, except that: corn starch is replaced by sweet potato starch.
Preparation example 3
This preparation is essentially the same as preparation 1, except that: corn starch was replaced with potato starch.
Examples
Example 1
The following description will be given by taking example 1 as an example. The embodiment discloses a preparation method of freeze-drying active probiotics, which specifically comprises the following steps:
s1, preparation of medium: adding 25g of soybean peptone, 5g of yeast extract powder, 20g of beef extract, 2.5g of magnesium sulfate and 75g of agar into 5L of distilled water, heating for dissolving, adjusting pH to 6.5, and sterilizing at 121 ℃ for 20min for later use;
s2, culturing probiotics: inoculating the preserved 25mL of bifidobacterium strains into the culture medium for activated culture, adding sterilized liquid paraffin into the culture medium, and culturing for 32 hours at 37 ℃ to form an activated culture solution; inoculating 25mL of activated culture solution into the culture medium for amplification culture, culturing at 37 ℃ for 32h to obtain an amplification culture solution, and standing the amplification culture solution at 10 ℃ for 2h to obtain a fermentation broth;
s3, collecting thalli: performing pipeline type centrifugation on the fermentation liquor for 10min at the temperature of 4 ℃ and the rotation speed of 5000r/min, discarding the supernatant to obtain a precipitate, performing centrifugation on the precipitate with equal volume of sterile physiological saline at the rotation speed of 3000r/min for 7min, and discarding the supernatant to obtain probiotic bacterial sludge;
s4, preparing a protective agent: preparing 10kg of protective agent, wherein 0.1kg of soluble starch, 0.1kg of trehalose, 0.3kg of skim milk, 0.05kg of sodium citrate and the balance of distilled water; sterilizing at 115 deg.C for 20 min; wherein, the soluble starch is prepared by the preparation example 1;
s5, vacuum freeze drying: uniformly stirring the 5kg of probiotic bacteria mud and 5kg of protective agent to form emulsion; placing the emulsion in a vacuum drier, pre-freezing at-40 deg.C for 1 hr, starting a vacuum pump, vacuumizing to vacuum degree of 60Pa, vacuum freeze-drying for 20 hr to obtain lyophilized powder, vacuum packaging, and storing.
Example 2
This embodiment is substantially the same as embodiment 1 except that: s1, adding 50g of soybean peptone, 15g of yeast extract powder, 10g of beef extract, 0.5g of magnesium sulfate and 50g of agar into 5L of distilled water, heating to dissolve, adjusting the pH to 6.5, and sterilizing at 121 ℃ for 20min for later use.
Example 3
This embodiment is substantially the same as embodiment 1 except that: s2, inoculating 50mL of preserved Lactobacillus bulgaricus into the culture medium for activation culture, adding sterilized liquid paraffin into the culture medium, and culturing at 43 ℃ for 24h to form an activation culture solution; inoculating 50mL of activated culture medium into the culture medium for amplification culture, culturing at 43 deg.C for 24 hr to obtain amplification culture medium, and standing at 15 deg.C for 4 hr to obtain fermentation broth.
Example 4
This embodiment is substantially the same as embodiment 1 except that: in the step S2, 50mL of preserved streptococcus thermophilus is inoculated into the culture medium for activation culture, sterilized liquid paraffin is added into the culture medium, and the activated culture solution is formed after 24 hours of culture at 43 ℃; inoculating 50mL of activated culture medium into the culture medium for amplification culture, culturing at 43 deg.C for 24 hr to obtain amplification culture medium, and standing at 20 deg.C for 6 hr to obtain fermentation broth.
Example 5
This embodiment is substantially the same as embodiment 1 except that: and S3, performing pipeline type centrifugation on the fermentation liquor for 8min at the temperature of 10 ℃ and the rotation speed of 7000r/min, discarding the supernatant to obtain a precipitate, performing centrifugation on the precipitate for 4min by using sterile physiological saline with the same volume at the rotation speed of 5000r/min to obtain the probiotic bacterial paste.
Example 6
This embodiment is substantially the same as embodiment 1 except that: in step S4, the soluble starch prepared in preparation example 2 was used.
Example 7
This embodiment is substantially the same as embodiment 1 except that: in step S4, the soluble starch prepared in preparation example 3 was used.
Example 8
This embodiment is substantially the same as embodiment 1 except that: in the step of S4, 10kg of protective agent is prepared, wherein 0.3kg of soluble starch, 0.5kg of trehalose, 0.5kg of skim milk, 0.1kg of sodium citrate and the balance of distilled water.
Example 9
This embodiment is substantially the same as embodiment 1 except that: in the step S4, 10kg of protective agent is prepared, wherein 0.1kg of soluble starch, 0.1kg of trehalose, 0.3kg of skim milk, 0.05kg of sodium acetate and the balance of distilled water.
Example 10
This embodiment is substantially the same as embodiment 1 except that: in the step S4, preparing 10kg of protective agent, wherein 0.1kg of soluble starch, 0.1kg of trehalose, 0.3kg of skim milk, 0.05kg of diammonium phosphate and the balance of distilled water; in this embodiment, the inorganic salt is diammonium phosphate, but is not limited thereto.
Example 11
This embodiment is substantially the same as embodiment 1 except that: in the step S5, 5kg of probiotic bacteria mud and 10kg of protective agent are uniformly stirred to form the emulsifier.
Example 12
This embodiment is substantially the same as embodiment 1 except that: in the step S5, 5kg of probiotic bacteria mud and 15kg of protective agent are uniformly stirred to form the emulsifier.
Example 13
This embodiment is substantially the same as embodiment 1 except that: s5, placing the emulsion in a vacuum drier, pre-freezing for 1h at-30 ℃, starting a vacuum pump, vacuumizing to 80Pa, freeze-drying for 40h in vacuum to form freeze-dried powder, and storing after vacuum packaging.
Comparative example 1
This comparative example differs from example 1 in that: in the step S4, 10kg of protective agent is prepared, wherein 0.55kg of trehalose and the balance of distilled water.
Comparative example 2
This comparative example differs from example 1 in that: in the step S4, 10kg of protective agent is prepared, wherein the protective agent comprises 0.1kg of trehalose, 0.45kg of skim milk and the balance of distilled water.
Comparative example 3
This comparative example differs from example 1 in that: in the step of S4, 10kg of protective agent is prepared, wherein 0.1kg of trehalose, 0.3kg of skim milk, 0.15kg of sodium citrate and the balance of distilled water.
Performance test
The same weight of the lyophilized powder obtained in examples 1 to 13 was used as the control samples 1 to 13, and the same weight of the lyophilized powder obtained in comparative examples 1 to 3 was used as the control samples 1 to 3. The test sample and the control sample were subjected to performance measurement, and the results are shown in Table 1.
Determination of survival rate: naturally thawing the freeze-dried bacterial powder, adding sterilized normal saline for rehydration for 30min, sucking 1mL of bacterial liquid which is equal to the bacterial liquid before freeze-drying by using a micropipettor, and diluting and coating the bacterial liquid for counting. The number of viable bacteria before and after freeze-drying is measured, and finally the freezing survival rate is calculated, wherein the survival rate (%) — the total number of viable bacteria after sample freeze-drying/the total number of viable bacteria before sample freeze-drying is multiplied by 100%.
Table 1 table of performance testing data
Referring to table 1, in combination with example 1 and comparative examples 1 to 3, it can be seen that the protective agent formed by compounding soluble starch, trehalose, skim milk and inorganic salt has better protective effect on the frozen probiotics than the protective agent formed by compounding trehalose, trehalose and skim milk, and trehalose, skim milk and sodium citrate (inorganic salt); the soluble starch can not enter cells, the trehalose can enter cell walls and can not enter cell membranes, and the skim milk can enter the cell walls and the cell membranes, so that the three have different protection effects on probiotics on different permeability mechanisms; soluble starch, trehalose and skim milk all belong to the micromolecular substance, and inorganic salt belongs to macromolecular substance, and in inorganic salt added to the protective agent, not only self can form the protective layer on the cell surface, can stimulate soluble starch, trehalose and skim milk to play the guard action simultaneously, therefore four have good synergistic effect, have improved the survival rate of probiotic after freezing.
Referring to Table 1, in combination with examples 1-2, it can be seen that the frozen microbial cells still had good survival rates by varying the contents of the respective components in the medium within appropriate ranges.
Referring to table 1, in combination with examples 1, 3 and 4, it can be seen that the frozen species still have good survival rate by replacing bifidobacterium with lactobacillus bulgaricus or streptococcus thermophilus and appropriately adjusting the amount of the strain, the culture temperature, the culture time and the like; the optimum growth temperature of the bifidobacteria is 37 ℃, so the bifidobacteria are cultured at 37 ℃; the optimal growth temperature of Lactobacillus bulgaricus and Streptococcus thermophilus is 40-45 deg.C, so that the culture is performed at 43 deg.C.
Referring to table 1, in combination with examples 1 and 5, it can be seen that when the cells were collected, the survival rate of the freeze-dried powder was still high by changing the parameters of the centrifugation speed, temperature and time within the appropriate range.
Referring to table 1, in combination with examples 1, 6 and 7, it can be seen that when the soluble starch is prepared, the corn starch is replaced by sweet potato starch or potato starch, the performance of the formed soluble starch is not changed greatly, so that the protective agent formed by different soluble starches still has good protective effect on the thallus.
Referring to Table 1, in combination with examples 1 and 8, the frozen cells still had good survival rates by varying the contents of the respective components in the protective agent within appropriate ranges.
Referring to table 1, in combination with examples 1, 9 and 10, it can be seen that the inorganic salt in the protecting agent is replaced by sodium citrate or diammonium acetate, and the formed protecting agent still has good protection effect on the thallus during freezing.
Referring to table 1, in combination with examples 1, 11 and 12, it can be seen that, by changing the mass ratio of the protective agent to the probiotic bacterial sludge, when the mass ratio of the protective agent to the probiotic bacterial sludge is 2:1, the protective effect of the protective agent on the bacterial cells is the best, and the bacterial cells have excellent survival rates; when the mass ratio of the protective agent to the probiotic bacterial mud is 1:1, the relative content of the protective agent is less, the wrapping property to the probiotics is poor, and the number of the viable bacteria is lower; when the mass ratio of the protective agent to the probiotic bacteria mud is 3:1, the protective agent is relatively excessive, and the probiotics are dispersed in more protective agents, so that the number of the viable bacteria in the measured freeze-dried powder is also low.
Referring to table 1, in combination with examples 1 and 13, it can be seen that freeze-dried powder prepared by properly adjusting the freezing time, temperature and degree of vacuum in the vacuum freeze-drying step all had excellent survival rate.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. A preparation method of freeze drying of active probiotics is characterized by comprising the following steps: the method comprises the following steps:
preparation of a culture medium: the culture medium is prepared and sterilized by the following raw materials in parts by weight: 5-10 parts of soybean peptone, 1-3 parts of yeast extract powder, 2-4 parts of beef extract, 0.1-0.5 part of magnesium sulfate, 10-15 parts of agar and the balance of distilled water;
culturing probiotics: inoculating the preserved probiotic strains into the culture medium for culturing, and cooling and standing the culture medium when the culture medium is cultured to a logarithmic phase to obtain probiotic fermentation liquor;
and (3) collecting thalli: centrifuging the probiotic fermentation liquor, and removing supernatant to obtain probiotic mud;
preparing a protective agent: the protective agent is prepared and sterilized by the following raw materials in parts by weight: 1-3 parts of soluble starch, 1-5 parts of trehalose, 3-5 parts of skim milk, 0.5-1 part of inorganic salt and the balance of distilled water;
vacuum freeze drying: mixing the probiotic bacteria mud with a protective agent to prepare an emulsion; and (3) carrying out vacuum freeze drying on the emulsion to prepare freeze-dried fungus powder, and storing after vacuum packaging.
2. The method for freeze-drying active probiotics according to claim 1, characterized in that: the preparation method of the soluble starch comprises the following steps: reacting starch with dry hydrogen chloride under a vacuum condition to obtain acidic soluble starch, neutralizing the acidic soluble starch, and crushing to obtain soluble starch; the starch is one of corn starch, sweet potato starch or potato starch.
3. The method for freeze-drying active probiotics according to claim 1, characterized in that: the inorganic salt is one of sodium citrate, sodium acetate or phosphate.
4. The method for freeze-drying active probiotics according to claim 1, characterized in that: the mass ratio of the probiotic bacteria mud to the protective agent is 1: (1-3).
5. The method for freeze-drying active probiotics according to claim 1, characterized in that: the probiotic strain is one of bifidobacterium, lactobacillus bulgaricus and streptococcus thermophilus.
6. The method for freeze-drying active probiotics according to claim 1, characterized in that: when the probiotics are cultured, activation culture is firstly carried out, and then expansion culture is carried out, specifically: inoculating the preserved probiotic bacteria into the culture medium according to the inoculation amount of 5-10%, culturing at 37-43 ℃ for 24-32h to obtain an activated culture solution, inoculating 5-10% of the activated culture solution into the culture medium again, culturing at 37-43 ℃ for 24-32h to obtain an expanded culture solution, and cooling and standing the expanded culture solution to obtain the probiotic fermentation broth.
7. The method for freeze-drying active probiotics according to claim 6, characterized in that: the cooling temperature of the amplification culture solution is 10-20 ℃, and the standing time is 2-6 h.
8. The method for freeze-drying active probiotics according to claim 1, characterized in that: and (2) freeze-drying the emulsion by using a vacuum freeze dryer, which specifically comprises the following steps: placing the emulsifier in a vacuum freeze dryer, pre-freezing for 1h at-40 ℃ to-30 ℃, starting a vacuum pump, vacuumizing until the vacuum degree is 60-80Pa, and freeze-drying for 20-40h in vacuum.
9. The method for freeze-drying active probiotics according to claim 1, characterized in that: the probiotic fermentation liquor is centrifuged by adopting a pipeline type centrifuge, wherein the temperature is 4-10 ℃, the rotating speed is 5000-7000r/min, and the time is 8-10 min.
10. The method for freeze-drying active probiotics according to claim 9, characterized in that: the probiotic fermentation liquor further comprises secondary centrifugation, and specifically comprises the following steps: washing the precipitate obtained by pipeline type centrifugation with sterile normal saline with the same volume, centrifuging at the rotating speed of 3000-5000r/min for 4-7min, and discarding the supernatant to obtain the probiotic bacterial mud.
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