CN115478017A - Cryoprotectant for improving activity of lactobacillus bulgaricus, freezing method and application - Google Patents
Cryoprotectant for improving activity of lactobacillus bulgaricus, freezing method and application Download PDFInfo
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- CN115478017A CN115478017A CN202211243216.1A CN202211243216A CN115478017A CN 115478017 A CN115478017 A CN 115478017A CN 202211243216 A CN202211243216 A CN 202211243216A CN 115478017 A CN115478017 A CN 115478017A
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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
- C12N1/04—Preserving or maintaining viable microorganisms
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- 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
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Abstract
The invention provides a cryoprotectant for improving the activity of lactobacillus bulgaricus, a freezing method and application thereof, wherein the cryoprotectant comprises 4-20 parts of glycerol, 0.2-5 parts of L-linoleic acid and 0.2-5 parts of alpha-linolenic acid in parts by mass. The invention creatively applies the combination of the glycerol, the L-linoleic acid and the alpha-linolenic acid to the cryopreservation of the lactobacillus bulgaricus, the three supplement each other, and the invention has the synergistic effect in the aspects of improving and maintaining the short-term and long-term survival rate and the thallus activity. The invention uses the cryoprotectant containing glycerol, L-linoleic acid and alpha-linolenic acid, realizes the great improvement of the viable count and the viable stability of the lactobacillus bulgaricus, and can provide reference thought for large-scale production.
Description
Technical Field
The invention belongs to the technical field of microbial cryopreservation, and particularly relates to a cryoprotectant for improving the activity of lactobacillus bulgaricus, a freezing method and application.
Background
The lactobacillus bulgaricus is gram-positive, anaerobic or facultative anaerobic and bacillus-free, and is widely applied to dairy product fermented food. The lactobacillus bulgaricus has longer length than common lactobacillus, and has size of (0.6-1) × (2-20) μm, and has poor freezing resistance and poor freezing stability due to damage to cell membrane during cryopreservation, thereby affecting activity of seed liquid and stability of production batch. At present, the method for preserving the lactobacillus bulgaricus mainly adopts a glycerol method, the preservation method is mature, but in the freezing storage process, the lactobacillus bulgaricus cells die quickly, and are not easy to preserve for a long time, and the influence on the industrialized production is large. There is a need to develop a method for prolonging the storage time of lactobacillus bulgaricus glycerol tubes. At present, the mechanism of damage of the lactobacillus bulgaricus thallus by freezing is not completely clear. Two mechanisms of damage that depend on the rate of cooling have been proposed, one being that at low freezing rates, cell damage is mainly caused by osmotic pressure on the cells (so-called "solution effect"). The second is the formation of extracellular ice crystals during freezing, which induce high solute concentrations in the medium, thereby destroying the cell membrane.
Patent document CN111107877a discloses a method and composition for culturing and preserving bacteria, wherein the cultured and preserved bacteria exhibit improved viability/growth compared to bacteria preserved/stored by means other than the subject method. More specifically, the invention discloses methods and compositions for improving bacterial viability after cryopreservation. The method comprises the following steps: a) Combining a first bacterial species with at least one second bacterial species to produce a bacterial mixture, wherein the first bacterial species is a member of the family aminoacidococcaceae or an aminoacidococcaceae, wherein the first bacterial species is present in the bacterial mixture in an amount sufficient to impart cryoprotection to the at least one second bacterial species, and wherein the member of the family aminoacidococcaceae species is spirochete mobilis; b) Culturing the bacterial mixture to produce a cultured bacterial mixture, wherein the culturing is for a period of time sufficient to confer the cryoprotection to the at least one second bacterial species in the cultured bacterial mixture; and c) cryopreserving the cultured bacterial mixture to produce a cryopreserved bacterial culture; wherein in the bacterial proliferation assay, the cryopreserved bacterial culture exhibits at least 10 x increased bacterial proliferation of the at least one second bacterial species relative to the bacterial proliferation of a cryopreserved bacterial culture comprising the at least one second bacterial species and lacking the first bacterial species after reconstitution.
The lactobacillus bulgaricus has larger thallus, and cell membrane damage is easy to occur in the process of cryopreservation, so that the lactobacillus bulgaricus has poor freezing resistance and stability. In the prior art, the method for preserving the lactobacillus bulgaricus mainly adopts a glycerol method, the lactobacillus bulgaricus has fast cell decay, is not easy to preserve for a long time, and has larger influence on industrialized production, so that the method for exploring, optimizing and improving the low-temperature freezing of the lactobacillus bulgaricus is very meaningful.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a cryoprotectant, a freezing method and application, and particularly provides the cryoprotectant for improving the activity of lactobacillus bulgaricus, the freezing method and the application.
In order to achieve the purpose, the invention adopts the following technical scheme:
according to the first aspect, the invention provides a cryoprotectant for improving the activity of lactobacillus bulgaricus, which comprises 4-20 parts of glycerol, 0.2-5 parts of L-linoleic acid and 0.2-5 parts of alpha-linolenic acid in parts by mass.
The glycerol extract can be 4 parts, 6 parts, 8 parts, 10 parts, 12 parts, 14 parts, 16 parts, 18 parts or 20 parts by weight.
The L-linoleic acid can be 0.2 part, 0.8 part, 1.4 parts, 2 parts, 2.8 parts, 3.6 parts, 4.4 parts or 5 parts by weight and the like.
The alpha-linolenic acid can be 0.2 part, 0.8 part, 1.4 parts, 2 parts, 2.8 parts, 3.6 parts, 4.4 parts or 5 parts by weight and the like.
Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
The cryoprotectant comprises glycerol, L-linoleic acid and alpha-linolenic acid, wherein the glycerol has small molecular weight and high solubility, is easy to penetrate through bacteria, plays a role in lowering the freezing point, reduces the formation of ice crystals under the low-temperature condition and plays a role in anti-freezing protection of thalli; the L-linoleic acid and the alpha-linolenic acid are important organic acids on the cell membrane of the lactobacillus bulgaricus, and the appropriate proportion of the linoleic acid and the linolenic acid can reduce the damage of the cell membrane caused by the change of osmotic pressure and the lipid oxidation of the cell membrane in the process of cryopreservation; the invention creatively applies the glycerol, the L-linoleic acid and the alpha-linolenic acid to improve the activity of the lactobacillus bulgaricus in cryopreservation, the three supplement each other, and the invention has synergistic effect in improving and maintaining the short-term and long-term survival rate and the thallus activity. The invention uses the cryoprotectant containing glycerol, L-linoleic acid and alpha-linolenic acid, realizes the great improvement of the viable count and the stability of the lactobacillus bulgaricus, and can provide a reference idea for large-scale production.
Preferably, the cryoprotectant further comprises an antifreeze protein.
Preferably, the cryoprotectant further comprises 0.02 to 2 parts by mass of an antifreeze protein.
The mass portion of the antifreeze protein can be 0.02 portion, 0.2 portion, 0.6 portion, 1 portion, 1.4 portions, 1.8 portions or 2 portions, etc. Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
The cryoprotectant also comprises antifreeze protein, and the antifreeze protein is a protein for improving the low-temperature adaptability of organisms. The antifreeze protein found at present can avoid the damage to organisms caused by low-temperature stress, and the action mechanism for improving the adaptability of organisms to low temperature mainly comprises the following steps: lowering freezing point, changing ice crystal morphology, inhibiting ice crystal growth, etc. The sources of antifreeze proteins are mainly fish, insects and plants. The cryoprotectant provided by the invention is added with the antifreeze protein which can be matched with 3 components of glycerol, L-linoleic acid and alpha-linolenic acid for synergistic interaction, and has stronger functions in reducing cell damage of lactobacillus bulgaricus and enhancing acidification activity and survival rate of lactobacillus bulgaricus; when the part of the antifreeze protein is less than 0.02 part, the joint application effect of the antifreeze protein and 3 components is not remarkably enhanced, and when the specific part of the antifreeze protein is 0.02-2 parts, the cryoprotectant has more excellent antifreeze activity and is more economical and practical.
In a second aspect, the use of a cryoprotectant as described in the first aspect in the cryopreservation of lactobacillus gasseri.
In a third aspect, the freezing method for improving the activity of lactobacillus bulgaricus comprises the steps of mixing lactobacillus bulgaricus liquid with the cryoprotectant in the first aspect uniformly, cooling and freezing.
The lactobacillus bulgaricus liquid is firstly mixed with the cryoprotectant uniformly, then cooled and frozen, and finally placed and stored for a long time. And the addition of the cooling and freezing step is favorable for slowing down the nucleation of a lactobacillus bulgaricus bacterial liquid freezing system and the formation of small ice crystals inside and outside cells, so that the whole freezing process is less cell damage, and the lactobacillus bulgaricus has higher short-term and long-term storage activity and survival rate.
Preferably, the lactobacillus bulgaricus bacterial liquid is prepared by the following method: inoculating and culturing the lactobacillus bulgaricus strain to obtain the lactobacillus bulgaricus bacterial liquid.
Preferably, the medium used for inoculation of lactobacillus bulgaricus species comprises MRS medium.
The culture medium used for inoculation of the lactobacillus bulgaricus strain comprises an MRS culture medium, wherein the MRS culture medium is a conventional culture medium, and mainly comprises peptone, beef extract powder, yeast extract powder, glucose, tween-80, dipotassium phosphate, sodium acetate, triammonium citrate, magnesium sulfate, manganese sulfate and agar powder.
Preferably, the culture temperature for inoculation of the Lactobacillus bulgaricus strain is 30-40 ℃.
The specific value of 30-40 deg.C may be 30 deg.C, 32 deg.C, 34 deg.C, 36 deg.C, 38 deg.C or 40 deg.C etc. Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
Preferably, the lactobacillus bulgaricus strain is inoculated for a culture period of 1 to 3 days.
The specific value of 1 to 3 days can be 1 day, 2 days or 3 days.
Preferably, the inoculation is carried out by streaking on the surface of a solid medium to obtain a single colony of lactobacillus bulgaricus.
Preferably, the scoring is repeated 1-3 times.
The specific value in the 1 to 3 times may be 1 time, 2 times or 3 times.
Preferably, the culturing includes the step of picking up the single colony and inoculating the single colony to a liquid medium for culturing.
Preferably, the temperature of the culture is 30-40 ℃, and the time of the culture is 6-12h.
The specific value of 30-40 deg.C may be 30 deg.C, 32 deg.C, 34 deg.C, 36 deg.C, 38 deg.C or 40 deg.C etc. Other specific values within the above ranges can be selected, and are not described in detail herein.
The specific numerical value of 6-12h can be 6h, 7h, 8h, 9h, 10h, 11h or 12h, etc. Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
Preferably, the raw materials of the culture medium include glycine, sorbitol, betaine, and sodium chloride.
The raw materials of the culture medium comprise glycine, sorbitol, betaine and sodium chloride, wherein the glycine, sorbitol and betaine can enter lactobacillus bulgaricus cells, ice crystals formed inside the cells in the freezing process are reduced, the formation of large ice crystals inside the cells is effectively controlled, the intracellular damage caused by the ice crystals inside the cells is further reduced, and the cell survival rate and the cell activity are improved; the addition of the sodium chloride can effectively maintain the intracellular osmotic pressure of the lactobacillus bulgaricus and promote glycine, sorbitol and betaine to enter the cells to play a protective role under the stress condition of high osmotic pressure. Glycine, sorbitol and betaine, three components supplement each other, can synergize, and then cooperate with the enhancement effect of sodium chloride, the culture medium containing 4 raw materials has stronger effects of reducing cell damage and improving the activity and viable count of lactobacillus bulgaricus under short-term and long-term freezing conditions.
Preferably, the raw materials of the culture medium comprise 1-8 parts of glycine, 3-40 parts of sorbitol, 1-14 parts of betaine and 30-60 parts of sodium chloride in parts by mass.
The glycine may be present in an amount of 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, or the like by mass.
The sorbitol can be 3 parts, 5 parts, 8 parts, 11 parts, 14 parts, 17 parts, 20 parts, 23 parts, 26 parts, 29 parts, 32 parts, 35 parts, 38 parts or 40 parts by weight.
The betaine can be 1 part, 2 parts, 4 parts, 6 parts, 8 parts, 10 parts, 12 parts or 14 parts by weight.
The sodium chloride can be 30 parts, 35 parts, 40 parts, 45 parts, 50 parts, 55 parts or 60 parts by mass.
Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
Preferably, the raw materials of the medium further include a carbon source, a nitrogen source, inorganic salts, and a surfactant.
Preferably, the carbon source includes any one or a combination of at least two of glucose, fructose, or sucrose, such as a combination of glucose and fructose, a combination of fructose and sucrose, a combination of glucose and sucrose, and the like, and other combinations can be selected, which is not described in detail herein.
Preferably, the nitrogen source includes any one or a combination of at least two of peptone, yeast extract or corn steep liquor, such as a combination of peptone and yeast extract, a combination of yeast extract and corn steep liquor, a combination of peptone and corn steep liquor, and the like, and other combinations can be selected, and are not described in detail herein.
Preferably, the inorganic salt includes any one of diammonium citrate, dipotassium hydrogen phosphate, magnesium sulfate or manganese sulfate or a combination of at least two of the diammonium citrate, the dipotassium hydrogen phosphate and the magnesium sulfate, the magnesium sulfate and the manganese sulfate, and the like, and other combinations can be selected, and are not described in detail herein.
Preferably, the surfactant includes any one of or a combination of at least two of tween-80, tween-40 or tween-20, such as a combination of tween-80 and tween-40, a combination of tween-40 and tween-20, or a combination of tween-80 and tween-20, and the like, and other combinations can be selected, and are not described in detail herein.
Preferably, the mass ratio of the lactobacillus bulgaricus bacterial liquid to the cryoprotectant is (0.5-10): 1.
The specific value of (0.5-10) can be selected from 0.5, 1, 3, 5, 7, 9 or 10, etc. Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
When the mass ratio of the lactobacillus bulgaricus bacterial liquid to the cryoprotectant is (0.5-10): 1, the cryoprotectant has stronger protective effect on the lactobacillus bulgaricus, and the lactobacillus bulgaricus has higher activity and more viable count under short-term and long-term freezing conditions.
Preferably, the cooling speed of the cooling is 2-10 ℃/min, and the temperature is reduced to-5 ℃.
The specific numerical value of 2-10 deg.C/min can be selected from 2 deg.C/min, 4 deg.C/min, 6 deg.C/min, 8 deg.C/min or 10 deg.C/min. Other specific point values within the above numerical ranges can be selected, and are not described in detail herein.
When the cooling rate is between 2 and 10 ℃/min during cooling, the cooling is in a slow cooling state, the nucleation of a system containing the lactobacillus bulgaricus is slower, the formation of large ice crystals inside and outside cells is less, the cell damage is less, and the acidification activity and the viable count of the lactobacillus bulgaricus are higher.
Compared with the prior art, the invention has the following beneficial effects:
the cryoprotectant comprises glycerol, L-linoleic acid and alpha-linolenic acid, wherein the glycerol has small molecular weight and high solubility, is easy to penetrate through bacteria, plays a role in lowering the freezing point, reduces the formation of ice crystals under the low-temperature condition and plays a role in anti-freezing protection of thalli; the L-linoleic acid and the alpha-linolenic acid are important organic acids on the cell membrane of the lactobacillus bulgaricus, and the appropriate proportion of the linoleic acid and the linolenic acid can reduce the damage of the cell membrane caused by the change of osmotic pressure and the lipid oxidation of the cell membrane in the process of cryopreservation; the invention creatively applies the glycerol, the L-linoleic acid and the alpha-linolenic acid to improve the activity of the lactobacillus bulgaricus in cryopreservation, the three supplement each other, and the invention has synergistic effect in improving and maintaining the short-term and long-term survival rate and the thallus activity. The invention uses the cryoprotectant containing glycerol, L-linoleic acid and alpha-linolenic acid, realizes the great improvement of the viable count and the stability of the lactobacillus bulgaricus, and can provide a reference idea for large-scale production.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
The antifreeze proteins involved in the following examples, comparative examples and test examples are Anfei biological antifreeze protein products from Nanjing Anfei Biotech Ltd; the commercial MRS solid medium was a product purchased from Haibo Biotech, inc. with model number HB 0384-5.
Preparation example 1
The preparation example provides a single colony of lactobacillus bulgaricus, and the specific preparation method comprises the following steps: after melting a lactobacillus bulgaricus ATCC BAA-365 glycerin tube, streaking on a commercial MRS solid culture medium by using an inoculating loop, culturing in an incubator at 37 ℃, after 2 days, selecting a single colony on a solid plate, streaking on the commercial MRS solid culture medium again, culturing in the incubator at 37 ℃ for 2 days, and selecting the single colony on the solid plate to obtain the single colony of the lactobacillus bulgaricus ATCC BAA-365.
Example 1
The embodiment provides a lactobacillus bulgaricus cryopreservation system, which comprises a bacterial liquid and a cryoprotectant, and the preparation method comprises the following steps: preparing a culture medium, wherein the components of the culture medium are as follows: 2 parts of glycine, 5 parts of sorbitol, 2 parts of betaine, 35 parts of sodium chloride, 20 parts of peptone, 5 parts of yeast extract, 40 parts of glucose, 2 parts of diammonium citrate, 1 part of tween-80, 6 parts of magnesium sulfate, 2 parts of manganese sulfate and 2 parts of dipotassium phosphate, and sterilizing at 121 ℃ for 20min after the components of the culture medium are proportionally prepared. The single colony of Lactobacillus bulgaricus prepared in preparation example 1 was inoculated into a culture medium, incubated at 37 ℃ for 10h at constant temperature, and left at 4 ℃ for 15min to obtain the Lactobacillus bulgaricus bacterial solution.
And (3) uniformly mixing 6 parts of glycerol, 1 part of L-linoleic acid, 0.8 part of alpha-linolenic acid and 0.2 part of antifreeze protein to obtain the cryoprotectant.
And (2) uniformly mixing 5 parts of lactobacillus bulgaricus liquid and 5 parts of cryoprotectant, cooling to-5 ℃ from 4 ℃ at the speed of 2 ℃/min by using a temperature-controlled refrigerator, and refrigerating at-80 ℃ to obtain the lactobacillus bulgaricus cryopreservation system.
Example 2
The embodiment provides a lactobacillus bulgaricus cryopreservation system, which comprises a bacterial liquid and a cryoprotectant, and the preparation method comprises the following steps: preparing a culture medium, wherein the components of the culture medium are as follows: 1 part of glycine, 3 parts of sorbitol, 8 parts of betaine, 40 parts of sodium chloride, 18 parts of peptone, 4 parts of yeast extract, 36 parts of glucose, 2 parts of diammonium citrate, 1 part of tween-80, 6 parts of magnesium sulfate, 1 part of manganese sulfate and 2 parts of dipotassium phosphate, and sterilizing at 121 ℃ for 20min after the components of the culture medium are proportionally prepared. And inoculating the single bacterial colony of the lactobacillus bulgaricus prepared in the preparation example 1 into a culture medium, culturing at constant temperature of 35 ℃ for 8h, and standing at 4 ℃ for 15min to obtain the lactobacillus bulgaricus bacterial liquid.
And 4 parts of glycerol, 1.5 parts of L-linoleic acid, 0.7 part of alpha-linolenic acid and 0.02 part of antifreeze protein are mixed uniformly to obtain the cryoprotectant.
And (3) uniformly mixing 4 parts of lactobacillus bulgaricus liquid and 6 parts of cryoprotectant, cooling to-5 ℃ from 4 ℃ at the speed of 3 ℃/min by using a temperature-controlled refrigerator, and refrigerating at-70 ℃ to obtain the lactobacillus bulgaricus cryopreservation system.
Example 3
The embodiment provides a lactobacillus bulgaricus cryopreservation system, which comprises a bacterial liquid and a cryoprotectant, and the preparation method comprises the following steps: preparing a culture medium, wherein the components of the culture medium are as follows: 8 parts of glycine, 10 parts of sorbitol, 1 part of betaine, 30 parts of sodium chloride, 18 parts of peptone, 6 parts of yeast extract, 41 parts of glucose, 2 parts of diammonium citrate, 1 part of tween-80, 6 parts of magnesium sulfate, 2 parts of manganese sulfate and 1 part of dipotassium hydrogen phosphate, and sterilizing at 121 ℃ for 20min after the components of the culture medium are proportionally prepared. The single colony of Lactobacillus bulgaricus prepared in preparation example 1 was inoculated into a culture medium, incubated at 38 ℃ for 11h, and left at 4 ℃ for 15min to obtain the Lactobacillus bulgaricus bacterial solution.
And (3) uniformly mixing 15 parts of glycerol, 0.5 part of L-linoleic acid, 1.8 parts of alpha-linolenic acid and 0.4 part of antifreeze protein to obtain the cryoprotectant.
And (3) uniformly mixing 6 parts of lactobacillus bulgaricus liquid and 4 parts of cryoprotectant, cooling to-5 ℃ from 4 ℃ at the speed of 5 ℃/min by using a temperature-controlled refrigerator, and refrigerating at-80 ℃ to obtain the lactobacillus bulgaricus cryopreservation system.
Example 4
This example provides a lactobacillus bulgaricus cryopreservation system that differs from example 1 only in that the medium does not contain glycine, and the parts by mass of glycine are apportioned to sorbitol, betaine, all as in example 1.
Example 5
This example provides a lactobacillus bulgaricus cryopreservation system that differs from example 1 only in that no sorbitol is contained in the medium and the parts by mass of sorbitol are apportioned to glycine, betaine and the rest is the same as example 1.
Example 6
This example provides a lactobacillus bulgaricus cryopreservation system that differs from example 1 only in that the medium does not contain betaine and the parts by mass of betaine are apportioned to glycine and sorbitol, all as in example 1.
Example 7
This example provides a lactobacillus bulgaricus cryopreservation system that differs from example 1 only in that glycine and sorbitol are not contained in the medium, and the parts by mass of glycine and sorbitol are all allocated to betaine, all the rest being the same as example 1.
Example 8
This example provides a lactobacillus bulgaricus cryopreservation system that differs from example 1 only in that the medium does not contain glycine and betaine, and the parts by mass of glycine and betaine are all assigned to sorbitol, all the other being the same as in example 1.
Example 9
This example provides a lactobacillus bulgaricus cryopreservation system that differs from example 1 only in that the medium does not contain betaine and sorbitol, and the parts by mass of betaine and sorbitol are all assigned to glycine, and the rest is the same as example 1.
Example 10
This example provides a Lactobacillus bulgaricus cryopreservation system which differs from example 1 only in that the cryoprotectant does not contain an antifreeze protein, and the parts of the antifreeze protein are apportioned to glycerol, L-linoleic acid, alpha-linolenic acid, and the rest are the same as in example 1.
Example 11
This example provides a lactobacillus bulgaricus cryopreservation system, which differs from example 1 only in that 5 parts of lactobacillus bulgaricus solution and 5 parts of cryoprotectant (the mass ratio of lactobacillus bulgaricus solution to cryoprotectant is 1:1) are replaced with 2 parts of lactobacillus bulgaricus solution and 8 parts of cryoprotectant (the mass ratio of lactobacillus bulgaricus solution to cryoprotectant is 0.25).
Example 12
This example provides a lactobacillus bulgaricus cryopreservation system, which differs from example 1 only in that 5 parts of lactobacillus bulgaricus solution and 5 parts of cryoprotectant (the mass ratio of lactobacillus bulgaricus solution to cryoprotectant is 1:1) are replaced with 9.2 parts of lactobacillus bulgaricus solution and 0.8 part of cryoprotectant (the mass ratio of lactobacillus bulgaricus solution to cryoprotectant is 11.5.
Example 13
The embodiment provides a lactobacillus bulgaricus cryopreservation system, and the cryopreservation system is different from the cryopreservation system in the embodiment 1 only in that the cooling speed is 1 ℃/min, the temperature is reduced to-5 ℃, and the rest is the same as the embodiment 1.
Example 14
The embodiment provides a lactobacillus bulgaricus cryopreservation system, which is different from the embodiment 1 only in that the cooling rate is 11 ℃/min, the temperature is reduced to-5 ℃, and the rest is the same as the embodiment 1.
Comparative example 1
This comparative example provides a lactobacillus bulgaricus cryopreservation system which differs from example 1 only in that the cryoprotectant does not contain glycerol and the parts by mass of glycerol are apportioned to L-linoleic acid and alpha-linolenic acid, the remainder being the same as in example 1.
Comparative example 2
This comparative example provides a lactobacillus bulgaricus cryopreservation system which differs from example 1 only in that the cryoprotectant does not contain L-linoleic acid and the mass fraction of L-linoleic acid is apportioned to glycerol and alpha-linolenic acid, the remainder being the same as in example 1.
Comparative example 3
This comparative example provides a lactobacillus bulgaricus cryopreservation system which differs from example 1 only in that the cryoprotectant does not contain alpha-linolenic acid, and the mass fraction of alpha-linolenic acid is apportioned to glycerol and L-linoleic acid, the remainder being the same as in example 1.
Comparative example 4
This comparative example provides a Lactobacillus bulgaricus cryopreservation system which differs from example 1 only in that the cryoprotectant contains only 7.8 parts glycerol, 0.2 parts antifreeze protein, and the remainder is the same as example 1.
Comparative example 5
This comparative example provides a Lactobacillus bulgaricus cryopreservation system which differs from example 1 only in that the cryoprotectant contains only 7.8 parts L-linoleic acid, 0.2 parts antifreeze protein, the remainder being the same as in example 1.
Comparative example 6
This comparative example provides a lactobacillus bulgaricus cryopreservation system which differs from example 1 only in that the cryoprotectant contained only 7.8 parts alpha-linolenic acid, 0.2 parts antifreeze protein, and the remainder being the same as example 1.
Comparative example 7
This comparative example provides a lactobacillus bulgaricus cryopreservation system that differs from example 1 only in that no cryoprotectant is added, the remainder being the same as example 1.
Comparative example 8
This comparative example provides a Lactobacillus bulgaricus cryopreservation system which differs from example 1 only in that the cryoprotectant contained no glycerol, L-linoleic acid, alpha-linolenic acid, only 8 parts of antifreeze protein, and the remainder being the same as in example 1.
Test example 1
The freezing survival rate of lactobacillus bulgaricus in the lactobacillus bulgaricus freezing and storing system prepared in the examples and the comparative examples is evaluated by the following specific evaluation method: according to the test method of GB 4789.35-2016, (1) a mixed system of the Bulgaria lactobacillus liquid which is not refrigerated on the same day after the temperature of examples 1-14 and comparative examples 1-8 is reduced and a cryoprotectant is taken, 1mL of the mixed system is sucked, 9mL of physiological saline is added, vortex mixing is carried out, the operation is repeated once, and the live bacteria quantity of the day prepared in examples 1-14 and comparative examples 1-8 is the initial live bacteria quantity; the lactobacillus bulgaricus cryopreservation systems prepared in examples 1 to 14 and comparative examples 1 to 8 were thawed after being left for 1 day and 6 months, and 1mL of the liquid was aspirated and added to 9mL of physiological saline, vortexed and mixed uniformly, and the process was repeated once. (2) Diluting the uniformly mixed liquid by 10 times, 20 times and 50 times respectively by using normal saline to obtain mixed bacterial liquids with different concentration gradients. (3) Sucking 1ml of mixed bacterial liquid, adding the mixed bacterial liquid into a flat plate, pouring a culture medium which is melted and cooled to about 45 ℃, slightly rotating the flat plate to uniformly mix the bacterial liquid and the culture medium, inverting after cooling, culturing at 30 ℃, taking out after 48 hours, and counting to obtain the number of viable bacteria. Survival rate (%) at 1 day/6 month of freezing = viable count at 1 day/6 months of freezing (cfu/mL)/initial viable count (cfu/mL).
TABLE 1
As can be seen from the data in Table 1, the culture media for example 4-9, which lack one or only one of glycine, sorbitol and betaine, have lower survival rates after being frozen for 1 day and 6 months than example 1, indicating that the three glycine, sorbitol and betaine in the culture media are synergistic, and have significant improvement in short-term increase in freezing resistance and long-term maintenance of Lactobacillus bulgaricus survival rate.
The cryoprotectant described in example 10 does not contain the antifreeze protein, and the cryoprotectant described in comparative example 8 only contains the antifreeze protein, so that the survival rates of the cryoprotectant after being frozen for 1 day and 6 months are lower than those of the cryoprotectant described in example 1, and the cryoprotectant has stronger synergistic effect on the aspect of enhancing the freezing survival rate of lactobacillus bulgaricus when the antifreeze protein is used in combination with glycerol, L-linoleic acid and alpha-linolenic acid. The mass ratio of the lactobacillus bulgaricus liquid to the cryoprotectant in the examples 11 and 12 is not in the range of (0.5-10): 1, and the survival rate of the lactobacillus bulgaricus liquid after being frozen for 1 day and 6 months is lower than that of the example 1, which shows that when the mass ratio of the lactobacillus bulgaricus liquid to the cryoprotectant is in the range of (0.5-10): 1, the lactobacillus bulgaricus liquid has better freezing resistance and higher survival rate; the cooling speed of the cooling in examples 13 and 14 is not in the range of 2-10 ℃/min, and the survival rates of the freezing in 1 day and 6 months are lower than those in example 1, which shows that when the cooling speed is in the range of 2-10 ℃/min, the nucleation of a protection system is delayed by gradient cooling of a temperature-controlled refrigerator, and the number of frozen viable bacteria can be effectively increased.
Comparative example 7, in which no cryoprotectant was added, the survival rates at day 1 and month 6 were worse than those of example 1, indicating that the addition of cryoprotectant can effectively improve the freezing resistance and survival rate of lactobacillus bulgaricus; comparative examples 1 to 6 lack one component of glycerol, L-linoleic acid and alpha-linolenic acid or only contain one component of glycerol, L-linoleic acid and alpha-linolenic acid, and the survival rate of the comparative example 1 is low, which shows that the combination of the three components of glycerol, L-linoleic acid and alpha-linolenic acid has remarkable synergistic effect in improving the freezing survival rate of lactobacillus bulgaricus.
Test example 2
The lactobacillus bulgaricus acidification activity in the lactobacillus bulgaricus cryopreservation system prepared in the examples and the comparative examples is evaluated by the following specific evaluation method: (1) Lactobacillus bulgaricus obtained in step (3) of test example 1 was inoculated into boiled sterilized skim milk in an amount of 2%v/v and cultured at 42 ℃. (2) The time required for skim milk pH to reach the lowest pH =4.4 was measured to indicate the acidifying activity of the bacterial species. The lactobacillus bulgaricus liquid and cryoprotectant mixed system on the day obtained in examples 1 to 14 and comparative examples 1 to 8 without refrigeration was initially acidified. Difference in acidification activity of 1 day or 6 months of freezing = (acidification activity of 1 day or 6 months of freezing-initial acidification activity)/initial acidification activity.
TABLE 2
As can be seen from the data in Table 2, the results of the culture media described in examples 4-9, which lack one or only one of glycine, sorbitol, and betaine, respectively, show that the differential acidification rates after 1 day and 6 months of freezing are significantly increased compared to example 1, i.e., the acidification activity after freezing is significantly reduced compared to example 1, indicating that the three components glycine, sorbitol, and betaine in the culture media are synergistically enhanced, and the antifreeze capacity of Lactobacillus bulgaricus liquid is increased in a short term and the activity of Lactobacillus bulgaricus is maintained in a long term.
The cryoprotectant described in example 10 does not contain the antifreeze protein, and the cryoprotectant described in comparative example 8 only contains the antifreeze protein, the difference rate of the acidification activities of the cryoprotectant for 1 day and 6 months is higher than that of the cryoprotectant in example 1, so that the cryoprotectant has stronger synergistic effect on enhancing the frost resistance of lactobacillus bulgaricus and increasing the short-term and long-term cryopreservation activities by combining the antifreeze protein with glycerol, L-linoleic acid and alpha-linolenic acid. The mass ratio of the lactobacillus bulgaricus liquid to the cryoprotectant in the examples 11 and 12 is not in the range of (0.5-10): 1, the acidification activity difference rate of the lactobacillus bulgaricus liquid after being frozen for 1 day and 6 months is higher than that of the example 1, namely, the acidification activity after being frozen is obviously reduced compared with the example 1, and the results show that when the mass ratio of the lactobacillus bulgaricus liquid to the cryoprotectant is in the range of (0.5-10): 1, the antifreeze performance of the lactobacillus bulgaricus is better, and the activity maintenance state is better; the cooling speed of the cooling in examples 13 and 14 is not in the range of 2-10 ℃/min, and the difference rate of the acidification activities of the ice crystals after being frozen for 1 day and 6 months is higher than that in example 1, which shows that when the cooling speed is in the range of 2-10 ℃/min, the ice crystals with larger size can be reduced to damage cells through slow cooling, and the activity of the lactobacillus bulgaricus can be effectively maintained.
Comparative example 7 only no cryoprotectant was added, and the difference rate of the acidification activities of day 1 and month 6 was higher than that of example 1, i.e. the acidification activity after freezing was reduced more significantly, indicating that the addition of cryoprotectant can effectively improve the survival rate of lactobacillus bulgaricus and the cells still maintain better activity; comparative examples 1 to 6 lack one component of glycerol, L-linoleic acid and alpha-linolenic acid or only contain one component of glycerol, L-linoleic acid and alpha-linolenic acid, and the difference rate of the acidification activities of the frozen lactobacillus bulgaricus after being frozen for 6 months is higher than that of the comparative example 1, which shows that the combination of the three components of glycerol, L-linoleic acid and alpha-linolenic acid has synergistic effect in improving the long-time freezing activity maintenance of lactobacillus bulgaricus.
The applicant states that the present invention is illustrated by the above examples of the process of the present invention, but the present invention is not limited to the above process steps, i.e. it is not meant that the present invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which fall within the scope and disclosure of the present invention, are contemplated.
Claims (10)
1. A cryoprotectant for improving the activity of lactobacillus bulgaricus is characterized by comprising, by mass, 4-20 parts of glycerol, 0.2-5 parts of L-linoleic acid and 0.2-5 parts of alpha-linolenic acid.
2. The cryoprotectant of claim 1, wherein the cryoprotectant further comprises an antifreeze protein.
3. The cryoprotectant of claim 1 or 2, further comprising 0.02 to 2 parts by mass of an antifreeze protein.
4. Use of a cryoprotectant according to any one of claims 1 to 3 for cryopreservation of lactobacillus gasseri.
5. A freezing method for improving the activity of lactobacillus bulgaricus, which is characterized by comprising the steps of mixing lactobacillus bulgaricus liquid with the cryoprotectant as claimed in any one of claims 1 to 3, cooling and freezing.
6. A freezing method according to claim 5, wherein the Lactobacillus bulgaricus liquid is prepared by the following method:
inoculating and culturing the lactobacillus bulgaricus strain to obtain the lactobacillus bulgaricus bacterial liquid.
7. A freezing method according to claim 6, wherein the raw material of the culture medium for Lactobacillus bulgaricus bacterial suspension culture comprises glycine, sorbitol, betaine and sodium chloride.
8. A freezing method according to claim 7, wherein the raw materials of the medium comprise, by mass, 1 to 8 parts of glycine, 3 to 40 parts of sorbitol, 1 to 14 parts of betaine, and 30 to 60 parts of sodium chloride.
9. The freezing method according to any one of claims 5 to 8, wherein the mass ratio of the Lactobacillus bulgaricus liquid to the cryoprotectant is (0.5-10): 1;
preferably, the temperature of the culture is 30-40 ℃, and the time of the culture is 6-12h.
10. A method of freezing as claimed in any one of claims 5 to 9 wherein the rate of reduction of the temperature is from 2 to 10 ℃/min to-5 ℃.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115558620A (en) * | 2022-09-30 | 2023-01-03 | 微康益生菌(苏州)股份有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146580A (en) * | 2013-03-07 | 2013-06-12 | 西北民族大学 | Anaerobic ultralow temperature strain preserving agent |
| CN107287121A (en) * | 2017-08-11 | 2017-10-24 | 洛阳泽达慧康医药科技有限公司 | A kind of lactic acid bacteria freeze drying protective agent and preparation method thereof and application method |
| CN107365705A (en) * | 2017-07-14 | 2017-11-21 | 江苏微康生物科技有限公司 | A kind of oil-soluble probiotics freezes freeze drying protectant and its application of bacterium powder |
-
2022
- 2022-10-11 CN CN202211243216.1A patent/CN115478017B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146580A (en) * | 2013-03-07 | 2013-06-12 | 西北民族大学 | Anaerobic ultralow temperature strain preserving agent |
| CN107365705A (en) * | 2017-07-14 | 2017-11-21 | 江苏微康生物科技有限公司 | A kind of oil-soluble probiotics freezes freeze drying protectant and its application of bacterium powder |
| CN107287121A (en) * | 2017-08-11 | 2017-10-24 | 洛阳泽达慧康医药科技有限公司 | A kind of lactic acid bacteria freeze drying protective agent and preparation method thereof and application method |
Non-Patent Citations (3)
| Title |
|---|
| 何宗柏;孙瑞胤;鄂晶晶;麻丽丽;张晓宁;王俊国;: "油酸对植物乳杆菌LIP-1生长及冻干存活率的影响及其机理", 食品科学, no. 10, pages 68 - 74 * |
| 王亚珍: "乳酸杆菌的保藏方法", 生物技术, vol. 08, no. 01, pages 41 - 42 * |
| 蒲丽丽;刘宁;: "保加利亚乳杆菌冻干保护剂保护作用的研究", 中国酿造, no. 05, pages 46 - 48 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115558620A (en) * | 2022-09-30 | 2023-01-03 | 微康益生菌(苏州)股份有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
| CN115558620B (en) * | 2022-09-30 | 2023-10-20 | 微康益生菌(苏州)股份有限公司 | Lactic acid bacteria freeze-drying protective agent and preparation method and application thereof |
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