CN113201459A - Method for quick freeze-drying preservation of microorganisms - Google Patents
Method for quick freeze-drying preservation of microorganisms Download PDFInfo
- Publication number
- CN113201459A CN113201459A CN202110672936.9A CN202110672936A CN113201459A CN 113201459 A CN113201459 A CN 113201459A CN 202110672936 A CN202110672936 A CN 202110672936A CN 113201459 A CN113201459 A CN 113201459A
- Authority
- CN
- China
- Prior art keywords
- freeze
- drying
- microorganisms
- hydroxyethyl
- polysaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N1/20—Bacteria; Culture media therefor
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of microorganism preservation, in particular to a method for rapid freeze-drying preservation of microorganisms, which comprises the following steps: activating, culturing and enriching microbes to obtain bacteria with content of 1 × 108cfu/mL～1×109cfu/mL of bacterial suspension; adding the microorganism freeze-drying protection of any one of claims 1-3 into the bacterial suspension, and uniformly mixing to obtain a mixed bacterial liquid; adding the vaterite calcium carbonate into the mixed bacterial liquid according to the weight-volume ratio of 1g to 0.8-2 mL, uniformly mixing, quickly freezing, and drying to obtain the freeze-dried powder. The method is simple and convenient to operate, and can quickly finish freeze-drying preservation of the microorganisms; lyophilized by the methods of the present applicationThe survival rate of the stored microorganisms is not less than 90 percent, the storage period of the microorganisms is obviously prolonged, the processing, the storage and the transfusion of microbial preparations are facilitated, in addition, the acid-producing activity of freeze-dried acid-producing bacteria is facilitated to be improved, and the biological activity of the acid-producing bacteria is facilitated to be maintained compared with the conventional freeze-dried storage method.
Description
Technical Field
The invention relates to the technical field of microorganism preservation, in particular to a method for quick freeze-drying preservation of microorganisms.
Background
The application of microorganisms in food health mainly comprises two aspects, one is to utilize the effect of beneficial microorganisms to prepare fermented food, the microorganisms are commonly applied in modern fermentation engineering, and the other is to prevent harmful microorganisms from polluting food so as to ensure the safety of the food.
Lactic Acid Bacteria (LAB) are a general term for a group of bacteria that can utilize fermentable carbohydrates to produce large amounts of lactic acid. It is widely distributed in nature and has high application value in industry and agriculture, food processing industry, medicine industry and the like. However, the activity of common lactic acid bacteria is weak, the requirements on nutrient components and external environment are high, the survival rate and the activity of the common lactic acid bacteria are reduced rapidly under long-term storage, and the application of the common lactic acid bacteria is severely limited.
At present, lactic acid bacteria are stored in a freeze-drying storage mode, but the activity of the lactic acid bacteria is seriously influenced when the lactic acid bacteria are directly freeze-dried, so that a freeze-drying protective agent is added to protect the microbial activity. For example, in the invention patent with the application number of CN2013103232152, tetrahydropyrimidine, trehalose, skim milk and sodium glutamate are used as freeze-drying protective agents, in the invention patent with the application number of CN2018106931842, glycerol, trehalose, skim milk powder, vitamin C and acacia are used as vacuum freeze-drying protective agents, and in the invention patent with the application number of 2019106154468, single-component carboxymethyl pachyman is used as freeze-drying protection to freeze-dry lactic acid bacteria.
The above background disclosure is only for the purpose of assisting understanding of the inventive concept and technical solutions of the present invention, and does not necessarily belong to the prior art of the present patent application, and should not be used for evaluating the novelty and inventive step of the present application in the case that there is no clear evidence that the above content is disclosed at the filing date of the present patent application.
Disclosure of Invention
Technical problem to be solved
In order to solve at least one technical problem in the prior art, the application provides a microbial freeze-drying protective agent capable of remarkably improving the survival rate of microorganisms, and the components of the microbial freeze-drying protective agent comprise theanine and hydroxyethyl polysaccharide. The invention also provides a rapid microbial freeze-drying preservation method which is beneficial to improving the survival rate of microbes and the acid production activity of freeze-drying acid-producing bacteria and is simple to operate.
(II) technical scheme
In order to solve the above technical problems or to achieve the above technical object, the present invention provides the following technical solutions.
A microbial freeze-drying protective agent comprises theanine and hydroxyethyl polysaccharide in a weight ratio of 1: 4.5-6.
The weight ratio of theanine to hydroxyethyl polysaccharide is more preferably 1: 5-5.5, and most preferably 1: 5.5.
The hydroxyethyl polysaccharide is at least one selected from hydroxyethyl Hericium Erinaceus polysaccharide, hydroxyethyl pachymaran or hydroxyethyl lentinan.
The hydroxyethyl polysaccharide is preferably hydroxyethyl Hericium erinaceus polysaccharide.
The microorganism comprises at least one of lactic acid bacteria or butyric acid bacteria.
The lactobacillus comprises at least one of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei or Lactobacillus bulgaricus.
This application the microorganism freeze-drying protective agent can be used to including lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus casei, lactobacillus bulgaricus etc. and quick freeze-drying of butyric acid bacterium preserve and/or freeze-drying powder preparation, this application freeze-drying protective agent can use alone, also can use with other conventional freeze-drying protective agent such as skim milk, alginic acid, lactalbumin etc. collocation, has the effect of the survival rate of apparent promotion freeze-drying preservation microorganism, compares in using conventional protective agent, and the survival rate of microorganism obtains improving by a wide margin.
Application of theanine and hydroxyethyl polysaccharide in rapid freeze-drying preservation of microorganisms.
The microorganism comprises at least one of lactic acid bacteria or butyric acid bacteria.
The lactobacillus comprises at least one of Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei or Lactobacillus bulgaricus.
The use comprises increasing the survival rate of the freeze-dried lactic acid bacteria.
The use comprises increasing the acid-producing activity of the freeze-dried lactic acid bacteria.
The application theanine and hydroxyethyl polysaccharide are favorable for quick freeze-drying preservation of probiotics such as lactic acid bacteria or butyric acid bacteria, can be independently applied, can also be matched with other conventional freeze-drying protective agents for application, and can exert the effect of remarkably improving the survival rate of freeze-drying preservation microorganisms.
A method for rapid lyophilization preservation of a microorganism, comprising:
s1, activating, culturing and enriching the microbes to obtain the bacteria with the content of 1 multiplied by 108cfu/mL～1×109cfu/mL of bacterial suspension;
s2, adding theanine in an amount of 0.5-1.5 g/100mL, adding hydroxyethyl polysaccharide in an amount of 2.25-9 g/100mL, and uniformly mixing to obtain a mixed bacterial liquid;
s3, adding the vaterite calcium carbonate into the mixed bacterial liquid according to the weight-volume ratio of 1g to 0.8-2 mL, uniformly mixing, quickly freezing, and drying to obtain the freeze-dried powder.
The microorganism comprises at least one of lactic acid bacteria or butyric acid bacteria.
When the microorganism is lactic acid bacteria, the enrichment is carried out after the culture reaches the growth stationary phase, and the bacteria content is 1-2 multiplied by 108Bacterial suspension of cfu/mLAnd (4) liquid.
When the microorganism is butyric acid bacteria, the enrichment is carried out after the culture reaches the logarithmic phase, and the bacteria content is 5-10 multiplied by 108cfu/mL of bacterial suspension.
The theanine and the hydroxyethyl polysaccharide can be added in the form of aqueous solution.
The weight ratio of theanine to hydroxyethyl polysaccharide is 1: 4.5-6, more preferably 1: 5-5.5, most preferably 1: 5.5.
Besides theanine and hydroxyethyl polysaccharide, the mixed bacterial liquid also contains 5-10 g/100mL of skimmed milk powder and 5-10 g/100mL of trehalose.
The hydroxyethyl polysaccharide is at least one selected from hydroxyethyl Hericium Erinaceus polysaccharide, hydroxyethyl pachymaran or hydroxyethyl lentinan.
The vaterite calcium carbonate is prepared according to the following steps: quickly mixing a sodium carbonate solution and a calcium chloride solution with equal molar concentrations in equal volumes, stirring at 1000-1200 r/min for 20-30 s, finishing the reaction, alternately washing with distilled water and absolute ethyl alcohol, centrifuging for 2-3 times, and drying to obtain the sodium carbonate/calcium chloride composite material. The method for preparing the vaterite calcium carbonate has the advantages of simple and easy operation steps, complete reaction process, high content of the ellipsoidal vaterite calcium carbonate in the obtained product, uniform granularity, granularity between 3 and 5 mu m, no obvious agglomeration phenomenon, suitability for adsorbing and protecting microorganisms, and contribution to the survival of freeze-dried and stored microorganisms compared with common calcium carbonate particles.
And the quick freezing is to cool the mixture to-45 to-60 ℃ at the speed of 20-30 ℃/min for at least 12 hours.
The freeze drying is carried out for at least 24h under 1-10 Pa.
The lyophilized preparation of microorganisms obtained according to the aforementioned method.
In the method, the microorganism is activated and cultured, and then the microorganism suspension is enriched, then the microorganism freeze-drying protective agent comprising theanine and hydroxyethyl polysaccharide is added, finally the vaterite calcium carbonate and the mixed bacterial liquid are mixed uniformly, and the mixture is frozen and dried after being cooled to low temperature at a specific cooling speed, so that the rapid freeze-drying preservation of the microorganism can be completed, the method is simple and convenient to operate, and the freeze-drying preservation of the microorganism can be rapidly completed; the survival rate of the microorganism freeze-dried and stored by the method is not less than 90 percent, the storage period of the microorganism is obviously prolonged, the processing, the storage and the transportation of a microbial preparation are facilitated, in addition, the method is also beneficial to the improvement of the acid production activity of freeze-dried acid-producing bacteria, and the method is beneficial to the maintenance of the biological activity of the acid-producing bacteria compared with the conventional freeze-dried and stored method; therefore, the method can be applied to large-scale, automatic and continuous modern production of microorganisms and has wide prospect.
The above-described preferred conditions may be combined with each other to obtain a specific embodiment, in accordance with common knowledge in the art.
The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.
(III) advantageous effects
The technical scheme of the invention has the following advantages:
1) the freeze-drying protective agent can be used for quick freeze-drying preservation and/or freeze-drying powder preparation of lactic acid bacteria and butyric acid bacteria, can be independently applied and can also be matched with other conventional freeze-drying protective agents for use, and has the effect of remarkably improving the survival rate of freeze-drying preserved microorganisms;
2) the theanine and the hydroxyethyl polysaccharide which are used as the protective agents are beneficial to improving the acid-producing activity of freeze-dried acid-producing bacteria and maintaining the biological activity of the acid-producing bacteria, and the possible reason is that the theanine and the hydroxyethyl polysaccharide can induce acid-producing bacteria cells to generate resistant substances in the freeze-drying process, so that the immunity of the acid-producing bacteria cells in the freeze-drying process is improved, and the genetic property and the stability of the acid-producing activity of the acid-producing bacteria are facilitated;
3) the method has the advantages that the vaterite type calcium carbonate with uniform granularity and no obvious agglomeration phenomenon is used for adsorbing and protecting the microorganisms, the method is more favorable for the survival of freeze-dried preserved microorganisms compared with common calcium carbonate particles, the mixed solution of the bacteria solution and the calcium carbonate is cooled to low temperature at a specific cooling speed and then is frozen and dried, the quick freeze-drying preservation of the microorganisms can be completed, the method is simple and convenient to operate, the freeze-drying preservation of the microorganisms can be quickly completed, the preservation period of the microorganisms is remarkably prolonged, and the processing, the storage and the transfer of microbial preparations are facilitated.
The invention adopts the technical scheme for achieving the purpose, makes up the defects of the prior art, and has reasonable design and convenient operation.
Drawings
The foregoing and/or other objects, features, advantages and embodiments of the invention will be more readily understood from the following description taken in conjunction with the accompanying drawings in which:
FIG. 1 is an infrared spectrum of Hericium erinaceum polysaccharide before and after modification, wherein A represents before modification and B represents after modification;
figure 2 is an XRD pattern of vaterite calcium carbonate, c represents the calcite crystalline form and v represents the vaterite crystalline form;
FIG. 3 is a schematic diagram of the acidogenic activity of microorganisms;
FIG. 4 is a schematic representation of the viability of microorganisms;
FIG. 5 is a schematic representation of bovine bile salt stress.
Detailed Description
Those skilled in the art can appropriately substitute and/or modify the process parameters to implement the present disclosure, but it is specifically noted that all similar substitutes and/or modifications will be apparent to those skilled in the art and are deemed to be included in the present invention. While the products and methods of making described herein have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the products and methods of making described herein may be made and utilized without departing from the spirit and scope of the invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The present invention uses the methods and materials described herein; other suitable methods and materials known in the art may be used. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting. All publications, patent applications, patents, provisional applications, database entries, and other references mentioned herein, and the like, are incorporated by reference herein in their entirety. In case of conflict, the present specification, including definitions, will control.
The materials, methods, and examples described herein are illustrative only and not intended to be limiting unless otherwise specified. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
All percentages, parts, ratios, etc., are by weight unless otherwise indicated; additional instructions include, but are not limited to, "wt%" means weight percent, "mol%" means mole percent, "vol%" means volume percent.
The hericium erinaceus polysaccharide is obtained through the following steps: dissolving commercially available Hericium erinaceus polysaccharide in water at 20KHz and 0.4W/cm2Leaching for 2h at 40 ℃ under the action of ultrasonic waves, filtering, concentrating in vacuum, precipitating with 90% ethanol, removing protein by a Sevage method after redissolving, washing after decolorizing with 5% hydrogen peroxide, and finally freeze-drying to obtain the protein.
The lactobacillus plantarum described herein was purchased from special medical use formula food ltd, denuran, shandong.
The carboxymethyl pachyman described in this application is purchased from Moore Biotech, Inc. of Shaanxi.
The present invention is described in detail below.
Example 1
Preparing hydroxyethyl hericium erinaceus polysaccharide: adding 1 weight part of hericium erinaceus polysaccharide into 50 weight parts of solution containing 1% of sodium hydroxide and 1% of sodium chloride, stirring at room temperature of 120r/min for 30min, and cooling to 0 ℃; dropwise adding 1.5 parts by weight of ethylene oxide, heating to 30 ℃, stirring at 180r/min for reaction for 36h, neutralizing with 1mol/L hydrochloric acid to neutrality, adding 5 times of acetone, centrifuging to obtain precipitate, dissolving in water, dialyzing with deionized water as a medium by applying a 1KDa dialysis bag for 3d, changing water every day, and freeze-drying to obtain the product.
Example 2
Preparation of vaterite calcium carbonate: preparing 0.5mol/L sodium carbonate solution and 0.5mol/L calcium chloride solution, quickly mixing in equal volume, stirring at 1200r/min for 30s, finishing the reaction, alternately washing with distilled water and absolute ethyl alcohol, centrifuging for 3 times, and drying at 80 ℃ to constant weight.
Example 3
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, activating lactobacillus plantarum, culturing to a growth stable stage, and enriching to obtain the strain with the content of 1 multiplied by 108cfu/mL of bacterial suspension;
s2, dissolving theanine and the hydroxyethyl hericium erinaceus polysaccharide obtained in the example 1 in water respectively, sequentially adding 1.4g of theanine, 7.7g of hydroxyethyl hericium erinaceus polysaccharide, 6g of skimmed milk powder and 6g of trehalose into the bacterial suspension per 100mL of the bacterial suspension, and uniformly mixing to obtain a mixed bacterial liquid;
s3, adding the vaterite calcium carbonate obtained in the embodiment 2 into the mixed bacterial liquid according to the weight-volume ratio of 1g:1mL, uniformly mixing, cooling to-60 ℃ at the speed of 25 ℃/min, freezing for 12h, and then freezing and drying for 24h under 5Pa to obtain the freeze-dried powder.
Example 4
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, activating lactobacillus plantarum, culturing to a growth stable stage, and enriching to obtain 2 multiplied by 10 bacteria content8cfu/mL of bacterial suspension;
s2, dissolving theanine and the hydroxyethyl hericium erinaceus polysaccharide obtained in the example 1 in water respectively, sequentially adding 1g of theanine, 5.5g of hydroxyethyl hericium erinaceus polysaccharide, 8g of skimmed milk powder and 5g of trehalose into the bacterial suspension per 100mL of the bacterial suspension, and uniformly mixing to obtain a mixed bacterial liquid;
s3, adding the vaterite calcium carbonate obtained in the example 2 into the mixed bacterial liquid according to the weight-volume ratio of 1g:1.5mL, uniformly mixing, cooling to-55 ℃ at the speed of 30 ℃/min, freezing for 12h, and then freezing and drying for 24h under 4Pa to obtain the freeze-dried powder.
Example 5
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, activating lactobacillus plantarum, culturing to a growth stable stage, and enriching to obtain lactobacillus with the bacterium content of 4 multiplied by 108cfu/mL of bacterial suspension;
s2, same as S2 of example 4;
s3, same as S3 of example 4.
Example 6
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, the same as step S1 of example 4;
s2, dissolving theanine in water, sequentially adding 6.5g of theanine, 8g of skimmed milk powder and 5g of trehalose into the bacterial suspension per 100mL of the bacterial suspension, and uniformly mixing to obtain a mixed bacterial liquid;
s3, the same as step S3 of example 4.
Example 7
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, the same as step S1 of example 4;
s2, dissolving the hydroxyethyl hericium erinaceus polysaccharide obtained in the example 1 in water, sequentially adding 6.5g of hydroxyethyl hericium erinaceus polysaccharide, 8g of skim milk powder and 5g of trehalose into each 100mL of bacterial suspension, and uniformly mixing to obtain a mixed bacterial liquid;
s3, the same as step S3 of example 4.
Example 8
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, the same as step S1 of example 4;
s2, sequentially adding 8g of skimmed milk powder and 5g of trehalose into each 100mL of bacterial suspension, and uniformly mixing to obtain a mixed bacterial liquid;
s3, the same as step S3 of example 4.
Example 9
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, the same as step S1 of example 4;
s2, respectively dissolving sodium glutamate and the hydroxyethyl hericium erinaceus polysaccharide obtained in the embodiment 1 in water, sequentially adding 1g of sodium glutamate, 5.5g of hydroxyethyl hericium erinaceus polysaccharide, 8g of skimmed milk powder and 5g of trehalose into the bacterial suspension per 100mL of the bacterial suspension, and uniformly mixing to obtain a mixed bacterial liquid;
s3, the same as step S3 of example 4.
Example 10
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, the same as step S1 of example 4;
s2, respectively dissolving theanine and carboxymethyl pachyman in water, sequentially adding 1g of theanine, 5.5g of carboxymethyl pachyman, 8g of skimmed milk powder and 5g of trehalose into each 100mL of bacterial suspension, and uniformly mixing to obtain a mixed bacterial liquid;
s3, the same as step S3 of example 4.
Example 11
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, the same as step S1 of example 4;
s2, dissolving theanine and the hydroxyethyl hericium erinaceus polysaccharide obtained in the example 1 in water respectively, sequentially adding 1g of theanine, 5.5g of hydroxyethyl hericium erinaceus polysaccharide and 5g of trehalose into the bacterial suspension per 100mL of the bacterial suspension, and uniformly mixing to obtain mixed bacterial liquid;
s3, the same as step S3 of example 4.
Example 12
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, same as S1 of example 4;
s2, dissolving theanine and the hydroxyethyl hericium erinaceus polysaccharide obtained in the example 1 in water respectively, sequentially adding 1g of theanine, 5.5g of hydroxyethyl hericium erinaceus polysaccharide and 8g of skimmed milk powder into the bacterial suspension per 100mL of the bacterial suspension, and uniformly mixing to obtain mixed bacterial liquid;
s3, the same as step S3 of example 4.
Example 13
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, the same as step S1 of example 4;
s2, dissolving theanine and the hydroxyethyl hericium erinaceus polysaccharide obtained in the example 1 in water respectively, sequentially adding 1g of theanine and 5.5g of hydroxyethyl hericium erinaceus polysaccharide into the bacterial suspension per 100mL of the bacterial suspension, and uniformly mixing to obtain mixed bacterial liquid;
s3, same as S3 of example 4.
Example 14
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, the same as step S1 of example 4;
s2, the same as step S2 of example 4;
and (3) cooling the mixed bacterial liquid obtained from S3 and S2 to-50 ℃ at the speed of 30 ℃/min, freezing for 12h, and then freezing and drying for 24h under 10Pa to obtain the freeze-dried powder.
Example 15
A rapid freeze-drying preservation method for lactobacillus plantarum comprises the following steps:
s1, same as S1 of example 4;
s2, same as S2 of example 4;
s3, adding calcium carbonate with the particle size of about 4 mu m into the mixed bacterial liquid according to the weight-volume ratio of 1g to 1.5mL, uniformly mixing, cooling to-50 ℃ at the speed of 30 ℃/min, freezing for 12h, and then freezing and drying for 24h under 10Pa to obtain the freeze-dried powder.
Experimental example 1
Respectively weighing a certain amount of hericium erinaceus polysaccharide before and after modification in example 1, tabletting with potassium bromide at 4000-500 cm-1Infrared spectral scanning was performed over the range and the image was obtained as in figure 1.
As can be seen from FIG. 1, 3330cm-1And 2920cm-1Wide peaks appear nearby and belong to O-H stretching vibration characteristic peak and-CH respectively2Characteristic peak of antisymmetric telescopic vibration, 1045cm-1And 1640cm-1Respectively presenting C-O and C ═ O, wherein the polysaccharide shows the performance before and after modification, and the modified polysaccharide is 1457-1363 cm-1in-between-CH2Facing the bending vibration peak at 2920cm-1near-CH2The peak of antisymmetric stretching vibration of the resin is strengthened, indicating that the modification is successful.
Experimental example 2
The phase analysis of the vaterite calcium carbonate obtained in example 2 was performed, and the XRD pattern thereof was shown in FIG. 2. It can be seen from fig. 2 that the proportion of the vaterite crystal form is high, and the large specific surface area, the high solubility and dispersibility and the light specific gravity of the vaterite crystal form are all suitable for the adsorption and protection of microorganisms.
Experimental example 3
Respectively vacuumizing the freeze-dried lactobacillus plantarum obtained in the embodiment 3-15, and then storing at 4 ℃; the method for testing lactobacillus by using GB 4789.35-2010 food microbiology is used for verifying the survival rate after freeze-drying and the survival rate after being stored at 4 ℃ for 6 months after vacuumizing, the survival rate is calculated by the ratio of the total number of the microorganisms after revival to the total number of the microorganisms before freeze-drying and storage, and the measurement results are shown in Table 1.
TABLE 1 Freeze drying survival Rate
| Examples | Survival after lyophilization/%) | Survival after 6 months/%) |
| 3 | 94.3 | 94.1 |
| 4 | 94.5 | 94.2 |
| 5 | 92.6 | 91.4 |
| 6 | 86.1 | 82.8 |
| 7 | 85.4 | 82.6 |
| 8 | 82.5 | 80.4 |
| 9 | 90.4 | 89.5 |
| 10 | 89.9 | 89.1 |
| 11 | 93.8 | 93.4 |
| 12 | 94.0 | 93.8 |
| 13 | 93.6 | 93.4 |
| 14 | 87.6 | 85.8 |
| 15 | 89.5 | 89.0 |
As can be seen from Table 1, rapid lyophilization preservation of microorganisms is achieved in the manner described herein, with the preferred embodiments of examples 3 and 4 resulting in a post-lyophilization survival rate and a post-vacuum cryopreservation survival time of 6 monthsThe rate is not lower than 94%, so that the high-efficiency and high-survival-rate preservation of the lactic acid bacteria is realized; example 5 shows that too high a content of microorganisms in the bacterial suspension is detrimental to the survival of lactic acid bacteria, and therefore 1-2X 10 described herein8cfu/mL is the better freeze-drying preservation concentration of the lactic acid bacteria, the survival of the lactic acid bacteria is not facilitated when the concentration is too high, and the best use of the lactic acid bacteria cannot be achieved when the concentration is too low, so that the preservation cost is improved; by combining the theanine and the hydroxyethyl hericium erinaceus polysaccharide in the embodiment 6-10, the survival rate of the freeze-dried preserved microorganisms can be improved, and the survival rate is improved to a certain extent compared with that of a conventional freeze-drying protective agent sodium glutamate or carboxymethyl pachymaran; from examples 11 to 13, it can be seen that the freeze-drying protective agent containing theanine and hydroxyethyl hericium erinaceus polysaccharide can be used independently, and can also be used in combination with a conventional freeze-drying protective agent; the freeze-drying of the bacterial suspension directly containing the freeze-drying protective agent in example 14 and the freeze-drying of the ordinary calcium carbonate powder in example 15 cannot ensure the high survival rate of the microorganisms, which indicates that the vaterite type calcium carbonate is beneficial to the preservation of the microorganisms, and the application is not disclosed in the relevant way.
Experimental example 4
The microorganisms of experimental example 3, which were subjected to vacuum cryopreservation, were reactivated, activated and inoculated in MRS liquid medium at an inoculation rate of 2 vol%, incubated at 37 ℃ for 36 hours, and the microorganisms which were not subjected to freeze-drying preservation were used as a control, and their relative acid-producing activities were measured by measuring pH, and the results are shown in fig. 3.
As can be seen from fig. 3, the microorganisms obtained after the microbial inoculum is reactivated in embodiments 3 to 5 of the present application have relatively excellent acid production activity, and compared to the schemes in which theanine and/or hydroxyethyl hericium erinaceus polysaccharide is not added to the freeze-drying protective agent, and in which sodium glutamate is used instead of theanine, and carboxymethyl pachymaran is used instead of hydroxyethyl hericium erinaceus polysaccharide, the scheme in which theanine is used in combination with hydroxyethyl hericium erinaceus polysaccharide has a significant effect of improving the acid production activity of freeze-dried lactic acid bacteria, and the possible reason is that the preservation method of the present application is beneficial to the survival of microorganisms, and the microorganisms can obtain relatively good growth capacity after being reactivated, so that the acid production capacity can be relatively well maintained. It can also be seen from fig. 3 that the acid-producing ability of the microorganisms was not significantly affected by the addition of skim milk powder and/or trehalose, and the choice of vaterite calcium carbonate also did not have a significant gain in acid-producing activity of the microorganisms.
Experimental example 5
On the basis of example 4, a freeze-drying protective agent is controlled to comprise 8g of skimmed milk powder, 5g of trehalose and a mixture of theanine and hydroxyethyl hericium erinaceus polysaccharide accounting for 6.5g in total, a series of microbial freeze-dried preparations are obtained by changing the weight ratio of the theanine to the hydroxyethyl hericium erinaceus polysaccharide mixture and according to the same method as example 4; and the survival rate of the microorganism after revival of the lyophilized preparation was measured according to the same method as in experimental example 3, and the statistical results are shown in fig. 4. As can be seen from FIG. 4, by changing the weight ratio of theanine to hydroxyethyl hericium erinaceus polysaccharide in the cryoprotectant, the survival rate of the microorganisms in the lyophilized preparation after revival has a certain fluctuation, which is specifically shown in that when the weight ratio of theanine to hydroxyethyl polysaccharide is 1: 4.5-6, the survival rate of the microorganisms preserved by lyophilization is excellent and is 93.7-94.5%, and the excessive or insufficient ratio of theanine to hydroxyethyl polysaccharide is not beneficial to the survival of the microorganisms, which indicates that the two components exert a synergistic effect under the condition of a special weight ratio to improve the survival rate of the microorganisms preserved by lyophilization.
Experimental example 6
The microorganisms of Experimental example 3, which were vacuum-stored at a low temperature, were reactivated and then inoculated at an inoculation rate of 2 vol% into MRS medium containing 0.1g/100mL of bovine bile salt, incubated at a constant temperature of 37 ℃ and the microorganisms which were not subjected to lyophilization storage were sampled at 0, 2, 4, 6, and 8 hours, respectively, and the viable cell count was measured by a plate count method, and the measurement results are shown in FIG. 5.
As can be seen from FIG. 5, under the stress of 0.1g/100mL of bovine bile salt, the growth of Lactobacillus plantarum described in the present application has a certain inhibitory effect, and comparative analysis examples 4, 14, and 15 show that freeze-drying preservation of bacterial suspension containing only a freeze-drying protective agent and freeze-drying preservation of microbial solution with ordinary calcium carbonate powder are not beneficial to improving the tolerance of lyophilized microorganisms to bovine bile salt. It is known that the freeze-drying process of microorganisms is accompanied by the formation of ice crystals in cells and the change of cell membrane permeability, which specifically includes the increase of salt concentration in cytoplasm, the change of pH value and ionic strength, and the influence of cell membrane includes the decrease of membrane stability, the appearance of heteromorphic mixture in membrane and the increase of membrane permeability, besides, the freeze-drying can also cause the loss of part of cell protein, hydrolase and inhibitor, and the release of inhibitor can cause the damage of cell metabolism, thereby reducing the activity and tolerance of cells. As can be seen from fig. 5, the microorganism of the preferred embodiment example 4 of the present application has only slight tolerance loss under stress of bovine bile salts compared to the microorganism which is not freeze-dried and still has quite excellent tolerance to the stress of bovine bile salts, probably because the presence of the vaterite calcium carbonate with large specific surface area, higher solubility and dispersibility is beneficial to maintain the integrity of the cell membrane of the microorganism, reduce the cell damage during the freeze-drying process and improve the tolerance of the lyophilized microorganism to the bovine bile salts.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
While the above detailed description has shown, described, and pointed out novel features as applied to various embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the device or method illustrated may be made without departing from the spirit of the disclosure. In addition, the various features and methods described above may be used independently of one another, or may be combined in various ways. All possible combinations and sub-combinations are intended to fall within the scope of the present disclosure. Many of the embodiments described above include similar components, and thus, these similar components are interchangeable in different embodiments. While the invention has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the invention extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and obvious modifications and equivalents thereof. Accordingly, the invention is not intended to be limited by the specific disclosure of preferred embodiments herein.
The invention is not the best known technology.
Claims (9)
1. A microbial freeze-drying protective agent comprises theanine and hydroxyethyl polysaccharide in a weight ratio of 1: 4.5-6.
2. The microbial lyoprotectant of claim 1, wherein: the weight ratio of theanine to hydroxyethyl polysaccharide is more preferably 1: 5-5.5, and most preferably 1: 5.5.
3. The microbial lyoprotectant of claim 1 or 2, wherein: the hydroxyethyl polysaccharide is at least one selected from hydroxyethyl Hericium Erinaceus polysaccharide, hydroxyethyl pachymaran or hydroxyethyl lentinan.
4. Application of theanine and hydroxyethyl polysaccharide in rapid freeze-drying preservation of microorganisms.
5. Use according to claim 4, characterized in that:
the use comprises increasing the survival rate of the freeze-dried lactic acid bacteria; and/or
The use comprises increasing the acid-producing activity of the freeze-dried lactic acid bacteria.
6. A method for rapid freeze-drying preservation of microorganisms is characterized by comprising the following steps:
s1, activating, culturing and enriching the microbes to obtain the bacteria with the content of 1 multiplied by 108cfu/mL～1×109cfu/mL of bacterial suspension;
s2, adding the microorganism freeze-drying protection of any one of claims 1-3 into the bacterial suspension, and uniformly mixing to obtain a mixed bacterial liquid;
s3, adding the vaterite calcium carbonate into the mixed bacterial liquid according to the weight-volume ratio of 1g to 0.8-2 mL, uniformly mixing, quickly freezing, and drying to obtain the freeze-dried powder.
7. The method of claim 6, wherein: the vaterite calcium carbonate is prepared according to the following steps: quickly mixing a sodium carbonate solution and a calcium chloride solution with equal molar concentrations in equal volumes, stirring at 1000-1200 r/min for 20-30 s, finishing the reaction, alternately washing with distilled water and absolute ethyl alcohol, centrifuging for 2-3 times, and drying to obtain the sodium carbonate/calcium chloride composite material.
8. The method according to claim 6 or 7, characterized in that: and the quick freezing is to cool the mixture to-45 to-60 ℃ at the speed of 20-30 ℃/min for at least 12 hours.
9. A lyophilized microbial preparation obtained by the method according to any one of claims 6 to 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110672936.9A CN113201459B (en) | 2021-06-17 | 2021-06-17 | Method for quick-freezing, drying and preserving microorganisms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110672936.9A CN113201459B (en) | 2021-06-17 | 2021-06-17 | Method for quick-freezing, drying and preserving microorganisms |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113201459A true CN113201459A (en) | 2021-08-03 |
| CN113201459B CN113201459B (en) | 2023-06-16 |
Family
ID=77022387
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110672936.9A Active CN113201459B (en) | 2021-06-17 | 2021-06-17 | Method for quick-freezing, drying and preserving microorganisms |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113201459B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114451485A (en) * | 2022-01-26 | 2022-05-10 | 江苏省淡水水产研究所 | Traditional Chinese medicine polysaccharide extract freeze-drying protective agent, direct-vat-set starter and preparation method thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0431315A (en) * | 1990-05-28 | 1992-02-03 | Maruo Calcium Co Ltd | Monodispersed spherical or elliptic vaterite calcium carbonate and production thereof |
| JPH11314915A (en) * | 1999-02-15 | 1999-11-16 | Maruo Calcium Co Ltd | Production of spherical or spheroidal vaterite calcium carbonate |
| JP2007126333A (en) * | 2005-11-04 | 2007-05-24 | New Raimu Kenkyusha:Kk | Vaterite type disk-shaped calcium carbonate and its production method |
| JP2009284820A (en) * | 2008-05-29 | 2009-12-10 | Morinaga Milk Ind Co Ltd | Agent for suppressing decrease in freeze-dried useful bacterial cell powder and method for suppressing decrease in freeze-dried useful bacterial cell powder |
| JP2012056921A (en) * | 2010-09-13 | 2012-03-22 | Kaneka Corp | Probiotic preparation |
| CN106188324A (en) * | 2016-07-13 | 2016-12-07 | 武汉大学 | The preparation of hydroxyethyl pachyman and as the application of slow releasing tablet framework material |
| CN107722133A (en) * | 2017-10-30 | 2018-02-23 | 福建农林大学 | One kind improves the immunocompetent hydroxyethylation method of hericium erinaceum polysaccharide |
| CN108676759A (en) * | 2018-07-09 | 2018-10-19 | 广州珍健生物科技有限公司 | A kind of lactic acid bacteria freeze drying protective agent and preparation method thereof |
-
2021
- 2021-06-17 CN CN202110672936.9A patent/CN113201459B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0431315A (en) * | 1990-05-28 | 1992-02-03 | Maruo Calcium Co Ltd | Monodispersed spherical or elliptic vaterite calcium carbonate and production thereof |
| JPH11314915A (en) * | 1999-02-15 | 1999-11-16 | Maruo Calcium Co Ltd | Production of spherical or spheroidal vaterite calcium carbonate |
| JP2007126333A (en) * | 2005-11-04 | 2007-05-24 | New Raimu Kenkyusha:Kk | Vaterite type disk-shaped calcium carbonate and its production method |
| JP2009284820A (en) * | 2008-05-29 | 2009-12-10 | Morinaga Milk Ind Co Ltd | Agent for suppressing decrease in freeze-dried useful bacterial cell powder and method for suppressing decrease in freeze-dried useful bacterial cell powder |
| JP2012056921A (en) * | 2010-09-13 | 2012-03-22 | Kaneka Corp | Probiotic preparation |
| CN106188324A (en) * | 2016-07-13 | 2016-12-07 | 武汉大学 | The preparation of hydroxyethyl pachyman and as the application of slow releasing tablet framework material |
| CN107722133A (en) * | 2017-10-30 | 2018-02-23 | 福建农林大学 | One kind improves the immunocompetent hydroxyethylation method of hericium erinaceum polysaccharide |
| CN108676759A (en) * | 2018-07-09 | 2018-10-19 | 广州珍健生物科技有限公司 | A kind of lactic acid bacteria freeze drying protective agent and preparation method thereof |
Non-Patent Citations (5)
| Title |
|---|
| 吴满刚;庄涛;王小兰;吴雪燕;陈洋洋;于海;葛庆丰;汪志君;: "植物乳杆菌增殖培养及其浓缩型冻干发酵剂的制备" * |
| 崔树茂;赵建新;陈卫;张灏;: "乳酸菌代谢保护剂中糖产酸对冷冻保护的影响" * |
| 欧阳碧妍;崔树茂;毛丙永;唐鑫;马方励;赵建新;张灏;陈卫;: "益生菌干酪乳杆菌CCFM711耐酸冻干粉的制备" * |
| 秦韬，罗洋，龙瑶，刘晓盼，丘富安，陈仕雄，宋玉龙，马玉芳，李健，任喆，黄一帆: "羟乙基化猴头菇多糖对小鼠树突状细胞和自然杀伤细胞相互作用的影响" * |
| 胡筱红;贾建波;: "微生菌制剂的制备研究" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114451485A (en) * | 2022-01-26 | 2022-05-10 | 江苏省淡水水产研究所 | Traditional Chinese medicine polysaccharide extract freeze-drying protective agent, direct-vat-set starter and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113201459B (en) | 2023-06-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108570421B (en) | Lactobacillus plantarum strain and silage starter thereof | |
| CN102660461B (en) | Microbial preparation for shortening tobacco fermentation period and application of microbial preparation | |
| CN110151795B (en) | Clostridium butyricum viable bacteria preparation and production process thereof | |
| CN106754524B (en) | Lactobacillus paracasei N1115 culture medium and application thereof | |
| CN102453681B (en) | Protective agent for vacuum freeze drying of lactobacillus johnsonii, and application thereof | |
| CN110468050B (en) | Freeze-drying method for improving survival rate of lactobacillus plantarum by utilizing polysaccharide | |
| CN109468259B (en) | Culture medium for promoting spore generation | |
| CN111621443B (en) | Freeze-drying protective agent and application thereof in freeze-drying lactobacillus reuteri | |
| CN112126599B (en) | High-density culture method of lactobacillus helveticus, preparation of high-activity bacterium powder and application of high-density culture method | |
| CN112694976A (en) | Preparation method of lactobacillus acidophilus powder with high viable count | |
| CN113201459B (en) | Method for quick-freezing, drying and preserving microorganisms | |
| CN113151140B (en) | Method for improving freeze-drying survival rate and storage stability of lactobacillus | |
| CN108018211B (en) | Culture medium for improving freezing tolerance of lactobacillus bulgaricus and application method thereof | |
| CN1844362A (en) | Method for high density culture of lactic acid bacteria for kraut | |
| CN112195103A (en) | Freeze-drying protective agent, coprinus comatus freeze-dried product and preparation method thereof | |
| KR20150012445A (en) | Microorganism additives compositions for fermentation of foods with enhanced survival rate of the microorganism comprising alginate beads galic crush and lactic acid bacteria embedded therein and method of preparing the same | |
| CN114958655B (en) | Clostridium Ding Suanxing produced from pit mud produced by white spirit brewing and application thereof | |
| CN111621461B (en) | Method for improving activity and tolerance of bacillus coagulans BC99 and application thereof | |
| KR20150125918A (en) | Microorganism additives compositions using glutinous rice paste as a cryoprotectant for fermentation of foods with enhanced survival rate of the microorganisms and method of preparing the same | |
| CN113584122B (en) | Liquid medium for recovering dry injury from Cronobacter and Salmonella | |
| CN113293101B (en) | Inactivation method and application of lactic acid bacteria | |
| CN110373351B (en) | Freeze-drying protective agent and application thereof in preparation of lactic acid bacteria freeze-dried powder | |
| CN113234632A (en) | Method for improving freeze-drying survival rate of lactobacillus reuteri through fermentation control | |
| CN115322937B (en) | Culture method for improving stability of bifidobacterium lactis, product and application | |
| CN111575223B (en) | Method for reducing secretion of surface substances of lactobacillus rhamnosus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |