A method for thawing a cryogenic bacterial culture

Abstract

This invention provides a method for thawing cryogenic bacterial strains, comprising the following steps: first, placing the frozen cryogenic bacterial strains at room temperature for 10-30 minutes; then, immersing the cryogenic bacterial strains in an environment of 28±1℃ for 30-35 minutes to obtain thawed cryogenic bacterial strains. The thawing method of this invention can significantly improve the survival rate and viability of cryogenic bacterial strains after thawing, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a method for thawing cryogenic bacterial strains. Background Technology

[0002] Cryogenic strains are commonly used for long-term preservation of microorganisms to ensure their genetic stability and biological activity. However, the thawing process of cryogenic strains has a significant impact on their survival rate and viability.

[0003] Currently, the conventional thawing method involves directly immersing the bacterial bags containing the inoculum in a water bath at room temperature or slightly higher until completely thawed. For example, CN 107751186 A discloses a method for rapid cryopreservation and thawing of cells, which discloses the thawing steps for cryopreserved cells, including: rapidly immersing the cryopreserved cells taken from liquid nitrogen into conventional thawing water at 38-40°C, and rapidly shaking until completely thawed. The cells can be used directly without removing the cryopreservation solution.

[0004] This thawing method often leads to a high mortality rate of microorganisms and a reduction in the number and viability of live bacteria, especially in cryogenic microorganisms packaged in EVA and PE materials, where this problem is particularly prominent.

[0005] Therefore, it is of great significance to develop an effective method for thawing cryogenically preserved microbial strains in order to ensure the viability of the strains after thawing and reduce the mortality rate of the strains. Summary of the Invention

[0006] The purpose of this invention is to provide a method for thawing cryogenic bacterial strains, which can effectively reduce the mortality rate of the strains and ensure their viability during the thawing process.

[0007] To achieve the above objectives, the present invention provides a method for thawing cryogenic bacterial strains, the method comprising the following steps:

[0008] First, place the frozen cryogenic bacterial samples at room temperature for 10-30 minutes;

[0009] Then, the cryogenic bacterial sample was placed in an environment of 28±1℃ and thawed for 30-35 minutes to obtain the thawed cryogenic bacterial strain.

[0010] According to a specific embodiment of the present invention, preferably, the method further includes, after obtaining the thawed cryogenic strain, immediately cooling the cryogenic strain to 0-8°C for storage and later use.

[0011] According to a specific embodiment of the present invention, preferably, the cryogenic bacterial strain sample is stored at a freezing temperature of -45±1℃.

[0012] According to a specific embodiment of the present invention, preferably, the cryogenic bacterial sample is a sample packaged in a bag, and the material of the bag is EVA and / or PE.

[0013] According to a specific embodiment of the present invention, preferably, the weight of each unit packaging bag of the cryogenic strain is 35.5g-36.5g.

[0014] According to a specific embodiment of the present invention, preferably, the thickness of the packaging bag is 0.35-0.36 mm.

[0015] According to a specific embodiment of the present invention, preferably, the net weight of the cryogenic strain in each unit package is 955g-965g.

[0016] According to a specific embodiment of the present invention, preferably, the bacterial strain is lactic acid bacteria.

[0017] According to a specific embodiment of the present invention, preferably, the lactic acid bacteria include one or more of Lactobacillus, Bifidobacterium, and Streptococcus thermophilus.

[0018] According to specific embodiments of the present invention, the Bifidobacterium genus includes, but is not limited to, one or more of the following: *Bifidobacterium animalis* subsp. *animal*, *Bifidobacterium bifidum*, *Bifidobacterium breve*, *Bifidobacterium longum* subsp. *long*, and *Bifidobacterium longum* subsp. *infant*. For example, the strain may be one or more of the following: *Bifidobacterium animalis* subsp. *animal* Bb-12, *Bifidobacterium animalis* subsp. *animal* HN019, *Bifidobacterium animalis* subsp. *animal* Bi-07, *Bifidobacterium breve* M-16V, *Bifidobacterium longum* subsp. *infant* R0033, *Bifidobacterium bifidum* R0071, and *Bifidobacterium longum* subsp. *BB536.

[0019] According to a specific embodiment of the present invention, preferably, the *Lactobacillus* genus includes, but is not limited to, one or more of *Lactobacillus acidophilus*, *Lactobacillus curvatureii*, *Lactobacillus delbrueckii* subsp. bulgaricus, *Lactobacillus delbrueckii* subsp. lactis, *Lactobacillus gasseri*, *Lactobacillus helveticus*, *Lactobacillus johnsonii*, and *Lactobacillus maltii* subsp. maltii. For example, the strain can be one or more of *Lactobacillus acidophilus* NCFM, *Lactobacillus rhamnosus* GG, *Lactobacillus rhamnosus* HN001, *Lactobacillus rhamnosus* MP108, *Lactobacillus reuteri* DSM 17938, *Lactobacillus fermentatus* CECT 5716, and *Lactobacillus helveticus* R0052.

[0020] According to a specific embodiment of the present invention, preferably, the thermophilic streptococcus includes the thermophilic subspecies of Streptococcus salivarius.

[0021] In some specific embodiments of the present invention, the bacterial strain is *Lactobacillus rhamnosus* HN001, which is cryogenically stored at -45°C. The packaging bag is made of EVA material, weighs 36.0g, has a wall thickness of 0.352mm, and contains 960g of cryogenically stored bacterial strain. When thawing the sample, it is first placed at room temperature (20-25°C) for 10-30 minutes, then the bacterial bag is immersed in a water bath preheated to 28±1°C for 30-35 minutes. After thawing, the bacterial bag is immediately cooled to 0-8°C, and the viable count after thawing is 8.0 × 10⁻⁶. 10 -6.8×10 11 CFU / g; Preferably, the inoculum is first placed at room temperature (20°C) for 10 minutes, then the inoculum bag is immersed in a water bath preheated to 28°C for 30 minutes to thaw; after thawing, the inoculum bag is immediately cooled to 4°C, and the viable count after thawing is 1.0 × 10⁻⁶. 11 CFU / g.

[0022] In some specific embodiments of the present invention, the bacterial strain is *Lactobacillus rhamnosus* HN001, which is cryogenically stored at -45°C. The packaging bag is made of EVA material, weighs 36.2g, has a wall thickness of 0.355mm, and contains 962g of cryogenically stored bacterial strain. When thawing the sample, it is first placed at room temperature (20-25°C) for 10-30 minutes, then the bacterial bag is immersed in a water bath preheated to 28±1°C for 30-35 minutes. After thawing, the bacterial bag is immediately cooled to 0-8°C, and the viable count after thawing is 8.0 × 10⁻⁶. 10 -6.8×10 11 CFU / g; Preferably, the inoculum is first placed at room temperature (22°C) for 20 minutes, then the inoculum bag is immersed in a water bath preheated to 28°C for 30 minutes to thaw; after thawing, the inoculum bag is immediately cooled to 5°C, and the viable count after thawing is 9.0 × 10⁻⁶. 10 CFU / g.

[0023] In some specific embodiments of the present invention, the bacterial strain is *Lactobacillus rhamnosus* HN001, which is cryogenically stored at -45°C. The packaging bag is made of PE material, weighs 36.2g, and has a wall thickness of 0.360mm. The cryogenic bacterial strain weighs 965g. When thawing this sample, it is first placed at room temperature (20-25°C) for 10-30 minutes, and then the bacterial bag is immersed in a water bath preheated to 28±1°C for 30-35 minutes. After thawing, the bacterial bag is immediately cooled to 0-8°C, and the viable count after thawing is 8.0 × 10⁻⁶. 10 -6.8×10 11 CFU / g; Preferably, the inoculum is first placed at room temperature (25°C) for 15 minutes, then the inoculum bag is immersed in a water bath preheated to 28°C for 30 minutes to thaw; after thawing, the inoculum bag is immediately cooled to 8°C, and the viable count after thawing is 8.0 × 10⁻⁶.10 CFU / g.

[0024] In some specific embodiments of the present invention, the bacterial strain is *Lactobacillus rhamnosus* HN001, which is cryogenically stored at -45°C. The packaging bag is made of PE material, weighs 36.0g, and has a wall thickness of 0.350mm. The cryogenic bacterial strain weighs 965g. When thawing this sample, it is first placed at room temperature (20-25°C) for 10-30 minutes, and then the bacterial bag is immersed in a water bath preheated to 28±1°C for 30-35 minutes. After thawing, the bacterial bag is immediately cooled to 0-8°C, and the viable count after thawing is 8.0 × 10⁻⁶. 10 -6.8×10 11 CFU / g; Preferably, the inoculum is first placed at room temperature (24°C) for 20 minutes, then the inoculum bag is immersed in a water bath preheated to 28°C for 30 minutes to thaw; after thawing, the inoculum bag is immediately cooled to 6°C, and the viable count after thawing is 6.8 × 10⁻⁶. 11 CFU / g.

[0025] According to a specific embodiment of the present invention, preferably, the viable count of the thawed cryogenic strain is 8.0 × 10⁻⁶. 10 -6.8×10 11 CFU / g.

[0026] According to a specific embodiment of the present invention, preferably, the room temperature condition is 20-25°C.

[0027] According to a specific embodiment of the present invention, the 28±1℃ environment is preferably achieved by a water bath or a constant temperature chamber; preferably, the water quality of the water bath meets the requirements for drinking water microbial parameters.

[0028] The advantages and effects of the present invention are as follows: Compared with cryogenic bacterial bags thawed at room temperature, (1) the thawing time of the step-by-step thawing method of the present invention is significantly shortened; (2) the mortality rate of the bacterial strain is significantly reduced; and (3) the number and viability of live bacteria after thawing are effectively guaranteed. Attached Figure Description

[0029] Figure 1 This is a flowchart of the lactic acid bacteria detection procedure. Detailed Implementation

[0030] The present invention will now be described in detail with reference to specific embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0031] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art in the relevant field.

[0032] In addition, to avoid repetition, the following lists the general steps and detection methods required for the experiments in each embodiment, such as the storage conditions of cryogenic strains and the methods for detecting strains.

[0033] The storage conditions for the cryogenic strains in this invention are -45±1℃, and the packaging material for the cryogenic strains is EVA and / or PE.

[0034] The room temperature condition in this invention is 20-25℃.

[0035] The water quality used in the water bath in this invention meets the requirements for drinking water microbiological parameters, referring to the "Standards for Drinking Water Quality" (GB 5749-2022).

[0036] The method for detecting viable bacteria count should be performed according to GB 4789.35, and the procedure for detecting lactic acid bacteria is as follows: Figure 1 The specific testing steps are as follows:

[0037] 1 Sample Preparation

[0038] (1) The entire sample preparation process should follow aseptic operation procedures.

[0039] (2) The diluent should be preheated at 36℃±1℃ for 15min~30min before the test.

[0040] (3) Frozen samples can be thawed at 2℃~5℃ for no more than 18 hours, or at a temperature not exceeding 45℃ for no more than 15 minutes.

[0041] (4) Solid and semi-solid samples: Weigh 25g of sample aseptically and place it in a sterile homogenizing cup containing 225mL of diluent. Homogenize at 8000×g~10000×g for 1min~2min to prepare a 1:10 sample homogenate; or place it in a sterile homogenizing bag containing 225mL of diluent and beat it with a tapping homogenizer for 1min~2min to prepare a 1:10 sample homogenate.

[0042] (5) Liquid samples: Liquid samples should be thoroughly shaken first, and then 25 mL of the sample should be drawn with a sterile pipette and placed into a sterile conical flask (with an appropriate number of sterile glass beads pre-placed in the flask) or a homogenizing bag containing 225 mL of diluent. Shake thoroughly or use a tapping homogenizer for 1 min to 2 min to prepare a 1:10 sample homogenate.

[0043] (6) Food samples containing lactic acid bacteria that have been treated with special techniques (such as embedding techniques) should undergo effective pretreatment under the corresponding technical / process requirements.

[0044] 2. Dilution and Incubation

[0045] (1) Use a 1mL sterile pipette or micropipette to draw 1mL of 1:10 sample homogenate and slowly pour it along the tube wall into a sterile test tube containing 9mL of diluent (be careful not to let the tip of the pipette or micropipette touch the diluent). Shake the test tube or use a sterile pipette to repeatedly blow and mix it to make a 1:100 sample homogenate.

[0046] (2) Take another 1 mL sterile pipette or micropipette tip and perform 10-fold incremental sample homogenization according to the above operation sequence. Replace with a 1 mL sterile pipette or tip for each incremental dilution.

[0047] (3) Foods containing lactic acid bacteria that have been treated with special techniques (such as encapsulation techniques) should be diluted in accordance with the corresponding technical / process requirements.

[0048] 3. Lactic acid bacteria count

[0049] 3.1 Total Lactic Acid Bacteria Count

[0050] The selection of culture conditions and the results of total lactic acid bacteria count are shown in Table 1.

[0051] Table 1. Selection of culture conditions and results for total lactic acid bacteria count.

[0052]

[0053]

[0054] 3.2 Bifidobacterium count

[0055] Based on the estimated Bifidobacterium content in the sample, select 2-3 consecutive suitable dilutions. For each dilution, pipette 1 mL of the homogenate into a sterile Petri dish, preparing two Petri dishes for each dilution. After transferring the dilution into the Petri dishes, pour 15-20 mL of modified MRS agar medium (mupirocin lithium and cysteine ​​salts cooled to 48-50°C) into each dish, rotating to ensure thorough mixing. After the medium solidifies, invert the dish for anaerobic incubation at 36°C ± 1°C. Based on the growth characteristics of Bifidobacterium, incubation for 48 hours is generally recommended. If no colonies grow or the growth is small, incubation can be extended to 72 hours. After incubation, count all colonies on the plate. The entire process from sample dilution to plate pouring should be completed within 15 minutes.

[0056] 3.3 Count of thermophilic streptococci

[0057] Based on the estimated viable count of *Streptococcus thermophilus* in the sample, select 2-3 consecutive suitable dilutions. For each dilution, pipette 1 mL of the homogenate into a sterile Petri dish, preparing two Petri dishes for each dilution. After transferring the dilution into the Petri dishes, promptly pour 15-20 mL of MC agar medium cooled to 48-50°C into each dish, rotating to ensure thorough mixing. After the medium solidifies, invert the dish and incubate aerobically at 36°C ± 1°C. Based on the growth characteristics of *Streptococcus thermophilus*, incubation for 48 hours is generally recommended. If no colony growth or small colonies are observed, incubation can be extended to 72 hours. The colony characteristics of *Streptococcus thermophilus* on MC agar plates are: medium to small colonies, smooth, reddish colonies with regular edges, 2 mm ± 1 mm in diameter, and a pinkish reverse side.

[0058] 3.4 Lactobacillus count

[0059] Based on the estimated total viable count of the sample, select 2-3 consecutive suitable dilutions. For each dilution, pipette 1 mL of the homogenate into a sterile Petri dish, preparing two Petri dishes for each dilution. After transferring the dilution into the Petri dishes, pour 15-20 mL of MRS agar medium cooled to 48-50°C into each dish, rotating the dish to ensure thorough mixing. After the medium solidifies, invert the dish for anaerobic incubation at 36°C ± 1°C. Based on the growth characteristics of Lactobacillus, incubation for 48 hours is generally recommended; if no colonies grow or the colonies are small, incubation up to 72 hours can be performed. The entire process from sample dilution to plate pouring should be completed within 15 minutes.

[0060] 4. Colony count

[0061] 4.1 Visual inspection is acceptable; if necessary, use a magnifying glass or colony counter. Record the dilution factor and the corresponding number of colonies. Colony counts are expressed in colony forming units (CFU).

[0062] 4.2 Select plates with colony counts between 30 CFU and 300 CFU and no spreading colony growth to count the total number of colonies. For plates with colony counts below 30 CFU, record the specific colony count; for plates with colony counts above 300 CFU, record as too many to count.

[0063] 4.3 If one of the plates has large, sheet-like colonies, it should not be used. Instead, the plate without large, sheet-like colonies should be used as the colony count for that dilution. If the sheet-like colonies are less than half the size of the plate, but the colonies in the other half are evenly distributed, the colony count for half the plate can be multiplied by 2 to represent the colony count for one plate.

[0064] 4.4 When chain-like growth without clear boundaries appears on the plate, each single chain is counted as a colony.

[0065] 5. Expression of Results

[0066] 5.1 If the number of colonies on only one dilution plate is within the appropriate counting range, calculate the average of the colony counts on the two plates, and then multiply the average by the corresponding dilution factor to obtain the total number of colonies per g (ml) of sample.

[0067] 5.2 If the number of colonies on plates with two consecutive dilutions is within the appropriate counting range, calculate according to formula (1).

[0068] N = ∑C / [(n1 + 0.1n2)d] (1)

[0070] N: Number of colonies in the sample;

[0071] ∑C: The sum of colony counts on the plate (including plates with colony counts within the appropriate range);

[0072] n1: Number of plates at the first dilution (lowest dilution factor);

[0073] n2: Number of plates at the second dilution (highest dilution factor);

[0074] d: Release factor (first dilution).

[0075] 5.3 If the colony count on all dilution plates is greater than 300 CFU, then count the plate with the highest dilution. Other plates can be recorded as uncountable. The result is calculated by multiplying the average colony count by the highest dilution factor.

[0076] 5.4 If the plate colony count of all dilutions is less than 30 CFU, the calculation should be based on the average colony count of the lowest dilution multiplied by the dilution factor.

[0077] 5.5 If no colonies grow on any of the dilutions (including the original liquid sample), calculate using less than 1 multiplied by the lowest dilution factor.

[0078] 5.6 If the plate colony counts of all dilutions are not between 30 CFU and 300 CFU, and some of them are less than 30 CFU or greater than 300 CFU, then the average colony count closest to 30 CFU or 300 CFU shall be multiplied by the dilution factor.

[0079] Example 1

[0080] This embodiment provides a method for thawing cryogenic bacterial strains, employing a stepped thawing condition:

[0081] Take a cryogenic culture bag containing 36.0g of EVA (ethylene-vinyl acetate copolymer) packaging material and a wall thickness of 0.352mm. The bag contains 960g of the cryogenic strain *Lactobacillus rhamnosus* HN001. First, place the bag at room temperature (20℃) for 10 minutes. Then, immerse the bag in a water bath preheated to 28℃ for 30 minutes to thaw. Immediately after thawing, cool the bag to 4℃ and test according to GB 4789.35 method. The viable count is 1.0 × 10⁻⁶. 11 CFU / g.

[0082] Example 2

[0083] This embodiment provides a method for thawing cryogenic bacterial strains, employing a stepped thawing condition:

[0084] Take a cryogenic culture bag containing 36.2g of EVA packaging material and a wall thickness of 0.355mm. The bag contains 962g of the cryogenic strain *Lactobacillus rhamnosus* HN001. First, place the bag at room temperature (22℃) for 20 minutes. Then, immerse the bag in a water bath preheated to 28℃ for 30 minutes to thaw. Immediately after thawing, cool the bag to 5℃ and test according to GB 4789.35 method. The viable count was 9.0 × 10⁻⁶. 10 CFU / g.

[0085] Example 3

[0086] This embodiment provides a method for thawing cryogenic bacterial strains, employing a stepped thawing condition:

[0087] Take a cryogenic microbial culture bag made of PE (polyethylene), weighing 36.2g, with a wall thickness of 0.360mm. The bag contains 965g of the cryogenic strain *Lactobacillus rhamnosus* HN001. First, place the bag at room temperature (25℃) for 15 minutes. Then, immerse the bag in a water bath preheated to 28℃ to thaw for 30 minutes. Immediately after thawing, cool the bag to 8℃ and test according to GB 4789.35 method. The viable count is 8.0 × 10⁻⁶. 10 CFU / g.

[0088] Example 4

[0089] This embodiment provides a method for thawing cryogenic bacterial strains, employing a stepped thawing condition:

[0090] Take a cryogenic microbial culture bag with a PE packaging material, a packaging weight of 36.0g, and a packaging wall thickness of 0.350mm. The bag contains 962g of the cryogenic strain *Lactobacillus rhamnosus* HN001. First, place the bag at room temperature (24℃) for 20 minutes. Then, immerse the bag in a water bath preheated to 28℃ for 30 minutes to thaw. Immediately after thawing, cool the bag to 6℃ and test according to GB4789.35 method. The viable count was 6.8 × 10⁻⁶. 11 CFU / g.

[0091] Comparative Example 1

[0092] This comparative example uses a traditional thawing method. A cryogenic culture bag containing 960g of *Lactobacillus rhamnosus* HN001, packaged in EVA material with a wall thickness of 0.355mm, was taken and thawed at 22℃. After 4 hours, it was completely thawed. The viable count was determined according to GB 4789.35 method, and was 6.1 × 10⁻⁶. 10 CFU / g.

[0093] Comparative Example 2

[0094] This comparative example uses a traditional thawing method. A cryogenic culture bag containing 960g of *Lactobacillus rhamnosus* HN001 (containing EVA packaging material, 0.36mm wall thickness) was thawed at 24℃. After 3 hours, the culture was completely thawed and tested according to GB 4789.35 method. The viable count was 6.5 × 10⁻⁶. 10 CFU / g.

[0095] Comparative Example 3

[0096] This comparative example uses a traditional thawing method. A cryogenic culture bag containing 962g of *Lactobacillus rhamnosus* HN001 (a PE-packaged, 0.355mm thick cryogenic strain) was taken and directly immersed in a 25°C water bath for 30 minutes. The viable count was determined according to GB 4789.35 method, and was 7.0 × 10⁻⁶. 10 CFU / g.

[0097] Comparative Example 4

[0098] This comparative example uses a traditional thawing method. A cryogenic culture bag containing 965g of *Lactobacillus rhamnosus* HN001 (containing PE packaging material, 0.36mm wall thickness) was taken and directly immersed in a 28℃ water bath for 30 minutes. The viable count was determined according to GB 4789.35 method, and was 7.5 × 10⁻⁶. 10CFU / g.

[0099] In summary, this invention provides an effective method for thawing cryogenic bacterial strains, which can significantly improve the survival rate and viability of cryogenic bacterial strains after thawing, and has broad application prospects.

[0100] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for thawing cryogenic bacterial strains, comprising the following steps: First, place the frozen cryogenic bacterial samples at room temperature for 10-30 minutes; Then, the cryogenic bacterial sample was placed in an environment of 28±1℃ and thawed for 30-35 minutes to obtain the thawed cryogenic bacterial strain.

2. The thawing method according to claim 1, wherein, The method also includes immediately cooling the thawed cryogenic strain to 0-8°C for storage after obtaining it.

3. The thawing method according to claim 1, wherein, The cryogenic bacterial strain samples were stored at a freezing temperature of -45±1℃.

4. The thawing method according to claim 1, wherein, The cryogenic bacterial samples were packaged in bags made of EVA and / or PE.

5. The thawing method according to claim 4, wherein, The weight of each unit of the cryogenic strain packaging bag is 35.5g-36.5g; the thickness of the packaging bag is 0.35-0.36mm.

6. The thawing method according to claim 5, wherein, The net weight of the cryogenic strain in each unit package is 955g-965g.

7. The thawing method according to any one of claims 1-6, wherein, The bacterial strain is lactic acid bacteria; Preferably, the lactic acid bacteria include one or more of Lactobacillus, Bifidobacterium, and Streptococcus thermophilus.

8. The method for thawing cryogenic bacteria according to claim 7, wherein, The viable count of the cryogenic strain after thawing was 8.0 × 10⁻⁶. 10 -6.8×10 11 CFU / g.

9. The thawing method according to claim 1, wherein, The room temperature condition is 20-25℃.

10. The thawing method according to claim 1, wherein, The 28±1℃ environment is achieved through a water bath or a constant temperature chamber; preferably, the water quality of the water bath meets the requirements for drinking water microbiological parameters.

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