CN111662860A - Method for improving survival rate of tetragenococcus halophilus under extreme conditions - Google Patents
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Abstract
The invention discloses a method for improving the survival rate of tetragenococcus halophilus under extreme conditions, and relates to the technical field of microorganisms. The method is characterized in that the halophil tetracoccus before inoculation is subjected to thermal pre-adaptation treatment, and a microbial stress protection mode is utilized, so that when somatic cells are cultured in an optimal growth condition and a lethal growth environment, a protection effect induced by an adaptive reaction is generated, the cells can be protected from a more severe extreme environment, and the survival rate of the halophil tetracoccus under the extreme condition is obviously improved; the method is simple and reliable, has good applicability and low cost, and is suitable for large-scale popularization and application.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a method for improving the survival rate of tetragenococcus halophilus under extreme conditions.
Background
Tetragenococcus halophilus belongs to the Tetragenococcus in the family of lactic acid bacteria, and is widely applied to the field of high-salt food fermentation, such as soy sauce, fish paste, bean products and the like. In the food fermentation process, the content of flavor substances such as organic acids, aldehydes, esters and the like in food can be obviously improved by adding the halophilic tetragenococcus, so that the method has high application value. However, in the production process of traditional fermentation products, the extreme environment (high salt, low acid and high temperature) seriously inhibits the growth performance of the thalli and the activity of related metabolic enzymes, thereby affecting the normal physiological activity of thalli cells, resulting in low survival rate and poor reproductive capacity of the thalli in the extreme environment, and seriously affecting the quality of the products in the application process. Therefore, the method has important significance for improving the survival rate of the tetragenococcus halophilus under extreme environmental conditions and ensuring the normal physiological activity of thallus cells.
In order to ensure the quality of a fermentation product of the halophilic tetragenococcus, improve the survival rate of the halophilic tetragenococcus under extreme environmental conditions and ensure the normal physiological activity of bacterial cells, people either increase the inoculation amount of the halophilic tetragenococcus during fermentation, weaken the extreme environmental conditions or select or culture a new strain with better stress resistance, however, although the method can ensure the quality of the fermentation product and improve the survival rate of the halophilic tetragenococcus to a certain extent, the production cost can be obviously increased or the product quality can be reduced, thereby seriously limiting the application range and the field of the halophilic tetragenococcus.
Disclosure of Invention
The invention aims to provide a method for improving the survival rate of the halophilic tetragenococcus under the extreme condition, the method can obviously improve the survival rate of the halophilic tetragenococcus under the extreme condition, and the method is simple and reliable, has good practicability and has a positive effect on large-scale application of the halophilic tetragenococcus.
A method of increasing survival of tetragenococcus halophilus under extreme conditions comprising the steps of: before the tetragenococcus halophilus is inoculated to an extreme condition for culture, carrying out thermal pre-adaptation treatment on the tetragenococcus halophilus; the heat pre-adaptation treatment is to culture the tetragenococcus halophilus for 1-5h at 40-50 ℃.
According to the method for improving the survival rate of the halophilic tetragenococcus under the extreme condition, the bacterial cells are cultured under the optimal growth condition and the lethal growth environment by means of thermal pre-adaptation treatment before inoculation and the stress protection mode of microorganisms, so that a protection effect induced by adaptive reaction is generated, the cells can be protected against the severe extreme environment, and the survival rate of the halophilic tetragenococcus under the extreme condition is obviously improved; the method is simple and reliable, has good applicability and low cost, and is suitable for large-scale popularization and application.
In the process of thermal pre-adaptation treatment, the temperature is a main influence factor for improving the survival rate of the tetragenococcus halophilus under extreme conditions, and when the temperature is too low (< 40 ℃), the temperature is at the optimal growth temperature of the tetragenococcus halophilus, the tetragenococcus halophilus cannot generate adaptive reaction, so that cells cannot be protected from being in a worse extreme environment, and the survival rate of the tetragenococcus halophilus under extreme conditions is improved; when the temperature is too high (more than 50 ℃), the halophilic tetragenococcus can die in a large amount in a short time, and the amount of the halophilic tetragenococcus after the thermal pretreatment is little, so that the method is not beneficial to practical application; preferably, the heat pre-adaptation treatment is to culture the tetragenococcus halophilus at the temperature of 43-48 ℃; most preferably, the heat pre-adaptation treatment is to culture the tetragenococcus halophilus at 45 ℃; by preference, the resulting heat-preconditioned tetragenococcus halophilus is high in number and has a high survival rate under extreme conditions.
Wherein, the time of the thermal pre-adaptation treatment is preferably 1 to 3 hours; the method has the advantages of long time, long culture period, large culture medium consumption, no contribution to large-scale popularization and application, and too short time, and incapability of producing adaptive reaction by the halophilic tetragenococcus; most preferably, the thermal preconditioning time is 1.5 hours.
Wherein in the process of thermal pre-adaptation treatment, a culture solution for culturing the tetragenococcus halophilus is a conventional culture solution suitable for the propagation and growth of the tetragenococcus halophilus; preferably, the culture solution comprises the following components: 10g/L peptone, 4g/L yeast extract powder, 5g/L sodium acetate trihydrate, 8g/L beef extract, 20g/L glucose, 1ml Tween 80, 2g/L dipotassium phosphate, 2g/L triammonium citrate, 0.2g/L magnesium sulfate heptahydrate, 0.05g/L manganese sulfate tetrahydrate, 60g/L sodium chloride and the balance of water; the pH value is 6.2; the preferable components of the culture solution are more suitable for the growth of the tetragenococcus halophilus and are beneficial to the generation of adaptive reaction of the tetragenococcus halophilus.
Wherein, preferably, the pH value of the culture solution is 6.0-6.5; the preferable pH value of the culture solution is more suitable for the growth of the halophilic tetragenococcus, and is beneficial to the halophilic tetragenococcus to generate adaptive reaction; most preferably, the pH of the culture broth is 6.2.
Wherein, preferably, the inoculation amount of the tetragenococcus halophilus in the process of thermal pre-adaptation treatment is 1.0 × 106-1.0×107cfu/mL, preferably the inoculation amount of the tetrahydratophilus, the growth state of the tetrahydratophilus is better, the tetrakiss halophilus is favorable for generating adaptive reaction, and most preferably, the inoculation amount is 5.0 × 10 during the culture of the tetrakiss halophilus6cfu/mL。
Wherein, the preferred preservation number of the tetragenococcus halophilus is CGMCC No. 3792.
Wherein the method for improving the survival rate of the tetragenococcus halophilus under the extreme conditions further comprises the following steps: a method for separating the pretreated halophilic tetragenococcus from the culture solution; the separation method is centrifugal treatment.
Wherein, preferably, the centrifugal rotation speed is 7000-9000r/min, the centrifugal temperature is 3-5 ℃, and the centrifugal time is 3-8 min; the preferable centrifugal condition has better separation effect on the tetragenococcus halophilus and better activity of the obtained tetragenococcus halophilus; most preferably, the centrifugal rotation speed is 8000r/min, the centrifugal temperature is 4 ℃, and the centrifugal time is 5 min.
Wherein, the extreme condition refers to an environment with a certain inhibiting effect on the growth of the tetragenococcus halophilus, which can reduce the biological activity and the propagation speed of the tetragenococcus halophilus to a certain extent and possibly cause a certain amount of tetragenococcus halophilus to die, and is a living environment between the optimal growth condition and the lethal condition of the tetragenococcus halophilus.
Among them, it is preferable that the extreme conditions include, but not limited to, one or more of a high temperature environment, an acidic environment, a high salt environment, an ethanol environment, and/or a hydrogen peroxide environment.
Wherein, preferably, the temperature of the high-temperature environment is 55 ℃; the high temperature can directly cause the death of the halophilic tetragenococcus, and the low temperature can not play a role in stressing the halophilic tetragenococcus.
Wherein, preferably, the pH value of the acidic environment is 2.5; the death of the halophilic tetracoccus can be directly caused by the excessively low pH value, and the stress effect on the halophilic tetracoccus cannot be realized due to the excessively high pH value.
Wherein, the mass concentration of the sodium chloride in the high-salt environment is preferably 35% (g/100 ml); the high concentration can directly cause the death of the halophilic tetrads coccus, and the low concentration can not play a role in stressing the halophilic tetrads coccus.
Wherein, preferably, the mass concentration of the hydrogen peroxide in the hydrogen peroxide environment is 0.03%; the high concentration can directly cause the death of the halophilic tetrads coccus, and the low concentration can not play a role in stressing the halophilic tetrads coccus.
Wherein, preferably, the volume percentage of ethanol in the ethanol environment is 10%; the high concentration can directly cause the death of the halophilic tetrads coccus, and the low concentration can not play a role in stressing the halophilic tetrads coccus.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the halophilic tetragenococcus before inoculation is subjected to thermal pre-adaptation treatment, so that the halophilic tetragenococcus has a protective effect induced by adaptive reaction, cells can be protected against severe extreme environments, and the survival rate of the halophilic tetragenococcus under various extreme conditions is obviously improved.
2. The method is simple and reliable, has good applicability and low cost, and is suitable for large-scale popularization and application.
Detailed Description
The present invention will be described in further detail with reference to test examples and specific embodiments. It should be understood that the scope of the above-described subject matter is not limited to the following examples, and any techniques implemented based on the disclosure of the present invention are within the scope of the present invention.
In the specific embodiment of the invention, the adopted Tetragenococcus halophilus is CGMCC No.3792 which is collected to-80 ℃ and stored in glycerol storage liquid. The strain is separated from soy sauce mash by a laboratory, is subjected to morphological, physiological and biochemical characteristic tests and 16S rDNA sequencing identification, and is preserved in China General Microbiological Culture Collection center (CGMCC).
In a specific embodiment of the present invention, the first culture solution has the same composition as the seed culture medium, and comprises: 10g/L peptone, 4g/L yeast extract powder, 5g/L sodium acetate trihydrate, 8g/L beef extract, 60g/L sodium chloride, 20g/L glucose, 1ml Tween 80, 2g/L dipotassium hydrogen phosphate, 2g/L triammonium citrate, 0.2g/L magnesium sulfate heptahydrate, 0.05g/L manganese sulfate tetrahydrate and the balance of water, wherein the pH value is 6.2;
the solid plate culture medium comprises: 10g/L peptone, 4g/L yeast extract powder, 5g/L sodium acetate trihydrate, 8g/L beef extract, 20g/L glucose, 1ml Tween 80, 2g/L dipotassium phosphate, 2g/L triammonium citrate, 60g/L sodium chloride, 0.2g/L magnesium sulfate heptahydrate, 0.05g/L manganese sulfate tetrahydrate, 20g/L agar, and the balance of water, wherein the pH value is 6.2.
Example 1
(1) Thawing the Tetragenococcus halophilus at 1.0 × 106Inoculating the cfu/mL inoculum size in a seed culture medium, performing static culture at 30 ℃ for 24h to a middle logarithmic phase, and performing amplification culture twice to obtain a seed culture solution with stable growth;
(2) thermal pre-adaptation treatment at 5.0 × 106Inoculating the seed culture solution subjected to the secondary expanding culture into the first culture solution according to the inoculation amount of cfu/mL, and culturing for 1.5h at the temperature of 45 ℃;
(3) and centrifuging the culture solution subjected to the thermal pretreatment for 5min at 8000r/min and 4 ℃ to collect thalli, thus obtaining the thalli of the thermoadaptative tetragenococcus halophilus.
Example 2
(1) Thawing the Tetragenococcus halophilus at 1.0 × 106Inoculating the cfu/mL inoculum size in a seed culture medium, performing static culture at 30 ℃ for 24h to a middle logarithmic phase, and performing amplification culture twice to obtain a seed culture solution with stable growth;
(2) thermal pre-adaptation treatment at 1.0 × 107Inoculating the seed culture solution subjected to the secondary expanding culture into the first culture solution according to the inoculation amount of cfu/mL, and culturing for 1h at the temperature of 50 ℃;
(3) and centrifuging the culture solution subjected to the thermal pre-adaptation treatment at 7000r/min and 5 ℃ for 8min to collect thalli, thereby obtaining the thalli of the thermoadaptative tetragenococcus halophilus.
Example 3
(1) Thawing the Tetragenococcus halophilus at 1.0 × 106Inoculating the cfu/mL inoculum size in a seed culture medium, performing static culture at 30 ℃ for 24h to a middle logarithmic phase, and performing amplification culture twice to obtain a seed culture solution with stable growth;
(2) thermal pre-adaptation treatment at 1.0 × 106Inoculating the seed culture solution subjected to secondary expanding culture into the first culture solution according to the inoculation amount of cfu/mL, and culturing for 3h at 40 ℃;
(3) centrifuging the culture solution subjected to thermal pre-adaptation treatment at 9000r/min and 3 ℃ for 3min to collect thalli, and obtaining the thalli of the thermoadaptative tetragenococcus halophilus.
Comparative example 1
(1) Thawing the Tetragenococcus halophilus at 1.0 × 106Inoculating the cfu/mL inoculum size in a seed culture medium, performing static culture at 30 ℃ for 24h to a middle logarithmic phase, and performing amplification culture twice to obtain a seed culture solution with stable growth;
(2) thermal pre-adaptation treatment at 5.0 × 106Inoculating the seed culture solution subjected to secondary expanding culture into the first culture solution according to the inoculation amount of cfu/mL, and culturing for 1.5h at 35 ℃;
(3) and centrifuging the culture solution subjected to the thermal pretreatment for 5min at 8000r/min and 4 ℃ to collect thalli, thus obtaining the thalli of the thermoadaptative tetragenococcus halophilus.
Comparative example 2
(1) Thawing the Tetragenococcus halophilus at 1.0 × 106Inoculating the cfu/mL inoculum size in a seed culture medium, performing static culture at 30 ℃ for 24h to a middle logarithmic phase, and performing amplification culture twice to obtain a seed culture solution with stable growth;
(2) thermal pre-adaptation treatment at 5.0 × 106Inoculating cfu/mL of the inoculum size to the first culture solution, and inoculating the second seed culture solution at 45 deg.CCulturing for 0.5 h;
(3) and centrifuging the culture solution subjected to the thermal pretreatment for 5min at 8000r/min and 4 ℃ to collect thalli, thus obtaining the thalli of the thermoadaptative tetragenococcus halophilus.
Comparative example 3
(1) Thawing the Tetragenococcus halophilus at 1.0 × 106Inoculating the cfu/mL inoculum size in a seed culture medium, performing static culture at 30 ℃ for 24h to a middle logarithmic phase, and performing amplification culture twice to obtain a seed culture solution with stable growth;
(2) centrifuging the seed culture solution at 8000r/min and 4 deg.C for 5min to collect thallus to obtain Tetragenococcus halophilus thallus.
Experimental example:
1. high temperature environmental stress experiment:
(1) the above-obtained Tetragenococcus halophilus strain was mixed at a ratio of 1.0 × 106The inoculum size of cfu/mL was resuspended in the first medium and incubated at 60 ℃ for 2.5 h.
(2) Centrifuging the cultured culture solution at 8000r/min (4 deg.C) for 5min, collecting thallus, suspending thallus in sterile water, spreading 10 μ L of thallus in solid plate culture medium, standing at 30 deg.C for 72h, counting colony number and calculating cell survival rate.
2. Acid environment stress experiment:
(1) adjusting pH of the first culture solution to 2.5 with lactic acid, and mixing the obtained Tetragenococcus halophilus with the amount of 1.0 × 106The inoculum size of cfu/mL was cultured in culture broth at 30 ℃ for 2.5 h.
(2) Centrifuging the cultured culture solution at 8000r/min (4 deg.C) for 5min, collecting thallus, suspending thallus in sterile water, spreading 10 μ L of thallus in solid plate culture medium, standing at 30 deg.C for 72h, counting colony number and calculating cell survival rate.
3. High salt environmental stress experiment:
(1) sodium chloride was added to the first culture solution to adjust the mass concentration to 35%, and the amount of the above-prepared Tetragenococcus halophilus cells was adjusted to 1.0 × 106The inoculum size of cfu/mL was cultured in culture broth at 30 ℃ for 2.5 h.
(2) Centrifuging the cultured culture solution at 8000r/min (4 deg.C) for 5min, collecting thallus, suspending thallus in sterile water, spreading 10 μ L of thallus in solid plate culture medium, standing at 30 deg.C for 72h, counting colony number and calculating cell survival rate.
4. Ethanol environmental stress experiment:
(1) ethanol with a mass concentration of 10% was added to the first culture solution, and the above-prepared tetragenococcus halophilus cells were cultured at a concentration of 1.0 × 106The inoculum size of cfu/mL was cultured in culture broth at 30 ℃ for 2.5 h.
(2) Centrifuging the cultured culture solution at 8000r/min (4 deg.C) for 5min, collecting thallus, suspending thallus in sterile water, spreading 10 μ L of thallus in solid plate culture medium, standing at 30 deg.C for 72h, counting colony number and calculating cell survival rate.
5. Hydrogen peroxide environmental stress experiment:
(1) adding hydrogen peroxide with mass concentration of 0.03% into the first culture solution, and mixing the obtained Tetragenococcus halophilus with the amount of 1.0 × 106The inoculum size of cfu/mL was cultured in culture broth at 30 ℃ for 2.5 h.
(2) Centrifuging the cultured culture solution at 8000r/min (4 deg.C) for 5min, collecting thallus, suspending thallus in sterile water, spreading 10 μ L of thallus in solid plate culture medium, standing at 30 deg.C for 72h, counting colony number and calculating cell survival rate.
6. Blank group experiment:
(1) the above-obtained Tetragenococcus halophilus strain was mixed at a ratio of 1.0 × 106The inoculum size of cfu/mL was resuspended in the first medium and incubated at 30 ℃ for 2.5 h.
(2) Centrifuging the cultured culture solution at 8000r/min (4 deg.C) for 5min, collecting thallus, suspending thallus in sterile water, spreading 10 μ L of thallus in solid plate culture medium, standing at 30 deg.C for 72h, counting colony number and calculating cell survival rate.
Survival statistics (survival calculated as a ratio to the blank for both examples and comparative examples below):
| experimental group | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| High temperature environment | 11.65％ | 11.87％ | 11.28％ | 3.16％ | 3.63％ | 3.12％ |
| Acid environment | 2.17％ | 2.25％ | 2.03％ | 0.35％ | 0.41％ | 0.32％ |
| High salt environment | 67.26％ | 67.55％ | 65.29％ | 36.43％ | 37.12％ | 36.21％ |
| Ethanol environment | 2.21％ | 2.31％ | 2.16％ | 0.24％ | 0.31％ | 0.22％ |
| Hydrogen peroxide environment | 14.28％ | 14.83％ | 13.97％ | 9.92％ | 10.15％ | 9.74％ |
| Blank group | 100％ | 100％ | 100％ | 100％ | 100％ | 100％ |
According to the statistical results, the survival rate of the tetragenococcus halophilus under various extreme conditions is remarkably improved after the heat pre-adaptation method disclosed by the invention is used for treatment. In example 1, the survival rate in high-temperature environment was increased by 3.68 times, the survival rate in acid (ph2.5) environment was increased by 6.2 times, the survival rate in high-salt (35%) environment was increased by 1.85 times, the survival rate in hydrogen peroxide (0.03%) environment was increased by 1.44 times, and the survival rate in alcohol (10%) environment was increased by 7.13 times.
Claims (10)
1. A method of increasing survival of tetragenococcus halophilus under extreme conditions comprising the steps of: before the tetragenococcus halophilus is inoculated to an extreme condition for culture, carrying out thermal pre-adaptation treatment on the tetragenococcus halophilus; the heat pre-adaptation treatment is to culture the tetragenococcus halophilus for 1-5h at 40-50 ℃.
2. The method of claim 1, wherein the heat pre-conditioning is culturing Tetragenococcus halophilus at 43-48 ℃.
3. The method of claim 1, wherein the thermal preconditioning time is between 1 and 3 hours.
4. The method according to claim 1, wherein the culture solution for culturing Tetragenococcus halophilus in the thermal pre-adaptation treatment process comprises the following components: 10g/L peptone, 4g/L yeast extract powder, 5g/L sodium acetate trihydrate, 8g/L beef extract, 20g/L glucose, 1ml Tween 80, 2g/L dipotassium phosphate, 2g/L triammonium citrate, 0.2g/L magnesium sulfate heptahydrate, 0.05g/L manganese sulfate tetrahydrate, 60g/L sodium chloride and the balance of water; the pH of the culture broth was 6.2.
5. The method according to claim 4, wherein the pH of the culture medium is 6.0 to 6.5.
6. The method of claim 1, wherein the amount of inoculum for the culture of Tetragenococcus halophilus in the thermal preconditioning step is 1.0 × 106-1.0×107cfu/mL。
7. The method according to claim 1, wherein the Tetragenococcus halophilus has a accession number of CGMCCNo.3792.
8. The method of claim 1, further comprising: a method for separating the pretreated halophilic tetragenococcus from the culture solution; the separation method is centrifugal treatment.
9. The method as claimed in claim 7, wherein the centrifugation speed is 7000-9000r/min, the centrifugation temperature is 3-5 ℃ and the centrifugation time is 3-8 min.
10. The method of claim 1, wherein the extreme conditions include, but are not limited to, one or more of a high temperature environment, an acidic environment, a high salt environment, an ethanol environment, and/or a hydrogen peroxide environment.
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