CN111647521A - Lactobacillus GM _1 and breeding method thereof - Google Patents
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Abstract
The invention belongs to the technical field of biology, and discloses a lactobacillus GM _1 and a breeding method thereof, wherein the breeding method of the lactobacillus GM _1 comprises the following steps: activating and culturing the lactobacillus strain to be bred; preparing lactobacillus inoculation liquid; adding nitrosoguanidine mother solution and sterilized phosphoric acid buffer solution, centrifuging, washing and diluting to obtain nitrosoguanidine treated bacterial solution; culturing the nitrosoguanidine-treated bacterial liquid to obtain a lactobacillus mutant group; after selecting bacterial colonies with regular morphology and obvious characteristics and bacterial colonies obviously larger than other bacterial colonies, inoculating and culturing, freezing and centrifuging, and crushing freeze-dried powder to obtain the lactobacillus GM _1 freeze-dried powder after breeding. The large-scale lactobacillus mutant group developed by the chemical mutagenesis method has no biological safety risk, stable heredity, short time consumption and low cost, can be used as a new lactobacillus genetic germplasm resource library for screening high-quality lactobacillus strains with any characters, and can meet the production requirements of various characteristic products.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a lactobacillus GM _1 and a breeding method thereof.
Background
Currently, lactobacillus is one of probiotics closely related to human life, and is widely applied to the fields of food fermentation, industrial lactic acid fermentation and medical care. Meanwhile, a large number of animal and human body experiments show that the lactobacillus GM _1 has strong probiotic functions of inhibiting pathogenic bacteria, reducing blood fat, improving organism oxidation resistance, protecting liver, preventing impaired glucose tolerance and type II diabetes, regulating organism immunity, resisting tumors and the like.
The existing lactobacillus breeding method is mainly used for breeding high-quality lactobacillus strains by means of establishing a strain library, radiation mutagenesis, genetic engineering improvement and the like. Establishing a large-scale strain resource pool is time-consuming and costly, and the required high-quality strain resources cannot be collected necessarily. The genetically engineered improved bacteria are mainly used for specific purposes in the aspects of industry and the like, but have little application in the breeding aspect of microbial strains. The radiation mutagenesis is a physical mutagenesis, and the mutagen mainly comprises ultraviolet rays, X-rays, gamma-rays, fast neutrons and the like, is commonly used in mutagenesis microbial strains, but the bred high-quality probiotic strains are still very limited. Therefore, a new method for breeding lactobacillus is needed to breed high-quality lactobacillus strains with high activity, regular morphology, obvious characteristics, rapid growth and easy high-density culture.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) in the existing method for breeding lactobacillus strains by establishing the strain library, the establishment of a large-scale strain resource library is time-consuming and high in cost, and required high-quality strain resources cannot be collected necessarily.
(2) The existing improved bacteria by genetic engineering are mainly used for specific purposes in the aspects of industry and the like, but have little application in the breeding aspect of microbial strains.
(3) The existing radiation mutagenesis is commonly used in mutagenic microorganism strains, but the bred high-quality probiotic strains are still very limited.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a lactobacillus GM _1 and a breeding method thereof.
The invention is realized in such a way that a breeding method of lactobacillus GM _1 comprises the following steps:
step one, strain activation culture: inoculating a lactobacillus strain to be bred into an MRS liquid culture medium, carrying out activation culture for 24h in the dark under the conditions of 37 ℃ and aerobic condition, carrying out subculture for 3 generations, streaking the obtained culture solution onto an MRS plate culture medium, and culturing for 48h at 37 ℃;
selecting a single colony from an MRS plate culture medium as an initial strain, inoculating the initial strain into 5ml of an MRS liquid culture medium, performing static culture at 37 ℃ for 24-48 h, and performing subculture for 3 generations; inoculating the strain in an MRS liquid culture medium according to the inoculation amount of 3%, performing static culture at 37 ℃ for 24-36 hours to obtain activated bacterial liquid, and performing freeze-drying preservation;
step three, preparing inoculation liquid: placing the lactobacillus single colony obtained in the step two into 5-8 mL of physiological saline, and uniformly mixing to obtain lactobacillus suspension; diluting the bacterial suspension with an LSM culture solution until the OD value is 0.15-0.2 under the condition of the wavelength of 600-800 nm to obtain a lactobacillus inoculation solution;
absorbing 2.5ml of lactobacillus inoculation liquid in a sterile centrifuge tube, adding nitrosoguanidine mother liquid and a sterile phosphate buffer solution to fix the volume to 5.0ml, placing the centrifuge tube on a shaking bed, uniformly mixing at the temperature of 36.5-37 ℃, centrifuging at 5000r/min for 6min, removing supernatant, washing, and diluting to obtain nitrosoguanidine treated bacterial liquid;
step five, coating the nitrosoguanidine-treated bacterial liquid on an MRS plate culture medium, culturing for 24h at 37 ℃, selecting a single colony to inoculate in the liquid culture medium, culturing for 24h at 37 ℃ to obtain lactobacillus mutant monomers, and forming a mutant group by each treated new germ plasm;
step six, streaking the mutant monomer obtained in the step five on an MRS plate culture medium, culturing for 24-36 h at 37 ℃, and selecting a colony which is regular in morphology, obvious in characteristic and obviously larger than other colonies to obtain a new and rapidly-growing lactobacillus GM _ 1;
step seven, inoculating the lactobacillus GM _1 obtained by breeding in the step six into an MRS culture medium for culture, and then carrying out refrigerated centrifugation to obtain a precipitate; and adding a freeze-drying protective agent into the precipitate, freeze-drying, taking out, and crushing to powder in an aseptic environment to obtain the bred lactobacillus GM _1 freeze-dried powder.
Further, in the first step, the MRS liquid culture medium is prepared from 20g/L glucose, 12g/L beef extract, 10g/L peptone, 5g/L yeast powder, 5g/L anhydrous sodium acetate, 2g/L dipotassium hydrogen phosphate, 1.5g/L diamine citrate, 0.5ml/L Tween-80, 0.25g/L manganese sulfate and KH with the concentration of 0.25-1.0 mol/L2PO4And (4) buffer solution.
Further, the preparation method of the MRS liquid culture medium comprises the following steps:
(1) mixing glucose, beef extract, peptone, yeast powder, anhydrous sodium acetate, dipotassium hydrogen phosphate, diamine citrate, Tween-80 and manganese sulfate;
(2) adding KH into the mixed solution2PO4Buffer solution with constant volume of 1000 mL;
(3) adjusting the pH value to 7 by using 0.1mol/L NaOH;
(4) sterilizing at 120 deg.C for 10min to obtain MRS liquid culture medium.
Further, the MRS plate culture medium is prepared by adding 25.0g/L of agar on the basis of an MRS liquid culture medium and carrying out autoclaving for 20-30 min.
Further, in the fourth step, the dilution method is as follows: after discarding the supernatant, washing the thalli for 2-3 times by using a sterilized phosphate buffer, adding an equal volume of the sterilized phosphate buffer to suspend the thalli, and properly diluting.
Further, in the fifth step, 20% of sterilized glycerol is added into the lactobacillus mutant population, and the lactobacillus mutant population is stored for a long time at the temperature of minus 80 ℃.
Further, in the sixth step, the colony size is more than 2 times of the colony size without mutagenesis in the same culture time.
Further, in the seventh step, after the lactobacillus GM _1 is cultured for 48 hours at 37 ℃, refrigerated centrifugation is carried out at 4 ℃ and 6000 rpm; freeze-drying in freeze dryer for 48 hr, and pulverizing.
Further, in the seventh step, the freeze-drying protective agent is composed of 15-20 parts of glycerol, 10-12 parts of lactose, 8-10 parts of trehalose, 5-8 parts of casein and 50-55 parts of sterile water.
The invention also aims to provide the lactobacillus GM _1 obtained by breeding by applying the breeding method of the lactobacillus GM _1, wherein the strain of the lactobacillus GM _1 is preserved by the general microorganism center of China Committee for culture Collection of microorganisms of China academy of sciences, China institute of microbiology, No.1, North Chen Lu, No. 3, the prefecture of the Korean district, Beijing, within 2015, at 07-17 days, and the preservation number is CGMCC No. 11131.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention develops a large-scale lactobacillus mutant group by using a chemical mutagenesis method, and then breeds a lactobacillus strain capable of rapidly growing by using a high-throughput microorganism screening technology, and the chemically mutagenized strain has no biological safety risk and is stable in heredity. The lactobacillus mutant group subjected to chemical mutagenesis can be used as a new lactobacillus genetic germplasm resource library for screening high-quality lactobacillus strains with any characters, and can meet the production requirements of various characteristic products. The breeding method of the lactobacillus provided by the invention has the advantages of short time consumption, low cost, no need of establishing a large-scale strain resource library, regular shape and obvious characteristics of the bred high-quality lactobacillus strain, obvious bacterial colony and quick growth.
Drawings
FIG. 1 is a flow chart of a method for breeding Lactobacillus GM _1 according to an embodiment of the present invention.
Fig. 2 is a flowchart of a method for preparing a MRS liquid medium according to an embodiment of the present invention.
FIG. 3 is a schematic diagram of Lactobacillus GM _1 mutant population provided by the embodiments of the present invention.
FIG. 4 is a schematic diagram of a novel Lactobacillus GM _1 that can be bred and grown rapidly according to embodiments of the present invention.
FIG. 5 is a schematic diagram of an electron microscope of Lactobacillus GM _1 provided by the embodiments of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a lactobacillus GM _1 and a breeding method thereof, and the invention is described in detail with reference to the attached drawings.
As shown in fig. 1, the method for breeding lactobacillus GM _1 provided by the embodiment of the present invention includes the following steps:
s101, strain activation culture: inoculating a lactobacillus strain to be bred into an MRS liquid culture medium, carrying out activation culture for 24h in the dark under the conditions of 37 ℃ and aerobic condition, carrying out subculture for 3 generations, streaking the obtained culture solution onto an MRS plate culture medium, and culturing for 48h at 37 ℃;
s102, selecting a single colony from an MRS plate culture medium as an initial strain, inoculating the initial strain into 5ml of an MRS liquid culture medium, performing static culture at 37 ℃ for 24-48 h, and performing subculture for 3 generations; inoculating the strain in an MRS liquid culture medium according to the inoculation amount of 3%, performing static culture at 37 ℃ for 24-36 hours to obtain activated bacterial liquid, and performing freeze-drying preservation;
s103, preparing a seed inoculation liquid: placing the lactobacillus single colony obtained in the step two into 5-8 mL of physiological saline, and uniformly mixing to obtain lactobacillus suspension; diluting the bacterial suspension with an LSM culture solution until the OD value is 0.15-0.2 under the condition of the wavelength of 600-800 nm to obtain a lactobacillus inoculation solution;
s104, sucking 2.5ml of lactobacillus inoculation liquid in a sterile centrifuge tube, adding nitrosoguanidine mother liquid and a sterile phosphate buffer solution to fix the volume to 5.0ml, placing the centrifuge tube on a shaking bed, uniformly mixing at the temperature of 36.5-37 ℃, centrifuging at 5000r/min for 6min, discarding supernatant, washing, and diluting to obtain nitrosoguanidine treated bacterial liquid;
s105, coating the nitrosoguanidine-treated bacterial liquid on an MRS plate culture medium, culturing for 24h at 37 ℃, selecting a single colony, inoculating the single colony in the liquid culture medium, culturing for 24h at 37 ℃ to obtain lactobacillus mutant monomers, and forming a mutant group by each treated new germ plasm;
s106, streaking the mutant monomer obtained in the fifth step on an MRS plate culture medium, culturing for 24-36 h at 37 ℃, and selecting a colony which is regular in morphology, obvious in characteristic and obviously larger than other colonies to obtain a new and rapidly-growing lactobacillus GM _ 1;
s107, inoculating the lactobacillus GM _1 obtained by breeding in the sixth step into an MRS culture medium for culture, and then carrying out refrigerated centrifugation to obtain a precipitate; and adding a freeze-drying protective agent into the precipitate, freeze-drying, taking out, and crushing to powder in an aseptic environment to obtain the bred lactobacillus GM _1 freeze-dried powder.
As shown in fig. 2, the preparation method of the MRS liquid medium provided by the embodiment of the present invention is:
s201, mixing glucose, beef extract, peptone, yeast powder, anhydrous sodium acetate, dipotassium hydrogen phosphate, diamine citrate, Tween-80 and manganese sulfate;
s202, adding KH into the mixed solution2PO4800mL of buffer solution;
s203, adjusting the pH value to 7 by using 0.1mol/L NaOH;
and S204, sterilizing at 120 ℃ for 10min to obtain the MRS liquid culture medium.
In step S101 provided by the embodiment of the invention, the MRS liquid medium is composed of 20g/L glucose, 12g/L beef extract, 10g/L peptone, 5g/L yeast powder, 5g/L anhydrous sodium acetate, 2g/L dipotassium hydrogen phosphate, 1.5g/L diamine citrate, 0.5ml/L Tween-80 and 0.25g/L manganese sulfate.
In step S101 provided by the embodiment of the invention, the MRS plate culture medium is prepared by adding 25.0g/L agar on the basis of an MRS liquid culture medium and performing autoclaving for 20-30 min.
In step S104 provided in the embodiment of the present invention, the diluting method includes: after discarding the supernatant, washing the thalli for 2-3 times by using a sterilized phosphate buffer, adding an equal volume of the sterilized phosphate buffer to suspend the thalli, and properly diluting.
In step S105 provided in the embodiment of the present invention, 20% of sterilized glycerol is added to the lactobacillus mutant population, and the lactobacillus mutant population is stored at-80 ℃ for a long period of time.
In step S106 provided in the embodiments of the present invention, the colony size is more than 2 times of the colony size without mutagenesis in the same culture time.
In step S107 provided in the embodiment of the present invention, after culturing lactobacillus GM _1 at 37 ℃ for 48 hours, performing refrigerated centrifugation at 4 ℃ and 6000 rpm; freeze-drying in freeze dryer for 48 hr, and pulverizing.
In step S107 provided by the embodiment of the invention, the freeze-drying protective agent is composed of 15-20 parts of glycerol, 10-12 parts of lactose, 8-10 parts of trehalose, 5-8 parts of casein and 50-55 parts of sterile water.
The lactobacillus GM _1 strain provided by the embodiment of the invention is preserved in the general microbiological center of China Committee for culture Collection of microorganisms of China academy of sciences, China national institute of microbiology No. 3, West Lu No.1, the south China, in the region of the rising of Beijing at 17.07.2015, and the preservation number is CGMCC No. 11131.
The technical solution of the present invention is further described with reference to the following specific examples.
The MRS medium used in the experiment consisted of the following amounts of components, calculated as 1L:
inoculating the lactobacillus GM _1 strain to an MRS culture medium, and sealing and culturing by gauze to obtain a fermentation strain.
Through detection, the MRS culture medium is beneficial to the multiplication culture of the lactobacillus GM _1 strain.
The bred Lactobacillus GM _1 strain is mixed with freeze-dried powder of lactococcus lactis and leuconostoc mesenteroides in a ratio of 2: 1: 0.5 for storage, the reduction speed of the pH value in storage is slow, and the storage time of the mixture at the storage temperature of 4 ℃ can reach 110 days.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A breeding method of lactobacillus GM _1 is characterized by comprising the following steps:
step one, strain activation culture: inoculating a lactobacillus strain to be bred into an MRS liquid culture medium, carrying out activation culture for 24h in the dark under the conditions of 37 ℃ and aerobic condition, carrying out subculture for 3 generations, streaking the obtained culture solution onto an MRS plate culture medium, and culturing for 48h at 37 ℃;
selecting a single colony from an MRS plate culture medium as an initial strain, inoculating the initial strain into 5ml of an MRS liquid culture medium, performing static culture at 37 ℃ for 24-48 h, and performing subculture for 3 generations; inoculating the strain in an MRS liquid culture medium according to the inoculation amount of 3%, performing static culture at 37 ℃ for 24-36 hours to obtain activated bacterial liquid, and performing freeze-drying preservation;
step three, preparing inoculation liquid: placing the lactobacillus single colony obtained in the step two into 5-8 mL of physiological saline, and uniformly mixing to obtain lactobacillus suspension; diluting the bacterial suspension with an LSM culture solution until the OD value is 0.15-0.2 under the condition of the wavelength of 600-800 nm to obtain a lactobacillus inoculation solution;
absorbing 2.5ml of lactobacillus inoculation liquid in a sterile centrifuge tube, adding nitrosoguanidine mother liquid and a sterile phosphate buffer solution to fix the volume to 5.0ml, placing the centrifuge tube on a shaking bed, uniformly mixing at the temperature of 36.5-37 ℃, centrifuging at 5000r/min for 6min, removing supernatant, washing, and diluting to obtain nitrosoguanidine treated bacterial liquid;
step five, coating the nitrosoguanidine-treated bacterial liquid on an MRS plate culture medium, culturing for 24h at 37 ℃, selecting a single colony to inoculate in the liquid culture medium, culturing for 24h at 37 ℃ to obtain lactobacillus mutant monomers, and forming a mutant group by each treated new germ plasm;
step six, streaking the mutant monomer obtained in the step five on an MRS plate culture medium, culturing for 24-36 h at 37 ℃, and selecting a colony which is regular in morphology, obvious in characteristic and obviously larger than other colonies to obtain a new and rapidly-growing lactobacillus GM _ 1;
step seven, inoculating the lactobacillus GM _1 obtained by breeding in the step six into an MRS culture medium for culture, and then carrying out refrigerated centrifugation to obtain a precipitate; and adding a freeze-drying protective agent into the precipitate, freeze-drying, taking out, and crushing to powder in an aseptic environment to obtain the bred lactobacillus GM _1 freeze-dried powder.
2. The method for selectively breeding lactobacillus GM _1 as claimed in claim 1, wherein in step one, the MRS liquid medium is prepared from 20g/L glucose, 12g/L beef extract, 10g/L peptone, 5g/L yeast powder, 5g/L anhydrous sodium acetate, 2g/L dipotassium hydrogen phosphate, 1.5g/L diamine citrate, 0.5ml/L Tween-80, 0.25g/L manganese sulfate and KH with concentration of 0.25-1.0 mol/L2PO4And (4) buffer solution.
3. The breeding method of lactobacillus GM _1 according to claim 2, wherein the preparation method of MRS liquid medium is:
(1) mixing glucose, beef extract, peptone, yeast powder, anhydrous sodium acetate, dipotassium hydrogen phosphate, diamine citrate, Tween-80 and manganese sulfate;
(2) adding KH into the mixed solution2PO4Buffer solution with constant volume of 1000 mL;
(3) adjusting the pH value to 7 by using 0.1mol/L NaOH;
(4) sterilizing at 120 deg.C for 10min to obtain MRS liquid culture medium.
4. The breeding method of lactobacillus GM _1 according to claim 1, wherein in step one, the MRS plate medium is supplemented with 25.0g/L agar based on MRS liquid medium and autoclaved for 20-30 min.
5. The method for breeding lactobacillus GM _1 according to claim 1, wherein in step four, the dilution method is: after discarding the supernatant, washing the thalli for 2-3 times by using a sterilized phosphate buffer, adding an equal volume of the sterilized phosphate buffer to suspend the thalli, and properly diluting.
6. The method for breeding lactobacillus GM _1 according to claim 1, wherein in step five, 20% of sterilized glycerol is added to the lactobacillus mutant population and the lactobacillus mutant population is stored for a long period of time at-80 ℃.
7. The method for selectively breeding Lactobacillus GM _1 according to claim 1, wherein in step six, the colony size is more than 2 times the colony size without mutagenesis in the same culture time.
8. The method for breeding lactobacillus GM _1 according to claim 1, wherein in step seven, the lactobacillus GM _1 is subjected to refrigerated centrifugation at 6000rpm at 4 ℃ after being cultured for 48 hours at 37 ℃; freeze-drying in freeze dryer for 48 hr, and pulverizing.
9. The breeding method of lactobacillus GM _1 as claimed in claim 1, wherein in step seven, the freeze-drying protective agent is composed of 15-20 parts of glycerol, 10-12 parts of lactose, 8-10 parts of trehalose, 5-8 parts of casein, and 50-55 parts of sterile water.
10. A lactobacillus GM _1 obtained by breeding by the method for breeding lactobacillus GM _1 according to any of claims 1 to 9, wherein the strain of lactobacillus GM _1 has been deposited by the general microbiology center of the institute of microbiology, china academy of sciences, china institute of microbiology, No.1, north chen west way, No. 3, north township, north america, on 17 months at 2015, 07-17 days, with a collection number of CGMCC No. 11131.
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| CN114774403A (en) * | 2022-04-27 | 2022-07-22 | 湖州师范学院 | Targeting breeding method of agricultural lactic acid bacteria |
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| CN105002125A (en) * | 2015-08-19 | 2015-10-28 | 内蒙古农业大学 | High resistant gentamycin lactobacillus casei GM-1 and breeding method thereof |
| WO2017037046A1 (en) * | 2015-08-31 | 2017-03-09 | Chr. Hansen A/S | Lactobacillus fermentum bacteria with antifungal activity |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114774403A (en) * | 2022-04-27 | 2022-07-22 | 湖州师范学院 | Targeting breeding method of agricultural lactic acid bacteria |
| CN114774403B (en) * | 2022-04-27 | 2023-08-08 | 至农科技发展(浙江)有限公司 | Targeted breeding method for agricultural lactic acid bacteria |
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Application publication date: 20200911 |