CN102533591A - High temperature resisting and high-glucose resisting lactic acid bacteria - Google Patents

Abstract

The invention discloses a strain of lactic acid bacteria resistant to high temperature and high sugar. The invention belongs to the field of lactic acid bacteria, in particular to strains resistant to high temperature and high sugar. The lactic acid bacterium provided by the present invention is Lactobacillus rhamnosus (Lactobacillus rhamnosus), and this bacterial strain is preserved in China Microorganism Culture Preservation Management Committee Common Microorganism Center, and preservation number is CGMCC No.4430, and bacterial strain CGMCC No.4430 glucose tolerance concentration can be It reached 270g/L, an increase of 95% compared with the original bacteria. After the fermentation, the lactic acid content was 50g/L, which was 158% higher than that of the original bacteria. The performance indicators tracked by the fermenter experiment show that the strain has normal metabolism, strong ability to produce L-lactic acid, and low content of heteroacids. It is an excellent strain of Lactobacillus rhamnosus with high temperature resistance and high sugar tolerance.

Description

A strain of lactic acid bacteria resistant to high temperature and high sugar

Technical field:

The invention belongs to the field of lactobacilli, in particular to high-temperature-resistant and high-sugar-resistant lactobacillus strains.

Background technique:

In recent years, due to some special functions, the research of extremophiles has attracted more and more attention. Among them, high temperature resistant and hypertonic lactic acid bacteria are particularly prominent due to their wide application in various industries such as food, medicine and chemical industry. At present, its main applications include the following aspects: (1) Lactic acid: Lactic acid is an important food and chemical raw material, and can be used to prepare a degradable polymer material - polylactic acid, so it has attracted much attention in recent years. (2) Biological preservatives: Nisin is a biological preservative widely used in food and beverages in recent years. Compared with chemical preservatives, it is harmless to the human body and has a wider antibacterial spectrum. (3) Food: Yogurt, cheese, kimchi, tempeh, etc. are all functional foods that are widely loved by various countries in the world. They are all fermented with lactic acid bacteria. Under the conditions of hypertonicity and high temperature, it is beneficial to increase the concentration of substrates and products. In addition, it can reduce the contamination of miscellaneous bacteria and reduce the risk of production during production.

For the microbial industry, how to obtain an excellent strain is very critical, so the isolation and selection of lactic acid bacteria is very important, but it is not easy to obtain an excellent strain. Although genetic engineering and other technical means can now be used to add foreign genes to cells, or to delete a certain gene. However, as far as the current technology is concerned, it is not easy to obtain a strain with excellent traits only by the above simple genetic manipulation, and deleting a certain gene or introducing a foreign gene may destroy the metabolic balance in the lactic acid bacteria, thereby affecting the normal state of the cells. metabolic growth. In addition, for the food industry, the use of genetically engineered bacteria may have high safety risks and is prohibited in many countries. Therefore, traditional mutation breeding is still the most important and effective technique for most industrial microorganism breeding.

The methods currently used for microbial mutation breeding include physical and chemical mutagenesis, wherein physical mutagenesis includes physical methods such as ultraviolet rays, lasers, X-rays, gamma rays, and fast neutrons, and chemical mutagenesis mainly includes various alkylating agents (sulfuric acid) Diethyl ester, nitrosoguanidine and ethyl methanesulfonate, etc.).

Alkylating agent, also known as alkylating agent, is a highly active class of chemical substances that can transfer small hydrocarbon groups to other molecules. Generally, the introduced alkyl groups are attached to atoms such as nitrogen, oxygen, and carbon. Alkylating agents are often mutagenic because they alter nucleotides in deoxyribonucleic acid. Several different chemotherapeutic drugs are known to be alkylating agents. They have one or two alkyl groups, which are called monofunctional or bifunctional alkylating agents, which can alkylate with nucleophilic groups in DNA, RNA or proteins of cells, often forming cross-links or causing Depurination breaks the DNA chain, and in the next replication, it can cause base pairing errors, resulting in damage to the structure and function of DNA, and in severe cases, cell death. Belongs to cell cycle non-specific drugs. Commonly used alkylating agents are olefins, haloalkanes, alkyl sulfates, etc.

These alkylating agents are widely used in traditional mutagenesis operations due to their good mutagenesis breeding effects and simple operating conditions.

Invention content:

The technical problem to be solved by the present invention is to provide a new lactobacillus strain and its application in lactic acid fermentation.

The lactobacillus provided by the present invention is Lactobacillus rhamnosus (Lactobacillus rhamnosus). No. 3, No. 1 West Road, Institute of Microbiology, Chinese Academy of Sciences, Zip code 100101. The date of deposit was December 8, 2010.

The characteristics of the strain are as follows: observed under a microscope, the strain is rod-shaped, with a width of less than 1 μm, and 2 to 3 bacilli are easy to connect together; on a solid medium, the bacterial colony is milky white, with a smooth, moist, sticky surface. Thick, with neater edges. Compared with the original strain, the mutant strain was obviously smaller than the starting strain in morphology.

The starting strain Lactobacillus rhamnosus CGMCC No.1.2134 was purchased from the General Microbiology Center of China Committee for the Collection of Microorganisms.

Lactobacillus rhamnosus of the present invention adopts following procedure to select and breed:

Original starting strain → test tube activation → high temperature acclimatization → diethyl sulfate (DES) mutagenesis → high sugar plate screening → nitrosoguanidine (NTG) mutagenesis screening → high temperature bacteria screening → shake flask re-screening → passage stability test →5L fermenter test

The optimal fermentation temperature of the original bacterial strain used in the present invention is 37°C. In order to improve its high temperature resistance, the method of gradually increasing the temperature is first adopted for domestication. Until it can grow on the plate at 45°C. Then adopt DES to carry out further mutagenesis to the high-temperature bacteria obtained, after the mutagenesis, carry out preliminary screening through medium A high sugar plate (250g/L glucose), then continue to carry out NTG mutagenesis to the bacterial strain that is bred, through medium A high Sugar plate (250g/L glucose) for primary screening of high-temperature bacteria (tolerance to 55°C), and then re-screening with 250mL Erlenmeyer flask to select and breed excellent Lactobacillus strains, and then do subculture experiments to evaluate their genetic stability, and use liquid phase Chromatography and GC-MS were used to determine the metabolite content in the fermentation broth, and finally a 5L fermenter was used to evaluate the experimental effect.

The results of the genetic stability of the strain CGMCC No.4430 showed that after ten consecutive passages, all performance indicators were relatively stable, heredity was good, and the traits did not recover. Therefore, the strain CGMCC No.4430 was selected as the target strain obtained by selection.

The target strain CGMCC No.4430 was tested in a 5L lactic acid fermenter, and the results showed that: compared with the starting strain, the glucose tolerance concentration of CGMCC No.4430 could reach 270g/L, which was 95% higher than that of the original strain; , the lactic acid content is 60g/L, which is 158% higher than that of the original bacteria.

Beneficial effect:

1) In this study, Lactobacillus rhamnosus was bred using the combination of DES and NTG mutagenesis techniques, and an excellent strain CGMCC No.4430 was obtained. The mutant strain can grow well at 55°C, and the fermentation medium does not need to be sterilized. The glucose tolerance is 270g/L, which is 95% higher than that of the original bacteria. This bacterial strain has good stable inheritance, and in the process of ten consecutive subcultures, the traits have not recovered, and all performance indicators are normal.

2) The various performance indicators tracked by the fermenter experiment show that the strain has normal metabolism, strong ability to produce L-lactic acid, and low heteroacid content.

Detailed ways:

The following examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

Example 1:

The specific process is as follows:

1. High temperature acclimatization

1) Take a ring of Lactobacillus rhamnosus CGMCC No.1.2134 on the slant of the test tube on the ultra-clean bench, insert it into a 250mL Erlenmeyer flask filled with 50mL of medium A (no agar), culture at 200rpm, 37°C for about 12h, Make the bacteria in the early logarithmic growth stage.

Medium A: (casein peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, glucose 5.0g, sodium acetate 5.0g, diamine citrate 2.0g, Tween 80 1.0g, dipotassium hydrogen phosphate 2.0g, heptahydrate Magnesium sulfate 0.2g, manganese sulfate monohydrate 0.05g, calcium carbonate 20.0g, agar 15.0g, distilled water 1.0L, pH6.8).

2) Take 5 mL of the bacterial liquid, centrifuge at 5000 rpm for 10 minutes to collect the bacterial cells, and wash twice with normal saline.

3) Dilute with normal saline to ¹⁰⁷ /mL bacterial suspension.

4) Dilute and spread on a plate containing medium A. After culturing at 39°C for 2 to 3 days, pick the strain with the largest colony and mark it as H1 bacteria.

5) Repeat the above method, and spread the screened bacterial dilution on the plate containing medium A. After culturing at 41°C for 2 to 3 days, pick the strain with the largest colony and label it as H2 bacteria.

6) Repeat the above operations, increasing the culture temperature by 2°C each time until a strain capable of growing at 45°C is screened out, labeled as H bacteria.

2. DES mutagenesis selection

1) Take the H-ring of Lactobacillus rhamnosus on the slant of the test tube on the ultra-clean bench, insert it into a 250mL Erlenmeyer flask containing 50mL medium A (no agar) medium, and incubate at 200rpm at 45°C for about 12h, so that The bacteria are in the early logarithmic growth stage.

2) Take 5 mL of the bacterial liquid, centrifuge at 5000 rpm for 10 minutes to collect the bacterial cells, and wash twice with normal saline.

3) Dilute with pH 7.0 phosphate buffer to 10 ⁷ /mL bacterial suspension.

4) Take 32mL of pH7.0 potassium phosphate buffer solution, 8mL of bacterial suspension, and 0.4mL of DES in a 150mL Erlenmeyer flask pre-placed in the rotor and mix thoroughly so that the final concentration of DES is 1% (v/v).

5) React in a shaker at 30° C. at 150 rpm for 30 min, take 1 mL of the mixed solution, and add 0.5 mL of 25% Na ₂ S ₂ O ₃ solution to stop the reaction.

6) Dilute and spread in medium A screening medium plate containing 250g/L glucose. After culturing at 45°C for 2 to 3 days, pick the strain with the largest colony and label it as HG bacteria.

3. Nitrosoguanidine Mutagenesis

1) Take the ring of Lactobacillus rhamnosus HG on the slant of the test tube on the ultra-clean bench, insert it into a 250mL Erlenmeyer flask containing 50mL medium A (agar-free) medium (glucose concentration is 250g/L), 200rpm , Cultivate at 45°C for about 12 hours, so that the bacteria are in the early logarithmic growth stage.

2) Take 5 mL of the bacterial solution and centrifuge at 5000 rpm for 10 minutes to collect the bacterial cells, and wash twice with normal saline.

3) Dilute with pH 6.0 phosphate buffer to 10 ⁷ /mL bacterial suspension.

4) Transfer 10 mL of bacterial suspension to a 100 mL Erlenmeyer flask, add 10 mg of NTG to prepare an NTG solution with a final concentration of 10 mg/mL, and add 4-5 drops of acetone to facilitate the dissolution of NTG.

5) Shake and react at 200 rpm for 30 min at 30° C., centrifuge at 5000 rpm for 10 min to collect the bacterial cells, wash with sterile saline several times, and terminate the reaction.

6) Appropriately dilute the coating, take 0.2mL of the final dilution of the bacterial solution, and spread it on the medium A screening medium plate containing 250g/L glucose. After culturing at 55°C for 2 to 3 days, pick 100 colonies with larger colonies.

4. Shake flask re-screening

1) Take a ring of Lactobacillus rhamnosus on the slant of each test tube on the ultra-clean bench, and insert it into a 250mL Erlenmeyer flask containing 50mL medium A (agar-free) medium (glucose concentration: 250g/L), 200 rpm, 30°C for about 15 hours, so that the bacteria are in the mid-logarithmic growth phase.

2) Take 5mL of bacterial liquid, insert it into a 250mL Erlenmeyer flask containing 50mL of high-sugar medium A (no agar) (glucose concentration is 250g/L), culture at 200rpm, 30°C for 3-4 days, and detect the glucose concentration every day and L-lactic acid concentration changes. After the fermentation, the glucose consumption rate and L-lactic acid production rate, the conversion rate of glucose to L-lactic acid and the heteroacid content of the 100 strains were compared.

3) Select the strain with fast glucose consumption rate, low final residual sugar concentration, high L-lactic acid concentration, high conversion rate of glucose to L-lactic acid and low heteroacid content as the final strain, named as HGN bacteria.

5. Genetic stability test

The HGN bacteria were successively subcultured on the slant for ten times, and the fermentation conditions after each subculture were detected by re-screening in shake flasks. The experiment found that after ten consecutive passages on the slant, the traits of the strain did not change significantly, and all performance indicators were normal, indicating that the strain had strong genetic stability.

6.5L fermenter test

1) Take a ring of Lactobacillus rhamnosus on the slant, insert it into a 250mL Erlenmeyer flask filled with 50mL medium A (no agar) (glucose concentration: 150g/L) medium, culture at 200rpm, 30°C for about 12h, Keep the cells in the mid-logarithmic growth phase.

2) Insert the strains in the logarithmic phase into a 5L fermenter equipped with 3L fermentation broth. The inoculum size is 10%, 100rpm at 30°C, the dissolved oxygen control is 10% in the early logarithmic period (0.5L/min aeration), and the anaerobic culture is cultivated for 3-4 days in the late period.

3) After the fermentation, the residual sugar content is 90g/L and the L-lactic acid content is 50g/L, which is 158% higher than that of the original bacteria.

Claims (3)

1. A strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) resistant to high temperature and high sugar, the preservation number is CGMCC No.4430. 2. As described in 1, Lactobacillus rhamnosus CGMCC No.4430, which is resistant to high temperature and high glucose, grows well at 55°C and tolerates 250g/L glucose. 3. The application of high temperature resistance and high sugar tolerance Lactobacillus rhamnosus CGMCC No.4430 in lactic acid production as described in right 1.