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Fused lactobacillus capable of utilizing xylose for fermenting lactic acid and resisting high temperature and breeding method of fused lactobacillus

A technology of lactic acid bacteria and high temperature resistance, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve problems such as loss, gene destruction, and affecting the acquisition of good genes of fusion sons, and achieve the effect of avoiding damage

Inactive Publication Date: 2014-06-11
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, during the mutagenesis process, the genes related to the excellent traits of the parents may be destroyed or lost, thereby affecting the acquisition of the excellent genes of the fusion

Method used

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  • Fused lactobacillus capable of utilizing xylose for fermenting lactic acid and resisting high temperature and breeding method of fused lactobacillus
  • Fused lactobacillus capable of utilizing xylose for fermenting lactic acid and resisting high temperature and breeding method of fused lactobacillus
  • Fused lactobacillus capable of utilizing xylose for fermenting lactic acid and resisting high temperature and breeding method of fused lactobacillus

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0036] Example 1, the preparation of protoplasts of Lactobacillus brevis, please refer to figure 1 .

[0037] 1. Pick Lactobacillus brevis (preserved in the General Microbiology Center of the China Microbiological Cultures Management Committee, the preservation number is CGMCC3051, and the public can purchase it at the General Microbiology Center of the Chinese Microbiological Cultures Management Committee). Single colonies were transferred to the modified MRSX medium, and cultured overnight at 37°C.

[0038] 2. Take an appropriate amount of the overnight culture into the fresh culture medium PMRSX to make its OD reach 0.2, and culture it at 37°C for 3~4 hours until the OD reaches 0.6.

[0039] 3. Collect the culture and wash with Tris-LBP buffer 3 times.

[0040] 4. Resuspend in Tris-LBP buffer to make the cell volume reach 109, add 5mg / ml lysozyme, 20U / ml mutanolysin, 37°C, enzymatic hydrolysis for 30~60min. Microscopic examination of the reaction system, when the protopl...

example 2

[0046] Example two, the preparation of plantaractobacillus protoplast

[0047] 1. Pick Lactobacillus plantarum (preserved in the General Microbiology Center of the China Microbiological Strains Management Committee, the preservation number is CGMCC1.556, and the public can purchase it at will from the General Microbiology Center of the China Microbiological Strains Management Committee) and drop it into the modified MRSX culture cultured overnight at 37°C.

[0048] 2. Take an appropriate amount of the overnight culture into the fresh culture medium PMRSX to make its OD reach 0.2, and culture it at 37°C for 3~4 hours until the OD reaches 0.6.

[0049] 3. Collect the culture and wash with Tris-LBP buffer 3 times.

[0050] 4. Resuspend in Tris-LBP buffer to make the cell volume reach 109, add 8mg / ml lysozyme, 25U / ml mutanolysin, 39°C, enzymatic hydrolysis for 30~60min. Microscopic examination of the reaction system, when the protoplast formation rate reached 90% (see figure 1 ...

example 3

[0056] Example three, the preparation of Lactobacillus pentosus protoplast

[0057] 1. Pick Lactobacillus plantarum and drop it into the modified MRSX medium, and culture it overnight at 37°C.

[0058] 2. Take an appropriate amount of the overnight culture into the fresh culture medium PMRSX to make its OD reach 0.2, and culture it at 37°C for 3~4 hours until the OD reaches 0.6.

[0059] 3. Collect the culture and wash with Tris-LBP buffer 3 times.

[0060] 4. Resuspend in Tris-LBP buffer buffer to make the cell volume reach 109, add 3mg / ml lysozyme, 15U / ml mutanolysin, 35°C, and enzymatically hydrolyze for 30~60min. Microscopic examination of the reaction system, when the protoplast formation rate reached 90% (see figure 1 ), 3500rpm, 15min, collect the cells, wash once with HEPE-LBP buffer, and terminate the enzyme reaction. Resuspend in 50uL HEPE-LBP buffer and set aside.

[0061] The media and reagents involved in the experiment consisted of the following.

[0062] MR...

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Abstract

The invention provides a breeding method specific to lactobacillus capable of utilizing xylose. In the method, according to the characteristic that mesophilic lactobacillus with xylose metabolism capability can utilize the xylose but is not resistant to high temperature, the characteristic, of which utilizes the xylose, of the mesophilic lactobacillus is used as a genetic marker. Lactobacillus capable of tolerating high temperature but not utilizing the xylose is selected, and the high-temperature tolerance of the lactobacillus is utilized to serve as the genetic marker for the lactobacillus. Under an appropriate condition, protoplasts of two parents are prepared and fused. High temperature and the xylose are utilized as screening pressure so as to regenerate a fusant of the two parents under the appropriate condition, and thus, a fusant capable of utilizing the xylose and resisting certain high temperature is obtained finally. The fusant obtained by using the method can utilize 40g / l glucose and xylose at the temperature of 45 DEG C respectively, and the yield of the lactic acid can reach 25.14g / l and 20.96g / l.

Description

technical field [0001] The invention relates to a breeding method of lactic acid bacteria, in particular to a breeding method of fusion lactic acid bacteria capable of utilizing xylose to ferment and resistant to high temperature. Background technique [0002] Lactic acid is widely used in the fields of medicine, food, and daily chemical industry. It is an important platform compound that can be converted into various compounds such as acrylic acid, propylene glycol, acetaldehyde, and acetylacetone. In recent years, with the application of lactic acid and its derivatives in biodegradable plastics and green solvents, the industrial demand for lactic acid has gradually increased. [0003] At present, the annual output of lactic acid in the world is about 200,000-250,000 tons, 90% of which are produced by breeding specific microorganisms by fermentation. Lactic acid fermentation mostly uses starchy raw materials such as rice and corn for fermentation, but the production cost o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/03C12N1/20C12R1/225
Inventor 陈树林郭蔚马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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