Lactobacillus plantarum with fast acid production and high acid yield and application of lactobacillus plantarum

A technology of Lactobacillus plantarum and acid amount, applied in the field of microorganisms, can solve the problems of unsatisfactory acid production rate and acid production amount, and achieve the effects of high positive mutation rate, quality improvement and growth inhibition.

Active Publication Date: 2021-02-26
HEBEI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a kind of plant lactobacillus with fast acid production and high acid production and its application, so as to solve the problem of unsatisfactory acid production rate and acid production of existing plant lactobacillus

Method used

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  • Lactobacillus plantarum with fast acid production and high acid yield and application of lactobacillus plantarum
  • Lactobacillus plantarum with fast acid production and high acid yield and application of lactobacillus plantarum
  • Lactobacillus plantarum with fast acid production and high acid yield and application of lactobacillus plantarum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Compound mutagenesis

[0027] 1. DES mutagenesis

[0028] Lactobacillus plantarum HBU03 was used as the starting strain, which was isolated from the sediment of the Baiyangdian River Basin in Xiong'an New District and preserved in the Microbial Diversity Research and Application Laboratory of Hebei Province. Take 9.4mL of the bacterial suspension of the strain into a sterile Erlenmeyer flask, add 10mL of pH7.0 sterile phosphate buffer solution and 0.6mL of DES solution, shake evenly, place it in a constant temperature shaker at 37°C, and add it from the DES solution Start timing, process for 35min, add 10mL25%Na 2 S 2 o 3 to stop the reaction. Take the mutagenized bacterial solution and dilute it to a suitable gradient, spread it on bromocresol purple medium, turn the culture dish upside down, and place it in a dark cardboard box, place it in a 39°C incubator for cultivation, and take it out after 4 to 6 days , Use a ruler to measure the diameter of the c...

Embodiment 2

[0033] Example 2 Preparation of protoplasts

[0034] The mutant strain HBU03DV with high acid production and the mutant strain HBU03DV15 with the shortest time to reach the highest acid production were selected as two parental strains for protoplast fusion, in order to achieve the traits of large acid production and short acid production time in the same mutant strain .

[0035] 1. Take the mutant strains HBU03DV and HBU03DV15 cultured for 8 hours (mid-to-late logarithm), collect the bacteria by centrifugation (4000r / min, 5min), wash twice with LPB buffer, resuspend in LPB, and the concentration of bacteria is about 10 7 -10 8 CFU / mL, add lysozyme at a final concentration of 100mg / mL, and bathe in water at 37°C for 30min.

[0036] 2. Protoplast inactivation

[0037]Ultraviolet inactivation: Pour the protoplast body fluid of the above-mentioned mutagenized strain HBU03DV into a glass petri dish with a diameter of 6 cm, put a magnetic needle in the petri dish as a rotor and p...

Embodiment 3

[0039] Example 3 Protoplast fusion and screening

[0040] 1. Fusion of protoplasts: Take 500 μl of protoplast body fluids from the two inactivated parents, mix them, centrifuge at 3000 r / min for 20 minutes at low speed, and discard the supernatant. Resuspend in 500μL LPB, add 9 times the volume of 40% PEG6000, react at room temperature for 20min, immediately add 5mL LPB to dilute to remove the effect of PEG6000, centrifuge twice at 3000r / min, 10min at low speed, and resuspend in 100μL in the buffer. After fusion, it was spread on regeneration plate and cultured in 37°C incubator for 48h. At the same time, uninactivated protoplast fluid was applied as a control.

[0041] 2. Screening of fusion strains: After the colonies grow out, pick a single fusion strain with a sterilized toothpick into the MRS liquid medium, and after static fermentation at 37°C for 42-48 hours, use the mutant strains HBU03DV, HBU03DV15 and the starting strain HBU03 was used as a control to detect the a...

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Abstract

The invention provides lactobacillus plantarum with fast acid production and high acid yield and an application of the lactobacillus plantarum. The lactobacillus plantarum is lactobacillus plantarum WG03, the preservation unit is China General Microbiological Culture Collection Center, the preservation number is CGMCC 20876, and the preservation date is October 12, 2020. The invention provides a microbial agent containing the lactobacillus plantarum. The lactobacillus plantarum or the microbial inoculum can be applied to silage fermentation. The lactobacillus plantarum with rapid acid production and high acid yield is obtained by combining a protoplast fusion technology with a traditional mutagenesis method, and the lactobacillus plantarum is used as a silage additive, so that the growth of spoilage bacteria can be well inhibited, the anaerobic fermentation process is quickly started, and the quality of silage is improved.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a lactobacillus plantarum with fast acid production and high acid production and application thereof. Background technique [0002] Lactic acid bacteria is a general term for a large group of bacteria that ferment sugar to produce a large amount of lactic acid, mainly including Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus, Enterococcus, Leuconostoc, Pediococcus, Bacillus and other genera. Lactic acid bacteria have high application value in important fields closely related to human life, such as medicine, industry, agriculture, etc. It is used in clinical medicine; Lactobacillus plantarum is used in silage. During the silage fermentation process, microorganisms such as lactic acid bacteria attached to raw materials use carbohydrates to form organic acids such as lactic acid, and create an anaerobic acidic environment, thereby inhibiting the growth of spoilage...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N15/03A23K30/18C12R1/25
CPCC12N1/20C12N15/03A23K30/18C12R2001/25C12N1/205A23V2400/169
Inventor 汤晖王艺静宫妍昌艳萍刘桂霞张秀敏
Owner HEBEI UNIVERSITY
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