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Method of separating and purifying phenyl lactic acid from lactobacillus plantarum

A technology of Lactobacillus plantarum and phenyllactic acid, applied in the field of food biology, can solve the problems of limited application scope and narrow antibacterial spectrum.

Active Publication Date: 2018-10-09
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, only one bacteriocin, Nisin, has been commercially used. Due to its narrow antibacterial spectrum, it can only inhibit some Gram-positive bacteria, which greatly limits the scope of application.

Method used

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  • Method of separating and purifying phenyl lactic acid from lactobacillus plantarum
  • Method of separating and purifying phenyl lactic acid from lactobacillus plantarum
  • Method of separating and purifying phenyl lactic acid from lactobacillus plantarum

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1: Activation of Lactobacillus plantarum ZJ316 strain and preparation of fermentation supernatant

[0023] Activation of Lactobacillus plantarum ZJ316 strain: take the strain out from -80°C, thaw at 4°C, streak on MRS solid medium, and culture at 37°C for 24 hours; pick a single colony and place it in MRS liquid medium at 37°C Cultured for 24 hours, successively subcultured twice to obtain seed solution. The seed liquid was inserted into the fermentation medium (MRS liquid) with an inoculation amount of 2% by volume for expansion cultivation, and cultured statically at 37° C. for 24 hours to obtain a fermentation liquid. The fermentation broth was centrifuged at 8000rpm for 15min (4°C) to obtain a supernatant, which was stored at 4°C until use.

Embodiment 2

[0024] Example 2: Separation and purification of phenyllactic acid in the fermentation supernatant of Lactobacillus plantarum ZJ316

[0025] The centrifuged supernatant was adsorbed by XAD-2 macroporous resin, and the flow-through was collected and filtered through a microporous membrane (0.22 μm). The filtrate was separated and purified twice by high performance liquid chromatography. HPLC conditions for the first separation and preparation: the chromatographic column model is YMC-Pack ODS-AQ 150×20mm L.D., the mobile phase: 0.05% trifluoroacetic acid / water (A) and 0.05% trifluoroacetic acid / methanol (B), and the ultraviolet detection conditions: Detector model Waters 2998, detector wavelength: 215nm, column temperature: 25°C, flow rate: 4mL / min, injection volume: 5mL. Gradient elution method: 0, 5, 25, 30, 40, 46 minutes eluent A / B volume ratio compositions are 95:5, 95:5, 5:95, 5:95, 95:5, 95, respectively :5. The chromatographic peak whose retention time is 24.854min is...

Embodiment 3

[0027] Example 3: Phenyllactic acid production and isolation purity in the supernatant of Lactobacillus plantarum ZJ316

[0028] Test the purity of phenyl lactic acid (see image 3 ), analytical HPLC conditions: column model YMC-PackODS-AQ 150×4.6mm L.D., UV detection conditions: detector model Waters 2498, detector wavelength: 215nm, column temperature: 25°C, flow rate: 0.8mL / min, injection volume: 50 μL. The mobile phase and gradient elution method are the same as the second high performance liquid chromatography separation and purification in Example 2.

[0029] Using the same method, a series of concentration gradients (0, 0.05mg / mL, 0.10mg / mL, 0.25mg / mL, 0.50mg / mL, 0.75mg / mL) of phenyl lactic acid standard (purchased from Sigma company) Determination. Establish the standard curve of peak retention time and peak area, and calculate regression equation (see Figure 4 ).

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Abstract

The invention discloses a method of separating and purifying phenyl lactic acid from lactobacillus plantarum and belongs to the field of food biotechnology. Lactobacillus plantarum ZJ316 is already collected at the China Center for Type Culture Collection under a collection number of CCTCC NO:M 208077. Lactobacillus plantarum ZJ316 is used as a target strain; without optimization under any condition, content of phenyl lactic acid in a MRS liquid culture medium fermentation supernatant reaches 108.87mg / mL(0.66mmol / L) through high performance liquid chromatography, and purity of purified phenyllactic acid is up to 98.14%. Chiral chromatographic column-high performance liquid chromatography shows that mass percentage of L-phenyl lactic acid is 92-98%. A product obtained by the method is safeand reliable, can effectively inhibit food-borne pathogenic bacteria and putrefying bacteria and can be applied in medicinal intermediates, food additives and cosmetics.

Description

technical field [0001] The invention belongs to the field of food biotechnology, and specifically relates to a method for separating and purifying phenyllactic acid from the fermentation supernatant of Lactobacillus plantarum ZJ316 (Lactobacillus plantarum ZJ316) strain with the preservation number CCTCC NO: M 208077. Background technique [0002] In the development of the food industry, antimicrobial agents are essential in preventing food spoilage and prolonging the shelf life of food. The types and quantities of chemically synthesized antibacterial agents, especially non-food-sourced chemical antibacterial agents, have multiplied. While promoting the development of the food industry, serious food safety hazards have been buried. Long-term studies have found that some synthetic preservatives are carcinogenic, teratogenic and easily cause food poisoning. The development of broad-spectrum, high-efficiency, safe, stable and natural antibacterial agents is an inevitable requi...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P7/40A23L33/10C12R1/25
CPCA23L33/10C12P7/40C12N1/205C12R2001/25
Inventor 顾青郦萍周青青
Owner ZHEJIANG GONGSHANG UNIVERSITY
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