Method for purifying indole-3-lactic acid in plant lactobacillus fermentation supernatant

A technology of plant lactic acid bacteria and fermentation supernatant is applied in the field of extracting indole-3-lactic acid from plant lactic acid bacteria fermentation supernatant, and can solve the problems of different mechanical properties of filters, differences in indole-3-lactic acid content and purity, etc.

Active Publication Date: 2022-02-15
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The size of molecules retained by ultrafiltration centrifugation has a certain range, and substances with similar molecular weights are all retained by centrifugation, and the difference in mechanical properties of filters can easily lead to differences in the content and purity of fractionated indole-3-lactic acid

Method used

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  • Method for purifying indole-3-lactic acid in plant lactobacillus fermentation supernatant
  • Method for purifying indole-3-lactic acid in plant lactobacillus fermentation supernatant
  • Method for purifying indole-3-lactic acid in plant lactobacillus fermentation supernatant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Determination of indole-3-lactic acid content in the fermentation supernatant of Lactobacillus plantarum ZJ316

[0051] (1) Streak the Lactobacillus plantarum ZJ316 strain on the MRS solid medium, culture at 37°C for 36h, pick a single colony and culture it in 10mL MRS liquid medium at 37°C for 24h;

[0052] (2) Inoculate the culture solution obtained in step (1) into 150 mL MRS liquid medium with 3% inoculum size (volume concentration) and continue to culture at 37° C. for 24 hours;

[0053] (3) The culture solution obtained in step (2) was inoculated with 3% inoculation amount (volume concentration) in 5L MRS liquid medium for fermentation. Fermentation conditions: temperature 37°C, rotation speed 180rpm, time 24h.

[0054] The obtained fermentation broth was centrifuged: 8000rpm, 25min, 4°C, and the fermentation supernatant was stored at 4°C until use.

[0055] (4) Ultraviolet detection conditions: detector model Waters 2498, detector wavelength: 280n...

Embodiment 2

[0063] Example 2: Separation, purification and identification of indole-3-lactic acid

[0064] Step 1: Adsorption and elution of macroporous resin XAD-16

[0065] (1) 5L Lactobacillus plantarum ZJ316 fermentation supernatant (gained in step (3) of embodiment 1) is adsorbed by 500g macroporous resin XAD-16, and the flow rate is 1mL / min; Then use 2L ultrapure water, 30% methanol , 50% methanol (37.5% concentrated hydrochloric acid to adjust pH=7), the flow rate is 1mL / min, collect the eluent (about 2L) corresponding to 50% methanol with pH=7;

[0066] (2) Concentrate the eluent (50% methanol eluent) obtained in step (1) to 200mL with a rotary evaporator to obtain a concentrated solution; rotary evaporation conditions: water bath temperature 37°C, pressure 30±5mbar, rotation speed 60rpm .

[0067] Step 2: Separation on Sephadex G25 Sephadex Gel Column

[0068] (1) Sephadex G25 Sephadex column was used for column separation of the concentrate obtained in step 1 (column height 8...

Embodiment 3

[0094] Example 3: Purity determination of isolated and purified indole-3-lactic acid

[0095] Take the concentrated sample "H4" of Step 3 of Example 2, and obtain the chromatogram of indole-3-lactic acid according to the analytical HPLC detection conditions and elution procedures described in Step (5) of Example 1, as shown in image 3 shown. Table 4 shows the starting and ending time (min), peak area (μV·s) and peak area ratio (%) of the main component peak (ie, indole-3-lactic acid) and two miscellaneous peaks. According to the peak area normalization method, the main component peak (i.e., indole-3-lactic acid) area is A, and the sum of the main component peak and other miscellaneous peak peak areas is ΣA, and the indole-3-lactic acid purity (%) is obtained. Calculation formula:

[0096] Indole-3-lactic acid purity (%)=A / ∑A×100%.

[0097] Table 4, the peak area of ​​HPLC chromatographic peak

[0098]

[0099] Therefore, through the separation and purification of steps...

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Abstract

The invention discloses a method for purifying indole-3-lactic acid from plant lactobacillus fermentation supernatant. The method utilizes a fermentation supernatant of lactobacillus plantarum ZJ316 with the preservation number of CCTCC NO: M 20807, and comprises the following steps: adsorbing the fermentation supernatant with macroporous resin XAD-16, then eluting, collecting an eluent corresponding to 50% methanol with the pH value of 7, and concentrating the eluent to obtain a concentrated solution; separating the concentrated solution through sephadex G25 to generate a G25-2 component; and carrying out reversed-phase high performance liquid chromatography purification on the G25-2 component, and concentrating the collected eluent to obtain the indole-3-lactic acid. The purity of the obtained indole-3-lactic acid is 99.00%, and the indole-3-lactic acid has a broad-spectrum antibacterial characteristic.

Description

technical field [0001] The invention belongs to the field of food biotechnology, and in particular relates to a method for extracting indole-3-lactic acid from the fermentation supernatant of plant lactic acid bacteria. Background technique [0002] Indole-3-lactic acid (ILA), a metabolite of tryptophan with an indole ring, has a molecular weight of 205 Da and a molecular formula of C 11 h 11 NO 3 , has the structure shown in formula I. Lactic acid bacteria such as Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium breve, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus reuteri, etc. can metabolize indole-3-lactic acid. Indole-3-lactic acid, the only tryptophan metabolite produced by Bifidobacteria, was found in relatively high levels in Bifidobacterium strains isolated from the intestinal tract of human infants compared with other strains, It is 22.17~33.12μg / mL. However, only Lactobacillus plantarum UM55, Lactobacillus plantarum dy-1, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D209/18C12P17/10C12R1/25
CPCC07D209/18C12P17/10C12R2001/25B01D15/08
Inventor 顾青周青青郦萍顾容铖
Owner ZHEJIANG GONGSHANG UNIVERSITY
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