CN101914477A - Lactobacillus plantarum strain and application thereof - Google Patents

Abstract

The invention discloses a high-yielding CLA (conjugated linoleic acid) lactobacillus plantarum strain, which is named as 'Lactobacillus plantarum' lp15-2-1 and collected in China General Microbiological Culture Collection Center (CGMCC) (collection date: April 27th, 2010; collection NO.: CGMCC NO. 3782). The strain of the invention can convert linoleic acid into conjugated linoleic acid, wherein the maximum yield of the conjugated linoleic acid in resting cells is 1,023.55 mu g/ml, and the conversion rate of the linoleic acid by direct shaking culture reaches 21.32 percent; therefore, the strain of the invention can be applied to the industrial production of acidophilous milk, pickles, fruit drinks and the like.

Description

One lactobacillus plantarum strain and uses thereof

Technical field

The invention belongs to the food microorganisms fermentation technical field, relate in particular to lactobacillus plantarum strain and uses thereof.

Background technology

Conjugated linolic acid (Conjugated linoleic acid is hereinafter to be referred as CLA) is the octadecadienoic acid that contains cis or trans conjugated double bond, is the one group of position of linolic acid (Linoleic acid is hereinafter to be referred as LA) and the general name of conformer.Wherein two of CLA kinds of isomer are along 9, anti-11-CLA (c9, t11-CLA) and anti-10, along 12-CLA (t10, c12-CLA) has the important physical function, such as anticancer (colorectal carcinoma, cancer of the stomach, mammary cancer, prostate cancer), the generation that improves cellular immunization, reduction body fat content, prevent diabetes and inhibition atherosclerosis etc.

Occurring in nature CLA is mainly derived from cud animal meat and milk preparation, and the content of CLA along with the change of animal varieties, feeding manner and the condition of raising difference; CLA content is 0.29%～0.71% in the human milk, and the horse Ruzhong is 0.05%～0.12%, and the pig Ruzhong is 0.19%～0.27%, and content is lower in sea-food and the rapeseed oil, is respectively 0.03%～0.06% and 0.01%～0.07%.Among milk and the contained CLA of meat product, c9, the t11-CLA isomer accounts for 75%～90%; And have 50% to be c9 in the rapeseed oil approximately, t11-CLA, the 40%th, t10, c12-CLA.

Commercially producing CLA at present mainly is to carry out the base catalysis isomery with the seed oil (as sunflower oil (64%LA), Semen Maydis oil (57%LA), Oleum Gossypii semen (53%LA), soya-bean oil (51%LA) etc.) that is rich in LA to make.Its mechanism is: under the highly basic condition, the 11st carbon of LA is seized a proton, forms carbanion, then because the thermodynamics factor causes the carbanion migration, thereby forms different conjugation products.Adopt the chemical process principle fairly simple, but can produce a series of CLA isomer mixtures, saturated or unsaturated fatty acids and toxic substance in reaction process, active CLA purifying technique is loaded down with trivial details, and application cost is higher.

Found that some cud bacterium, bacterium acidi propionici and milk-acid bacteria can synthesize CLA.The synthetic CLA of microorganism cultivates and flexibly, does not need High Temperature High Pressure; The isomer that generates is with c9, and t11-CLA is main, and purification procedures is simple relatively.But the cud bacterium is a strictly anaerobic bacterium, cultivates difficulty, is difficult to realize scale operation.And milk-acid bacteria is the human body probiotic bacterium mostly, produces CLA with it and can directly apply to food, or be refined into medicine, and therefore using milk-acid bacteria production CLA will have broad application prospects.

Summary of the invention

The invention provides a kind of lactobacterium plantarum strain, this bacterial strain can be converted into CLA with LA efficiently.

One plant height produces the lactobacterium plantarum strain of CLA, called after plant lactobacillus (Lactobacillusplantarum) lp15-2-1, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) that is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date is on April 27th, 2010, and preserving number is CGMCC NO.3782.

The above-mentioned bacterial strains major physiological is characterized as: well-grown on the MRS substratum, form white, translucent, circular bacterium colony, and the bacterium colony surface wettability, ne ar is rod-short, the two ends ellipse, the gramstaining reaction is positive, and no mobility is not produced gemma.

The present invention also provides above-mentioned bacterial strains in the application that LA is converted among the CLA.

Soon above-mentioned bacterial strains is inoculated in and directly shakes (grown cell) cultivation or resting cell cultivation in the MRS nutrient solution that contains substrate LA, the concentration of substrate LA can be controlled at 0.2mg/ml and 25mg/ml respectively, certainly this concentration can be adjusted, as long as not serious cell growth inhibiting is preferably at 0.2～25mg/ml.

Plant lactobacillus of the present invention can be converted into CLA with LA, and directly the transformation efficiency cultivated of concussion is up to 21.32%, and the highest CLA output 1023.55 μ g/ml of resting cell can be used for the suitability for industrialized production of sour milk, pickles, nectar etc.

Description of drawings

Fig. 1 is the phylogenetic tree of the 16S rRNA sequence of bacterial strain of the present invention.

Fig. 2 is the 16s rRNA sequence pcr amplification gel electrophoresis spectrum of bacterial strain of the present invention and plant lactobacillus ACCC10171.

Embodiment

The separation and purification of bacterial strain

Buy separation and the screening of carrying out bacterial classification the pickles that obtain from the different supermarkets of city of Hangzhou, concrete operations are for after adopting pickles the sterilized water washing, and the washings weight percent concentration is 0.8% stroke-physiological saline solution gradient dilution (10 ^-5), be coated on then on the MRS flat board, all flat boards are divided into 8 groups, after placing differing temps (30 ℃ or 37 ℃), different oxygen level (anaerobism or little oxygen) to cultivate different time (24 or 48 hours), picking list bacterium colony inserts in the MRS liquid nutrient medium, is kept in the MRS slant medium after cultivating under the condition identical with separation condition.

Be inoculated in the MRS liquid nutrient medium that contains 0.2mg/ml LA after the bacterial strain activation that above-mentioned separation is obtained, 180rpm utilizes two volumes normal hexane extraction CLA after cultivating 24h, utilize the resultant quantity of CLA in the determined by ultraviolet spectrophotometry substratum then, according to the CLA resultant quantity, relatively each bacterial strain conversion LA is the ability of CLA, chooses the 10 strain bacterial strains of CLA resultant quantity greater than 20 μ g/ml.

The activation of above-mentioned 10 strain bacterial strains is placed on makes suspension in the sterilized water, with concentration 1% ethyl sulfate mutagenesis 30min, be coated on after the dilution on the MRS flat board, select the bacterial strain that does not have serious variation, the bacterial strain part of picking is freezing, and part is inoculated in the MRS liquid nutrient medium and activates 2 times under 30 ℃ of conditions, is inoculated into then in the MRS substratum that contains 0.2mg/ml LA, detect CLA content with ultraviolet spectrophotometry behind the two volumes normal hexane extraction CLA, the bacterial strain of screening forward variation.

The bacterial strain activation of above-mentioned forward variation is placed on makes suspension in the sterilized water, shine 20s with the 30W ultraviolet lamp apart from 30cm, be coated on after the dilution on the MRS flat board, select the bacterial strain that does not have serious variation, the bacterial strain part of picking is freezing, part is inoculated in the MRS liquid nutrient medium and activates 2 times under 30 ℃ of conditions, be inoculated into then in the MRS substratum that contains 0.2mg/ml LA, behind two volumes normal hexane extraction CLA, adopt ultraviolet spectrophotometry to detect CLA content, screening forward dissociant is selected a strain and is transformed the strongest bacterial strain of LA ability.

Screen used various substratum:

Isolation medium (g/L): MRS;

Liquid seed culture medium (g/L): MRS;

Fermention medium (g/L): MRS+LA

The MRS medium component:

Peptone 10.0g/L, extractum carnis 10.0g/L, yeast extract 5.0g/L, glucose 20.0g/L, anhydrous sodium acetate 5.0g/L, triammonium citrate 2.0g/L, Tween 801.0ml/L, dipotassium hydrogen phosphate 2.0g/L, sal epsom 0.02g/L, manganous sulfate 0.05g/L.

The evaluation of bacterial strain

Above-mentioned strain separated is well-grown on the MRS substratum, forms white, translucent, circular bacterium colony, the bacterium colony surface wettability, and ne ar is rod-short, the two ends ellipse, the gramstaining reaction is positive, and no mobility is not produced gemma.

Carry out the fermenting experiment that bacterial strain utilizes 49 kinds of carbohydrate with API 50CHL test kit (French Mei Liai company), the result is as shown in the table:

Annotate :+represent positive ,-represent feminine gender, d represents uncertain.

Above-mentioned experimental result is identified to it with APILAB Plus automated interpretation system qualification result shows that this bacterial strain is the plant lactobacillus (Lactobacillus plantarum) of genus lactubacillus, evaluation value 99.9%.Consult uncle Jie Shi systematic bacteriology identification handbook simultaneously and find that the colonial morphology of plant lactobacillus is consistent with the colonial morphology of above-mentioned separating obtained bacterial strain.

16s rRNA sequence to above-mentioned bacterial strains and plant lactobacillus ACCC10171 increases, and the pcr amplification product size is about 1500bp (as shown in Figure 2), and sequencing result shows that the 16SrRNA total length of bacterial strain is 1471bp, and the Genbank accession number is: FJ763580; Plant lactobacillus ACCC10171 16S rRNA total length is 1468bp.Through comparison, bacterial strain and plant lactobacillus ACCC10171 16S rRNA sequence homology reach 99.2%, and drawing system is grown tree behind the Blast, the result shows that bacterial strain belongs to plant lactobacillus (Lactobacillus plantarum) (as shown in Figure 1), and is consistent with the gained result of API Bacteria Identification system.Therefore can assert that this bacterium is a plant lactobacillus, and called after plant lactobacillus (Lactobacillus plantarum) lp15-2-1, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), preservation date is on April 27th, 2010, and preserving number is CGMCC NO.3782.

Transform LA

Utilize the cultural method of plant lactobacillus CGMCC No.3782 production CLA of the present invention as follows:

At first it is inoculated in the MRS nutrient solution and activates (1% inoculum size access 10ml MRS, 30 ℃ of cultivation 24h; Double), again bacterial classification is forwarded in the MRS nutrient solution, adds substrate LA and carry out fermentation culture, can divide following dual mode:

Directly shake (grown cell) cultural method: leavening temperature is 30 ℃, and rotating speed is 180r/min, and substrate LA concentration is 0.2mg/ml, stops fermentation behind the cultivation 48h, and obtaining CLA output is 44.72 μ g/ml, and transformation efficiency is 21.32%.

Resting cell cultural method: after interpolation 0.5%LA induces in fermented liquid, cultivate 24h.Behind the 24h, 10000rpm, 4 ℃, frozen centrifugation 5～20min collects thalline, and washs three times with physiological saline.The wet cell that weighing obtains, with the citric acid-sodium citrate damping fluid dissolving of pH5.5, the LA25.0mg/ml after the adding emulsification, 120h is cultivated in concussion, and obtaining CLA output is 1023.55 μ g/ml.

Claims (5)

1. lactobacterium plantarum strain, it is characterized in that: called after plant lactobacillus (Lactobacillus plantarum) lp15-2-1, preserving number is CGMCC NO.3782.

2. the described lactobacterium plantarum strain of claim 1 is in the application that linolic acid is converted in the conjugated linolic acid.

3. application according to claim 2 is characterized in that, may further comprise the steps:

Lactobacterium plantarum strain CGMCC NO.3782 is inoculated in contains in the linoleic substratum, directly concussion is cultivated or resting cell is cultivated 48～120h.

4. application according to claim 2 is characterized in that: described substratum is the MRS substratum.

5. application according to claim 2 is characterized in that: described linolic acid concentration is 0.2mg/ml～25mg/ml.