CN109295126B

CN109295126B - Lactobacillus plantarum exopolysaccharide with immunoregulatory activity and preparation method thereof

Info

Publication number: CN109295126B
Application number: CN201811009262.9A
Authority: CN
Inventors: 刘书亮; 祝元婷; 潘婉舒; 王兴洁; 周康
Original assignee: Sichuan Agricultural University
Current assignee: Sichuan Agricultural University
Priority date: 2018-08-31
Filing date: 2018-08-31
Publication date: 2021-01-08
Anticipated expiration: 2038-08-31
Also published as: CN109295126A

Abstract

The invention belongs to the technical field of polysaccharide in microbiology, and particularly relates to microbial exopolysaccharideRelates to lactobacillus polysaccharide with immunoregulation activity from lactobacillus plantarum and a preparation method thereof. The preparation method comprises the steps of preparing the lactobacillus plantarum (A)Lactobacillus plantarumDL 7X) fermenting to obtain extracellular polysaccharide, extracting crude polysaccharide, separating and purifying extracellular polysaccharide to obtain extracellular polysaccharide homogeneous component with immunoregulation activity. The invention adopts ion exchange and molecular sieve chromatographic separation and purification to obtain a uniform component EPS 3; an inflammatory cell model is constructed by stimulating macrophage RAW264.7 of a mouse with lipopolysaccharide, and EPS3 (20-100 mu g/ml) can inhibit NO production of RAW264.7 induced by lipopolysaccharide in a dose-dependent manner; and the strain has negative regulation effect on iNOS and TNF-alpha mRNA on the transcription level, which shows that DL7X exopolysaccharide EPS3 is effective immunomodulatory polysaccharide, and the results provide a new choice for better applying the strain to the fermented food industry and preparing functional foods.

Description

Lactobacillus plantarum exopolysaccharide with immunoregulatory activity and preparation method thereof

Technical Field

The invention belongs to the technical field of polysaccharide in microbiology, and particularly relates to microbial exopolysaccharide, in particular to lactobacillus exopolysaccharide with immunoregulatory activity and a preparation method thereof.

Background

Exopolysaccharides (EPS) of Lactic Acid Bacteria (LAB) are a general term for mucus or capsular polysaccharides secreted outside the cell wall by lactic acid bacteria during growth and metabolism. The lactobacillus which produces exopolysaccharides in nature has wide sources, such as dairy products, fermented foods, animal intestines and the like. In recent years, researches show that extracellular polysaccharide from lactic acid bacteria is a good source of food-grade polysaccharide, LAB EPS has important technological functions, can be used as a thickening agent, a stabilizer, a water holding agent and the like to be applied to the fields of foods, medicines, biochemical products and the like, and particularly has important influence on the rheology, texture, mouthfeel and flavor of yogurt, cheese and most fermented dairy products in the production and application of fermented dairy products. In addition, researches show that EPS has multiple physiological functions, including antioxidation, blood pressure reduction, tumor resistance, intestinal micro-ecological environment improvement, human immunity enhancement and the like. Among them, lactobacillus is often used as an auxiliary starter in the fermented food industry. During the fermentation process of lactobacillus, EPS is generated in situ, so that the quality and the taste of the product can be improved, and the product can be endowed with nutrition and health care effects. Therefore, a great deal of research work is carried out at home and abroad in recent years on the development and application of the high-yield exopolysaccharide lactic acid bacteria.

Among them, research on extracellular polysaccharides of lactic acid bacteria having immunoregulatory activity has been receiving wide attention, and LAB EPS generally enhances the immunoprotection of a host by enhancing cell-mediated immune reactions, such as promotion of T/B lymphocyte proliferation, monocyte phagocytic ability, release of cytokines, and the like. However, researches show that the EPS produced by different strains has very different immunocompetence and is influenced by various factors such as monosaccharide composition, molecular weight, functional groups, charged charges and the like, for example, researches show that acidic polysaccharides with smaller molecular weight often show immunostimulating activity, and neutral polysaccharides with larger molecular weight show immunosuppressive action. Because the LAB EPS has strain specificity, the polysaccharide produced by different strains has great difference, and different polysaccharides have respective specific functions, the research on the immunological activity of the LAB EPS of a newly separated strain is necessary, and theoretical basis can be provided for the application of functional lactobacillus strains, the development of functional foods and the like.

The lactobacillus plantarum DL7X is a high-yield exopolysaccharide lactic acid bacterium screened from Sichuan traditional home-made pickle. Classification name of strain: lactobacillus plantarumLactobacillus plantarumThe microbial inoculum is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation place is No. 3 Xilu No.1 Beijing of the Chaoyang district, and the preservation numbers are as follows: CGMCC number 13331, date of preservation: 2016, 11, 22 months.

Disclosure of Invention

The invention discloses a lactobacillus plantarum exopolysaccharide which is prepared from lactobacillus plantarumL. plantarumDL7X strain. The preferred method is as follows: mixing Lactobacillus plantarumL. plantarumStanding DL7X strain in semisynthetic chemical culture medium (SDM) at 37 deg.C for constant temperature culture, inactivating the fermentation liquid in boiling water bath for 15min, centrifuging to obtain extracellular polysaccharide supernatant, and separating with Tris-D-Removing protein with chloroacetic acid, precipitating with ethanol, dialyzing to obtain crude polysaccharide, and purifying by DEAE Sepharose CL-6B ion exchange chromatography and Sephadex G-100 gel column chromatography. More preferred methods are: standing and culturing lactobacillus plantarum DL7X (CGMCC number 13331) in an SDM culture medium at a constant temperature of 37 ℃ for 18-24 h, inactivating the culture solution in a boiling water bath for 15min, centrifuging to remove thalli to obtain a supernatant, removing protein from the supernatant by a trichloroacetic acid method, centrifuging to remove protein precipitate, precipitating with 3-5 times volume of precooled ethanol, and freeze-drying in vacuum to obtain crude polysaccharide; purifying the crude polysaccharide by DEAE Sepharose CL-6B ion exchange chromatography, wherein the mobile phase comprises 20mM Tris-HCl (pH 7.60) and 0.2-1.0M NaCl, and the flow rate is 1.0 mL/min; dialyzing the eluted components, vacuum drying and the like to obtain primarily purified polysaccharide components, and then evaluating the anti-inflammatory activity of each component by using macrophage RAW264.7 induced by lipopolysaccharide; based on the activity evaluation result, selecting the component with the strongest activity to perform Sephadex G-100 molecular sieve chromatography, wherein the mobile phase is ultrapure water, and the flow rate is 0.25 mL/min; dialyzing the eluted components for 3 days by a dialysis bag with the molecular weight cutoff of 8000-14000 Da, and carrying out vacuum freeze drying on the dialyzed trapped fluid to obtain the final product.

The strain is subjected to constant-temperature static culture in 1L of SDM medium, and the yield of crude polysaccharide can reach 362.5 mg/L. Purifying the cultured crude polysaccharide by DEAE Sepharose CL-6B ion exchange chromatography and Sephadex G-100 molecular sieve chromatography to obtain water-soluble extracellular polysaccharide component EPS3 with acidic ionic group and molecular weight of 2.02 × 10⁴Da. In vitro activity experiments show that in an inflammatory cell model constructed by stimulating macrophage RAW264.7 of a mouse with lipopolysaccharide, NO generated by cells in an inflammatory state is detected by a Griess Kit, and the anti-inflammatory activity of the NO is preliminarily identified; next, the inhibition of inflammatory factors iNOS and TNF-. alpha.mRNA by EPS3 was further examined by PCR. The result shows that the exopolysaccharide component EPS3 produced by the lactobacillus plantarum DL7X can obviously inhibit NO production and iNOS and TNF-alpha mRNA expression in lipopolysaccharide-induced macrophages, and has wide application value in the aspects of development of immunosuppressants, anti-inflammatory drugs and related functional foods.

Anti-inflammatory activity tests of in vitro macrophage inflammation models show that the lactobacillus plantarum exopolysaccharide EPS3 has the activity of remarkably inhibiting NO production in macrophages induced by lipopolysaccharide and inhibiting mRNA expression of related inflammatory factors (iNOS and TNF-alpha). The macrophage is a murine macrophage RAW264.7 cell.

The following are in vitro activity tests and results of the polysaccharides of the present invention:

lipopolysaccharide is used for stimulating macrophage RAW264.7 of a mouse to construct an inflammatory cell model, and the anti-inflammatory activity of extracellular polysaccharide is evaluated. The cells were cultured in RPMI-1640 medium (37 ℃ C., 5% CO) containing 10% fetal bovine serum₂) The mouse macrophage line RAW264.7 was cultured and grouped. Blank control group: no LPS treatment and no polysaccharide intervention; model control group: adding 0.5 mu g/mL LPS for intervention; experimental groups: 0.5. mu.g/mL LPS was co-treated with different concentrations of the Lactobacillus plantarum exopolysaccharide fraction EPS3 (

final concentrations

25, 50, 100. mu.g/mL, respectively).

Determination of NO secretion levels: by Griess method on NO₂Quantitative indirect detection of NO production. The cells are inoculated on a 96-well culture plate, after each group of cells act for 24 hours under different treatment factors, 50 mu L of cell culture solution is taken from each well, and the cell culture solution is tested by Griess reagent, and the light absorption value is measured under 550 nm of an enzyme-labeling instrument. According to NaNO₂The standard curve prepared was used to calculate the amount of NO produced in the medium. The results are shown in FIG. 2.

The lactobacillus plantarum exopolysaccharide EPS3 has no statistically significant influence on macrophage activity in the metering range of 25-100 mu g/ml. Therefore, the maximum dose of 100. mu.g/ml selected in this experiment is safe for macrophages. As can be seen from fig. 2, the amount of NO produced by macrophages under normal conditions is at basal level. Upon stimulation with EPS, NO production increased significantly (compared to the normal group,P< 0.01). LPS-induced NO secretion was dose-dependently inhibited after incubation of RAW264.7 macrophages at different concentrations of EPS3, and was able to inhibit 40% of LPS-induced NO secretion when EPS3 was at a concentration of 100. mu.g/ml.

Determination of cytokine mRNA expression levels: real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR) technology is adopted to analyze the LPS induction of the lactobacillus plantarum extracellular polysaccharide EPS3The iNOS, IL-6 mRNA expression of the cells was measured by adding cells in the exponential growth phase to a 24-well cell culture plate and adjusting the cell suspension concentration to 2.5X 10⁵cells/well at 37 ℃ with 5% CO₂The culture was carried out in an incubator for 24h, after which 25, 50, 100. mu.g/ml EPS3 was added per well and incubated for 2 h, then LPS was added to a final concentration of 0.5. mu.g/ml and the incubation was continued at 37 ℃ for 24 h. Extracting total RNA of cells by Trizol reagent, reverse transcribing to cDNA, amplifying by specific primer and corresponding primer and cDNA on fluorescent quantitative PCR instrument in steps of 2^−ΔΔCtGene expression analysis was performed by relative quantitation.

Effect of EPS3 on LPS-induced cytokine mRNA expression in macrophages: as shown in FIG. 3, under normal conditions, iNOS and TNF-. alpha.mRNA were expressed only in a small amount, and the expression level was significantly increased after LPS stimulation. The lactobacillus plantarum exopolysaccharide EPS3 has obvious inhibition on iNOS mRNA expression and concentration dependence. The inhibition rate of EPS3 on iNOS mRNA expression at a dose of 100. mu.g/ml was about 50%. Furthermore, although EPS3 exhibited some degree of inhibition of TNF α mRNA expression, it did not have concentration dependency, and had a lower inhibitory effect than iNOS mRNA, with an inhibition rate of 28.6% at a dose of 100 μ g/ml. Many studies indicate that NO is mainly produced in macrophages under the catalysis of Inducible Nitric Oxide Synthase (iNOS), and high-expression iNOS and overproduced NO are involved in the occurrence and development of inflammatory diseases. In the research, RAW264.7 macrophage stimulated by lipopolysaccharide is taken as an inflammatory cell model, and release of macrophage NO activated by LPS by lactobacillus plantarum exopolysaccharide EPS3 is discussed. The results show that EPS3 dose-dependently inhibited lipopolysaccharide-induced macrophage NO production in the 25-100 μ g/ml dose range. Further studies have found that EPS3 has a negative regulatory effect on iNOS and TNF-. alpha.mRNA at the transcriptional level.

Drawings

FIG. 1 is the elution profile of Lactobacillus plantarum DL7X on a DEAE Sepharose CL-6B anion exchange column and EPS-3 on a Sephadex G-100 gel column chromatography.

FIG. 2 is EPS3 inhibiting lipopolysaccharide-induced macrophage NO production.

FIG. 3 shows that EPS3 inhibits the expression of macrophage iNOS and TNF-. alpha.mRNA.

Detailed Description

Fermentation preparation of lactobacillus plantarum DL7X exopolysaccharide EPS3 with immunoregulatory activity

1. Fermenting and culturing lactobacillus plantarum exopolysaccharide DL7X in a 2L fermentation bottle

(1) Cultivation of seed liquid

The strain required by the experiment is streaked from a glycerol tube preserved at minus 80 ℃ until an MRS plate is activated, the strain is placed in a 37 ℃ incubator to be cultured for 24 hours, then a single colony is picked up to 5ml of MRS liquid culture medium by using an inoculating loop, the strain is placed in the 37 ℃ incubator to be cultured for 16 hours, then the strain is switched to 50 ml of MRS liquid culture medium, and the strain is placed in the 37 ℃ culture medium to be cultured for 16 hours for standby.

(2) Preparation of fermentation medium

Preparation of 2L semisynthetic chemical Medium (SDM): the method is improved on the basis of MRS culture, wherein a yeast basic nitrogen source (5 g/L) and peptone (10 g/L) are used for replacing beef extract, yeast extract and peptone in an MRS culture medium formula, other components and contents are consistent, the beef extract, the yeast extract and the peptone are sterilized at the high pressure of 121 ℃ for 15min, and the beef extract, the yeast extract and the peptone are placed at the normal temperature for standby after cooling.

(3) Fermentation culture of SDM medium

Inoculating a 16 h seed culture in an MRS liquid culture medium into 2L SDM culture medium in an inoculation amount (v/v) of 3% -5%, and placing the culture medium in a constant temperature incubator at 37 ℃ for 24 h.

2. Extraction of crude extracellular polysaccharide of lactobacillus plantarum

Heating 2L fermentation liquid in boiling water bath for 15min to inactivate residual thallus. Then 2L of the fermentation broth of the inactivated bacteria was taken out and cooled to room temperature, and centrifuged at 12000 Xg at 4 ℃ for 20 min to remove the precipitate. Adding trichloroacetic acid with the mass fraction of 80% into the supernatant until the final concentration is 4% by mass, standing overnight in a refrigerator at 4 ℃, centrifuging at 12000 Xg at 4 ℃ for 20 min, and collecting the supernatant. Adding 3-5 times volume of precooled 95% ethanol into the supernatant, fully oscillating, separating out polysaccharide in a flocculent precipitate, and standing overnight in a refrigerator at 4 ℃. After the polysaccharide is fully precipitated in the form of flocculent precipitates, centrifuging at 12000 Xg at 4 ℃ for 20 min to collect polysaccharide precipitates. Dissolving the polysaccharide precipitate with hot water, transferring into dialysis bag, dialyzing with deionized water at 4 deg.C for 3 d, and changing water at 4h interval. And after the dialysis is finished, carrying out vacuum freeze drying on the polysaccharide solution to obtain dry white fluffy crude polysaccharide.

3. Preparation of lactobacillus plantarum extracellular refined polysaccharide

DEAE-Sepharose CL-6B ion gel was loaded onto a chromatography column (D2.6X 30 cm) and the column was equilibrated with Tris-HCl eluent (20 mM, pH 7.60). Dissolving crude polysaccharide with Tris-HCl buffer solution, loading the sample with the concentration of 10 mg/mL and 70 mg of the sample, and sequentially performing gradient elution with Tris-HCl eluent and eluent containing 0.2, 0.4, 0.6, 0.8 and 1.0 mol/L NaCl at the flow rate of: 2.5 mL/min, collection volume per tube: 5 mL. Detecting polysaccharide content in the collecting tube by phenol-sulfuric acid method, combining and collecting peak components, dialyzing, concentrating, and freeze-drying to obtain polysaccharide sample. The results are shown in FIG. 1A, from which it can be seen that the extracellular polysaccharide of Lactobacillus plantarum DL7X is mainly composed of four acidic polysaccharide components. Then, based on the result of the evaluation of the immunoregulatory activity, the polysaccharide component EPS-3 with the strongest activity is obtained.

The fractions obtained above were further separated on Sephadex G-100 gel column (D1.0X 100 cm), respectively. Eluent: ultrapure water; sample loading concentration: 10 mg/mL, loading: 20-25 mg. Flow rate: 0.20 mL/min, collection volume per tube: 3.0 mL. Detecting polysaccharide content in the collecting pipe by phenol-sulfuric acid method, combining and collecting peak components, dialyzing, concentrating, and freeze-drying to obtain polysaccharide sample. The results are shown in FIG. 1B, from which it can be seen that the salt-washed fraction EPS-3 eluted from NaCl in DEAE Sepharose CL-6B anion exchange column was further purified by Sephadex G-100 gel column chromatography to obtain a single, symmetrical elution peak, indicating that Lactobacillus plantarum exopolysaccharide EPS3 is a homogeneous monosaccharide fraction, which was detected to be protein-free.

And (3) dialysis and freeze-drying: selecting a dialysis bag with the molecular weight cut-off of 8000-14000 Da, adding the collected liquid obtained by elution of the molecular sieve, dialyzing for 3 days, carrying out vacuum freeze drying to obtain purified polysaccharide EPS3, weighing, marking and storing. The molecular weight is 2.02 × 10 by analyzing physical and chemical properties⁴Da, the polysaccharide content is 85.36%, the water content is 10.41%, and protein and phosphate radical are not contained.

The above examples are intended to illustrate embodiments of the invention only and not to limit the scope of the invention, and modifications and variations of the above description will occur to those skilled in the art, but all such modifications and variations are intended to be within the scope of the invention as defined in the appended claims.

Claims

1. The Lactobacillus plantarum DL7X for producing extracellular polysaccharide is characterized in that the Lactobacillus plantarum DL7X is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation place is No. 3 Xilu No.1 Beijing, the sunward area of Beijing, and the preservation numbers are as follows: CGMCC No.13331, preservation date: 2016, 11, 22 months.

2. Exopolysaccharide obtained by fermentation of Lactobacillus plantarum DL7X according to claim 1, which was prepared by the method: inoculating Lactobacillus plantarum DL7X into an SDM culture medium, standing and culturing at a constant temperature of 37 ℃ for 18-24 h, inactivating a culture solution for 15min by using a boiling water bath, centrifuging to remove thalli to obtain a supernatant, precipitating proteins in the supernatant by using a trichloroacetic acid method, centrifuging to remove protein precipitates, precipitating by using precooled ethanol with the volume of 3-5 times that of the supernatant, and performing vacuum freeze drying to obtain crude polysaccharide; purifying the crude polysaccharide by DEAE Sepharose CL-6B ion exchange chromatography, wherein the mobile phase is 20mM Tris-HCl pH 7.60 and 0.2-1.0M NaCl, and the flow rate is 1.0 mL/min; dialyzing the eluted components, drying in vacuum to obtain primarily purified solid, and then evaluating the anti-inflammatory activity of each component by using lipopolysaccharide-induced macrophage RAW 264.7; based on the activity evaluation result, selecting the component EPS-3 with the strongest activity to perform Sephadex G-100 molecular sieve chromatography, wherein the mobile phase is ultrapure water, and the flow rate is 0.25 mL/min; dialyzing the eluted components for 3 days by a dialysis bag with the molecular weight cutoff of 8000-14000 Da, and carrying out vacuum freeze drying on the dialyzed trapped fluid to obtain the uniform polysaccharide component EPS3 with the immunoregulatory activity.