CN107523514B - Extracellular polysaccharide-producing lactobacillus plantarum capable of effectively adsorbing phthalic acid monoester - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to the field of production and development of functional lactic acid bacteria. In particular to an exopolysaccharide lactobacillus plantarum capable of effectively adsorbing phthalic acid monoester. The strain number of the Lactobacillus plantarum (Lactobacillus plantarum) capable of effectively adsorbing the phthalic acid monoester and producing the exopolysaccharide is RS20D, and the strain number is registered and preserved in China general microbiological culture Collection center (CGMCC); the preservation number is CGMCC No.13272, and the preservation date is 2016, 11 and 14 days. The lactobacillus plantarum RS20D provided by the invention is a functional lactic acid bacterium, has acid resistance, grows well under the environment condition of pH 3.0-9.0, and particularly has good characteristics in the aspects of adsorbing phthalic acid monoester and producing exopolysaccharide, and if the lactobacillus plantarum RS20D is developed into a medicine, intestinal flora can be improved after long-term administration, and the harm of phthalic acid ester plasticizer to human body exposure is reduced.

Description

An exopolysaccharide-producing Lactobacillus plantarum efficiently adsorbing phthalic acid monoesters

technical field

The invention relates to the field of biotechnology, in particular to the field of production and development of functional lactic acid bacteria. In particular, it relates to an extracellular polysaccharide-producing Lactobacillus plantarum which effectively adsorbs phthalic acid monoesters.

Background technique

Lactic acid bacteria exopolysaccharide (LAB EPS) is a mucus or capsular polysaccharide secreted outside the cell during growth and metabolism of lactic acid bacteria. The exopolysaccharide-producing lactic acid bacteria strains can be directly applied to the production of fermented milk to replace or reduce the use of thickeners, improve the viscosity, texture and taste of yogurt products, and prevent shrinkage and whey precipitation. At the same time, Lactobacillus exopolysaccharides also have good physiological activities, such as anti-tumor, immune regulation and anti-oxidation, and can also be used as prebiotics to promote the growth of other probiotics in the intestinal tract and optimize the intestinal microecological environment. Therefore, the development of lactic acid bacteria with high exopolysaccharide production has obvious economic significance.

Phthalates (phthalate esters, PAEs) are a class of commonly used chemical industrial products, mainly used as plasticizers. The annual global use of PAEs has exceeded 4 million tons. The toxicity of PAEs has attracted extensive attention in recent years. The toxicity studies mainly focus on the reproductive endocrine toxicity of PAEs as endocrine disruptors to animals and humans. The routes of human exposure to PAEs include the esophagus, skin or respiratory tract, and PAEs ingested through food media are the main routes for human exposure to PAEs, especially those with relatively large molecular weights. Phthalates in food mainly come from the migration of plastic products to food in food production, processing, transportation and food packaging.

With the deepening of research, people have a further understanding of the primary metabolites of phthalate-phthalate monoesters (PMEs). For PAEs entering the body through the esophagus, its hydrolysis is more likely to occur in the intestine. Enteric esterase in mammalian intestinal mucosal cells and extracellular enzymes in the small intestine can hydrolyze phthalic acid diesters into monoesters. Therefore, the PAEs entering the body through the esophagus are mainly absorbed into the systemic circulation through the intestinal mucosa in the form of monoesters produced by esterase hydrolysis. In vitro and in vivo studies of phthalates have shown that phthalates are more biologically active and more toxic when metabolized to monoesters. It has been reported that phthalic acid monoester has various toxicological effects such as reproductive and developmental toxicity, embryo toxicity, and influence on hormones in the body. Esters can directly or indirectly affect the structure and function of Sertoli cells and Leydig cells, and the external manifestations are the reduction of anogenital distance (AGD) and anogenital index (AGI). The effect on female reproduction is to affect ovarian function, and the main site of action is ovarian granulosa cells.

Lactic acid bacteria is a general term for a type of bacteria that ferment carbohydrates and produce lactic acid, which are widely found in naturally fermented foods such as kimchi, fermented milk, etc. Scientific research has shown that probiotics represented by lactic acid bacteria are indispensable to the human body and play an important role in the health of the host. They can improve the intestinal microflora, reduce the number of harmful bacteria, improve the body's immunity, and improve the body's antioxidant capacity. ability, anti-aging and anti-cancer functions. Therefore, in addition to the application of lactic acid bacteria in fermented dairy products, more and more researches have begun to focus on the development of lactic acid bacteria functional foods, which are of great significance to human nutrition and health.

At present, in the field of environmental engineering, photolysis, microbial degradation, and physical adsorption methods based on cyclodextrin, biomass carbon, and chitosan have been studied to effectively remove phthalates from sewage and other environments. pollutants. However, in the food industry, there is no effective method for removing phthalate contamination in food. At present, there is no literature or patent report on the use of lactic acid bacteria to remove phthalates and their toxic metabolites—phthalic acid monoesters. In recent years, studies have shown that lactic acid bacteria have a good adsorption effect on a variety of mycotoxins, heavy metal ions, benzopyrene, heterocyclic amines and other food pollutants. Therefore, lactic acid bacteria, as a food-grade microorganism, have the potential to become functional microorganisms that can effectively adsorb plasticizers and their metabolites.

Therefore, lactic acid bacteria were widely isolated and purified from traditional fermented food and animal feces samples, and myxogenic strains were screened by colony drawing method and fermented milk viscosity method. Based on this, a lactic acid bacteria with excellent adsorption capacity of phthalate monoester, the main metabolite of terephthalate, was further screened, which not only has broad application prospects, but also has important social significance. .

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an exopolysaccharide-producing Lactobacillus plantarum that effectively adsorbs phthalic acid monoesters, so as to be further developed and utilized.

The bacterial strain provided by the present invention is Lactobacillus plantarum RS20D, which is preserved in the General Microorganism Center of China Microorganism Culture Collection and Management Committee, and the preservation place is No. 1 Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.13272 , the deposit date is November 14, 2016.

Described Lactobacillus plantarum RS20D is a kind of functional lactic acid bacteria and has the following properties:

(1) It has acid resistance and grows well under pH 3.0-9.0 environmental conditions;

(2) It has good colony drawing characteristics, can significantly increase the viscosity of fermented milk, improve the texture and sensory characteristics of fermented milk, and has the characteristics of high production of extracellular polysaccharides;

(3) Incubating in an aqueous solution containing phthalic acid monoester in vitro, the phthalic acid monoester has a good adsorption capacity.

(4) It is expected to be developed into a drug, and long-term use can improve the intestinal flora and reduce the harm of phthalate plasticizers to human exposure.

Another object of the present invention is to invent the preparation method of above-mentioned Lactobacillus plantarum RS20D, comprising the following steps:

(1) Lactic acid bacteria are obtained by separating and screening from traditional Sichuan home-made pickles, fermented dairy products, fermented meat products and animal feces;

(2) by colony drawing method, fermented milk viscosity measurement and phenol-sulfuric acid method, screen to obtain lactic acid bacteria with stronger viscosity-producing ability;

(3) incubating in an aqueous solution containing phthalic acid monoester in vitro, and further screening to obtain exopolysaccharide-producing lactic acid bacteria with good adsorption capacity for phthalic acid monoester;

(4) It was identified as Lactobacillus plantarum by Gram staining, catalase test, cell morphology and 16S rDNA sequencing.

Description of drawings

Figure 1 shows the colony drawing of Lactobacillus plantarum RS20D and the RS20D fermented milk with better viscosity.

Figure 2 is a liquid chromatogram of the adsorption of monobutyl phthalate by Lactobacillus plantarum RS20D.

Fig. 3 is the colony morphology and microscopic morphology of Lactobacillus plantarum RS20D.

Detailed ways

1. Sample source

Screening samples of functional lactic acid bacteria include traditional Sichuan home-made pickles, fermented dairy products, fermented meat products and animal feces. The strain in this case is derived from Sichuan traditional home-made pickles. Sichuan kimchi is a traditional fermented vegetable product with Chinese characteristics, which is rich in lactic acid bacteria.

2. Isolation and screening of lactic acid bacteria from different samples

Take different samples such as kimchi, fermented milk, animal feces, etc., homogenize them in a sterile homogenizing bag at a ratio of 1:10, take 1 mL of the homogenate and add 9 mL of sterile normal saline for 10-fold gradient dilution, and take a sample with an appropriate dilution. 100 μL was spread on MRS-CaCO ₃ medium plate and cultured at 37°C for 24h-48h. Pick colonies with calcification circles and typical characteristics of lactic acid bacteria, carry out multiple streak purification, and then save them for future use.

3. Through the colony drawing method, fermented milk viscosity measurement and phenol-sulfuric acid method, the lactic acid bacteria with strong viscosity-producing ability were screened and obtained

The above purified strains were streaked on the MRS screening medium (on the basis of MRS solid culture, glucose was changed to 5.0 g, and 50.0 g of sucrose was added) to obtain single colonies, anaerobic at 25 ° C for 48 h, and the characteristics of the colonies were observed and recorded, and sterile toothpicks were used. Gently pull outward, leave vertically to form continuous wire drawing on the medium, repeat the operation for 4-5 colonies, measure the maximum length (cm) of colony wire drawing, and the results are expressed as average values.

On the basis of obtaining the lactic acid bacteria with the above-mentioned drawing properties, the method of measuring the viscosity of fermented milk is further adopted to screen out the lactic acid bacteria that can significantly increase the viscosity of fermented milk. According to the inoculum amount of 3% (concentration of 10 ⁸ CFU mL ^-1 ), the lactic acid bacteria culture solution was respectively added to the skim milk medium, anaerobic fermentation at 30°C to curd, and then refrigerated at 4°C and cooked for 24 hours. The viscosity of each fermented milk was measured by the instrument, measured 3 times in parallel, and the average value was taken. Conditions for the determination of fermented milk texture characteristics: The British TA.XTplus physical property tester was used for analysis, and the hardness, consistency, cohesion, viscosity index and other physical properties of the test samples were obtained through the processing of the tester-related software. Probe selection is suitable for viscous food test probe mold: A/BE, backextrusion cell; probe platen diameter: 45mm; test mode: compressive stress mode; selection method: start-return start; probe running speed before test: 1.0mm /s; Test rate: 1.0mm/s; Post-test rate: 10.0mm/s; Penetration test distance: 30mm; Trigger type (sensing force): Auto-1.0g; Data acquisition rate: 400pps; Measurement temperature: room temperature .

The exopolysaccharide-producing ability of lactic acid bacteria was further verified by the phenol-sulfuric acid method. Lactobacillus with good colony drawing properties and significantly increasing the viscosity of fermented milk were inoculated into MRS liquid medium for activation, and cultured at 37 °C for 10 h. Then, it was inoculated into skim milk medium according to the inoculum amount of 1% (v/v) for expansion culture, and it was fermented at 30°C for 24h. Take 20 mL of fermentation broth, take a boiling water bath for 10 min, take out and cool to room temperature, and centrifuge at 12000 × g for 20 min at 4°C to remove the precipitate. Trichloroacetic acid with a mass fraction of 80% was added to the supernatant to a final concentration of 4% (m/v), placed in a refrigerator at 4°C overnight, and centrifuged at 12,000 × g at 4°C for 20 min to collect the supernatant. Add 3-5 times the volume of 95% ethanol (ethanol pre-cooling) to the supernatant, shake fully (polysaccharide is precipitated in flocculent form), and let stand overnight in a refrigerator at 4°C. After the polysaccharide was fully precipitated in flocculent form, the polysaccharide precipitate was collected by centrifugation at 12,000 × g at 4°C for 20 min. The polysaccharide precipitate was dissolved in hot water and then transferred to a dialysis bag, placed in a 4°C refrigerator and dialyzed with deionized water for 3 days, and the water was changed every 4 hours. After the dialysis was completed, the exopolysaccharide solution of each strain was fixed in 50 mL of distilled water, and then the polysaccharide content was determined by the phenol-sulfuric acid method. The results showed that RS20D had better drawing properties (15cm), the viscosity index of fermented milk was 253.997g·s, and the exopolysaccharide content in the obtained fermented milk reached 250mg/L, which was significantly better than other strains. See Figure 1 for the colony drawing of the RS20D strain and the better viscosity of the fermented fermented milk.

4. In vitro screening to obtain exopolysaccharide-producing lactic acid bacteria with good adsorption capacity for phthalic acid monoesters

Preparation of bacterial suspension: the above screened high exopolysaccharide-producing lactic acid bacteria strains are further used for in vitro screening of strains that efficiently adsorb phthalic acid monoesters. Take out 30 μL of glycerol-preserved lactic acid bacteria solution, put it into 5 mL of MRS medium, and cultivate at 37 °C for 16-24 hours, and then continue to activate it once at 2% of the inoculum, and expand the activated strain at 37 °C for 18- 22h. Centrifuge the strain culture solution (8 000r/min, 4°C, 15min), collect the bacteria, wash twice with sterile saline and resuspend, adjust the cell concentration of the strain to 1.0×10 ¹⁰ CFU/mL, and store the bacteria at 4°C. Suspension, ready to use within 12h.

Centrifuge 1 mL of the prepared bacterial suspension of 1.0 × 10 ¹⁰ CFU/mL at 4°C for 15 min (8000 r/min), then remove the supernatant, add 1 mL of phthalic acid monoester solution, and finally obtain 10 mg/L of ophthalmic acid. Phthalate Monoester Bacterial Suspension. The mixture was incubated at 37° C. with shaking for 24 h, then centrifuged for 15 min (8000 r/min), and the supernatant was collected for use. The content of phthalic acid monoester was quantitatively detected by high performance liquid chromatography, and the blank control was 1 mL of 10 mg/L phthalic acid monoester solution containing no lactic acid bacteria cells. The adsorption rate of lactic acid bacteria to phthalic acid monoester is calculated according to the following formula: adsorption rate (%)=(C ₀ -C)/C ₀ × 100%, where: C ₀ is the phthalic acid monoester in the blank control C is the concentration (mg/L) of the residual phthalic acid monoester after adsorption in the adsorption system. The liquid chromatograms of lactic acid bacteria strain RS20D before and after adsorption of monobutyl phthalate are shown in Figure 2, where a is the liquid chromatogram of monobutyl phthalate (MBP) in the blank control (10mg/L), b is the residual concentration of monobutyl phthalate after adsorption in the adsorption system. Strain RS20D also has obvious adsorption effect on high concentration of monobutyl phthalate (100mg/L). The adsorption rate reaches 25.42% in 6h, and a higher adsorption rate can be achieved within 15min. In addition, the strain is resistant to other species of phthalate monoesters, including monomethyl phthalate (MMP), monoethyl phthalate (MEP), and monoethylhexyl phthalate (MEHP) All of them have better adsorption effect, among which MEHP has the highest adsorption rate, reaching 60.91%.

The HPLC detection method is: LC-10A2010C HT type liquid chromatography system, reverse-phase Shimadzu InertSustain C18 chromatographic column (4.6mm×250mm, 5μm), acetonitrile: 0.1% acetic acid aqueous solution as mobile phase, flow rate 1.0mL/min, column The temperature was 25 °C, the injection volume was 20 μL, and the UV detector was used to detect at 224 nm.

V. Identification of strains obtained from screening

(1) Gram stain, catalase test

Gram staining: Pick colonies on the plate for smearing, fixation, crystal violet primary staining, mordant staining, destaining, water washing, safranine counterstaining, drying, and microscopic examination.

Catalase test: the bacteria to be tested were exposed to the air for 30min first, and a small amount of 3% H ₂ O ₂ was drawn on the surface of the plate with a capillary tube to grow colonies. If no bubbles are produced, it is negative for catalase.

(2) Colony morphology, microscopic observation of bacterial cells and physiological and biochemical identification

The colony morphology characteristics of the strains were observed with naked eyes, and the observation results are shown in Figure 3a. The colony color of this strain is milky white, the colony edge is neat, round and convex, the surface is smooth, sticky and moist, the colony diameter is about 2mm, which conforms to the characteristics of lactic acid bacteria colony; The morphological characteristics of the cells are shown in Figure 3b. The cell morphology is rod-shaped and the Gram staining is positive, so the strain RS20D is preliminarily judged to be Lactobacillus. Physiological and biochemical identification was carried out with reference to the method in the Manual for the Identification of Common Bacterial Systems.

(3) 16S rDNA sequence analysis and identification

Extraction of total DNA from lactic acid bacteria strains: Pick a purified single colony and inoculate it in 5 mL of MRS liquid medium, culture at 37°C for 12 h, and centrifuge at 10,000 r/min for 5 min to collect bacteria. The total DNA of the strains was extracted using TIAN GEN Bacteria DNA Kit.

PCR amplification of 16S rDNA: PCR reaction system (50 μL) includes 2 μL (10 μM) of upstream and downstream primers (two synthetic universal primers: 16s 27F: GAGAGTTTGATCCTGGCTCAG; 16s 1492R: CGGCTACCTTGTTACGACTT), template DNA 2 μL, 10×PCR Buffer (Mg ²⁺ Free) 5 μL, MgCl ₂ (25 mM) 3 μL, 4 μL dNTP mixture, Taq ^™ (2.5 U/μL) 1 μL, 31 μL ultrapure water.

PCR reaction program: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 1 min, annealing at 56 °C for 45 s, extension at 72 °C for 2 min, 30 cycles; extension at 72 °C for 10 min. After the PCR reaction, 2 μL of the reaction product was sampled, and electrophoresed on a 1% agarose gel (prepared with TAE buffer) at 120 V for 30 min. After the end, the results were observed in a gel imaging system. The detected amplification products were sequenced by Chengdu Qingke Zixi Biotechnology Co., Ltd. Using the BLAST program on the NCBI website, the 16S rDNA gene sequencing results were compared with the sequences in the gene bank for homology, and it was finally identified as Lactobacillus plantarum.

The above examples are only to describe the embodiments of the present invention, but not to limit the scope of the present invention. For those skilled in the art, the above description can be improved or deformed, but all these improvements or deformations should be It falls into the protection scope determined by the claims of the present invention.

Claims (2)

1. Extracellular polysaccharide-producing lactobacillus plantarum (L.) capable of effectively adsorbing phthalic acid monoesterLactobacillus plantarum) RS20D, characterized by: the lactobacillus plantarum RS20D has good exopolysaccharide production capacity and good adsorption capacity on phthalic acid monoester; said Lactobacillus plantarum: (Lactobacillus plantarum) RS20D has been preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation place is No. 3 of Xilu No.1 of Beijing, chaoyang, the preservation number is CGMCC No.13272, and the preservation date is 2016, 11 and 14 days.

2. Use of exopolysaccharide-producing lactobacillus plantarum RS20D, effective for the adsorption of phthalic acid monoesters, according to claim 1, characterized in that: is used for preparing medicines with the function of adsorbing phthalic monoester.

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