CN107523514B - Extracellular polysaccharide-producing lactobacillus plantarum capable of effectively adsorbing phthalic acid monoester - Google Patents
Extracellular polysaccharide-producing lactobacillus plantarum capable of effectively adsorbing phthalic acid monoester Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61K35/74—Bacteria
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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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
Technical Field
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.
Background
Extracellular polysaccharide of lactic acid bacteria (LAB EPS) is a mucus or capsular polysaccharide secreted outside cells by lactic acid bacteria during growth, metabolism. The lactobacillus strain for producing the exopolysaccharides can be directly applied to the production of fermented milk to replace or reduce the use of a thickening agent, improve the viscosity, the texture and the mouthfeel of the sour milk product and prevent shrinkage and whey precipitation. Meanwhile, the extracellular polysaccharide of the lactobacillus also has good physiological activity, such as anti-tumor, immunoregulation and antioxidation, and can be used as a prebiotic to promote the growth of other probiotics in the intestinal tract and optimize the intestinal micro-ecological environment. Therefore, the development of the lactobacillus with high extracellular polysaccharide has obvious economic significance.
Phthalates (PAEs) are a commonly used class of chemical industry, primarily as plasticizers. The worldwide annual use of PAEs has exceeded 400 million. The toxicity of PAEs has attracted considerable attention in recent years. The toxicity research mainly focuses on that PAEs as endocrine disruptors cause reproductive endocrine toxicity to animals and human bodies. The path of the PAEs exposed to the human body comprises esophagus, skin or respiratory tract, and the PAEs which are taken in through food media are the main path of the PAEs exposed to the human body, particularly the PAEs with relatively large molecular mass. The phthalic acid esters in the food mainly come from the migration of plastic products which are contacted in the links of food production, processing, transportation, food packaging and the like to the food.
With the progress of research, there is further knowledge of the primary metabolites of phthalates, the Phthalate Monoesters (PMEs). For PAEs that enter the body via the esophagus, hydrolysis is more likely to occur in the intestine, and intestinal esterases in the intestinal mucosal cells of mammals and extracellular enzymes in the small intestine hydrolyze the phthalate diester to a monoester. Thus, PAEs that enter the body via the esophagus are absorbed into the systemic circulation through the intestinal mucosa, mainly in the form of monoesters produced by the hydrolysis of their esterases. In vitro and in vivo studies on phthalate esters have shown that phthalate esters are more biologically active and toxic when metabolized to monoesters. It has been reported that phthalic acid monoester has various toxicological effects such as reproductive development toxicity, embryotoxicity, influence on hormone in vivo and the like, wherein in the research on reproductive endocrine toxicity, the research on male reproductive toxicity is the most, and phthalic acid monoester can directly or indirectly influence the structure and function of testicular supporting cells and interstitial cells and is externally expressed by reduction of anogenital distance (AGD) and anogenital index (AGI). The effect on female reproduction is to influence the function of ovary, and the acting site is mainly ovary granular cells.
Lactic acid bacteria are a general term for a group of bacteria that ferment carbohydrates and produce lactic acid, and are widely present in naturally fermented foods such as kimchi, fermented milk, etc., and in the human intestinal tract. Scientific research shows that probiotics represented by lactic acid bacteria are indispensable to human bodies and have important effects on host health, and the probiotics can improve intestinal microbial flora and reduce the number of harmful bacteria; the immunity of the organism is improved; improving the oxidation resistance of the organism, delaying senility, preventing cancer, and the like. Therefore, in addition to the application of lactic acid bacteria to fermented dairy products, more and more research is focused on the development of functional foods of lactic acid bacteria, which have important significance for human nutrition and health.
Currently, in the field of environmental engineering, studies have been made to use photolysis, microbial degradation and physical adsorption methods based on cyclodextrin, biomass carbon, chitosan and the like for effectively removing phthalate pollutants in environments such as sewage and the like. However, in the food industry, there is no effective way to remove the phthalate type contamination from food. There is no literature or patent report on the use of lactic acid bacteria to remove phthalate esters and their toxic metabolites, phthalate monoesters. In recent years, researches show that lactic acid bacteria have a good adsorption effect on various food pollutants such as mycotoxins, heavy metal ions, benzopyrene, heterocyclic amine and the like. Therefore, lactic acid bacteria, which are food-grade microorganisms, have a great potential to become functional microorganisms that can effectively adsorb plasticizers and metabolites thereof.
Therefore, lactic acid bacteria were widely isolated and purified from conventional fermented foods and animal waste samples, and slime-producing strains were preliminarily screened by the colony drawing method and the fermented milk viscosity method. Based on the method, the lactic acid bacteria with excellent adsorption capacity to the phthalic acid monoester, which is the main metabolite of the phthalic acid ester, is further screened, so that the method has wide application prospect and important social significance.
Disclosure of Invention
The invention aims to provide an exopolysaccharide lactobacillus plantarum capable of effectively adsorbing phthalic acid monoester, and further development and utilization are expected.
The strain provided by the invention is Lactobacillus plantarum (Lactobacillus plantarum) RS20D, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation place is No.1 Hospital, xilu, north Chen, of the area from the south of the republic of Beijing, the preservation number is CGMCC No.13272, and the preservation date is 2016, 11 and 14 days.
The lactobacillus plantarum RS20D is a functional lactic acid bacterium and has the following properties:
(1) Has acid resistance and good growth under the environment condition of pH 3.0-9.0;
(2) The bacterial colony wiredrawing property is good, the viscosity of the fermented milk can be obviously increased, the texture and the sensory property of the fermented milk are improved, and the high-yield extracellular polysaccharide property is achieved;
(3) The compound has good adsorption capacity to the phthalic monoester after being incubated in an in-vitro aqueous solution containing the phthalic monoester.
(4) Is expected to be developed into a medicine, and can improve intestinal flora and reduce the harm of phthalate plasticizer to human body after long-term administration.
The invention also aims to invent the preparation method of the lactobacillus plantarum RS20D, which comprises the following steps:
(1) Separating and screening from Sichuan traditional home-made pickles, fermented dairy products, fermented meat products and animal wastes to obtain lactic acid bacteria;
(2) Screening to obtain lactobacillus with strong viscosity-producing capability by a colony drawing method, fermented milk viscosity measurement and a phenol-sulfuric acid method;
(3) Incubating in an in vitro aqueous solution containing the phthalic acid monoester, and further screening to obtain the exopolysaccharide lactic acid bacteria with good adsorption capacity to the phthalic acid monoester;
(4) The lactobacillus plantarum is identified by gram staining, catalase experiments, thallus morphology and 16S rDNA sequencing.
Drawings
FIG. 1 shows the colony stringiness of Lactobacillus plantarum RS20D and RS20D fermented milk showing superior viscosity.
FIG. 2 is a liquid chromatogram of Lactobacillus plantarum RS20D adsorbing monobutyl phthalate.
FIG. 3 is a colony morphology and a cell microscopic morphology of Lactobacillus plantarum RS 20D.
Detailed Description
1. Sample source
The functional lactic acid bacteria screening samples comprise Sichuan traditional home-made pickles, fermented dairy products, fermented meat products, animal wastes and the like. The strain is derived from Sichuan traditional homemade pickles. Sichuan pickles are traditional fermented vegetable products with Chinese characteristics and contain rich lactobacillus flora.
2. Separating and screening lactic acid bacteria from different products
Taking different samples such as pickle, fermented milk, animal waste and the like, homogenizing in a sterile homogenizing bag according to the proportion of 1Diluting with water in 10 times gradient, and coating MRS-CaCO with 100 μ L of sample with appropriate dilution 3 Culturing the medium plate at 37 ℃ for 24-48 h. And (4) picking the bacterial colony with the calcium dissolving ring and the typical bacterial colony characteristics of the lactic acid bacteria, carrying out streaking purification for multiple times, and then storing for later use.
3. Lactic acid bacteria with strong viscosity producing capability are obtained by screening through a colony drawing method, fermented milk viscosity measurement and a phenol-sulfuric acid method
The purified strain is streaked on an MRS screening culture medium (on the basis of MRS solid culture, glucose is changed into 5.0g, 50.0g of cane sugar is added) to obtain a single colony, the single colony is subjected to anaerobic treatment at 25 ℃ for 48 hours, the characteristics of the colony are observed and recorded, an aseptic toothpick is used for slightly pulling outwards to form continuous wiredrawing on the culture medium in a vertical separation mode, 4-5 colonies are repeatedly operated, the maximum length (cm) of the wiredrawing of the colony is measured, the result is expressed by an average value, and the lactobacillus with the wiredrawing characteristic is obtained through primary screening.
In addition to the lactic acid bacteria having the above stringiness, lactic acid bacteria capable of significantly increasing the viscosity of fermented milk were selected by a method of measuring the viscosity of fermented milk. The inoculation amount is 3% (the concentration is 10) 8 CFU mL -1 ) Respectively inoculating the lactic acid bacteria culture solution into skim milk culture medium, performing anaerobic fermentation at 30 deg.C to curd, refrigerating at 4 deg.C, aging for 24 hr, measuring the viscosity of each fermented milk with texture analyzer, measuring for 3 times, and taking the average value. Fermented milk texture characteristics measurement conditions: XTplus physical property tester in UK is used for analysis, and the relevant software of the tester is used for processing to obtain various physical property indexes of hardness, consistency, cohesiveness, viscosity index and the like of the test sample. The probe is selected to be suitable for testing the probe mould of the viscous food: A/BE, back exclusion cell; diameter of the probe platen: 45mm; and (3) a test mode: a compressive stress mode; the selection mode is as follows: start-regression start; probe running pre-measurement rate: 1.0mm/s; and (3) testing rate: 1.0mm/s; and (3) measuring the rate: 10.0mm/s; penetration test distance: 30mm; trigger type (induction force): auto-1.0g; data acquisition rate: 400pps; measuring temperature: and (4) room temperature.
And further verifying the exopolysaccharide production capacity of the lactic acid bacteria by adopting a phenol-sulfuric acid method, inoculating the lactic acid bacteria which are obtained by screening and have good colony drawing characteristics and can obviously increase the viscosity of the fermented milk into an MRS liquid culture medium for activation, and culturing for 10 hours at 37 ℃. Then inoculating into skim milk culture medium according to the inoculation amount of 1% (v/v) for propagation, and fermenting at 30 ℃ for 24h. 20mL of fermentation liquid is taken, boiled in a water bath for 10min, taken out, cooled to room temperature, and centrifuged at 12000 Xg for 20min at 4 ℃ to remove the precipitate. Adding trichloroacetic acid with a mass fraction of 80% into the supernatant to a final concentration of 4% (m/v), standing overnight in a refrigerator at 4 ℃, centrifuging at 12000 Xg for 20min at 4 ℃, and collecting the supernatant. Adding 95% ethanol (precooled by ethanol) with the volume of 3-5 times of that of the supernatant, fully oscillating (polysaccharide is precipitated in a flocculent shape), and standing overnight in a refrigerator at 4 ℃. After the polysaccharide is fully precipitated in the form of flocculent precipitates, centrifuging at the temperature of 4 ℃ of 12000 Xg for 20min and collecting the polysaccharide precipitates. Dissolving the polysaccharide precipitate with hot water, transferring into dialysis bag, dialyzing with deionized water at 4 deg.C for 3d, and changing water at 4h interval. After dialysis, the extracellular polysaccharide solution of each strain is subjected to constant volume in 50mL of distilled water, and then the polysaccharide content is measured by adopting a phenol-sulfuric acid method. The result shows that RS20D has excellent wire drawing property (15 cm), the viscosity index of the fermented milk is 253.997 g.s, the content of extracellular polysaccharide in the obtained fermented milk reaches 250mg/L, and the fermented milk is obviously superior to other strains. The colony stringiness of the RS20D strain and the relatively superior viscosity of the fermented milk obtained by fermentation are shown in FIG. 1.
4. Exopolysaccharide-producing lactic acid bacteria with good adsorption capacity on phthalic acid monoester obtained by in vitro screening
Preparation of bacterial suspension: the screened high-yield exopolysaccharide lactobacillus strain is further used for in vitro screening of strains capable of efficiently adsorbing phthalic monoester compounds. Taking out 30 mu L of the lactobacillus liquid preserved by the glycerol, respectively inoculating the lactobacillus liquid into 5mL of MRS culture medium, culturing for 16-24h at 37 ℃, then continuously activating once according to the inoculum size of 2%, and carrying out amplification culture on the activated strain for 18-22h at 37 ℃. Centrifuging the strain culture solution (8 000r/min,4 deg.C, 15 min), collecting thallus, centrifuging and washing with sterile physiological saline for 2 times, and resuspending to adjust the cell concentration of the strain to 1.0 × 10 10 And (5) storing the bacterial suspension at 4 ℃ in a CFU/mL manner for 12h for later use.
The prepared 1.0 x 10 10 1mL of CFU/mL bacterial suspension was centrifuged at 4 ℃ for 15min (8000 r/min) and thenAnd removing the supernatant, and adding 1mL of phthalic monoester solution to finally obtain 10mg/L of phthalic monoester bacterial suspension. The mixture was cultured with shaking at 37 ℃ for 24 hours, and then centrifuged for 15min (8000 r/min), and the supernatant was collected for further use. The content of the phthalic acid monoester was quantitatively determined by high performance liquid chromatography, and the blank control was 1mL of 10mg/L phthalic acid monoester solution containing no cells of the lactic acid bacteria strain. The adsorption rate of the lactic acid bacteria to the phthalic monoester is calculated according to the following formula: adsorption rate (%) = (C) 0 -C)/C 0 X 100%, wherein: c 0 The concentration (mg/L) of the phthalic acid monoester in the blank control; c is the concentration (mg/L) of the phthalic acid monoester residue after adsorption in the adsorption system. The liquid chromatogram before and after the lactobacillus strain RS20D adsorbs the monobutyl phthalate is shown in figure 2, wherein a is the liquid chromatogram of the monobutyl phthalate (MBP) in a blank control (10 mg/L), and b is the concentration of the monobutyl phthalate residue after adsorption in the adsorption system. The strain RS20D also has an obvious adsorption effect on high-concentration monobutyl phthalate (100 mg/L), the adsorption rate reaches 25.42% in 6 hours, and the high adsorption rate can be realized within 15 min. In addition, the strain has excellent adsorption effect on other types of phthalic acid monoesters, including monomethyl phthalate (MMP), monoethyl phthalate (MEP) and ethylhexyl phthalate (MEHP), wherein the MEHP has the highest adsorption rate which reaches 60.91%.
The HPLC detection method comprises the following steps: LC-10A2010C HT type liquid chromatography system, reverse-phase Shimadzu InertSustain C18 column (4.6 mm. Times.250mm, 5 μm), acetonitrile: 0.1% acetic acid water solution as mobile phase, flow rate 1.0mL/min, column temperature 25 deg.C, sample size 20 μ L, and detection at 224nm with ultraviolet detector.
5. Identifying the strains obtained by screening
(1) Gram stain, catalase assay
Gram staining: selecting bacterial colonies on the plate for smear, fixing, crystal violet initial staining, mordant staining, decoloring, washing, safranine counterstaining, drying and microscopic examination.
Catalase assay: exposing the bacteria to be tested in air for 30minCapillary suction of a small amount 3%H 2 O 2 Drop on the colony grown on the surface of the plate. Catalase was negative if no bubbles were formed.
(2) Colony morphology, thallus microscopic morphology observation and physiological and biochemical identification
The colony morphology of the strain was visually observed, and the observation results are shown in FIG. 3a. The bacterial colony of the strain is milky white, the edges of the bacterial colony are neat, the bacterial colony is in a circular convex shape, the surface is smooth, the bacterial colony is viscous and moist, the diameter of the bacterial colony is about 2mm, and the bacterial colony accords with the characteristics of lactobacillus bacterial colony; after gram staining, the cell morphology characteristics of the strain are observed under an oil microscope of an optical microscope, the observation result is shown in figure 3b, the cell morphology is rod-shaped, gram staining is positive, and therefore the strain RS20D is preliminarily judged to be lactobacillus. The physiological and biochemical identification is carried out according to the method of 'handbook for identifying common bacteria system'.
(3) 16S rDNA sequence analysis and identification
Extracting total DNA of lactobacillus strains: the purified single colony is selected and inoculated in 5mL of MRS liquid culture medium, cultured at 37 ℃ for 12h, and centrifuged at 10000r/min for 5min to collect thalli. Extracting the total DNA of the strain by using a TIAN GEN Bacteria DNA Kit.
PCR amplification of 16S rDNA: the PCR reaction system (50. Mu.L) included 2. Mu.L (10. Mu.M) each of the upstream and downstream primers (two synthetic universal primers: 1696 27F 2+ Free)5μL，MgCl 2 (25mM)3μL，4μL dNTP mixture，Taq TM 1 μ L of (2.5U/. Mu.L) 31 μ L ultrapure water.
PCR reaction procedure: pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 1min, annealing at 56 ℃ for 45s, extension at 72 ℃ for 2min,30 cycles; extension at 72 ℃ for 10min. After the PCR reaction, 2. Mu.L of the reaction product was loaded and electrophoresed in 1% agarose gel (prepared in TAE buffer) at 120V for 30min, and the results were observed in a gel imaging system after the end of the PCR reaction. The detected amplification product is sent to Chengdu Zhixi biotechnology limited company for sequencing. And carrying out homology comparison on the sequencing result of the 16S rDNA gene and the sequence in the gene library by utilizing a BLAST program of an NCBI website, and finally identifying the lactobacillus plantarum.
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 (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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