CN117106666A - Pediococcus pentosaceus LL-07, pediococcus pentosaceus LL-07 extracellular polysaccharide, and production method and application thereof - Google Patents
Pediococcus pentosaceus LL-07, pediococcus pentosaceus LL-07 extracellular polysaccharide, and production method and application thereof Download PDFInfo
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- CN117106666A CN117106666A CN202311243885.3A CN202311243885A CN117106666A CN 117106666 A CN117106666 A CN 117106666A CN 202311243885 A CN202311243885 A CN 202311243885A CN 117106666 A CN117106666 A CN 117106666A
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- 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
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- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P17/18—Antioxidants, e.g. antiradicals
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
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Abstract
The invention provides Pediococcus pentosaceus LL-07, pediococcus pentosaceus LL-07 extracellular polysaccharide, and a production method and application thereof, and belongs to the technical field of microorganisms. The classification of Pediococcus pentosaceus LL-07 of the invention is named: pediococcus pentosaceus, deposited in China center for type culture Collection, with the deposit address: the preservation date of the university of Wuhan, china is: 2023, 7 and 11, deposit number: cctccc M20231264. The Pediococcus pentosaceus LL-07 provided by the invention can be fermented by taking glucose, fructose and lactose as carbon sources respectively to obtain corresponding extracellular polysaccharide GEPS, LEPS, FEPS, and the three extracellular polysaccharides have strong antioxidant activity, alpha amylase activity inhibition and moisture retention performance, so that technical support is provided for research and development of Pediococcus pentosaceus and extracellular polysaccharide thereof in products such as foods, medicines and cosmetics.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to Pediococcus pentosaceus LL-07, pediococcus pentosaceus LL-07 extracellular polysaccharide, a production method and application thereof.
Background
Pediococcus pentosaceus (Pediococcus pentosaceus) has recently been increasingly playing an important role as one of lactic acid bacteria in the application of lactic acid bacteria. In the 90 s of the 20 th century, it has been demonstrated that some Pediococcus pentosaceus can be used in addition to fermentation as a biological promoter for animal growth, but most of the characteristics of Pediococcus pentosaceus have not been studied intensively at that time. Over the years of technological development, and deep mining of pediococcus pentosaceus, many other previously undiscovered features have been demonstrated. Besides the function of lactic acid bacteria for acid production, pediococcus pentosaceus can also improve the flavor, quality and safety of fermented products, and more research results show that Pediococcus pentosaceus has potential as probiotics.
Extracellular Polysaccharide (EPS) is a secondary metabolite produced by microorganisms during growth and metabolism, is a macromolecular carbohydrate substance which exists in extracellular matrix and is not covalently linked with cell membranes, is widely distributed in nature, and has a well-known distribution in animals, plants, fungi and bacteria. Many studies have found that EPS has various physiological functions such as antioxidation, antitumor, cholesterol reduction, intestinal flora balance promotion, etc., and is often used as a thickener, stabilizer, gel, emulsifier, etc. in the industries of foods, medicines, cosmetics, etc. In the growth and metabolism process of Pediococcus pentosaceus, besides active substances such as bacteriocin and the like, EPS with an active function is also produced, and the functions of Pediococcus pentosaceus source EPS are continuously verified and deeply excavated by scientific researchers, so that the potential connection between the probiotic function and special physicochemical properties of Pediococcus pentosaceus and EPS is shown.
At present, research on lactobacillus source EPS is mainly focused on lactobacillus plantarum and streptococcus thermophilus, but research on pediococcus pentosaceus source EPS is not yet in depth.
Disclosure of Invention
In view of the above, the present invention aims to provide Pediococcus pentosaceus LL-07, pediococcus pentosaceus LL-07 exopolysaccharide, and its production method and application, wherein Pediococcus pentosaceus LL-07 can ferment with glucose, fructose and lactose as carbon sources to obtain corresponding exopolysaccharide, has strong antioxidant activity, inhibits alpha amylase activity and moisture retention, and provides technical support for the research and development of Pediococcus pentosaceus and exopolysaccharide thereof in products such as food, medicine and cosmetics.
In order to achieve the above object, the present invention provides the following technical solutions:
pediococcus pentosaceus LL-07, which has classification name Pediococcus pentosaceus and is deposited in China center for type culture collection, with the deposit address: the preservation date of the university of Wuhan, china is: 2023, 7 and 11, deposit number: cctccc M20231264.
In certain embodiments, the Pediococcus pentosaceus LL-07 is isolated from Guizhou sour meat.
In certain embodiments, the specific nucleotide sequence of the 16S rDNA of Pediococcus pentosaceus LL-07 is set forth in SEQ ID NO: 1.
The invention also provides Pediococcus pentosaceus LL-07 exopolysaccharide, and Pediococcus pentosaceus LL-07 exopolysaccharide GEPS, LEPS, FEPS is obtained by fermenting Pediococcus pentosaceus LL-07 with glucose, lactose and fructose as carbon sources.
In certain embodiments, the Pediococcus pentosaceus LL-07 exopolysaccharide GEPS, LEPS, FEPS has a number average molecular weight of 14.694kDa, 13.237kDa, 39.031kDa, respectively; the weight average molecular weight is 29.781kDa, 56.892kDa and 83.224kDa respectively; the z-average molecular weight is 86.429kDa, 1014.057kDa and 147.623kDa respectively, and the molecular weight distribution width is 2.027, 4.298 and 2.132 respectively.
In certain embodiments, the Pediococcus pentosaceus LL-07 extracellular polysaccharide GEPS includes three polysaccharides, GEPS-1M, GEPS-2M, GEPS-3M; the LEPS comprises three polysaccharides of LEPS-1M, LEPS-2M, LEPS-3M; the FEPS comprises FEPS-1M, FEPS-2M polysaccharide; wherein the monosaccharide composition of the GEPS comprises 0.74 mol% of L-fucose, 0.30 mol% of L-arabinose, 12.47 mol% of D-galactose, 17.44 mol% of D-glucose, 67.47 mol% of D-mannose and 1.58 mol% of D-ribose; the monosaccharide composition of the LEPS comprises, in mol%, 0.37% of L-fucose, 0.21% of L-arabinose, 17.79% of D-galactose, 15.59% of D-glucose, 64.88% of D-mannose, 1.17% of D-ribose; the monosaccharide composition of FEPS comprises, in mol%, 0.13% L-fucose, 0.65% L-arabinose, 1.32% D-galactose, 26.75% D-glucose, and 71.14% D-mannose.
In certain embodiments, GEPS-1M, LEPS-1M, FEPS-1M is a neutral polysaccharide; GEPS-2M, GEPS-3M, LEPS-2M, LEPS-3M, FEPS-2M is an acidic polysaccharide.
In certain embodiments, the monosaccharide composition of the GEPS-1M comprises, in mol%, 0.46% L-fucose, 0.24% L-arabinose, 5.75% D-galactose, 15.72% D-glucose, 75.37% D-mannose, 2.24% D-ribose; the monosaccharide composition of the GEPS-2M comprises 1.79 mol percent of L-fucose, 0.42 mol percent of L-arabinose, 13.45 mol percent of D-galactose, 12.45 mol percent of D-glucose and 71.90 mol percent of D-mannose; the monosaccharide composition of the GEPS-3M comprises 0.69 mol percent of L-fucose, 0.49 mol percent of L-arabinose, 57.07 mol percent of D-galactose, 37.31 mol percent of D-glucose and 4.44 mol percent of D-mannose; the monosaccharide composition of the LEPS-1M comprises 0.19 mol% of L-fucose, 0.13 mol% of L-arabinose, 12.24 mol% of D-galactose, 14.74 mol% of D-glucose, 70.92 mol% of D-mannose and 1.77 mol% of D-ribose; the monosaccharide composition of the LEPS-2M comprises 0.79 mol% of L-fucose, 0.27 mol% of L-arabinose, 10.64 mol% of D-galactose, 8.29 mol% of D-glucose and 80.01 mol% of D-mannose; the monosaccharide composition of the LEPS-3M comprises 0.58 mol% of L-fucose, 0.51 mol% of L-arabinose, 60.08 mol% of D-galactose, 32.92 mol% of D-glucose and 5.91 mol% of D-mannose; the monosaccharide composition of the FEPS-1M comprises 1.01 mol percent of L-fucose, 1.11 mol percent of L-arabinose, 13.10 mol percent of D-galactose, 11.51 mol percent of D-glucose and 73.28 mol percent of D-mannose; the monosaccharide composition of the FEPS-2M comprises 0.09% of L-fucose, 0.07% of L-arabinose, 0.64% of D-galactose, 3.07% of D-glucose and 96.13% of D-mannose in mol%.
The invention also provides a production method of the Pediococcus pentosaceus LL-07 exopolysaccharide in the technical scheme, which comprises the following steps:
s1, inoculating Pediococcus pentosaceus LL-07 in the technical scheme into a liquid culture medium with glucose, lactose and fructose as carbon sources according to the inoculum size of 2-4% by volume ratio, and culturing and fermenting at the constant temperature of 32-37 ℃ for 24-48 hours to obtain a fermentation culture solution;
s2, placing the fermentation culture solution in a water bath at 90-95 ℃ for 10-30 min at constant temperature, centrifuging the fermentation solution after constant temperature to obtain supernatant, adding trichloroacetic acid water solution with the concentration of 0.75-0.85 g/mL to ensure that the final concentration of trichloroacetic acid is 0.035-0.045 g/mL, standing, centrifuging to obtain supernatant, concentrating, adding pre-cooled absolute ethyl alcohol with the volume of 4-6 times that of concentrated solution, standing, centrifuging to obtain precipitate, adding sterile water into the precipitate, centrifuging again, concentrating to dryness, and adding sterile water for re-dissolving to obtain crude polysaccharide solution;
s3, dialyzing the crude polysaccharide solution in deionized water for 48-72 hours by using a dialysis bag of 8000-14000 Da at the dialysis temperature of 2-4 ℃, and freeze-drying after dialysis to obtain pediococcus pentosaceus LL-07 extracellular polysaccharide GEPS, LEPS, FEPS respectively;
s4, separating the Pediococcus pentosaceus LL-07 extracellular polysaccharide GEPS, LEPS, FEPS by a Cellulose DE-52 chromatographic column and purifying by a Sepharose CL-6B gel chromatographic column to obtain a corresponding purified polysaccharide component of the Pediococcus pentosaceus LL-07 extracellular polysaccharide GEPS, LEPS, FEPS.
In certain embodiments, the size of the Cellulose DE-52 column is preferably 30 cm. Times.Φ2.6cm.
In certain embodiments, the Cellulose DE-52 chromatographic column separation is specifically: according to the feed liquid ratio of 10 mg/mL-20 mg/mL, respectively dissolving Pediococcus pentosaceus LL-07 extracellular polysaccharide GEPS, LEPS, FEPS in deionized water; loading the supernatant onto a Cellulose DE-52 chromatographic column, and eluting with deionized water and NaCl solution with concentration of 0.1mol/L, 0.3mol/L and 0.6mol/L as eluent at flow rate of 1mL/min; collecting eluate every 6min, measuring EPS content, collecting 8 polysaccharides, dialyzing, concentrating, freeze drying to obtain primarily purified polysaccharides GEPS-1, GEPS-2, GEPS-3, LEPS-1, LEPS-2, LEPS-3, FEPS-1, FEPS-2.
In certain embodiments, the size of the Sepharose CL-6B gel chromatography column is 100 cm. Times.phi.1.6 cm.
In certain embodiments, the Sepharose CL-6B gel chromatography column purification specifically comprises the steps of: dissolving the primarily purified polysaccharide in deionized water according to the feed liquid ratio of 10 mg/mL-20 mg/mL, centrifuging, and taking supernatant; filtering the supernatant with 0.45 μm filter membrane, loading into Sepharose CL-6B gel chromatographic column, eluting with deionized water as eluent at flow rate of 0.3mL/min, and collecting eluate every 6min to determine EPS content; concentrating and freeze-drying the obtained eluent to obtain the corresponding purified polysaccharide component GEPS-1M, GEPS-2M, GEPS-3M, LEPS-1M, LEPS-2M, LEPS-3M, FEPS-1M, FEPS-2M.
The invention also provides the Pediococcus pentosaceus LL-07 in the technical scheme and/or the application of the Pediococcus pentosaceus LL-07 extracellular polysaccharide in the technical scheme in foods, medicines, cosmetics or health care products.
In certain embodiments, the invention is not particularly limited in terms of the dosage form or type of the food, pharmaceutical, cosmetic or health product, and Pediococcus pentosaceus LL-07 is employed, and/or the dosage form or type of Pediococcus pentosaceus LL-07 extracellular polysaccharide is acceptable in the corresponding product. The preparation method of the food, the medicine, the cosmetic or the health care product is not particularly limited, and the preparation method of corresponding dosage forms or types is adopted. The invention is not particularly limited to the content of Pediococcus pentosaceus LL-07 and/or the extracellular polysaccharide of Pediococcus pentosaceus LL-07 in the food, the medicine, the cosmetic or the health care product, and the content of the conventional active substances in the food, the medicine, the cosmetic or the health care product can be adopted.
The invention also provides an application of the Pediococcus pentosaceus LL-07 exopolysaccharide in the technical scheme in preparing products for resisting oxidization and/or inhibiting alpha amylase, wherein the Pediococcus pentosaceus LL-07 exopolysaccharide is one or two of GEPS and LEPS.
In certain embodiments, the product preferably has a GEPS and LEPS content of greater than or equal to 10mg/mL, and more preferably has a GEPS content of 50mg/mL. When the concentration of the GEPS obtained by the invention is 50mg/mL, the clearance capacity of the GEPS to DPPH is equivalent to Vc.
In certain embodiments, the invention is not particularly limited as to the type of product in question, as long as the Pediococcus pentosaceus LL-07 extracellular polysaccharides GEPS, LEPS are of a type acceptable in the corresponding products. The preparation method of the product is not particularly limited, and the corresponding type of preparation method is adopted.
The invention also provides an application of the Pediococcus pentosaceus LL-07 exopolysaccharide in the technical scheme in preparation of moisturizing products, wherein the Pediococcus pentosaceus LL-07 exopolysaccharide is FEPS.
In certain embodiments, the invention is not particularly limited as to the type of product in question, as long as the Pediococcus pentosaceus LL-07 exopolysaccharide FEPS is of a type acceptable in the corresponding product. The preparation method of the product is not particularly limited, and the corresponding type of preparation method is adopted. The invention has no special limitation on the content of the Pediococcus pentosaceus LL-07 exopolysaccharide FEPS in the product, and the content of the conventional active substances in the corresponding product is adopted. The moisture retention performance of the Pediococcus pentosaceus LL-07 exopolysaccharide FEPS is superior to that of glycerol, and is similar to that of sodium hyaluronate in RH43%, and can reach 87.3%.
The beneficial technical effects are as follows: the invention provides Pediococcus pentosaceus LL-07, and the classification name of Pediococcus pentosaceus LL-07 is: pediococcus pentosaceus Pediococcus pentosaceus deposited in China center for type culture collection with the following deposit address: the preservation date of the university of Wuhan, china is: 2023, 7 and 11, deposit number: cctccc M20231264. The Pediococcus pentosaceus LL-07 provided by the invention can be fermented by taking glucose, fructose and lactose as carbon sources respectively to obtain corresponding extracellular polysaccharide GEPS, LEPS, FEPS, and in vitro experiments show that the three extracellular polysaccharides have strong antioxidant activity, alpha amylase activity inhibition and moisture retention performance, and provide technical support for research and development of Pediococcus pentosaceus and extracellular polysaccharide thereof in products such as foods, medicines and cosmetics.
Drawings
FIG. 1 is a phylogenetic tree of 16S rDNA of Pediococcus pentosaceus (Pediococcus pentosaceus) LL-07;
FIG. 2 shows the identification result of the test strip of the LL-07API50 of Pediococcus pentosaceus (Pediococcus pentosaceus);
FIG. 3 is an elution profile of the extracellular polysaccharide of Pediococcus pentosaceus (Pediococcus pentosaceus) LL-07; wherein G1, G2 and G3 in the upper diagram of FIG. 3 represent GEPS-1, GEPS-2 and GEPS-3 respectively, L1, L2 and L3 represent LEPS-1, LEPS-2 and LEPS-3 respectively, F1 and F2 represent FEPS-1 and FEPS-2 respectively, and DE-52 represents DEAE-52; g1, G2, G3 in the lower diagram of FIG. 3 represent GEPS-1M, GEPS-2M, GEPS-3M, L1, L2, L3 represent LEPS-1M, LEPS-2M, LEPS-3M, F1, F2 represent FEPS-1M, FEPS-2M, respectively;
FIG. 4 shows DPPH scavenging ability of the extracellular polysaccharide LL-07 of Pediococcus pentosaceus (Pediococcus pentosaceus);
FIG. 5 shows the hydroxyl radical clearance of the extracellular polysaccharide of Pediococcus pentosaceus (Pediococcus pentosaceus) LL-07;
FIG. 6 shows the inhibitory capacity of Pediococcus pentosaceus (Pediococcus pentosaceus) LL-07 exopolysaccharide alpha amylase;
FIG. 7 shows the moisturizing properties of the LL-07 extracellular polysaccharide from Pediococcus pentosaceus (Pediococcus pentosaceus).
Detailed Description
For a better understanding of the present invention, the following examples are further illustrated, but are not limited to the following examples.
The experiments and methods described in the examples were performed substantially in accordance with conventional methods well known in the art and described in various references unless specifically indicated. For example, for the conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention, reference may be made to Sambrook (Sambrook), friech (Fritsch) and manitis (Maniatis), molecular cloning: laboratory Manual (MOLECULAR CLONING: ALABORATORY MANUAL), edit 2 (1989); the handbook of contemporary molecular biology (CURRENT PROTOCOLS IN MOLECULAR BIOLOGY) (edited by f.m. ausubel (f.m. ausubel) et al, (1987)); series (academic publishing company) of methods in enzymology (METHODS IN ENZYMOLOGY): PCR2: practical methods (PCR 2: APRACTICAL APPROACH) (M.J. MaxFrson (M.J. MacPherson), B.D. Hemsl (B.D. Hames) and G.R. Taylor (G.R. Taylor) editions (1995)), and animal cell CULTURE (ANIMAL CELL CULTURE) (R.I. French Lei Xieni (R.I. Freshney) editions (1987)).
In addition, the specific conditions are not specified in the examples, and the process is carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. The entire disclosures and other references herein are incorporated by reference in their entirety.
EXAMPLE 1 isolation and identification of Pediococcus pentosaceus LL-07 Strain
1.1 isolation of Pediococcus pentosaceus LL-07 Strain
1.1.1 configuration of solid Medium:
20g/L of glucose, 10g/L of tryptone, 5g/L of beef extract powder, 4g/L of yeast powder, 5g/L of sodium acetate, 2g/L of triamine citrate, 1g/L of tween 80, 2g/L of dipotassium hydrogen phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate and 14g/L of agar, adding double distilled water to fix the volume to 1L, and adjusting the pH to be 6.5+/-0.2. Sterilizing at high temperature for use.
1.1.2 isolation and purification of Pediococcus pentosaceus LL-07 Strain
Taking 10g of Guizhou sour meat sample (purchased from Miao county, miao nationality, qian, guizhou) under aseptic condition, mincing, placing into an aseptic homogenizing bag, addingAdding 90mL of sterile physiological saline (0.9%) into a sterile beating homogenizer, beating for 5min, adding 1mL of sample solution into 9mL of sterile physiological saline for dilution, and continuously diluting the sample to 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Gradient. The resulting sample dilutions were then coated with CaCO 3 Is cultured at 37℃for 48 hours. After the cultivation is finished, selecting a strain with viscosity-producing and wiredrawing properties as a target strain, selecting a single colony for re-streaking, repeating the process for 4 to 5 times, and observing whether the strain is purified or not through gram staining. Preserving the purified strain.
The preservation information is as follows:
preservation name: pediococcus pentosaceus (Pediococcus pentosaceus) LL-07, classified nomenclature: pediococcus pentosaceus Pediococcus pentosaceus, accession number: china center for type culture collection, preservation address: chinese, university of armed chinese, accession number: CCTCC M20231264, time of preservation: 2023, 7, 11.
1.2 identification of Pediococcus pentosaceus LL-07
1.2.1 molecular biological identification
1) Bacterial strain DNA extraction:
the genome was extracted according to the instructions of the bacterial genome DNA extraction kit (Tiangen Biochemical technology (Beijing) Co., ltd.).
2) 16S rDNA amplification:
PCR amplification primer:
forward primer 27F (SEQ ID NO: 2): 5'-AGAGTTTGATCCTGGCTCAG-3';
reverse primer 1492R (SEQ ID NO: 3): 5'-GGTTACCTTGTTACGACTT-3'.
The PCR amplification system was appropriately adjusted by referring to the instructions of 2X Taq PCR MasterMix II (Tiangen Biochemical technologies (Beijing) Co., ltd.).
Amplification system: stencil (< 1 μg): 4. Mu.L; primer F (10 μm): 2. Mu.L; primer R (10 μm): 2. Mu.L; 2X Taq PCR MasterMix II: 15. Mu.L; ddH 2 O was added to 30. Mu.L.
PCR amplification conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, elongation at 72℃for 1min for 30s,30 cycles; and extending at 72 ℃ for 10min.
And (3) passing the agarose gel inspection of the PCR amplification product, and sending the PCR amplification product to a worker for sequencing, wherein the sequencing result is shown as SEQ ID NO: 1.
SEQ ID NO：1：
AGCATGGCGGGTGCTATAATGCAGTCGAACGAACTTCCGTTAATTGATTATGACGTACTTGTACTGATTGAGATTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTAGGGGATAACACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCATGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTAAGAGTAACTGTTTACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGTTTCCGCTCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGACAGTCTAAGAGATTAGAGGTTCCCTTCGGGGACAGAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTAAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGACCGCGAGGTTAAGCTAATCTCTTAAAACCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGGGGTAACCTTTTAGGAGCTAGCCGTCTAAGGTGACAGATA。
3) Sequence similarity analysis and phylogenetic tree construction
The results after the assay were logged into the national center for biotechnology (National Center for Biotechnology Information, NCBI) for BLAST alignment and phylogenetic trees were constructed using MEGA software (fig. 1).
From the results of FIG. 1, LL-07 was Pediococcus pentosaceus (Pediococcus pentosaceus).
1.2.2API50CH identification experiment
1) Strain culture:
200. Mu.L of stock bacteria were aspirated into 10mL of MRS liquid medium, subcultured 2 times at 37℃and inoculated into MRS agar.
2) Preparation of the test strip:
a incubator was prepared, and about 10mL of distilled water was poured into the alveoli of the tray to create a wet chamber, strain numbers were written on the edge of the tray, and the test strips were removed from the package and placed in the incubator tray.
3) Preparation of inoculum:
bacterial liquid is collected from MRS solid culture medium by using cotton swab and added into physiological saline to prepare high-concentration bacterial suspension. The suspension was added with a high concentration of bacterial suspension to prepare a bacterial suspension of 2McF, and the number of drops n was recorded. The API50CHL culture medium ampoule is opened, 2n drops of bacteria liquid are added for inoculation, and the mixture is uniformly mixed for standby.
4) Inoculation of test strips
The bacterial liquid is inoculated into each small hole of an incubation plate by a sterile sample feeder, and is cultured for 48 hours at 36+/-2 ℃, and the test strip results are read and recorded at 24 hours and 48 hours of culture respectively. After the cultivation is finished, strain identification is carried out by using an identification software APIweb, and the identification result is shown in figure 2. As can be seen from FIG. 2 and the results of the identification, LL-07 is Pediococcus pentosaceus (Pediococcus pentosaceus).
EXAMPLE 2 extraction, purification and analysis of Pediococcus pentosaceus LL-07 exopolysaccharide
2.1 preparation of culture medium:
liquid medium: 20g/L of glucose, 10g/L of tryptone, 5g/L of beef extract powder, 4g/L of yeast powder, 5g/L of sodium acetate, 2g/L of triamine citrate, 1mL/L of tween 80, 2g/L of dipotassium hydrogen phosphate, 0.2g/L of magnesium sulfate heptahydrate and 0.05g/L of manganese sulfate, adding double distilled water to fix the volume to 1L, and adjusting the pH to be 6.5+/-0.2. Sterilizing at high temperature for use.
2.2, strain activation and preparation of fermentation liquor:
and (3) activating and culturing: sterilizing with ultra-clean bench for 30min, taking out Pediococcus pentosaceus (Pediococcus pentosaceus) LL-07 cryopreservation tube stored at-80deg.C, and thawing at normal temperature. 200 mu L of frozen bacteria liquid is absorbed and placed in 10mL of liquid culture medium, placed in a constant temperature incubator at 37 ℃ and cultured for 24-48 hours to activate the bacteria, and the activation culture is repeated for 2 times.
Preparing fermentation liquid: after 2 generations of activation culture, inoculating the culture medium with an inoculum size of 3% by volume, and culturing in a constant temperature incubator at 37 ℃ for 24-48 hours (preferably 24 hours) to obtain fermentation culture solution.
2.3 extraction of Pediococcus pentosaceus LL-07 exopolysaccharide
1) Placing the fermentation broth in water bath at 90deg.C for 15min to inactivate thallus and polysaccharide decomposing enzyme.
2) Centrifuging the fermentation liquor after constant temperature to obtain supernatant, adding trichloroacetic acid aqueous solution with the concentration of 0.75 g/mL-0.85 g/mL into the supernatant obtained by centrifugation, enabling the final concentration of trichloroacetic acid to be 0.035 g/mL-0.045 g/mL, and standing for 12 h-16 h at the temperature of 2 ℃ to 4 ℃; centrifuging to remove precipitate to obtain deproteinized supernatant;
3) Concentrating the supernatant after protein removal to 1/2-1/3 of the original volume under the conditions of 50mBar vacuum degree and 55 ℃ rotary evaporation temperature, adding 2-4 ℃ precooled absolute ethyl alcohol which is 4-6 times of the volume of the concentrated solution, standing for 12-24 hours at 2-4 ℃, centrifuging, adding sterile water into the precipitate obtained by centrifugation, centrifuging again, taking supernatant, concentrating the supernatant to dryness under 50mBar vacuum degree, and adding sterile water for re-dissolving to obtain a crude polysaccharide solution;
4) Dialyzing the crude polysaccharide solution in deionized water for 48-72 h by using a dialysis bag of 8000-14000 Da, wherein the dialysis temperature is 2-4 ℃, deionized water is replaced every 4h before 24h during dialysis, and deionized water is replaced every 8h after 24h until the dialysis is finished; concentrating the dialyzate, and freeze-drying to obtain Pediococcus pentosaceus LL-07 exopolysaccharide.
1L of fermentation culture solution taking glucose as a carbon source can finally obtain 561.12mg of Pediococcus pentosaceus LL-07 extracellular polysaccharide GEPS.
5) Extraction of LEPS and FEPS: the carbon source glucose in the 2.1 culture medium is replaced by lactose and fructose respectively, and the dosage is kept unchanged; the strain activation and the preparation of fermentation broth, the extraction of extracellular polysaccharide is the same as 2.2 and 2.3, and the final obtained results are as follows:
when the carbon source is lactose, 534.10mg of Pediococcus pentosaceus LL-07 extracellular polysaccharide LEPS can be finally obtained from 1L of fermentation culture solution;
when the carbon source is fructose, 428.85mg of Pediococcus pentosaceus LL-07 extracellular polysaccharide FEPS can be finally obtained from 1L of fermentation culture broth.
2.4 isolation and purification of the extracellular polysaccharide of Pediococcus pentosaceus LL-07
2.4.1DEAE-52 column chromatography for separating and purifying crude polysaccharide of Pediococcus pentosaceus LL-07 Extracellular Polysaccharide (EPS)
1) Pretreatment of Cellulose DEAE-52: and (3) placing DEAE-52 powder into deionized water, slightly stirring with a glass rod, standing, pouring off cellulose monomer and impurity fragments floating on the upper layer, and repeating for 2-3 times. Adding a proper amount of hot water for swelling, and then using deionized water for suction filtration for 3-4 times to remove ethanol in the original filler.
2) Column loading and balancing: a chromatographic column with the specification of 30cm multiplied by phi 2.6cm is vertically fixed on an iron stand, and a small amount of deionized water is added at the lower end of the column to remove air in the column. Closing the water outlet at the lower end of the column, reserving a small amount of deionized water in the column, draining activated DEAE-52 filler by using a glass rod close to the column wall, slowly and continuously pouring into the column, and preventing air bubbles from being brought in. In the sedimentation process, the liquid level of the filler is ensured to be always below the water surface, when the filler naturally subsides until the liquid level is not changed, a peristaltic pump is started to punch the column bed at the elution flow rate of 5 times, and deionized water is balanced for 24 hours for standby.
3) Loading and eluting: respectively weighing 10mg of GEPS, 10mg of LEPS and 10mg of FEPS, respectively dissolving in 10mL of deionized water, respectively loading the solutions to a Cellulose DE-52 chromatographic column, and respectively eluting with deionized water and NaCl solutions with the concentration of 0.1mol/L, 0.3mol/L and 0.6mol/L as eluents at the flow rate of 1mL/min; collecting eluate at intervals of 6min, measuring EPS content, collecting total 8 polysaccharides, dialyzing, concentrating, freeze drying, and respectively obtaining primarily purified polysaccharides (GEPS-1, GEPS-2, GEPS-3, LEPS-1, LEPS-2, LEPS-3, FEPS-1, FEPS-2).
As is clear from the elution profile of the extracellular polysaccharide DEAE-52 (abbreviated as DE-52 in the figure) shown in FIG. 3, the elution profile has a sharp peak and a narrow peak width, which indicates that the separation effect after elution is good. GEPS, LEPS, FEPS and separating by DEAE-52 column to obtain 3, 3 and 2 components respectively. Wherein GEPS-1, LEPS-1 and FEPS-1 are neutral polysaccharides; the rest components are acidic polysaccharide.
2.4.2Sepharose CL-6B gel column purification
1) Pretreatment of Sepharose CL-6B: the Sepharose CL-6B gel is placed in deionized water, gently stirred with a glass rod, left to stand, the upper insoluble material is decanted, and repeated 2-3 times. Filtering with deionized water for 3-4 times, removing ethanol in the original filler, adding deionized water for swelling, and preparing into homogenate.
2) Column loading and balancing: a chromatographic column with the specification of 100cm multiplied by phi 1.6cm is vertically fixed on an iron stand, and a small amount of deionized water is added at the lower end of the column to remove air in the column. Closing the water outlet at the lower end of the column, reserving a small amount of deionized water in the column, draining the activated Sepharose CL-6B gel by using a glass rod close to the column wall, slowly and continuously pouring the gel into the column, and preventing bubbles from being brought in. In the sedimentation process, the liquid level of the filler is ensured to be always below the water surface, when the filler naturally subsides until the liquid level is not changed, a peristaltic pump is started to punch the column bed at the elution flow rate of 5 times, and deionized water is balanced for 24 hours for standby.
3) Loading and eluting: the components of EPS separated as described above (initially purified polysaccharides GEPS-1, GEPS-2, GEPS-3, LEPS-1, LEPS-2, LEPS-3, FEPS-1, FEPS-2, FEPS-3) were each 10mg, and were each subjected to the following procedure: dissolving with 10mL deionized water, and filtering with a water film of 0.45 μm; loading the sample into a Sepharose CL-6B gel chromatographic column, eluting with deionized water as an eluent at a flow rate of 0.3mL/min, and collecting the eluent every 6min to determine EPS content; the obtained eluent is concentrated and freeze-dried to obtain the corresponding purified polysaccharide component (GEPS-1M, GEPS-2M, GEPS-3M, LEPS-1M, LEPS-2M, LEPS-3M, FEPS-1M, FEPS-2M, FEPS-3M). The elution curve of the polysaccharide Sepharose CL-6B is shown in the lower graph of FIG. 3.
2.5 analysis of the composition of the extracellular polysaccharide of Pediococcus pentosaceus LL-07
2.5.1 Standard preparation
After each standard is accurately weighed, water is added to prepare a standard solution mother solution single standard of 10mg/mL, and the required series of standard products are prepared according to the concentration gradient of the table 1.
TABLE 1 monosaccharide mix gradient concentration information
2.5.2 sample pretreatment
A clean chromatographic vial was taken, an appropriate amount of polysaccharide sample was weighed, 1mL of 2M TFA acid solution was added, and heated at 121℃for 2h. And (5) introducing nitrogen and drying. Adding 99.99% methanol for cleaning, drying, and repeating the methanol cleaning for 2-3 times. Adding sterile water for dissolving, and transferring into chromatographic bottle for testing.
2.5.3 instrument parameters
The monosaccharide components were analyzed using a Thermo ICS 5000+ ion chromatography system (ICS5000+, thermo Fisher Scientific, USA) using an electrochemical detector.
Using Dionex TM CarboPac TM PA20 (150 x 3.0mm,10 μm) liquid chromatography column; the sample loading was 5. Mu.L. Mobile phase A (H) 2 O), mobile phase B (0.1M NaOH), mobile phase C (0.1MNaOH,0.2M NaAc), flow rate 0.5mL/min; the column temperature is 30 ℃; elution gradient: 0min A/B/C (95:5:0, V/V/V), 26min A/B/C (85:5:10, V/V/V), 42min A/B/C (85:5:10, V/V), 42.1min A/B/C (60:0:40, V/V/V), 52min A/B/C (60:40:0, V/V/V), 52.1min A/B/C (95:5:0, V/V/V), 60min A/B/C (95:5:0, V/V/V).
2.5.4 monosaccharide composition content
The monosaccharide composition content of each component is shown in table 2.
Table 2 monosaccharide composition content of the components
2.6 determination of molecular weight of the extracellular polysaccharide of Pediococcus pentosaceus LL-07
2.6.1 sample pretreatment
Dissolving the sample in 0.1M NaNO 3 Aqueous solution (containing 0.02% NaN) 3 W/w) was 1mg/mL and was detected by filtration through a filter having a pore size of 0.45 μm and then on-line.
2.6.2 instrument parameters
The gel chromatography-differential-multi-angle laser light scattering system was used, the liquid phase system was U3000 (Thermo, USA), the differential detector was Optilab T-rEX (Wyatt technology, calif., USA), and the laser light scattering detector was DAWN HELEOS II (Wyatt technology, calif., USA). The gel exclusion chromatographic column (Ohpak SB-805HQ (300X 8 mm), ohpak SB-804HQ (300X 8 mm) and Ohpak SB-803HQ (300X 8 mm) were used in series, the column temperature was 45 ℃, the sample injection amount was 100. Mu.L, and the mobile phase A (0.02% NaN) 3 ，0.1M NaNO 3 ) Flow rate 0.4mL/min, elution gradient: isocratic for 100min.
Molecular weight measurement result of 2.6.3 Pediococcus pentosaceus LL-07 exopolysaccharide
The molecular weights of the extracellular polysaccharides of Pediococcus pentosaceus LL-07 were determined and are shown in Table 3.
TABLE 3 polysaccharide molecular weight scale
EXAMPLE 3 antioxidant, alpha-amylase activity inhibition, moisturizing Performance test
3.1 in vitro antioxidant Activity experiment
3.1.1DPPH clearance determination:
preparing 3 extracellular polysaccharide samples (GEPS, LEPS, FEPS) into sample solutions with different concentrations of 1mg/mL, 5mg/mL, 10mg/mL, 25mg/mL and 50mg/mL by using sterile water, taking 2mL of sample solution, adding 2mL of 0.2mmol/L (0.04 mg/mL) DPPH-99.9% ethanol solution, uniformly mixing, placing in a dark place at room temperature (25 ℃) for 30min, centrifuging for 5min 5000r/min, taking the supernatant and measuringAbsorbance at 517nm at V C For positive control, each sample was repeated 3 times, averaged, and DPPH radical scavenging was calculated as follows.
DPPH clearance (%) = [1- (a) S -A SB )/A C ]*100
A S : absorbance of 2mL of sample solution+2 mL of DPPH sample; a is that SB : absorbance of 2mL of sample solution+2 mL of ethanol sample; a is that C : absorbance of 2mL water+2 mL DPPH sample.
The DPPH radical scavenging results of Pediococcus pentosaceus LL-07 exopolysaccharide are shown in FIG. 4. As can be seen from FIG. 4, the extracellular polysaccharide GEPS and LEPS of Pediococcus pentosaceus LL-07 have better DPPH free radical scavenging effect, and when the concentration of the GEPS and the LEPS is more than 10mg/mL, the DPPH free radical scavenging rate is more than 98 percent. When the GEPS concentration is more than 50mg/mL, the effect is equivalent to the DPPH free radical scavenging effect of Vc and reaches 99.7 percent.
3.1.2 determination of hydroxyl radical clearance:
3 extracellular polysaccharide samples (GEPS, LEPS, FEPS) are prepared into sample solutions with different concentrations of 1mg/mL, 5mg/mL, 10mg/mL, 25mg/mL and 50mg/mL by using sterile water, and 9mmol/L FeSO is respectively added into 1mL of the sample 4 And 9mmol/L salicylic acid-ethanol solution 2mL each was added with 2mL 1.2mmol/L H last 2 O 2 Starting reaction, reacting at 37deg.C for 30min, centrifuging at 5000r/min for 5min, collecting supernatant, zeroing with deionized water, and measuring sample concentration absorbance A at wavelength of 510nm 1 The method comprises the steps of carrying out a first treatment on the surface of the In addition, deionized water replaces H 2 O 2 Repeating the above operation to determine absorbance A of sample 2 Simultaneously, water is used for replacing the sample solution to determine the absorbance A 0 。V C Is a positive control. Each sample was repeated 3 times, averaged, and the hydroxyl radical scavenging rate was calculated according to the following formula.
Hydroxyl radical clearance (%) = [1- (a) 1 -A 0 )/A 2 ]*100
The results of hydroxyl radical clearance of Pediococcus pentosaceus LL-07 exopolysaccharide are shown in FIG. 5. As can be seen from FIG. 5, the extracellular polysaccharides GEPS and LEPS of Pediococcus pentosaceus LL-07 have good hydroxyl radical scavenging rates of 59.9% and 58.8%, respectively, when the polysaccharide concentration is 50mg/mL.
In conclusion, pediococcus pentosaceus (Pediococcus pentosaceus) LL-07 extracellular polysaccharide GEPS and LEPS have better in-vitro antioxidant activity.
3.2 experiments to inhibit alpha Amylase Activity
3 extracellular polysaccharide samples (GEPS, LEPS, FEPS) were prepared with sterile water as sample solutions of different concentrations of 1mg/mL, 5mg/mL, 10mg/mL, 25mg/mL, 50mg/mL. Taking 500 mu L of each sugar solution with different concentrations, adding 500 mu L of alpha-amylase (0.5 mg/mL), standing for 100min at room temperature, adding 500 mu L of starch solution with 1g/100mL for reaction for 10min (the reaction temperature is the optimal activity temperature of the alpha-amylase), adding 1mL of DNS reagent, and carrying out boiling water bath for 5min. Cooling, diluting with distilled water to constant volume of 10mL, measuring absorbance at 540nm wavelength, and recording as A 1 The method comprises the steps of carrying out a first treatment on the surface of the In addition, the operation was repeated with sterile water instead of the alpha amylase solution, and the absorbance was measured as a control group and designated A 2 The method comprises the steps of carrying out a first treatment on the surface of the The extracellular polysaccharide sample liquid is replaced by sterile water with the same volume, repeated operation is used as a blank group to determine the absorbance value, and is marked as A 3 The method comprises the steps of carrying out a first treatment on the surface of the The absorbance value of the starch solution was designated A 4 . Acarbose was used as a positive control. Each sample was repeated 3 times, and the inhibition ratio of the alpha-amylase activity was calculated by taking an average value.
Alpha amylase activity inhibition (%) = [1- (a) 1 -A 2 )/(A 3 -A 4 )]*100
The inhibition of alpha amylase by Pediococcus pentosaceus LL-07 exopolysaccharide is shown in FIG. 6. As can be seen from FIG. 6, the inhibition of alpha amylase by LEPS at a concentration of 50mg/mL can reach 94.68%, which is close to that of alpha amylase at the same concentration of acarbose.
In conclusion, pediococcus pentosaceus LL-07 extracellular polysaccharide GEPS and LEPS have better alpha amylase inhibition capability.
3.3 moisture Property experiments
The dried flat weighing bottle with plug (outer diameter 50mm, height 15 mm) was taken and placed in an incubator with a temperature of 25 ℃ + -1 ℃ and a humidity of 43% and 81% one day before the experiment. Weighing a proper amount of sample (GEPS, LEPS, FEPS), placing in a weighing bottle, and weighing with mass of m 1 . By weight ofAdding 10% deionized water, precisely weighing to be m 2 . Placing the weighing bottle with bottle stopper in a constant temperature incubator with temperature of 25deg.C+ -1deg.C and relative humidity of 43% and a drier with silica gel as desiccant for 5 days, and weighing the weight of m 3 . Glycerin and sodium hyaluronate were used as controls.
Moisture retention (%) = (m) 3 -m 2 )/(m 2 -m 1 )*100％
The moisturizing rate of Pediococcus pentosaceus LL-07 extracellular polysaccharide is shown in FIG. 7. As can be seen from FIG. 7, the FEPS has a moisture retention rate close to that of hyaluronic acid in a constant temperature incubator with a relative humidity of 43%, which can reach 87.3%, and has good moisture retention performance.
In conclusion, the Pediococcus pentosaceus LL-07 provided by the invention can be fermented by taking glucose, fructose and lactose as carbon sources respectively to obtain corresponding extracellular polysaccharide GEPS, LEPS, FEPS, and in vitro experiments show that the three extracellular polysaccharides have strong antioxidant activity, alpha amylase activity inhibition and moisture retention performance, and technical support is provided for research and development of Pediococcus pentosaceus and extracellular polysaccharide thereof in products such as foods, medicines and cosmetics.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. Pediococcus pentosaceus LL-07, characterized in that the classification of Pediococcus pentosaceus LL-07 is named: pediococcus pentosaceus, deposited in China center for type culture Collection, with the deposit address: the preservation date of the university of Wuhan, china is: 2023, 7 and 11, deposit number: cctccc M20231264.
2. The method for preparing the exopolysaccharide of Pediococcus pentosaceus LL-07 is characterized in that Pediococcus pentosaceus LL-07 is fermented by taking glucose, lactose and fructose as carbon sources respectively to obtain the exopolysaccharide GEPS, LEPS, FEPS of Pediococcus pentosaceus LL-07.
3. The pediococcus pentosaceus LL-07 exopolysaccharide according to claim 2, wherein the number average molecular weight of pediococcus pentosaceus LL-07 exopolysaccharide GEPS, LEPS, FEPS is 14.694kDa, 13.237kDa, 39.031kDa, respectively; the weight average molecular weight is 29.781kDa, 56.892kDa and 83.224kDa respectively; the z-average molecular weight is 86.429kDa, 1014.057kDa and 147.623kDa respectively, and the molecular weight distribution width is 2.027, 4.298 and 2.132 respectively.
4. The pediococcus pentosaceus LL-07 exopolysaccharide according to claim 2 or 3, wherein the pediococcus pentosaceus LL-07 exopolysaccharide GEPS comprises three polysaccharides GEPS-1M, GEPS-2M, GEPS-3M; the LEPS comprises three polysaccharides of LEPS-1M, LEPS-2M, LEPS-3M; the FEPS comprises two polysaccharides FEPS-1M, FEPS-2M.
5. The extracellular polysaccharide of Pediococcus pentosaceus LL-07 of claim 4, wherein GEPS-1M, LEPS-1M, FEPS-1M is a neutral polysaccharide; GEPS-2M, GEPS-3M, LEPS-2M, LEPS-3M, FEPS-2M is an acidic polysaccharide.
6. The pediococcus pentosaceus LL-07 extracellular polysaccharide according to claim 4, wherein the simple sugar composition of GEPS-1M comprises, in mol%, 0.46% of L-fucose, 0.24% of L-arabinose, 5.75% of D-galactose, 15.72% of D-glucose, 75.37% of D-mannose, 2.24% of D-ribose; the monosaccharide composition of the GEPS-2M comprises 1.79 mol percent of L-fucose, 0.42 mol percent of L-arabinose, 13.45 mol percent of D-galactose, 12.45 mol percent of D-glucose and 71.90 mol percent of D-mannose; the monosaccharide composition of the GEPS-3M comprises 0.69 mol percent of L-fucose, 0.49 mol percent of L-arabinose, 57.07 mol percent of D-galactose, 37.31 mol percent of D-glucose and 4.44 mol percent of D-mannose; the monosaccharide composition of the LEPS-1M comprises 0.19 mol% of L-fucose, 0.13 mol% of L-arabinose, 12.24 mol% of D-galactose, 14.74 mol% of D-glucose, 70.92 mol% of D-mannose and 1.77 mol% of D-ribose; the monosaccharide composition of the LEPS-2M comprises 0.79 mol% of L-fucose, 0.27 mol% of L-arabinose, 10.64 mol% of D-galactose, 8.29 mol% of D-glucose and 80.01 mol% of D-mannose; the monosaccharide composition of the LEPS-3M comprises 0.58 mol% of L-fucose, 0.51 mol% of L-arabinose, 60.08 mol% of D-galactose, 32.92 mol% of D-glucose and 5.91 mol% of D-mannose; the monosaccharide composition of the FEPS-1M comprises 1.01 mol percent of L-fucose, 1.11 mol percent of L-arabinose, 13.10 mol percent of D-galactose, 11.51 mol percent of D-glucose and 73.28 mol percent of D-mannose; the monosaccharide composition of the FEPS-2M comprises 0.09% of L-fucose, 0.07% of L-arabinose, 0.64% of D-galactose, 3.07% of D-glucose and 96.13% of D-mannose in mol%.
7. A method for producing an extracellular polysaccharide of pediococcus pentosaceus LL-07 according to any one of claims 2 to 6, comprising the steps of:
s1, inoculating Pediococcus pentosaceus LL-07 in claim 1 into a liquid culture medium with glucose, lactose and fructose as carbon sources according to the inoculum size of 2-4% by volume ratio, and culturing and fermenting at a constant temperature of 32-37 ℃ for 24-48 hours to obtain a fermentation culture solution;
s2, placing the fermentation culture solution in a water bath at 90-95 ℃ for 10-30 min at constant temperature, centrifuging the fermentation solution after constant temperature to obtain supernatant, adding trichloroacetic acid water solution with the concentration of 0.75-0.85 g/mL to ensure that the final concentration of trichloroacetic acid is 0.035-0.045 g/mL, standing, centrifuging to obtain supernatant, concentrating, adding pre-cooled absolute ethyl alcohol with the volume of 4-6 times that of concentrated solution, standing, centrifuging to obtain precipitate, adding sterile water into the precipitate, centrifuging again, concentrating to dryness, and adding sterile water for re-dissolving to obtain crude polysaccharide solution;
s3, dialyzing the crude polysaccharide solution in deionized water for 48-72 hours by using a dialysis bag of 8000-14000 Da at the dialysis temperature of 2-4 ℃, and freeze-drying after dialysis to obtain pediococcus pentosaceus LL-07 extracellular polysaccharide GEPS, LEPS, FEPS respectively;
s4, separating the Pediococcus pentosaceus LL-07 extracellular polysaccharide GEPS, LEPS, FEPS by a Cellulose DE-52 chromatographic column and purifying by a Sepharose CL-6B gel chromatographic column to obtain a corresponding purified polysaccharide component of the Pediococcus pentosaceus LL-07 extracellular polysaccharide GEPS, LEPS, FEPS.
8. Use of pediococcus pentosaceus LL-07 according to claim 1 and/or the pediococcus pentosaceus LL-07 extracellular polysaccharide according to any one of claims 2 to 6 in food, pharmaceutical, cosmetic or health products.
9. Use of an extracellular polysaccharide of pediococcus pentosaceus according to any one of claims 2 to 6 for the preparation of a product for antioxidant and/or alpha amylase inhibition, characterized in that the extracellular polysaccharide of pediococcus pentosaceus is one or both of GEPS and LEPS.
10. Use of an extracellular polysaccharide of pediococcus pentosaceus according to any one of claims 2 to 6 for the preparation of a moisturizing product, characterized in that the extracellular polysaccharide of pediococcus pentosaceus is FEPS.
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