CN106222103B - The Pediococcus pentosaceus of one plant of extracellular polysaccharide and its application - Google Patents

The Pediococcus pentosaceus of one plant of extracellular polysaccharide and its application Download PDF

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CN106222103B
CN106222103B CN201610578363.2A CN201610578363A CN106222103B CN 106222103 B CN106222103 B CN 106222103B CN 201610578363 A CN201610578363 A CN 201610578363A CN 106222103 B CN106222103 B CN 106222103B
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pediococcus pentosaceus
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polysaccharide
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陈佩
兰新哲
党辉
季晓辉
裴金金
金文刚
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SHAANXI RADIO AND TELEVISION UNIVERSITY
Shaanxi University of Technology
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    • 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
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    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/38—Other non-alcoholic beverages
    • A23L2/382—Other non-alcoholic beverages fermented
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

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Abstract

The invention discloses the Pediococcus pentosaceus of one plant of extracellular polysaccharide and its applications, belong to microorganism field.For Pediococcus pentosaceus Pediococcus pentosaceus of the invention in preservation on December 11 in 2015 and Chinese microorganism strain collection, deposit number is CGMCC No:11857.The bacterial strain can high-yield extracellular polysaccharide, polysaccharide yield 153.60mg/L.The bacterial strain is to NaCl, cholate, gastro-intestinal Fluid have very strong tolerance, there is stronger adhesive capacity to Caco-2 cell, and there is the oxidation resistance for improving HepG2 cell under oxidative stress status, it can be as probiotics or oxidation resistant product for fields such as food, medicine, cosmetics.

Description

The Pediococcus pentosaceus of one plant of extracellular polysaccharide and its application
Technical field
The present invention relates to the Pediococcus pentosaceus of one plant of extracellular polysaccharide and its applications, belong to microorganism field.
Background technique
Polysaccharide is the most abundant biopolymer of content in nature, has multiple functions, is a kind of in the food industry Most common food additives；With good bioactivity, it is adjustable the immune function of body, is ideal immunological regulation Agent.Currently, a variety of polysaccharide are used for clinical test, it is mainly used for the research of the drugs such as anti-aging, antiviral and antitumor.With people Further investigation to the synthesis of polysaccharide, preparation, structure, pharmacology and clinical medicine, develop polyose medicament and health care product have become For one of the research hotspot of scientists instantly.Polyose also will be with boundless prospect.
Probiotics (probiotics) is that a kind of balance by improving host intestine microbial flora plays a role Active microorganism is the important physiology bacterium of human body intestinal canal, can promote the ecological balance of gut microflora, has a variety of lifes Function is managed, can such as improve intestinal microflora, promote the proliferation of beneficial bacterium in enteron aisle while inhibiting the growth of harmful bacteria, raising machine It is high can to prevent and treat three for body immunity.It is ground so carrying out development and application of the probiotics in functional food this year and being increasingly becoming one Study carefully hot spot, while consumer also has become increasingly aware of probiotics to the important function of human health.Probiotic lactobacillus is probiotics In most important family, in food application it is very extensive, many physiological activity of lactic acid bacteria are considered and its exocellular polysaccharide (extracellular polymeric substances, EPS) is directly related, such as makees to the improvement of Yoghourt and cheese quality With hypoglycemic, adjust the effects of blood pressure, antitumor, immunological regulation.
Compared with other polysaccharide, the research of the EPS of lactic acid bacteria is more significant and application value: (1) research shows that lactic acid The EPS of bacterium has important physiological activity and physiological function；(2) EPS of lactic acid bacteria does not have to be separately separated to be purified, Ke Yizhi It connects and lactic acid bacteria is applied in fermented food；(3) lactic acid bacteria is the generally acknowledged microorganism with grade-safe, EPS Belong to natural products；4) EPS of lactic acid bacteria can be generated directly in fermented food, improve Food Texture, and mouthfeel etc. does not need it Its additive can reduce product cost.
Currently, the main problem in sour milk products is the problems such as whey is easily precipitated, grumeleuse is easily broken.It is commonly solved in production Certainly mode is to add skimmed milk powder, filter cream evaporation and concentration or film, these measures all undoubtedly increase production cost.It is another Kind method is to add stabilizer also to mask the true taste of Yoghourt to a certain extent simultaneously although having reached desired effect Road, and some countries, European Union clearly forbid adding thickener and stabilizer in the fermented dairy products such as Yoghourt.With consumer Improvement of living standard, more stringent requirements are proposed for the qualities of dairy products by people.The application of lactic acid bacteria EPS, may replace and add Add the use of agent, EPS can be improved stickiness and curd strength, keep gel structure, whey be prevented to be precipitated, can reduce product at This, from market, angle has pulled the research to EPS.
The further investigation of the metabolites such as lactic acid bacteria EPS has weight to fermented dairy product quality and optimization technological design is improved Big meaning.In addition, with the development of biotechnology, lactic acid bacteria EPS medical treatment, cosmetics, cell and in terms of all have There is potential Development volue.
What majority used in present country's Dairy Production is all external patented strain, with independent intellectual property rights prebiotic Bacterium only accounts for seldom a part.In addition, the yield of the exocellular polysaccharide of lactic acid bacteria is not generally high at present.So this research is intended to from tradition Fermented food in screening have it is preferable produce viscous polysaccharide, while being with the lactic acid bacteria strains of certain biological function again Future, development functionality leavening laid the foundation.
Summary of the invention
The first purpose of the invention is to provide the Pediococcus pentosaceus Pediococcus of one plant of extracellular polysaccharide Pentosaceus P36 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on December 10th, 2015 Center, deposit number are CGMCC No.11857, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
A second object of the present invention is to provide the Pediococcus pentosaceus Pediococcuspentosaceus P36 to make Application in standby oxidation resistant product.
Third object of the present invention is to provide the Pediococcus pentosaceus Pediococcuspentosaceus P36 to eat The application of product, medicine, chemical field.
The application includes preparing dairy products.
The application includes preparing fermented beverage.
The application includes preparing food additives.
The application includes preparing cosmetics, preparing Medicines and Health Product.
The utility model has the advantages that Pediococcus pentosaceus Pediococcuspentosaceus P36 can high-yield extracellular polysaccharide, it is extracellular more Candy output is 153.60mg/L.The bacterial strain has very strong tolerance to NaCl, cholate, gastro-intestinal Fluid, has to Caco-2 cell Stronger adhesive capacity, and there is the oxidation resistance for improving HepG2 cell under oxidative stress status, it can be used as probiotics In preparing oxidation resistant product or for the fields such as food, medicine, cosmetics.
Detailed description of the invention
Fig. 1 is the colonial morphology of Pediococcus pentosaceus P36；
Fig. 2 is the cellular morphology of Pediococcus pentosaceus P36 (400 times of amplification factor) under the microscope；
Fig. 3 is influence of the pH to Pediococcus pentosaceus P36 thalli growth；
Fig. 4 is survival rate of the Pediococcus pentosaceus P36 in simulate the gastric juice；
Fig. 5 is survival rate of the Pediococcus pentosaceus P36 in simulated intestinal fluid (pH 8.0)；
Fig. 6 is influence of the NaCl to Pediococcus pentosaceus P36 thalli growth；
Fig. 7 be Pediococcus pentosaceus P36 under the microscope (400 times of amplification factor) to the adherency form of Caco-2 cell.
Specific embodiment
The screening and identification of 1 Pediococcus pentosaceus of embodiment
29 parts of pickle juice sample are adopted altogether in Southern Shausi, carry out bacterial strain screening separation.Specific step is as follows:
Sample pretreatment: sample 5ml is respectively taken to be respectively put into the sterile water equipped with 45ml 0.1% with the liquid-transfering gun of sterilizing (contain bead) in triangular flask, stands 20min after rocking 1min, it is spare.
Pretreated sample is equivalent to raw sample and dilutes 10 times, is successively diluted sample step by step with same method, Each dilution takes 0.1mL dilute sample, is coated on MRS solid medium, at 37 DEG C, constant temperature incubation 48h, with sterile tooth Label picking is sticky and has the single colonie of obvious wire drawing.It crosses and separates on MRS solid medium again, obtain pure single colonie, and Carry out Morphological Identification, including the experiment of Gram's staining, microscopy, catalase, curdled milk experiment.As the result is shown: Gram's staining sun Property, catalase experiment feminine gender, energy curdled milk primarily determine as lactic acid bacteria.
Phend-sulphuric acid surveys polyoses content: by the above isolated strain inoculated into MRS fluid nutrient medium, 37 DEG C Constant temperature incubation for 24 hours, takes 10ml streptococcus acidi lactici fermented solution, and boiling water bath 10min is cooled to room temperature, addition 80% trichloroacetic acid of 5ml, and 4 It DEG C stands overnight.10000r/min is centrifuged 20min deproteination matter and other big molecular impurities.Supernatant after centrifugation moves to In 14000 bag filter.A water is changed with distilled water dialysis 3d, every 8h.After three days, polysaccharide is in cotton-shaped precipitation, constant volume.Using sulphur Acid-phynol method measurement exocellular polysaccharide content.
It isolates to obtain 101 plants of lactic acid bacteria, wherein the 20 of high polysaccharide plant.It is to wherein one plant of polysaccharide yield 153.60mg/L carries out morphological analysis.For the bacterial strain in MRS culture medium, naked eyes can be formed after 37 DEG C of constant temperature incubation 48h can The obvious bacterium colony seen, for colonial morphology as shown in Figure 1, colonial morphology is rounded, milky is opaque, and surface is smooth, there is protrusion, Neat in edge, not chromogenesis, picking energy wire drawing.After choosing single colonie smear, carries out Gram's staining and observe under the microscope Its morphosis.Gram's staining purple as the result is shown shows that this bacterial strain is gram-positive bacteria, and strain morphology is spherical (Fig. 2).
The genome of the bacterial strain is extracted, and carries out 16S rDNA amplification, obtains the sequence as shown in SEQ IN NO.1.By SEQ Sequence shown in ID NO.1 is compared in Genbank database, determines that lactic acid bacteria P36 is Pediococcus pentosaceus (Pediococcus pentosaceus), is named as Pediococcuspentosaceus P36.The bacterial strain is in 2015 12 The moon is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC on the 10th No.11857。
Influence of the embodiment 2pH to Pediococcus pentosaceus P36 strain growth
Adjusting MRS fluid nutrient medium pH value with HCl (1mol/L) is respectively 2.0,2.5,3.0,4.0,5.0,6.0,6.5 and 7.0, the Pediococcus pentosaceus P36 of logarithmic growth phase are inoculated with 2% inoculum concentration, are sufficiently mixed uniformly, are cultivated at 37 DEG C Absorbance value OD of its bacterium solution under 600nm wavelength is measured after 18h600.Growing state of the Pediococcus pentosaceus P36 in different pH See Fig. 3, the results showed that the bacterial strain can still be grown in the acidic environment of pH2, illustrate that it has preferable acid resistance.
Tolerance of the 3 Pediococcus pentosaceus P36 of embodiment to simulation gastro-intestinal Fluid
Manual simulation's gastro-intestinal Fluid needs Fresh.The pepsin for being 3g/L with PBS (pH 3.0) configuration concentration, 0.22 μm Required simulate the gastric juice is prepared into after membrane filtration.The trypsase for being 1g/L with PBS (pH 8.0) configuration concentration, 0.22 μm of filter membrane Simulated intestinal fluid is prepared into after filtering.
Pediococcus pentosaceus P36 is resuspended with physiological saline, and its bacterium solution density is adjusted in simulate the gastric juice (pH 3.0) To 1 × 109CFU/mL is mixed well, and detects viable count after 37 DEG C of 1,2 and 3h of culture.After 3h, simulate the gastric juice (pH 3.0) is taken In culture solution 1ml be added into the simulated intestinal fluid of 9ml (pH 8.0), mix well, after 37 DEG C of 2,4 and 8h of culture detect live Bacterium number.With 37 DEG C of culture 48h of MRS solid medium, the survival rate of lactic acid bacteria is calculated with following formula:
Survival rate (%)=log cfu N1/log cfu N0× 100%
N1=the viable count after simulation gastro-intestinal Fluid processing, N0=without the viable count before gastro-intestinal Fluid processing
Lactic acid bacteria will enter enteron aisle and play its prebiotic effect, should have the ability of tolerance gastric juice first.Fig. 4 shows penta Sugar-tablet coccus P36 can survive 3h in artificial simulation gastric juices (pH 3.0), and survival rate is greater than 85%.Fig. 5 shows pentose piece Coccus P36 can survive 8h in simulated intestinal fluid (pH 8.0), and survival rate can reach 92% or more.
Tolerance of the 4 Pediococcus pentosaceus P36 of embodiment to cholate
The Pediococcus pentosaceus P36 of logarithmic growth phase, is inoculated in respectively with 2% inoculum concentration with or without 0.3% gallbladder In MRS-THIO (MRS contains 0.2% sodium thioglycolate) culture of salt, it is sufficiently mixed uniformly, is cultivated at 37 DEG C.Record training The time needed for each group when base light absorption value reaches 0.3 unit is supported, the time difference that two groups of cholate of cholate and addition is not added is penta The delay time (LT) that sugar-tablet coccus P36 is grown in cholate.The length of delay time is able to reflect lactic acid bacteria to the resistance to of cholate By ability.
The effect that 1 cholate of table grows P36
The important indicator whether evaluation bacterial strain can survive and colonize in enteron aisle is exactly bacterial strain for the resistance to of cholate By ability.Shown in table 1, Pediococcus pentosaceus P36 is 0.96h for the delay time of cholate, illustrates that this bacterial strain has cholate Good tolerance.
Tolerance of the 5 Pediococcus pentosaceus P36 of embodiment to NaCl
The P36 of logarithmic growth phase, with 2% inoculum concentration be respectively connected to NaCl mass fraction be 0%, 2%, 4%, 6%, 7%, it in 8%, 9% MRS fluid nutrient medium, is sufficiently mixed uniformly, measures its bacterium solution OD after cultivating 18h at 37 DEG C600Value.
As shown in fig. 6, Pediococcus pentosaceus P36 still can survive when NaCl concentration is 9%, illustrate Pediococcus pentosaceus P36 has stronger tolerance to NaCl.
6 Pediococcus pentosaceus P36 of embodiment influences the adhesive capacity of Caco-2 cell
To contain 20% fetal calf serum, 1%L- glutamine, 1% nonessential amino acid and 1% dual anti-DMEM in high glucose are complete Full culture medium culture Caco-2 cell, 37 DEG C, 5%CO2Incubator in cultivated, change liquid every other day, to cell Proliferation merge When rate reaches 80% or so, passed on after pancreatin digestion according to 1:3.The cell of logarithmic growth phase carries out lower step experiment.
By Caco-2 cell with 4 × 104The hole cell/ is inoculated in 6 orifice plates, and 37 DEG C of cultures are for 24 hours.By culture to logarithmic phase It is rinsed 3 times, is resuspended in DMEM in high glucose complete medium with PBS (pH 7.2) after Pediococcus pentosaceus P36 centrifugation, adjustment bacterium solution is dense Degree is 1 × 109CFU/mL is added in the Caco-2 cell monolayer grown, 37 DEG C of culture 2h.Cell is rinsed with PBS again Single layer 3 times, after with methanol fix 30min, then carry out Gram's staining.20 visual field numbers, 100 cells are looked at random, with averagely every The P36 bacterium number that adheres on a cell evaluates its adhesive capacity.
Caco-2 cell is a kind of people clone colon adenocarcinoma cell, and the small intestine epithelium for being structurally and functionally similar to differentiation is thin Born of the same parents can preferably simulate the environment in human body intestinal canal.It is analyzed by adhesive capacity of the bacterial strain to Caco-2 cell, it can be evaluated Whether strain excellent can be become.It can be seen that in Fig. 7, Pediococcus pentosaceus P36 has certain adhesive capacity to Caco-2 cell, According to experiment statistics the result shows that adhesion rate is 15/cell.Thus speculate, Pediococcus pentosaceus P36 should have in human body intestinal canal There is preferable colonization ability.
Influence of the 7 Pediococcus pentosaceus P36 of embodiment to HepG2 cells and supernatant anti-oxidation function
The HepG2 cell that growth is in logarithmic growth phase is collected with 0.25% trypsin digestion, with 2 × 105A/mL Cell density be inoculated in 6 well culture plates, every hole 2mL culture solution, 37 DEG C, 5%CO2, culture is for 24 hours.Packet transaction: blank control 2mL DMEM culture solution is added in group；H containing 0.10mmol/L is added in model group2O2DMEM culture solution；Lactic acid bacteria group adds simultaneously Enter to contain H2O2DMEM culture solution and P36 bacterium solution (1 × 109CFU/mL), continue to cultivate 12h and for 24 hours.
When 12h, compared with the control group, the vigor of the GSH-Px in model group cell supernatant significantly increase 74% (P < 0.05), POD vigor significantly reduces 24% (P<0.05), and there was no significant difference (P>0.05) for the content of SOD, GSH, CAT and MDA, Show that model group cell does not enter oxidative stress status also at this time.Compared with model group, after adding Pediococcus pentosaceus P3612h, carefully T-AOC, SOD, GSH-Px and CAT vigor in born of the same parents' supernatant is all significantly increased, and 59%, 23%, 28% and has been respectively increased 126% (P < 0.05), the vigor of POD significantly reduce 44% (P < 0.05), and MDA content increases 19%, show that P36 can be mentioned High cellular anti-oxidant capacity, but also have certain stimulation to cell.After for 24 hours, compared with the control group, on model group cell T-AOC in clear liquid significantly reduces 33% (P < 0.05), the vigor of SOD, GSH-Px and CAT significantly reduces 11% respectively, 52% and 47% (P < 0.05), while POD vigor dramatically increases 32% (P < 0.05), MDA content dramatically increase 81% (P < 0.05), show that cell has shown as oxidative stress status at this time.Compared with model group, after adding P3612h, in cell supernatant T-AOC, GSH-Px and CAT vigor be all significantly increased, be respectively increased 80%, 30% and 124% (P < 0.05), SOD is living Power increases, but not significant (P>0.05), and the vigor of POD significantly reduces 40% (P<0.05), and MDA content increases 24%, show that cellular anti-oxidant capacity can be improved in Lactobacillus casei, but also has certain stimulation to cell.After for 24 hours, The vigor of T-AOC and SOD in cell supernatant significantly increase 145% and 123% (P < 0.05), GSH-Px, CAT and POD Vigor dramatically increase (P < 0.05), the content of GSH and MDA significantly reduce 28% and 49% (P < 0.05) respectively.There is research Show lactic acid bacteria have efficient scavenging hydroxyl and super oxide anion free radical ability, and can be improved human body SOD and GSH-Px vigor reduces enteron aisle oxidative stress, enhances oxidation resistance.Herein the result shows that P36 improves the conjunction of antioxidase At and secretion, enhance the oxidation resistance of cell.
Antioxidant activity when 2 cell supernatant of table is in 12h and for 24 hours
Note: control group is not added with H2O2；Model group adds H2O2；Experimental group adds Pediococcus pentosaceus P36
Influence of the 8 Pediococcus pentosaceus P36 of embodiment to HepG2 cell pyrolysis liquid antioxidant activity
Cell continue cultivate 12h and for 24 hours afterwards draw culture supernatant loading centrifuge tube, -80 DEG C freeze it is spare.Every hole PBS Washing 3 times, then every hole adds the Triton X-100 of 1mL 1% sufficiently to be blown with suction pipe even, and 2000r/min is centrifuged 15min, collects Supernatant is cell pyrolysis liquid, -80 DEG C freeze it is spare.
Antioxidant activity when 3 cell pyrolysis liquid of table is in 12h and for 24 hours
Note: control group is not added with H2O2；Model group adds H2O2；Experimental group adds Pediococcus pentosaceus P36
Shown in table 3, after 12h, compared with the control group, SOD vigor content decreases in model group cell pyrolysis liquid, but poor Different not significant (P>0.05), GSH content significantly reduce (P<0.05), and the vigor of POD significantly improves 92% (P<0.05).As a result Show that cell just starts to be stimulated at this time, POD vigor intracellular increases to protect cells from damage.After adding P3612h, cream In the cell pyrolysis liquid of sour bacterium group SOD vigor significantly increase 14% and 17% respectively compared with control group and model group (P < 0.05)；The vigor of POD significantly increases 102% (P < 0.05) compared with the control group to be compared with model group also increased；GSH Content (P < 0.05) is dramatically increased compared with model group.Show oxidation stimulation of the cell by lactic acid bacteria at this time, it is intracellular Oxidizing ferment content increase.After for 24 hours, compared with the control group, SOD vigor and GSH content in model group cell pyrolysis liquid 44% and 61% (P < 0.05) is significantly increased respectively；POD vigor also increased.The SOD vigor of addition P36 group compares control group It increased, but significantly reduce 15% (P < 0.05) than model group；POD vigor dramatically increases respectively than control group and model group 114% and 94% (P < 0.05)；GSH content significantly increases 72% (P < 0.05) than control group, also has compared with model group Increased.The result shows that by strong oxidative stress, intracellular polyphenoils starts largely the cell of model group at this time Synthesis, to protect cells from damage.In addition, P36 improves the vigor of intracellular antioxidase by stimulation, cell is enhanced Antioxidation has certain protective effect to cell.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

Claims (7)

1. Pediococcus pentosaceus (Pediococcus pentosaceus) P36 of one plant of extracellular polysaccharide, on December 10th, 2015 It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.11857, is protected Hiding address is BeiJing, China, Institute of Microorganism, Academia Sinica.
2. a kind of dairy products, which is characterized in that its active constituent contains bacterial strain described in claim 1.
3. a kind of fermented tea beverage, which is characterized in that the exocellular polysaccharide containing the production of bacterial strain described in claim 1.
4. a kind of medical product, which is characterized in that the medical product be oral preparation, including tablet, capsule, oral solution and Freeze dried powder；The ingredient of the oral preparation contains the exocellular polysaccharide of the production of bacterial strain described in claim 1.
5. a kind of food additives, which is characterized in that the exocellular polysaccharide containing bacterial strain described in claim 1 and its production.
6. bacterial strain described in claim 1 is preparing the application in oxidation resistant product.
7. bacterial strain described in claim 1 prepare food, the application in medical product.
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CN110964673B (en) * 2019-12-30 2022-03-04 上海应用技术大学 Pediococcus pentosaceus with high oxidation resistance and whitening effect and application thereof
CN113388540A (en) * 2021-05-21 2021-09-14 深圳市华大农业应用研究院 Pediococcus pentosaceus HG-9 strain and application thereof
CN114085875B (en) * 2021-11-10 2023-04-25 四川大学 Extracellular polysaccharide, preparation method and application thereof
CN117757658A (en) * 2023-11-13 2024-03-26 江苏科技大学 Pediococcus pentosaceus from silkworm intestinal tract and application thereof

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