CN108330086A - A kind of extracellular polysaccharide spatial plant lactobacillus SS18-33 and its application in improving biological antioxidant activity - Google Patents
A kind of extracellular polysaccharide spatial plant lactobacillus SS18-33 and its application in improving biological antioxidant activity Download PDFInfo
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- CN108330086A CN108330086A CN201810111945.9A CN201810111945A CN108330086A CN 108330086 A CN108330086 A CN 108330086A CN 201810111945 A CN201810111945 A CN 201810111945A CN 108330086 A CN108330086 A CN 108330086A
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- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000004562 water dispersible granule Substances 0.000 description 1
- 239000004563 wettable powder Substances 0.000 description 1
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Abstract
The invention discloses a kind of extracellular polysaccharide spatial plant lactobacillus SS18 33 and its applications in improving biological antioxidant activity.Spatial plant lactobacillus SS18 33 disclosed by the invention is CGMCC No.15151 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.The 33 extracellular polysaccharide of institute of spatial plant lactobacillus SS18 of the present invention has to DPPH Scavenging activities, to ultra-oxygen anion free radical (O2 ‑) Scavenging activity, to Fe2+Sequestering power and the total reducing power or total antioxidant capacity for blocking peroxide to be formed to providing hydrogen atom substance；In addition, the bacterial strain also has gastrointestinal tract inverse ring border resistance characteristics, practical basis is provided for application of the exocellular polysaccharide in improving biological antioxidant activity.The present invention has filled up the research blank of space food microorganisms engineering bacteria.
Description
Technical field
The present invention relates in biotechnology, a kind of extracellular polysaccharide spatial plant lactobacillus SS18-33 and its carrying
Application in high biological antioxidant activity.
Background technology
Exopolysaccharides Produced by Lactic Acid Bacteria (exopolysaccharides, EPS) is lactic acid bacteria under the conditions of sub- Optimal Growing, to support
Anti- poor environment factor secretion is its important secondary metabolite, molecule in the mucilage polysaccharides or capsular polysaccharide of cell surface
Amount is 4.0 × 104~6.0 × 106Between Da.In the natural environment, EPS usually has the function of protecting microbial cell, such as keeps away
Exempt from cell drying and dehydrating, from macrophage invasion or phage-infect and resist the effect of antibiotic or noxious material, may be used also
Steady seepage pressure participates in cellular informatics transmission and cell composition etc..Many studies have shown that EPS has immunoregulation effect, anti-grease
Matter peroxidative activity, antitumor action or cancer cell specific induction of apoptosis promote intestinal flora balance, reduce serum cholesterol and glycerine
Three esters are horizontal, and fatty liver is inhibited the different physiological roles such as to form.In addition, EPS is often by as stabilizer, thickener, gel
Agent and emulsifier etc. and applied in food additives.
Space microorganism is due to by space microgravity effect, high vacuum, extreme difference in temperature, low-intensity magnetic field and high energy particle (electricity
Son, proton, heavy ion) mutagenesis such as radiation, it is remarkably improved the frequency of mutation and producer is mutated, its biology will be caused
Character (such as individual morphology, colony characteristics, physio-biochemical characteristics, immunogenicity), fermenting and producing performance (such as biomass, product
Amount, enzyme activity, potency, fermenting speed etc.) it changes.It is carried using No. 11 spaceships of Heavenly Palace 2 and divine boat and returns to ground
Lactobacillus plantarum after space flight is compared with the lactobacillus plantarum that ground is original, selects the height that forward mutation assay occurs
The bacterial strain of EPS is produced, inheritance stability is recycled and the higher spatial plant lactobacillus detections of production EPS is produced under optimization of fermentation conditions
The antioxidant activity and gastrointestinal tract inverse ring border resistance characteristics of EPS carries for applications of the EPS in improving biological antioxidant activity
For practical basis.At present domestic and foreign scholars to the research of space microorganism be concentrated mainly on space pathogen, space corrosion bacterium and
In terms of microbiological pharmacy, and the research of space food microorganisms engineering bacteria is in space state.
Invention content
Resist the object of the present invention is to provide a kind of extracellular polysaccharide spatial plant lactobacillus SS18-33 and its improving biology
Application in oxidation activity.
Lactobacillus plantarum SS18-33 provided by the invention is lactobacillus plantarum (Lactobacillus plantarum)
Fullarton-H-SS18-33, in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center
For CGMCC No.15151.
The present invention also provides a kind of microbial inoculum, the active constituent of the microbial inoculum is the lactobacillus plantarum
(Lactobacillus plantarum)Fullarton-H-SS18-33。
The purposes of the microbial inoculum can be to prepare exocellular polysaccharide.
The microbial inoculum can be by the lactobacillus plantarum (Lactobacillus plantarum) Fullarton-H-
The culture that SS18-33 is cultivated in MRS fluid nutrient mediums.
In above-mentioned microbial inoculum, the microbial inoculum can also include carrier.The carrier can be solid carrier or liquid-carrier.It is described
Solid carrier can be glycitols, protide or vitamin substances；The glycitols carrier can be extra large bath sugar, lactose, sucrose, wheat
At least one of bud dextrin, maltose, sucrose, fructose, mannitol and sorbierite；The Protion carrier be skimmed milk powder,
At least one of whey powder, yeast powder and casein；The vitamins carrier can be vitamin C and/or vitamin E.It is described
Liquid-carrier can be glycerine, vegetable oil or water.In the microbial inoculum, the active constituent can be with the living cells of culture, living cells
Zymotic fluid, cell culture filtrate or the form of cell and the mixture of filtrate exist.The dosage form of the composition can be
A variety of dosage forms, such as liquor, emulsion, suspending agent, pulvis, granule, wettable powder or water dispersible granules.
The present invention also provides the preparation method of exocellular polysaccharide, the method includes：Utilize the lactobacillus plantarum
Exocellular polysaccharide is prepared in (Lactobacillus plantarum) Fullarton-H-SS18-33.
The method may include cultivating the lactobacillus plantarum (Lactobacillus plantarum) Fullarton-H-
SS18-33 obtains zymotic fluid；Extraction obtains exocellular polysaccharide from the zymotic fluid.
The fermentation medium of the culture can be the MRS fluid nutrient mediums.The pH of the MRS fluid nutrient mediums can be
7.0。
The MRS fluid nutrient mediums are made of solute and solvent, and the solvent is distilled water, the solute and its concentration point
It is not：Casein peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, glucose 10g/L, sodium acetate 5g/L, dibasic ammonium citrate
2g/L, Tween-80 1mL/L, K2HPO4 2g/L、MgSO4·7H2O 0.2g/L、MnSO4·H2O0.05g/L。
The temperature of the culture can be 37 DEG C.The time of the culture can be 20 hours.It can be by institute when carrying out the culture
It states microbial inoculum to be seeded in the fermentation medium, the inoculum concentration of the microbial inoculum can be 3% (percent by volume).
Extraction obtains exocellular polysaccharide and may include from the zymotic fluid：The zymotic fluid is centrifuged, exocellular polysaccharide is made
Into in supernatant, supernatant is collected, exocellular polysaccharide is obtained from the supernatant.
It is above-mentioned from obtaining exocellular polysaccharide in the supernatant and may also include to remove in the supernatant using trichloroacetic acid
Exocellular polysaccharide in supernatant described in 95% cold ethanol precipitation of impurity and utilization, obtains exocellular polysaccharide.
The method, which may additionally include, to be removed the impurity in the supernatant using trichloroacetic acid and utilizes 95% cold ethyl alcohol
It dialyses after precipitating the exocellular polysaccharide in the supernatant.
The present invention also provides the exocellular polysaccharides obtained using the method.
The present invention also provides the lactobacillus plantarum (Lactobacillus plantarum) Fullarton-H-
SS18-33, or, application of the microbial inoculum in preparing following any product：
A1) there is the product for the inhibition of lipid peroxidation function of improving organism or biological cell；
A2) there is the product for stablizing biological cell osmotic pressure function；
A3) there is the product for improving people and/or animal immune ability function；
A4) there is the functional product for protecting biological cell to be influenced from dehydration；
A5) there is antitumor and/or cancer cell specific induction of apoptosis function product；
A6) there is the product for promoting people and/or microbial population of animal intestinal tract equilibrium function；
A7) there is the product for reducing people and/or animal blood serum cholesterol and/or triglyceride levels function；
A8) have and people and/or animal tallow liver is inhibited to form the product of function；
A9) there is the product of tolerance people and/or animal gastrointestinal tract against environmental functional；
A10) stabilizer；
A11) thickener；
A12) gelling agent；
A13) emulsifier.
The present invention also provides the lactobacillus plantarum (Lactobacillus plantarum) Fullarton-H-
SS18-33, or, the microbial inoculum, or, the lactobacillus plantarum (Lactobacillus plantarum) Fullarton-H-
Following any applications of the exocellular polysaccharide of SS18-33 secretions：
B1 the inhibition of lipid peroxidation of organism or biological cell) is improved；
B2) stablize biological cell osmotic pressure；
B3 people and/or animal immune ability) are improved；
B4 influence of the biological cell from dehydration) is protected；
B5) antitumor and/or cancer cell specific induction of apoptosis；
B6) promote people and/or microbial population of animal intestinal tract balance；
B7 people and/or animal blood serum cholesterol and/or triglyceride levels) are reduced；
B8) people and/or animal tallow liver is inhibited to be formed；
B9 people and/or animal gastrointestinal tract inverse ring border) are resistant to；
B10) it is used as stabilizer；
B11) it is used as thickener；
B12) it is used as gelling agent；
B13) it is used as emulsifier.
The present invention also provides the product with following any function, the active constituent of the product is the plant breast bar
Bacterium (Lactobacillus plantarum) Fullarton-H-SS18-33 or described microbial inoculums or the lactobacillus plantarum
The exocellular polysaccharide of (Lactobacillus plantarum) Fullarton-H-SS18-33 secretions：
B1 the inhibition of lipid peroxidation of organism or biological cell) is improved；
B2) stablize biological cell osmotic pressure；
B3 people and/or animal immune ability) are improved；
B4 influence of the biological cell from dehydration) is protected；
B5) antitumor and/or cancer cell specific induction of apoptosis；
B6) promote people and/or microbial population of animal intestinal tract balance；
B7 people and/or animal blood serum cholesterol and/or triglyceride levels) are reduced；
B8) people and/or animal tallow liver is inhibited to be formed；
B9 people and/or animal gastrointestinal tract inverse ring border) are resistant to.
In the present invention, the antioxidant activity may be embodied in DPPH Scavenging activities, to ultra-oxygen anion free radical
(O2 -) Scavenging activity, to Fe2+Sequestering power and/or the total also proper energy for blocking peroxide to be formed to providing hydrogen atom substance
Power or total antioxidant capacity.
In the present invention, the biology can be human or animal, and the biological cell can be people and/or zooblast.
The present invention carries the plant breast bar for returning to ground after space flight using No. 11 spaceships of Heavenly Palace 2 and divine boat
Bacterium is compared with the lactobacillus plantarum that ground is original, selects the spatial plant lactobacillus for the high yield EPS that forward mutation assay occurs
SS18-33 bacterial strains, the bacterial strain inheritance stability and production EPS amount height, and its produced EPS's has high anti-oxidation activity：Spatial plant
The produced EPS of lactobacillus SS18-33 are 78.80% to DPPH clearance rates in a concentration of 2mg/mL；In a concentration of 10mg/mL,
To ultra-oxygen anion free radical (O2 -) clearance rate be 35.70%；In a concentration of 10mg/mL, Fe2+Sequestering power is
36.30%；In a concentration of 4mg/mL, total reducing power absorbance value (A700nm) it is 0.5140.In addition, the bacterial strain also has stomach and intestine
Road inverse ring border resistance characteristics provide practical basis for applications of the EPS in improving biological antioxidant activity.The present invention has filled up sky
Between food microorganisms engineering bacteria research blank.
Biomaterial preservation explanation
The Classification And Nomenclature of biomaterial：Lactobacillus plantarum (Lactobacillus plantarum)
The strain number of biomaterial：Fullarton-H-SS18-33
Depositary institution's title of biomaterial：China Committee for Culture Collection of Microorganisms's common micro-organisms center
The depositary institution of biomaterial is referred to as：CGMCC
The depositary institution address of biomaterial：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism
Research institute, postcode：100101
The preservation date of biomaterial：On 01 02nd, 2018
The collection of biomaterial is registered on the books number：CGMCC No.15151
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
MRS fluid nutrient mediums in following embodiments are made of solute and solvent, and solvent is distilled water, solute and its concentration
Respectively：Casein peptone 10g/L, beef extract 10g/L, yeast powder 5g/L, glucose 10g/L, sodium acetate 5g/L, dibasic ammonium citrate
2g/L, Tween-80 1mL/L, K2HPO4 2g/L、MgSO4·7H2O 0.2g/L、MnSO4·H2O0.05g/L, pH 7.0.MRS
Solid medium is the culture medium that agar powder 1.7g/L is added into MRS fluid nutrient mediums and obtains.
Embodiment 1, the screening to the spatial plant lactobacillus strain of high yield EPS
(1) bacterial strain isolates and purifies
1, bacterial strain
Spatial plant lactobacillus (Lactobacillus plantarum) SS18：By floor vegetation lactobacillus
(Lactobacillus plantarum) GS18 (China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation
Number：CGMCC 1.485) the space flight strain through No. 11 Spaceship Carryings return of Heavenly Palace 2 and Divine Land, it is preserved in Fuller and gives birth suddenly
Object engineering science and technology (Beijing) Co., Ltd space flight microorganism fungus kind library.In order to distinguish spaceship-carried front and back strain differences, by ground
Space flight bacterial strain after face lactobacillus plantarum (Lactobacillus plantarum) GS18 is carried is denoted as spatial plant breast
Bacillus (Lactobacillus plantarum) SS18.
2, bacterial strain activates
It will freeze in the floor vegetation lactobacillus GS18 and spatial plant lactobacillus SS18 glycerol stocks pipes point of -80 DEG C of refrigerator-freezers
An not be in 2%~3% access 5mL MRS fluid nutrient medium first, 37 DEG C of culture 16h are used for follow-up test after continuous three generations activation.
3, bacterial strain isolates and purifies
Floor vegetation lactobacillus GS18 is returned through Heavenly Palace 2 and the Spaceship Carrying of Divine Land 11, obtains space flight strain
SS18 is control with the individual morphology of floor vegetation lactobacillus GS18 and colony characteristics, from space in MRS solid medium first
It isolates and purifies to obtain 120 plants of bacterial strains in mutagenic species SS18, labeled strain code name is SS18-1 to SS18-120 respectively.
Isolation and purification method：The culture solution 1mL after space S S18 actication of culture is taken, with 9mL sterile salines+0.1%
Tween 80 prepares dilution bacterium solution, after whirlpool fully vibrates, is inoculated in MRS solid medium first tablets with plate streak, is placed in
Picking stickiness is higher after 37 DEG C of cultures, and the single bacterium colony purifying to differ greatly with the colony characteristics of ground G S18 strains is incubated at
MRS solid medium first slant tubes, and microscopy observation individual morphology and purity.By culture switching continuous passage culture, protect
Kind is spare.
MRS fluid nutrient medium first is that the pH of MRS fluid nutrient mediums is replaced with 6.5 obtained culture mediums by 7.0.MRS is solid
Body culture medium first is the solid medium that 17g/L agar powders are added into MRS fluid nutrient medium first and obtain.
(2) selection and breeding of spatial plant lactobacillus high-yield extracellular polysaccharide strains
1, the fermentation test of spatial plant lactobacillus production EPS
Above-mentioned 120 isolated and purified plant spatial plant lactobacillus slant culture is inoculated in MRS fluid nutrient mediums respectively
In, Zengjing Granule activated for 3~4 generations, then was transferred in 10mL MRS fluid nutrient mediums with 2% inoculum concentration, and 37 DEG C of culture 14h are obtained
The zymotic fluid of EPS is produced to spatial plant lactobacillus.
2, in spatial plant breast bar zymotic fluid EPS extraction
Extract the EPS in above-mentioned 120 plants of spatial plants lactobacillus ferment liquid respectively by the following method：Zymotic fluid is transferred to
In 10mL centrifuge tubes (centrifuge tube is intended to sterilizing rear and can be used), use refrigerated centrifuge with 8000r/min, 4 DEG C of centrifugations
10min collects supernatant；The 10g/100mL trichloroacetic acids that the pH5.0 for being 1/5 volume of supernatant is added into supernatant are water-soluble
Liquid obtains treatment fluid in 4 DEG C of oscillation treatment 30min；Treatment fluid is collected into supernatant with 10000r/min, 4 DEG C of centrifugation 10min；
Into supernatant, addition is that 95% cold ethanol water of 3 times of volumes of supernatant handles 11~12h in 4 DEG C, centrifuges (10000r/
Min, 4 DEG C of centrifugation 10min), collect the polysaccharide material of precipitation；Polysaccharide material (is added into the amount of distilled water with sterile distilled water dissolving
For the half of Preliminary fermentation liquid product), then with pretreated bag filter (8000~14000Da of molecular cut off) in 4 DEG C of dialysis
12h (period changes 3 sterile distilled waters), dialyzate is polysaccharide extraction liquid.
Wherein, the model CR3i of CR3i refrigerated centrifuges, the production of Thermo companies of the U.S..
3, the making of glucose standard curve
Standard glucose 100mg accurately is weighed in 500mL volumetric flasks, adds water constant volume.Various reagents are measured as shown in table 1
The uniform hybrid manipulation in colorimetric cylinder stands 10min, shakes up, be placed at room temperature for after 20min in 490nm measure EPS light absorption values (with
2.0mL distilled water is blank tune 0 by same color operation).With glucose content (100mg/L) for abscissa, absorbance
(A490nm) be ordinate draw standard curve be y=0.5985x+0.0223 (R2=0.9991).
1 phend-sulphuric acid of table surveys polysaccharide standard curve parameter list
4, the measurement of sample EPS contents
The sugar-containing concentration method of polysaccharide extraction liquid is measured using phend-sulphuric acid：The polysaccharide extraction liquid after dialysis is taken to steam
Its constant volume to 10mL is taken 2mL in 25mL colorimetric cylinders by distilled water, and 6% phenol 2mL is added into colorimetric cylinder, is rapidly added dense
Sulfuric acid 10mL after mixing, is stored at room temperature 20min, and same color operation is pressed as blank tune 0, using ultraviolet point using 2.0mL distilled water
Light photometer measures EPS absorbance values (A at 490nm490nm), sample absorbance y values will be measured and substitute into glucose standard curve
Regression equation y=0.5985x+0.0223 (R2=0.9991) to get to polyoses content x values (g/L) in, EPS productions are screened accordingly
Measure higher spatial plant lactobacillus strain.Spatial plant lactobacillus extracellular polysaccharide A490nmTesting result is as shown in table 2.
Wherein, the model UV-2600 of ultraviolet specrophotometer, the production of unico companies of the U.S..
The Polysaccharide A of 2 spatial plant lactobacillus exocellular polysaccharide of table490nmTesting result
As can be seen from Table 2, the lactobacillus plantarum GS18 original with ground extracellular polysaccharide A490nmIt compares, produces for the first time
Sugar fermentating test filters out the higher 23 bacterial strain spatial plant lactobacillus of EPS yield, and sugar fermentation verification test sieve is produced through second
Select output of sugar higher SS18-4, SS18-15, SS18-23, SS18-24, SS18-30, SS18-33, SS18-37, SS18-
82, SS18-116, SS18-117, SS18-118, SS18-119 spatial plant lactobacillus illustrates 12 plants of spatial plant lactobacillus
Direct mutation has occurred in related gene under the conditions of space environment.Wherein SS18-33 bacterial strains extracellular polysaccharide A490nmHighest,
It is secondary be SS18-119 bacterial strains extracellular polysaccharide A490nmIt is higher.
(3) genetic stability of spatial plant lactobacillus strain
By above-mentioned reinspection card screening obtain the higher lactobacillus reuteri SS18-4, SS18-15 of production EPS, SS18-23,
SS18-24, SS18-30, SS18-33, SS18-37, SS18-82, SS18-116, SS18-117, SS18-118, SS18-119 are empty
Between lactobacillus plantarum, in 50 generation of 5mL MRS fluid nutrient mediums continuous passage (inoculum concentration 2%~3%, 37 DEG C culture 16h), then
The fermentation test that spatial plant lactobacillus produces EPS is carried out by step (2), extracts the EPS in zymotic fluid, and use phenolsulfuric acid
Method measures the sugar content (A of EPS extracting solutions490nm), the results are shown in Table 3.Sample absorbance y values will be measured and substitute into Glucose standards
Regression Equations y=0.5985x+0.0223 (R2=0.9991) to get to polysaccharide yield x values (g/L) in, as a result such as table 4
It is shown.
The A of the spatial plant lactobacillus exocellular polysaccharide of 3 50 generation of table passage490nmAs a result
The yield result of the spatial plant lactobacillus exocellular polysaccharide of 4 50 generation of table passage
By table 3 and table 4 as it can be seen that after 50 generations passed on, by production EPS verification tests of fermenting three times, 12 plants of spatial plant breasts
Although the EPS yield of bar is higher than ground G S18 bacterial strains, wherein there are 3 plants of bacterium (SS18-4, SS18-23, SS18-117 bacterium
Strain) EPS A490nmThere is different degrees of decline, the Polysaccharide A of remaining 9 plants of bacterium490nmNumerical value obviously higher than ground G S18 bacterial strains,
And the A of EPS490nmIt is basicly stable.Wherein SS18-119 bacterial strains output of sugar highest, polysaccharide crude yield are 2.0g/L, compare ground
GS18 bacterial strains improve 53.85%；Followed by SS18-33 bacterial strains, polysaccharide crude yield are 1.9g/L, are carried than ground G S18 bacterial strains
It is high by 46.15%.Show that 2 bacterial strain is fermentation production EPS higher and the preferable bacterial strain of inheritance stability.
Conclusion：Spatial plant lactobacillus SS18-33 is best for genetic stability, and the extracellular higher bacterium of polysaccharide of fermenting
Strain can become prebiotic food of fungi most potential bacterial strain of the exploitation with antioxidant activity.
(4) Morphological Identification of space lactobacillus reuteri is identified with molecules
SS18-33 bacterial strains are subjected to Gram's staining, as a result show that SS18-33 bacterial strains are gram-positive bacteria.Morphology
The thalline of observation, SS18-33 bacterial strains is in rod-short or club-shaped, single or short catenation；In MRS solid medium tablets
Bacterium colony size be 1~2mm, canescence, the smooth moistening in translucent, flat, surface, edge are irregular.
The 16S rDNA of SS18-33 bacterial strains are detected, sequencing result is as shown in the sequence 1 of sequence table.16s rDNA identification knots
Fruit shows that SS18-33 bacterial strains and the similitude of lactobacillus plantarum Lactobacillus plantarum reach 100%.By shape
State and 16S rDNA identifications, it may be determined that SS18-33 bacterial strains belong to lactobacillus plantarum.
(5) preservation of spatial plant lactobacillus SS18-33
It is lactobacillus plantarum (Lactobacillus plantarum) Fullarton-H- by SS18-33 Strain Designations
SS18-33 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 01 02nd, 2018 (referred to as
CGMCC；Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；Postcode：100101),
Deposit number is CGMCC NO.15151.Lactobacillus plantarum (Lactobacillusplantarum) Fullarton-H-SS18-
33 referred to as spatial plant lactobacillus SS18-33.
The antioxidant activity detection of embodiment 2, spatial plant lactobacillus SS18-33 productions EPS
(1) preparation of spatial plant lactobacillus SS18-33 leavenings
Spatial plant lactobacillus SS18-33 is activated in MRS fluid nutrient mediums, the MRS test tubes after being activated
MRS tube cultures after activation are filled 100mL sterilizing MRS liquid by culture with the immigration of 3% (percentage by volume) inoculum concentration
In the triangular flask of culture medium, after 37 DEG C of culture 16h, spatial plant lactobacillus SS18-33 leavenings are obtained.The hollow inter-planting of leavening
The number of viable of object lactobacillus SS18-33 is (2.0~5.0) × 109CFU/mL。
(2) spatial plant lactobacillus SS18-33 produces the fermentation test of EPS
By spatial plant lactobacillus SS18-33 leavenings by 3% (percentage by volume) inoculum concentration access 1000mL liquid MRS
In fluid nutrient medium (it is 7.0 that culture medium, which originates pH), production EPS fermentation 20h are carried out in 37 DEG C, obtain zymotic fluid.
(3) preparation of the EPS crude products of spatial plant lactobacillus SS18-33
The zymotic fluid of step (2) large capacity tabletop refrigerated centrifuge is centrifuged, with 8000r/min, 4 DEG C of centrifugations
10min collects supernatant；The 10g/100mL trichloroacetic acids that the pH5.0 for being 1/5 volume of supernatant is added into supernatant are water-soluble
Liquid obtains treatment fluid in 4 DEG C of oscillation treatment 30min；Treatment fluid is collected into supernatant with 10000r/min, 4 DEG C of centrifugation 10min；
Into supernatant, addition is that 95% cold ethyl alcohol of 3 times of volumes of supernatant handles 11~12h in 4 DEG C, centrifuge (10000r/min, 4 DEG C
Centrifuge 10min), collect the precipitation containing polysaccharide material；Precipitation is dissolved with sterile distilled water, then with pretreated bag filter profit
With sterile distilled water dialysed overnight (period changes 3 sterile distilled waters), obtained dialyzate is polysaccharide extraction liquid；By 50~
100mL polysaccharide extraction liquids inject in the aseptic freeze-dried bottles of 500mL, prior to -35 DEG C under the conditions of pre-freeze to frozen state, then utilize true
Under conditions of -55 DEG C, 0.16~0.18mBar of vacuum degree, 36~72h is lyophilized in the EPS extracting solutions of pre-freeze by vacuum freecing-dry machine
To completely dried state, white (or the slightly pale yellow) crystalline state of EPS crude products after drying.
Wherein, the model Biofuge stratos of large capacity tabletop refrigerated centrifuge, the production of power ＆ light company of the U.S.；Very
The model 6L LABCONCO of vacuum freecing-dry machine, it is produced in USA.
(4) antioxidant activity of the EPS of detection spatial plant lactobacillus SS18-33
1, measurement of the EPS of spatial plant lactobacillus SS18-33 to DPPH Scavenging activities
The DPPH ethanol solutions that a concentration of 0.01mmol/L of DPPH are prepared with absolute ethyl alcohol, take 2mL various concentrations respectively
EPS solution [EPS crude products that step (3) obtains are configured to a concentration of 0 with deionized water, 0.4,0.8,1.2,1.6,2.0mg/
The EPS solution of mL], DPPH solution 2mL are added into the EPS solution of each concentration, dark reacts down 30min after mixing, with nothing
For water-ethanol as blank control tune 0, it is the absorbance value (A at 517nm to measure wavelength517nm).It is each in experiment that concentration is parallel does 3
It is secondary, its average value is taken, calculates clearance rate as follows, the results are shown in Table 5.
In formula：AiFor EPS solution+DPPH ethanol solution absorbance values；
AjFor EPS solution+absolute ethyl alcohol absorbance value；
AoFor DPPH ethanol solutions+distilled water absorbance value.
Clearance rates of 5 the produced EPS of SS18-33 bacterial strains of table to DPPH
DPPH (1,1- diphenyl -2- trinitrophenyl-hydrazines) free radical is a kind of artificial synthesized, the stabilization centered on nitrogen
Organic compound, ethanol solution is dark purple, and has maximum absorption band in 515~520nm ranges, by free radical scavenger plus
Enter wherein, DPPH ethanol solutions become light yellow or colourless from darkviolet, therefore can be clear to free radical by the variation of absorbance
Except agent carries out quantitative analysis.As shown in Table 5, the ability of the produced EPS removings DPPH of spatial plant lactobacillus SS18-33 and polysaccharide are dense
Degree, which is positively correlated, is, as a concentration of 2mg/mL of EPS, DPPH clearance rates are 78.80%.As it can be seen that spatial plant lactobacillus SS18-
33 produced EPS are in higher level to the Scavenging activity of DPPH free radicals.
2, the EPS of spatial plant lactobacillus SS18-33 is to ultra-oxygen anion free radical (O2 -) Scavenging activity measurement
Being separately added into the EPS solutions of 1mL various concentrations in test tube, [the EPS crude products for obtaining step (3) are with deionization
Water is configured to the solution of a concentration of 0,2,4,6,8,10mg/mL], the super oxygen measured by the following method under different EPS concentration is cloudy
Ion radical (O2 -) Scavenging activity：It is added pH8.2 into 1mL EPS solutions, the Tris- hydrochloride buffers of 0.05mol/L,
10min is kept the temperature in 25 DEG C of water-baths, the pyrogallol of the 30mmol/L of same pre-temperature heat treatment is then added into reaction system again
12 μ L of aqueous solution accurately react 4min after mixing, are terminated and are reacted with 0.5mL concentrated hydrochloric acids, using Tris- hydrochloride buffers as blank
Control adjusts 0, measures the absorbance value (A at 320nm320nm), clearance rate is calculated as follows, and the results are shown in Table 6.
In formula：AiThe absorbance value after EPS solution is added for reaction system (Tris- hydrochloric acid+pyrogallol)；
AjFor the absorbance value of EPS solution；
AoFor the absorbance value of reaction system (Tris- hydrochloric acid+pyrogallol).
Clearance rates of 6 the produced EPS of SS18-33 bacterial strains of table to ultra-oxygen anion free radical
Ultra-oxygen anion free radical (O2 -) in organism be all active oxygen radicals precursor, can be converted into other
The active oxygen radical and body injury to organism, aging and disease have significant impact.As shown in Table 6, spatial plant breast bar
The ability that the produced EPS of bacterium SS18-33 remove ultra-oxygen anion free radical increases with the raising of polysaccharide concentration, works as polysaccharide concentration
For 10mg/mL when, be 75.60% to the clearance rate of ultra-oxygen anion free radical.As it can be seen that spatial plant lactobacillus SS18-33 institutes
Produce EPS has good Scavenging activity to ultra-oxygen anion free radical.
3, the EPS of spatial plant lactobacillus SS18-33 is to Fe2+Sequestering power measures
The EPS solution of 3mL various concentrations is taken [to be configured to deionized water EPS crude products that step (3) obtains dense respectively
Degree is the solution of 0,2,4,6,8,10mg/mL], the Fe under different EPS concentration is measured by the following method2+Sequestering power：To 3mL
The 5mmol/L phenanthrene hello piperazine aqueous solutions of the 2mmol/L ferrous chloride aqueous solutions and 0.2mL of 0.05mL are added in EPS solution, through oscillation
10min is stored at room temperature after mixing.Using distilled water as blank control tune 0, reaction system after reaction is measured at 562nm
Absorbance value (A562nm), the Fe of the produced EPS of spatial plant lactobacillus SS18-33 is calculated as follows2+Sequestering power, knot
Fruit is as shown in table 7.
In formula：AoFor the absorbance value (substituting EPS solution using distilled water) of control group；
AiFor the absorbance value of sample；
AjFor the absorbance value (substituting solution of ferrous chloride using distilled water) of blank group.
7 the produced EPS of SS18-33 bacterial strains of table is to Fe2+Sequestering power
Metal ion energy inducing lipids peroxidating in body causes to damage to body biomembrane and nucleic acid, therefore chelates
Fe2+Ability be also an important Antioxidant Indexes.As shown in Table 7, the Fe of the produced EPS of spatial plant lactobacillus SS18-332 +Sequestering power is also positively correlated with polysaccharide concentration, when polysaccharide concentration is 10mg/mL, Fe2+Sequestering power is 36.30%.By
This is as it can be seen that the produced EPS of spatial plant lactobacillus SS18-33 have chelating Fe2+It is certain anti-to show that it has when concentration is higher for ability
Oxidation.
4, measurement of the EPS of spatial plant lactobacillus SS18-33 to total reducing power (total antioxidant capacity)
The EPS solution of 1mL various concentrations is taken [to be configured to deionized water EPS crude products that step (3) obtains dense respectively
Degree is the solution of 0,2,4,6,8,10mg/mL], total reducing power under different EPS concentration is measured by the following method：To 1mL
The phosphate buffer 2.5mL and mass fraction that addition 0.2mol/L pH are 6.6 in EPS solution are 1% potassium ferricyanide solution
2.5mL, 50 DEG C of heat preservation 20min of water-bath add 10g/100mL trichloroacetic acid solutions after flowing water cools down after mixing
2.5mL obtains mixed liquor；Supernatant 2.5mL is taken after mixed liquor is centrifuged 10min with 3000r/min, into 2.5mL supernatants
It is 0.1% ferric chloride solution that 2.5mL distilled water and 2.5mL mass fractions, which is added, and mixing is stored at room temperature 10min, is with distilled water
The sample of no reducing power measures the absorbance value (A at 700nm as blank control tune 0700nm), the results are shown in Table 8.
The total reducing powers of the produced EPS of 8 SS18-33 of table
Total reducing power is to measure an important indicator of oxidation resistance, and the substance with reducing power can be by providing hydrogen
Atom blocks peroxide to be formed, to make free chain reaction that can not carry out.As shown in Table 8, spatial plant lactobacillus
Total reducing power of the produced EPS of SS18-33, which is also positively correlated with polysaccharide concentration, is, total to restore when polysaccharide concentration is 10mg/mL
Ability absorbance value (A700nm) it is 0.5140.Wavelength is that 700nm absorbance values are higher, shows that total antioxidant capacity is better, thus
As it can be seen that the total antioxidant capacity of the EPS of spatial plant lactobacillus SS18-33 enhances with the raising of polysaccharide concentration.
In conclusion Study on Its Antioxidant Activity in Vitro shows the produced EPS of spatial plant lactobacillus SS18-33 a concentration of
It is 78.80% to DPPH clearance rates when 2mg/mL；In a concentration of 10mg/mL, to ultra-oxygen anion free radical (O2 -) remove
Rate is 35.70%；In a concentration of 10mg/mL, Fe2+Sequestering power is 36.30%；In a concentration of 4mg/mL, total reducing power
Absorbance value (A700nm) it is 0.5140, display space lactobacillus plantarum SS18-33 has stronger antioxidant activity.
Embodiment 3, the spatial plant resistance to gastrointestinal tract of lactobacillus SS18-33 bacterial strains are against environmental test
Artificial simulation gastric juices：0.2g/100mL containing NaCl, pepsin (enzyme activity 250U/mg) 0.3g/100mL are used
1mol/L HCl tune pH to 3.0, surplus are deionized water, spare after 0.22 μm of membrane filtration degerming.
Artificial simulation intestinal juice：Trypsase (enzyme activity 1655U/mg) is dissolved in 0.1mol/L phosphate buffers (pH
8.0) in, make its final concentration of 1g/L, bovine bile is added and makes its a concentration of 0.3g/100mL, through 0.22 μm of membrane filtration degerming
It is spare afterwards.
Strain to be tested：Floor vegetation lactobacillus GS18 and spatial plant lactobacillus SS18-33.
1, strain to be tested is inoculated in 10mL MRS fluid nutrient mediums (pH 7.0), 37 DEG C, stationary culture 18h (thalline
A concentration of 2.0 × 109CFU/mL~8.0 × 109CFU/mL), 6000r/min centrifuges 10min and collects precipitation.
2, the precipitation obtained using 0.85% brine steps 1 of 10mL, 6000r/min, which centrifuges 10min and collects, to sink
It forms sediment.
3, be resuspended the obtained precipitation of step 2 using 1mL artificial simulation gastric juices, 37 DEG C, stationary culture, respectively at 0h and 3h into
Row viable bacteria counting number simultaneously counts survival rate, as a result as shown in table 9 and table 1.
4, after completing step 3, whole system is taken, 6000r/min centrifuges 10min and collects precipitation, using 9mL manual simulation's intestines
Precipitation is resuspended in liquid, and 37 DEG C of stationary cultures carry out viable bacteria counting number respectively at 0h and 8h and count survival rate, as a result such as table 9 and table
Shown in 10.
Viable bacteria counting number and survival rate computational methods are：Take the incremental dilution of 10 times of 1mL bacterium solution 9mL sterile salines
Afterwards, wherein 2~3 suitable dilution bacterium solutions are taken, are inoculated on MRS solid mediums with tilt-pour process, to life after 37 DEG C of culture 48h
Long bacterium colony is counted, and counts viable count, and calculate survival rate as follows.
Strain survival rate (%)=[log N1/log N0]×100
In formula：N1=by simulate the gastric juice or intestinal juice treated viable count (CFU/mL)
N0=untreated preceding viable count (CFU/mL)
The 9 spatial plant resistance to gastrointestinal tract of lactobacillus SS18-33 bacterial strains of table is against environmental test viable count result
As shown in Table 9, spatial plant lactobacillus SS18-33 bacterial strains by the viable count after artificial simulation gastric juices be (3.7 ±
0.13)×109CFU/mL；It is (7.8 ± 0.79) × 10 by the viable count after artificial simulation intestinal juice7CFU/mL.Spatial plant breast
Bacillus SS18-33 bacterial strains are more than 10 by the viable count of stomach and intestine simulated solution6CFU/mL shows spatial plant lactobacillus SS18-33
Bacterial strain can pass through gastrointestinal tract and play health-care efficacy.
The 10 spatial plant resistance to gastrointestinal tract of lactobacillus SS18-33 bacterial strains of table is against environmental test survival results
As shown in Table 10, spatial plant lactobacillus SS18-33 bacterial strains survival rate after artificial simulation gastric juices 3h is
92.28%, 13.50% is improved compared with floor vegetation lactobacillus GS18 bacterial strains, survival rate is after artificial simulation intestinal juice 8h
82.46%, improve 9.89% compared with the bacterial strain GS18 of ground.Display space lactobacillus plantarum SS18-33 bacterial strains are resistant to stomach and intestine
Road is stronger against environment capacity.
<110>Fuller gives birth suddenly object engineering science and technology（Beijing）Co., Ltd
<120>A kind of extracellular polysaccharide spatial plant lactobacillus SS18-33 and its application in improving biological antioxidant activity
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 2206
<212> DNA
<213>Lactobacillus plantarum（Lactobacillus plantarum）
<400> 1
agtgatgggg ggtgctatac atgcagtcga acgaactctg gtattgattg gtgcttgcat 60
catgatttac atttgagtga gtggcgaact ggtgagtaac acgtgggaaa cctgcccaga 120
agcgggggat aacacctgga aacagatgct aataccgcat aacaacttgg accgcatggt 180
ccgagtttga aagatggctt cggctatcac ttttggatgg tcccgcggcg tattagctag 240
atggtggggt aacggctcac catggcaatg atacgtagcc gacctgagag ggtaatcggc 300
cacattggga ctgagacacg gcccaaactc ctacgggagg cagcagtagg gaatcttcca 360
caatggacga aagtctgatg gagcaacgcc gcgtgagtga agaagggttt cggctcgtaa 420
aactctgttg ttaaagaaga acatatctga gagtaactgt tcaggtattg acggtattta 480
accagaaagc cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt 540
tgtccggatt tattgggcgt aaagcgagcg caggcggttt tttaagtctg atgtgaaagc 600
cttcggctca accgaagaag tgcatcggaa actgggaaac ttgagtgcag aagaggacag 660
tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca gtggcgaagg 720
cggctgtctg gtctgtaact gacgctgagg ctcgaaagta tgggtagcaa acaggattag 780
ataccctggt agtccatacc gtaaacgatg aatgctaagt gttggagggt ttccgccctt 840
cagtgctgca gctaacgcat taagcattcc gcctggggag tacggccgca aggctgaaac 900
tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagctac 960
gcgaagaacc ttaccaggtc ttgacatact atgcaaatct aagagattag acgttccctt 1020
cggggacatg gatacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1080
taagtcccgc aacgagcgca acccctaata acattgttca cttacgcggc tggttcctaa 1140
aaggttaccc caccgacttt gggtgttaca aactctcatg gtgtgacggg cggtgtgtac 1200
aaggcccggg aacgtattca ccgcggcatg ctgatccgcg attactagcg attccgactt 1260
catgtaggcg agttgcagcc tacaatccga actgagaatg gctttaagag attagcttac 1320
tctcgcgagt tcgcaactcg ttgtaccatc cattgtagca cgtgtgtagc ccaggtcata 1380
aggggcatga tgatttgacg tcatccccac cttcctccgg tttgtcaccg gcagtctcac 1440
cagagtgccc aacttaatgc tggcaactga taataagggt tgcgctcgtt gcgggactta 1500
acccaacatc tcacgacacg agctgacgac aaccatgcac cacctgtatc catgtccccg 1560
aagggaacgt ctaatctctt agatttgcat agtatgtcaa gacctggtaa ggttcttcgc 1620
gtagcttcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca attcctttga 1680
gtttcagcct tgcggccgta ctccccaggc ggaatgctta atgcgttagc tgcagcactg 1740
aagggcggaa accctccaac acttagcatt catcgtttac ggtatggact accagggtat 1800
ctaatcctgt ttgctaccca tactttcgag cctcagcgtc agttacagac cagacagccg 1860
ccttcgccac tggtgttctt ccatatatct acgcatttca ccgctacaca tggagttcca 1920
ctgtcctctt ctgcactcaa gtttcccagt ttccgatgca cttcttcggt tgagccgaag 1980
gctttcacat cagacttaaa aaaccgcctg cgctcgcttt acgcccaata aatccggaca 2040
acgcttgcca cctacgtatt accgcggctg ctggcacgta gttagccgtg gctttctggt 2100
taaataccgt caatacctga acagttactc tcagatatgt tcttctttaa caacagagtt 2160
ttacgagccg aaacccttct tcactcacgc ggcgttgctc cctcgg 2206
Claims (10)
- Lactobacillus plantarum 1. (Lactobacillus plantarum) Fullarton-H-SS18-33, in China Microbiological bacterium The deposit number of kind preservation administration committee common micro-organisms center is CGMCC No.15151.
- 2. a kind of microbial inoculum, active constituent is lactobacillus plantarum described in claim 1 (Lactobacillus plantarum) Fullarton-H-SS18-33。
- 3. microbial inoculum according to claim 2, it is characterised in that：The purposes of the microbial inoculum is to prepare exocellular polysaccharide.
- 4. the preparation method of exocellular polysaccharide, including：Utilize lactobacillus plantarum (Lactobacillus described in claim 1 Plantarum) exocellular polysaccharide is prepared in Fullarton-H-SS18-33.
- 5. according to the method described in claim 4, it is characterized in that：The method includes cultivating plant described in claim 1 Lactobacillus (Lactobacillus plantarum) Fullarton-H-SS18-33, obtains zymotic fluid；From the zymotic fluid Extraction obtains exocellular polysaccharide.
- 6. the exocellular polysaccharide obtained using the method described in claim 4 or 5.
- 7. lactobacillus plantarum (Lactobacillus plantarum) Fullarton-H-SS18-33 described in claim 1, Or, application of the microbial inoculum according to claim 2 or 3 in preparing following any product：A1) there is the product for the inhibition of lipid peroxidation function of improving organism or biological cell；A2) there is the product for stablizing biological cell osmotic pressure function；A3) there is the product for improving people and/or animal immune ability function；A4) there is the functional product for protecting biological cell to be influenced from dehydration；A5) there is antitumor and/or cancer cell specific induction of apoptosis function product；A6) there is the product for promoting people and/or microbial population of animal intestinal tract equilibrium function；A7) there is the product for reducing people and/or animal blood serum cholesterol and/or triglyceride levels function；A8) have and people and/or animal tallow liver is inhibited to form the product of function；A9) there is the product of tolerance people and/or animal gastrointestinal tract against environmental functional；A10) stabilizer；A11) thickener；A12) gelling agent；A13) emulsifier.
- 8. lactobacillus plantarum (Lactobacillus plantarum) Fullarton-H-SS18-33 described in claim 1, Or, microbial inoculum according to claim 2 or 3, or, lactobacillus plantarum (Lactobacillus described in claim 1 Plantarum) following any applications of the exocellular polysaccharide of Fullarton-H-SS18-33 secretions：B1 the inhibition of lipid peroxidation of organism or biological cell) is improved；B2) stablize biological cell osmotic pressure；B3 people and/or animal immune ability) are improved；B4 influence of the biological cell from dehydration) is protected；B5) antitumor and/or cancer cell specific induction of apoptosis；B6) promote people and/or microbial population of animal intestinal tract balance；B7 people and/or animal blood serum cholesterol and/or triglyceride levels) are reduced；B8) people and/or animal tallow liver is inhibited to be formed；B9 people and/or animal gastrointestinal tract inverse ring border) are resistant to；B10) it is used as stabilizer；B11) it is used as thickener；B12) it is used as gelling agent；B13) it is used as emulsifier.
- 9. the product with following any function, active constituent is lactobacillus plantarum described in claim 1 (Lactobacillus plantarum) Fullarton-H-SS18-33 or according to claim 2 or 3 microbial inoculums or right It is required that described in 1 lactobacillus plantarum (Lactobacillus plantarum) Fullarton-H-SS18-33 secretion it is extracellular more Sugar：B1 the inhibition of lipid peroxidation of organism or biological cell) is improved；B2) stablize biological cell osmotic pressure；B3 people and/or animal immune ability) are improved；B4 influence of the biological cell from dehydration) is protected；B5) antitumor and/or cancer cell specific induction of apoptosis；B6) promote people and/or microbial population of animal intestinal tract balance；B7 people and/or animal blood serum cholesterol and/or triglyceride levels) are reduced；B8) people and/or animal tallow liver is inhibited to be formed；B9 people and/or animal gastrointestinal tract inverse ring border) are resistant to.
- 10. the product described in application according to claim 7 or 8 or claim 9, it is characterised in that：It is described anti-oxidant Activity is embodied in DPPH Scavenging activities, to ultra-oxygen anion free radical (O2 -) Scavenging activity, to Fe2+Sequestering power and/or The total reducing power or total antioxidant capacity for blocking peroxide to be formed to providing hydrogen atom substance.
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| CN110272846A (en) * | 2019-07-08 | 2019-09-24 | 江苏省农业科学院 | Lactobacillus plantarum and its application with ABTS free radical and PTIO free radical scavenging activity |
| CN110846244A (en) * | 2019-10-15 | 2020-02-28 | 江南大学 | Lactobacillus with high extracellular polysaccharide yield and application thereof in yogurt production |
| CN111100810A (en) * | 2019-12-20 | 2020-05-05 | 华南农业大学 | Lactobacillus plantarum DNB1, and extracellular polysaccharide and application thereof |
| CN111100810B (en) * | 2019-12-20 | 2022-04-05 | 华南农业大学 | Lactobacillus plantarum DNB1, and extracellular polysaccharide and application thereof |
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