CN105859898B - A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity - Google Patents
A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity Download PDFInfo
- Publication number
- CN105859898B CN105859898B CN201610202526.7A CN201610202526A CN105859898B CN 105859898 B CN105859898 B CN 105859898B CN 201610202526 A CN201610202526 A CN 201610202526A CN 105859898 B CN105859898 B CN 105859898B
- Authority
- CN
- China
- Prior art keywords
- lactobacillus plantarum
- exocellular polysaccharide
- polysaccharide
- final concentration
- exocellular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention improves its negative electrical charge supply capacity on the basis of lactobacillus plantarum exocellular polysaccharide, by sulfonating reaction, with DPPH free radicals, OH‑Free radical, O2‑Free radical scavenging ability is evaluated, and gained sulfonated products have more preferably antioxidant activity in itself compared with lactobacillus plantarum exocellular polysaccharide.And this method of modifying applicability in all kinds of lactobacillus plantarum exocellular polysaccharides is wider.On this basis, unconspicuous problem is promoted for antioxidant activity after the lactobacillus plantarum exocellular polysaccharide sulfonating reaction of partial category, the further preferred raw material exocellular polysaccharide for being suitable for the method for modifying of the present invention, the sulfonating reaction of the polysaccharide is abundant, product is uniform, structure is clear and definite, while has prominent antioxidant activity.In addition, the polysaccharide raw material characterized with preparation method, yield, purity are higher, are conducive to subsequent modified-reaction.The method of the present invention flow is shorter, cost is relatively low, operates relative ease, meets the technological requirement of large-scale production, great promotion potential.
Description
Technical field
The present invention relates to carbohydrate chemistry technical field, further to the chemical modification technique of polysaccharide, and in particular to a kind of
Improve the chemical modification method of lactobacillus plantarum exocellular polysaccharide antioxidant activity.
Background technology
Exopolysaccharides Produced by Lactic Acid Bacteria (Exopolysaccharide, EPS), refers in lactic acid bacteria metabolic process and is secreted into cell
Outer saccharide compound.Since Exopolysaccharides Produced by Lactic Acid Bacteria has good rheological characteristic and can assign fermented food good mouth
Sense and flavor, therefore it can be widely used in breast at present as the gelling agent of food industry, stabilizer, antistaling agent and emulsifier etc.
The production of the food such as product, beverage, cake, candy, ice cream.In addition, some researches show that there is EPS antitumor, reduction courage to consolidate
Alcohol, anti-aging adjust the effects that intestinal flora balance, therefore EPS may be one of material base of lactic acid bacteria prebiotic effect.
Antioxidant activity refers to the ability that substance eliminates oxygen radical in human body.Some researches show that internal oxygen radical contains
It measures excessively high will generate cell directly, slowly to damage, organ function will be involved over time, accelerate body aging, even
Lead to lesion.Therefore if the substance with antioxidant activity can be taken in diet link, it will help it is free to reduce internal oxygen
Base content, plays prebiotic effect.
In the prior art, it is recognized that there is the health products master compared with strong anti-oxidative activity (more than ORAC value 3000umol TE/g)
Come from plant extracts, the tealeaves including blueberry extract, grape seed extract, high-quality propolis, high content tea polyphenol carries
Take object, resveratrol, Co-Q10, wolfberry fruit extract, emblic extract etc..Since the production cycle of plant product is longer, kind
The links such as plant, harvesting are complex, therefore such product cost is higher in the prior art.Different from plant product, microorganism
On the one hand culture is not limited by region, space, another aspect production efficiency is high, product easily harvests, culture and purifying process
It also allows for standardizing, so if a kind of microbe-derived anti-oxidation active substance can be developed, only consumer does not provide
A kind of new selection, while production cost will be significantly reduced.
Lactobacillus plantarum (Lactobacillus plantarum), belongs to homofermentative lactic bacteria, and Gram-positive is facultative
It is aerobic.Some researches show that the extracellular polysaccharide of strain institute has anti-pathogenic bacteria, adjusts intestinal flora balance and the works such as anti-oxidant
With, however test and find, the antioxidant activity of such exocellular polysaccharide in itself is generally relatively low, as antioxidant activity-based health care
Product, effect are difficult to ensure that.
Invention content
The present invention is directed to be directed to the technological deficiency of the prior art, a kind of raising lactobacillus plantarum exocellular polysaccharide antioxygen is provided
Change the chemical modification method of activity, asked with solving the relatively low technology of the lactobacillus plantarum exocellular polysaccharide antioxidant activity of the prior art
Topic.
Another technical problem to be solved by the present invention is that when using method of sulfonating improved plant lactobacillus exocellular polysaccharide, no
The sulfonation effect of generic lactobacillus plantarum exocellular polysaccharide is inhomogenous, partial category lactobacillus plantarum exocellular polysaccharide sulfonation again
Its antioxidant activity promotes unobvious afterwards.
The invention solves another technical problem be when using method of sulfonating improved plant lactobacillus exocellular polysaccharide when, fit
It is difficult to prepare in the polysaccharide of sulfonation.
The invention solves another technical problem be when using method of sulfonating improved plant lactobacillus exocellular polysaccharide when, fit
It is relatively low in the purity of polysaccharide of sulfonation.
For realization more than technical purpose, the present invention uses following technical scheme：
A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity, includes the following steps：
1) the lactobacillus plantarum exocellular polysaccharide is taken, is dissolved with anhydrous dimethyl formamide；
2) sulfur trioxide pyridine complex is added in after 15~45min thereto to final concentration of 5~15mg/mL, reaction 2~
4h。
Preferably, further include step 3)：It is dialysed obtained by step 2) using the bag filter that molecular cut off is 8~14KDa
Product takes bag filter content to dry.On this basis it is further preferred that being first adjusted to step 2) products therefrom pH before dialysis
6.5~7.5 dialyse again；More preferably, pH value is 7.0.On this basis it is further preferred that the adjusting of the pH is to utilize
What the NaOH solution of 4mol/L was realized.On this basis it is further preferred that the drying is freeze-drying.On this basis into
One step is preferred, and the duration of the dialysis is 4~6d；More preferably, the duration of dialysis is 5d.
Preferably, the temperature reacted described in step 2) is 75~85 DEG C, it is cooled to 20~30 DEG C after reaction；Its
In more preferably reaction temperature be 80 DEG C, more preferably cooling temperature is 25 DEG C.Preferred on this basis, the cooling procedure is logical
Cross what ice-water bath mode was realized.
Preferably, the lactobacillus plantarum exocellular polysaccharide being dissolved in step 1) in anhydrous dimethyl formamide is final concentration of
3~7mg/mL.
Preferably, lactobacillus plantarum exocellular polysaccharide be dissolved in anhydrous dimethyl formamide after, add in sulfur trioxide pyrrole
Before pyridine compound, solution is in concussion or stirring.
Preferably, in the step 2) reaction process, solution is in concussion or stirring.
Preferably, the lactobacillus plantarum exocellular polysaccharide is prepared by the following method：
A) lactobacillus plantarum zymotic fluid is taken, separation of solid and liquid takes supernatant；
B) ethyl alcohol, alcohol precipitation are added in into the step a) supernatants, separation of solid and liquid takes precipitation, and molecule is retained with 8~14KDa
The bag filter dialysis of amount, takes bag filter content to dry, as Thick many candies；
C) to contain 40~60mM Tris-HCl, 5~15mM MgSO4·7H2The solution of O is molten as Thick many candies lysate
Step b) the Thick many candies are solved, then add in nuclease DNAse type-I to 2~3 μ g/mL of final concentration, hydrolysis；
D) protease P ronase E to 40~60 μ g/mL of final concentration, enzymolysis are then added in；
E) trichloroacetic acid is then added in final concentration 10~15% (w/v), and separation of solid and liquid takes supernatant after 20~40min, thoroughly
Analysis, it is the lactobacillus plantarum exocellular polysaccharide to take bag filter content.
Preferably, the zymotic fluid when zymotic fluid described in step a) is lactobacillus plantarum 22~26h of anaerobic fermentation；It is more excellent
, fermentation time is for 24 hours.
Preferably, the addition of ethyl alcohol is 1.5~2.5 times of the supernatant volume in step b)；More preferably, ethyl alcohol
Addition is 2 times of of the supernatant volume
Preferably, in step c) Thick many candies final concentration of 4~6mg/L；More preferably, it is 5mg/L.
Preferably, the step c) hydrolysis is to continue 4~8h under the conditions of 35~39 DEG C；More preferably, it is in 37 DEG C of items
Continue 6h under part.
Preferably, the step d) enzymolysis is to continue 16~20h under the conditions of 35~39 DEG C；More preferably, it is at 37 DEG C
Under the conditions of continue 18h.
Preferably, before step e) dialysis, supernatant pH is first adjusted to 4~5, then dialyse；More preferably, pH to 4.5 is adjusted；
Optimal, described adjust is realized using the NaOH solution of 10mol/L.
Preferred on the basis of any of the above technical solution, the lactobacillus plantarum described in step a) is that deposit number is
The lactobacillus plantarum of CCTCC M 2014170.The polysaccharide has exact safety assurance for human body at this time
Preferred on the basis of any of the above technical solution, described separation of solid and liquid is turned with 10000~14000rpm
15~25min of speed centrifugation.
Preferred on the basis of any of the above technical solution, the duration of dialysis is 60~84h；More preferably, dialysis
Duration is 72h.On this basis it is further preferred that replacing the ultra-pure water 2 times outside bag filter during dialysis daily.
Preferably, the zymotic fluid described in step a), is to ferment to obtain with MRS culture mediums.
Preferably, the zymotic fluid described in step a), is to ferment to obtain under the conditions of 35~39 DEG C；More preferably fermentation temperature
Spend is 37 DEG C.
Preferably, the drying described in step b) is freeze-drying.
Preferably, the pH value of step c) the Thick many candies lysates is 7.2~7.8；More preferably, pH 7.5.
Preferably, final concentration of 12% (w/v) after trichloroacetic acid adds in step e), continues after adding in trichloroacetic acid
30min carries out separation of solid and liquid again.
In above technical scheme, deposit number is that the depositary institution of the biomaterial of CCTCC M 2014170 is " China
Type Tissue Collection ", address be " No. 299 Wuhan Universitys of Wuhan City, Hubei Province Wuchang District Bayi Road are in the school ", the life
The preservation date of object material is on April 28th, 2014, and Classification And Nomenclature is lactobacillus plantarum (Lactobacillus
plantarum)。
In above technical scheme, the chemical modification refers to by way of chemical reaction to the molecular structure of substance
It is modified, makes products therefrom that there is the method for specific physicochemical property.In above technical scheme, the sulfur trioxide pyridine is answered
It closes object and is also known as sulfur trioxide pyridine complex, molecular formula C5H5NO3S can be bought from market.
The present invention improves its negative electrical charge on the basis of lactobacillus plantarum exocellular polysaccharide, by sulfonating reaction and supplies energy
Power, with DPPH free radicals, OH-Free radical, O2-Free radical scavenging ability evaluation finds that gained sulfonated products are compared with lactobacillus plantarum born of the same parents
Exo polysaccharides have more preferably antioxidant activity in itself.And this method of modifying applicability in all kinds of lactobacillus plantarum exocellular polysaccharides
It is wider, definite effect.On this basis, for antioxidant activity after the lactobacillus plantarum exocellular polysaccharide sulfonating reaction of partial category
Promote unconspicuous problem, the further preferred raw material exocellular polysaccharide for being suitable for the method for modifying of the present invention, the sulfonation of the polysaccharide
Reaction is fully, product is uniform, structure is clear and definite, while has prominent antioxidant activity.In addition, it is somebody's turn to do with what preparation method was characterized
Polysaccharide raw material, yield, purity are higher, are conducive to subsequent modified-reaction.The method of the present invention flow is shorter, cost is relatively low, behaviour
Make relative ease, meet the technological requirement of large-scale production, great promotion potential.
Description of the drawings
Fig. 1 is infrared spectrum (FT-IR) collection of illustrative plates of exocellular polysaccharide for extracting, purifying in the embodiment of the present invention 1.
Fig. 2 is infrared spectrum (FT-IR) collection of illustrative plates of exocellular polysaccharide modified product in the embodiment of the present invention 1.
Fig. 3 is the DPPH free radical scavenging activities of lactobacillus plantarum exocellular polysaccharide and its modified product in the embodiment of the present invention 1
Testing result figure.
Fig. 4 is the OH of lactobacillus plantarum exocellular polysaccharide and its modified product in the embodiment of the present invention 1-Free radical scavenging activity
Testing result figure.
Fig. 5 is the O of lactobacillus plantarum exocellular polysaccharide and its modified product in the embodiment of the present invention 12-Free radical scavenging activity is examined
Survey result figure.
Specific embodiment
The specific embodiment of the present invention will be described in detail below.In order to avoid excessive unnecessary details,
It will not be described in detail in following embodiment to belonging to well known structure or function.
Approximating language used in following embodiment can be used for quantitative expression, show in the feelings for not changing basic function
Quantity is allowed to have certain variation under condition.Therefore, it is not limited to this accurately with the modified numerical value of the language such as " about ", " left and right " institute
Numerical value is in itself.In some embodiments, the range for allowing its modified numerical value in positive and negative 10 (10%) " about " is represented
Interior variation, for example, what " about 100 " represented can be any numerical value between 90 to 110.In addition, " the about first numerical value arrives
In the statement of second value ", the first and second numerical value two values are at about corrected.In some cases, approximating language
It may be related with the precision of measuring instrument.
In addition to being defined, technical and scientific term used in following embodiment has and fields technology people of the present invention
The identical meanings that member is commonly understood by.
Test reagent consumptive material used in following embodiment is conventional biochemical reagent unless otherwise specified；The experiment
Method is conventional method unless otherwise specified；Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result
It is averaged；% in following embodiment is mass percentage unless otherwise instructed.
Embodiment 1
Extraction, the purifying of 1.1 exocellular polysaccharides
For 24 hours, 12000 × g centrifugation 20min abandon precipitation, supernatant to 37 DEG C of Anaerobic culturels to lactobacillus plantarum in MRS culture mediums
The bag filter for being 8-14KDa with molecular cut off is dialysed 3 days, changes ultra-pure water daily 2 times.After freeze-drying, with 50mM Tris-
HCl、10mM MgSO4·7H2O (pH 7.5) dissolves Thick many candies (final concentration of 5mg/mL), using final concentration of 2.5 μ
6h is hydrolyzed to nucleic acid at 37 DEG C in the nuclease DNAse type-I (D5025, Sigma) of g/mL.Thick many candies after hydrolysis are adopted
Enzymolysis 18h is carried out to protein at 37 DEG C with the protease P ronase E (165921, Roche) of final concentration of 50 μ g/mL.Egg
After white enzymatic treatment, 30min is handled with final concentration of 12% trichloroacetic acid, the supernatant that 12000 × g centrifugations 20min is obtained needs to adjust
PH to 4.0~5.0 is saved, continues dialysis 3 days, changes water daily 2 times, is desired polysaccharide after the drying of bag filter content.
Exocellular polysaccharide is analyzed using infrared spectrum (FT-IR)：Solid sample is worn into less than 2 μm of powder, be suspended in easily
In the solvent of volatilization, then this suspension is dripped and is paved in KBr chip bases, forms uniform powder thin layer after the solvent is volatilized, then
Further analysis.
The infrared spectrogram of the polysaccharide is as shown in Figure 1, its infrared absorption spectrum has typical O-H (34177.12), C-H
(2932.40) and the stretching vibration Absorption Characteristics peak of C=O (1641) etc..
1.2 lactobacillus plantarum exocellular polysaccharides it is structurally-modified
The exocellular polysaccharide that 50mg is taken to purify, with 10mL anhydrous DMFs, room temperature handles 30min, and constantly shakes up, and adds 100mg tri-
Sulfur oxide pyridine complex reacts 3h in 80 DEG C of prolonged agitations, and 25 DEG C are then cooled in ice water.It is adjusted with the NaOH of 4mol/L
PH to 7.0 uses molecular cut off to dialyse 5 days for the bag filter of 8-14KDa, is finally freeze-dried.
The exocellular polysaccharide derivative obtained using infrared spectrum (FT-IR) analysis：Solid sample is worn into less than 2 μm of powder
End is suspended in volatile solvent, is then dripped this suspension and is paved in KBr chip bases, is formed after the solvent is volatilized uniform
Powder thin layer, is further analyzed.
The infrared spectrogram of modified product is as shown in Fig. 2, its infrared absorption spectrum shakes in addition to flexible containing exocellular polysaccharide
Outside dynamic Absorption Characteristics peak, the strong O=S=O stretching vibration Absorption Characteristics peak also containing there are one shows that the modified product belongs to
State the sulfonated products of exocellular polysaccharide.In addition, the collection of illustrative plates is in 1243.65cm-1Place has the special absorption peak of sulfate group, the group
More negative electrical charges on carbon atom can be provided, be conducive to radical conversion into inactive ingredient.
1.3 modified products are to the measure of DPPH (1,1- diphenyl -2- trinitrophenyl-hydrazines) Scavenging activity
A concentration of 0,0.5,1.0,1.5,2.0 and 2.5mg/mL exocellular polysaccharides for taking 0.5mL are added in the centrifuge tube of 15mL
In.The often DPPH solution (being dissolved with 95% absolute ethyl alcohol) of pipe plus 3.5mL 0.2nmol/L is reacting at room temperature 30min,
Measure the light absorption value at 517nm.Shown in the calculation formula of DPPH clearance rates such as formula (1)：
Ao is blank group in formula (1), and Ai is experimental group, and Aj is control group.
Fig. 3 shows the testing result of lactobacillus plantarum exocellular polysaccharide and its modified product to DPPH free radical scavenging activities,
By Fig. 3 it can be found that the DPPH free radical scavenging abilities of exocellular polysaccharide sulfonated products are apparently higher than exocellular polysaccharide in itself, in concentration
Under conditions of 1.0,1.5 and 2.0mg/mL, there is significant difference.
1.4 modified products are to the measure of hydroxyl radical free radical Scavenging activity
Take a concentration of 0,0.5,1.0,1.5, the 2.0 of 0.5mL and 2.5mg/mL exocellular polysaccharides add in take 6 15mL from
Heart pipe, often pipe addition 1.0mL PBS buffer solution (pH7.4,0.02mol/mL), is subsequently added into the 1 of 0.5mL 2.5mmol/mL,
The hydrogen peroxide of 10- Phens, the ferrous sulfate of 0.5mL 2.5mmol/mL and 0.5mL 20mmol/mL, is finally separately added into
A concentration of 0,0.5,1.0,1.5, the 2.0 of 0.5mL and 2.5mg/mL exocellular polysaccharides.37 DEG C are reacted 1 hour, are measured at 536nm
Light absorption value.OH-Clearance rate calculation formula such as formula (2) shown in：
Ao is blank group in formula (2), and Ai is experimental group, and Aj is control group.
Fig. 4 shows lactobacillus plantarum exocellular polysaccharide and its modified product to OH-The testing result of free radical scavenging activity, by
Fig. 4 is it can be found that when concentration is less than 2.0mg/mL, and modified product is to OH-Free radical scavenging ability is significantly better than plant breast bar
In itself, and even if under the low consistency conditions of 0.5mg/mL, modified product also shows exact OH to bacterium exopolysaccharide-From
By base removal ability.
1.5 modified products are to the measure of superoxide anion Scavenging activity
Take a concentration of 0,0.5,1.0,1.5, the 2.0 of 0.1mL and 2.5mg/mL exocellular polysaccharides add in take 6 15mL from
Heart pipe, then add 340 μ L Tris-HCl buffer solutions (50mmol/L, pH8.2) and 50 μ L 25mmol/L pyrogallols respectively in room
Temperature reaction 4min, then terminates reaction with the hydrochloric acid of 10 μ L 8.0nmol/L, measures the light absorption value at 320nm.O2-Clearance rate
Calculation formula such as formula (3) shown in：
Ao is blank group in formula (3), and Ai is experimental group, and Aj is control group.
Fig. 5 shows lactobacillus plantarum exocellular polysaccharide and its modified product to O2-The testing result of free radical scavenging activity, by
Fig. 5 is it can be found that under each concentration conditions, the O of modified product2-Free radical scavenging activity is superior to lactobacillus plantarum exocellular polysaccharide sheet
Body.
Embodiment 2
A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity, includes the following steps：
1) the lactobacillus plantarum exocellular polysaccharide is taken, is dissolved with anhydrous dimethyl formamide；
2) sulfur trioxide pyridine complex is added in after 15min thereto to final concentration of 5mg/mL, reacts 2h.
On the basis of above technical scheme, meet the following conditions：
Further include step 3)：Using the bag filter dialysis step 2) products therefrom that molecular cut off is 8KDa, bag filter is taken
Content is dried.Step 2) products therefrom pH first is adjusted to 6.5 before dialysis to dialyse again.
The temperature reacted described in step 2) is 75 DEG C, is cooled to 20 DEG C after reaction.
The final concentration of 3mg/mL of lactobacillus plantarum exocellular polysaccharide being dissolved in step 1) in anhydrous dimethyl formamide.
The lactobacillus plantarum exocellular polysaccharide is prepared by the following method：
A) lactobacillus plantarum zymotic fluid is taken, separation of solid and liquid takes supernatant；
B) ethyl alcohol, alcohol precipitation are added in into the step a) supernatants, separation of solid and liquid takes precipitation, with 8KDa molecular cut offs
Bag filter is dialysed, and bag filter content is taken to dry, as Thick many candies；
C) to contain 40mM Tris-HCl, 5mM MgSO4·7H2The solution of O is as Thick many candies lysate, dissolving step b)
The Thick many candies then add in nuclease DNAse type-I to 2 μ g/mL of final concentration, hydrolysis；
D) protease P ronase E to 40 μ g/mL of final concentration, enzymolysis are then added in；
E) trichloroacetic acid is then added in final concentration 10% (w/v), and separation of solid and liquid takes supernatant after 20min, dialyses, takes dialysis
Bag content is the lactobacillus plantarum exocellular polysaccharide.
The addition of ethyl alcohol is 1.5 times of the supernatant volume in step b).
The final concentration of 4mg/L of Thick many candies in step c).
Before step e) dialysis, supernatant pH is first adjusted to 4, then dialyse.
Lactobacillus plantarum described in step a) is the lactobacillus plantarum that deposit number is CCTCC M 2014170.
Embodiment 3
A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity, includes the following steps：
1) the lactobacillus plantarum exocellular polysaccharide is taken, is dissolved with anhydrous dimethyl formamide；
2) sulfur trioxide pyridine complex is added in after 45min thereto to final concentration of 15mg/mL, reacts 4h.
On the basis of above technical scheme, meet the following conditions：
Further include step 3)：Using the bag filter dialysis step 2) products therefrom that molecular cut off is 14KDa, bag filter is taken
Content is dried.Step 2) products therefrom pH first is adjusted to 7.5 before dialysis to dialyse again.
The temperature reacted described in step 2) is 85 DEG C, is cooled to 30 DEG C after reaction.
The final concentration of 7mg/mL of lactobacillus plantarum exocellular polysaccharide being dissolved in step 1) in anhydrous dimethyl formamide.
The lactobacillus plantarum exocellular polysaccharide is prepared by the following method：
A) lactobacillus plantarum zymotic fluid is taken, separation of solid and liquid takes supernatant；
B) ethyl alcohol, alcohol precipitation are added in into the step a) supernatants, separation of solid and liquid takes precipitation, with 14KDa molecular cut offs
Bag filter dialysis, bag filter content is taken to dry, as Thick many candies；
C) to contain 60mM Tris-HCl, 15mM MgSO4·7H2The solution of O is as Thick many candies lysate, dissolving step
B) Thick many candies then add in nuclease DNAse type-I to 3 μ g/mL of final concentration, hydrolysis；
D) protease P ronase E to 60 μ g/mL of final concentration, enzymolysis are then added in；
E) trichloroacetic acid is then added in final concentration 15% (w/v), and separation of solid and liquid takes supernatant after 40min, dialyses, takes dialysis
Bag content is the lactobacillus plantarum exocellular polysaccharide.
The addition of ethyl alcohol is 2.5 times of the supernatant volume in step b).
The final concentration of 6mg/L of Thick many candies in step c).
Before step e) dialysis, supernatant pH is first adjusted to 5, then dialyse.
Lactobacillus plantarum described in step a) is the lactobacillus plantarum that deposit number is CCTCC M 2014170.
Embodiment 4
A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity, includes the following steps：
1) the lactobacillus plantarum exocellular polysaccharide is taken, is dissolved with anhydrous dimethyl formamide；
2) sulfur trioxide pyridine complex is added in after 30min thereto to final concentration of 10mg/mL, reacts 3h.
On the basis of above technical scheme, meet the following conditions：
Further include step 3)：Using the bag filter dialysis step 2) products therefrom that molecular cut off is 11KDa, bag filter is taken
Content is dried.
The temperature reacted described in step 2) is 80 DEG C, is cooled to 25 DEG C after reaction.
The lactobacillus plantarum exocellular polysaccharide is prepared by the following method：
A) lactobacillus plantarum zymotic fluid is taken, separation of solid and liquid takes supernatant；
B) ethyl alcohol, alcohol precipitation are added in into the step a) supernatants, separation of solid and liquid takes precipitation, with 12KDa molecular cut offs
Bag filter dialysis, bag filter content is taken to dry, as Thick many candies；
C) to contain 50mM Tris-HCl, 10mM MgSO4·7H2The solution of O is as Thick many candies lysate, dissolving step
B) Thick many candies then add in nuclease DNAse type-I to 2.5 μ g/mL of final concentration, hydrolysis；
D) protease P ronase E to 50 μ g/mL of final concentration, enzymolysis are then added in；
E) trichloroacetic acid is then added in final concentration 12% (w/v), and separation of solid and liquid takes supernatant after 30min, dialyses, takes dialysis
Bag content is the lactobacillus plantarum exocellular polysaccharide.
The addition of ethyl alcohol is 2 times of the supernatant volume in step b).
Before step e) dialysis, supernatant pH is first adjusted to 4.5, then dialyse.
Embodiment 5
A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity, includes the following steps：
1) the lactobacillus plantarum exocellular polysaccharide is taken, is dissolved with anhydrous dimethyl formamide；
2) sulfur trioxide pyridine complex is added in after 40min thereto to final concentration of 12mg/mL, reacts 3.5h.
On the basis of above technical scheme, meet the following conditions：
Further include step 3)：Using the bag filter dialysis step 2) products therefrom that molecular cut off is 8~14KDa, take
Analyse the drying of bag content.Step 2) products therefrom pH first is adjusted to 7.2 before dialysis to dialyse again.
Embodiment 6
A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity, includes the following steps：
1) the lactobacillus plantarum exocellular polysaccharide is taken, is dissolved with anhydrous dimethyl formamide；
2) sulfur trioxide pyridine complex is added in after 20min thereto to final concentration of 8mg/mL, reacts 2.5h.
The embodiment of the present invention is described in detail above, but the content is only presently preferred embodiments of the present invention,
It is not intended to limit the invention.All all any modification, equivalent and improvement done in the application range of the present invention etc., should all
It is included within protection scope of the present invention.
Claims (3)
1. a kind of chemical modification lactobacillus plantarum exocellular polysaccharide is preparing the application in removing DPPH drugs；Change described in the application
Learn a concentration of 1mg/mL of improved plant lactobacillus exocellular polysaccharide or 1.5mg/mL or 2mg/mL；
Wherein, the chemical modification lactobacillus plantarum exocellular polysaccharide, is prepared by the following method：
1) lactobacillus plantarum exocellular polysaccharide is taken, is dissolved with anhydrous dimethyl formamide；
2) sulfur trioxide pyridine complex is added in after 15~45min thereto to final concentration of 5~15mg/mL, reacts 2~4h；
3) using the bag filter dialysis step 2) products therefrom that molecular cut off is 8~14KDa, bag filter content is taken to dry；
Wherein, step 2) products therefrom pH first is adjusted to 6.5~7.5 before dialysing to dialyse again；
The temperature reacted described in step 2) is 75~85 DEG C, is cooled to 20~30 DEG C after reaction；
Final concentration of 3~the 7mg/mL of lactobacillus plantarum exocellular polysaccharide being dissolved in step 1) in anhydrous dimethyl formamide；
Lactobacillus plantarum exocellular polysaccharide described in step 1) is prepared by the following method：
A) zymotic fluid of lactobacillus plantarum that deposit number is CCTCC M 2014170 is taken, separation of solid and liquid takes supernatant；
B) ethyl alcohol, alcohol precipitation are added in into the step a) supernatants, separation of solid and liquid takes precipitation, with 8~14KDa molecular cut offs
Bag filter is dialysed, and bag filter content is taken to dry, as Thick many candies；
C) to contain 40~60mM Tris-HCl, 5~15mM MgSO4·7H2The solution of O is as Thick many candies lysate, dissolving step
Rapid b) described Thick many candies then add in nuclease DNAse type-I to 2~3 μ g/mL of final concentration, hydrolysis；
D) protease P ronase E to 40~60 μ g/mL of final concentration, enzymolysis are then added in；
E) trichloroacetic acid is then added in 10~15%w/v of final concentration, and separation of solid and liquid takes supernatant after 20~40min, dialyses, takes
It is the lactobacillus plantarum exocellular polysaccharide to analyse bag content；
Final concentration of 4~6mg/L of Thick many candies in step c).
2. application according to claim 1, it is characterised in that the addition of ethyl alcohol is the supernatant volume in step b)
1.5~2.5 times.
3. application according to claim 1, it is characterised in that before step e) dialysis, supernatant pH is first adjusted to 4~5, then
Dialysis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610202526.7A CN105859898B (en) | 2016-04-05 | 2016-04-05 | A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610202526.7A CN105859898B (en) | 2016-04-05 | 2016-04-05 | A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105859898A CN105859898A (en) | 2016-08-17 |
| CN105859898B true CN105859898B (en) | 2018-06-19 |
Family
ID=56626817
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610202526.7A Active CN105859898B (en) | 2016-04-05 | 2016-04-05 | A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105859898B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108165512B (en) * | 2018-02-05 | 2019-12-13 | 富乐顿生物工程科技（北京）有限公司 | Extracellular polysaccharide-producing space lactobacillus plantarum SS18-119 and application thereof in improving biological antioxidant activity |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255752A (en) * | 2015-04-27 | 2016-01-20 | 南昌大学 | Lactobacillus plantarum ZDY2013 with high acid resistance |
-
2016
- 2016-04-05 CN CN201610202526.7A patent/CN105859898B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255752A (en) * | 2015-04-27 | 2016-01-20 | 南昌大学 | Lactobacillus plantarum ZDY2013 with high acid resistance |
Non-Patent Citations (3)
| Title |
|---|
| Antioxidant and antibacterial activities of exopolysaccharides from Bifidobacterium bifidum WBIN03 and Lactobacillus plantarum R315;Shengjie Li et al.;《Journal of Dairy Science》;20141003;第97卷(第12期);摘要，结论部分，EPS的提取、纯化、检测部分 * |
| Antioxidant and antibacterial activities of sulphated polysaccharides from Pleurotus eryngii and Streptococcus thermophilus ASCC 1275;Siqian Li et al.;《Food Chemistry》;20140529;第165卷;摘要，第2.1、2.4、4节 * |
| Characterisation of the exopolysaccharide (EPS)-producing Lactobacillus paraplantarum BGCG11 and its non-EPS producing derivative strains as potential probiotics;Milica Nikolic et al.;《International Journal of Food Microbiology》;20120720;第158卷;2.2节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105859898A (en) | 2016-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | Chemical and physical characteristics and antioxidant activities of the exopolysaccharide produced by Tibetan kefir grains during milk fermentation | |
| AU2011230685B2 (en) | Anti-allergic agent | |
| Hooshdar et al. | A review on production of exopolysaccharide and biofilm in probiotics like lactobacilli and methods of analysis | |
| CN102876748B (en) | Method for preparing oligomeric hyalurate by digestion method, and oligomeric hyalurate and application thereof | |
| CN109182162B (en) | Lactobacillus plantarum with antioxidant capacity and application thereof | |
| CN104957610B (en) | A kind of plant enzyme and its production and use | |
| CN111534455B (en) | Preparation of lactobacillus sporolysis product and application of lactobacillus sporolysis product in cosmetics | |
| CN106222158B (en) | Preparation method of lactobacillus-embedding microcapsule | |
| JP2009507863A (en) | New use of white jellyfish miscellaneous polysaccharide or its extract | |
| Guan et al. | Simulated digestion and in vitro fermentation of a polysaccharide from lotus (Nelumbo nucifera Gaertn.) root residue by the human gut microbiota | |
| CN100375754C (en) | Method for producing extracellular mucopoly-saccharide using rhamnose lactobacillus and product | |
| CN107920575A (en) | A kind of manufacture method of Synbiotics tunning and the Synbiotics tunning thus manufactured | |
| CN110627919B (en) | Intestinal prebiotics black skin termitomyces albuminosus polysaccharide ORP-1 and preparation method and application thereof | |
| CN106834379A (en) | A kind of preparation method and applications of new fine jade tetrose | |
| CN105859898B (en) | A kind of chemical modification method for improving lactobacillus plantarum exocellular polysaccharide antioxidant activity | |
| CN104231106B (en) | Paenibacillus exopolysaccharide and preparation method and application of paenibacillus exopolysaccharide | |
| CN116077415B (en) | Ternary probiotic factor composition for regulating skin microecological balance | |
| CN104382162A (en) | Method for preparing bifidobacterium infantis ATCC 15697 and aloe co-fermented beverage | |
| CN105063128B (en) | A kind of preparation method for the low molecular weight konjac oligosaccharides adjusting intestinal flora | |
| CN113041200B (en) | Cosmetic containing multiple fermentation liquor of custard apple and preparation method thereof | |
| CN115887343A (en) | Fermentation formula and fermentation method using birch juice as solvent | |
| CN109169899A (en) | A kind of preparation method of the shield stomach acidified milk containing Hericium erinaceus beta glucan | |
| CN114891699A (en) | Lactobacillus salivarius for regulating flora balance | |
| CN102198057B (en) | Oat extract, preparation method thereof and application thereof | |
| CN105837699B (en) | A kind of lactobacillus plantarum exocellular polysaccharide and its purposes for antagonism bacillus cereus enterotoxin toxicity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230809 Address after: 221000 Office Area 203, Kangtian Science and Technology Park, Shizhuang Village, Zizhuang Town, Jiawang District, Xuzhou City, Jiangsu Province Patentee after: Xuzhou Tianrenxue Technology Co.,Ltd. Address before: Building C, No.888, Huanhu West 2nd Road, Lingang New District, China (Shanghai) pilot Free Trade Zone, Pudong New Area, Shanghai Patentee before: Shanghai ruishenglian Information Technology Co.,Ltd. Effective date of registration: 20230809 Address after: Building C, No.888, Huanhu West 2nd Road, Lingang New District, China (Shanghai) pilot Free Trade Zone, Pudong New Area, Shanghai Patentee after: Shanghai ruishenglian Information Technology Co.,Ltd. Address before: 999 No. 330031 Jiangxi province Nanchang Honggutan University Avenue Patentee before: Nanchang University |
|
| TR01 | Transfer of patent right |