CN103875667B - A kind of antioxidant and application thereof - Google Patents
A kind of antioxidant and application thereof Download PDFInfo
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- CN103875667B CN103875667B CN201210562694.9A CN201210562694A CN103875667B CN 103875667 B CN103875667 B CN 103875667B CN 201210562694 A CN201210562694 A CN 201210562694A CN 103875667 B CN103875667 B CN 103875667B
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Abstract
A kind of antioxidant of the present invention and application thereof relate to chlorella polysaccharide application in preparing green plant pesticide and food additive. Described chlorella polysaccharide is one group of molecular weight obtaining after enzymolysis, deproteinization and precipitate with ethanol from chlorella water-insoluble fraction (algae-residue) polysaccharide at 10.6 ~ 13.6kDa, and its monosaccharide composition is mainly glucose, galactose, arabinose, fucose, rhamnose. Described chlorella polysaccharide can significantly improve plant cell antioxidase (SOD, POD and CAT) and external Scavenging action to hydroxyl free radical, reducing power, DPPH clearance rate isoreactivity, can as a plant growth regulators and natural additive for foodstuff.
Description
Technical field
The present invention relates to the bioactivity research of polysaccharide, have studied the biological activity of water-insoluble polysaccharide in a kind of marine algae and application thereof in detail, belong to glycobiology field.
Background technology
Existing result of study shows that biotic and abiotic stress can cause degeneration-resistant reaction in plant, such as disease-resistant, cold-resistant, drought resisting and antioxidation etc. Wherein, oxidation reaction is mainly manifested in the generation of active oxygen ROS (reactiveoxygenspecies) in higher plant, and main species has superoxide anion (O2 -), singlet oxygen (1O2), hydroxyl radical free radical (HO), hydrogen peroxide (H2O2) etc. ROS under these naturalness is growth and development of plants and the second message,second messenger to environment-stress, it is possible to cause the physiological effecies such as cytoprotective reaction, seed germination, root gravitropism and stomatal closure. And excessive ROS can result in the oxidative damage of protein, DNA and lipid, and finally cause cell death. Antioxidase [mainly includes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT)] and is ROS scavenging system main in plant; excessive ROS finally can be degraded to water, play the effect of protection body normal physiological function.
The existing Natural antioxidant is mainly vitamin substances, and such as vitamin A/C/E, carotenoid etc., but it is expensive, and range of application is limited; And the antioxidant of synthetic, although price has superiority but there is potential safety hazard during application in food, medicine, feedstuff, environment friendly agricultural etc. Therefore, find and a kind of there is natural, nontoxic, cheap antioxidant as a kind of additive or adjuvant, and be applied to the field such as environment friendly agricultural, food additive, contribute to realizing the social development target that ecological ideas environmental protection, food safety are nontoxic.
Chlorella (Chlorella) is unicellular alga, is a general natural disposition monoplast green alga of class, and its kind is many, and ecologicaI distribution is wide, it is easy to cultivating, fast growth, containing substantial amounts of bioactive substance, such as carbohydrate, protein and vitamin etc. Additionally, chlorella has powerful Lipid-producing ability, of great interest currently as a kind of energy microalgae. Existing research shows, chlorella water soluble polysaccharide has good antioxidation and anti-aging effects, and there is not been reported for the correlational study of polysaccharide in algae-residue remaining after chlorella oil-producing.
Summary of the invention
It is an object of the invention to the exploitation of chlorella algae-residue polysaccharide anti-oxidative activity and application thereof, a kind of antioxidant economic, effective and biological pesticide can be provided for food service industry and plant husbandry, be conducive to the higher value application of microalgal polysaccharide simultaneously.
For achieving the above object, the technical solution used in the present invention is:
1. described in, chlorella algae-residue polysaccharide obtains through enzymolysis, it is mainly made up of 7 kinds of monosaccharide such as rhamnose, fucose, glucose and galactose to adopt gas chromatography determination, and mol ratio is Rha:Fuc:Arb:Xyl:Man:Glc:Gal=7.03:15.23:6.34:1:3.276:17.2 3:16.54; Employing high performance gel filtration chromatography records its relative molecular mass scope and is: 10.6 ~ 13.6kDa.
2. described in, cellular anti-oxidant activity is with plant such as Semen Tritici aestivi, Oryza sativa L., arabidopsiss for object of study, measure chlorella algae-residue polysaccharide to the impact of antioxidase (SOD, POD and CAT) activity in different plants and same plant different tissues, find that this polysaccharide can significantly improve Semen Tritici aestivi and the antioxidant activity of arabidopsis two kind of plant cell, at 0 ~ 200 μ g mL-1In range of concentrations, Semen Tritici aestivi SOD, three kinds of enzymes of POD and CAT relatively matched group alive improve 5% ~ 30%; And three kinds of enzymes work are respectively increased 5 ~ 25% in arabidopsis, there is good internal antioxidant effect.
3. the antioxidation activity in vitro described in, it is with external Scavenging action to hydroxyl free radical, reducing power, DPPH clearance rate for evaluation index, investigate the antioxidant effect of variable concentrations chlorella algae-residue polysaccharide, screen the best use of concentration, experimental result finds: present concentration-dependant in this polysaccharide Antioxidtive Activities in Vitro, Scavenging action to hydroxyl free radical and DPPH clearance rate are in 5% ~ 70% scope, and reducing power (OD700nm), between 0.01 ~ 1.70, has good Antioxidtive Activities in Vitro.
4. application described in is characterized in that: chlorella algae-residue polysaccharide has good plant cell antioxidant activity, can improve activities of antioxidant enzymes in plant, thus changing the content of ROS in plant. And existing research finds, ROS in plant can as second message,second messenger, to the growth of plant, growth and the reaction of environment-stress is played an important role. Therefore, can as the effective plant growth regulator of one. Additionally, chlorella algae-residue polysaccharide has good antioxidation activity in vitro, can prevent oils and fats autoxidation in food from becoming sour and food enzymatic oxidative browning etc., also act as natural additive for foodstuff.
Present invention have the advantage that
1 present invention demonstrates that chlorella algae-residue polysaccharide has antioxidant activity, is the main composition of chlorella, has multiple biological activity, originate natural, have no side effect, can be developed into green plant pesticide or food additive.
2 chlorella water-insoluble polysaccharide raw material sources are extensive, cheap, and the present invention is conducive to the comprehensive utilization of chlorella algae-residue and improves its added value.
3 technical operations of the present invention are simple, only need to be mixed by a certain percentage with fertilizer or food by chlorella algae-residue polysaccharide, it is possible to play enhancing antioxidation.
Accompanying drawing explanation
The impact on Semen Tritici aestivi, arabidopsis SOD activity of Fig. 1 variable concentrations chlorella algae-residue polysaccharide; * expression process group and matched group are at the horizontal significant difference of P < 0.05;
The impact on reducing power of Fig. 2 variable concentrations chlorella algae-residue polysaccharide;
The impact on Hydroxyl radical-scavenging ability of Fig. 3 variable concentrations chlorella algae-residue polysaccharide;
The impact on DPPH Scavenging activity of Fig. 4 variable concentrations chlorella algae-residue polysaccharide.
Detailed description of the invention
The chlorella algae-residue polysaccharide that the present invention adopts is the one group of polysaccharide obtained through Enzymatic Extraction by chlorella water-insoluble fraction.
Adopting neutral protease A that algae-residue (5.0g) is carried out water-insoluble polysaccharide extraction, extraction conditions is: concentration of substrate is 10%, enzyme addition 1%, temperature 55 DEG C, extraction time 24h;
By said extracted liquid at 50 DEG C, rotate when rotating speed 60rpm and be evaporated to the 1/4 of original solution volume, add 5 times of volume 95% ethanol, standing overnight, then the centrifugal 15min of 5000rpm, is precipitated part, finally it is made directly dry, obtains chlorella water-insoluble polysaccharide solid.
Adopting high performance gel filtration chromatography to measure this polysaccharide relative molecular mass is 10.6 ~ 13.6kDa, adopting its monosaccharide of gas chromatography determination composition and mol ratio is Rha:Fuc:Arb:Xyl:Man:Glc:Gal=7.03:15.23:6.34:1:3.276:17.2 3:16.54, and finds that chlorella polysaccharide has antioxidant activity by the experiment of plant cell antioxidation and antioxidation in vitro experiment.
The present invention evaluates this polysaccharide anti-oxidative activity by measuring wheat cell antioxidase (SOD, POD and CAT) and external Scavenging action to hydroxyl free radical, reducing power, DPPH clearance rate isoreactivity, thus for this polysaccharide in the application certain foundation of offer.
Described cellular anti-oxidant activity is with plant such as Semen Tritici aestivi, Oryza sativa L., arabidopsiss for object of study, measure chlorella algae-residue polysaccharide to the impact of antioxidase (SOD, POD and CAT) activity in different plants and same plant different tissues, find that this polysaccharide can significantly improve Semen Tritici aestivi and the antioxidant activity of arabidopsis two kind of plant cell, at 0 ~ 200 μ g mL-1In range of concentrations, Semen Tritici aestivi SOD, three kinds of enzymes of POD and CAT relatively matched group alive improve 5% ~ 30%; And three kinds of enzymes work are respectively increased 5 ~ 25% in arabidopsis, there is good internal antioxidant effect.
Described antioxidation activity in vitro, it is with external Scavenging action to hydroxyl free radical, reducing power, DPPH clearance rate for evaluation index, investigate the antioxidant effect of variable concentrations chlorella algae-residue polysaccharide, screen the best use of concentration, experimental result finds: present concentration-dependant in this polysaccharide Antioxidtive Activities in Vitro, Scavenging action to hydroxyl free radical and DPPH clearance rate are in 5% ~ 70% scope, and reducing power (OD700nm), between 0.01 ~ 1.70, has good Antioxidtive Activities in Vitro.
Embodiment 1 plant cell antioxidation experiment one
(1) plant seed soaking experiment: adopt the western agriculture of Semen Tritici aestivi " 9871 " to carry out antioxidation experiment:
A) respectively by full for kind of grain, wheat seed of uniform size wraps in gauze, running water 10min, it is possible to removes the impurity of the surface of the seed and stops the material germinateed.
B) seed immersing immersion 5min in mass concentration 75% ethanol, removes the protein of the surface of the seed, lipid, the surface of the seed is carried out disinfection prevents the pollution of mycete simultaneously.
C) with 5,50,200 μ g mL-1Chlorella algae-residue polysaccharide solution, deionized water respectively the seed disinfected is carried out seed soaking, under room temperature soak 10h.
D) seed expanded after in leaching put in the culture dish of lower berth 2 metafiltration paper cultivate, each culture dish amplifies about 30 seeds, each concentration do three parallel, cultivate 11d. Condition of culture: cultivation temperature 25 DEG C, intensity of illumination 2000-2500lux, Light To Dark Ratio 12h:12h.
(2) prepared by wheat cell antioxidase: by the wheat plant of each process, randomly select 15 strains and rinse well with deionized water, surface moisture is blotted in absorbent paper, weigh stage casing, blade face and be about 0.5g sample, shred, in mortar, add 3mL0.05mol/L (pH7.8) sodium phosphate buffer (PBS) of pre-cooling, ice bath grinds in homogenate, being transferred in 10mL graduated centrifuge tube, constant volume is to 5mL, at 10000r/min-1At 4 DEG C, centrifugal 20min, takes supernatant, is divided into 3 parts, saves backup in-20 DEG C.
(3) mensuration of enzymatic activity: SOD activity adopts NBT (NBT) photochemical reduction to be measured, to suppress the 50% of NBT photoreduction as 1 enzyme activity unit (U), unit is U g-1FW·h-1; POD activity adopts guaiacol-hydrogen peroxide method to be measured, and increases by 1 for 1 enzyme activity unit (U) with OD470nm per minute, and unit is U g-1FW·h-1; CAT activity adopts peroxide decomposition method to be measured, and reduces 1 for 1 enzyme activity unit (U) with interior OD240nm per minute, and unit is U g-1FW·h-1。
Embodiment 2 plant cell antioxidation experiment two
(1) plant sprays cultivation: adopt arabidopsis to carry out antioxidation experiment:
A) arabidopsis seed puts into addition sterilized water 2ml centrifuge tube from 4 DEG C of refrigerators after taking out;
B) then plant in soil, cover preservative film and put into (Light To Dark Ratio is 12/12, temperature 22 ± 2 DEG C) in incubator.
C) about 4d sprouts and takes off film, and about 21d can grow 2 true leaves (relevant with light application time), 31,33, to spray 3 concentration be 0,5,50,200 μ g mL to 35d-1Test polysaccharide, 38d samples and tests.
(2) prepared by arabidopsis cell antioxidase: with reference to " prepared by wheat cell antioxidase ".
(3) mensuration of enzymatic activity: with operation described in " plant cell antioxidation experiment one ".
(4) result: the chlorella algae-residue polysaccharide impact on plant anti-oxidation enzyme: as can be seen from Figure 1 compared with blank group, plant anti-oxidation enzyme is had a significant effect by chlorella algae-residue polysaccharide, and different plant action effect is different: be 50 μ g mL in concentration-1In time, improve maximum to Semen Tritici aestivi SOD activity, reaches 22.51%; Arabidopsis SOD activity is presented concentration dependent change, at 200 μ g mL-1Improve 15.23%; POD and CAT determination data is unlisted.
The impact on Semen Tritici aestivi, arabidopsis SOD activity of Fig. 1 variable concentrations chlorella algae-residue polysaccharide; * expression process group and matched group are at the horizontal significant difference of P < 0.05;
Embodiment 3 antioxidation in vitro is tested
(1) reducing power measures: take certain density chlorella algae-residue polysaccharide solution 1mL and 1mL0.2mol L-1The phosphate buffer mixing of pH6.6, is subsequently adding the potassium ferricyanide of 1mL0.1%, bathes 20min at the Water Unders of 50 DEG C. Reaction cools down after terminating rapidly, terminates reaction with 1ml10%TCA. Take this reactant liquor of 2ml and add 2ml distilled water and 0.8ml0.1%FeCl3Mixing, measures the mixed liquor light absorption value A when 700nm after 10min. Sample is replaced to do blank with 1ml distilled water, with 90 μ g mL-1Vc does positive control. Three Duplicate Samples made by each sample, average. A700More big, it was shown that the reducing power of sample is more strong. Measurement result is as shown in Figure 2: the impact on reducing power of Fig. 2 variable concentrations chlorella algae-residue polysaccharide; (2) Hydroxyl radical-scavenging ability measures: take 1mL9mmol L-1FeSO4Solution, 1mL9mmol L-1Salicylic acid-alcoholic solution and 1mL finite concentration sample solution mix in test tube, then add 0.8mL8.8mmol L-1H2O2Start reaction, mixed liquor is placed in 37 DEG C of reaction 30min. Replace sample to do blank with equal-volume distilled water, replace H with equal-volume distilled water2O2Do the impact of sample background color, do reference with distilled water, the assaying reaction liquid light absorption value As when 510nm simultaneously. With 250 μ g mL-1Vc replaces sample to do positive control. Three Duplicate Samples made by each sample, average. As is more little, it was shown that the hydroxyl radicals of sample is more strong. Measurement result is as shown in Figure 3:
——As: sample and the Vc light absorption value at 510nm place
A0: sample background color is at the light absorption value at 510nm place
Ac: blank is at the light absorption value at 510nm place
The impact on Hydroxyl radical-scavenging ability of Fig. 3 variable concentrations chlorella algae-residue polysaccharide;
(3) DPPH removes power mensuration: the 0.3mmol L of 1mL-1DPPH alcoholic solution (the 95% fresh preparation of ethanol) be added in 2.5mL sample solution. Room temperature reaction 30min, measures light absorption value at 517nm place. With 100 μ g mL-1Vc oppose and do positive control. Three Duplicate Samples made by each sample, average. Measurement result is as shown in Figure 4:
——A0: the blank group light absorption value containing only DPPH
A1: the light absorption value of sample and DPPH mixed liquor
As: there is no the sample liquid light absorption value of DPPH
The impact on DPPH Scavenging activity of Fig. 4 variable concentrations chlorella algae-residue polysaccharide;
The result of study of above-mentioned antioxidation activity in vitro shows, this polysaccharide has good antioxidant activity and shows concentration-dependant change, during 10mg/mL, DPPH clearance rate when external Scavenging action to hydroxyl free radical, reducing power (OD700nm) and 500 μ g/ml is maximum, respectively reach 56.47 ± 1.05%, 1.479 ± 0.0149 and 39.13 ± 0.74%.
Claims (3)
1. an antioxidant, it is with chlorella water-insoluble polysaccharide for active ingredient, described chlorella water-insoluble polysaccharide adopts gas chromatography determination: it is made up of rhamnose, fucose, arabinose, xylose, mannose, glucose and 7 kinds of monosaccharide of galactose, and mol ratio is rhamnose (Rha): fucose (Fuc): arabinose (Arb): xylose (Xyl): mannose (Man): glucose (Glc): galactose (Gal)=7.03:15.23:6.34:1:3.276:17.23:16.54; Employing high performance gel filtration chromatography records its relative molecular mass scope and is: 10.6～13.6kDa.
2. antioxidant according to claim 1, its preparation process:
First chlorella is carried out breaking cellular wall, and with hot water extraction method, its water soluble polysaccharide is fully extracted, and centrifugal be precipitated, then water is utilized repeatedly to clean this precipitation to remove remaining soluble polysaccharide therein, again by one or two or more kinds in neutral protease and glycoside hydrolase to its extraction carrying out water-insoluble polysaccharide, eventually pass concentration, precipitate with ethanol and lyophilization and obtain chlorella water-insoluble polysaccharide;
The process of hot water extraction chlorella water soluble polysaccharide, first chlorella is carried out breaking cellular wall, again with water soluble polysaccharide in 40～90 DEG C of hot water extraction chlorellas, centrifugal 1～3 time of 1000～5000rpm after extraction, each 10～20min, collect precipitation and with deionized water wash 3 times, obtain chlorella water-insoluble after drying and precipitate;
The described employing enzyme process to the extraction of water-insoluble polysaccharide, above-mentioned precipitation is mixed with enzyme, it is 1:10～100 (m/m) at enzyme-to-substrate mass ratio, concentration of substrate is 1～50%, temperature is 40～120 DEG C, carry out the extraction of chlorella water-insoluble polysaccharide when time is 0.5～24h, be then centrifuged for collecting supernatant;
The process of the concentration of described polysaccharide extraction liquid and precipitate with ethanol, by said extracted liquid at 30～60 DEG C, rotate when rotating speed 40～120rpm and be evaporated to the 1/3～1/8 of original solution volume, add 3～8 times of volume 95% ethanol of concentrated solution, 5000rpm is centrifuged 15min, it is precipitated part, is finally made directly dry, obtain chlorella water-insoluble polysaccharide solid;
The mass ratio of chlorella and water is 1:5～50;
Described glycoside hydrolase is one or two or more kinds in cellulase, pectase.
3. the application of antioxidant described in a claim 1, it is characterised in that:
Described antioxidant can as plant-biological pesticide or food additive.
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| CN113243477B (en) * | 2021-05-14 | 2022-10-28 | 中国海洋大学 | Composition for preventing browning of beverage and application thereof |
| CN114946860B (en) * | 2022-05-18 | 2023-06-20 | 山西农业大学 | Method for improving disease resistance of arabidopsis thaliana |
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