CN103820512B - A kind of preparation method and application of frost resistance K- carrageenan oligosaccharides - Google Patents
A kind of preparation method and application of frost resistance K- carrageenan oligosaccharides Download PDFInfo
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- CN103820512B CN103820512B CN201410040800.6A CN201410040800A CN103820512B CN 103820512 B CN103820512 B CN 103820512B CN 201410040800 A CN201410040800 A CN 201410040800A CN 103820512 B CN103820512 B CN 103820512B
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Abstract
The present invention relates to a kind of preparation method and applications of frost resistance K carrageenan oligosaccharides.Carragheen is digested using modern biotechnology, Sevage methods removing protein and dialysis desalination are carried out to the carrageenan oligosaccharide of degradation, the carrageenan oligosaccharide obtained to purifying is studied.The preparation method is compared with the preparation method of existing carrageenan oligosaccharide, the carrageenan oligosaccharide prepared has freezing tolerance by verification, be with a wide range of applications, can be used as cold storage of cell, freeze during additive, while can be used to play the antifreeze effect of skin in skin care item.
Description
Technical field
The present invention relates to a kind of preparation method and applications of frost resistance K- carrageenan oligosaccharides.
Background technology
Carrageenan oligosaccharide is the catabolite of carragheen, and molecular weight is smaller, and dissolubility is preferable, is easy to absorb, stability and
Safety all makes moderate progress, while because the active group on strand fully exposes, so that its activity is significantly improved compared with carragheen, opening up
The wide application field of carragheen.Currently, since carrageenan oligosaccharide type is different, ingredient is different, needs respectively corresponding
Preparation method needs different control conditions and reagent.The preparation method of carrageenan oligosaccharide includes Physical, chemical method and micro- life
Object enzymatic isolation method, currently, being obtained using enzymatic isolation method has the preparation method of bioactivity carrageenan oligosaccharide very much, such as China Patent No.
03138975.9 a kind of preparation method, the China Patent No. of galactan sulfate with anti-tumor activity be
200610070792.5 a kind of preparation method, the China Patent No. of the carrageenan oligosaccharide sulfuric ester with antioxidant activity are
A kind of 200810238763.4 preparation methods of the carrageenan sulfated oligosaccharide with antiviral activity, but there has been no about utilizing enzyme
Solution prepares the report with frost resistance K- carrageenan oligosaccharides and its application.
The freeze proof of cell mainly either forms layer protecting film by reducing the freezing point of intracellular fluid in cell peripheral
Two methods, and the dissolubility of carrageenan oligosaccharide is good, film forming is poor, so mainly being improved by reducing intracellular fluid freezing point
The frost resistance of cell.
Invention content
The present invention is to provide a kind of preparation of frost resistance K- carrageenan oligosaccharides to make up defect of the existing technology
Method and application.
To achieve the goals above, the technical solution adopted in the present invention is：A kind of system of frost resistance K- carrageenan oligosaccharides
Preparation Method, it is characterised in that：
（1）It is prepared by seed liquor：By N5-3（Cellulophaga lyticastrain）Strain is inoculated in fluid nutrient medium
In, condition of culture is 30 DEG C, 150rpm, incubation time 20h；Fluid nutrient medium group becomes：Kappa-Carraginan：0.5%；Peptone：
0.3%；FeSO4.7H2O：0.002%；Old sea appropriate amount of water.
（2）The preparation of crude enzyme liquid：Seed liquor is inoculated in zymotic fluid by 2% inoculum concentration, and 12h after fermentation will fermentation
For liquid under the conditions of 4 DEG C, 5000r/min centrifuges 30min, and gained supernatant is crude enzyme liquid；The medium component of zymotic fluid：K- OK a karaoke clubs
Glue：0.5%；Peptone：0.3%；FeSO4.7H2O：0.002%.
（3）The preparation of Kappa-Carraginan oligosaccharides crude product：According to 0.5% carragheen：Crude enzyme liquid：Distilled water=2:1:1 ratio is matched
After making, it is 30 DEG C to be placed in condition of culture, the shaking table top fermentation 48h of 150rpm, and 5000r/min centrifugations 30min is gone under the conditions of 4 DEG C
Fall undegradable carragheen, takes supernatant to rotate evaporation and concentration at 70 DEG C, obtain carrageenan oligosaccharide semifinished product concentrate.
（4）The purifying of Kappa-Carraginan oligosaccharides semifinished product：Chlorine is added according to the 1/5 of the gained carrageenan oligosaccharide volume of the concentrated liquid
It is imitative, the n-butanol mixing of 1/5 chloroform volume is then added, 160r/min shakes 30min, takes out and centrifuges 5000r/ by 10min
Min, it takes supernatant to repeat aforesaid operations twice, rotary evaporation concentration is carried out at 70 DEG C to supernatant, be placed in ventilation volatilization
8-10h, the carrageenan oligosaccharide semifinished product concentrate for the protein that is removed；The concentrate of gained is packed into 500 molecular cut offs
Bag filter dialyse desalination, extracellular fluid dialysis is distilled water, 4 DEG C of dialysis is placed in, until AgNO is added in extracellular fluid dialysis3Nothing after solution
Until white precipitate generates, dialyzate is subjected to rotary evaporation concentration at 70 DEG C, gained concentrate is placed at -20 DEG C and is freezed
In drying on vacuum freezedrying instrument after for 24 hours, more pure carrageenan oligosaccharide powder is finally obtained.
A kind of application of frost resistance K- carrageenan oligosaccharides, it is characterised in that carrageenan oligosaccharide can be used for cold storage of cell, freeze
Freeze proof protection additive in the process, other than long-term preservation, it may also be used for the transport of above-mentioned organism is bought, represents；It needs
Freezen protective mainly has cell, tissue, organ, limbs, above-mentioned freezen protective that can also add carragheen widow into frozen stock solution
Icing Sugar end, is configured to frozen stock solution and comes freeze-stored cell, tissue, organ, limbs；Carrageenan oligosaccharide can also be added to skin care item
In, such as hand lotion, toner, moisturizer, face cream, improve the freezing tolerance of skin-protection product.
For the preparation method compared with the preparation method of existing carrageenan oligosaccharide, the carrageenan oligosaccharide prepared passes through verification
With freezing tolerance, be with a wide range of applications, can be used as cold storage of cell, freeze during additive, while can use
The antifreeze effect of skin is played in skin care item.
Description of the drawings
Cell relative viability after carrageenan oligosaccharide is handled 24 hours at 4 DEG C of Fig. 1.
Cell relative viability after carrageenan oligosaccharide is handled 1.5 hours at Fig. 2-20 DEG C.
Fig. 3 carrageenan oligosaccharides combine the influence of the cell relative viability of p- 20 DEG C of processing 3h with 10%DMSO.
LDH leakage rates in cell culture fluid after carrageenan oligosaccharide is handled 24 hours at 4 DEG C of Fig. 4.
LDH leakage rates in cell culture fluid after carrageenan oligosaccharide processing 2h at Fig. 5-20 DEG C.
Specific implementation mode
A kind of preparation method of frost resistance K- carrageenan oligosaccharides, it is characterised in that：
（1）It is prepared by seed liquor：By N5-3（Cellulophaga lytica strain）Strain is inoculated in fluid nutrient medium
In, condition of culture is 30 DEG C, 150rpm, incubation time 20h；The fluid nutrient medium group becomes：Kappa-Carraginan：0.5%；
Peptone：0.3%；FeSO4.7H2O：0.002%；Old sea appropriate amount of water.
（2）The preparation of crude enzyme liquid：Seed liquor is inoculated in zymotic fluid by 2% inoculum concentration, and 12h after fermentation will fermentation
For liquid under the conditions of 4 DEG C, 5000r/min centrifuges 30min, and gained supernatant is crude enzyme liquid；The medium component of the zymotic fluid：
Kappa-Carraginan：0.5%；Peptone：0.3%；FeSO4.7H2O：0.002%；Old sea appropriate amount of water.
（3）The preparation of Kappa-Carraginan oligosaccharides crude product：According to 0.5% carragheen：Crude enzyme liquid：Distilled water=2:1:1 ratio is matched
After making, it is 30 DEG C to be placed in condition of culture, the shaking table top fermentation 48h of 150rpm, and 5000r/min centrifugations 30min is gone under the conditions of 4 DEG C
Fall undegradable carragheen, takes supernatant to rotate evaporation and concentration at 70 DEG C, obtain carrageenan oligosaccharide semifinished product concentrate.
（4）The purifying of Kappa-Carraginan oligosaccharides semifinished product：Chlorine is added according to the 1/5 of the gained carrageenan oligosaccharide volume of the concentrated liquid
It is imitative, the n-butanol mixing of 1/5 chloroform volume is then added, 160r/min shakes 30min, takes out and centrifuges 5000r/ by 10min
Min, it takes supernatant to repeat aforesaid operations twice, rotary evaporation concentration is carried out at 70 DEG C to supernatant, be placed in ventilation volatilization
8-10h, the carrageenan oligosaccharide semifinished product concentrate for the protein that is removed；The concentrate of gained is packed into 500 molecular cut offs
Bag filter dialyse desalination, extracellular fluid dialysis is distilled water, 4 DEG C of dialysis is placed in, until AgNO is added in extracellular fluid dialysis3Nothing after solution
Until white precipitate generates, dialyzate is subjected to rotary evaporation concentration at 70 DEG C, gained concentrate is placed at -20 DEG C and is freezed
In drying on vacuum freezedrying instrument after for 24 hours, more pure carrageenan oligosaccharide powder is finally obtained.
The present invention has found that carrageenan oligosaccharide can significantly improve the freezing tolerance of cell by acting on Human normal hepatocyte.
Therefore, another aspect of the present invention provides the side for being able to demonstrate that K- carrageenan oligosaccharides have cell freezing tolerance
Method, specific implementation are as follows：
1、MTT:By the Human normal hepatocyte in exponential phase with 5 × 104A/ml is inoculated in 96 orifice plates, per hole
It is inoculated with 200ul, is placed in 37 DEG C, 5%CO2Incubator in continue culture for 24 hours, the carrageenan oligosaccharide solution of various concentration is added
（10、25、50、 100、 150、200、 250、 300 ug/ml）, it is control group to be not added with oligosaccharides（0）, every group sets 6 and puts down
96 orifice plates are put into 4 DEG C of refrigerators for 24 hours by row sample, and 20ul MTT are added per hole later, are put and are continued to cultivate 4h in incubator, then will
Liquid is sucked out in hole, and 150ul DMSO are added, and surveys the OD values in every hole under 570nm with microplate reader.It was found that carrageenan oligosaccharide is added
The OD values of group are bigger than the OD values of control group, illustrate that cell viability improves, carrageenan oligosaccharide can improve low temperature（4℃）The work of lower cell
Power.
By the Human normal hepatocyte in exponential phase with 5 × 104A/ml is inoculated in 96 orifice plates, is inoculated with per hole
200ul is placed in 37 DEG C, 5%CO2Incubator in continue culture for 24 hours, the carrageenan oligosaccharide solution of various concentration is added（10、25、
50、100、 150、 200、 250、300 ug/ml）, it is control group to be not added with oligosaccharides（0）, every group sets 6 Duplicate Samples, by 96
Orifice plate is put into 1.5h in -20 DEG C of refrigerators, and liquid in fast melt hole, 20ul MTT are added per hole, put incubator later after 1.5h
In continue to cultivate 4h, then liquid in hole is sucked out, 150ul DMSO is added, survey the OD values in often hole under 570nm with microplate reader.Hair
The OD values that carrageenan oligosaccharide group is now added are bigger than the OD values of control group, illustrate that cell viability improves, carrageenan oligosaccharide can improve low
Temperature（-20℃）The vigor of lower cell.
By the Human normal hepatocyte in exponential phase with 5 × 104A/ml is inoculated in 96 orifice plates, is inoculated with per hole
200ul is placed in 37 DEG C, 5%CO2Incubator in continue culture for 24 hours, the carrageenan oligosaccharide solution of various concentration is added（10、
25、 50、100、150、 200、 250、300 ug/ml）（Contain 10%DMSO）, it is control group to be not added with oligosaccharides（10%
DMSO）, every group sets 6 Duplicate Samples, and 96 orifice plates are put into 3h in -20 DEG C of refrigerators, liquid in fast melt hole after 3h, later per hole
20ul MTT are added, puts and continues to cultivate 4h in incubator, then liquid in hole is sucked out, addition 150ul DMSO, with microplate reader in
The OD values per hole are surveyed under 570nm.It was found that the OD values that carrageenan oligosaccharide group is added are bigger than the OD values of control group, illustrate that cell viability carries
Height, carrageenan oligosaccharide can improve low temperature（-20℃）The vigor of lower cell.
2, in cell culture fluid LDH leakages rate measurement：Human normal hepatocyte in exponential phase is digested
Afterwards with 1 × 105A/ml is inoculated in 24 orifice plates, and 1ml is inoculated with per hole, is placed in 37 DEG C, 5%CO2Incubator in continue culture for 24 hours,
The carrageenan oligosaccharide solution of various concentration is added（25、50、100、150、200 ug/ml）, it is control group to be not added with oligosaccharides
（0）, every group sets three Duplicate Samples, and culture plate is put into 4 DEG C of refrigerators and is freezed for 24 hours（Or it is changed to serum-free after -20 DEG C of freezing 2h
Culture medium continues culture for 24 hours）Afterwards, the supernatant per hole is collected, and is marked, then addition and supernatant volume phase in every hole
Deng cell pyrolysis liquid lytic cell 30min after, collect and correspond to supernatant per hole lysate and mark.Put 4 DEG C it is spare.
Lactic dehydrogenase in colorimetric method for determining cell culture fluid（LDH）Leakage rate：Operating procedure is as follows：
It mixes well, reacts at room temperature colorimetric after 3-5mim, colorimetric wavelength 440nm is returned to zero with blank tube, measures each pipe extinction
Value（OD values）.
LDH leakages rate in cell culture fluid（%）=culture solution OD values/(culture solution OD values+cell pyrolysis liquid OD values) × 100
Containing lactic acid 4.328ml in per 200ml Matrix buffers, diethanol amine 3.86ml adds distilled water 160ml, with
The NaOH solution of 1mol/L adjusts pH to 8.8, and then plus distilled water is settled to 200ml.NAD solution：11.3mmol/L, i.e., often
Contain 15mgNAD, 4 DEG C of preservations in 2ml distilled waters.2,4 dinitrophenyl hydrazine solution：1mmol/L, 20mg2,4- dinitrophenylhydrazine
It is dissolved in the hydrochloric acid of 25ml 4mol/L, 100ml is added water to after dissolving.NaOH solution：0.4mol/L.
3, using Apoptosis after the bis- dye method detection process of flow cytometer FITC-PI, it is characterised in that cell is added not
With the carrageenan oligosaccharide solution of concentration（0、50、100、150、200ug/ml）Afterwards after -80 DEG C of freezing processings after 30d, collect thin
Born of the same parents 1-5 × 105It is a；It is secondary that cell is washed with PBS（Centrifuge 2000rpm, 5min）；The Annexin V of 500uL are added
Binding Buffer suspension cells；After 5uL Annexin V-FITC mixings are added, 5 uL Propidium are added
Iodide, mixing；It is protected from light, reacts at room temperature 10min；Up flow type detects（Ex=488 nm; Em=530 nm）Apoptosis situation.
Analysis of experimental results：
1、（1）The influence of the cell relative viability of 4 DEG C of processing of carrageenan oligosaccharide pair for 24 hours, as shown in Figure 1.
After plating cells are adherent, it is respectively 0,10,25,50,100,150,200,250,300 that concentration, which is added,
The carrageenan oligosaccharide solution of ug/ml is placed in 4 DEG C of processing for 24 hours, carries out MTT experiments, finds the carrageenan oligosaccharide that various concentration is added
The relative activity of cell is bigger than the cell viability for not adding carrageenan oligosaccharide group afterwards, and significant difference（*：P<
0.05;**：P<0.01）.
（2）The influence of the cell relative viability of the p- 20 DEG C of processing 1.5h of carrageenan oligosaccharide, as shown in Figure 2.
After plating cells are adherent, be added concentration be respectively 0,50,100,150,200, the carrageenan oligosaccharide of 300ug/ml it is molten
Liquid is placed in -20 DEG C of processing 1.5h, carries out MTT experiments, find the relative activity of cell after the carrageenan oligosaccharide of addition various concentration
Cell viability than not adding carrageenan oligosaccharide group is big, and significant difference（*：P<0.05;**：P<0.01）.
（3）Carrageenan oligosaccharide combines the influence of the cell relative viability of p- 20 DEG C of processing 3h with 10%DMSO, such as Fig. 3 institutes
Show.
After plating cells are adherent, the card that concentration is respectively 0,10,25,50,100,150,200,250,300ug/ml is added
Glue oligosaccharide solution is drawn, -20 DEG C of processing 3h is placed in, carries out MTT experiments, finds to be added cell after the carrageenan oligosaccharide of various concentration
Relative activity is bigger than the cell viability for not adding carrageenan oligosaccharide group, and significant difference（*：P<0.05;**：P<0.01,
Compare with 10% DMSO）.
2, the influence that carrageenan oligosaccharide protects cellular damage,
（1）For 24 hours, the variation of LDH leakages rate is as shown in Figure 4 in cell culture fluid for carrageenan oligosaccharide processing cell at 4 DEG C.
Addition carrageenan oligosaccharide group is reduced than not adding LDH leakage rates in carrageenan oligosaccharide group cell culture fluid, is illustrated thin
Born of the same parents' extent of damage reduces, and carrageenan oligosaccharide can reduce damage of 4 DEG C of low temperature to cell.
（2）Carrageenan oligosaccharide handles cell 2h at -20 DEG C, and the variation of LDH leakage rates is as shown in Figure 5 in cell culture fluid.
Addition carrageenan oligosaccharide group is reduced than not adding LDH leakage rates in carrageenan oligosaccharide group cell culture fluid, is illustrated thin
Born of the same parents' extent of damage reduces, and carrageenan oligosaccharide can reduce damage of -20 DEG C of low temperature to cell.
3, influence of the carrageenan oligosaccharide to Apoptosis
Carrageenan oligosaccharide handles cell 30d at -80 DEG C, and changes of cell apoptosis is as shown in the table, adds carrageenan oligosaccharide group
Increase than not adding carrageenan oligosaccharide group viable count, late apoptic number is reduced, and early apoptosis number is reduced, meronecrosis rate drop
It is low, illustrate that carrageenan oligosaccharide can also play the role of protecting cell at -80 DEG C.
Claims (1)
1. a kind of application of frost resistance K- carrageenan oligosaccharides, which is characterized in that frost resistance K- carrageenan oligosaccharides for cold storage of cell,
Freeze proof protection additive during freezing adds carrageenan oligosaccharide powder into frozen stock solution, matches during refrigerating, freezing
Frozen stock solution is made and comes freeze-stored cell, tissue, organ, limbs.
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| CN105274162B (en) * | 2015-09-18 | 2019-03-15 | 集美大学 | A kind of preparation method of different polymerization degree kappa-carrageenin oligose |
| CN105695387A (en) * | 2016-03-08 | 2016-06-22 | 大连大学 | Stress-resistant protectant based on carrageenan oligosaccharides as well as preparation method and application of protectant |
| CN109007776B (en) * | 2018-06-13 | 2021-08-03 | 福建农林大学 | Compound water-retaining agent for Chinese meat dishes as well as preparation method and application of compound water-retaining agent |
| CN109007779B (en) * | 2018-06-13 | 2021-07-23 | 福建农林大学 | Frozen minced fillet compound water-retaining agent and preparation method and application thereof |
| CN110724208A (en) * | 2019-10-30 | 2020-01-24 | 临沂艾德森生物科技有限公司 | Anti-freezing carrageenan oligosaccharide and preparation method and application thereof |
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| CN103114113A (en) * | 2013-01-06 | 2013-05-22 | 威海康博尔生物药业有限公司 | Preparation method for k-carrageenan oligosaccharide with low polymerization degree |
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| CN103114113A (en) * | 2013-01-06 | 2013-05-22 | 威海康博尔生物药业有限公司 | Preparation method for k-carrageenan oligosaccharide with low polymerization degree |
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