CN106117332A - A kind of method extracting ice structural protein from Herba Medicaginis - Google Patents
A kind of method extracting ice structural protein from Herba Medicaginis Download PDFInfo
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- CN106117332A CN106117332A CN201610726905.6A CN201610726905A CN106117332A CN 106117332 A CN106117332 A CN 106117332A CN 201610726905 A CN201610726905 A CN 201610726905A CN 106117332 A CN106117332 A CN 106117332A
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Abstract
A kind of method extracting ice structural protein from Herba Medicaginis, relates to a kind of method extracting ice structural protein from Herba Medicaginis.The present invention is to solve that in prior art, ice structural protein extraction raw material is limited, extraction process is complicated, and the ice structural protein that relatively costly, extraction obtains can not be in frozen food and the most wide variety of problem.Extracting method: one, take alfalfa hay leaf, pulverize and sieve；Two, diameter 2cm ice hockey is prepared；Three, alfalfa meal mixes with phosphate buffered solution, is centrifuged；Four, ice hockey adds supernatant extraction；Five, answering of ice structural protein carries.The inventive method extracting concentration is high, and impurity is few, the cost-effective time, can directly carry out SDS PAGE and measure its molecular weight, be not required to cold-induced, and raw material is conveniently easy to get, low cost, and extraction process simply can be commonly used in frozen food.The present invention is be applicable to extracting ice structural protein from Herba Medicaginis.
Description
Technical field
The present invention relates to a kind of method extracting ice structural protein from Herba Medicaginis.
Background technology
Ice structural protein (ice structuring proteins, ISPs), is also called and does not freezes albumen, antifreeze protein
(antifreeze proteins, AFPs) is a class by some Fish, insecticide, plant, fungus and antibacterial is the extraneous ring of opposing
Polypeptide produced by the stress of border, has and stops ice crystal formation, modification ice crystal form and the ability of suppression recrystallization, and it has
Certain heat stagnation activity, i.e. can reduce the freezing point of solution, but the impact on fusing point is less with non-colligative property form, so that molten
Between freezing point and the fusing point of liquid, difference occurs.The distinctive character of ice structural protein ensures biology existence under cryogenic,
Have and carry out specific binding ability with ice.2006, China's Ministry of Public Health was announced ice structural protein and can be added as novel foodstuff
Add agent to be applied in chilled food.
Limited owing to extracting ISPs raw material, mostly extract from cold water fish, the factors such as extraction process is complicated, cause its cost
Higher, expensive, not in frozen food and the most commonly used, the development to ISPs causes restriction.How to make
Produce the main task that simple, the cheap ISPs of extraction process is ISPs product development.The structure of plant ISPs and function
Complex, there is low grade fever stickiness (the most about 0.1～0.5 DEG C) and high recrystallization inhibition activity (exceeds Fish and insecticide ISPs
About 10～100 times) feature, a small amount of plant ISPs just has the brilliant inhibitory activity of higher weight.
Perennial Herba Medicaginis freezing tolerance is strong, and protein content is high.Herba Medicaginis (Medicago sativa), is commonly called as Herba Loti Tenuis, SANYE
Grass, area, Shanghai, Zhejiang is referred to as toothed burclover or grass is purple.Herba Medicaginis is pulse family Medicago herbaceos perennial.Well developed root system, stem and leaf enriches, point
The features such as branch ability is strong, has yield high, strong stress resistance.Plant height 30-70cm.Winter resistance is extremely strong, through winter extreme low temperature (-
Less than 45 DEG C) still can have more than 90% regrowth rate, there is extremely strong freezing tolerance.The gross protein value of Herba Medicaginis is generally
15%-22%, its protein concentrates leaf protein is considered one by FAO (Food and Agriculture Organization of the United Nation) (FAO) and has high exploitation valency
The novel protein resource of value.At present, alfalfa exploitation, study, utilize the study hotspot becoming each field.Use
Strong, the cheap Herba Medicaginis of frost resistance is as raw material, and its Antifreeze protein constituent content enriches, and has exploitation application greatly latent
Power, possesses the raw material advantage that industrial separation is extracted.
Document was only mentioned the method extracting ice structural protein from the Folium Ligustri Lucidi with auxiliary trash ice, but the method existed not
Definitiveness and shortcoming, complex process, and raw material need to carry out cold-induced, cold-induced principle: ice structural protein is at some
Biological cylinder accumulation and elimination have seasonal variety, typically disappear in summer, and autumn occurs, after Cold exposed, winter reaches
Peak, does not carry out strict control, and then can not strictly control extraction ratio or yield and quality trash ice usage amount；Fructus Ligustri Lucidi
The protein product of leaf will could express through low temperature induction, synthesize and accumulate.
Summary of the invention
The present invention is to solve that in prior art, ice structural protein extraction raw material is limited, extraction process is complicated, relatively costly,
Extracting the ice structural protein obtained can not be in frozen food and the most wide variety of problem, it is proposed that one carries from Herba Medicaginis
The method taking ice structural protein.
The present invention extracts the method for ice structural protein from Herba Medicaginis and sequentially includes the following steps:
One, alfalfa hay processes
Take alfalfa hay, and remove the impurity in alfalfa hay and dust, discard grass blade, retain alfalfa hay leaf, pulverize
Rear mistake 80 mesh sieve is standby；The moisture content of described alfalfa hay is 1.3%；
Two, the preparation of ice hockey
Place the glass bead of a diameter of 1cm in each ice hole site of silica gel ice tray, note distilled water to ice hole, then put
In the refrigerator of-18 DEG C, freezing, obtains the ice hockey of a diameter of 2cm；
Described silica gel ice tray be heatproof be the silica gel ice tray of-40 DEG C, silica gel ice tray is tabular, and a length of 24cm is a width of
7.3cm, plate is provided with 10 × 2 spherical ice holes, and the internal diameter in each ice hole is 2cm；
Three, prepared by supernatant
It being 1g:(5～30mL by solid-liquid ratio) alfalfa meal step one prepared is mixed to get with phosphate buffered solution
Mixed liquor, puts at room temperature by the mixed liquor obtained, and is stirred extracting 0.5～3.0h, and the mixed liquor after then extracting is put
In centrifuge, in centrifuge, centrifugal 14～16min obtain supernatant；
The pH of described phosphate buffered solution is 6.0～8.5, and phosphate concentration is 10～70mmol/L；Described stirring carries
Using magnetic stirring apparatus when taking, the rotating speed of magnetic stirring apparatus is 800～1000r/min；Described centrifugal time centrifuge rotating speed be
8000～10000r/min；
Four, ice hockey step 2 prepared adds upper surface and the supernatant liquid level phase in supernatant to the superiors' ice hockey
Flat, it is subsequently placed in-18 DEG C of refrigerators, extracts 2～5min, now ice hockey absorption ISPs reaches saturated, leaches ice hockey, and ice hockey melts
After obtain ice structural protein solution, obtain remaining supernatant after leaching ice hockey；
Five, answering of ice structural protein ISPs carries
In remaining supernatant, add ice hockey prepared by step 2, repetition step 4 2～4 times, collect and extract every time
The ice structural protein solution arrived, i.e. completes.
The present invention possesses following beneficial effect:
One, in the inventive method, the diameter of institute's ice hockey is 2cm, and circular surface improves contact area, improves ISPs and inhales
Freezing the glass bead that diameter is 1cm in attached amount ice hockey, after ice hockey melts, liquor capacity reduces, and the absorption of ISPs
Measure constant, add the concentration of ISPs, it is also possible to reduce the volume of ice hockey, cost-effective and operating time；
Two, in prior art, ISPs typically uses extraction, ammonium sulfate precipitation, ion exchange mechansim process, gel mistake
Five steps such as the separation process of filter chromatographic separation process and high performance liquid chromatography, method is loaded down with trivial details, and non-freeze proof active component is more,
Impurity multimass is poor, especially ammonium sulfate precipitation, isolated and purified brings the biggest difficulty to follow-up；And the present invention is according to ice structure
Specificity that albumen is combined with ice and irreversibility, be reduced to ice by the first two step and carry, crude separation out freeze proof for having
The ice structural protein of component, method is easy, and the ISPs purity that present invention extraction obtains is high, impurity is few, and avoids follow-up point
From purification, can directly carry out SDS-PAGE and measure its molecular weight；
Three, the present invention uses alfalfa hay to extract ice structural protein, is not required to cold-induced, and Alfalfa raw material sources are wide, convenient
It is easy to get, low cost, and extraction process simply can be commonly used in frozen food；
Four, the present invention uses phosphate buffer to combine ice lifting manipulation, by strict solid-liquid ratio, the pH of phosphate buffer, phosphoric acid
The concentration of buffer and ice hockey addition, it is possible to accurately control and improve the extraction ratio of ISPs, suitable for scale production；
Five, the extraction ratio of ice structural protein of the present invention is 2.15%～4.26%.
Detailed description of the invention:
Technical solution of the present invention is not limited to act detailed description of the invention set forth below, also includes between each detailed description of the invention
Arbitrarily reasonable combination.
Detailed description of the invention one: present embodiment is extracted the method for ice structural protein from Herba Medicaginis and sequentially included the following steps:
One, alfalfa hay processes
Take alfalfa hay, and remove the impurity in alfalfa hay and dust, discard grass blade, retain alfalfa hay leaf, pulverize
Rear mistake 80 mesh sieve is standby；
Two, the preparation of ice hockey
Place the glass bead of a diameter of 1cm in each ice hole site of silica gel ice tray, note distilled water to ice hole, then put
In the refrigerator of-18 DEG C, freezing, obtains the ice hockey of a diameter of 2cm；
Three, prepared by supernatant
It being 1g:(5～30mL by solid-liquid ratio) alfalfa meal step one prepared is mixed to get with phosphate buffered solution
Mixed liquor, puts at room temperature by the mixed liquor obtained, and is stirred extracting 0.5～3.0h, and the mixed liquor after then extracting is put
In centrifuge, in centrifuge, centrifugal 14～16min obtain supernatant；
Four, ice hockey step 2 prepared adds upper surface and the supernatant liquid level phase in supernatant to the superiors' ice hockey
Flat, it is subsequently placed in-18 DEG C of refrigerators, extracts 2～5min, now ice hockey absorption ISPs reaches saturated, leaches ice hockey, and ice hockey melts
After obtain ice structural protein solution, obtain remaining supernatant after leaching ice hockey；
Five, answering of ice structural protein carries
In remaining supernatant, add ice hockey prepared by step 2, repetition step 4 2～4 times, collect and extract every time
The ice structural protein solution arrived, i.e. completes.
Present embodiment possesses following beneficial effect:
One, in present embodiment method, the diameter of institute's ice hockey is 2cm, and circular surface improves contact area, improves
Freezing the glass bead that diameter is 1cm in ISPs adsorbance ice hockey, after ice hockey melts, liquor capacity reduces, and ISPs
Adsorbance constant, add the concentration of ISPs, it is also possible to reduce ice hockey volume, the cost-effective and operating time；
Two, in prior art, ISPs typically uses thick extraction, ammonium sulfate precipitation, ion exchange mechansim process, gel
Five steps such as the separation process of filtering chromatogram separation process and high performance liquid chromatography, method is loaded down with trivial details, and non-freeze proof active component is relatively
Many, impurity multimass is poor, especially ammonium sulfate precipitation, isolated and purified brings the biggest difficulty to follow-up；And present embodiment root
The specificity being combined with ice according to ice structural protein and irreversibility, be reduced to ice by the first two step and carry, crude separation out be
Having the ice structural protein of freeze resisting component, method is easy, and the ISPs purity that present embodiment extraction obtains is high, impurity is few, and
Avoid later separation purification, can directly carry out SDS-PAGE and measure its molecular weight；
Three, present embodiment uses alfalfa hay to extract ice structural protein, is not required to cold-induced, and Alfalfa raw material sources are wide,
Conveniently it is easy to get, low cost, and extraction process simply can be commonly used in frozen food；
Four, present embodiment use phosphate buffer combine ice lifting manipulation, by strict solid-liquid ratio, the pH of phosphate buffer,
The concentration of phosphate buffer and ice hockey addition, it is possible to accurately control and improve the extraction ratio of ISPs, suitable for scale production；
Five, the extraction ratio of present embodiment ice structural protein is 2.15%～4.26%.
Detailed description of the invention two: present embodiment is unlike detailed description of the invention one: alfalfa hay described in step one
Moisture content be 1.3%.Other steps are identical with detailed description of the invention one with parameter.
Detailed description of the invention three: present embodiment is unlike detailed description of the invention one or two: silica gel described in step 2
Ice tray be heatproof be the silica gel ice tray of-40 DEG C, silica gel ice tray is tabular, and a length of 24cm, a width of 7.3cm are provided with 10 × 2 on plate
Individual spherical ice hole, the internal diameter in each ice hole is 2cm.Other steps are identical with detailed description of the invention one or two with parameter.
Detailed description of the invention four: present embodiment is unlike one of detailed description of the invention one to three: described in step 3
The pH of phosphate buffered solution is 6.0～8.5, and phosphate concentration is 10～70mmol/L.Other steps and parameter are real with concrete
Execute one of mode one to three identical.
Detailed description of the invention five: present embodiment is unlike one of detailed description of the invention one to four: step 3 step
Stirring described in three uses magnetic stirring apparatus when extracting, and the rotating speed of magnetic stirring apparatus is 800～1000r/min.Other steps and ginseng
Number is identical with one of detailed description of the invention one to four.
Detailed description of the invention six: present embodiment is unlike one of detailed description of the invention one to five: described in step 3
Time centrifugal, the rotating speed of centrifuge is 8000～10000r/min.Other steps and parameter and one of detailed description of the invention one to five phase
With.
For checking beneficial effects of the present invention, carry out tests below:
Embodiment 1
The present embodiment extracts the method for ice structural protein from Herba Medicaginis and sequentially includes the following steps:
One, alfalfa hay processes
Take alfalfa hay, and remove the impurity in alfalfa hay and dust, discard grass blade, retain alfalfa hay leaf, pulverize
Rear mistake 80 mesh sieve is standby；The moisture content of described alfalfa hay is 1.3%；
Two, the preparation of ice hockey
Place the glass bead of a diameter of 1cm in each ice hole site of silica gel ice tray, to the water filling of ice hole, be subsequently placed in-18
DEG C refrigerator in freezing, obtain the ice hockey of a diameter of 2cm；
Described silica gel ice tray be heatproof be the silica gel ice tray of-40 DEG C, silica gel ice tray is tabular, and a length of 24cm is a width of
7.3cm, plate is provided with 10 × 2 spherical ice holes, and the internal diameter in each ice hole is 2cm；
Three, prepared by supernatant
It is that the alfalfa meal step one prepared of 1g:20mL is mixed to get with phosphate buffered solution and mixes by solid-liquid ratio
Liquid, puts at room temperature by the mixed liquor obtained, and is stirred extracting 2h, and the mixed liquor after then extracting is placed in a centrifuge,
In centrifuge, centrifugal 15min obtains supernatant；Add Coomassie brilliant blue after taking 1mL supernatant distilled water diluting 100 times to survey
Its light absorption value (Coomassie Brilliant Blue survey protein content)；
Described stirring uses magnetic stirring apparatus when extracting, and the rotating speed of magnetic stirring apparatus is 1000r/min；Described centrifugal time from
The rotating speed of scheming is 8000r/min；The pH of described phosphate buffered solution is 7.99, and phosphate concentration is 61.06mmol/L；
Four, ice hockey step 2 prepared adds upper surface and the supernatant liquid level phase in supernatant to the superiors' ice hockey
Flat, it is subsequently placed in-18 DEG C of refrigerators, extracts 2min, now ice hockey absorption ISPs and reach saturated, leach ice hockey, after ice hockey melts
Obtain ice structural protein solution, after leaching ice hockey, obtain remaining supernatant；
Five, answering of ice structural protein carries
In remaining supernatant, add ice hockey prepared by step 2, repeat step 42 times, collect what extraction every time obtained
Ice structural protein solution, i.e. completes.
The present embodiment possesses following beneficial effect:
One, in the present embodiment method, the diameter of institute's ice hockey is 2cm, and circular surface improves contact area, improves ISPs
Freezing the glass bead that diameter is 1cm in adsorbance ice hockey, after ice hockey melts, liquor capacity reduces, and the suction of ISPs
Attached amount is constant, adds the concentration of ISPs, it is also possible to reduce the volume of ice hockey, cost-effective and operating time；
Two, in prior art, ISPs typically uses thick extraction, ammonium sulfate precipitation, ion exchange mechansim process, gel
Five steps such as the separation process of filtering chromatogram separation process and high performance liquid chromatography, method is loaded down with trivial details, and non-freeze proof active component is relatively
Many, impurity multimass is poor, especially ammonium sulfate precipitation, isolated and purified brings the biggest difficulty to follow-up；And the present embodiment according to
Specificity that ice structural protein is combined with ice and irreversibility, be reduced to ice by the first two step and carry, crude separation out for tool
Having the ice structural protein of freeze resisting component, method is easy, and the ISPs purity that present invention extraction obtains is high, impurity is few, and avoids
Later separation purification, can directly carry out SDS-PAGE and measure its molecular weight；
Three, the present embodiment uses alfalfa hay to extract ice structural protein, is not required to cold-induced, and Alfalfa raw material sources are wide, side
Easy, low cost, and extraction process simply can be commonly used in frozen food；
Four, the present embodiment uses phosphate buffer to combine ice lifting manipulation, by strict solid-liquid ratio, the pH of phosphate buffer, phosphorus
The concentration of acid buffer and ice hockey addition, it is possible to accurately control and improve the extraction ratio of ISPs, suitable for scale production；
Five, use differential scanning calorimetry to analyze heat stagnation activity (THA) of Herba Medicaginis ISPs prepared by the present embodiment, adopt simultaneously
By heat stagnation activity (THA) of differential scanning calorimetry analytical standard bovine serum albumin (BSA) as a comparison, the heat stagnation of BSA is lived
Property be 0.00 DEG C～0.01 DEG C, almost without heat stagnation activity, and the heat stagnation of Herba Medicaginis ISPs prepared by the present embodiment activity (THA) is respectively
It is 0.52～0.54, illustrates that the Activity of Antifreeze of Herba Medicaginis ISPs prepared by the present embodiment is significantly stronger than standard bovine serum albumin
, and impurity is less, purity is higher (BSA)；
Six, the Herba Medicaginis ISPs prepared the present embodiment carries out amino acid analysis, and result is as shown in table 1, and Herba Medicaginis ISPs contains
17 kinds of aminoacid, total amino acid content is 12.52%, by can be calculated the ratio knowing hydrophilic amino acid with hydrophobic amino acid
For 7:4, illustrating that Herba Medicaginis ISPs hydrophilic amino acid is more than hydrophobic amino acid, Herba Medicaginis ISPs has stronger hydrophilic, with water
Molecule affinity is relatively strong, further relates to the reasonability of the present embodiment method, and illustrates prepared by the present embodiment by testing result
ISPs can be directly used for ice structural protein amino acid composition detection.
Seven, the Herba Medicaginis ISPs prepared the present embodiment carries out SDS-PAGE detection, and testing result is, Herba Medicaginis ISPs's is relative
Molecular weight is about 52KD, and main band is clear, also has band at 30KD to 35KD place, but is not clearly；It can be said that bright, this
Herba Medicaginis ISPs prepared by embodiment can be directly used for the detection of molecular weight.
Eight, the extraction ratio of the present embodiment method ice structural protein can reach 4.26%, and extraction ratio is: the ice knot finally given
The quality of structure albumen accounts for the percentage ratio of the gross mass of the multiple proteins contained in raw material；
Table 1
Claims (6)
1. the method extracting ice structural protein from Herba Medicaginis, it is characterised in that the method sequentially includes the following steps:
One, alfalfa hay processes
Take alfalfa hay, and remove the impurity in alfalfa hay and dust, discard grass blade, retain alfalfa hay leaf, mistake after pulverizing
80 mesh sieves are standby；
Two, the preparation of ice hockey
Place the glass bead of a diameter of 1cm in each ice hole site of silica gel ice tray, note distilled water to ice hole, be subsequently placed in-18
DEG C refrigerator in freezing, obtain the ice hockey of a diameter of 2cm；
Three, prepared by supernatant
It being 1g:(5～30mL by solid-liquid ratio) alfalfa meal step one prepared is mixed to get with phosphate buffered solution and mixes
Liquid, puts at room temperature by the mixed liquor obtained, and is stirred extracting 0.5～3.0h, the mixed liquor after then extracting be placed in from
In scheming, in centrifuge, centrifugal 14～16min obtain supernatant；
Four, ice hockey step 2 prepared adds in supernatant equal with supernatant liquid level, so to the upper surface of the superiors' ice hockey
Being placed in-18 DEG C of refrigerators, extract 2～5min, now ice hockey absorption ISPs reaches saturated, leaches ice hockey, and ice hockey obtains after melting
To ice structural protein solution, after leaching ice hockey, obtain remaining supernatant；
Five, answering of ice structural protein carries
In remaining supernatant, add ice hockey prepared by step 2, repetition step 4 2～4 times, collect what extraction every time obtained
Ice structural protein solution, i.e. completes.
A kind of method extracting ice structural protein from Herba Medicaginis the most according to claim 1, it is characterised in that: step one institute
The moisture content stating alfalfa hay is 1.3%.
A kind of method extracting ice structural protein from Herba Medicaginis the most according to claim 1, it is characterised in that: step 2 institute
State silica gel ice tray be heatproof be the silica gel ice tray of-40 DEG C, silica gel ice tray is tabular, a length of 24cm, a width of 7.3cm, on plate arrange
Having 10 × 2 spherical ice holes, the internal diameter in each ice hole is 2cm.
A kind of method extracting ice structural protein from Herba Medicaginis the most according to claim 1, it is characterised in that: step 3 institute
The pH stating phosphate buffered solution is 6.0～8.5, and phosphate concentration is 10～70mmol/L.
A kind of method extracting ice structural protein from Herba Medicaginis the most according to claim 1, it is characterised in that: step 3 institute
Stating when stirring is extracted and use magnetic stirring apparatus, the rotating speed of magnetic stirring apparatus is 800～1000r/min.
A kind of method extracting ice structural protein from Herba Medicaginis the most according to claim 1, it is characterised in that: step 3 institute
When stating centrifugal, the rotating speed of centrifuge is 8000～10000r/min.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104650199A (en) * | 2015-01-12 | 2015-05-27 | 江南大学 | Method for quickly and specifically purifying ice structure protein in barley grains |
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2016
- 2016-08-25 CN CN201610726905.6A patent/CN106117332A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104650199A (en) * | 2015-01-12 | 2015-05-27 | 江南大学 | Method for quickly and specifically purifying ice structure protein in barley grains |
Non-Patent Citations (3)
| Title |
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| 刘尚 等: ""一种女贞叶抗冻蛋白的分离纯化"", 《植物学通报》 * |
| 张超 等: ""特异性亲和吸附法纯化冬小麦麸皮抗冻蛋白"", 《中国粮油学报》 * |
| 曲敏 等: ""苜蓿冰结构蛋白的提取及对冷冻面团的影响"", 《食品科学》 * |
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