CN104650199A - Method for quickly and specifically purifying ice structure protein in barley grains - Google Patents

Abstract

The invention relates to a method for quickly and specifically purifying ice structure protein in barley grains. The method is characterized by comprising the following steps: (1) impurity removal of a solution and adjustment of the concentration of protein; (2) determination of a freezing curve; (3) programmed cooling; (4) introduction of a crystal nucleus; (5) affinity adsorption; (6) separation; and (7) freeze-drying. The method for quickly and specifically purifying the ice structure protein in the barley grains has the characteristics of short consumed time and high efficiency by introducing the crystal nucleus at the stage of solution supercooling to produce a large amount of small ice crystals to specifically purify the ice structure protein through using the characteristic that the ice structure protein of the barley grains can be specifically affined to ice crystals, and therefore the novel method for specifically purifying the ice structure protein in the barley grains is established.

Description

The quick specificity purification process of ice structural protein in a kind of barley grain

Technical field: the present invention relates to Food Engineering Development field, is specifically related to the quick specificity purification process of ice structural protein in a kind of barley grain.

Background technology:

After cold induction, a series of physiology, biochemistry and molecular biological change can occur in plant materials, comprise cold temperature induced protein, carbohydrate, the synthesis of osmotic adjustment and secretion, membrane structure and composition change the appearance etc. with some new enzymes.Ice structural protein (ISPs) is the one in plant low temperature inducible protein, the characteristic of have heat stagnation activity, regulate protoplastis supercooled state, modify ice crystal form and suppression recrystallization.Different from fish, insect ISPs, the antifreeze properties of plant ISPs is that low heat stagnation is active and high recrystallization inhibition is active, makes it be more suitable for being applied in frozen product, to reduce the frozen product quality deterioration preserved and cause because of temperature fluctuation in transportation.

The purifying major part of current ISPs adopts conventional series of columns chromatographic technique to carry out, and only has the report of a small amount of utilization " freezing cold finger " (Icefinger) method specificity purifying ISPs.Conventional series of columns chromatography method is applied widely, but it is loaded down with trivial details to there is technique, the low and shortcoming that long processing period, treatment capacity are little of the rate of recovery.And in purge process, usually select active relatively high component to carry out next step purification process, therefore there is undetected possibility.Absorption-the inhibitory theory of ISPs thinks that ISPs is adsorbed onto ice crystal surface by self thus suppresses the growth of ice crystal." freezing cold finger " method specificity purifying is the ISPs's that purifying is combined with ice from crude extract based on the absorption-inhibitory theory of ISPs.The defect that " freezing cold finger " method can avoid conventional column chromatography undetected, can obtain multiple ISPs simultaneously, but higher to the requirement of equipment and operational condition.And will lower the temperature to " freezing cold finger " on the one hand in purge process, again hyperthermic treatment is carried out to crude extract system on the other hand and to conduct heat the reduction of the system temperature caused to make up " freezing cold finger ", there is energy dissipation problem.In addition, because " freezing cold finger " upper effective ice adsorption surface is limited, operation required time is longer.

Freezing solution comprises cooling and crystallization two processes.In temperature-fall period, temperature not to be solidified or the liquid of crystallization is called supercooled liquid yet lower than zero pour.Supercooled liquid be in metastable, as long as have small sample perturbations formed the nucleus of condensation just can bring out crystallization, form a large amount of little ice crystal instantaneously.The feature of active according to the low heat stagnation of plant ISPs in high recrystallization inhibition activity, the present invention utilizes ISPs to be adsorbed onto ice crystal surface by self thus suppresses the characteristic of the growth of ice crystal, establish the novel quick specificity purification process that a kind of ice crystal utilizing solution self to produce carries out ISPs affinity purification, utilize the method can specific from barley grain ISP crude extract purifying ISPs, there is high specificity, require low to equipment and operational condition, energy conservation and treatment capacity are large, short feature consuming time.

Summary of the invention:

The object of the invention is to avoid the deficiencies in the prior art, a kind of quick specificity purification process of barley grain ice structural protein is provided.The present invention, to be separated the ISPs crude extract that obtains by vacuum infiltration centrifuging for purifying object from barley grain, by controlling solution rate of temperature fall and solution temperature, make solution self produce a large amount of ice crystal and with ISPs, affine absorption occur, then can obtain forming relatively simple and that activity is higher ISPs through solid-liquid separation, thus provide technical support for widening ISPs further in the application in each field.

The technical scheme adopted for realizing object of the present invention is:

(1) solution removal of impurities and protein concentration regulate: the millipore filtration adopting 0.22 μm, the graininess impurity in removing ISPs crude extract, and Function protein concentration is 0.1 ~ 1.0mg/mL;

(2) freezing curve measures: utilize thermopair, and measure the freezing curve of solution, determine freezing point temperature and supercooling temperature, rate of temperature fall is 0.5 ~ 3 DEG C/min;

(3) programmed cooling: adopt low-temperature refrigeration circulating device to carry out programmed cooling to solution, rate of temperature fall is 0.5 ~ 3 DEG C/min, cooling terminal temperature is-10 ~ 0 DEG C;

(4) nucleus is introduced and ice crystal formation: can deliver directly mode using devitrified glass as nucleus and add nucleus, also bring out generation nucleus by concussion, the mode of tapping, ice crystal is formed in a large number;

(5) affine absorption: the frozen water suspension formed in (4) is stirred 1 ~ 5min with the rotating speed of 10-100r/min at-10 ~ 0 DEG C;

(6) solid-liquid separation: under low temperature, use the sand core funnel of precooling that the suspension obtained in (5) is carried out solid-liquid separation, the ice slag of acquisition, except the protein solution carried secretly in deicing slag, is put in the phosphate buffered saline buffer of 50mM, is made it melt by vacuum filtration;

(7) lyophilize: the solution lyophilize obtained in (6) is namely obtained the barley grain ice structural protein after ice affinity purification.

The advantage of the inventive method:

1, the present invention carries out the affine absorption of ice of ISPs with the ice crystal that protein solution self produces, and substantially increases the requirement reduced equipment, can reduce the cost of ice structural protein.

2, the present invention utilizes solution to introduce the affine absorption of ice that principle that nucleus can produce a large amount of little ice crystal carries out ISPs spending cold stage, and substantially increasing can the quantity of ice crystal of affine ISPs and surface-area, shortens affine required time, raising purification efficiency.

3, the present invention does not want the plant and instrument of any special expensive, and experiment condition is simple and easy to realize, and meets the requirement of industrialization scale operation completely.

Accompanying drawing illustrates:

Figure 1 shows that the quick specificity purification process schema of barley grain ISPs in the embodiment of the present invention 1;

Figure 2 shows that the freezing curve figure of barley grain ISPs in the embodiment of the present invention 1;

To Figure 3 shows that in the embodiment of the present invention 1 the active comparison diagram of heat stagnation before and after the quick specificity purifying of barley grain ISPs;

To Figure 4 shows that in the embodiment of the present invention 1 molecular weight distribution comparison diagram before and after the quick specificity purifying of barley grain ISPs.

Embodiment:

Embodiment 1

(1) solution removal of impurities and protein concentration regulate: the millipore filtration adopting 0.22 μm, the graininess impurity in removing crude extract, and Function protein concentration is 0.5mg/mL;

(2) freezing curve measures: utilize thermopair, measures the freezing curve of protein solution, rate of temperature fall about 1 DEG C/min;

(3) programmed cooling: adopt low-temperature refrigeration circulating device to carry out programmed cooling to protein solution, rate of temperature fall is 1 DEG C/min, cooling terminal temperature is-7 DEG C;

(4) nucleus is introduced: bring out generation nucleus by tapping mode, ice crystal is formed in a large number;

(5) affine absorption: the frozen water suspension formed in (4) is stirred 3min with the rotating speed of 50r/min at-7 DEG C;

(6) be separated: at-7 DEG C, use the sand core funnel of precooling that the suspension obtained in (5) is carried out solid-liquid separation, the ice slag of acquisition, except the protein solution carried secretly in deicing slag, is put in the phosphate buffered saline buffer of 50mM, is made it melt by vacuum filtration;

(7) lyophilize: the solution lyophilize obtained in (6) is namely obtained the barley grain ice structural protein after ice affinity purification.

Active by the heat stagnation of barley grain ISPs extract after determine with dsc method ice affinity purification, and compare with the heat stagnation activity of original barley grain extract, result is as Fig. 3.As can be seen from the figure, after ice affinity purification, ISPs Solution Active is greatly improved.

Adopt Superdex-G75 gel column to carry out wash-out to protein solution before and after ice affinity purification, result is as Fig. 4.As can be seen from the figure, after ice affinity purification, the molecular weight distribution of ISPs solution there occurs change, proves that the non-ISPs of part is removed.

Claims (1)

1. the quick specificity purification process of ice structural protein in barley grain, its feature comprises the following steps:

(1) crude extract removal of impurities and protein concentration regulate: the millipore filtration adopting 0.22 μm, the graininess impurity in removing crude extract, and Function protein concentration is 0.1 ~ 1.0mg/mL;

(2) freezing curve measures: utilize thermopair, and measure and record the freezing curve of (1) middle solution, determine freezing point temperature and supercooling temperature, rate of temperature fall is 0.5 ~ 3 DEG C/min;

(3) programmed cooling: adopt low-temperature refrigeration circulating device to carry out programmed cooling to solution in (1), rate of temperature fall is 0.5-3 DEG C/min, cooling terminal temperature is-10 ~ 0 DEG C;

(4) nucleus is introduced: using devitrified glass as nucleus, and employing is delivered directly mode and added solution, also brings out generation nucleus by concussion, the mode of tapping, ice crystal is formed in a large number;

(5) affine absorption: the frozen water suspension formed in (4) is stirred 1 ~ 5min with the rotating speed of 10 ~ 100r/min at a constant temperature;

(6) be separated: under low temperature, use the sand core funnel of precooling that the suspension obtained in (5) is carried out solid-liquid separation, the ice slag of acquisition, except the solution carried secretly in deicing slag, is placed in the phosphate buffered saline buffer of 50mM, makes it melt by vacuum filtration;

(7) lyophilize: the solution lyophilize obtained in (6) is namely obtained the barley grain ice structural protein after ice affinity purification.