CN1101942A - Process for making F-1-P crystal protein from fresh tobacco leaves - Google Patents

Abstract

The process for preparing F-1-P crystal protein includes homogenating fresh tobacco leaves in the presence of reducer, press filtering, centrifugal separation to obtain homogenated clear liquid direct precipitation of raw F-1-P, re-dissolving raw F-1-P and recrystallizing to obtain F-1-P protein with special geometry.

Description

Process for making F-1-P crystal protein from fresh tobacco leaves

The present invention relates to the processing method of extracting effective components in green plants, exactly is a kind of novel method of producing the F-1-P crystallin in fresh tobacco leaf.

Contain water-soluble and water-insoluble substance in the dry (accounting for 10～20%) that the bright leaf of green plants is left after dewatering, the latter mostly is the fibrous texture material, the former is divided into two classes again, one class is that (molecular weight is no more than 10 to low molecule, 000) compound is as sugar, amino acid etc., another kind of is protein (molecular weight is more than 30,000).Protein divides two kinds again, a kind of be molecular weight 30,000～100, the mixed protein between 000, general designation F-2-P albumen is (hereinafter to be referred as P ₂), another kind is single albumen, molecular weight 550,000 claims F-1-P albumen (hereinafter to be referred as P ₁), it is a kind of enzyme relevant with photosynthesis, claims 1.5-bisphosphate ribose ketone carboxylase, Rubisco etc. again.Soluble proteins accounts for 20% of dry, P in the fresh tobacco leaf ₁And P ₂Respectively account for about 10%.

Highly purified P ₁Colourless, tasteless, performances such as its amino acid composition, protein efficiency ratio, PER, water-soluble, gelation, emulsifying property, suction oil suction all are better than other food protein additives, especially be fit to renal function disease patient and protein metabolism imbalance patient take, so its application prospect are very wide.The seventies is found by obtaining crystalline P in the fresh tobacco leaf ₁, and take from the generally non-crystallizable of other green plantss.

In fresh tobacco leaf, extract P ₁At first all need bright leaf and phyllopodium homogenate (promptly mashing, grind to form pasty state) to discharge effective ingredient wherein, are separated homogenate then,, obtain being dissolved with P with thicker solid matter of filtering (fiber-like) and pigment material ₁And P ₂Brown juice.To this juice, 1, use filter membrane and dialysis tubing to carry out ultrafiltration, dialysis, form crystal after holding back macromole.2, Sephadex G-25 or G-50 column chromatography.3, ammonium sulfate precipitation, polyethylene glycol system crystallization.4, isoelectric precipitation, percrystallization again after the redissolution.Above method industrialization is had any problem.United States Patent (USP) 4268632(applying date 79.9.4) a kind of thermal induction crystalline extraction process is disclosed, bright leaf heats (48～52 ℃) to homogenate after homogenate, separate after being cooled to room temperature, elimination fiber-like and pigment material get brown juice, then juice are cooled to 8 ℃, leave standstill after 24 hours and just obtain P ₁The octagon crystal.Heating can promote the cohesion of pigment material, separate more thorough, the P that crystallization is separated out ₁Purer.But consumes energy is wanted in heating, and Heating temperature wants strict control, otherwise will cause protein denaturation, forms the insoluble protein precipitation.

The purpose of this invention is to provide and a kind ofly simple and effectively just can obtain high-purity crystals P at normal temperatures ₁The preparation method, this method can realize industrialization, and easy to operate, be easy to control.

The present invention is achieved in that and at first directly precipitates the preparation crude product in the homogenate clear liquid, recrystallize after making it to redissolve by the ionic strength that changes solution then, thereby the crystal P that obtains having geometry in particular ₁Detailed process is as follows:

1, gets fresh tobacco leaf and phyllopodium, under the condition that reductive agent exists, carry out homogenate.Reductive agent can be to dredge basic ethanol, xitix, Sodium Metabisulfite etc., suppresses polyphenol oxidase product and proteinic covalent attachment.Homogenate in the pasty state, the disperse phase (B) of the solid of including fiber class (A), pigment, P ₁And P ₂And other impurity such as reductive agent and Polyphenols.

2, separate homogenate.A is removed in press filtration, and centrifugal or fractionation by adsorption is removed B, obtains brown or henna homogenate clear liquid, includes P ₁, P ₂With residual B and other impurity.

3, the homogenate clear liquid left standstill 1～5 hour, and isoelectric precipitation also separates P ₁, this precipitation is amorphous, is crude product.

4, crude product is purified.The variation of the ingenious land productivity salts solution of contriver ionic strength in the present invention comes protein purification.(〉=0.05M) sodium chloride solution stirs the crude product input, transfers PH to 7.5 simultaneously, P to get finite concentration ₁Dissolving, and other impurity do not dissolve, and separate and remove impurity.With clear liquid dilution (more than 1 times, promptly reducing the ionic strength of solution) or transfer pH value to 5.0～6.0 o'clock, the P of redissolution ₁Recrystallize is separated out, and separates to obtain pure product crystal P ₁, crystal is regular hexagon dodecahedron or square two pyramids of octagon.

Experiment showed, that except that sodium-chlor the soluble inorganic salt of sodium sulfate, SODIUMNITRATE, yellow soda ash, sodium sulphite, sodium-acetate etc. and sylvite, ammonium salt and calcium and magnesium all can be used to purifying P ₁PH is 7.5 during redissolution, when the ionic strength that reduces solution or when transferring pH value 5.0～6.0, and P ₁Recrystallize is separated out.

5, isolate crystal P ₁After clear liquid when regulating PH to 4, P ₂Precipitation is separated out, separated and collected P ₂

The present invention is preparation of industrialization high-purity crystals P ₁Opened up new approach, technology is simple, stable operation, control, not consumes energy easily.

Embodiment:

One, crude product P ₁Preparation.

Get the 1000g fresh tobacco leaf, add 500ml1% Sodium Metabisulfite solution grinding homogenate and get the pasty state homogenate.Homogenate is removed A with the squeezer press filtration, and filtrate was removed most of B in 10 minutes in the 4000rpm centrifugation, gets the homogenate clear liquid.The homogenate clear liquid left standstill four hours, and isoelectric precipitation is separated out P ₁,, get crude product P in 4000rpm centrifugation 5 minutes ₁

Two, crude product is purified.

1, gets 0.5MNaCl solution 200ml, with crude product P ₁Drop into and stir, transfer PH7.5, make P with 0.1NNaOH ₁Dissolving, in 4000rpm centrifugation 5 minutes, elimination impurity, filtrate is transferred PH5.0～6.0, leaves standstill P 2～4 hours ₁Recrystallize is separated out.Centrifugation gets 4～6g crystal P ₁, crystalline form is square two pyramids of octagon.

2, get 0.3MNaCl solution 200ml, with crude product P ₁Drop into and stir, transfer PH7.5, make P with 0.1NNaOH ₁Dissolving, later operation transfer PH5.0～5.5 to get 4～6g crystal P with embodiment 1 ₁

3, get 0.1MNaCl solution 200ml, with crude product P ₁Drop into and stir, transfer PH7.5, make P with 0.1NNaOH ₁Dissolving, impurity is fallen in centrifugation, and P was left standstill in 2 times of filtrate water dilutions 10 hours ₁Recrystallize is separated out.Centrifugation gets 4～6g crystal P ₁, crystalline form is the regular hexagon dodecahedron.

4, get 0.05NaCl solution 200ml, with crude product P ₁Drop into and stir, transfer PH7.5, make P with 0.1NNaOH ₁Dissolving, later operation is with embodiment 3, but only dilutes 1 times during dilute with water.Get 4～6g crystal P ₁

Concerning the NaCl solution of same concentration, after crude product redissolves, or dilution, or transfer PH, but equal recrystallize, productive rate basically identical, stable operation.

NaCl is the easiest to be obtained, and cheap, is suitable for industrial use most.In fact the soluble salt of other salt of sodium and sylvite, ammonium salt and calcium, magnesium all can be used to the P that purifies ₁, because the ionic strength of salts solution or PH can control.

Three, reclaim P ₂

Isolate crystallization P ₁After clear liquid transfer PH to 4.0, P ₂Precipitation is separated out, and centrifugation gets P ₂P ₂It also is a kind of food protein additive.

Claims (5)

1, a kind of novel method of producing the F-1-P crystallin in fresh tobacco leaf is made up of homogenate, precipitation, crystallization, sepn process, it is characterized in that:

(1), obtains the F-1-P crude product by directly precipitating to separate out and separate in the homogenate clear liquid;

(2), crude product is dissolved in concentration more than or equal to 0.05M, PH is the soluble inorganic salt solution of the salt of 7.5 sodium or potassium or ammonium or calcium or magnesium, remove by filter impurity;

(3), remove filtrate water dilution behind the impurity more than 1 times or transfer pH value between 5.0～6.0, separate obtaining the F-1-P crystallin that crystallization is separated out.

2, a kind of novel method of producing the F-1-P crystallin in fresh tobacco leaf according to claim 1, it is characterized in that: described inorganic salt are sodium-chlor or Repone K.

3, a kind of novel method of producing the F-1-P crystallin in fresh tobacco leaf according to claim 1 and 2, it is characterized in that: described inorganic salt are sodium-chlor.

4, a kind of novel method of producing the F-1-P crystallin in fresh tobacco leaf according to claim 1 is characterized in that: 2 times of the filtrate water dilutions behind the removal impurity.

5, a kind of novel method of producing the F-1-P crystallin in fresh tobacco leaf according to claim 1 is characterized in that: the filtrate behind the removal impurity is transferred pH value 5.0～5.5.