CN104178534B

CN104178534B - A kind of method of preparing TYR taking krill as raw material

Info

Publication number: CN104178534B
Application number: CN201410459646.6A
Authority: CN
Inventors: 薛长湖; 王彦超; 常耀光; 徐杰; 王静凤; 唐庆娟; 王玉明
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2014-09-10
Filing date: 2014-09-10
Publication date: 2016-05-18
Anticipated expiration: 2034-09-10
Also published as: CN104178534A

Abstract

The object of the present invention is to provide a kind of method of preparing TYR taking krill as raw material, the method has following operating procedure: krill powder, after endo protease and exoproteinase continuous hydrolysis, obtains the enzymolysis solution that contains free tyrosine; After the pH value of enzymolysis solution is adjusted to 5.5～5.7, through Vacuum Concentration, induced crystallization, in enzymolysis solution, be that adularescent crystal is separated out; Enzymolysis solution, after centrifugal treating, is got precipitation, with deionized water washing precipitation three times, after will precipitating and being dried, pulverizes, and obtains TYR. The invention provides a kind of TYR preparation method easy and simple to handle, with low cost. The method gentleness, production process is pollution-free, products obtained therefrom purity is higher.

Description

A kind of method of preparing TYR taking krill as raw material

Technical field

The invention belongs to amino acid preparing technical field, be specifically related to one and prepare L-taking krill as raw materialThe method of tyrosine.

Background technology

TYR is the primary raw material of the medicines such as synthetic peptide hormone, antibiotic, dopamine, extensively shouldFor fields such as medicine, food additives, biochemical industry and feeds. At present, the topmost life of TYRProduct method is taking human hair, feather etc. as raw material, after strong acid is hydrolyzed into amino acid, hydrolyzate is neutralized with alkaliTo faintly acid, amino acid is separated successively from mixture, recrystallization obtains amino acid sterling, this kind of methodTechnique is loaded down with trivial details, production cost is higher.

Japan Patent 2007-010821 discloses a kind of method of extracting TYR from soy sauce residues, the partyMethod adopts and adds alkali, heat treated residue of soya, and the then method of acid neutralization, need to add a large amount of chemical substances,Leaching process is comparatively loaded down with trivial details. Chinese patent CN101182297A discloses a kind of from producing the row of CYSTINEIn tapping, reclaim the method for TYR, in the method, mixed liquor is successively through activated-charcoal column absorption and hydroxideSodium solution desorption process, separates and obtains tyrosine. Chinese patent CN102863366A discloses a kind of from angleIn the cystine of dietary protein origin and tyrosine mixture, separate the method for tyrosine, the heating of the method employing acid adding,Add the mode of Organic Alcohol, separate and obtain tyrosine. Chinese patent CN103012179A discloses a kind of utilizationOne one-step refining method is extracted the method for tyrosine from hair, during the method employing is quick by cystine refinement mother liquorWith to after higher pH, through separating, acid-soluble, decolouring, refining neutralization obtain tyrosine fine work. But above-mentionedAll there is step complex operation in method, the low inferior problem of productive rate.

Summary of the invention

The object of the present invention is to provide a kind of method of preparing TYR taking aquatic products as raw material, particularlyPrepare a method for TYR taking krill as raw material, thereby supplement the deficiencies in the prior art, simplifyThe production technology of TYR.

The method of preparing TYR in the present invention taking krill as raw material, comprises the steps:

1) in the krill meal of the South Pole, add distilled water to make the aqueous solution, regulate the pH value of mixed liquor, then to mixedClose in liquid and add protease A, constant temperature stirs, and hydrolysis finishes rear heating enzymolysis reaction, centrifugal, getsClear liquid, obtains enzymolysis solution for the first time;

2) to adding Cathepsin B in enzymolysis solution for the first time, constant temperature stirs, and hydrolysis temperature is 40～60 DEG C,Hydrolysis time is 2～6h, and hydrolysis finishes rear heating enzymolysis reaction, centrifugal, gets supernatant, obtains secondInferior enzymolysis solution;

3), to adding 2MHCl in enzymolysis solution for the second time, then enzymolysis solution heating is for the second time carried out denseAfter contracting, obtain concentrate;

4) in concentrate, add crystal seed, be then cooled to adularescent crystal in solution and separate out;

5) by step 4) solution of adularescent crystal after centrifugal treating, get precipitation, wash by deionized waterPrecipitation, will precipitate be dried after, pulverize, obtain TYR.

Step 1) in protease A be food stage papain, trypsase, neutral proteinase or alkalescenceOne in protease, the pH value of mixed liquor is adjusted to 6.0～10.0, the quality of protease A and krill mealThan being 1:200～3:200, hydrolysis temperature is 40～60 DEG C, and hydrolysis time is 2～6h;

Step 1) in protease A preferred food product level trypsase, the pH of mixed liquor is adjusted to 8.0,Hydrolysis temperature is 50 DEG C, and hydrolysis time is 6h;

Step 2) in Cathepsin B be food stage flavor protease, the pH value of enzymolysis solution A is adjusted to7.0～9.0, the mass ratio of Cathepsin B and krill meal is 1:200～3:200, and hydrolysis temperature is 40～60 DEG C, waterThe solution time is 2～6h;

Step 3) in for the second time the pH value of enzymolysis solution be adjusted to 5.5～5.7, mode of heating is decompression heating,Heating-up temperature is 40～80 DEG C, and vacuum is 0.06～0.08Mpa;

Step 4) in crystal seed be TYR powder, the mass ratio of addition and krill meal is 0.01～0.05:100;

Step 4) in solution C be cooled to 20～30 DEG C, time of repose is 6～48h;

Step 5) in precipitation drying mode be freeze drying or heat drying.

The invention provides a kind of TYR preparation method easy and simple to handle, with low cost. The method gentleness,Production process is pollution-free, products obtained therefrom purity is higher. Krill powder is through endo protease and exoproteinaseAfter continuous hydrolysis, obtain the aqueous solution that contains free tyrosine, through Vacuum Concentration, induced crystallization, obtain pureSpend higher TYR crystal, yield is 0.8～1.6%, purity >=90%. Adopt krill manufactureWhen tyrosine, can also obtain the krill active peptide product that essential amino acids content is abundant.

Brief description of the drawings

Fig. 1: the present invention prepares the amino acid composition analysis figure of gained TYR crystal;

Fig. 2: powder X-ray-diffracting spectrum of preparing gained TYR crystal in the present invention.

Detailed description of the invention

Krill is a class oceanic invertebrate, is distributed in antarctic waters. According to statistics, krillReserves are hundred million tons of 6.5-10, are the huge protein resource treasure-houses of the mankind. In krill dry weight, protein content approximately65-75%, its amino acid composition rationally, is the important source material of carrying out protein higher value application.

The present invention is in the time preparing TYR with krill, and the yield that TYR is prepared in discovery is very low, because ofThis, applicant adopts the mode of twice enzymolysis in the process of preparation, makes tyrosine fully from albumen middle reachesFrom release; Meanwhile, in enzymolysis solution, add tyrosine crystal seed, the crystallization of free tyrosine in inducing solution,Thereby the yield that TYR is provided greatly, has shortened crystallization time.

Below in conjunction with embodiment, method of the present invention is described in detail.

Embodiment 1

1) get krill powder 5kg, in krill meal, add distilled water 60L, in mixed liquor, add 2MHClRegulate pH of mixed value to 7.5, be warming up to 45 DEG C, in mixed liquor, add neutral proteinase 25g, constant temperatureStirring reaction 5h, is heated to 100 DEG C and stops enzyme reaction, centrifugal, gets supernatant, obtains solution A;

2) in solution A, add 2MNaOH, the pH value to 7.5 of regulator solution A, is warming up to 50 DEG C,In solution A, add flavor protease 50g, constant temperature stirring reaction 4h, is heated to 100 DEG C and stops enzyme reaction,Centrifugal, get supernatant, obtain solution B;

3) in solution B, add 2MHCl, the pH value to 5.6 of regulator solution A, by solution B at 65 DEG CUnder condition, it is 20% that decompression is heated to solid content, and vacuum is 0.08MPa, obtains solution C;

4) in solution C, add TYR 2.5g, be cooled to 25 DEG C, constant temperature leaves standstill 48h, solution CIn be that adularescent crystal is separated out;

5) solution C is after centrifugal treating, and rotating speed is 5000r/min, gets precipitation; Precipitation is washed through deionizationWash after three times, as for dry in 85 DEG C of baking ovens; After dry, pulverize, obtain TYR.

In the present embodiment, gained TYR is white powder crystal, and yield is 1.05%. As 1, accompanying drawingShow, content >=90% of TYR in product of the present invention, purity is high; Figure 2 shows that TYR productX-diffraction pattern, shown in be all the characteristic crystal peak of TYR.

Embodiment 2

1) get krill powder 10kg, in krill meal, add 95% ethanol 100L, in reactor, stir6h, centrifuging and taking precipitation, vacuum drying obtains degreasing krill meal;

2) extracting degreasing shrimp med 5kg adds distilled water 100L in degreasing shrimp med, is warming up to 50 DEG C, to mixingIn liquid, add 2MHCl to regulate pH of mixed value to 8.0, in mixed liquor, add trypsase 50g, constant temperatureStirring reaction 6h, is heated to 100 DEG C and stops enzyme reaction, centrifugal, gets supernatant, obtains solution A;

2) in solution A, add 2MHCl, the pH value to 7.5 of regulator solution A, is warming up to 45 DEG C,In solution A, add flavor protease 50g, constant temperature stirring reaction 6h, is heated to 100 DEG C and stops enzyme reaction,Centrifugal, get supernatant, obtain solution B;

3) in solution B, add 2MHCl, the pH value to 5.7 of regulator solution A, by solution B at 70 DEG CUnder condition, it is 25% that decompression is heated to solid content, and vacuum is 0.08MPa, obtains solution C;

4) in solution C, add TYR 2.0g, be cooled to 30 DEG C, constant temperature leaves standstill 32h, solution CIn be that adularescent crystal is separated out;

5) solution C is after centrifugal treating, and rotating speed is 5000r/min, gets precipitation; Precipitation is washed through deionizationWash after three times, through freeze drying, pulverize, obtain TYR.

In the present embodiment, gained TYR is white powder crystal, and yield is 1.25%, purity >=90%.

Embodiment 3

3) in solution A, add 2MHCl, the pH value to 5.7 of regulator solution A, by solution A at 70 DEG CUnder condition, it is 25% that decompression is heated to solid content, and vacuum is 0.08MPa, obtains solution B;

4) by solution B, be cooled to 30 DEG C, constant temperature leaves standstill 48h, is that adularescent crystal is separated out in solution B;

5) solution B is after centrifugal treating, and rotating speed is 5000r/min, gets precipitation; Precipitation is washed through deionizationWash after three times, through freeze drying, pulverize, obtain TYR.

In the present embodiment, gained TYR is white powder crystal, and yield is 0.65%.

Claims

1. a method of preparing TYR taking krill as raw material, is characterized in that, described methodComprise the steps:

1) in the krill meal of the South Pole, add distilled water to make the aqueous solution, regulate the pH value of aqua liquid, then to waterIn solution, add protease A, constant temperature stirs, and hydrolysis finishes rear heating enzymolysis reaction, centrifugal, getsClear liquid, obtains enzymolysis solution for the first time;

5) by step 4) solution of adularescent crystal after centrifugal treating, get precipitation, wash by deionized waterPrecipitation, will precipitate be dried after, pulverize, obtain TYR;

Described step 1) in protease A be papain, trypsase, neutral proteinase, alkalescenceAny one or several in protease;

Described step 2) in Cathepsin B be flavor protease, the pH value of enzymolysis solution is conditioned for the first timeBe 7.0～9.0, the mass ratio of Cathepsin B and krill meal is 1:200～3:200, and hydrolysis temperature is 40～60 DEG C,Hydrolysis time is 2～6h;

Described step 4) in crystal seed be TYR powder, the mass ratio of addition and krill meal is0.01～0.05:100。

2. the method for claim 1, is characterized in that described step 1) in the pH of the aqueous solutionValue is adjusted to 6.0～10.0.

3. the method for claim 1, is characterized in that described step 1) middle protease A and phosphorusThe mass ratio of shrimp med is 1:200～3:200, and hydrolysis temperature is 40～60 DEG C, and hydrolysis time is 2～6h.

4. method as claimed in claim 3, is characterized in that described step 1) in protease A beTrypsase, and the pH of the aqueous solution is adjusted to 8.0, and hydrolysis temperature is 50 DEG C, and hydrolysis time is 6h.

5. the method for claim 1, is characterized in that described step 3) in enzymolysis is molten for the second timeThe pH value of liquid is adjusted to 5.5～5.7, and mode of heating is decompression heating, and heating-up temperature is 40～80 DEG C, vacuumDegree is 0.06～0.08Mpa.

6. the method for claim 1, is characterized in that described step 4) in solution be cooled to20～30 DEG C, time of repose is 6～48h.

7. the method for claim 1, is characterized in that described step 5) the middle dry side of precipitatingFormula is freeze drying or heat drying.