CN110467669A - The extracting method of Zinc metal sulfur protein zinc in one seed oyster - Google Patents
The extracting method of Zinc metal sulfur protein zinc in one seed oyster Download PDFInfo
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- CN110467669A CN110467669A CN201910660927.0A CN201910660927A CN110467669A CN 110467669 A CN110467669 A CN 110467669A CN 201910660927 A CN201910660927 A CN 201910660927A CN 110467669 A CN110467669 A CN 110467669A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/825—Metallothioneins
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Abstract
The extracting method of Zinc metal sulfur protein in a seed oyster disclosed by the invention, comprising the following steps: take fresh oyster, 24-48h is impregnated in brine ice, peel off and take oyster meat, and shear the fragment for being 1-3cm to diameter；Tissue lysates, dithiothreitol (DTT) are added, obtain homogenate through glass homogenizer homogenate 10-20min；It is added in fat hydrolase, cellulase, polysaccharide hydrolase and homogenate, filtering；80 DEG C of heating water baths digest mixture, and plant acid for adjusting pH is added in filtrate and obtains PHYTIC ACID ZINC SALT precipitating to 3.5-4.5, then adjusts pH to 1 for filtering, and filtering removal phytic acid cadmium obtains metallothionein solution；Metallothionein is separated using molecular sieve gel chromatography；PHYTIC ACID ZINC SALT is taken to precipitate, adjust pH to 8-10, filtering, gained filtrate is mixed with gained metallothionein, 50-70 DEG C is heated to, is stirred to react to obtain Zinc metal sulfur protein, utilize different conditions biological zinc, Zinc metal sulfur protein chelate is obtained, heavy metal free residual, purity is high, foreign protein is few, utilization rate of zinc is high, recovery rate is high.
Description
Technical field
The present invention relates to oysters to extract field, and in particular to the extracting method of Zinc metal sulfur protein in a seed oyster.
Background technique
Zinc is the indispensable microelement of our human bodies, and the zinc supplementation reagent suitable for human body experienced three kinds of forms: such as sulphur
The inorganic zinc of sour zinc, zinc chloride, zinc nitrate etc., such as grape saccharic acid zinc, licorzinc, zinc acetate, zinc citrate, amino-acid zinc, lactic acid
The organic zinc of zinc etc., the biological zinc of the object that boils together of protein and zinc, biological zinc are called zinc albumen, zinc-binding protein for another example, care for name
Think the conjugate that justice is protein and zinc, it is without any side effects to human body for zinc albuminate is compared to inorganic zinc, organic zinc, it can
Bfore meals, activity is high, can effectively facilitate absorption and utilization of the human body to various nutrients, and will not the nutrients such as antagonism calcium iron
Absorption, to be finally reached the effect for making human body and supplementing from diet various nutrients, the connection between them either
With the compound form of Covalent bonding together, it is also possible to the chelate based on coordinate bond, the kind of protein in the Nature
Class is up to millions of kinds, therefore theoretically, and zinc albuminate can also have millions of kinds of even more, and Zinc metal sulfur proteins
It (Zn-MT) is a kind of low molecular weight protein rich in zinc and sulfydryl, while zinc supplementation, in heavy metal detoxification, micro member
Element metabolism, removing free radical, antagonism lipid peroxidation etc. play an important role.
Oyster also known as raw oyster, also cry oyster, and fresh oyster meat claims oyster " sea in Bluish white, soft delicacy, European people
In milk ", be in all foods containing zinc it is the most abundant, every 100g oyster meat, aqueous 87.1%, 71.2mg containing zinc, how male
It is a key subjects that zinc in oyster, which is used as biological zinc extraction for human body, in the prior art, such as patent
The shortcomings that CN201010234452 discloses from oyster the synchronous method for extracting taurine, biological zinc and polypeptide, the method be
Since sonioation method is to generate bubble when acting on solution using high frequency machinery wave, bubble parameters and broken cavitation occur existing
As, and shock wave caused by cavitation phenomenon and shearing force make cell rupture, ultrasonic wave itself causes shock wave and shearing force not properly
Tropism and molecular cleavage ability, therefore free biological zinc can only be released, it can not with the biological zinc of zinc protein structure for major part
It releases, the biological zinc molecular weight that dissociates is different, causes the disadvantages of bio-absorbable utilization rate is low, absorption is slow, therefore to the patent
Improvement patent CN105476014A free biological zinc and zinc albumen are extracted using physical grinding and enzymatic isolation method, however, and being somebody's turn to do
Method can only obtain amino-acid zinc or polypeptide zinc, amino-acid zinc belong to organic zinc, they are that weak acid and mild base salt and gastric acid combine,
Zinc chloride can be still generated, zinc chloride is highly corrosive agents, to the irritating effect of gastrointestinal tract, causes nausea and vomiting etc., therefore have one
Fixed side effect, and because zinc content is higher, the absorption of the nutrients such as energy antagonism calcium iron cannot be with mending, and long-term use can cause to lack
The symptoms such as calcium anaemia, in addition this method molecule sieve separation biological zinc, such as more because molecular sieve can only act on macromolecular separation
Peptide, protein, nucleic acid, polysaccharide etc. can enter all holes of gel with mobile phase for the zinc ion of small molecule such as free state
Hole is finally flowed out from column, and the zinc of free state is caused to mix the loss of the impurity such as enzymic hydrolysates, and recovery rate is low.
Summary of the invention
To solve the above problems, the present invention provides the extracting method of Zinc metal sulfur protein in a seed oyster, different shapes are utilized
The biological zinc of state targetedly obtains Zinc metal sulfur protein chelate, and heavy metal free residual, purity is high, foreign protein are few, zinc utilizes
Rate is high, recovery rate is high.
The technical solution that the present invention solves the problems, such as is to provide the extracting method of Zinc metal sulfur protein in a seed oyster, including
Following steps: (1) taking fresh oyster, 24-48h impregnated in brine ice, peels off and takes oyster meat, and shearing to diameter is 1-3cm
Fragment；(2) Tissue lysates, dithiothreitol (DTT) are added, obtain homogenate through glass homogenizer homogenate 10-20min；(3) add
Enter in fat hydrolase, cellulase, polysaccharide hydrolase and homogenate, filter, obtains enzymatic hydrolysis liquid mixture；(4) 80 DEG C of water-baths
Heating enzymatic hydrolysis mixture, filtering are added in filtrate and plant acid for adjusting pH to 3.5-4.5, obtain PHYTIC ACID ZINC SALT precipitating, then adjust pH to
1, filtering removal phytic acid cadmium obtains metallothionein solution；(5) metallothionein is separated using molecular sieve gel chromatography；(6) it takes
PHYTIC ACID ZINC SALT precipitating, adjusts pH to 8-10, and gained filtrate is mixed with metallothionein obtained by step (5), is heated to 50- by filtering
70 DEG C, it is stirred to react to obtain Zinc metal sulfur protein.
Preferably, the Tissue lysates include the Triton X- of the Tris-base of 50 mmol/L Ph 7.4,1%
100,0.25% deoxysodium cholate, the EDTA of NaCl, 1mmol/L of 150mmol/L, the leupeptin of 1 μ g/ml, 1 μ g/ml
Aprotinin, 1 μ g/ml stomach cardia inhibitor, 1mmol/L PMSF.
Preferably, the additional amount of the Tissue lysates is 750-1000ml, in terms of 100g oyster meat.
Preferably, the additional amount of the dithiothreitol (DTT) is 10-20g, in terms of 100g oyster meat.
Preferably, the fat splitting enzyme activity is 5-7U/g, and additional amount 5-10mg, the cellulase activity is 4-
7U/g, additional amount 1-5mg, the polysaccharide hydrolysis enzyme activity be 20-30 U/g, additional amount 2-8mg, wherein additional amount with
100g oyster meat meter.
Preferably, gel used in the molecular sieve gel chromatography is sephadex Sephadex G-25, elution used
Liquid is Tris- glycine.
Preferably, the step (3) and step (4) further include step (3.1): addition enzyme activity is 10-20U/g 20mg
Proteinase, by oyster meat for 100g in terms of.
Oyster is due to the pollution of marine environment, and there are heavy metal such as Cd2+The case where pollution, and together with Zn existing for itself2+
The expression of metallothionein in Oysters is induced, therefore there are the metallothioneins of rich content in oyster, it contains 61 ammonia
Base acid, wherein 21 amino acid are cysteine, each MT molecule in this way can at most combine seven metallic atoms, practical
In situation, induce synthesis metallothionein will not each molecule combine full metal ion, with chelating metal ion
Zn-MT, Cd-MT form and non-chelating metal ion MT form exist, wherein the zinc or cadmium per molecule quantity of chelating are not
Deng, they belong to soluble metal albumen, it is easy to take in the MT containing heavy metal if directly extracting the albumen containing MT, draw
Play human poisoning, it is therefore desirable to the Cd of removal and MT chelating2+, and in order to improve the recovery rate of Zinc metal sulfur protein, as far as possible
By the Zn in oyster2+Extract completely and with metallothionein chelating and remove unrelated foreign protein.
Zinc metal sulfur protein is that a kind of positive tetrahedron metal sulfydryl is endured and race, and crystallization structure is mutually quite stable, but it can still lead to
It crosses and zinc is provided and activates many that can supplement trace element zinc and protein necessary to mammal using zinc as the enzyme of prothetic group, and
Good absorbing, antagonism hazardous trace elements lead, cadmium etc., activates a variety of antioxidases, to eliminate the oxidative damage of free radical, keeps
The stabilization of cell membrane；The immune function that mammal can be enhanced has and promotes the normal dual regulation of endocrine, in solution shape
State easily generates dimer or Multimeric forms when being oxidized, and this programme quickly completes Zn-MT extraction at low ambient temperatures, and
Dithiothreitol (DTT), which is added, prevents the formation of dimer as reducing agent.
MT is free of alpha-helix and beta-pleated sheet, there is very firm conformation, and chemical property is stablized, can be in 80 DEG C of water
It is middle to survive 30 minutes, it survives 3 minutes and non-inactivation in 100 DEG C of water, with very strong heat resistance and resists protease digestion
Ability removes unrelated foreign protein using the property in this programme.
Although MT molecule itself is acidproof, heat-resisting, MT-Zn in pH3.5-4.5 legibility from MT-Cd is easy when pH is less than 1
Dissociation, and phytic acid has a large amount of active groups such as carboxyl and phosphate, is a kind of multiple tooth chelating agent of metal, when with metal complex
When, multiple chelating rings are easily formed, are formed by that Stability of Metal Complexes is extremely strong, and immediately in strong acid environment, phytic acid can also be formed surely
Fixed complex compound, and phytic acid acts on metal ion and is also confined to calcium, magnesium, iron, aluminium, shorter mention heavy metal ion, we
Phytic acid and MT competition coordination Zn in case2+, gained complex compound is white flock precipitate, then obtains high-purity by molecular sieve gel method
MT molecule, zinc and metallothionein in oyster are utilized to greatest extent, obtains Zinc metal sulfur protein.
This programme has the beneficial effect that
1. low temperature environment induces the synthesis of metallothionein in oyster, the load of sufficient amount is provided for subsequent full utilization biological zinc
Body avoids the waste of Zn-ef ficiency in oyster, while salt water plays the role of sterilization, avoids microbial bacterial to biological zinc or oyster egg
It is white to degrade；
2. cracking Oyster Tissue using Tissue lysates, homogenizer homogenate is avoided using the epistasis means such as ultrasound, more complete to obtain
To the protein ingredient in biological zinc and oyster, while in homogenization process, dithiothreitol (DTT), which is added, prevents Zinc metal sulfur protein shape
At two innings or polymer, it is not easy to zinc and is separated with metallothionein；
3. fat hydrolase, cellulase, polysaccharide hydrolysis enzyme hydrolysis Oyster Tissue homogenate, sufficiently release biological zinc, and be convenient for
Subsequent step (6) alkali removes hydrolysate fatty acid, and utilizes 80 DEG C of denaturation foreign proteins of metallothionein high-temperature stability, obtains
To the metallothionein of high-purity；
4. the metallic zinc of dissociation and metallothionein chelating forms PHYTIC ACID ZINC SALT precipitating, then adjusts pH when pH is less than 3.5-4.5
To 1, dissociating metals sulfoprotein and cadmium ion remove phytic acid cadmium, obtain the metallothionein of non-chelated metal ions, and separation
PHYTIC ACID ZINC SALT and phytic acid cadmium, prevent the loss of zinc in extraction process, obtain the metallothionein of dissociating metals ion；
5. separating metallothionein using molecular sieve gel chromatography, precision is high, avoids the loss and macromolecular of metallothionein
Impurity is mixed into；
6. extracting, overall process is nontoxic, and gained Zinc metal sulfur compound is safe and healthy.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further, but the present invention is not limited only to this.
Embodiment 1
Configure Tissue lysates: the Tris-base of 50 mmol/L Ph 7.4,1% Triton X-100,0.25% deoxidation gallbladder
Sour sodium, the EDTA of NaCl, 1mmol/L of 150mmol/L, the leupeptin of 1 μ g/ml, the aprotinin of 1 μ g/ml, 1 μ g/
Stomach cardia inhibitor, the 1mmol/L PMSF of ml mixes up to RIPA Tissue lysates.
It collects fresh oyster to be cleaned, removes silt, peel off and take oyster meat, 100g oyster meat addition salt solution is existed
It is impregnated in ice water for 24 hours, then drained after being rinsed with clear water, oyster meat is cut into the fragment that diameter is 1cm, 750ml RIPA group is added
Lysate is knitted, 20g is added to the dithiothreitol (DTT) for preventing Zn-MT from polymerizeing, is homogenized 10min with glass homogenizer, obtains oyster
Complex enzyme: lipase is added in homogenate, and wherein fat splitting enzyme activity is 5U/g, additional amount 5mg, cellulase, wherein fine
Tieing up plain enzyme activity is 4U/g, and additional amount 5mg, polysaccharide hydrolase, wherein polysaccharide hydrolysis enzyme activity is 30 U/g, and additional amount is
8mg, obtains enzymatic hydrolysis liquid mixture at 30 degrees Celsius of enzymatic hydrolysis half an hour；
80 DEG C of heating water baths digest mixture, and filtering is added 1mol/L phytic acid and adjusts homogenate pH to 4, makes impurity protein
Irreversible inactivation, MT discharge Zn2+, phytic acid and MT compete, form PHYTIC ACID ZINC SALT precipitating, filtering, filtrate is again by 1mol/L phytic acid
Adjust pH to 1, refilter, take filtrate, using molecule sieve separation, gel used be sephadex Sephadex G-25 with
Tris- glycine elution, obtains metallothionein, and gained PHYTIC ACID ZINC SALT is precipitated and adjusts pH to 8, phytic acid and Zn2+Dissociation, is filtered to take
Zn2+Filtrate mixes the Zn2+Filtrate and metallothionein are heated to 50 DEG C, are stirred to react to obtain Zinc metal sulfur protein.
Embodiment 2
It collects fresh oyster to be cleaned, removes silt, peel off and take oyster meat, salt solution is added in ice water in 100g oyster meat
Middle immersion 48h, then drained after being rinsed with clear water, oyster meat is cut into the fragment that diameter is 1cm, 800ml RIPA tissue is added and splits
Liquid is solved, 15g is added to the dithiothreitol (DTT) for preventing Zn-MT from polymerizeing, is homogenized 10min with glass homogenizer, obtains oyster homogenate
Complex enzyme: lipase is added in liquid, and wherein fat splitting enzyme activity is 6U/g, additional amount 7mg, cellulase, wherein cellulose
Enzyme activity is 5U/g, additional amount 2mg, polysaccharide hydrolase, and wherein polysaccharide hydrolysis enzyme activity is 25 U/g, additional amount 5mg, 30
Degree Celsius enzymatic hydrolysis half an hour, obtain enzymatic hydrolysis liquid mixture；
80 DEG C of heating water baths digest mixture, and filtering is added 1mol/L phytic acid and adjusts homogenate pH to 4.5, sends out impurity protein
Raw irreversible inactivation, MT discharge Zn2+, phytic acid and MT compete, form PHYTIC ACID ZINC SALT precipitating, filtering, filtrate is planted by 1mol/L again
Acid for adjusting pH is refiltered, takes filtrate to 1, using molecule sieve separation, gel used be sephadex Sephadex G-25 with
Tris- glycine elution, obtains metallothionein, and gained PHYTIC ACID ZINC SALT is precipitated and adjusts pH to 8, phytic acid and Zn2+Dissociation, is filtered to take
Zn2+Filtrate mixes the Zn2+Filtrate and metallothionein are heated to 50 DEG C, are stirred to react to obtain Zinc metal sulfur protein.
Embodiment 3
It collects fresh oyster to be cleaned, removes silt, peel off and take oyster meat, salt solution is added in ice water in 100g oyster meat
Middle immersion 48h, then drained after being rinsed with clear water, oyster meat is cut into the fragment that diameter is 1cm, 1000ml RIPA tissue is added
Lysate is added 15g to the dithiothreitol (DTT) for preventing Zn-MT from polymerizeing, is homogenized 10min with glass homogenizer, it is even to obtain oyster
Slurries, be added complex enzyme: rouge lipase, wherein fat splitting enzyme activity be 7U/g, additional amount 10mg, cellulase, wherein
Cellulase activity is 7U/g, and additional amount 1mg, polysaccharide hydrolase, wherein polysaccharide hydrolysis enzyme activity is 20 U/g, and additional amount is
2mg, obtains enzymatic hydrolysis liquid mixture at 30 degrees Celsius of enzymatic hydrolysis half an hour；
80 DEG C of heating water baths digest mixture, and filtering is added 1mol/L phytic acid and adjusts homogenate pH to 3.5, sends out impurity protein
Raw irreversible inactivation, MT discharge Zn2+, phytic acid and MT compete, form PHYTIC ACID ZINC SALT precipitating, filtering, filtrate is planted by 1mol/L again
Acid for adjusting pH is refiltered, takes filtrate to 1, using molecule sieve separation, gel used be sephadex Sephadex G-25 with
Tris- glycine elution, obtains metallothionein, and gained PHYTIC ACID ZINC SALT is precipitated and adjusts pH to 8, phytic acid and Zn2+Dissociation, is filtered to take
Zn2+Filtrate mixes the Zn2+Filtrate and metallothionein are heated to 50 DEG C, are stirred to react to obtain Zinc metal sulfur protein.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (7)
1. the extracting method of Zinc metal sulfur protein zinc in a seed oyster, which comprises the following steps:
(1) fresh oyster is taken, 24-48h is impregnated in brine ice, peels off and takes oyster meat, and shearing to diameter is the broken of 1-3cm
Piece；
(2) Tissue lysates, dithiothreitol (DTT) are added, obtain homogenate through glass homogenizer homogenate 10-20min；
(3) it is added in fat hydrolase, cellulase, polysaccharide hydrolase and homogenate, filtering, obtains enzymatic hydrolysis liquid mixture；
(4) 80 DEG C of heating water baths digest mixture, and filtering is added in filtrate and plants acid for adjusting pH to 3.5-4.5, obtains PHYTIC ACID ZINC SALT
Precipitating, then pH to 1 is adjusted, filtering removal phytic acid cadmium obtains metallothionein solution；
(5) metallothionein is separated using molecular sieve gel chromatography；
(6) it taking PHYTIC ACID ZINC SALT to precipitate, adjusts pH to 8-10, filtering mixes gained filtrate with metallothionein obtained by step (5),
It is heated to 50-70 DEG C, is stirred to react to obtain Zinc metal sulfur protein.
2. the extracting method of Zinc metal sulfur protein zinc in a seed oyster according to claim 1, which is characterized in that described group
Knit Tris-base, 1% Triton X-100,0.25% deoxysodium cholate that lysate includes 50 mmol/L Ph 7.4,
The EDTA of NaCl, 1mmol/L of 150mmol/L, the leupeptin of 1 μ g/ml, the aprotinin of 1 μ g/ml, 1 μ g/ml stomach
Protein inhibitor, 1mmol/L PMSF.
3. the extracting method of Zinc metal sulfur protein zinc in a seed oyster according to claim 1, which is characterized in that described group
The additional amount for knitting lysate is 750-1000ml, in terms of 100g oyster meat.
4. the extracting method of Zinc metal sulfur protein zinc in a seed oyster according to claim 1, which is characterized in that described two
The additional amount of sulphur threitol is 10-20g, in terms of 100g oyster meat.
5. the extracting method of Zinc metal sulfur protein zinc in a seed oyster according to claim 1, which is characterized in that the rouge
Fat hydrolysis activity is 5-7U/g, and additional amount 5-10mg, the cellulase activity is 4-7U/g, additional amount 1-5mg, institute
Stating polysaccharide hydrolysis enzyme activity is 20-30 U/g, and additional amount 2-8mg, wherein additional amount is in terms of 100g oyster meat.
6. the extracting method of Zinc metal sulfur protein zinc in a seed oyster according to claim 1, which is characterized in that described point
Gel used in son sieve gel chromatography is sephadex Sephadex G-25, and eluent used is Tris- glycine.
7. the extracting method of Zinc metal sulfur protein zinc in a seed oyster according to claim 1, which is characterized in that the step
Suddenly (3) and step (4) further include step (3.1): the proteinase that enzyme activity is 10-20U/g 20mg are added, with oyster meat
For 100g meter.
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Application publication date: 20191119 |
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