CN101585879A - Method of quickly inducing pork liver metallothionein - Google Patents

Method of quickly inducing pork liver metallothionein Download PDF

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Publication number
CN101585879A
CN101585879A CNA2009100621684A CN200910062168A CN101585879A CN 101585879 A CN101585879 A CN 101585879A CN A2009100621684 A CNA2009100621684 A CN A2009100621684A CN 200910062168 A CN200910062168 A CN 200910062168A CN 101585879 A CN101585879 A CN 101585879A
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Prior art keywords
metallothionein
carried out
pork liver
days
liver
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CNA2009100621684A
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Chinese (zh)
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李丽立
张彬
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Institute of Subtropical Agriculture of CAS
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Institute of Subtropical Agriculture of CAS
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Priority to CNA2009100621684A priority Critical patent/CN101585879A/en
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Abstract

The invention discloses a method of quickly inducing pork liver metallothionein, including the following steps: (1) induction: pig is fed with feeding stuffs of zinc additive, and then the induction in progressive increase of dose is carried out in abdominal injection method by using ZnSO4 solution; (2) diction: slaughtering is carried out after induction, the content of metallothionein of fetched fresh pork liver sample is directly detected according to mercapto color development method; (3) rough extraction: the fresh pork liver that is qualified after detection is smashed, mixing homogenate is carried out to pour in a beaker, alcohol-chloroform mixed solution is then added, low-temperature centrifugation is carried out, water bath is carried out after putting in water bath pot, cooling is carried out till room temperature after taking out, and the supernatant fluid after centrifugation is metallothionein crude extract; (4) purification: the crude extract is ultrafiltered and condensed to obtain concentrated solution, alcohol is added in the concentrated solution and disposed overnight, deposit is taken out for centrifugation, deposition, filtrate chromatography, elution, collection and lyophilization are carried out so as to obtain freeze-dried powder. The invention has low cost, good effect, high yield, broad source of revulsive; and pure products are manufactured by MT, and the content is more than 95%.

Description

A kind of method of quickly inducing pork liver metallothionein
Technical field
The present invention relates to the preparation method of metallothionein(MT), more specifically relate to a kind of method of rapid induction pork liver metallothionein(MT).
Background technology
Metallothionein(MT) (MT) has the effect that intensive is eliminated free radical; can be used as nutritional additive and use separately or be used, can suppress lipid peroxidation injury, protection cell, blood viscosity lowering effectively, improve various biological functions such as blood circulation and enhance immunity with gsh (GSH), vitamins C (VC), vitamin-E (VE) etc.Metallothionein(MT) can also use as a kind of antioxidant, prevents that food or feed oxide are rotten.In addition, metallothionein(MT) can combine with many metal ions, participates in the metabolism of body metal and regulates, and balance body metal ion stable state participates in the metal ion running and drains, and mineral matter nutritional is played an important role.Metallothionein(MT) as the additive of health care, protection and cosmetics and some treatment of diseases medicine in every field widespread uses already such as medical science, food-processing, trophology.
The metallothionein(MT) (Zn-MT) that contains zinc is an a kind of positive tetrahedron metal sulfydryl chelating family, crystallization structure phase quite stable, but it can be the enzyme of prothetic group with Zn by providing Zn to activate many still.Therefore carrying out zinc induces the research of metallothionein(MT) synthetic to have important theory to be worth and wide application prospect.
The more existing pork liver metallothionein(MT) of the present invention extracting method induction time is short, the induction method that adopts first food to inject again, pig only stress be little, extract the more existing pork liver metallothionein(MT) of engineering extracting method and reduced the centrifugal number of times, and induce the metallothionein(MT) content of extraction suitable with the metallothionein(MT) content of existent method extraction.
Summary of the invention
Purpose of the present invention just is to overcome the problem and shortage that original technology exists, and has been to provide a kind of method of quickly inducing pork liver metallothionein.The induction method that adopts first food to inject again, become 3 times centrifugal into 2 times centrifugal, but about every gram pork liver containing metal sulfoprotein 8mg, to extract the metallothionein(MT) amount suitable with original technological guide.
The object of the present invention is achieved like this: use suckling pig as donor, the pig starter feed that contained autogamy trace element zinc additive through food 4-6 days, abdominal injection is induced then, slaughter after inducing for 2-4 time, separation and Extraction is a kind of from the liver of pig has a good stability, the metallothionein(MT) that purity is high, induction time " a kind of pork liver metallothionein(MT) extracting method " shortens 1/3rd, and food induce can alleviate greatly in 4-6 days pig stress and side effect, reduce injection inductive number of times and time, leaching process is simplified, and quite (patent No. is: 200410013212.X) and yield is with " a kind of pork liver metallothionein(MT) extracting method ".It is low to have more cost, effective, the output height, and inductor source is wide, and characteristics such as have no side effect.And pig is the very general animal of a kind of raising, and the source is wide, metallothionein(MT) can be used for human health care, radioprotective, beauty treatment, wound repair and animal and fowl fodder anti-oxidant with the anti-stress effect in.After inducing the extraction metallothionein(MT), pork can be edible as usual.
A kind of method of quickly inducing pork liver metallothionein, its step is as follows:
1. inducement on metallothionein in liver of pig
Select for use healthy piggy to close in cage, fed piggy 4-6 days, use zinc sulfate (ZnSO again with the pig starter feed that contains 1500-2500ppm zinc sulfate additive of autogamy 4) the solution abdominal injection, carrying out 2-4 time the inducing of dosage escalation, each dosage is the 50-150mg/kg body weight.
Adopt first food zinc, the solution that increases progressively gradually of zinc dosage carries out abdominal injection and induces again, the zinc of the food 1500ppm-2500ppm of elder generation, when zinc concentration increases considerably in the body, inject zinc again, can strengthen tolerance and the adaptability of pig to zinc, overworked dead phenomenon can not take place in piggy, thereby developed piggy MT has been induced synthetic potential, guaranteed to have a large amount of of competent temporal induction MT to synthesize again.
2. the detection of metallothionein(MT):
Induced after 1-3 days and butchered, and getting fresh liver sample has directly been detected by the content of sulfydryl development process to metallothionein(MT); Simultaneously each sample freezing temp is controlled at-18 ℃--22 ℃, take out after 10-15 days metallothionein(MT) content is detected again.Require extractible metallothionein(MT) 〉=5mg in every 1g pork liver.
3. slightly carry:
Smash to pieces detecting qualified fresh pig liver, stir homogenate, pour beaker into, add 500mLTris-HCl and 250mL ethanol-chloroform mixing solutions again, stirred 8-12 minute with Tris-HCl, and low-temperature centrifugation (10000r/min, 4 ℃, 20min).Get supernatant liquor in another centrifuge tube, put into 78-82 ℃ water-bath water-bath 4-6min, be cooled to room temperature (20 ℃-25 ℃) after the taking-up rapidly, low-temperature centrifugation (10000r/min, 4 ℃, 20min), supernatant liquor after centrifugal is the metallothionein(MT) crude extract, be positioned over 4 ℃ standby.
4. purifying:
With crude extract through ultrafiltration and concentration, concentrated solution, be in metallothionein(MT) crude extract in to add ethanol at 1: 3 to place and spend the night by volume in concentrated solution, getting precipitation, centrifugal (10000r/min, 4 ℃, 20min), the precipitation after centrifugal is with the NH of 0.1mol/L 4HCO 3Solution is dissolved to 3 times of volumes, filter paper filtering.Filtrate is carried out Sephedex G-100 column chromatography, painted at cylinder adding MT colouring reagents (sulfhydryl reagent) to the MT component; Use 0.1mol/LNH 4HCO 3Carry out wash-out, flow rate control 20mL/min collected 1 and manages in per 5 minutes.The coloured part of collecting is carried out Sepharose Fast Flow post, and (chromatography of 30mm * 300mm) is used 0.01M-0.30NH 4HCO 3Carry out gradient elution, automatic collector is in charge of collection, every pipe 5ml, and two peak nose part elutriants are collected in ultraviolet 254nm monitoring; Follow procedure intensification lyophilize is 25 hours in freeze drying box, and the obtained freeze-drying powder is MT and makes pure product by oneself, and content is more than 95%.
5. packing
The lyophilized powder packing is stand-by.
The present invention has the following advantages and positively effect:
1, to extract metallothionein(MT) from pork liver be to induce through 5 times to prior art, and the present invention has shortened induction time, shortens into 3 times by original 5 times, extracts syntheticly also to have optimized, and is optimized to low-temperature centrifugation 2 times by 3 times original low-temperature centrifugations.
2, generally induce and be single subcutaneous injection or abdominal injection because frequency injection is many, not only trivial operations, labour intensity is big and cost is high, and pig only stress intensity big, is prone to the phenomenon that the induced liquid diffusion is smooth, heap stagnates.Adopt the abdominal injection again of feeding earlier, strengthen pig tolerance and adaptability to zinc, overworked dead phenomenon can not take place in pig yet when zinc concentration increases considerably, thereby developed pig MT is induced synthetic potential, has guaranteed to have a large amount of of competent temporal induction MT to synthesize again.
3, yield height, but about every gram pork liver containing metal sulfoprotein 8mg;
4, good stability, storage time is longer under cold condition.
5, the product that the present invention extracted be applicable to human health care, radioprotective, beauty treatment, wound repair and animal and fowl fodder anti-oxidant with the anti-stress effect in.
Embodiment
Embodiment 1:
A kind of extracting method of pork liver metallothionein(MT), its step is as follows:
1. inducement on metallothionein in liver of pig
Select the market pig about healthy 5-10kg for use, close in cage.With feed piggy 4 days or 5 days or 6 days of the autogamy pig starter feed that contains the 1500-2500ppm zinc additive, use ZnSO again 4Solution adopts intraperitoneal injection, carries out the inducing of dosage escalation of 2 times or 3 times or 4 times, and each dosage is the 50-150mg/kg body weight.
With the ZnSO for preparing 4.7H 2O solution adopts intraperitoneal injection to induce, every other day induces once, and coinduction 2 times or 3 times or 4 times, each dosage is respectively 50mg/kg, 100mg/kg, 150mg/kg, accumulative total 300mg/kg.
With pig is donor, and the amount of the MT that makes in the pork liver after inducing by 3 times to be produced is 7.5mg/g, and is suitable with 5 inductive, but reduced pig only stress with in response to the death that swashs and solution diffusion causes slowly.
2. detect:
Induced to butcher in back second day and got liver, and getting fresh liver sample is directly carried out the detection of metallothionein(MT) content by the sulfydryl development process, it is freezing down simultaneously each sample to be placed-18 ℃ or-19 ℃ or-20 ℃ or-21 ℃ or-22 ℃, takes out after 10 days or 11 days or 12 days or 13 days or 14 days or 15 days and detects metallothionein(MT) content again.
Freezing under 18 ℃ or-19 ℃ or-20 ℃ or-21 ℃ or-22 ℃, after 10 days or 11 days or 12 days or 13 days or 14 days or 15 days, each amount of handling MT in the pork liver all decreases, but respectively handles fall little (P＞0.05).Therefore, it is considered herein that inducing the best zinc inductive dose of pork liver MT synthetic accumulative total is 330mg/kg, and metallothionein(MT) is more stable under the cold condition, it is slower to degrade, and is fit to cryopreservation below-4 ℃.
3. slightly carry:
Smash to pieces detecting qualified fresh liver, press fresh liver 25g and stir homogenate with 0.02MTris-HCl (pH8.6) 50ml, (10000r/min, 20min), gets supernatant liquor in another centrifuge tube to low-temperature centrifugation by 4 ℃; Get supernatant liquor in another centrifuge tube; Put into the pot water-bath 4 or 5 or 6min of 78 ℃ or 79 ℃ or 80 ℃ or 81 ℃ or 82 ℃, be cooled to room temperature (20-25 ℃) after the taking-up rapidly, frozen centrifugation (10000r/min again, 4 ℃, 20min), get supernatant liquor and supernatant liquor is placed semi-permeable membranes,, dialysed 24 hours with 10 times of volumes of PBS (pH7.4).Rest solution is the crude extract of metallothionein(MT) in the semi-permeable membranes, be positioned over 4 ℃ standby.
4. purifying
The metallothionein(MT) crude extract is through the dehydrated alcohol of 3 times of volumes precipitation, and 4 ℃ of placements are spent the night, and getting precipitation, centrifugal (10000r/min, 4 ℃, 20min), the precipitation after centrifugal is with the NH of 0.1mol/L 4HCO 3Solution is dissolved to 3 times of volumes, filter paper filtering.Filtrate is carried out Sephedex G-100 column chromatography, painted at cylinder adding MT colouring reagents (sulfhydryl reagent) to the MT component; Use 0.1mol/LNH 4HCO 3Carry out wash-out, flow rate control 20mL/min collected 1 and manages in per 5 minutes.The coloured part of collecting is carried out Sepharose Fast Flow post, and (chromatography of 30mm * 300mm) is used 0.01M-0.30NH 4HCO 3Carry out gradient elution, automatic collector is in charge of collection, every pipe 5ml, and two peak nose part elutriants are collected in ultraviolet 254nm monitoring; Follow procedure intensification lyophilize is 25 hours in freeze drying box, and the obtained freeze-drying powder is MT and makes pure product by oneself, and content is more than 95%.
5. packing
By every 25mg packing.

Claims (2)

1, a kind of method of quickly inducing pork liver metallothionein the steps include:
1., inducement on metallothionein in liver of pig:
Select piggy for use, fed piggy 4-6 days with the pig starter feed that contains 1500-2500ppm zinc sulfate additive, use the solution of zinc sulfate abdominal injection again, carry out 2-4 time the inducing of dosage escalation, dosage is the 50-150mg/kg body weight at every turn;
2., the detection of metallothionein(MT):
Induced after 1-3 days and butchered, and getting fresh liver sample directly detected by the content of sulfydryl development process to metallothionein(MT), simultaneously each sample freezing temp is controlled at-18 ℃---22 ℃, take out after 10-15 days and metallothionein(MT) content is detected the metallothionein(MT) 〉=5mg that extracts in every 1g pork liver again;
3., slightly carry:
Smash to pieces detecting qualified fresh pig liver, stir homogenate, pour beaker into Tris-HCl, add 500mLTris-HCl and 250mL ethanol-chloroform mixing solutions again, stirred low-temperature centrifugation 10000r/min 8-12 minute, 4 ℃, 20min gets supernatant liquor in another centrifuge tube, put into 78-82 ℃ water-bath water-bath 4-6min, be cooled to room temperature after the taking-up rapidly, low-temperature centrifugation 10000r/min, 4 ℃, 20min, the supernatant liquor after centrifugal is the metallothionein(MT) crude extract, be positioned over 4 ℃ standby;
4., purifying:
With crude extract through ultrafiltration and concentration, concentrated solution, be in metallothionein(MT) crude extract in to add ethanol at 1: 3 to place and spend the night by volume in concentrated solution, get the centrifugal 10000r/min of precipitation, 4 ℃, 20min, the precipitation after centrifugal is with the NH of 0.1mol/L 4HCO 3Solution is dissolved to 3 times of volumes, and filter paper filtering carries out Sephedex G-100 column chromatography to filtrate, and is painted to the MT component at cylinder adding MT colouring reagents; Use 0.1mol/LNH 4HCO 3Carry out wash-out, flow rate control 20mL/min collected 1 pipe, and with the painted Sepharose Fast Flow column chromatography of collecting that carries out, used 0.01M-0.30NH in per 5 minutes 4HCO 3Carry out gradient elution, automatic collector is in charge of collection, every pipe 5ml, and two peak point elutriants are collected in ultraviolet 254nm monitoring; Follow procedure intensification lyophilize is 25 hours in freeze drying box, the obtained freeze-drying powder.
2, by the extracting method of the described a kind of pork liver metallothionein(MT) of claim 1, it is characterized in that: described piggy is selected 5-10kg for use, and closing feeds in cage contains the feed of 2500ppm zinc, feeds continuously 4-6 days, with the ZnSO for preparing 4.7H 2O solution adopts intraperitoneal injection to induce, every other day induces once, and coinduction 2-4 time, each dosage is respectively 50mg/kg, 100mg/kg, 150mg/kg.
CNA2009100621684A 2009-05-19 2009-05-19 Method of quickly inducing pork liver metallothionein Pending CN101585879A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102731648A (en) * 2012-07-10 2012-10-17 陕西省动物研究所 Simple preparation of rabbit hepatocyte metallothionein
CN108522900A (en) * 2018-05-10 2018-09-14 浙江省海洋水产研究所 A kind of aquatic products anti-stress feed addictive
CN110229225A (en) * 2019-06-12 2019-09-13 山东师范大学 The preparation method of sulfur-rich and/or rich caesium albumen in a kind of krill
CN114409764A (en) * 2021-12-23 2022-04-29 中国农业大学 Method for efficiently extracting metallothionein from animal liver

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102731648A (en) * 2012-07-10 2012-10-17 陕西省动物研究所 Simple preparation of rabbit hepatocyte metallothionein
CN108522900A (en) * 2018-05-10 2018-09-14 浙江省海洋水产研究所 A kind of aquatic products anti-stress feed addictive
CN110229225A (en) * 2019-06-12 2019-09-13 山东师范大学 The preparation method of sulfur-rich and/or rich caesium albumen in a kind of krill
CN110229225B (en) * 2019-06-12 2021-07-20 山东师范大学 Preparation method of sulfur-rich and/or cesium-rich protein in antarctic krill
CN114409764A (en) * 2021-12-23 2022-04-29 中国农业大学 Method for efficiently extracting metallothionein from animal liver

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Application publication date: 20091125