CN1970784A - Method for extracting carnosine - Google Patents
Method for extracting carnosine Download PDFInfo
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- CN1970784A CN1970784A CN 200610161141 CN200610161141A CN1970784A CN 1970784 A CN1970784 A CN 1970784A CN 200610161141 CN200610161141 CN 200610161141 CN 200610161141 A CN200610161141 A CN 200610161141A CN 1970784 A CN1970784 A CN 1970784A
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Abstract
The invention discloses an extracting method of carnosine, which comprises the following steps: removing non-muscle tissue in the animal meat; washing; drying; grinding; adding water to stir into slurry; proceeding double-enzyme hydrolysis; proceeding enzyme inactivation; separating supernatant; adsorbing through hydroxy agustite chromatographic column; hyperfiltering through hyperfiltering film; collecting filtrate; spraying to dry carnosine liquid; collecting dried powder; packing in the vacuum.
Description
Technical field
The present invention relates to the production technique of medication chemistry, specifically from animals skeletal muscle, extract the method for carnosine.
Background technology
Carnosine is a kind of natural dipeptides, is present in the muscle, is made up of Beta-alanine and Histidine.Boldyrev report carnosines such as (1988) has antioxygenation, can suppress the fats oxidn reaction that is caused by iron, copper, myohaemoglobin and lipoxidase.Carnosine has the antioxygenation stronger than BHA, BHT, TBHQ, PG and alpha-tocopherol.Decker etc. (1990) discover that also carnosine can suppress in the body by iron, reduced hematin, lipoxidase and the catalytic oxidizing reaction of simple substance oxygen.Carnosine can also suppress the formation and the change in color of rancid flavor, the sample that adds carnosine is after storage 1 month, its rancid flavor significantly is lower than the sample that adds BHT, alpha-tocopherol and tripoly phosphate sodium STPP, and it to protect chromatic effect better than BHT, alpha-tocopherol and tripoly phosphate sodium STPP, BHT, alpha-tocopherol and tripoly phosphate sodium STPP are invalid (Decker etc., 1991 to the protection color; 1995).The shrimps that add carnosine are after storage 45 days, and its shrimp shell color, taste etc. are no change substantially.Carnosine is widely used in fields such as pharmaceutical adjuvant, foodstuff additive, makeup and fodder additives at present, is a kind of natural antioxidants, colour protecting agent of high-efficiency low-toxicity.
The preparation method of existing carnosine gets (F.J.Vinicket al. for utilizing chemical synthesis process, J.Org.Chem.1983,48 (3), 392 etc.), its shortcoming is to use a large amount of organic chemistry solvents: hydrazine compound, sulfide etc. is the chemical substance of severe toxicity, and triazo-compound is inflammable and explosive chemical, produce in must be between hoolivan, troublesome poeration, contaminate environment, infringement HUMAN HEALTH; And facility investment is big, and production efficiency is low, and the production cycle is long, the production cost height.
Summary of the invention
At the harm of existing carnosine preparation method existence and the problem that production efficiency is low, production cost is high, the object of the invention is to provide a kind of production technique of extracting carnosine from animals skeletal muscle.
The present invention adopts following technical solution:
(1) gets fresh or frozen healthy animal meat, remove non-muscle groups fabric, clean, drain with water for injection;
(2) be twisted into fragment with mincer earlier, levigate with colloidal mill, collect homogenate, add water for injection and stir evenly into slurries;
(3) slurries are heated to 42 ℃～45 ℃, transfer pH to 1.5～1.7, add stomach en-, or aspartic protease or papain, after stirring evenly, keep the condition hydrolysis of pH1.5～1.7,42 ℃～45 ℃ with acid;
(4) transfer pH to 7.5～7.7 with the slurries of alkali after with hydrolysis, add then after pancreatin or Sumizyme MP stir evenly, maintain 7.5～7.7,42 ℃～45 ℃ hydrolysis of pH, enzymolysis finishes post-heating and is warming up to 100 ℃, be incubated 5～15 minutes and carry out enzyme-deactivating, be cooled to below 30 ℃;
(5) transfer pH to 2.8～3.0, standing over night with acid;
(6) carry out solid-liquid separation, isolated clear liquid is with adjusting PH with base to 6.8～7.0, and is standby;
(7) with water for injection hydroxyapatite furnishing pasty state is adorned post, use the superphosphate buffer solution for cleaning behind the dress post, use acid phosphatase salt buffer balance again, hydroxyapatite chromatography post on the above-mentioned filtrate is adsorbed, use the superphosphate buffer solution elution;
(8) collect target compound, carry out ultrafiltration, collect filtrate, carnosine stoste is carried out spraying drying, collect xeraphium with filter membrane;
(9) microbial limit, assay, acidity detection are carried out in sampling.
Described animal meat can be beef, rabbit meat, chicken, the flesh of fish, mutton or pork etc.
The alkali that described adjust pH is used can be sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium bicarbonate or ammoniacal liquor etc.
The acid that described adjust pH is used can be hydrochloric acid, phosphoric acid, citric acid, sulfuric acid etc.
With the process that enzyme is hydrolyzed, can grasp the hydrolysis process by the control hydrolysis time, hydrolysis time is short, and the insufficient yield that causes of hydrolysis is low, can extend manufacture cycle again but hydrolysis time is long, increases production cost.Hydrolysis time can be 420～540 minutes in the general step (3).Equally, hydrolysis time is 120～240 minutes in the described step (4).
Described acid phosphatase salt buffer can be 10～30mmol/L pH5.8～6.8 phosphate buffered saline buffers.
For guaranteeing drying effect, described centrifugal spray drier feeding temperature can be made as 45 ℃, and out temperature can be 140 ℃～70 ℃, atomizer rotating speed 8000r/min.
It is the ultra-filtration membrane of 3～8KD that described ultrafiltration can be adopted molecular weight cut-off.
The present invention is hydrolyzed into micromolecule polypeptide by animals skeletal muscle is carried out double-enzyme hydrolysis with macro-molecular protein, uses hydroxyapatite chromatography post absorption and purification then, can Fractional Collections obtains single, purity than higher carnosine.This method is compared with synthetic method, has following advantage:
1, do not use severe toxicity, inflammable and explosive chemical such as hydrazine compound, sulfide, triazo-compound, fresh or frozen animal meat is directly extracted carnosine through production technique such as two enzyme enzymolysis, the tool safety non-pollution, less put into production equipment, reduce advantages such as production link.
2, double-enzyme hydrolysis muscle tissue homogenate adopt to extract, the centrifugal acquisition supernatant liquor of residue, after filtration, go up hydroxyapatite chromatography post absorption and purification, production technique such as ultrafiltration, hydrolysis is complete, can remove foreigh protein removing and macromole impurity, reaches good separating effect.
3, adopt two enzyme enzymolysis, extraction, the centrifugal acquisition supernatant liquor of residue, after filtration, go up hydroxyapatite chromatography post absorption and purification, production technique such as ultrafiltration, advantage be shorten greatly in production stage, cycle, yield improves, production cost reduces, production environment requires to reduce, easy and simple to handle, reduced medicine and be subjected to microbial contamination, rotten chance.
4, adopt centrifugal spray drying, can obtain faint yellow powdered granule, uniform particles, no brown impurity solvability is good.
5, preparation is simple, and raw material is easy to get, the reaction conditions gentleness, and the yield height, good stability, and also the cycle is short, and also very low to preparation equipment and place requirement, be suitable for large-scale industrial production.
Embodiment
Be described further below in conjunction with the extracting method of specific embodiment carnosine of the present invention.
Embodiment:
Fresh or the frozen healthy beef 100.0kg that learns from else's experience and be up to the standards removes non-muscle groups fabrics such as reticular tissue, grease, broken bone, cleans with water for injection, drains, and weighs.Earlier be twisted into fragment with mincer, scale to 200 purpose colloidal mill is levigate with regulating, and collects homogenate, and the water for injection that adds 150.0kg (1.5 times of weights) stirs evenly.Slurries are heated to 42 ℃～45 ℃, transfer pH to 1.5～1.7 with 4.5% hydrochloric acid, add the pepsic ratio of 10 million international units (than vigor) in every kg slurries, add 2500 million international units (than vigor) stomach en-, after stirring evenly, keep the condition hydrolysis 8 hours of pH1.5～1.7,42 ℃～45 ℃.With 18%NaOH the pepsin hydrolysis slurries are transferred pH to 7.5～7.7, the ratio that adds 4 million international units (than vigor) pancreatin in every kg slurries, add 1000 million international units (than vigor) pancreatin, after stirring evenly, maintain pH7.5～7.7,42 ℃～45 ℃ hydrolysis 4 hours, enzymolysis finishes post-heating and is warming up to 100 ℃, is incubated 15 minutes and carries out enzyme-deactivating, is cooled to below 30 ℃.Transfer pH2.8～3.0, standing over night with 4.5% hydrochloric acid.Suction filtration gets supernatant liquor 176.0kg, and residue is centrifugal with whizzer, collects supernatant liquor and gets 53.0kg, merges supernatant liquor, and 229.0kg carries out coarse filtration altogether, collects filtrate and gets 227.0kg, transfers pH to 6.8～7.0 with 18%NaOH, and is standby.With hydroxyapatite furnishing pasty state, adopt slurry type method dress post with water for injection, dress column pressure 0.5Mpa.Clean with 400mmol/L pH6.8 phosphate buffered saline buffer earlier behind the dress post, use 20mmol/L pH6.8 phosphate buffered saline buffer balance again, hydroxyapatite chromatography post on the above-mentioned filtrate is adsorbed, with 20mmol/L pH6.8 phosphate buffered saline buffer wash-out.Collect target compound and get 285.0kg, carry out ultrafiltration with the filter membrane that to molecular retention is 3K, collect filtrate and get 274.0kg, the centrifugal spray drier feeding temperature is made as 45 ℃, out temperature is 140 ℃～70 ℃, and atomizer rotating speed 8000r/min carries out spraying drying to carnosine stoste, collect xeraphium, get carnosine powder 26.38kg.Vacuum packaging, standby.Detections such as microbial limit, assay, acidity are carried out in sampling.
Six batches of pilot-scale experiment:
| Lot number | 20060801 | 20060802 | 20060803 | 20060804 | 20060805 | 20060806 |
| Skeletal muscle | Beef | Mutton | Pork | The flesh of fish | Chicken | Rabbit meat |
| Charging capacity | 100.0kg | 100.0kg | 100.0kg | 100.0kg | 100.0kg | 100.0kg |
| The carnosine powder | 26.3kg | 24.6kg | 25.8kg | 22.5kg | 26.1kg | 25.7kg |
| Proterties | Pale yellow powder | Pale yellow powder | Pale yellow powder | Pale yellow powder | Pale yellow powder | Pale yellow powder |
| Telling test | Up to specification | Up to specification | Up to specification | Up to specification | Up to specification | Up to specification |
| Weight loss on drying (%) | 0.53 | 0.65 | 0.58 | 0.68 | 0.64 | 0.70 |
| The pH value | 6.24 | 6.28 | 6.17 | 6.23 | 6.18 | 6.10 |
| Microorganism limits | Up to specification | Up to specification | Up to specification | Up to specification | Up to specification | Up to specification |
| Content of peptides (mg/g) | 94.79 | 93.90 | 94.00 | 94.86 | 93.87 | 94.61 |
Content of peptides is measured according to the method for cattle encephalon glycoside and ignotin injection content of peptides among the national drug standards WS-10001-(HD-0874)-2002.
Claims (10)
1, a kind of extracting method of carnosine, this method is made up of following process successively:
(1) gets fresh or frozen healthy animal meat, remove non-muscle groups fabric, clean, drain with water for injection;
(2) be twisted into fragment with mincer earlier, levigate with colloidal mill, collect homogenate, add water for injection and stir evenly into slurries;
(3) slurries are heated to 42 ℃～45 ℃, transfer pH to 1.5～1.7, add stomach en-, or aspartic protease or papain, after stirring evenly, keep the condition hydrolysis of pH1.5～1.7,42 ℃～45 ℃ with acid;
(4) transfer pH to 7.5～7.7 with the slurries of alkali after with hydrolysis, add then after pancreatin or Sumizyme MP stir evenly, maintain pH7.5～7.7,42 ℃～45 ℃ of hydrolysis, enzymolysis finishes post-heating and is warming up to 100 ℃, be incubated 5～15 minutes and carry out enzyme-deactivating, be cooled to below 30 ℃;
(5) transfer pH to 2.8～3.0, standing over night with acid;
(6) carry out solid-liquid separation, isolated clear liquid is with adjusting PH with base to 6.8～7.0, and is standby;
(7) with water for injection hydroxyapatite furnishing pasty state is adorned post, use the superphosphate buffer solution for cleaning behind the dress post, use acid phosphatase salt buffer balance again, hydroxyapatite chromatography post on the above-mentioned filtrate is adsorbed, use the superphosphate buffer solution elution;
(8) collect target compound, carry out ultrafiltration, collect filtrate, carnosine stoste is carried out spraying drying, collect xeraphium with filter membrane;
(9) microbial limit, assay, acidity detection are carried out in sampling.
2, the extracting method of carnosine as claimed in claim 1 is characterized in that: described animal meat is beef, rabbit meat, chicken, the flesh of fish, mutton or pork.
3, the extracting method of carnosine as claimed in claim 1 is characterized in that: the alkali that described adjust pH is used is sodium hydroxide, calcium hydroxide, potassium hydroxide, sodium bicarbonate or ammoniacal liquor.
4, the extracting method of carnosine as claimed in claim 1 is characterized in that: the acid that described adjust pH is used is hydrochloric acid, phosphoric acid, citric acid or sulfuric acid.
5, the extracting method of carnosine as claimed in claim 1 is characterized in that: hydrolysis time is 420～540 minutes in the described step (3).
6, the extracting method of carnosine as claimed in claim 1 is characterized in that: hydrolysis time is 120～240 minutes in the described step (4).
7, the extracting method of carnosine as claimed in claim 1 is characterized in that: in described step (6), first suction filtration gets supernatant liquor, and residue is centrifugal with whizzer, collects supernatant liquor, merges supernatant liquor, filters, and isolates clear liquid.
8, the extracting method of carnosine as claimed in claim 1 is characterized in that: described phosphate buffered saline buffer is 10～30mmol/L pH5.8～6.8 phosphate buffered saline buffers.
9, the extracting method of carnosine as claimed in claim 1 is characterized in that: described centrifugal spray drier feeding temperature is made as 45 ℃, and out temperature is 140 ℃～70 ℃, atomizer rotating speed 8000r/min.
10, the extracting method of carnosine as claimed in claim 1 is characterized in that: it is the ultra-filtration membrane of 3～8KD that molecular weight cut-off is adopted in described ultrafiltration.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006101611417A CN100465285C (en) | 2006-12-07 | 2006-12-07 | Method for extracting carnosine |
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| CNB2006101611417A CN100465285C (en) | 2006-12-07 | 2006-12-07 | Method for extracting carnosine |
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| CN100465285C CN100465285C (en) | 2009-03-04 |
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| CN103621761A (en) * | 2012-08-24 | 2014-03-12 | 天津科技大学 | Preparation for fat-reducing peptide |
| CN104232715A (en) * | 2014-08-20 | 2014-12-24 | 青岛贝尔特生物科技有限公司 | Preparation method of protein oligopeptide |
| CN105124319A (en) * | 2015-09-30 | 2015-12-09 | 江苏省协同医药生物工程有限责任公司 | Method for preparing dog food meat-flavor attractant |
| CN108157580A (en) * | 2017-12-29 | 2018-06-15 | 浙江大飞龙动物保健品股份有限公司 | A kind of pork functional polypeptide and preparation method thereof |
| CN110150449A (en) * | 2018-01-17 | 2019-08-23 | 浙江大飞龙动物保健品股份有限公司 | The purposes of pork functional polypeptide |
| CN110419618A (en) * | 2019-07-25 | 2019-11-08 | 李猷 | A kind of animal albumen powder and preparation method thereof rich in carnosine |
| CN113332414A (en) * | 2021-05-31 | 2021-09-03 | 北京四环制药有限公司 | Cerebroside carnosine injection with good stability, preparation method and application thereof |
| CN114836491A (en) * | 2022-05-30 | 2022-08-02 | 广州恒雅生物化工有限公司 | Preparation method of anti-aging skin-brightening carnosine and application of carnosine in cosmetics |
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| CN106366158B (en) * | 2016-08-31 | 2019-11-05 | 精晶药业股份有限公司 | A kind of method for crystallising of carnosine |
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| JP2001029042A (en) * | 1999-07-19 | 2001-02-06 | Nikken Foods Co Ltd | Edible composition containing natural carnosine |
| CN1318444C (en) * | 2003-03-19 | 2007-05-30 | 四川三高生化股份有限公司 | Method for producing carnosine |
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- 2006-12-07 CN CNB2006101611417A patent/CN100465285C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103621761A (en) * | 2012-08-24 | 2014-03-12 | 天津科技大学 | Preparation for fat-reducing peptide |
| CN104232715A (en) * | 2014-08-20 | 2014-12-24 | 青岛贝尔特生物科技有限公司 | Preparation method of protein oligopeptide |
| CN105124319A (en) * | 2015-09-30 | 2015-12-09 | 江苏省协同医药生物工程有限责任公司 | Method for preparing dog food meat-flavor attractant |
| CN108157580A (en) * | 2017-12-29 | 2018-06-15 | 浙江大飞龙动物保健品股份有限公司 | A kind of pork functional polypeptide and preparation method thereof |
| CN110150449A (en) * | 2018-01-17 | 2019-08-23 | 浙江大飞龙动物保健品股份有限公司 | The purposes of pork functional polypeptide |
| CN110419618A (en) * | 2019-07-25 | 2019-11-08 | 李猷 | A kind of animal albumen powder and preparation method thereof rich in carnosine |
| CN113332414A (en) * | 2021-05-31 | 2021-09-03 | 北京四环制药有限公司 | Cerebroside carnosine injection with good stability, preparation method and application thereof |
| CN113332414B (en) * | 2021-05-31 | 2022-02-11 | 北京四环制药有限公司 | Cerebroside carnosine injection with good stability, preparation method and application thereof |
| CN114836491A (en) * | 2022-05-30 | 2022-08-02 | 广州恒雅生物化工有限公司 | Preparation method of anti-aging skin-brightening carnosine and application of carnosine in cosmetics |
| CN114836491B (en) * | 2022-05-30 | 2023-03-14 | 广州恒雅生物化工有限公司 | Preparation method of anti-aging skin-brightening carnosine and application of carnosine in cosmetics |
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