CN101008026A - Method for preparing silkworm chrysalis polypeptide using complex enzyme for hydrolyzing silkworm chrysalis - Google Patents
Method for preparing silkworm chrysalis polypeptide using complex enzyme for hydrolyzing silkworm chrysalis Download PDFInfo
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- CN101008026A CN101008026A CN 200610166598 CN200610166598A CN101008026A CN 101008026 A CN101008026 A CN 101008026A CN 200610166598 CN200610166598 CN 200610166598 CN 200610166598 A CN200610166598 A CN 200610166598A CN 101008026 A CN101008026 A CN 101008026A
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- silkworm chrysalis
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Abstract
The invention discloses a method for preparing silkworm chrysalis polypeptide by taking defatted silkworm chrysalis as raw material. It is characterized in that: it selects defatted silkworm chrysalis as raw material, takes composite enzyme of bromelain and neuter protease as proteolytic enzyme, carries out hydrolysis under special condition, and gets silkworm chrysalis polypeptide after drying. The invention is characterized by simple process, low production cost, long perservation time, ability of checking polypeptide content in silkworm chrysalis polypeptide through polypeptide content checking method, and the product is characterized by stable quality and ensured nutriment and effect. The product is widely used in food and health care food field, and can be matched with other animal, plant raw material with normal method to prepare capsule, soft capsule, granula, tablet and powder medicine.
Description
Technical field
The present invention relates to a kind of preparation method of extract that obtains from animal body, particularly is the method for feedstock production silkworm chrysalis polypeptide with the degreasing silkworm chrysalis.
Background technology
Silkworm chrysalis (Silkworm Chrysalis) is the pupa of the pupa of Bombycidae (Bombycidae) silkworm (Bombyx moriLinnaeus) or Saturniidae (Saturniidae) tussah (Antheraea pernyi), Semen Ricini silkworm (Attacus cynthia ricini Boisduval).Measure by analysis and show, Protein content is more than 50% in the silkworm chrysalis, fat content is 20%～30%, all the other are chitin and inorganic salt etc., the contained protein of every double centner silkworm chrysalis is equivalent to 85 kilograms of thin porks, or 96 kilograms of eggs and 109 kilograms of protein that crucian is contained, and pupa albumen is a kind of full price albumen, the amino acid great variety of goods wherein contains 18 seed amino acids, and 8 seed amino acid total amounts of needed by human are 40.85%, 36% the standard that surpasses World Health Organization's defined, compare pork, mutton, egg, contained indispensable amino acid is high several times in the milk, and the indispensable amino acid harmony is good, and ratio is suitable mutually.Therefore, silkworm chrysalis is the very high edible animal source of a kind of nutritive value.Yet at present silkworm chrysalis except wherein sub-fraction is used as edible and medicinal, the overwhelming majority all is to be used as animal-feed, its major cause be since dried cocoon in the storage of long period, and in the silk reeling process afterwards, suffer stronger alkali, thermal treatment, pupa albumen often in sex change and the pupal cell owing to be rich in fat, corrupt, decomposition easily takes place, and then produce the stench flavor be difficult to eliminate, for extraction, separation, the application of pupa albumen are made troubles, its true value is difficult to embody, and resources advantage can not be converted into economic advantages.
At present, the deep processing and utilization to silkworm chrysalis mainly is to prepare silkworm compound amino acid with the silkworm chrysalis hydrolysis.But modern physiology and medical science are verified, to proteinic absorption, mainly are protein and the amino acid of thinking to the absorption rather than the tradition of polypeptide class in the human body.Protein must become polypeptide or amino acid could be the absorption of human body utilization through further hydrolysis, and polypeptide is compared amino acid and had and better see through power and diffusion effect under the equal conditions, and this mainly has benefited from the lower molecular weight section of polypeptide and specific space structure.In addition, polypeptide also has the not available assistance transportation function of amino acid, and promptly can be used as carrier is that body transports various nutritions; Polypeptide also has very high activity in addition, still can detect its physiologically active after some polypeptide dilutes 1,000,000 times.However, do not see that as yet the silkworm chrysalis polypeptide production marketing is arranged on the market now.
By retrieval, domestic existing people adopts the enzymatic hydrolysis silkworm chrysalis to prepare polypeptide, as the Chinese patent publication number is 00107796.1, denomination of invention is " a kind of preparation method of enzymolyzed silkworm product and a purposes ", and it discloses the method that the composite plant enzymic hydrolysis silkworm product of forming with papoid and bromeline prepares the small molecule active peptide; And for example the Chinese patent publication number is 02118706.1, and denomination of invention is " protective foods of silkworm chrysalis enzymolyzing liquid and functional oligose preparation ", prepares polypeptide liquid with Bacillus subtillis protease hydrolysis silkworm chrysalis; People such as Xiong Yanfei have delivered the article of " the enzymatic hydrolysis pupa albumen prepares the technical study of polypeptide " on " Hubei University's journal (natural science edition) " 1999 the 21st the 3rd phases of volume, the used enzyme of its hydrolysis is single trypsinase or the white enzyme of pawpaw or pancreatin or stomach en-.But composite plant enzyme that they are used or single enzyme are to hydrolyzed animal protein---the silkworm chrysalis effect is not ideal enough, and the solubility in acid polypeptide yield is generally lower.
Summary of the invention
The method that the purpose of this invention is to provide a kind of preparing silkworm chrysalis polypeptide using complex enzyme for hydrolyzing silkworm chrysalis, it can solve the practical problems that above-mentioned silkworm chrysalis polypeptide preparation exists, and has very strong specific aim.
The step that the present invention realizes is:
1, with degreasing silkworm chrysalis raw material, this material protein content is more than 60%, the silkworm chrysalis of removing impurity earlier and going mouldy, add an amount of clean water again and on colloidal mill, carry out homogenate 2～5 times, after the slurries of gained sieve through 100 purposes, get filtrate and adjust its concentration with clean water, the content that makes silkworm chrysalis is 0.5%～10%, promptly becomes the silkworm chrysalis liquid for the treatment of hydrolysis;
2, hydrolysis selects for use bromeline and neutral protease as the hydrolysis enzyme, and its proportioning is calculated as bromeline by enzyme activity: neutral protease=1～1: 8, and wherein the enzyme activity of bromeline is 35 * 10
4～100 * 10
4Ug
-1, the enzyme activity of neutral protease is 5 * 10
4～50 * 10
4Ug
-1, enzyme concentration is mixed in proportion above-mentioned two kinds of enzymes then by 0.2%～1% of the used silkworm chrysalis weight of hydrolysis, and is standby;
3, the silkworm chrysalis liquid that will treat hydrolysis places reactor, constant temperature is at 45 ℃～60 ℃, after treating its abundant swelling, pH value with 1mol/L sodium hydroxide and 1mol/L hydrochloric acid conditioning solution is 7.0 again, then adds the above-mentioned prozyme that has prepared and is hydrolyzed, and picks up counting after adding enzyme, a pH value was measured at every interval in 8～12 minutes, according to measurement result, keep its pH value stabilization 5.5～7.5 with sodium hydroxide and hydrochloric acid, hydrolysis time 2～5 hours;
4, after hydrolysis is finished, hydrolyzed solution is warming up to 80 ℃～90 ℃ and stablized 10～20 minutes, treat that hydrolyzed solution is cooled to 30 ℃～40 ℃ after, after 100 purposes sieve, get filtrate, add gac by 8%～15% of filtrate, centrifugal 15～20 minutes, collect supernatant liquor;
5, add dilute hydrochloric acid or dilute sulphuric acid in supernatant liquor, adjusting the pH value is 4.5, left standstill 20～30 minutes, and centrifugal 15～20 minutes, collect supernatant liquor, adjusting the pH value is 7;
6, adopt the conventional above-mentioned supernatant liquor that makes of drying means convection drying or in supernatant liquor, add an amount of auxiliary material, as press 10～30% adding food starchs of supernatant liquor, carry out the silkworm chrysalis polypeptide powder that spraying drying makes faint yellow or white powder then;
7, finished product.
Adopt following method the silkworm chrysalis polypeptide powder that makes to be carried out the mensuration of content of peptides:
One, principle:
Macromolecule protein is easily precipitated under acidic conditions, and the polypeptide of lower molecular weight is that acid-soluble protein dissolves in trichoroacetic acid(TCA) (TCA) solution (comprising peptide and total free aminoacids).Sample is after acidifying, and the sour molten protein content in the filtrate deducts the content that free aminoacid content is polypeptide.
Two, sour molten determining the protein quantity:
Get the 1g sample, the TCA solution 5ml of adding 15% mixes, and leaves standstill 10min, all move into solution in the centrifuge tube, 4000 rev/mins, centrifugal 10min gets whole supernatant liquors, pressing the method for GB/T5009.5-2003 regulation measures, the protein reduction factor is 6.25, and assay weight loss on drying is per sample converted and is butt.Getting sour molten proteic content is P
1
Three, free aminoacid content is measured:
Get the 1g sample, add 15% TCA solution 5ml, mix, leave standstill 10min, solution is all moved in the centrifuge tube, 4000 rev/mins, centrifugal 10min gets whole supernatant liquors, presses the method for GB/T5009.124-2003 regulation and measures.Getting free aminoacid content is P
2
Four, expression of results:
The mensuration content that the content P of silkworm chrysalis polypeptide equals acid-soluble protein deducts the mensuration content of total free aminoacids, that is: P=P
1-P
2
The inventive method compared with prior art, its advantage is: the inventive method has been used the prozyme of being made up of bromeline and neutral protease, made full use of like this that the neutral protease cleavage site is many, hydrolysis ability is strong, and the good advantage of bromelain enzyme modification, hydrolysis reaction is suitable, the pupa albumen residual quantity and the total free aminoacids amount that obtain are lower, and the Acid polypeptide yield is higher.The silkworm chrysalis polypeptide powder is widely used in fields such as food and protective foods, can adopt conventional method to cooperate separately or with other animal and plant raw material, makes capsule, soft capsule, granule, tablet and powder etc. as heath food or healthcare products.
Embodiment
Embodiment 1, extracting degreasing silkworm chrysalis, remove impurity earlier and go mouldy, take by weighing 5kg (its protein content is 62.3%) then, add the 10kg clean water and on colloidal mill, carry out homogenate 3 times, after the slurries of gained sieve through 100 purposes, get filtrate, be made into the filtrate that contains silkworm chrysalis 10% with the 35kg clean water, then these filtrates are placed the constant temperature stirred autoclave, after adjusting the temperature to 55 ℃, constant temperature, adjusting the pH value with 1mol/L dilute hydrochloric acid or rare white liquor is 7.0, add prozyme 25g, wherein bromeline (vigor 60 * 10
4Ug
-1) 3.6g,, neutral protease (vigor 20 * 10
4Ug
-1) 21.4g, reacted 3 hours, regulate pH with dilute hydrochloric acid or rare white liquor during this period and be stabilized in 7.0.After finishing, reaction is warming up to 85 ℃ of enzymes 20 minutes of going out.After treating that hydrolyzed solution is cooled to room temperature, cross 100 purposes sieve, get filtrate, add the 4kg activated carbon decolorizing, get supernatant liquor after centrifugal, supernatant liquor is placed carry out drying in the microwave dryer, obtain silkworm chrysalis polypeptide powder 2212g, protein recovery 71.01%, solubility in acid peptide yield 91.23% after testing.
Embodiment 2, extracting degreasing silkworm chrysalis, remove impurity earlier and go mouldy, take by weighing 1kg (its protein content is 71.6%) then, add the 3kg clean water and on colloidal mill, carry out homogenate 3 times, after the slurries of gained sieve through 100 purposes, get filtrate, be made into the filtrate that contains silkworm chrysalis 0.5% with the 196kg clean water, then these filtrates are placed the constant temperature stirred autoclave, after adjusting the temperature to 51 ℃, constant temperature, adjusting the pH value with 1mol/L dilute hydrochloric acid or rare white liquor is 7.0, add prozyme 2g, wherein bromeline (vigor 100 * 10
4Ug
-1) 0.4g,, neutral protease (vigor 25 * 10
4Ug
-1) 1.6g, reacted 2.6 hours, regulate pH with dilute hydrochloric acid or rare white liquor during this period and be stabilized in 7.0.After finishing, reaction is warming up to 85 ℃ of enzymes 20 minutes of going out.After treating that hydrolyzed solution is cooled to room temperature, cross 100 purposes sieve, get filtrate, add the 16kg activated carbon decolorizing, get supernatant liquor after centrifugal, supernatant liquor is placed carry out drying in the Vacuumdrier, obtain silkworm chrysalis polypeptide powder 489g, protein recovery 68.34%, solubility in acid peptide yield 90.17% after testing.
Embodiment 3, extracting degreasing silkworm chrysalis, remove impurity earlier and go mouldy, take by weighing 20kg (its protein content is 66.6%) then, add the 40kg clean water and on colloidal mill, carry out homogenate 3 times, after the slurries of gained sieve through 100 purposes, get filtrate, be made into the filtrate that contains silkworm chrysalis 5% with the 340kg clean water, then these filtrates are placed the constant temperature stirred autoclave, after adjusting the temperature to 60 ℃, constant temperature, adjusting the pH value with 1mol/L dilute hydrochloric acid or rare white liquor is 7.0, add prozyme 40g, wherein bromeline (vigor 40 * 10
4Ug
-1) 20g,, neutral protease (vigor 40 * 10
4Ug
-1) 20g, reacted 3.5 hours, regulate pH with dilute hydrochloric acid or rare white liquor during this period and be stabilized in 7.0.After finishing, reaction is warming up to 85 ℃ of enzymes 20 minutes of going out.After treating that hydrolyzed solution is cooled to room temperature, cross 100 purposes sieve, get filtrate, add the 60kg activated carbon decolorizing, get supernatant liquor after centrifugal, add and carry out spraying drying after food starch 80kg is made into suspension liquid, make silkworm chrysalis polypeptide powder 98.6kg.Solubility in acid peptide yield 9.46% after testing.
Claims (4)
1, a kind of method of preparing silkworm chrysalis polypeptide using complex enzyme for hydrolyzing silkworm chrysalis is characterized in that this method may further comprise the steps:
(1) be raw material with the degreasing silkworm chrysalis, add an amount of clean water and carry out homogenate on colloidal mill, get filtrate, with the concentration of clean water adjustment filtrate, the content that makes silkworm chrysalis is 0.5%～10%, promptly becomes the silkworm chrysalis liquid for the treatment of enzymolysis;
(2) hydrolysis selects for use bromeline and neutral protease as the hydrolysis enzyme, and its proportioning is calculated as bromeline by enzyme activity: neutral protease=1～1: 8, and wherein the enzyme activity of bromeline is 35 * 10
4～100 * 10
4Ug
-1, the enzyme activity of neutral protease is 5 * 10
4～50 * 10
4U g
-1, enzyme concentration is mixed in proportion above-mentioned two kinds of enzymes the disposable adding in back then and treats to be hydrolyzed in the silkworm chrysalis liquid of enzymolysis by 0.2%～1% of the used silkworm chrysalis weight of hydrolysis, and the condition of hydrolysis is respectively 45 ℃～60 ℃ of temperature, 2～5 hours time, pH value 5.5～7.5;
(3) after hydrolysis is finished, hydrolyzed solution is warming up to 80 ℃～90 ℃ and stablized 10～20 minutes, treat that hydrolyzed solution is cooled to 30 ℃～40 ℃ after, after 100 purposes sieve, get filtrate, add gac by 8%～15% of filtrate, centrifugal 15～20 minutes, collect supernatant liquor, standby;
(4) adjust the pH value.
2, the method for preparing silkworm chrysalis polypeptide using complex enzyme for hydrolyzing silkworm chrysalis according to claim 1 is characterized in that:
The protein content of described degreasing silkworm chrysalis raw material adds an amount of clean water and carry out homogenate 2～5 times on colloidal mill more than 60%, and the slurries of gained are got filtrate after sieving through 100 purposes.
3, the method for preparing silkworm chrysalis polypeptide using complex enzyme for hydrolyzing silkworm chrysalis according to claim 1 is characterized in that:
Described step also comprises drying, this drying adopts the conventional above-mentioned supernatant liquor that makes of drying means convection drying or add an amount of auxiliary material in supernatant liquor, as press 10～30% of supernatant liquor and add food starchs, carry out the silkworm chrysalis polypeptide powder that spraying drying makes faint yellow or white powder then.
4, according to the method for the described preparing silkworm chrysalis polypeptide using complex enzyme for hydrolyzing silkworm chrysalis of claim 1, it is characterized in that:
Described adjustment pH value is to add dilute hydrochloric acid or dilute sulphuric acid in supernatant liquor, and adjusting the pH value is 4.5, left standstill 20～30 minutes, and centrifugal 15～20 minutes, collect supernatant liquor, adjusting the pH value is 7.
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| CN101701240B (en) * | 2009-11-12 | 2012-05-23 | 常州康和生物技术有限公司 | Method for preparing antihypertensive peptide by utilizing combined enzyme enzymolysis silkworm chrysalis protein |
| CN102823715A (en) * | 2012-09-19 | 2012-12-19 | 昆明理工大学 | Method for preparing bioactive peptide through full-bionic digestion model method |
| CN103103243A (en) * | 2013-01-22 | 2013-05-15 | 广东环西生物科技股份有限公司 | Method for preparing silkworm chrysalis peptide by conducting combined hydrolysis on free enzyme and immobilized enzyme |
| CN103190524A (en) * | 2013-04-02 | 2013-07-10 | 江苏科技大学 | Method for improving silkworm chrysalis protein functional characteristics using ultrasonic wave coordinated with enzymolysis technology and product and application thereof |
| CN103540396A (en) * | 2013-10-30 | 2014-01-29 | 浙江汇能动物药品有限公司 | Method for producing silkworm chrysalis oil and extracting alpha-linolenic acid by adopting biological enzyme method |
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| CN106173185A (en) * | 2016-07-15 | 2016-12-07 | 安徽精准医疗产业研究院有限公司 | A kind of preparation method of the little peptide of Pupa bombycis |
| CN106723070A (en) * | 2016-11-24 | 2017-05-31 | 江苏爱西施科技服务咨询股份有限公司 | A kind of method for preparing compound amino acid as raw material with silkworm chrysalis |
| CN107761386A (en) * | 2017-09-07 | 2018-03-06 | 重庆大学 | A kind of preparation method for aoxidizing viscose rayon crosslinking silkworm chrysalis peptide |
| CN108192940A (en) * | 2018-02-10 | 2018-06-22 | 海盐县凌特生物科技有限公司 | The preparation method of silkworm chrysalis collagen |
| CN108543055A (en) * | 2018-02-06 | 2018-09-18 | 成都世煌生物科技有限责任公司 | A kind of hero silkworm egg white tiles production technology |
| CN109480054A (en) * | 2018-11-01 | 2019-03-19 | 苏州卫生职业技术学院 | A kind of Polypeptide-k composition and preparation method thereof |
| CN112048528A (en) * | 2020-09-16 | 2020-12-08 | 四川绵竹市瑞洋生物技术有限公司 | Method for preparing compound amino acid by enzymolysis process |
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| CN101701240B (en) * | 2009-11-12 | 2012-05-23 | 常州康和生物技术有限公司 | Method for preparing antihypertensive peptide by utilizing combined enzyme enzymolysis silkworm chrysalis protein |
| CN102823715A (en) * | 2012-09-19 | 2012-12-19 | 昆明理工大学 | Method for preparing bioactive peptide through full-bionic digestion model method |
| CN102823715B (en) * | 2012-09-19 | 2013-11-27 | 昆明理工大学 | Method for preparing bioactive peptide through full-bionic digestion model method |
| CN103103243A (en) * | 2013-01-22 | 2013-05-15 | 广东环西生物科技股份有限公司 | Method for preparing silkworm chrysalis peptide by conducting combined hydrolysis on free enzyme and immobilized enzyme |
| CN103190524A (en) * | 2013-04-02 | 2013-07-10 | 江苏科技大学 | Method for improving silkworm chrysalis protein functional characteristics using ultrasonic wave coordinated with enzymolysis technology and product and application thereof |
| CN103190524B (en) * | 2013-04-02 | 2014-06-04 | 江苏科技大学 | Method for improving silkworm chrysalis protein functional characteristics using ultrasonic wave coordinated with enzymolysis technology and product and application thereof |
| CN103540396A (en) * | 2013-10-30 | 2014-01-29 | 浙江汇能动物药品有限公司 | Method for producing silkworm chrysalis oil and extracting alpha-linolenic acid by adopting biological enzyme method |
| CN103540396B (en) * | 2013-10-30 | 2014-12-31 | 浙江汇能动物药品有限公司 | Method for producing silkworm chrysalis oil and extracting alpha-linolenic acid by adopting biological enzyme method |
| CN104921240A (en) * | 2015-05-27 | 2015-09-23 | 毛庆云 | High-protein silkworm chrysalis and bovine bone protein beverage and preparation method thereof |
| CN105613936A (en) * | 2016-03-24 | 2016-06-01 | 刘冬明 | Preparation method for spleen polypeptides |
| CN105779519A (en) * | 2016-05-16 | 2016-07-20 | 上海艾苛密进出口有限公司 | Preparation method of silkworm chrysalis amino acid |
| CN105779519B (en) * | 2016-05-16 | 2019-06-04 | 上海艾苛密进出口有限公司 | The preparation method of abundant amino acids from silkworm pupa |
| CN106173185A (en) * | 2016-07-15 | 2016-12-07 | 安徽精准医疗产业研究院有限公司 | A kind of preparation method of the little peptide of Pupa bombycis |
| CN106723070A (en) * | 2016-11-24 | 2017-05-31 | 江苏爱西施科技服务咨询股份有限公司 | A kind of method for preparing compound amino acid as raw material with silkworm chrysalis |
| CN107761386A (en) * | 2017-09-07 | 2018-03-06 | 重庆大学 | A kind of preparation method for aoxidizing viscose rayon crosslinking silkworm chrysalis peptide |
| CN108543055A (en) * | 2018-02-06 | 2018-09-18 | 成都世煌生物科技有限责任公司 | A kind of hero silkworm egg white tiles production technology |
| CN108192940A (en) * | 2018-02-10 | 2018-06-22 | 海盐县凌特生物科技有限公司 | The preparation method of silkworm chrysalis collagen |
| CN109480054A (en) * | 2018-11-01 | 2019-03-19 | 苏州卫生职业技术学院 | A kind of Polypeptide-k composition and preparation method thereof |
| CN112048528A (en) * | 2020-09-16 | 2020-12-08 | 四川绵竹市瑞洋生物技术有限公司 | Method for preparing compound amino acid by enzymolysis process |
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