CN105403708A - Method for screening deep sea fish skin collagen polypeptides with highly biological absorbing ability and high-bioavailability - Google Patents
Method for screening deep sea fish skin collagen polypeptides with highly biological absorbing ability and high-bioavailability Download PDFInfo
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- CN105403708A CN105403708A CN201510671249.XA CN201510671249A CN105403708A CN 105403708 A CN105403708 A CN 105403708A CN 201510671249 A CN201510671249 A CN 201510671249A CN 105403708 A CN105403708 A CN 105403708A
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention relates to fish-skin collagen polypeptides, and more specifically relates to a method for screening deep sea fish skin collagen polypeptides with highly biological absorbing ability and high-bioavailability. Deep sea fish skin collagen polypeptide segments which are selected from different sources and have different molecular weights are used as raw materials, molecular weights of raw material polypeptides are confirmed with HPLC; raw material polypeptides with confirmed molecule weights are prepared into a lavage solution with normal saline for carrying out a rat lavage experiment; after 24 hours, night soil, urine and feed intake of rats are collected, and absorptivity of hydroxyproline is determined; blood from eye sockets of rats at different time points after rat lavage are taken, content of hydroxyproline in blood is determined, the group with the highest content is selected, and deep sea fish skin collagen polypeptides with high-bioavailability and highly biological absorbing ability are obtained. A kind of collagen polypeptide with high-bioavailability and highly biological absorbing ability is selected, and the collagen polypeptide with highly biological absorbing ability has wide sources and can be widely applied to many fields, such as health products, cosmetics, biological dressings, medical haemostatic materials, animal feed, etc.
Description
Technical field
The present invention relates to fishskin collagen polypeptide, be specifically related to a kind of method of screening the deep-sea fish skin collagen polypeptide of high bio-absorbable availability.
Background technology
Collagen is the widest, content the is maximum a kind of protein that distributes in animal body, is present in widely in the connective tissues such as the skin of animal, bone, tendon, film.Collagen has triple-helix structure, and remains biologically active, and triple-helix structure thoroughly unclamps, and becomes 3 peptide chains freely, and is degraded into polydisperse peptide section, and therefore collagen is the potpourri of polypeptide.Research finds, in vital movement, various physiological change and biochemical reaction have the participation of polypeptide.
Traditional collagen obtains mostly from the mammiferous skin of land, but along with the frequent generation of livestock and poultry pestilence in recent years, with land skin of mammal for raw material sources are more and more dangerous, in addition, due to religious reasons, certain restriction is also received to the extraction of pigskin collagen.Therefore, seek a kind of collagen newly to have originated into a big hot topic.
China has long and narrow shore line and extremely richly endowed ocean resources, and living marine resources are very abundant, by the large important sources being collagen.The trade union that adds of marine prods leaves a large amount of leftover bits and pieces, and availability is inadequate, causes the waste of resource, also pollutes the environment.And collagen content can account for the 60-80% of total protein in discarded fish-skin, it is the part that protein content is the highest in the various organization of fish.Therefore deep-sea fish skin collagen can be used as a kind of novel collagen resource.
The focus that biologically active peptide Present Domestic is studied outward, it has multiple body metabolism and physiological regulation function, absorption easy to digest, have the effect such as Promote immunity, hormone control, antibacterial, antiviral, hypotensive, reducing blood lipid, and joint lubrication, wound healing, calcification, blood clotting and aging etc. there is close relationship.Therefore, study a kind of collagen polypeptide of high bio-absorbable availability, can be field of medicaments, healthcare field, field of food, cosmetic field application good technical support is provided.
But the collagen polypeptide of not all kind and molecular weight all can show stronger physiological action, body is to the degree that the absorbs difference of the polypeptide of variety classes different molecular weight ranges, therefore, select a kind of collagen polypeptide of high bio-absorbable availability significant.
Summary of the invention
The present invention is intended to the method for screening the deep-sea fish skin collagen polypeptide of high bio-absorbable availability from the deep-sea fish skin collagen polypeptide of different kinds and different molecular weight section.
For achieving the above object, the technical solution used in the present invention is:
A kind of method of screening the deep-sea fish skin collagen polypeptide of high bio-absorbable availability:
(1) the deep-sea fish skin collagen polypeptide segment choosing separate sources different molecular weight, as raw material, carries out by HPLC the molecular weight confirming raw material polypeptide;
(2) the raw material polypeptide normal saline of above-mentioned confirmation molecular weight is become gavage solution, to carrying out rat oral gavage experiment;
(3) collect ight soil, urine, the Feed consumption of rat after 24h, measure the absorptivity of hydroxyproline;
(4) get the blood that above-mentioned steps (2) carries out the different time points rat eye socket after rat oral gavage respectively, measure the hydroxyproline content in blood; According to formula absorptivity=hydroxyproline uptake/body weight, the group that after calculating gavage, hydroxyproline absorptivity is the highest, and measure hydroxyproline content in blood, select the group that content is the highest, namely obtain the deep-sea fish skin collagen polypeptide of high bio-absorbable availability.
The deep-sea fish skin collagen polypeptide of described raw material separate sources different molecular weight is molecular weight is be less than 1KDa respectively, the salmon collagen polypeptide of 1-3KDa, 3-6KDa; Molecular weight is be less than 1KDa respectively, the cod skin collagen polypeptide of 1-3KDa, 3-6KDa.
Described HPLC carries out confirming that the molecular weight of raw material polypeptide adopts exclusion chromatography, wherein, and chromatographic column TSKgelG2000SWXL7.8 × 300mm, 5 μm, TOSOH, Japan; Mobile phase A (0.1% trifluoroacetic acid), Mobile phase B (acetonitrile), time 30min, A:B=80:20, sample size 10 μ L, λ=220nm, temperature 25 DEG C.
Described gavage solution concentration is 40mg/mL.
Described step (4) get respectively above-mentioned steps (2) carry out rat oral gavage after 0.5h, the blood of 1h, 2h, 4h, 8h different time points rat eye socket.
Usefulness of the present invention:
The inventive method is adopted to obtain the higher deep-sea fish skin collagen polypeptide of absorptivity from two kinds of conventional deep-sea fish skin collagen polypeptide, in addition, in the good molecular weight section of absorptivity found at present for being less than in the scope of 6KDa, segmenting and have found the molecular weight section absorbing Du Genggao.To medicine produce, health products produce significant.
Accompanying drawing explanation
The high-efficient liquid phase chromatogram of the different molecular weight polypeptide sample that Figure 1A-F provides for the embodiment of the present invention, Figure 1A (ZJHS<1kDa), Figure 1B (ZJHS1-3kDa), Fig. 1 C (ZJHS3-6kDa), Fig. 1 D (QDKN<1kDa), Fig. 1 E (QDKN1-3kDa), Fig. 1 F (QDKN3-6kDa).
The polypeptide sample content variation diagram in time in blood that Fig. 2 provides for the embodiment of the present invention.
Embodiment
According to embodiment, the present invention is described in further detail below.
Screen the method for the deep-sea fish skin collagen polypeptide of high bio-absorbable availability:
(1) using the salmon bought and cod as screening deep-sea fish skin collagen polypeptide raw material, often kind of collagen is divided into three molecular weight sections, is less than 1KDa, 1-3KDa, 3-6KDa.
(2) HPLC exclusion chromatography is utilized to carry out the checking of molecular weight section, wherein, gel column is chromatographic column TSKgelG2000SWXL7.8 × 300mm, 5 μm of (TOSOH, Japan), mobile phase A (0.1% trifluoroacetic acid), Mobile phase B (chromatogram acetonitrile), A:B=80:20, temperature 25 DEG C, gradient elution 30min, gel chromatography figure are as shown in Figure 1A-F, and all polypeptide sample all meet its molecular weight section declared as calculated.
(3) 6 peptide species raw material physiological saline are mixed with the gavage solution that concentration is 40mg/mL respectively, gavage is carried out to rat.
(4) before gavage, 12h can't help water to Rat Fast, and every rat pours into 40mg polypeptide according to the every 100g of body weight.Record rat body weight before gavage, forage volume.
(5) every rat ight soil, urine is collected after 24h, and rat feed surplus, hydroxyproline content in ight soil, urine and feed is measured respectively with hydroxyproline testing cassete (Bioengineering Research Institute is built up in Nanjing), and calculate hydroxyproline absorptivity, as shown in table 1, can show that salmon collagen polypeptide molecular weight section is higher in the absorptivity of 1-3KDa, cod skin collagen polypeptide molecular weight is higher in the absorptivity of 1-3KDa and 3-6KDa.
The absorptivity of the polypeptide sample that table 1 provides for the embodiment of the present invention
Meanwhile, the gavage solution that above-mentioned steps (3) configures is carried out gavage to rat, but do not need to record forage volume, according to different time point 0.5h after gavage, 1h, 2h, 4h, 8h carry out eye socket and get blood, each about 1mL.
By the centrifugal blood obtained, 3500rpm/min, time 15min, get the content that upper serum hydroxyproline testing cassete (Bioengineering Research Institute is built up in Nanjing) measures hydroxyproline, as shown in Figure 2, the initial absorption amount of cod skin collagen polypeptide 1-3KDa and 3-6KDa is all higher than 25 μ g/mL, the initial absorption amount of all the other groups is then lower, all lower than 20 μ g/mL, but molecular weight section is very fast at the cod skin collagen polypeptide degradation speed ratio of 3-6KDa, after gavage during 2h, hydroxyproline content rapid drawdown is to about 5 μ g/mL, therefore determine that the oral absorption utilizing status of the cod skin collagen polypeptide of 1-3KDa is best.
Analyze the hydroxyproline content in hydroxyproline absorptivity and blood, hydroxyproline absorptivity and content higher, illustrative experiment animal is higher to its degree of absorbing, and therefore selection molecular weight section is the collagen polypeptide of cod skin collagen polypeptide as high bio-absorbable availability of 1-3KDa.
Claims (5)
1. screen a method for the deep-sea fish skin collagen polypeptide of high bio-absorbable availability, it is characterized in that:
(1) the deep-sea fish skin collagen polypeptide segment choosing separate sources different molecular weight, as raw material, carries out by HPLC the molecular weight confirming raw material polypeptide;
(2) the raw material polypeptide normal saline of above-mentioned confirmation molecular weight is become gavage solution, to carrying out rat oral gavage experiment;
(3) collect ight soil, urine, the Feed consumption of rat after 24h, measure the absorptivity of hydroxyproline;
(4) get the blood that above-mentioned steps (2) carries out the different time points rat eye socket after rat oral gavage respectively, measure the hydroxyproline content in blood, select the group that content is the highest, namely obtain the deep-sea fish skin collagen polypeptide of high bio-absorbable availability.
2. by the method for deep-sea fish skin collagen polypeptide of screening high bio-absorbable availability described in claim 1, it is characterized in that: the deep-sea fish skin collagen polypeptide of described raw material separate sources different molecular weight is molecular weight is be less than 1KDa respectively, the salmon collagen polypeptide of 1-3KDa, 3-6KDa; Molecular weight is be less than 1KDa respectively, the cod skin collagen polypeptide of 1-3KDa, 3-6KDa.
3. by the method for deep-sea fish skin collagen polypeptide of screening high bio-absorbable availability described in claim 1, it is characterized in that: described HPLC carries out confirming that the molecular weight of raw material polypeptide adopts exclusion chromatography, wherein, chromatographic column TSKgelG2000SWXL7.8 × 300mm, 5 μm, TOSOH, Japan; Mobile phase A (0.1% trifluoroacetic acid), Mobile phase B (acetonitrile), time 30min, A:B=80:20, sample size 10 μ L, λ=220nm, temperature 25 DEG C.
4., by the method for deep-sea fish skin collagen polypeptide of screening high bio-absorbable availability described in claim 1, it is characterized in that: described gavage solution concentration is 40mg/mL.
5. by the method for deep-sea fish skin collagen polypeptide of screening high bio-absorbable availability described in claim 1, it is characterized in that: described step (4) get respectively above-mentioned steps (2) carry out rat oral gavage after 0.5h, 1h, 2h, the blood of 4h, 8h different time points rat eye socket.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003284586A (en) * | 2002-03-28 | 2003-10-07 | Jellice Co Ltd | Method for producing collagen peptide and method for producing product utilizing the same |
| WO2009140573A1 (en) * | 2008-05-15 | 2009-11-19 | Purdue Research Foundation | Collagen peptide conjugates and uses therefor |
| CN102247589A (en) * | 2010-05-19 | 2011-11-23 | 李勇 | Application of marine collagen peptide in preparing medicament and food for delaying skin aging |
| CN102718832A (en) * | 2012-03-05 | 2012-10-10 | 蒋鹏 | Marine organism collagen peptide |
| CN103627761A (en) * | 2013-10-22 | 2014-03-12 | 浙江省海洋开发研究院 | Method for preparing collagen peptide rich in hydroxyproline |
-
2015
- 2015-10-13 CN CN201510671249.XA patent/CN105403708A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003284586A (en) * | 2002-03-28 | 2003-10-07 | Jellice Co Ltd | Method for producing collagen peptide and method for producing product utilizing the same |
| WO2009140573A1 (en) * | 2008-05-15 | 2009-11-19 | Purdue Research Foundation | Collagen peptide conjugates and uses therefor |
| CN102247589A (en) * | 2010-05-19 | 2011-11-23 | 李勇 | Application of marine collagen peptide in preparing medicament and food for delaying skin aging |
| CN102718832A (en) * | 2012-03-05 | 2012-10-10 | 蒋鹏 | Marine organism collagen peptide |
| CN103627761A (en) * | 2013-10-22 | 2014-03-12 | 浙江省海洋开发研究院 | Method for preparing collagen peptide rich in hydroxyproline |
Non-Patent Citations (2)
| Title |
|---|
| KOJI IWAI ET AL.: "Identification of Food-Derived Collagen Peptides in Human Blood after Oral Ingestion of Gelatin Hydrolysates", 《J.AGRIC.FOODCHEM.》 * |
| 张宁等: "胶原蛋白肽分子量对吸收过程的影响研究", 《生物学杂志》 * |
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