CN111234008B - I-type collagen with antigenicity removed, preparation method and application thereof, coconut candy and preparation method thereof - Google Patents
I-type collagen with antigenicity removed, preparation method and application thereof, coconut candy and preparation method thereof Download PDFInfo
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- CN111234008B CN111234008B CN202010214138.7A CN202010214138A CN111234008B CN 111234008 B CN111234008 B CN 111234008B CN 202010214138 A CN202010214138 A CN 202010214138A CN 111234008 B CN111234008 B CN 111234008B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
- A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
- A23G3/42—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds characterised by the carbohydrates used, e.g. polysaccharides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
- A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
- A23G3/44—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing peptides or proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
- A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
- A23G3/48—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing plants or parts thereof, e.g. fruits, seeds, extracts
Abstract
The invention belongs to the field of functional leisure food, and particularly relates to antigenicity-removed type I collagen, a preparation method and application thereof, coconut candy and a preparation method thereof. The method for removing the antigenicity of the type I collagen comprises the following steps: carrying out dynamic high-pressure microjet treatment on the type I collagen solution to obtain a treated type I collagen solution; uniformly mixing the treated type I collagen solution with glucose to obtain a mixed solution; the mixture is freeze-dried and reacted at 50-100 deg.C and 60-85% relative humidity to remove antigenicity of type I collagen. The method of the invention can better remove the antigenicity of the type I collagen, and the collagen coconut candy prepared by the method can be widely used by consumers.
Description
Technical Field
The invention belongs to the field of functional leisure food, and particularly relates to a method for removing antigenicity of type I collagen, the antigenicity-removed type I collagen prepared by the method, application of the antigenicity-removed type I collagen in preparation of food, cosmetics or biomedical materials, coconut candy and a preparation method thereof.
Background
The fish collagen is a source of high-quality collagen, but the immune globulin E in the fish collagen can cause allergic phenomena such as vomit, diarrhea and the like; type I collagen has been shown to be highly allergenic, with about 50% of patients allergic to fish being allergic to type I collagen. The type I collagen consists of 2 alpha 1 subunits and 1 alpha 2 subunit, is in a three-strand helical structure and has the molecular mass of about 300 kDa; the type I collagen has good biocompatibility, easy biodegradation, no toxicity and stable structure, and is widely applied to the fields of food, cosmetics, biomedical materials and the like. The antigenicity of the type I collagen is removed, and the application of the type I collagen in various fields can be enhanced.
With the improvement of living standard, the demand of people on beauty and health care products is improved, and various health care products rich in collagen are particularly favored by young beauty lovers. At present, common collagen health care products in the market comprise oral liquid, chewable tablets and capsules, and leisure food rich in collagen is not common. The collagen added into the beauty food is generally type I collagen, because the price of the type I collagen is low, but the defect of the type I collagen has stronger antigenicity and can not be accepted by wide consumers. Therefore, the antigenicity removing treatment of the collagen I can enhance the edible safety of the collagen and reduce the edible risk of sensitive people, and is an important way for improving the quality of collagen health-care food.
At present, the research on collagen health care products mostly focuses on the product preparation method, and the research on the antigenicity removing treatment of the products is not reported yet.
Disclosure of Invention
The invention aims to overcome the defect that no method for removing antigenicity of type I collagen exists in the prior art, and provides a method for removing antigenicity of type I collagen, the antigenicity-removed type I collagen prepared by the method, application of the antigenicity-removed type I collagen in preparation of food, cosmetics or biomedical materials, coconut candy and a preparation method thereof. The method of the invention can better remove the antigenicity of the type I collagen, and the collagen coconut candy prepared by the method can be widely used by consumers.
The inventor of the invention finds in research that the antigenicity of the type I collagen can be effectively removed by the method of carrying out physical modification and chemical modification at the same time by firstly carrying out dynamic high-pressure microjet treatment on the type I collagen and then modifying the type I collagen by using glucose at specific temperature and humidity, and the method is simple. The physical modification is to treat the collagen in a dynamic high-pressure micro-jet manner, and the high pressure causes the collagen to be subjected to comprehensive actions such as severe rotation, high-speed shearing, high-frequency oscillation and the like in the process of passing through the slit at a high speed to cause conformational change of the sensitized immunoglobulin, so that the antigenicity of the sensitized immunoglobulin is reduced; the chemical modification refers to that the I-type collagen with changed structure is glycosylated by glucose, and the external connection sugar molecule modifies the protein conformation by hindering the linear epitope of the protein, thereby achieving the purpose of reducing the antigenicity of the protein.
In order to achieve the above objects, one aspect of the present invention provides a method for removing antigenicity of collagen type I, comprising:
(1) carrying out dynamic high-pressure microjet treatment on the type I collagen solution to obtain a treated type I collagen solution;
(2) uniformly mixing the treated type I collagen solution with glucose to obtain a mixed solution;
(3) freeze-drying the mixture, and reacting at 50-100 deg.C and 60-85% relative humidity to remove antigenicity of type I collagen.
In a second aspect, the invention provides antigenically-removed type I collagen prepared by the method described above.
In a third aspect, the present invention provides the use of the antigenicity-removed type I collagen described above in the preparation of a food, a cosmetic or a biomedical material.
In a fourth aspect, the present invention provides a coconut candy prepared from the raw materials of the antigenicity-removed type I collagen described above, coconut milk, maltose, coconut candy and water;
the amount of coconut milk is 150-400 parts by weight, the amount of maltose is 30-70 parts by weight, the amount of coconut sugar is 30-70 parts by weight, and the amount of water is 30-70 parts by weight, relative to 100 parts by weight of the antigenicity-removed type I collagen.
The fifth aspect of the invention provides a preparation method of coconut candy, which comprises the following steps:
(1) uniformly mixing coconut milk, maltose and water, and heating a mixed system to 120-150 ℃;
(2) cooling the system of step (1) to 50-70 ℃, adding the antigenically removed type I collagen of claim 6, and cooling to obtain the coconut sugar;
wherein, relative to 100 weight portions of the antigenicity-removed type I collagen, the dosage of the coconut milk is 150-400 weight portions, the dosage of the maltose is 30-70 weight portions, the dosage of the coconut sugar is 30-70 weight portions, and the dosage of the water is 30-70 weight portions.
According to the invention, the type I collagen is subjected to dynamic high-pressure microjet treatment firstly, and then is subjected to glycosylation modification by using glucose at a specific temperature and humidity, so that the antigenicity of the type I collagen can be effectively removed, the method is simple, the traditional enzyme treatment step is omitted, and the cost is saved.
The products such as various foods, cosmetics or biomedical materials and the like prepared by the antigenicity-removed type I collagen can effectively reduce the allergic phenomenon caused by the antigenicity-removed type I collagen, so that the products have wider consumers.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.
FIG. 1 is a graph showing the effect of removing antigenicity of type I collagen treated in example 1 and comparative example.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In a first aspect, the present invention provides a method of removing antigenicity of collagen type I, the method comprising:
(1) carrying out dynamic high-pressure microjet treatment on the type I collagen solution to obtain a treated type I collagen solution;
(2) uniformly mixing the treated type I collagen solution with glucose to obtain a mixed solution;
(3) freeze-drying the mixture, and reacting at 50-100 deg.C and 60-85% relative humidity to remove antigenicity of type I collagen.
In the present invention, the removal of the antigenicity of type I collagen is not intended to completely remove the antigenicity thereof, as long as the antigenicity thereof is considered to be removed as long as the antigenicity thereof is reduced as compared with the original type I collagen, and in the present invention, the removal rate of the type I collagen is preferably at least 80%, and for example, may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
According to the present invention, in step (1), the conditions for the dynamic high pressure microjet treatment can be changed in a wide range, for example, the conditions can be changed according to the conventional operating conditions, as long as the operating conditions can change the structure of the type I collagen, but preferably, the pressure for the dynamic high pressure microjet treatment is 100-150MPa, preferably 110-130MPa (for example, 110MPa, 112MPa, 114MPa, 115MPa, 116MPa, 117MPa, 118MPa, 119MPa, 120MPa, 121MPa, 122MPa, 123MPa, 124MPa, 125MPa, 126MPa, 128MPa, 130MPa), and the cycle can be performed for 2-4 times, in order to improve the effect of removing antigenicity.
According to the present invention, in the step (1), the concentration of the type I collagen in the type I collagen solution may be changed in a wide range, and in order to further enhance the effect of removing antigenicity, the concentration of the type I collagen in the type I collagen solution is preferably 40 to 60% by weight, for example, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60% by weight.
According to the present invention, in the step (2), the amount of glucose may be changed within a wide range as long as it can sufficiently glycosylate the processed type I collagen obtained in the step (1), and it is preferable that the amount of glucose is 80 to 120 parts by weight, preferably 90 to 110 parts by weight, for example, 90 parts by weight, 92 parts by weight, 94 parts by weight, 96 parts by weight, 98 parts by weight, 100 parts by weight, 102 parts by weight, 104 parts by weight, 106 parts by weight, 108 parts by weight, and 110 parts by weight, based on 100 parts by weight of the type I collagen on a dry basis, in order to enhance the effect of removing antigenicity.
According to the present invention, the mixture is preferably freeze-dried and subjected to a reaction at 60 to 80 ℃ (for example, 60 ℃, 62 ℃, 64 ℃, 66 ℃, 68 ℃, 70 ℃, 72 ℃, 74 ℃, 76 ℃, 78 ℃, 80 ℃) and 70 to 80% (for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%) relative humidity.
According to the invention, in step (3), the time of the reaction can vary within wide limits, preferably from 4 to 8 hours, more preferably from 5 to 7 hours.
According to the present invention, the collagen I may be various types of commercially available collagen I, or may be a self-prepared collagen I. According to a preferred embodiment of the invention, the type I collagen is type I collagen from freshwater fish.
According to a preferred embodiment of the present invention, the method for preparing type I collagen from freshwater fish comprises:
(i) sequentially carrying out alkali impregnation, organic solvent impregnation and acid impregnation on the freshwater fish skin, and carrying out solid-liquid separation on the acid-impregnated material to obtain a liquid phase;
(ii) (ii) salting out the liquid phase obtained in the step (i), and carrying out solid-liquid separation on the obtained salting-out product to obtain a solid phase;
(iii) dissolving the solid phase in acid, and then dialyzing in acid solution and water in sequence to obtain the type I collagen from the freshwater fish.
By combining the preferred method for producing type I collagen, the effect of removing the antigenicity of type I collagen can be further improved.
According to the present invention, in the step (i), the freshwater fish skin may be various freshwater fish skins, which may be obtained from fish markets. After the freshwater fish skin is obtained, it is preferably first subjected to impurity removal to remove non-fish skin materials such as bone spurs and the like therein, and then subjected to washing.
According to the invention, before the alkali impregnation, the method further comprises shearing the fresh water fish skin, for example, shearing the fresh water fish skin into 0.3-0.8cm by 0.3-0.8cm of broken fish skin, so as to improve the impregnation efficiency.
According to the present invention, the base may be a conventionally used base. According to a preferred embodiment of the invention, the base is a sodium hydroxide solution, the concentration of which may be between 0.5 and 1.5mol/L, preferably between 0.8 and 1.2 mol/L.
The amount of lye used for alkali impregnation according to the present invention may vary within wide limits and is preferably from 15 to 25 parts by volume relative to 1 part by volume of fish skin.
According to the invention, the alkaline impregnation can be carried out at normal temperature, the impregnation time can be 30-40 hours, and the solution is preferably changed every 4-8 hours during the impregnation process.
After the alkali impregnation, most of the protein and pigments in the fish skin can be removed.
According to the invention, before the organic solvent impregnation, the fish skin after the alkali impregnation is preferably washed with water until the eluate is neutral.
According to the present invention, in the organic solvent impregnation, the kind of the organic solvent may be selected from a wide range as long as it can effectively remove fat in the fish skin, and preferably, the organic solvent is an alcohol, more preferably an alcohol of C2-C6, further preferably an alcohol of C3-C5, further preferably butanol, and most preferably n-butanol. The alcohol concentration may be 5-15 vol%, preferably 8-12 vol%.
According to the invention, the amount of organic solvent can vary within wide limits, preferably from 15 to 25 parts by volume, relative to 1 part by volume of fish skin.
According to the invention, the organic solvent impregnation can be carried out at normal temperature, the impregnation time can be 20-30 hours, and the liquid is preferably changed once every 4-8 hours during the impregnation process.
According to the invention, before the pickling, the fish skin after the organic solvent impregnation is preferably washed with water until the eluate is free of organic solvent.
According to the present invention, the acid may be a conventionally used acid. According to a preferred embodiment of the invention, the acid is an acetic acid solution, the concentration of which may be between 0.1 and 1mol/L, preferably between 0.4 and 0.6 mol/L.
According to the invention, the amount of acid used for pickling may vary within wide limits, preferably between 25 and 35 parts by volume with respect to 1 part by volume of fish skin.
According to the invention, the acid impregnation can be carried out at normal temperature, the impregnation time can be 60-100 hours, and continuous stirring is preferably carried out during the impregnation process.
According to the invention, after the acid impregnation, the impregnated material is preferably subjected to solid-liquid separation, for example, centrifugation at 6000-1000rpm for 10-20min to obtain a liquid phase.
In step (ii), the salt used for salting out the liquid phase obtained in step (I) may be a conventional salt capable of precipitating the protein, and in the present invention, the salt is preferably sodium chloride in order to obtain a more pure type I collagen. Among them, the amount of the salt added is preferably such that the concentration of the salt in the liquid phase is 0.7 to 0.9mol/L, more preferably 0.75 to 0.85 mol/L.
According to the present invention, the method of subjecting the salting-out product to solid-liquid separation may be a conventional method, for example, filtration, centrifugation or the like. According to a preferred embodiment of the present invention, the solid-liquid separation is centrifugation, for example, at 6000-10000rpm for 10-20 min.
According to the invention, in step (iii), the acid used for the reconstitution of the solid phase may be a conventional acid, acetic acid is preferred according to the invention, and a 0.4-0.6mol/L acetic acid solution is more preferred.
According to the invention, the acid solution used for dialysis is preferably acetic acid, more preferably an acetic acid solution of 0.8-1.2mol/L, and the dialysis time in the acetic acid can be 24-36 h.
According to the invention, the dialysis is preferably carried out in water for a period of 36 to 48 hours.
According to the present invention, preferably, the method for preparing type I collagen from freshwater fish further comprises lyophilizing the dialyzed material. The lyophilization may be carried out by a conventional process of freeze-drying.
According to the invention, the prepared type I collagen from the freshwater fish can be stored in a refrigerator at the temperature of-80 ℃ for standby and taken out when being used.
In a second aspect, the present invention provides antigenically-removed type I collagen prepared by the method described above.
In a third aspect, the present invention provides the use of the antigenicity-removed type I collagen described above in the preparation of a food, a cosmetic or a biomedical material.
Wherein the food, cosmetic and biomedical material may be any food, cosmetic and biomedical material in which type I collagen is used.
In a fourth aspect, the present invention provides a coconut candy prepared from the raw materials comprising the antigenicity-removed type I collagen as described above, coconut pulp, maltose, coconut candy and water;
the amount of coconut milk is 150-400 parts by weight, the amount of maltose is 30-70 parts by weight, the amount of coconut sugar is 30-70 parts by weight, and the amount of water is 30-70 parts by weight, relative to 100 parts by weight of the antigenicity-removed type I collagen.
Preferably, the amount of coconut milk is 200-300 parts by weight, the amount of maltose is 40-60 parts by weight, the amount of coconut sugar is 40-60 parts by weight, and the amount of water is 40-60 parts by weight, relative to 100 parts by weight of the antigenicity-removed type I collagen.
The term "coconut milk" is a white liquid obtained by grinding and pressing coconut white meat, and is an excellent cool and thirst-quenching beverage.
The term "coconut sugar" refers to a sugar obtained by filtering the juice extracted from all the trees of Palmae and decocting to remove water, and is a natural palm tree sugar, which is a raw sugar. The traditional Chinese medicine is commonly called coco sugar and palm sugar, and the official party should be called palm tree sugar.
According to the present invention, it is understood that the coconut sugar contains glucose as well, since glucose is also used in the process of preparing the antigenicity-removed type I collagen.
In a fifth aspect, the present invention provides a method for preparing coconut candy, comprising:
(1) uniformly mixing coconut milk, maltose and water, and heating a mixed system to 120-150 ℃;
(2) cooling the system of step (1) to 50-70 ℃, adding the antigenicity-removed type I collagen as described above, and cooling to obtain the coconut candy;
wherein, relative to 100 weight portions of the antigenicity-removed type I collagen, the dosage of the coconut milk is 150-400 weight portions, the dosage of the maltose is 30-70 weight portions, the dosage of the coconut sugar is 30-70 weight portions, and the dosage of the water is 30-70 weight portions.
Preferably, the coconut milk is used in an amount of 200-300 parts by weight, the maltose is used in an amount of 40-60 parts by weight, the coco saccharide is used in an amount of 40-60 parts by weight, and the water is used in an amount of 40-60 parts by weight, relative to 100 parts by weight of the antigenicity-removed type I collagen.
According to the present invention, in order to further improve the taste and flavor of the coconut candy prepared, preferably, the method of raising the temperature of the mixed system to 120-150 ℃ comprises: the temperature of the mixed system is raised to 90-110 ℃ at a temperature raising rate of 8-12 ℃/s, preferably 9-11 ℃/s (for example, 9 ℃/s, 9.2 ℃/s, 9.4 ℃/s, 9.6 ℃/s, 9.8 ℃/s, 10 ℃/s, 10.2 ℃/s, 10.4 ℃/s, 10.6 ℃/s, 10.8 ℃/s, 11 ℃/s), and then raised to 150 ℃ at a temperature raising rate of 3-5 ℃/s, preferably 3.5-4.5 ℃/s (for example, 3.5 ℃/s, 3.6 ℃/s, 3.7 ℃/s, 3.8 ℃/s, 3.9 ℃/s, 4 ℃/s, 4.1 ℃/s, 4.2 ℃/s, 4.3 ℃/s, 4.4 ℃/s, 4.5 ℃/s).
According to the present invention, the method of cooling the system of step (1) to 50 to 70 ℃ is preferably natural cooling.
According to the invention, the preparation method of the coconut candy further comprises the steps of adding the antigenicity-removed type I collagen, quickly pouring the obtained mixed material into a mould, cooling and demoulding to obtain the antigenicity-removed collagen coconut candy.
The present invention will be described in detail below by way of examples.
Preparation example
This preparation example is illustrative of the preparation of type I collagen
(1) Removing impurities from freshwater fish skin, cleaning, cutting into 0.5cm by 0.5cm pieces of fish skin, adding 20 times of 0.1mol/L NaOH solution, stirring, leaching for 36h, changing solution every 6h for 1 time, and removing impurity protein and pigment;
(2) repeatedly washing the fish skin soaked with the alkali by using distilled water until the eluate is neutral;
(3) after draining, adding 10 vol% n-butanol with 20 times of volume, stirring, leaching for 24h, changing the solution every 6h, and removing fat in the fish skin;
(4) repeatedly cleaning fish skin with distilled water, draining, adding 30 times volume of 0.5mol/L acetic acid solution, stirring, leaching for 72h, centrifuging at 8000rmp for 15min, and collecting supernatant;
(5) adding pre-ground NaCl powder for salting out, stirring until the final salt concentration is 0.8mol/L, centrifuging at 8000rmp for 15min after salting out, and collecting precipitate.
(6) Re-dissolving the precipitate in 0.5mol/L acetic acid, dialyzing in 0.1mol/L acetic acid solution for 36 hr and distilled water for 48 hr, freeze drying to obtain freshwater fish type I collagen, and storing in refrigerator at-80 deg.C.
Examples 1 to 1
This example demonstrates the removal of antigenicity of type I collagen
(1) Mixing 100 parts by weight of the type I collagen prepared in the preparation example with 100 parts by weight of deionized water, and magnetically stirring until the collagen is sufficiently dissolved;
(2) treating the solution in the step (1) by using dynamic high-pressure microjet, and circulating for 3 times under the pressure of 120 MPa;
(3) adding 100 parts by weight of glucose into the solution obtained in the step (2), and magnetically stirring until the glucose is fully dissolved;
(4) and (4) after the solution in the step (3) is frozen and dried, reacting for 6 hours at 70 ℃ and 75% relative humidity to obtain the antigenicity-removed type I collagen.
Examples 1 to 2
This example demonstrates the removal of antigenicity of type I collagen
(1) Mixing 100 parts by weight of the type I collagen prepared in the preparation example with 67 parts by weight of deionized water, and magnetically stirring until the collagen is sufficiently dissolved;
(2) treating the solution in the step (1) by using dynamic high-pressure microjet, and circulating for 3 times under the pressure of 110 MPa;
(3) adding 90 parts by weight of glucose into the solution obtained in the step (2), and magnetically stirring until the glucose is fully dissolved;
(4) and (4) after the solution in the step (3) is frozen and dried, reacting for 7 hours at 65 ℃ and 70% relative humidity to obtain the antigenicity-removed type I collagen.
Examples 1 to 3
This example demonstrates the removal of antigenicity of type I collagen
(1) Mixing 100 parts by weight of the type I collagen prepared in the preparation example with 150 parts by weight of deionized water, and magnetically stirring until the collagen is sufficiently dissolved;
(2) treating the solution in the step (1) by using dynamic high-pressure microjet, and circulating for 3 times under the pressure of 130 MPa;
(3) adding 110 parts by weight of glucose into the solution obtained in the step (2), and magnetically stirring until the glucose is fully dissolved;
(4) and (4) after the solution in the step (3) is frozen and dried, reacting for 5 hours at 80 ℃ and 80% relative humidity to obtain the antigenicity-removed type I collagen.
Examples 1 to 4
This example demonstrates the removal of antigenicity of type I collagen
(1) Mixing 100 parts by weight of the type I collagen prepared in the preparation example with 100 parts by weight of deionized water, and magnetically stirring until the collagen is sufficiently dissolved;
(2) treating the solution in the step (1) by using dynamic high-pressure microjet, and circulating for 3 times under the pressure of 100 MPa;
(3) adding 80 parts by weight of glucose into the solution obtained in the step (2), and magnetically stirring until the glucose is fully dissolved;
(4) and (4) after the solution in the step (3) is frozen and dried, reacting for 6 hours at 55 ℃ and 60% relative humidity to obtain the antigenicity-removed type I collagen.
Examples 1 to 5
This example demonstrates the removal of antigenicity of type I collagen
(1) Mixing 100 parts by weight of the type I collagen prepared in the preparation example with 100 parts by weight of deionized water, and magnetically stirring until the collagen is sufficiently dissolved;
(2) treating the solution in the step (1) by using dynamic high-pressure microjet, and circulating for 3 times under the pressure of 150 MPa;
(3) adding 120 parts by weight of glucose into the solution obtained in the step (2), and magnetically stirring until the glucose is fully dissolved;
(4) and (4) after the solution in the step (3) is frozen and dried, reacting for 6 hours at 90 ℃ and 85% relative humidity to obtain the antigenicity-removed type I collagen.
Examples 1 to 6
This example demonstrates the removal of antigenicity of type I collagen
The removal of antigenicity of type I collagen was carried out according to the method of example 1-1, except that type I collagen was obtained from the scientific and well-known Biotechnology Co., Ltd, of Wuhan Hua, whose catalog number is fish collagen type I9007-34-5.
Comparative examples 1 to 1
This preparation is illustrative of a reference method for removing antigenicity of type I collagen
The removal of antigenicity of type I collagen was carried out in the same manner as in example 1-1, except that the step (3) was not included, that is, the material obtained in the step (2) was directly subjected to lyophilization and then reacted at 70 ℃ and 75% relative humidity for 6 hours to obtain treated type I collagen.
Comparative examples 1 to 2
This preparation is illustrative of a reference method for removing antigenicity of type I collagen
The removal of antigenicity of type I collagen was carried out in the same manner as in example 1-1, except that the step (3) was not included, and glucose was directly added to the material obtained in the step (2), and the material was lyophilized and reacted at 70 ℃ and 75% relative humidity for 6 hours to obtain treated type I collagen.
Comparative examples 1 to 3
This preparation is illustrative of a reference method for removing antigenicity of type I collagen
The removal of antigenicity of type I collagen was carried out in the same manner as in example 1-1, except that the steps (2) and (3) were not included, and glucose was directly added to the type I collagen solution of step (1), and the resulting mixture was lyophilized and reacted at 70 ℃ and 75% relative humidity for 6 hours to obtain treated type I collagen.
Comparative examples 1 to 4
This preparation is illustrative of a reference method for removing antigenicity of type I collagen
Removal of antigenicity of collagen I was carried out in the same manner as in example 1-1, except that the solution of step (3) was not lyophilized, but the solution of step (3) was directly reacted at 70 ℃ under 75% relative humidity for 6 hours to give treated collagen I.
Comparative examples 1 to 5
This preparation is illustrative of a reference method for removing antigenicity of type I collagen
Removal of antigenicity of collagen type I was carried out in the same manner as in example 1-1, except that glucose was replaced with galactooligosaccharide.
Test example 1
This test example was conducted to test the antigenicity of the collagen type I produced
The antigenicity analysis of the collagen type I prepared in the examples and comparative examples was carried out by enzyme-linked immunosorbent assay. Type I collagen prepared in examples and comparative examples was diluted to 5. mu.g/ml with coating buffer, and 100. mu.l was added to each well of the plate labeled with enzyme, and coated overnight at 4 ℃. After removing the coating solution, 200. mu.l of PBST (PBS + Tween 20) was added to each well, washed 5 times for 1min each time, and then blotted dry after removing the washing solution. Mu.l of blocking solution (PBS + 3% BSA) was added to each well and blocked at 37 ℃ for 2 h. Removing the confining liquid, washing with washing liquid for 5 times, and patting dry. Mu.l of Anti-Collagen I antibody (dilution factor 4000) was added to each well and incubated at 37 ℃ for 1.5 h. The liquid in the microplate was removed, washed 5 times with PBST and patted dry. Mu.l of HRP-labeled goat anti-rabbit IgG antibody (dilution 6000) was added to each well and incubated at 37 ℃ for 1.5 h. The liquid in the microplate was removed, washed 5 times with PBST and patted dry. Mu.l of TMB developing solution was added to each well, and incubated at 37 ℃ for 20min in the absence of light. 50. mu.l of 2mol/L H per well2SO4The reaction was terminated, and the OD value at 450nm wavelength of each well was measured, and the antigenicity of each group of collagen type I was represented by OD450nm, and the antigenicity removal rate (control sample-test sample)/control sample was calculated by using as a control the collagen type I which had not been treated (prepared collagen type I or collagen type I purchased from Biotech, Inc., of Hua Jie, Inc., having the product number of fish collagen type I9007-34-5), and the results are shown in Table 1In the figure, the results of OD values of example 1 and comparative example are shown in FIG. 1.
TABLE 1
| OD value | Removal Rate (%) | |
| Examples 1 to 1 | 0.08 | 90.59％ |
| Examples 1 to 2 | 0.05 | 94.11％ |
| Examples 1 to 3 | 0.07 | 91.76％ |
| Examples 1 to 4 | 0.1 | 88.23％ |
| Examples 1 to 5 | 0.12 | 85.88％ |
| Examples 1 to 6 | 0.15 | 82.35％ |
| Comparative examples 1 to 1 | 0.2 | 76.47％ |
| Comparative examples 1 to 2 | 0.25 | 70.59％ |
| Comparative examples 1 to 3 | 0.35 | 58.82％ |
| Comparative examples 1 to 4 | 0.3 | 64.71％ |
As can be seen from Table 1, the method of the present invention can effectively remove antigenicity while simplifying the operation, and the removal effect is better with the preferred ratio.
Example 2-1
This example illustrates coconut candy and its preparation method
(1) 250 parts by weight of coconut milk (Jiale, Indonesia), 50 parts by weight of maltose, 50 parts by weight of coconut candy (more coconut candy, Thailand) and 50 parts by weight of water are uniformly mixed, heated at a heating rate of 10 ℃/s without stopping stirring, and when the temperature is increased to 100 ℃, the heating is continued to 130 ℃ at a heating rate of 4 ℃/s, and the heating is stopped.
(2) When the syrup in the step (1) is naturally cooled to 60 ℃, quickly and uniformly mixing the powder in the step (4);
(3) and (3) quickly pouring the sugar liquid obtained in the step (2) into a mould, cooling and demoulding to obtain the collagen coconut candy with antigenicity removed.
Examples 2 to 2
This example illustrates coconut candy and its preparation method
The coconut candy was prepared according to the method of example 2-1, except that the heating was continued to 130 ℃ at a heating rate of 5 ℃/s when the temperature was increased to 100 ℃ without stopping the stirring, and the heating was stopped at a heating rate of 8 ℃/s.
Examples 2 to 3
This example illustrates coconut candy and its preparation method
The coconut candy was prepared according to the method of example 2-1, except that the heating was continued to 130 ℃ at a heating rate of 3 ℃/s when the temperature was increased to 100 ℃ without stopping the stirring, and the heating was stopped at a heating rate of 12 ℃/s.
Examples 2 to 4
This example illustrates coconut candy and its preparation method
The coconut candy was prepared according to the method of example 2-1, except that the heating was continued to 130 ℃ at a temperature rising rate of 5 ℃/s when the temperature was raised to 100 ℃ without stopping the stirring, at a temperature rising rate of 12 ℃/s.
Test example 2
The viscosity of the coconut sugar was measured using an antopa rheometer (MCR102, Anton Paar, Graz, Austria) and the results are shown in table 2. The emulsion was measured by selecting a stainless steel conical plate (angle: 1 ℃ C., gap 0.1667mm)) having a diameter of 60mm, setting the shear rate in the range of 0.1 to 100,1/s, strain 1% and measuring temperature 25 ℃.
TABLE 2
| Viscosity (mPa. s) | |
| Example 2-1 | 364 |
| Examples 2 to 2 | 463 |
| Examples 2 to 3 | 481 |
| Examples 2 to 4 | 553 |
Therefore, the coconut candy prepared by the method has better performance.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (9)
1. A method for antigenically removing collagen type I, comprising:
(1) carrying out dynamic high-pressure microjet treatment on the type I collagen solution to obtain a treated type I collagen solution; wherein the pressure for processing the dynamic high-pressure micro-jet is 110-130 MPa;
(2) uniformly mixing the treated type I collagen solution with glucose to obtain a mixed solution, wherein the dosage of the glucose is 90-110 parts by weight relative to 100 parts by weight of type I collagen on a dry basis;
(3) freeze-drying the mixture, and reacting at 60-80 deg.C and 70-80% relative humidity to remove antigenicity of type I collagen.
2. The method according to claim 1, wherein in step (1), the concentration of type I collagen in the type I collagen solution is 40 to 60% by weight.
3. The method of claim 1 or 2 in which the type I collagen is type I collagen from freshwater fish.
4. The method of claim 3, wherein the type I collagen from freshwater fish is prepared by:
(i) sequentially carrying out alkali impregnation, organic solvent impregnation and acid impregnation on the freshwater fish skin, and carrying out solid-liquid separation on the acid-impregnated material to obtain a liquid phase;
(ii) (ii) salting out the liquid phase obtained in the step (i), and carrying out solid-liquid separation on the obtained salting-out product to obtain a solid phase;
(iii) dissolving the solid phase in acid, and then dialyzing in acid solution and water in sequence to obtain the type I collagen from the freshwater fish.
5. Antigenically removed type I collagen produced by the method of any one of claims 1 to 4.
6. Use of the antigenicity-removed type I collagen of claim 5 in the preparation of food, cosmetics or biomedical materials.
7. A coconut candy characterized by being prepared from the raw materials comprising the antigenicity-removed type I collagen of claim 5, coconut milk, maltose, coconut candy and water;
the amount of coconut milk is 150-400 parts by weight, the amount of maltose is 30-70 parts by weight, the amount of coconut sugar is 30-70 parts by weight, and the amount of water is 30-70 parts by weight, relative to 100 parts by weight of the antigenicity-removed type I collagen.
8. A method for preparing coconut candy is characterized by comprising the following steps:
(1) uniformly mixing the coconut milk, maltose, coconut sugar and water, and heating the mixed system to 120-150 ℃;
(2) cooling the system of step (1) to 50-70 ℃, adding the antigenically removed type I collagen of claim 5, and cooling to obtain the coconut sugar;
wherein, relative to 100 weight portions of the antigenicity-removed type I collagen, the dosage of the coconut milk is 150-400 weight portions, the dosage of the maltose is 30-70 weight portions, the dosage of the coconut sugar is 30-70 weight portions, and the dosage of the water is 30-70 weight portions.
9. The method as claimed in claim 8, wherein the step (1) of raising the temperature of the mixed system to 110-150 ℃ comprises: the temperature of the mixed system is raised to 90-110 ℃ at the temperature raising rate of 8-12 ℃/s, and then the temperature is raised to 120-150 ℃ at the temperature raising rate of 3-5 ℃/s.
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| CN101948533A (en) * | 2010-09-21 | 2011-01-19 | 苏州大学 | Preparation method of fish skin collagen |
| CN105613902A (en) * | 2014-10-27 | 2016-06-01 | 卢雨萍 | Tamarind fruit-coconut candy and production method thereof |
| CN109897879A (en) * | 2019-04-30 | 2019-06-18 | 江西师范大学 | A kind of preparation method of subsensitivety ovalbumin peptide |
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| CN101948533A (en) * | 2010-09-21 | 2011-01-19 | 苏州大学 | Preparation method of fish skin collagen |
| CN105613902A (en) * | 2014-10-27 | 2016-06-01 | 卢雨萍 | Tamarind fruit-coconut candy and production method thereof |
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