CN114617262A - Preparation method of flavor enzyme enzymolysis nanoparticles for resisting gastrointestinal digestion - Google Patents
Preparation method of flavor enzyme enzymolysis nanoparticles for resisting gastrointestinal digestion Download PDFInfo
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- CN114617262A CN114617262A CN202210380054.XA CN202210380054A CN114617262A CN 114617262 A CN114617262 A CN 114617262A CN 202210380054 A CN202210380054 A CN 202210380054A CN 114617262 A CN114617262 A CN 114617262A
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- nanoparticles
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- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 claims description 2
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- 239000005018 casein Substances 0.000 claims description 2
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/14—Vegetable proteins
- A23J3/16—Vegetable proteins from soybean
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
- A23L29/045—Organic compounds containing nitrogen as heteroatom
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/15—Vitamins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/15—Vitamins
- A23L33/155—Vitamins A or D
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
- A23P10/00—Shaping or working of foodstuffs characterised by the products
- A23P10/30—Encapsulation of particles, e.g. foodstuff additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses a preparation method of flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion, which comprises the following steps: preparing particles, preparing an organic phase, mixing the two phases, removing the organic phase and removing a small amount of insoluble substances. The zinc oxide has good shape and structure, good chemical zinc oxide, good anti-digestion performance, protection of fat-soluble cellulose from being damaged during digestion in vivo, contribution to absorption by human body, simple raw materials, low cost, simple and easily-controlled preparation process, economic use, greenness, safety and suitability for industrial continuous production.
Description
Technical Field
The invention belongs to the technical field of food additives, and particularly relates to a preparation method of flavor enzyme enzymolysis nanoparticles for resisting gastrointestinal digestion.
Background
Fat-soluble nutrients have low solubility in water and poor permeability to gastrointestinal mucus, and are difficult to pass through a water layer without agitation, and thus are difficult to absorb and utilize in the human body. In order to improve the bioavailability of the fat-soluble nutrient, the fat-soluble nutrient is prevented from being damaged and degraded in the digestive tract to the maximum extent, so that the fat-soluble nutrient can smoothly reach epithelial cells of small intestines to exert the health-care effect. If a carrier form is adopted to embed the fat-soluble nutrient, the solubility of the nutrient in water is greatly enhanced, and the utilization efficiency of the nutrient can be greatly improved.
Protein is a biological material with great potential value, but the protein molecule is highly compressed and has compact structure, hydrophobic active substances are difficult to wrap in the protein molecule, and the controlled release performance of the loaded fat-soluble functional components is poor. Thereby limiting their use in the field of functional carriers.
After the protein is hydrolyzed by protease, the polypeptide formed by hydrolysis has good interfacial activity and self-assembly capability, and new carriers such as nano-micelle, nano-fiber, nano-tube, nano-particle and the like are formed in a non-covalent combination mode to wrap hydrophobic active substances, so that the protein has great potential in the delivery of nutritional food and drugs. Meanwhile, the number of enzyme cutting sites of the nano-particles formed by enzymolysis in the gastrointestinal digestion process is reduced compared with that of original protein, so that the complete morphological structure of the nano-particles can be maintained more easily in the digestion process, fat-soluble nutrient substances wrapped in the nano-particles can be better protected, and the absorption and utilization of a human body are facilitated.
CN202110683000.6 adopts the combined action of electrostatic crosslinking and enzymolysis of sulfite to prepare nanoparticles, and sulfite or bisulfite is decomposed when meeting strong acid, so that the prepared particles can change along with the change of pH, thereby reducing the stability of the particles in the process of gastric digestion and failing to well protect the wrapped fat-soluble nutrients. CN201810009738.2 adopts an ultrasonic method to wrap curcumin in nanoparticles formed by enzymolysis, and the method is only suitable for some nutrients with weak non-polarity and is not suitable for nutrients with strong non-polarity, such as beta-carotene, vitamin E and the like, thereby limiting the application range of the method.
The flavor protease hydrolyzes hydrophilic groups, so that some hydrophilic amino acids on the surface can be hydrolyzed in the process of preparing the nano particles, the interior hidden in the interior and provided with a compact structure is exposed, the amphipathy of hydrolysate is enhanced, alkaline protease, trypsin, pepsin and the like are commonly used for preparing the enzyme for embedding the fat-soluble nutrient nano particles by an enzymolysis method, and the flavor protease has great application potential in the aspect.
Aiming at the defects of the prior art, the invention hopes to find an efficient and simple method, and the obtained product has small nano particles, good embedding effect, high stability and bioavailability, and effectively solves the adverse effect of gastrointestinal digestion of a human body on fat-soluble nutrients.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made keeping in mind the above and/or other problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide a preparation method of flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion comprises the following steps:
preparing particles, namely performing enzymolysis on a protein solution by using flavor enzyme, heating to inactivate the enzyme and simultaneously promoting self-assembly to prepare nano particles;
preparing an organic phase, namely mixing the fat-soluble nutrient with an organic solvent to prepare a fat-soluble nutrient solution;
mixing the two phases: mixing the fat-soluble nutrient solution and the nano particles by using a high-speed stirrer;
removing an organic phase: removing the organic solvent by using a nitrogen blowing instrument;
removing a small amount of insoluble substances, centrifuging the hydrolysate at a rotation speed of 10000r/min for 20 min.
The invention relates to a preferable scheme of a preparation method of flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion, which comprises the following steps: preparing an organic phase, wherein the organic solvent comprises one or more of dichloromethane, methanol, ethyl acetate, chloroform and acetonitrile. .
As a preferable scheme of the preparation method of the gastrointestinal digestion resistant flavourzyme enzymolysis nanoparticle, the preparation method comprises the following steps: in the preparation of the particles, the protein in the protein solution is one or more of soybean protein isolate, alpha-lactalbumin, rice protein, peanut protein isolate, bovine serum albumin, casein and corn protein.
The invention relates to a preferable scheme of a preparation method of flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion, which comprises the following steps: preparing organic phase, wherein the fat-soluble nutrient comprises one of beta-carotene, vitamin-E, lutein, curcumin, resveratrol and vitamin D.
The invention relates to a preferable scheme of a preparation method of flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion, which comprises the following steps: in the two-phase mixing, the fat-soluble nutrient solution and the nano particles are mixed according to the volume ratio of 1: 2-9.
The invention relates to a preferable scheme of a preparation method of flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion, which comprises the following steps: in the preparation of the particles, the concentration of the protein solution is 0.5-2% by weight.
The invention relates to a preferable scheme of a preparation method of flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion, which comprises the following steps: in the preparation of the granules, the ratio of the flavor enzyme to the substrate is 0.05-0.2% by weight.
The invention relates to a preferable scheme of a preparation method of flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion, which comprises the following steps: in the mixing of the two phases, the rotating speed of a high-speed stirrer is 8000rpm, and the stirring time is 4-8 min.
The invention relates to a preferable scheme of a preparation method of flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion, which comprises the following steps: in preparing the granules, the concentration of the protein solution was 2% by weight.
The invention relates to a preferable scheme of a preparation method of flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion, which comprises the following steps: the ratio of flavourzyme to substrate in the granulate was 0.1% by weight.
The invention provides a method for preparing flavor enzyme enzymolysis nano-particles for resisting gastrointestinal digestion, which has the following advantages:
1. the formed nanoparticles have good shape structure: spherical and at the same time uniform. The particle size is 80-100nm, and the particle size is small, so that the particles can smoothly enter epithelial cells of small intestines.
2. The prepared nano-particles have ideal anti-digestibility, so that the fat-soluble vitamins are protected from being damaged during digestion in vivo, and the absorption by a human body is facilitated.
3. Compared with the fat-soluble vitamin micelle dispersion, the water-soluble fat-soluble nutrient nano-particles prepared by the invention have higher chemical stability, and the retention rate of the fat-soluble nutrient is still kept above 50 percent after being placed for 15 days under illumination.
4. The invention has the advantages of simple raw materials, low raw material cost, simple and easily-controlled preparation process, economy, practicality, greenness, safety and suitability for industrial continuous production.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a flow chart of the preparation of the present invention; .
FIG. 2 is a graph showing the release of beta-carotene during gastric digestion in vitro in example 6 of the present invention and in comparative example 1;
FIG. 3 shows the release of beta-carotene during intestinal digestion in vitro in example 6 and comparative example 1 in accordance with the present invention;
FIG. 4 is a transmission electron micrograph of nanoparticles prepared in example 6;
fig. 5 is a projection electron micrograph of the nanoparticles of comparative example 1.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention more comprehensible, embodiments accompanying the present invention are described in detail below with reference to examples.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The nitrogen blowing instrument adopted in the embodiment of the invention is an American organization nitrogen blowing instrument N-EVAP 112 (without gears), and the stirrer used in the invention is a pre-sublimation heat collection type constant temperature heating magnetic stirrer DF-101S.
Example 1
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing 2% (w/w) protein solution and 0.1% (w/w) beta-carotene solution by using water and ethyl acetate, placing the protein solution in a heat collection type constant temperature heating magnetic stirrer, heating at the rotating speed of 200r/min and the temperature of 50 ℃ and keeping the constant temperature for 10 minutes, adding flavourzyme (Sigma P6110) into the protein solution, reacting for 30 minutes, and heating at the temperature of 85 ℃ for 10 minutes to inactivate the enzyme, wherein the ratio of the enzyme to a substrate is 0.1% (w/w). After cooling to room temperature, dropwise adding the beta-carotene solution into the enzyme-deactivated enzymolysis liquid under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 4min, and the ratio of the final organic solution to the water solution is 1: 5. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifuge at the rotating speed of 10000r/min for 20 min.
Example 2
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing 1% (w/w) protein solution and 0.1% (w/w) beta-carotene solution by using water and ethyl acetate, placing the protein solution in a heat collection type constant temperature heating magnetic stirrer, heating at the rotating speed of 200r/min and the temperature of 50 ℃ and keeping the constant temperature for 10 minutes, adding flavourzyme (Sigma P6110) into the protein solution, reacting for 30 minutes, and then heating at the temperature of 85 ℃ for 10 minutes to inactivate the enzyme. After cooling to room temperature, the beta-carotene solution is dropwise added into the enzymatic hydrolysate after enzyme deactivation under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 4min, and the ratio of the final organic solution to the water solution is 1: 5. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifuge at the rotating speed of 10000r/min for 20 min.
Example 3
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing 0.5% (w/w) protein solution and 0.1% (w/w) beta-carotene solution by using water and ethyl acetate, placing the protein solution in a heat collection type constant temperature heating magnetic stirrer, heating at the rotating speed of 200r/min and the constant temperature of 50 ℃ for 10 minutes, adding flavourzyme (Sigma P6110) into the protein solution, reacting for 30 minutes, and then heating at the temperature of 85 ℃ for 10 minutes to inactivate the enzyme. After cooling to room temperature, the beta-carotene solution is dropwise added into the enzyme-deactivated enzymolysis liquid under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 4min, and the ratio of the final organic solution to the water solution is 1: 5. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifuge at the rotating speed of 10000r/min for 20 min.
Example 4
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing 2% (w/w) of protein solution and 0.1% (w/w) of beta-carotene solution by using water and ethyl acetate, placing the protein solution in a heat collection type constant temperature heating magnetic stirrer, heating at the rotating speed of 200r/min and the temperature of 50 ℃ and keeping the constant temperature for 10 minutes, adding flavourzyme (Sigma P6110) into the protein solution, reacting for 30 inches, and then heating at the temperature of 85 ℃ for 10 minutes to inactivate the enzyme, wherein the ratio of the enzyme to a substrate is 0.05% (w/w). After cooling to room temperature, the beta-carotene solution is dropwise added into the enzyme-deactivated enzymolysis liquid under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 4min, and the ratio of the final organic solution to the water solution is 1: 2. Then a nitrogen blowing instrument is used for removing the ethyl acetate,
example 5
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing 2% (w/w) of protein solution and 0.1% (w/w) of beta-carotene solution by using water and ethyl acetate, placing the protein solution in a heat collection type constant temperature heating magnetic stirrer, heating at the rotating speed of 200r/min and the temperature of 50 ℃ and keeping the constant temperature for 10 minutes, adding flavourzyme (Sigma P6110) into the protein solution, reacting for 30 minutes, and then heating at the temperature of 85 ℃ for 10 minutes to inactivate the enzyme. After cooling to room temperature, the beta-carotene solution is dropwise added into the enzyme-deactivated enzymolysis liquid under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 4min, and the ratio of the final organic solution to the water solution is 1: 9. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifuge at the rotating speed of 10000r/min for 20 min.
Example 6
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing 2% (w/w) of protein solution and 0.1% (w/w) of beta-carotene solution by using water and ethyl acetate, placing the protein solution in a heat collection type constant temperature heating magnetic stirrer, heating at the rotating speed of 200r/min and the temperature of 50 ℃ and keeping the constant temperature for 10 minutes, adding flavourzyme (Sigma P6110) into the protein solution, reacting for 60 minutes, and then heating at the temperature of 85 ℃ for 10 minutes to inactivate the enzyme. After cooling to room temperature, the beta-carotene solution is dropwise added into the enzyme-deactivated enzymolysis liquid under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 6min, and the ratio of the final organic solution to the water solution is 1: 5. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifuge at the rotating speed of 10000r/min for 20 min.
Example 7
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing a 2% (w/w) protein solution and a 0.1% (w/w) beta-carotene solution by using water and ethyl acetate, placing the protein solution in a magnetic stirring pot at 50 ℃, heating and keeping the temperature constant for 10 minutes, adding flavourzyme (Sigma P6110) into the protein solution, reacting for 90 minutes, and then heating at 85 ℃ for 10 minutes to inactivate the enzyme. After cooling to room temperature, the beta-carotene solution is dropwise added into the enzyme-deactivated enzymolysis liquid under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 6min, and the ratio of the final organic solution to the water solution is 1: 5. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifugal machine at the rotating speed of 10000r/min for 20 min.
Example 8
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing 2% (w/w) of protein solution and 0.1% (w/w) of beta-carotene solution by using water and ethyl acetate, placing the protein solution in a heat collection type constant temperature heating magnetic stirrer, heating at the rotating speed of 200r/min and the temperature of 50 ℃ and keeping the constant temperature for 10 minutes, adding flavourzyme (Sigma P6110) into the protein solution, reacting for 60 minutes, and then heating at the temperature of 85 ℃ for 10 minutes to inactivate the enzyme. After cooling to room temperature, the beta-carotene solution is dropwise added into the enzyme-deactivated enzymolysis liquid under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 6min, and the ratio of the final organic solution to the water solution is 1: 2. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifuge at the rotating speed of 10000r/min for 20 min.
Example 9
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing 2% (w/w) of protein solution and 0.1% (w/w) of beta-carotene solution by using water and ethyl acetate, placing the protein solution in a heat collection type constant temperature heating magnetic stirrer, heating at the rotating speed of 200r/min and the temperature of 50 ℃ and keeping the constant temperature for 10 minutes, adding flavourzyme (Sigma P6110) into the protein solution, reacting for 60 minutes, and then heating at the temperature of 85 ℃ for 10 minutes to inactivate the enzyme. After cooling to room temperature, the beta-carotene solution is dropwise added into the enzyme-deactivated enzymolysis liquid under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 6min, and the ratio of the final organic solution to the water solution is 1: 9. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifuge at the rotating speed of 10000r/min for 20 min.
Example 10
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing 2% (w/w) of protein solution and 0.1% (w/w) of beta-carotene solution by using water and ethyl acetate, placing the protein solution in a heat collection type constant temperature heating magnetic stirrer, heating at the rotating speed of 200r/min and the temperature of 50 ℃ and keeping the constant temperature for 10 minutes, adding flavourzyme (Sigma P6110) into the protein solution, reacting for 60 minutes, and then heating at the temperature of 85 ℃ for 10 minutes to inactivate the enzyme. After cooling to room temperature, the beta-carotene solution is dropwise added into the enzyme-deactivated enzymolysis liquid under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 4min, and the ratio of the final organic solution to the water solution is 1: 9. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifugal machine at the rotating speed of 10000r/min for 20 min.
Example 11
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing 2% (w/w) of protein solution and 0.1% (w/w) of beta-carotene solution by using water and ethyl acetate, placing the protein solution in a heat collection type constant temperature heating magnetic stirrer, heating at the rotating speed of 200r/min and the temperature of 50 ℃ and keeping the constant temperature for 10 minutes, adding flavourzyme (Sigma P6110) into the protein solution, reacting for 60 minutes, and then heating at the temperature of 85 ℃ for 10 minutes to inactivate the enzyme. After cooling to room temperature, the beta-carotene solution is dropwise added into the enzyme-deactivated enzymolysis liquid under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 8min, and the ratio of the final organic solution to the water solution is 1: 9. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifuge at the rotating speed of 10000r/min for 20 min.
Comparative example 1
Accurately weighing soybean protein isolate and beta-carotene by using an analytical balance, preparing 2% (w/w) of protein solution and 0.1% (w/w) of beta-carotene solution by using water and ethyl acetate, placing the protein solution in a heat collection type constant temperature heating magnetic stirrer, heating at the rotating speed of 200r/min and the temperature of 50 ℃ and keeping the constant temperature for 10 minutes, adding alkaline protease (Novesson Alcalase 2.4L FG) into the protein solution, reacting for 60 minutes, and then heating at the temperature of 85 ℃ for 10 minutes to inactivate the enzyme. After cooling to room temperature, dropwise adding the beta-carrot solution into the enzyme-deactivated enzymolysis liquid under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding lutein into the granule with high speed stirrer at 8000rpm/min for 4min, and mixing the final organic solution and water at a ratio of 1: 5. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifuge at the rotating speed of 10000r/min for 20 min.
Comparative example 2
Accurately weighing the isolated soy protein and the beta-carotene by using an analytical balance, preparing 0.5% (w/w) protein solution by using water and ethyl acetate, and dropwise adding the beta-carotene solution into the isolated soy protein solution under the stirring condition of a high-speed stirrer at 8000 rpm/in. Embedding beta-carotene into the granules by using a high-speed stirrer, wherein the rotating speed is 8000rpm/min, the time is 4min, and the ratio of the final organic solution to the water solution is 1: 5. And removing ethyl acetate by using a nitrogen blowing instrument, and finally removing a small amount of insoluble substances by using a centrifugal machine at the rotating speed of 10000r/min for 20 min.
Example 12
Beta-carotene prepared in examples 1 to 11 and comparative examples 1 and 2 was used for detection, encapsulation efficiency thereof was measured and in vitro simulated digestion was performed.
The in vitro simulated digestion method is as follows:
5mL of the beta-carotene nanocarrier sample was mixed with an equal amount of ultrapure water, added to 10mL of simulated gastric fluid (containing 2g/L NaCl L, 3.2g/L pepsin, and 7mL/L concentrated HCl), and then the pH was adjusted to 2.0 with 0.5M NaOH or 0.5M HCl. The digest was stirred slowly at 37 ℃ for 1 hour. Immediately after the gastric phase digestion, the digested sample was adjusted to ph7.0 with 2.5M sodium hydroxide. Then 20mL of a simulated intestinal solution (containing 8mg/mL bile salts, 1mg/mL trypsin and 5mM calcium chloride, pH7.0 in PBS buffer) was added to the sample solution at a ratio of 1:1, and the reaction was carried out at 37 ℃ for 2 hours with slow stirring.
The encapsulation and bioavailability of the resulting digested digest β -carotene was determined and the results obtained are reported in table 1.
TABLE 1 encapsulation efficiency and bioavailability of beta-carotene prepared in examples 1-11 and comparative examples 1, 2
| Examples | Encapsulation efficiency (%) | Biological availability (%) |
| Example 1 | 62.1±0.23 | 66.1±0.32 |
| Example 2 | 52.3±0.49 | 56.3±0.83 |
| Example 3 | 39.9±0.65 | 45.1±0.54 |
| Example 4 | 42.1±0.39 | 45.6±0.67 |
| Example 5 | 52.1±0.42 | 57.1±0.92 |
| Example 6 | 92.1±0.19 | 95.2±0.44 |
| Example 7 | 82.1±0.39 | 85.2±0.56 |
| Example 8 | 42.1±0.41 | 42.3±0.87 |
| Example 9 | 82.1±0.98 | 86.1±0.76 |
| Example 10 | 62.1±0.43 | 63.2±0.23 |
| Example 11 | 72.1±0.78 | 78.1±0.98 |
| Comparative example 1 | 32.1±0.49 | 33.1±0.93 |
| Comparative example 2 | 10.12±0.32 | 13.3±0.42 |
As can be seen from table 1, the encapsulation efficiency and bioavailability of the β -carotene carrier prepared in example 6 were the highest, and the preferred example was example 6. Meanwhile, as can be seen from table 1, the encapsulation efficiency and the bioavailability of the examples are much higher than those of the comparative examples 1-2, which shows the superiority of the examples.
The highest encapsulation efficiency and bio-availability data were obtained for the beta-carotene carriers prepared in examples 1, 2, and 3, and the optimal concentration of the protein solution was 2% (w/w).
The best ratio of enzyme to protein was 0.1% (w/w), based on the highest encapsulation efficiency and bioavailability data obtained for the beta-carotene carriers prepared in examples 1, 4, 5.
The highest encapsulation efficiency and bioavailability data were obtained for the beta-carotene carriers prepared in examples 1, 6, and 7, and the optimal time for enzymatic hydrolysis was 60 min.
The highest maximum encapsulation efficiency and bioavailability data were obtained for the beta-carotene carriers prepared in examples 6, 8, and 9, and the optimal ratio condition for mixing the proteolytic enzymatic hydrolysate and the organic phase was 1: 5.
The highest encapsulation and bioavailability data were obtained for the beta-carotene carriers prepared in examples 6, 10, and 11, and the optimal time for high speed dispersion was 4 minutes.
Example 13
Secondary structures were measured by circular dichroism using the protein enzymatic hydrolysate and the protein solution prepared in example 6 and comparative examples 1 and 2, and the obtained results are recorded in table 2.
Table 2 secondary structure content of solutions prepared in example 6 and comparative examples 1 and 2
| Two-stage structure | Alpha-helix | beta-S sheet | Beta-turn angle | Random crimp |
| Example 6 | 22.60％ | 46.10％ | 16.30％ | 15.60％ |
| Comparative example 1 | 17.00％ | 52.50％ | 14.50％ | 14.20％ |
| Comparative example 2 | 13.40％ | 52.30％ | 21.10％ | 13.20％ |
As can be seen from table 2, the content of α -helix and random coil in example 6 is higher than that in comparative example 1 and comparative example 2, which indicates that the particles formed in comparative example 6 are more flexible and flexible in structure, which helps protein molecules to be closely adsorbed on the interface, so that the nanoparticles have better emulsibility, and the β -carotene is more easily encapsulated by the nanoparticles.
Example 14
The free amino acid content was determined using the solutions prepared in example 6 and comparative example 1 and compared to the SPI total amino acid content, and the results obtained are reported in table 3.
TABLE 3 content of free amino acids and amount of SPI total amino acids in the finally prepared solutions of EXAMPLE 6 and COMPARATIVE EXAMPLE 1
As can be seen from Table 3, the percentage difference between the release of free amino acids in example 6 and that in comparative example 1 was large, and the release of met (20.33%), phe (16.16%), leu (13.12%) was relatively large for each amino acid in example 6. Comparative example 1, however, contained a greater amount of free amino acids-cys-s (23.66%) and val (15.2%), leu (37, 97%), phe (19.65%) with the remaining amino acids not much exposed.
This is because natural soy protein isolate is a globular protein, usually located internally with most non-polar amino acids and polar amino acids on the outside. The polypeptide chains fold into a compact, globular conformation. These subunits in turn associate with each other to form a complex quaternary structure. The soybean protein molecules are highly compressed and have compact structures, so that hydrophobic active substances are difficult to wrap in the soybean protein molecules, and the controlled release performance of the soybean protein molecules on loaded fat-soluble functional ingredients is poor.
The alkaline protease hydrolyzes peptide bonds of hydrophobic amino acids, which are peptide bonds in the protein, and meanwhile, the hydrolysis specificity is low, and the peptide bonds of certain specific amino acids cannot be hydrolyzed, so that more hydrophobic amino acids are liberated, the hydrophobic amino acids in the particles are fewer, the contact positions of the particles and beta-carotene are reduced, and the encapsulation efficiency is lower.
The flavourzyme is characterized in that the endoprotease and the exoenzyme can hydrolyze peptide bonds on peptide chains, so that some hydrophilic amino acids on the surfaces and hydrophobic amino acids in the interior can be hydrolyzed in the process of preparing the nano particles, the interior with a compact structure hidden in the interior is exposed, the formed particles are more amphiphilic, and therefore beta-carotene can be embedded in the interior of the particles more easily, and the flavourzyme used in the embodiment has certain superiority in effect compared with alkaline protease.
Example 15
The nanoparticles of example 6 and comparative example 1 were photographed by transmission electron microscopy and subjected to in vitro simulated digestion, wherein gastric digestion was performed for 0, 5, 15, 30, and 60 minutes, intestinal digestion was performed for 0, 5, 15, 30, 60, and 90120 minutes, and the release rate of beta-carotene was measured, as shown in fig. 2, the release rate of beta-carotene was significantly lower in gastric digestion than in comparative example 1, demonstrating that the nanoparticles formed in example 6 have a certain resistance to digestion. As shown in fig. 2, the nanoparticles prepared in example 6 have a lower release rate of β -carotene than comparative example 1 during intestinal digestion, and achieve the desired sustained release effect.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A preparation method of flavor enzyme enzymolysis nanoparticles for resisting gastrointestinal digestion is characterized by comprising the following steps: the method comprises the following steps:
preparing particles, namely performing enzymolysis on a protein solution by using flavor enzyme, heating to inactivate the enzyme and simultaneously promoting self-assembly to prepare nano particles;
preparing an organic phase, namely mixing the fat-soluble nutrient with an organic solvent to prepare a fat-soluble nutrient solution;
mixing the two phases: mixing the fat-soluble nutrient solution and the nano particles by using a high-speed stirrer;
removing an organic phase: removing the organic solvent by using a nitrogen blowing instrument;
removing a small amount of insoluble substances, centrifuging the hydrolysate at a rotation speed of 10000r/min for 20 min.
2. The method for preparing flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion according to claim 1, wherein the method comprises the following steps: in the preparation organic phase, the organic solvent comprises one or more of dichloromethane, methanol, ethyl acetate, trichloromethane and acetonitrile.
3. The method for preparing flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion according to claim 1, wherein the method comprises the following steps: in the prepared particles, the protein in the protein solution is one or more of soybean protein isolate, alpha-lactalbumin, rice protein, peanut protein isolate, bovine serum albumin, casein and zein.
4. The method for preparing flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion according to claim 1, wherein the method comprises the following steps: in the organic phase, the fat-soluble nutrient comprises one of beta-carotene, vitamin-E, lutein, curcumin, resveratrol and vitamin D.
5. The method for preparing flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion according to claim 2, wherein the method comprises the following steps: in the two-phase mixing, the fat-soluble nutrient solution and the nano particles are mixed according to the volume ratio of 1: 2-9.
6. The method for preparing flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion according to claim 3, wherein the method comprises the following steps: in the prepared particles, the concentration of the protein solution is 0.5-2% by weight.
7. The method for preparing flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion according to claim 1, wherein the method comprises the following steps: in the prepared particles, the ratio of the flavor enzyme to the substrate is 0.05-0.2% by weight.
8. The method for preparing flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion according to claim 1, wherein the method comprises the following steps: in the two-phase mixing, the rotating speed of a high-speed stirrer is 8000rpm, and the stirring time is 4-8 min.
9. The method for preparing flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion according to claim 6, wherein the method comprises the following steps: in the preparation of granules, the concentration of the protein solution was 2% by weight.
10. The method for preparing flavourzyme enzymolysis nanoparticles for resisting gastrointestinal digestion according to claim 7, wherein the method comprises the following steps: in the prepared granules, the ratio of flavourzyme to substrate was 0.1% by weight.
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