CN116869166A - Fish oil emulsion and preparation method and application thereof - Google Patents

Fish oil emulsion and preparation method and application thereof Download PDF

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Publication number
CN116869166A
CN116869166A CN202310915735.6A CN202310915735A CN116869166A CN 116869166 A CN116869166 A CN 116869166A CN 202310915735 A CN202310915735 A CN 202310915735A CN 116869166 A CN116869166 A CN 116869166A
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fish oil
emulsion
oil
preparation
microcapsule
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CN116869166B (en
Inventor
夏秋瑜
禤俊勇
刘书成
孙钦秀
魏帅
王泽富
韩宗元
刘阳
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Guangdong Ocean University
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Guangdong Ocean University
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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
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • 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
    • A23L29/06—Enzymes
    • 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
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/231—Pectin; Derivatives thereof
    • 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
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/238—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seeds, e.g. locust bean gum or guar gum
    • 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
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/256—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seaweeds, e.g. alginates, agar or carrageenan
    • 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
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • A23L29/281—Proteins, e.g. gelatin or collagen
    • 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
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • A23L29/281—Proteins, e.g. gelatin or collagen
    • A23L29/284—Gelatin; Collagen
    • 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
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    • A23L29/294—Inorganic additives, e.g. silica
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/60—Fish, e.g. seahorses; Fish eggs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/04—Dispersions; Emulsions
    • A61K8/06—Emulsions
    • A61K8/062—Oil-in-water emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/11—Encapsulated compositions
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A61K8/925—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of animal origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5052—Proteins, e.g. albumin
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61Q19/08—Anti-ageing preparations
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    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/14—Polymerisation; cross-linking
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Abstract

The invention belongs to the technical field of biological material preparation, and particularly relates to a fish oil emulsion and a preparation method and application thereof. According to the invention, the fish oil zymolyte and the fish oil which is not subjected to enzymolysis are used as oil phases to prepare the fish oil emulsion for the first time, and an external emulsifier and an external antioxidant are not required to be added, so that the requirements of safety and environmental protection can be met, the stability and the nutritional value of the emulsion can be improved, the fish oil emulsion is further prepared into the fish oil microcapsule, and the obtained fish oil microcapsule can form a more compact embedding structure, so that the oxidation stability of the microcapsule is improved; the preparation process of the fish oil microcapsule is simple to operate, the reaction condition is mild, the fish oil microcapsule is green and safe, and the fish oil microcapsule has a high application value.

Description

Fish oil emulsion and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological material preparation. More particularly relates to a fish oil emulsion and a preparation method and application thereof.
Background
Fish oils have a variety of health effects due to the enrichment of Omega-3 polyunsaturated fatty acids (Omega-3 PUFAs). However, the properties of fish oil, such as easy oxidation, poor water solubility, heavy fishy smell, etc., limit its application in industry. Thus, microencapsulation of fish oil is an effective means to delay its oxidation and mask the fishy smell, enabling stable delivery of Omega-3 functional lipids. For example, chinese patent application CN111011860a discloses a fish oil microcapsule powder containing a natural antioxidant composition, which can effectively prevent oxidation of unsaturated fatty acid grease by external high temperature environment and oxygen, and improve shelf life and stability of the product. However, this method still requires the addition of a large amount of antioxidants to improve the stability of the fish oil. In addition, the content of omega-3 PUFAs in natural fish oil is low, the specific fatty acid proportion is not ideal enough, and the special requirements of groups such as food, medicine and the like are difficult to meet.
Therefore, there is an urgent need to provide a fish oil product which does not require additional antioxidants, is not easily oxidized, and has high nutritive value.
Disclosure of Invention
The invention aims to overcome the defects that the existing fish oil product still needs to be added with an exogenous antioxidant to improve the stability of the fish oil and the specific fat proportion in the fish oil is not ideal enough, and provides a preparation method of the fish oil emulsion.
The invention aims to provide the fish oil emulsion obtained by the preparation method.
The invention further aims to provide a preparation method of the fish oil microcapsule, which is used for further preparing the fish oil emulsion into the fish oil microcapsule, and the obtained fish oil microcapsule can form a more compact embedding structure, so that the oxidation stability of the microcapsule is improved.
The invention also aims to provide the fish oil microcapsule obtained by the preparation method.
It is another object of the present invention to provide the use of said fish oil emulsion or fish oil microcapsule.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a preparation method of fish oil emulsion, which comprises the following steps:
s1, adding a buffer solution into fish oil, and adding lipase for enzymolysis to obtain fish oil zymolyte;
s2, preparing an oil phase: adding the fish oil zymolyte obtained in the step S1 into fish oil, and fully and uniformly mixing to obtain a mixed oil phase;
s3, preparation of O/W emulsion: fully and uniformly mixing the mixed oil phase obtained in the step S2 with the protein solution to form fish oil emulsion;
wherein the lipase is Candida antarctica lipase (i.e., ADL lipase), which can improve the natural composition of fish lipids, has a stronger DHA enrichment, and exhibits the potential to synthesize Omega-3 PUFAs functional lipids due to its selective hydrolysis of saturated fatty acids and the production of higher levels of Omega-3 containing glycerides;
the hydrolysis degree of the fish oil zymolyte is 20-60%. Modification of fish oil quality can be realized by an enzymatic catalysis technology, so that structural lipid which is obviously improved in the aspects of physicochemical property, structural characteristics, nutritional characteristics and the like is obtained, and the stability and the nutritional value of the obtained fish oil emulsion are improved.
Preferably, the hydrolysis degree of the fish oil enzymatic hydrolysate is 25-30%.
Preferably, the addition amount of the lipase is 1.0-4.0 wt% of the mass of the fish oil.
More preferably, in step S1, the lipase is added in an amount of 1.5 to 2wt% based on the mass of the fish oil.
Preferably, in the step S2, the addition amount of the fish oil zymolyte is 10-50wt% of the mass of the mixed oil phase.
More preferably, in the step S2, the addition amount of the fish oil enzymatic hydrolysate is 12-13 wt% of the mass of the mixed oil phase.
Preferably, the protein comprises gelatin, soy protein or casein.
More preferably, the protein is gelatin.
Preferably, the final concentration of the protein in the fish oil microcapsule is 2.0 to 3.0wt%.
Further, in step S1, the buffer solution is a phosphate buffer solution.
Further, in the step S1, the addition amount of the buffer solution is 1.5-2 times of the mass of the fish oil.
Further, in step S1, the enzymolysis conditions are as follows: the pH value is 7.0-7.5, and the temperature is 40-45 ℃.
Preferably, in step S3, the conditions for sufficient mixing are: after pre-emulsifying for 3-5 min at 1000-1500 rpm, ultrasonic power of 300-350W is used for carrying out ultrasonic treatment on the mixed sample for 1-2 min, and then the pre-emulsified sample is emulsified for 12-15 min at 18000-20000 rpm.
The invention also protects the fish oil emulsion prepared by the preparation method. In the emulsion preparation process, a temperature-controlled circulating water bath is used for keeping the test temperature at 5-10 ℃ so as to avoid oxidation of fish oil due to high-speed homogenization.
The invention also provides a preparation method of the fish oil microcapsule, which comprises the following steps: adding complex coacervation inducer into the fish oil emulsion, fully and uniformly mixing, regulating pH value, implementing complex coacervation, adding cross-linking agent and cross-linking so as to obtain the invented fish oil microcapsule. Compared with other microencapsulation methods for bioactive substances such as omega-3 grease, the complex coacervation method has the advantages of high effective load, low surface oil content, high embedding rate and the like.
Preferably, the complex coacervation inducer comprises sodium hexametaphosphate, pectin, or sodium alginate.
More preferably, the complex coacervation inducer is sodium hexametaphosphate.
Preferably, the final concentration of the complex coacervation inducer in the fish oil microcapsules is 0.15 to 0.3wt%.
Preferably, the pH is 3.6 to 5.0. Since the optimal coagulation pH varies when different proteins and complex coagulation inducers undergo complex coagulation, in practice, the pH may be adjusted depending on the use of different proteins and complex coagulation inducers.
Preferably, the cross-linking agent is transglutaminase.
The invention also protects the fish oil microcapsule prepared by the preparation method. Compared with the non-enzymatic hydrolysis fish oil serving as an oil phase, the complex coacervation microcapsule prepared by mixing the fish oil enzymatic hydrolysate and the non-enzymatic hydrolysis fish oil together has more compact structural characteristics and higher oxidation stability.
The invention also protects the application of the fish oil emulsion or the fish oil microcapsule in preparing medicines, foods or cosmetics.
The invention has the following beneficial effects:
according to the invention, the fish oil zymolyte and the fish oil which is not subjected to enzymolysis are used as oil phases to prepare the fish oil emulsion for the first time, and an external emulsifier and an external antioxidant are not required to be added, so that the requirements of safety and environmental protection can be met, the stability and the nutritional value of the emulsion can be improved, the fish oil emulsion is further prepared into the fish oil microcapsule, and the obtained fish oil microcapsule can form a more compact embedding structure, so that the oxidation stability of the microcapsule is improved; the preparation process of the fish oil microcapsule is simple to operate, the reaction condition is mild, the fish oil microcapsule is green and safe, and the fish oil microcapsule has a high application value.
Drawings
Fig. 1 is a statistical plot of the data for the main fatty acid content of the different oil phases, wherein the differences are marked differently in the plot (P < 0.05) and the same letters indicate the differences are not marked (P > 0.05).
FIG. 2 is a data statistical chart of the composition and content of the glyceride (30% and 60% hydrolysis degree) of the tuna oil and its ADL enzymatic glyceride.
Fig. 3 is a statistical plot of the emulsifying activity and emulsifying stability of gelatin O/W emulsions of different oil phase compositions, wherein the differences are marked with lower-case letters (P < 0.05) and the differences are not marked with the same letters (P > 0.05).
FIG. 4 is a statistical plot of interfacial protein adsorption for gelatin O/W emulsions of different oil phase compositions, wherein the differences are marked with lower-case letters (P < 0.05) and the differences are not marked with the same letters (P > 0.05).
FIG. 5 is a data statistical plot of interfacial tension analysis of gelatin O/W emulsions of different oil phase compositions.
FIG. 6 is a data statistical plot of apparent viscosity analysis of gelatin O/W emulsions of different oil phase compositions.
FIG. 7 is a data statistical plot of storage modulus (G') of gelatin O/W emulsions of different oil phase compositions.
FIG. 8 is a data statistical plot of loss modulus (G ") analysis of gelatin O/W emulsions of different oil phase compositions.
FIG. 9 is a data statistical plot of protein secondary structure analysis of gelatin O/W emulsions of different oil phase compositions.
FIG. 10 is a graph of emulsion, complex coacervation, and microcapsule morphology of different oil phase compositions under an optical microscope;
a-1 to a-3: a tuna oil phase; b-1 to B-3:12.5% ADL enzymatic glyceride oil phase; c-1 to
C-3:1% mono-di-glycerol fatty acid ester oil phase; a-1, B-1 and C-1 are emulsions; a-2, B-2 and C-2 are complex coacervation; a-3, B-3 and C-3 are microcapsules.
Fig. 11 is a statistical plot of data from oxidation stability analysis of different oil phases and microcapsules prepared therefrom, wherein the differences are marked with lower-case letters (P < 0.05) and the differences are not marked with the same letters (P > 0.05).
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
The lipase Novocoadl (i.e., ADL lipase) derived from Candida antarctica (Candida antarctica); purchased from novelian limited (nosluks, new south wilt, australia); mono-di-glycerol fatty acid esters (GMS) from the biochemical science and technology company of galdeli, guangzhou, china; tuna oil is Tuna oil.
Example 1 preparation of enzymatic glyceride of tuna oil
20g of tuna oil is weighed and added into 40mL of phosphate buffer solution with pH 7.0, 0.4g of ADL lipase is added, and the mixture is mixed, and enzymolysis is carried out at the temperature of 40 ℃ at the speed of 350 r/min. The hydrolysis degree of the enzymatic hydrolysate is periodically detected, and enzymatic hydrolysate is extracted when the hydrolysis degree is approximately 30%. To the enzymatic hydrolysate, 0.5mol/L KOH 30% ethanol solution was added dropwise to neutralize the free fatty acids until the pH exceeded 9. Then extracting the enzymatic hydrolysis glyceride with diethyl ether and N-hexane sequentially, rotary evaporating at 50deg.C, N 2 Drying the residual organic solvent, and sealing and preserving at 4 ℃ to obtain the enzymatic hydrolysis glyceride named ADL enzymatic hydrolysis glyceride.
Example 2 preparation of Fish oil microcapsule
The preparation of the fish oil microcapsule comprises the following steps:
s1, preparing an oil phase: 2g of ADL enzymolysis glyceride is added into 14g of tuna oil, and the mixture is fully and uniformly mixed to obtain a mixed oil phase.
S2, preparation of O/W emulsion: mixing the mixed oil phase obtained in the step S1 with 120g of 8wt% gelatin solution, pre-emulsifying for 5min at 1200rpm, performing ultrasonic treatment on the mixed sample for 2min at 300W ultrasonic power, and emulsifying the pre-emulsified sample for 15min at 20000rpm by using a high-speed homogenizer to obtain an emulsion.
S3, preparing fish oil microcapsules: 105g of the O/W emulsion obtained in the step S2 was taken, an equal amount of 0.53wt% sodium hexametaphosphate solution was added, 50g of ultrapure water was added, stirring was performed at a speed of 800rpm, the pH of the mixed solution was adjusted to 4.80 by dropping 1% phosphoric acid to conduct complex coacervation, the initial temperature of the complex coacervation process was fixed at 50℃and then cooled to 5℃at a speed of 12℃per hour using a programmable refrigeration cycle water bath. After the sample was kept at 5℃for 30min, 50mL of a10 wt% transglutaminase solution was added. Then the temperature of the solution is increased to 25 ℃ at a heating rate of 5 ℃/h to activate the enzyme, and the solution is kept at 25 ℃ for 5h to complete crosslinking to obtain wet fish oil microcapsules, and freeze drying is carried out to obtain the dry fish oil microcapsules.
Example 3 preparation of Fish oil microcapsule
The preparation of the fish oil microcapsule comprises the following steps:
s1, preparing an oil phase: 2.5g of ADL enzymolysis glyceride is added into 17.5g of tuna oil, and the mixture is fully and uniformly mixed to obtain a mixed oil phase.
S2, preparation of O/W emulsion: mixing the mixed oil phase obtained in the step S1 with 150g of 8wt% gelatin solution, pre-emulsifying for 5min at 1200rpm, performing ultrasonic treatment on the mixed sample for 2min at 300W ultrasonic power, and emulsifying the pre-emulsified sample for 15min at 20000rpm by using a high-speed homogenizer to obtain an emulsion.
S3, preparing fish oil microcapsules: 150g of the O/W emulsion obtained in the step S2 was taken, and after adding an equal amount of 0.53wt% sodium hexametaphosphate solution, 50g of ultrapure water was added, and stirring was performed at a speed of 800rpm, the pH of the mixed solution was adjusted to 4.60 by dropping 1% phosphoric acid to conduct complex coacervation, the initial temperature of the complex coacervation process was fixed at 50℃and then cooled to 5℃at a speed of 12℃per hour using a programmable refrigeration cycle water bath. After the sample was kept at 5℃for 30min, 50mL of a10 wt% transglutaminase solution was added. Then the temperature of the solution is increased to 25 ℃ at a heating rate of 5 ℃/h to activate the enzyme, and the solution is kept at 25 ℃ for 5h to complete crosslinking to obtain wet fish oil microcapsules, and freeze drying is carried out to obtain the dry fish oil microcapsules.
Comparative example 1 preparation of Fish oil microcapsule
The preparation of the fish oil microcapsule comprises the following steps:
s1, preparing an oil phase: 16g of tuna oil was weighed.
S2, preparation of O/W emulsion: mixing the oil phase obtained in the step S1 with 120g of 8wt% gelatin solution, pre-emulsifying for 5min at 1200rpm, performing ultrasonic treatment on the mixed sample for 2min at 300W ultrasonic power, and emulsifying the pre-emulsified sample for 15min at 20000rpm by using a high-speed homogenizer to obtain an emulsion.
S3, preparing fish oil microcapsules: 105g of the O/W emulsion obtained in the step S2 was taken, an equal amount of 0.53wt% sodium hexametaphosphate solution was added, 50g of ultrapure water was added, stirring was performed at a speed of 800rpm, the pH of the mixed solution was adjusted to 4.80 by dropping 1% phosphoric acid to conduct complex coacervation, the initial temperature of the complex coacervation process was fixed at 50℃and then cooled to 5℃at a speed of 12℃per hour using a programmable refrigeration cycle water bath. After the sample was kept at 5℃for 30min, 50mL of a10 wt% transglutaminase solution was added. Then the temperature of the solution is increased to 25 ℃ at a heating rate of 5 ℃/h to activate the enzyme, and the solution is kept at 25 ℃ for 5h to complete crosslinking to obtain wet fish oil microcapsules, and freeze drying is carried out to obtain the dry fish oil microcapsules.
Comparative example 2 preparation of Fish oil microcapsule
The preparation of the fish oil microcapsule comprises the following steps:
s1, preparing an oil phase: to 15.84g of tuna oil, 0.16g of a fatty acid ester of mono-diglycerol was added and thoroughly mixed.
S2, preparation of O/W emulsion: mixing the oil phase obtained in the step S1 with 120g of 8wt% gelatin solution, pre-emulsifying for 5min at 1200rpm, performing ultrasonic treatment on the mixed sample for 2min at 300W ultrasonic power, and emulsifying the pre-emulsified sample for 15min at 20000rpm by using a high-speed homogenizer to obtain an emulsion.
S3, preparing fish oil microcapsules: 105g of the O/W emulsion obtained in the step S2 was taken, an equal amount of 0.53wt% sodium hexametaphosphate solution was added, 50g of ultrapure water was added, stirring was performed at a speed of 800rpm, the pH of the mixed solution was adjusted to 4.80 by dropping 1% phosphoric acid to conduct complex coacervation, the initial temperature of the complex coacervation process was fixed at 50℃and then cooled to 5℃at a speed of 12℃per hour using a programmable refrigeration cycle water bath. After the sample was kept at 5℃for 30min, 50mL of a10 wt% transglutaminase solution was added. Then the temperature of the solution is increased to 25 ℃ at a heating rate of 5 ℃/h to activate the enzyme, and the solution is kept at 25 ℃ for 5h to complete crosslinking to obtain wet fish oil microcapsules, and freeze drying is carried out to obtain the dry fish oil microcapsules.
The oil phase compositions in example 1 and comparative examples 1 to 2 and the corresponding emulsion and microcapsule designations are summarized in table 1, and the results are shown below.
Table 1 is the nomenclature of the oil phase compositions and corresponding emulsions and microcapsules in example 2 and comparative examples 1-2
Wherein the addition amount of the ADL enzymolysis glyceride or the mono-diglyceride fatty acid ester is the ratio of the total mass of the oil phase. Experimental example 1 analysis of lipid profile of enzymatic glyceride of tuna oil
1. Experimental method
For lipid species: isolation and content calculations were performed using SF-16A rod thin layer chromatography to determine Triglycerides (TAG), free Fatty Acids (FFA), diglycerides (DAG) and Monoglycerides (MAG) in tuna oil and its enzymatically hydrolyzed glycerides. 10. Mu.L of the sample was dissolved in 4mL of n-heptane, the spotting amount was 1. Mu.L, and the sample was developed in n-hexane/diethyl ether/formic acid (60:17:0.2, v/v/v) for 20min, and after drying for 5min, the results were scanned.
For fatty acid composition: the oil phase samples of example 2 and comparative examples 1 to 2 were weighed and mixed with 1mL of toluene, 200. Mu.L of 1mg/mL of an antioxidant (10 mg of BHT in 10mL of toluene) and 2mL of a 10% acetyl chloride-methanol solution, respectively, and then sealed overnight at 50 ℃. After cooling to room temperature, 5mL of NaCl solution (5%, m/v) and 5mL of n-hexane were added in this order, followed by shaking and standing, and the supernatant was taken. Adding 5mL KHCO into the supernatant 3 The solution (2%, m/v) was washed, the supernatant was taken, added with an appropriate amount of anhydrous sodium sulfate, vigorously shaken, filtered through a 0.22 μm filter, and stored at 4℃for gas chromatography.
The fatty acid composition in the oil phase was determined using a device equipped with InertTQ8050NX gas chromatograph-mass spectrometer for Pure-WAX quartz capillary column (30 mX0.25 mm,0.25 μm). GC analysis conditions: the carrier gas is helium, the pressure is 54.2kPa, the control mode is linear speed, the total flow is 41.7mL/min, and the chromatographic column flow is 0.70mL/min; and the sample is introduced in a split mode, and the split ratio is 50:1. Detector FID: the temperature of the sample inlet is 250 ℃, and the temperature of the detector is 250 ℃; chromatographic column temperature program: the temperature was kept at 130℃for 5min, and then at 4℃per min to 240℃for 30min. And (3) qualitative and quantitative determination of fatty acid in the oil phase sample by adopting a GC-MS mass spectrum library and combining fatty acid methyl ester mixed labeling.
For glyceride composition: 10mg of tuna oil and an enzymolysis glyceride sample thereof are respectively dissolved in 1mL of isopropanol, vortex is carried out for 30s, 200 mu L of the sample is dissolved in 800 mu L of isopropanol, vortex is carried out for 30s, an organic filter membrane with the thickness of 0.22 mu m is adopted, then 10 mu L of the sample is taken into an inner cannula filled with 80 mu L of isopropanol, 10 mu L of 10 mu g/mL of an internal standard substance is added, and the sample is put on a machine after ultrasound for 10 s.
Lipid composition was determined using a UPLC 30A system equipped with a Phenomenex Kinetex C18 column (100 mm. Times.2.1 mm,2.6 μm) in combination with a triple TOF 6600 system. The sample injection amount is 1 mu L, the flow rate is 0.40mL/min, and the column temperature is 60 ℃. Mobile phase a was a water/MeOH/ACN (1:1:1, v/v/v;5mM ammonium acetate) mixture and mobile phase B was an IPA/ACN (5:1, v/v;5mM ammonium acetate) mixture. Gradient elution conditions were as follows: 0-0.5min,20% B;0.5-1.5min,40% B;1.5-3min,60% B;3-13min,98% B;13-13.1min,20% B;13.1-17min,20% B.
2. Experimental results
(1) Lipid species analysis
Table 2 compares the lipid profile of tuna oil and the enzymatically hydrolyzed glyceride of fish oil prepared in example 1. As is clear from the table, tuna oil is almost triglyceride, and its relative content is 97% or more. After the fish oil is hydrolyzed and the free fatty acid in the reaction liquid is neutralized, a large amount of diglyceride and monoglyceride are generated in the enzymolysis glyceride, wherein the content of the diglyceride is high, and the residual free fatty acid is also slightly neutralized. The diglyceride and monoglyceride are easier to be absorbed by organisms than the triglyceride, so that the fish oil is added into the fish oil to carry out enzymolysis on the glyceride, and the formed fish oil microcapsule has higher nutritive value.
TABLE 2 lipid class analysis of tuna oil and its enzymatically hydrolyzed glycerides
Note that: the same lower case letter marked on the column indicates that the difference is significant (P < 0.05), the letter the same or no letter indicates that the difference is not significant (P > 0.05).
(2) Principal fatty acid composition analysis
The main fatty acid content of the different oil phases is shown in figure 1. The C22:6n3 (DHA) content of the tuna oil phase containing 12.5% ADL enzymatic glyceride is significantly increased compared to that of the tuna oil phase, whereas the tuna oil phase containing 1% mono-di-glyceride exhibits the opposite trend.
(3) Glyceride composition analysis
The data statistics of the composition and content of the tuna oil and its ADL enzymatic glyceride (30% and 60% hydrolysis degree) are shown in FIG. 2. Statistical analysis was performed on the Diglyceride (DAG) and Monoglyceride (MAG) component information of the tuna oil and ADL enzymatically hydrolyzed glycerides using a cluster thermogram analysis. And classifying the combination of the samples and the variables, taking the index variable as an ordinate, taking sample information as an abscissa, and visually presenting the multi-sample multivariable global change and clustering relation by adopting a transverse comparison method.
The tuna oil and its ADL enzymatically hydrolyzed glycerides were each detected as 85 diglycerides and 7 monoglyceride components. According to the color level of the cluster heat map, most of DAG and MAG contents are obviously improved after the tuna oil is hydrolyzed by lipase ADL. As the degree of hydrolysis increased, 21 DAGs, such as DAG (16:2-22:6), DAG (18:4-20:5) and DAG (20:3-22:6), increased in content, 20 of the 21 DAGs contained EPA and/or DHA, indicating that ADL had the potential to produce an Omga-3 enriched DAG. In addition, with the change in the degree of hydrolysis (30% and 60%), the effect of lipase ADL on monoglyceride content was small.
Therefore, the addition of 12.5% of ADL enzymatic glyceride to tuna oil can enhance the physicochemical properties and nutritive value of the fish oil.
Experimental example 2 measurement of particle size, zeta potential and surface hydrophobicity of O/W emulsion
1. Particle size analysis
After diluting the emulsion 400 times with ultrapure water, the average particle diameter of the emulsion was measured using a NanoZS-type malvern nanoparticle potentiometer.
2. Zeta potential analysis
After diluting the emulsion 400 times with ultrapure water, the zeta potential of the emulsion was measured using a NanoZS-type malvern nanoparticle potentiometer.
3. Emulsion Activity and emulsion stability analysis
The absorbance was measured using a UV-2550 type ultraviolet spectrophotometer. 0.5mL of the fresh emulsion was diluted 100-fold with a 0.1% Sodium Dodecyl Sulfate (SDS) solution, and the Emulsion Activity (EAI) of the emulsion was calculated by measuring absorbance at a wavelength of 500nm using the SDS solution as a blank solution. After standing for 12 hours, the absorbance was measured, and Emulsion Stability (ESI) was calculated. EAI (m) 2 The calculation formulas of the/g) and ESI (%) are as follows:
wherein A is 0 Absorbance at 500nm at 0 min; a is that 12 Absorbance at 500nm at 12 h; d is dilution multiple; c is the mass fraction of protein (g/mL);is the volume fraction of the oil phase.
4. Experimental results
TABLE 3 particle size, zeta potential and surface hydrophobicity analysis of gelatin O/W emulsions with different oil phase compositions
Note that: the same lower case letter marked on the column indicates that the difference is significant (P < 0.05), the letter the same or no letter indicates that the difference is not significant (P > 0.05).
(1) Particle size analysis
The smaller droplet size can cause the emulsion to deflocculate and coalesce, delay Brownian motion and gravitational field motion, and improve stability. The oil phase composition of the gelatin O/W emulsion of example 2 and comparative examples 1-2 has a particle size as shown in Table 3, and the average particle size of the enzymatically hydrolyzed glyceride emulsion and the GMS emulsion are both significantly smaller than that of the tuna oil emulsion (P < 0.05), wherein the enzymatically hydrolyzed glyceride emulsion has a minimum particle size of 550.37nm. This is probably because partial enzymatic hydrolysis of fish oil by lipase ADL produced DAG (diglyceride) and MAG (monoglyceride), improving the emulsification effect and contributing to the stability of the resulting fish oil emulsion.
(2) Potentiometric analysis
In table 3, both the Zeta potential of the ADL enzymatic glyceride emulsion and the mono-di-glyceride fatty acid ester emulsion are significantly smaller than that of the tuna oil emulsion (P < 0.05), which may be related to the emulsion droplet size, the smaller the emulsion droplet size, the larger the emulsion specific surface area, resulting in an increased ratio of oil phase to gelatin, and a reduced Zeta potential.
(3) Analysis of surface hydrophobicity
Surface hydrophobicity can be used to measure the exposure of hydrophobic groups on the surface of a protein. The surface hydrophobicity of the gelatin O/W emulsion composed of the oil phase of example 2 and comparative examples 1-2 is shown in Table 3, and the surface hydrophobicity of the enzymatic hydrolysis glyceride emulsion and the mono-diglyceride fatty acid ester emulsion are both significantly greater than that of the tuna oil emulsion (P < 0.05), wherein the surface hydrophobicity of the enzymatic hydrolysis glyceride emulsion is the maximum of 11.51. This may be a result of interaction of DAG and MAG in the enzymatically cleaved glycerides with interfacial proteins, which unfold the protein structure, thereby increasing the surface hydrophobicity of the emulsion. The exposure of hydrophobic groups of the emulsion is probably one of the reasons for the increase of EAI and ESI of the emulsion, the size of emulsion drops is reduced, the structure is unfolded, and the protein has better adsorption potential at the O-W interface. The larger the hydrophobic interaction of the emulsion, the stronger the acting force for driving the emulsion to carry out complex coacervation, and the more favorable the subsequent preparation of the fish oil microcapsule.
(4) Emulsifying Properties of O/W emulsion
The emulsion properties of the oil phase O/W emulsions of example 2 and comparative examples 1 to 2 are shown in FIG. 3. Emulsion Activity (EAI) refers to the adsorption capacity of a protein at the oil-water interface, and Emulsion Stability (ESI) can be used to reflect the ability of an emulsion to resist flocculation and coalescence upon storage. The result shows that the addition of the enzymolysis glyceride and the mono-diglyceride fatty acid ester can obviously improve the emulsifying activity (P < 0.05) of the emulsion.
The tuna oil emulsion had the lowest ESI, which was only 75.16%. The highest ESI of the emulsion added with the enzymatic hydrolysis glyceride is 92.00%, which is probably because DAG and MAG in the glyceride can be quickly adsorbed on an O-W interface in the emulsion forming process, so that the phenomena of droplet aggregation, instability and the like are slowed down. In addition, the smaller emulsion particle size (table 3) also suppressed flocculation and coalescence of the droplets to some extent, thereby improving emulsion stability.
Experimental example 3 determination of interfacial properties of emulsion
1. Interfacial protein adsorption assay
After centrifugation of the emulsion at 10000rpm for 30min at 4℃the clear lower supernatant was collected using a syringe and the supernatant was passed through a 0.45 μm filter to remove residual impurities. The initial protein content of the emulsion and the protein content of the clear liquid which is not adsorbed at the O/W interface are determined by using a Lowry method, and the interface protein content AP (%) is calculated by using the following formula:
wherein C is INI Is the initial protein content (mg/mL) of the emulsion, C SER Is the protein content (mg/mL) of the supernatant after centrifugation, which is not adsorbed at the O/W interface.
2. Interfacial tension analysis
Interfacial tension between different fish oils and gelatin solutions was determined using LSA100 type contact angle/surface tension measuring instrument and using the hanging drop method. Fish oil was added to the syringe, the stainless steel capillary needle of the syringe was immersed in a container filled with gelatin solution and squeezed out at the needle tip to form a drop of fish oil. The temperature of the test chamber was maintained at 40 ℃ using a temperature-controlled circulating water bath. The principle of water drop contour analysis used in this study is based on determining the coordinates of the drop from the video image and comparing these coordinates to the theoretical contour calculated from the Laplace-Young formula.
3. Experimental results
(1) Interfacial protein adsorption assay for O/W emulsions
The interfacial protein adsorption amounts of the 3 emulsions are shown in FIG. 4. The highest adsorption amount of interfacial protein of the tuna oil emulsion is 89.83%. This is probably due to the fact that DAG and MAG contained in enzymatically hydrolyzed glycerides competed for adsorption with gelatin protein at the O-W interface of the oil droplets and gradually replaced the original interface protein. The structure and stability of the interfacial protein film is dependent in part on the type of emulsifier adsorbed on the interface and the interactions between them, whereas both DAG and MAG are low molecular weight surfactants, with very strong mobility properties that may allow droplets to readily flocculate under hydrophobic interactions, which provides favorable conditions for the formation of subsequent complex coacervates.
(2) Interfacial tension analysis of gelatin O/W emulsion
The interfacial tension between different oil samples and gelatin solutions is measured, so that the adsorption behavior of DAG and MAG contained in the enzymatic hydrolysis glyceride on the O-W interface can be better understood. Fig. 5 shows the trend of interfacial tension between different oil samples and gelatin solutions over time. Over time, the interfacial tension tends to stabilize, indicating that the emulsifier is stable at the interface. Wherein, the interfacial tension of the enzymolysis glyceride and the mono-diglyceride fatty acid ester and the gelatin solution is obviously reduced compared with that of tuna oil. The interface tension of the sample containing 12.5% of enzymolysis glyceride is the lowest, and the lower interface tension is beneficial to improving the stability of the emulsion.
Experimental example 4 analysis of rheological Properties of emulsion
1. Dynamic shear test
Pouring 0.5mL fresh emulsion on an instrument plate by using a P35 probe, setting a measurement gap of 0.5mm, setting the temperature to 25 ℃, balancing for 5 minutes, and using a HAAKE MARS rheometer at a shearing rate of 0.1-100s -1 The apparent viscosity of the emulsion was measured as follows.
2. Oscillation frequency sweep
The Linear Viscoelasticity Region (LVR) was measured by dynamic strain scanning at a frequency of 10rad/s over a strain range of 0.01% to 100% and the strain value was fixed at 1%. The storage modulus (G ') and loss modulus (G') of the emulsions obtained in example 2 and comparative examples 1-2 were measured at an oscillation frequency of 0.1 to 10Hz using a P35 probe, pouring 0.5mL of fresh emulsion onto an instrument panel, setting a measurement gap of 0.5mm, a temperature of 25℃and balancing for 5 minutes, and then using a HAAKE MARS type rheometer.
3. Experimental results
(1) Apparent viscosity analysis of O/W emulsions
The apparent viscosity of the 3 gelatin O/W emulsion is shown in FIG. 6. All 3 emulsions exhibited shear thinning behavior and all exhibited pseudoplastic fluids. At the same shear rate, the viscosity of the experimental group was significantly increased compared to the tuna oil emulsion, with the apparent viscosity of the 12.5% adl enzymatic glyceride emulsion being the greatest. The increase in apparent viscosity may be a uniform particle size distribution of the emulsion, tending to be consistent, helping the emulsion to better resist external shear.
(2) Viscoelastic analysis of O/W emulsions
FIGS. 7 and 8 reflect the viscoelasticity of gelatin O/W emulsions of different lipid compositions. The results show that the storage modulus (G') is greater than the loss modulus (G ") at oscillation frequencies of 0.1-10Hz, exhibiting weak gel and solids-like behavior. Both G 'and G' of the emulsion increase with increasing frequency, and G 'is always greater than G', indicating that the viscoelasticity of the emulsion is dominant in elasticity. All emulsions were consistent in viscoelastic properties with apparent viscosity, with G' of 12.5% adl enzymatic glyceride emulsion and 1% mono-di-glyceride fatty acid ester emulsion being greater than tuna oil emulsion, which can be attributed to reduced droplet size, smaller particle size distribution, and formation and strengthening of a three-dimensional network of oil droplets and emulsifier molecules. At the same time, the interaction of glyceride and interfacial protein also increases the viscoelasticity of the emulsion, and the increased viscoelasticity can endow the emulsion with higher physical stability.
Experimental example 5 conformational analysis of emulsion proteins
1. Protein secondary structure analysis
Diluting the emulsion by using ultrapure water for 100 times, taking 200 mu L of sample, placing the sample in a quartz absorption tank with the optical path length of 5mm, measuring circular dichroism of protein in the emulsion by using a Chirascan V10 type circular dichroism spectrometer, setting the scanning wavelength range to be 180-260nm, setting the scanning speed to be 100nm/min, and carrying out three-time scanning to obtain the average value. The circular secondary maps were processed using Pro-Data Viewer software and the CDNN software was used to calculate the relative amounts of various secondary structures in the protein.
2. Analysis of surface hydrophobicity
The emulsion was diluted to 62.5, 125, 250, 500, 1000 times with 1-anilino-8-naphthalene sulfonic Acid (ANS) as fluorescent probe, 20. Mu.L of 8mM ANS (prepared with 0.1M PBS phosphate buffer solution at pH 7.0) was added to 4mL of O/W emulsion, and after mixing, the reaction was carried out in the dark for 30min to cause fluorescence of the hydrophobic site of the protein bound at the O/W interface with ANS, and then the fluorescence intensity (RFI) was measured using an RF-5301pc fluorescent spectrometer. The excitation wavelength is set to 390nm and the emission wavelength is set to 470nm. The Relative Fluorescence Intensity (RFI) for each concentration is calculated as follows:
wherein Fs and F 0 Fluorescence intensity of the protein-ANS conjugate and fluorescence intensity of ultrapure water, respectively; surface hydrophobicity is expressed as the initial slope of RFI as a function of protein concentration (mg/mL).
3. Experimental results
The protein secondary structure and relative content of the gelatin O/W emulsions with different oil phase compositions are shown in FIG. 9 and Table 4, respectively. Compared with tuna oil emulsion, the relative content of the alpha-helix structure and the beta-sheet structure in the enzymolysis glyceride emulsion and the mono-diglyceride fatty acid ester emulsion is improved, and the relative content of the beta-corner structure and the random coil structure is reduced. The effect of 1% of the mono-diglyceride fatty acid ester on the secondary structure of the emulsion protein is more obvious than that of 12.5% of the ADL enzymolysis glyceride. The decrease in the relative amount of alpha-helical structure indicates that the ordered structure of the protein in the emulsion is destroyed. The decrease in the relative content of beta-sheet structure and the increase in the relative content of random coil indicate that the protein molecules are unfolded and tend to be loose and disordered, the hydrophobic amino acids held in the internal structure are exposed, the flexibility of the overall conformation of the protein is correspondingly altered, and the results are consistent with the measurement results of the surface hydrophobicity (Table 3). The stretched protein structure may be more easily affected by the change of the internal conditions of the micro-environment of the emulsion, and when sodium hexametaphosphate emulsion with opposite charges is added into the emulsion, the emulsion is more induced to generate more severe aggregation behavior, so that the preparation of the fish oil microcapsule is facilitated.
TABLE 4 analysis of protein secondary Structure relative content of gelatin O/W emulsions of different lipid compositions
Note that: the same lower case letter marked on the column indicates that the difference is significant (P < 0.05), the letter the same or no letter indicates that the difference is not significant (P > 0.05).
Experimental example 6 characterization of morphology
1. Experimental method
The formation of the fish oil microcapsules was monitored using a microscope equipped with a camera (OLYMPUS CX43, OLYMPUS CORPORATION, TOKYO, JAPAN).
2. Experimental results
Optical microscopic morphology observations at the 3 stages of emulsion preparation, complex coacervation occurring and microcapsule formation during the preparation of microcapsules by complex coacervation using emulsion samples are shown in fig. 10. At pH value of 4.8-5.0, O/W emulsion drops (shown in figures A-1, B-1 and C-1) are aggregated under the induction of sodium hexametaphosphate to form complex coacervates (shown in figures A-2, B-2 and C-2), and after the procedure and the crosslinking, the aggregation degree of the drops is increased, and finally 'polynuclear' microcapsules (shown in figures A-3, B-3 and C-3) are formed. It can be seen that, compared with the loose complex coacervation morphology of tuna oil, the complex coacervation of the enzymatic glyceride emulsion and the mono-di-glyceride fatty acid ester emulsion is more severe, the morphology boundary is clear, a smooth coacervate is formed around the oil droplets, the particle size of the oil droplets contained in the enzymatic glyceride microcapsules (figure B-3) and the mono-di-glyceride fatty acid ester microcapsules (C-3) is smaller, the microcapsules are distributed more densely, and each microcapsule tends to be piled up and overlapped, which may be related to the increased surface hydrophobicity of the O/W emulsion stage, the reduced zeta potential and the more severe complex coacervation occurrence process, which means that the addition of the oil phase of the fish oil enzymatic glyceride is more favorable for driving the complex coacervation to occur, thereby forming the fish oil microcapsules with compact embedding structure.
Experimental example 7 determination of microcapsule embedding Properties
1. Experimental method
Determination of surface oil: 2g of freeze-dried solid fish oil microcapsules were dissolved in 30mL of n-hexane and vortexed for 60s. After centrifugation at 10000rpm for 30min at 4℃the n-hexane fraction was removed and the solvent was removed by rotary evaporation at 50 ℃. Then the mixture is placed at 90 ℃ to remove residual solvent and moisture, dried to constant weight, cooled to room temperature and the surface oil content is determined by a gravimetric method. Determination of total oil: 2g of the microcapsules were dissolved in 20mL of 4M hydrochloric acid, vortexed for 60s, then 20mL of n-hexane was added, and vortexed for 60 seconds. The mixture was mixed at room temperature for 14h to allow the oil to be sufficiently dissolved in n-hexane. After the mixture was centrifuged at 10000rpm at 4℃for 30min, the n-hexane fraction was removed and the solvent was removed by rotary evaporation at 50 ℃. Then the mixture is placed at 90 ℃ to remove residual solvent and moisture, and dried to constant weight, cooled to room temperature, and the total oil content is determined by a gravimetric method. Embedding rate (EE), payload (PL), and encapsulation Efficiency (EY) are shown in the following equations.
Wherein EE is the embedding rate (%), PL is the payload (%), EY is the encapsulation efficiency (%), W t And W is s Total oil and surface oil mass (g), W of the microcapsules, respectively i Mass (g), W of oil used for forming microcapsules m The mass (g) of the microcapsule is shown.
2. Experimental results
The embedding characteristics of the complex coacervated microcapsules prepared from the emulsions obtained in example 2 and comparative examples 1-2 are shown in table 5, and are characterized by a low surface oil content (< 2%), a high payload (> 50%) and an extremely high embedding rate (> 95%). The surface oil content of the glyceride microcapsules is slightly increased and the embedding rate is slightly reduced compared with the tuna oil microcapsules, which is probably caused by the fact that a small amount of free fatty acid exists in glyceride, but the encapsulation efficiency of the microcapsules added with the enzymolysis glyceride is not significantly different from that of the tuna oil microcapsules and is superior to that of the microcapsules formed by adding 1% of the mono-diglyceride fatty acid ester.
TABLE 5 embedding Properties of different Mixed oil phase microcapsules
Note that: the same lower case letter marked on the column indicates that the difference is significant (P < 0.05), the letter the same or no letter indicates that the difference is not significant (P > 0.05).
Experimental example 8 measurement of oxidative stability of core Material and microcapsule
1. Experimental method
Accelerated oxidation tests were performed on oil phases and microcapsules using a Metrohm 743 type Rancimat grease oxidation stability analyzer. 2g of the oil phase or microcapsules were heated in purified air at a flow rate of 20L/h at 80℃and the induction time of the samples tested was recorded as the Oxidation Stability Index (OSI).
2. Experimental results
This study uses the Rannimat accelerated oxidation method to determine the Oxidation Stability Index (OSI) of the oil phases and their microcapsules of 3 different oil phase compositions. The greater the oxidation stability index, the better the stability of the sample under accelerated storage conditions. Fig. 11 shows the oxidation stability index of different oil phases and their microcapsule samples. The OSI of the tuna oil phase is highest, while the other 2 phases are lower, which is related to the unsaturated fatty acid content contained in the oil phase. While the OSI of the tuna oil microcapsule is minimum for 15.97 hours, the enzymatic glyceride microcapsule and the mono-diglyceride fatty acid ester microcapsule exhibit higher oxidative stability, wherein the OSI of the enzymatic glyceride microcapsule is maximum for 17.28 hours. The higher oxidation stability of the microcapsules may be attributed to the smaller droplet size of the enzymatically hydrolyzed glyceride emulsion, stronger associated forces, and more severe complex coacervation that occurs, thereby forming a denser microcapsule embedding structure, thus reducing the diffusion of ambient oxygen and moisture through the microcapsule shell, and having better protective effect on the embedded oil phase.
In conclusion, the addition of the enzymolysis glyceride can reduce the O-W (oil-water) interfacial tension and the emulsion droplet size, improve the emulsion stability of the emulsion, and increase the apparent viscosity and the viscoelasticity of the emulsion, which is beneficial to the improvement of the emulsion stability in the microencapsulation process, and the stable emulsion can promote the generation of high-performance microcapsules. The enzymolysis glyceride is adsorbed on the O-W interface, so that the property of the interface protein film is changed, and the protein conformation is converted. The emulsion has reduced surface charge, increased surface hydrophobicity, and unfolded protein structure, and these improved properties and interface behavior provide good conditions for subsequent complex coacervation of the fish oil microcapsules to occur. The characteristic of the microcapsule is affected to a certain extent by the enzymolysis glyceride and the fish oil together, and the microcapsule prepared from the oil phase containing the enzymolysis glyceride can remarkably improve the oxidation stability of the oil phase on the basis that the embedding rate and the effective load are still kept at higher levels.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (10)

1. The preparation method of the fish oil emulsion is characterized by comprising the following steps of:
s1, adding a buffer solution into fish oil, and adding lipase for enzymolysis to obtain fish oil zymolyte;
s2, preparing an oil phase: adding the fish oil zymolyte obtained in the step S1 into fish oil, and fully and uniformly mixing to obtain a mixed oil phase;
s3, preparation of O/W emulsion: fully and uniformly mixing the mixed oil phase obtained in the step S2 with the protein solution to form fish oil emulsion;
wherein the lipase is ADL lipase; the hydrolysis degree of the fish oil zymolyte is 20-60%.
2. The method according to claim 1, wherein the lipase is added in an amount of 1.0 to 4.0wt% based on the mass of the fish oil in step S1.
3. The preparation method according to claim 1, wherein in the step S2, the addition amount of the fish oil enzymatic hydrolysate is 10-50 wt% of the mass of the mixed oil phase.
4. The method of claim 1, wherein the protein comprises gelatin, soy protein, or casein.
5. The fish oil emulsion prepared by the preparation method of any one of claims 1 to 4.
6. The preparation method of the fish oil microcapsule is characterized by comprising the following steps: adding complex coacervation inducer into the fish oil emulsion obtained in claim 5, fully and uniformly mixing, regulating pH value, carrying out complex coacervation, and adding cross-linking agent for cross-linking to obtain the fish oil microcapsule.
7. The method of claim 6, wherein the complex coacervation inducer comprises sodium hexametaphosphate, pectin, or sodium alginate.
8. The method according to claim 6, wherein the pH is 3.6 to 5.0.
9. Fish oil microcapsules prepared by the preparation method of any one of claims 6 to 8.
10. Use of the fish oil emulsion of claim 5 or the fish oil microcapsule of claim 9 for the preparation of a pharmaceutical, food or cosmetic product.
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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1454511A (en) * 2002-04-29 2003-11-12 李长青 Seafood soy-sauce containing 'DHA fatty ethyl ester' and its preparation method
CN101940240A (en) * 2010-07-01 2011-01-12 大连工业大学 Method for preparing fish oil ethyl ester microcapsule from fish pomace
CN103060076A (en) * 2012-11-02 2013-04-24 浙江省海洋开发研究院 Method for preparing fish oil by virtue of leftovers of tuna
US20160220521A1 (en) * 2015-01-29 2016-08-04 Singao (Xiamen)Agribusiness Development Co., Ltd. Microcapsule fat powder of omega-3 enriched meat, egg and milk, and preparation method thereof
CN106566849A (en) * 2016-11-04 2017-04-19 汕头市智丽科技有限公司 DHA brain-invigorating essence, and preparation method and application thereof
CN108208685A (en) * 2017-12-18 2018-06-29 丁玉琴 A kind of preparation method of fish oil soft capsule
CN110257446A (en) * 2019-07-08 2019-09-20 青岛和合汇途工程技术有限公司 A kind of preparation method of high-purity EPA glyceride and DHA glyceride
CN111011860A (en) * 2019-12-27 2020-04-17 南京泛成生物科技有限公司 Fish oil microcapsule powder containing natural antioxidant composition and preparation method thereof
CN111172211A (en) * 2020-03-20 2020-05-19 江南大学 Method for preparing long-chain polyunsaturated fatty acid glyceride rich in fish oil n-3 by enzyme method and product thereof
CN111616355A (en) * 2020-06-05 2020-09-04 广东健明生物科技有限公司 Protein peptide-polyphenol-fish oil emulsion and preparation method and application thereof

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1454511A (en) * 2002-04-29 2003-11-12 李长青 Seafood soy-sauce containing 'DHA fatty ethyl ester' and its preparation method
CN101940240A (en) * 2010-07-01 2011-01-12 大连工业大学 Method for preparing fish oil ethyl ester microcapsule from fish pomace
CN103060076A (en) * 2012-11-02 2013-04-24 浙江省海洋开发研究院 Method for preparing fish oil by virtue of leftovers of tuna
US20160220521A1 (en) * 2015-01-29 2016-08-04 Singao (Xiamen)Agribusiness Development Co., Ltd. Microcapsule fat powder of omega-3 enriched meat, egg and milk, and preparation method thereof
CN106566849A (en) * 2016-11-04 2017-04-19 汕头市智丽科技有限公司 DHA brain-invigorating essence, and preparation method and application thereof
CN108208685A (en) * 2017-12-18 2018-06-29 丁玉琴 A kind of preparation method of fish oil soft capsule
CN110257446A (en) * 2019-07-08 2019-09-20 青岛和合汇途工程技术有限公司 A kind of preparation method of high-purity EPA glyceride and DHA glyceride
CN111011860A (en) * 2019-12-27 2020-04-17 南京泛成生物科技有限公司 Fish oil microcapsule powder containing natural antioxidant composition and preparation method thereof
CN111172211A (en) * 2020-03-20 2020-05-19 江南大学 Method for preparing long-chain polyunsaturated fatty acid glyceride rich in fish oil n-3 by enzyme method and product thereof
CN111616355A (en) * 2020-06-05 2020-09-04 广东健明生物科技有限公司 Protein peptide-polyphenol-fish oil emulsion and preparation method and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
刘书成;章超桦;谢燕;洪鹏志;吉宏武;郝记明;张静;: "脂肪酶水解低值鱼油富集EPA和DHA甘油酯的研究", 中国粮油学报, no. 03, pages 79 - 83 *
禤俊勇，等: "选择性脂肪酶水解金枪鱼油制备改性水解甘油酯工艺优化", 《广东海洋大学学报》, vol. 42, no. 2, pages 104 - 111 *
陈莹: "富含二十二碳六烯酸鱼油微胶囊的绿色酶法制备、 体外消化及氧化稳定性研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》, no. 4, pages 1 - 110 *

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