CN117338944A - Carotenoid-loaded nano delivery system and preparation method and application thereof - Google Patents

Carotenoid-loaded nano delivery system and preparation method and application thereof Download PDF

Info

Publication number
CN117338944A
CN117338944A CN202311318535.9A CN202311318535A CN117338944A CN 117338944 A CN117338944 A CN 117338944A CN 202311318535 A CN202311318535 A CN 202311318535A CN 117338944 A CN117338944 A CN 117338944A
Authority
CN
China
Prior art keywords
carotenoid
sesame
delivery system
sesame protein
nano
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202311318535.9A
Other languages
Chinese (zh)
Inventor
赵磊
赵亮
艾欣
潘飞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Technology and Business University
Original Assignee
Beijing Technology and Business University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Technology and Business University filed Critical Beijing Technology and Business University
Priority to CN202311318535.9A priority Critical patent/CN117338944A/en
Publication of CN117338944A publication Critical patent/CN117338944A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/01—Hydrocarbons
    • A61K31/015—Hydrocarbons carbocyclic
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Dispersion Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Botany (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention relates to a nano delivery system for loading carotenoid, a preparation method and application thereof. The nano delivery system has small particle size, uniform particle size distribution, fine texture, strong stability, high encapsulation efficiency and good slow release characteristic in the gastrointestinal tract, solves the technical problems of large particle size, poor stability and the like of the traditional carotenoid delivery system, has good gastrointestinal slow release performance, can improve the bioavailability and tissue distribution of the carotenoid in the body, and ensures that the product has higher nutritive value.

Description

Carotenoid-loaded nano delivery system and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, relates to a carotenoid-loaded nano delivery system and a preparation method and application thereof, and in particular relates to a carotenoid nano delivery system based on sesame protein zymolyte and a preparation method and application thereof.
Background
Carotenoids are natural fat-soluble pigments widely found in nature, mainly from plants, fungi and bacteria, giving them yellow, orange, red, etc. Carotenoids are largely classified as non-oxygen containing carotenoids and oxygen containing carotenoids. Carotenoids have the functions of resisting oxidation, regulating lipid metabolism, protecting eyesight, slowing down neurodegenerative diseases, protecting cardiovascular system, preventing and resisting cancer and the like, and part of carotenoids are important sources of vitamin A in human bodies. However, the bioavailability of carotenoids is low, and the bioavailability of carotenoids in most fruit and vegetable foods is generally less than 10%. Studies have shown that the bioavailability of carotenoids in foods is affected by a number of factors, including food composition and structure, food processing methods, food that is digested, and physiological differences between individuals. Carotenoids are susceptible to different degrees of isomerization by light, heat, oxygen, acid, alkali and the like during food processing and storage, and are also susceptible to different degrees of chemical factors, enzymes and the like during gastrointestinal tract digestion, so that the stability and bioavailability of the carotenoids are greatly reduced. In addition, poor water solubility is also a major reason for limiting the use of carotenoids in food products. Therefore, in order to popularize the application of the carotenoid, certain measures must be taken to improve the stability and the dispersibility of the carotenoid in a food application system and further improve the bioavailability of the carotenoid in a human body.
The bioavailability of carotenoids can be improved by processing carotenoids through the preparation of nano-delivery systems and isomerization techniques. However, current methods of carotenoid isomerization all cause the carotenoids to degrade to varying degrees during the reaction. Meanwhile, chemical reagents introduced by the thermal isomerization method are difficult to remove, equipment of the photoisomerization method is difficult to be applied to actual production, and the carotenoid isomerization industry is difficult to popularize on a large scale in a short period of time.
In contrast, nano-delivery systems are widely used for entrapment and delivery of carotenoids due to their extremely high specific surface area. The more commonly used carotenoid nano-delivery systems mainly comprise microcapsules, liposomes, nanoemulsions and the like, and the selection of the proper nano-delivery system is important for improving the stability and bioavailability of the carotenoid. The microcapsule delivery system often has the problems of organic matter residue, larger particle size and the like; although the liposome has higher biocompatibility and safety, the liposome still has the problems of wide particle size distribution range, poor stability, easy oxidation of unsaturated fatty acid in a lipid layer and the like; nanoemulsions are highly unstable to droplet coalescence, and single or combined surfactants are often required to promote the formation of nanoemulsions and ensure their dynamic stability during storage.
The polypeptide has been widely applied to self-assembled nano-carriers, and hydrogen bonds between peptide chain main chains can drive peptide monomers to be longitudinally assembled into beta-sheets, so that the polypeptide has the advantages of simple structure, good biocompatibility, relatively stability and the like, has various biological activities, has quicker response compared with liposome, and has good environmental response specificity, so that the polypeptide has great potential in the aspect of being assembled into novel nano-delivery systems of foods and biomedicine, and receives attention of a plurality of researchers.
Sesame seed meal is used as a byproduct after sesame oil extraction, the protein content is high (about 50 percent), the amino acid types in the sesame seed meal are comprehensive, the content of lysine which is the first limiting amino acid is superior to that of other raw material meal, and the methionine content is about 2 times of that of soybean meal and corn meal, so that the sesame seed meal has potential high nutritional value. However, most of sesame seed meal is used as cheap animal and plant feed at present and is not fully utilized.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a nano delivery system for supporting carotenoid and a preparation method and application thereof, in particular to a nano delivery system for carotenoid based on sesame protein zymolyte and a preparation method and application thereof. The sesame polypeptide self-assembly in the sesame protein hydrolysate is utilized to form a stable nano micelle for delivering carotenoid, and the sesame protein hydrolysate has important significance for improving the gastrointestinal stability and the sustained release characteristic of the carotenoid, improving ulcerative colitis and maintaining visual health.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a carotenoid-loaded nano-delivery system comprising a carrier material sesame protein hydrolysate and carotenoids encapsulated in the sesame protein hydrolysate.
In view of the problems that carotenoids have strong hydrophobicity, are almost insoluble in water, have poor structural stability, are sensitive to conditions such as heat, light, oxygen and the like, are extremely easy to be oxidized to be seriously degraded or isomerized to form cis/trans isomers, and have low bioavailability and the like, the invention creatively takes sesame protein zymolyte as a carrier material, uses the sesame protein zymolyte to encapsulate the carotenoids, and forms a nano micelle delivery system, which can overcome the technical defects, thereby widening the application range of the carotenoids in foods.
Meanwhile, compared with sesame protein, the sesame protein zymolyte is rich in various bioactive peptides, and is hopeful to improve the stability and bioavailability of carotenoid as a delivery carrier, and can also play a synergistic effect with the carotenoid to promote the carotenoid to better play the bioactivity.
The nano delivery system has small particle size, uniform particle size distribution, fine texture, strong stability, high encapsulation efficiency and good slow release characteristic in the gastrointestinal tract, solves the technical problems of large particle size, poor stability and the like of the traditional carotenoid delivery system, has good gastrointestinal slow release performance, can improve the bioavailability and tissue distribution of the carotenoid in the body, and ensures that the product has higher nutritive value.
The test results of the invention show that compared with water-dispersible and oil-soluble carotenoids, the nano-delivery system provided by the invention increases the uptake and utilization of carotenoids by rats, and the bioavailability and tissue distribution are obviously improved. Compared with independent carotenoid, the carotenoid nano micelle has more obvious improvement effect on ulcerative colitis of mice and photo-oxidative damage of retina of rats, and the nano delivery system not only enhances the physiological activity of the carotenoid, but also has synergistic effect on improving ulcerative colitis and maintaining visual health after the carotenoid and sesame polypeptide form nano micelle.
Preferably, the sesame protein hydrolysate is prepared by a preparation method comprising the following steps:
mixing sesame protein and protease for enzymolysis reaction, inactivating enzyme, centrifuging, and freeze-drying supernatant to obtain the sesame protein hydrolysate.
Preferably, the protease comprises any one or a combination of at least two of pepsin, trypsin, cathepsin, papain, subtilisin, neutral protease or alkaline protease.
Preferably, the mass ratio of protease to sesame protein is 1 (50-200), e.g. 1:50, 1:70, 1:80, 1:100, 1:120, 1:140, 1:160, 1:180, 1:200, etc.
Preferably, the system pH of the enzymatic hydrolysis reaction is 1.5-8.5, e.g., ph=1.5, ph=2.5, ph=3, ph=3.5, ph=4, ph=5, ph=6.5, ph=7.5, ph=8.5, etc.; the temperature is 30-55deg.C, such as 30deg.C, 35deg.C, 40deg.C, 48deg.C, 50deg.C, 52 deg.C, 55deg.C, etc.
Preferably, the time of the enzymatic hydrolysis reaction is 2-10h, such as 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, etc.
Other specific point values within the above listed numerical ranges are all selectable, and will not be described in detail herein.
Preferably, the sesame protein is obtained by an extraction method comprising the following steps:
removing oil from the sesame raw material by adopting a solvent leaching method, and extracting sesame protein from the defatted sesame raw material by adopting an alkali extraction and acid precipitation method.
Preferably, the sesame raw material is sesame seed meal.
The sesame seed meal is used as the preparation raw material, so that the utilization rate of edible protein in the sesame seed meal serving as a byproduct in the sesame processing process is improved, the protein resource is fully developed and utilized, the added value of sesame products is improved, the pollution of the sesame processing byproducts to the environment is reduced, the sesame seed meal has great significance in development and utilization of the sesame resource, and a new idea is provided for the application of the sesame seed meal in the food field.
Preferably, the alkali extraction and acid precipitation method comprises the following steps: the protein is solubilized at a pH of 9.0-11.0 (e.g., ph=9, ph=9.5, ph=10, ph=10.5, ph=11, etc.), 50-55 ℃ (e.g., 50 ℃, 52 ℃, 53 ℃,54 ℃, 55 ℃, etc.), centrifuged, the supernatant pH is adjusted to 3.5-5.5 (e.g., ph=3.5, ph=4, ph=4.5, ph=5, ph=5.5, etc.), and the protein is precipitated by standing.
Other specific point values within the above listed numerical ranges are all selectable, and will not be described in detail herein.
Preferably, the carotenoid comprises any one or a combination of at least two of alpha-carotene, beta-carotene, gamma-carotene, lycopene, lutein, astaxanthin, capsanthin or zeaxanthin.
In a second aspect, the present invention provides a method for preparing a carotenoid-loaded nano-delivery system according to the first aspect, the method comprising:
and mixing the sesame protein hydrolysate dispersion liquid with carotenoid solution, and carrying out ultrasonic treatment to obtain the carotenoid-loaded nano delivery system.
The invention adopts the ultrasonic-assisted self-assembly technology to prepare the nano delivery system for loading the carotenoid, has simple process, safe operation and easy industrialized implementation, relates to sesame protein from sesame processing byproducts, has low price, good nutrition functionality and biodegradability, and has wide development prospect in the application of food industry.
Preferably, the concentration of the sesame protein hydrolysate dispersion is 0.25-2.5%, for example 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 1.0%, 1.5%, 2%, 2.5%, etc., preferably 0.25-0.45%.
Preferably, the carotenoid solution has a concentration of 0.5-1.5%, e.g., 0.5%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, etc.
Preferably, the mass ratio of sesame protein hydrolysate to carotenoid is 10:1-20:1, e.g. 10:1, 11:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, etc.
Preferably, the solvent of the carotenoid solution is selected from any one or a combination of at least two of petroleum ether, n-hexane, dichloromethane or acetone.
Preferably, the working parameters of the ultrasonic treatment are 50-250w, such as 50w, 80w, 100w, 150w, 200w, 250w, etc.; 30-45min, such as 30min, 33min, 35min, 38min, 40min, 42min, 45min, etc.
Preferably, the dispersion of sesame protein hydrolysate is further pH-adjusted to 4.0-8.0, for example ph=4.0, ph=5.0, ph=6.0, ph=6.5, ph=7.0, ph=7.5, ph=8.0, etc., preferably 6.0-8.0, before mixing with the carotenoid solution.
Preferably, the ultrasonic treatment is further performed after the completion of the treatment by removing the organic solvent by placing in a water bath at 50-70deg.C (e.g., 50deg.C, 55deg.C, 60deg.C, 65deg.C, 70deg.C, etc.) for 0.5-2 hours (e.g., 0.5 hours, 1 hour, 1.5 hours, 2 hours, etc.).
Other specific point values within the above listed numerical ranges are all selectable, and will not be described in detail herein.
In a third aspect, the present invention provides the use of a carotenoid-loaded nano-delivery system according to the first aspect for the manufacture of a medicament for the prevention, amelioration or treatment of ulcerative colitis.
In a fourth aspect, the present invention provides the use of a carotenoid-loaded nano-delivery system according to the first aspect for the preparation of a food, pharmaceutical or health-care product for maintaining visual health.
Compared with the prior art, the invention has the following beneficial effects:
the nano delivery system has small particle size, uniform particle size distribution, fine texture, strong stability, high encapsulation efficiency and good slow release characteristic in the gastrointestinal tract, solves the technical problems of large particle size, poor stability and the like of the traditional carotenoid delivery system, has good gastrointestinal slow release performance, can improve the bioavailability and tissue distribution of the carotenoid in the body, and ensures that the product has higher nutritive value.
The test results of the invention show that compared with water-dispersible and oil-soluble carotenoids, the nano-delivery system provided by the invention increases the uptake and utilization of carotenoids by rats, and the bioavailability and tissue distribution are obviously improved. Compared with independent carotenoid, the carotenoid nano micelle has more obvious improvement effect on ulcerative colitis of mice and photo-oxidative damage of retina of rats, and the nano delivery system not only enhances the physiological activity of the carotenoid, but also has synergistic effect on improving ulcerative colitis and maintaining visual health after the carotenoid and sesame polypeptide form nano micelle.
Drawings
FIG. 1 is a schematic illustration of the preparation flow of the carotenoid-loaded nanodelivery system of example 1;
FIG. 2 is a FTIR plot of each set of samples in test example 2;
FIG. 3 is a transmission electron microscope image of each set of samples in test example 2;
FIG. 4 is an X-ray diffraction pattern of each group of samples in test example 2;
FIG. 5 is a circular dichroism spectrum of each set of samples in test example 2;
FIG. 6 is a graph showing the results of in vitro sustained release experiments of carotenoid-loaded nano-delivery systems prepared in example 1;
fig. 7 is a graph of the pharmacokinetic results of the carotenoid-loaded nanodelivery system prepared in example 1.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1
The embodiment provides a nano delivery system for supporting carotenoid, the preparation flow diagram of which is shown in fig. 1, and the specific operation is as follows:
(1) Preparation of defatted sesame seed meal:
weighing 5.0g of sesame seed meal, crushing to obtain sesame seed meal powder, mixing the sesame seed meal powder with n-hexane according to a feed-liquid ratio (1:10, w/v) in a conical flask, sealing, magnetically stirring, leaching for 2h at 55 ℃, and filtering under reduced pressure to remove n-hexane extract; and repeatedly leaching the sesame seed meal cake for 2 times by the method, leaching the defatted sesame seed meal powder, filtering, removing the solvent in a vacuum drying oven at 60 ℃, and drying to obtain the defatted sesame seed meal.
(2) Preparation of sesame protein:
weighing a certain amount of defatted sesame meal powder, adding deionized water into the defatted sesame meal powder at a feed-liquid ratio of 1:10 for dispersion, adjusting the pH value to 11.0, magnetically stirring and extracting for 1h at 50 ℃, centrifuging (5000 r/min,15 min), collecting supernatant, adjusting the pH value of the supernatant to 4.5,4 ℃ by using 1mol/L HCl, standing overnight to fully precipitate protein, centrifuging (9000 r/min,15 min), collecting precipitate, adding deionized water for washing, adjusting the pH value to be neutral, and performing vacuum freeze drying to obtain sesame protein.
(3) Preparation of sesame protein zymolyte:
sesame protein was dispersed in deionized water at 5% (w/v), stirred for 2h, and hydrated overnight at 4 ℃. Adjusting pH to 8.0 with 1mol/L NaOH solution, adding alkaline protease (enzyme and substrate 1:100, w/w), performing enzymolysis in 50deg.C water bath, continuously adding 1mol/L NaOH solution to maintain pH 8.0, performing enzymolysis for 4 hr, immediately inactivating enzyme with boiling water for 10min, cooling, centrifuging (8000 rpm,15 min), and lyophilizing supernatant to obtain sesame protein hydrolysate.
(4) Preparation of beta-carotene-loaded nanodelivery systems:
preparing sesame protein zymolyte dispersion liquid with the concentration of 0.25% (w/v), regulating the pH value to 7.0 by using 1mol/L HCl solution, adding a certain amount of beta-carotene petroleum ether solution (1% w/v) into the sesame protein zymolyte dispersion liquid, enabling the mass ratio of the sesame protein zymolyte to the beta-carotene to be 12.5:1, magnetically stirring and uniformly mixing the sesame protein zymolyte dispersion liquid, performing ice bath ultrasonic treatment (150 w, working for 2s, gaps for 2s and 35 min), and volatilizing the sesame protein zymolyte dispersion liquid in a water bath at 60 ℃ for 1h to remove petroleum ether, thus obtaining the sesame protein zymolyte.
Example 2
The embodiment provides a nano delivery system for loading carotenoid, which specifically comprises the following operations:
(1) Preparation of defatted sesame seed meal:
as in example 1.
(2) Preparation of sesame protein:
as in example 1.
(3) Preparation of sesame protein zymolyte:
sesame protein was dispersed in deionized water at 5% (w/v), stirred for 2h, and hydrated overnight at 4 ℃. Regulating pH to 2.0 with 1mol/L HCl solution, adding pepsin (enzyme and substrate 1:70, w/w), performing enzymolysis in 37deg.C water bath, continuously adding 1mol/L HCl solution to maintain pH 2.0, performing enzymolysis for 6 hr, immediately inactivating enzyme with boiling water for 10min, cooling, centrifuging (8000 rpm,15 min), and lyophilizing supernatant to obtain sesame protein hydrolysate.
(4) Preparation of astaxanthin-loaded nano-delivery system:
preparing sesame protein hydrolysate dispersion liquid with the concentration of 0.25% (w/v), regulating the pH value to 7.0, adding a certain amount of astaxanthin normal hexane solution (1% w/v) into the sesame protein hydrolysate dispersion liquid to ensure that the mass ratio of the sesame protein hydrolysate to the astaxanthin is 12.5:1, carrying out ice bath ultrasonic treatment (200 w, working for 2s, and gaps for 2s and 35 min) after magnetic stirring and mixing uniformly, and volatilizing in a water bath to remove normal hexane to obtain the sesame protein hydrolysate.
Example 3
The embodiment provides a nano delivery system for loading carotenoid, which specifically comprises the following operations:
(1) Preparation of defatted sesame seed meal:
as in example 1.
(2) Preparation of sesame protein:
as in example 1.
(3) Preparation of sesame protein zymolyte:
sesame protein was dispersed in deionized water at 5% (w/v), stirred for 2h, and hydrated overnight at 4 ℃. Adjusting pH to 6.9 with 1mol/L HCl solution, adding trypsin (enzyme and substrate 1:150, w/w), performing enzymolysis in 37deg.C water bath, continuously adding 1mol/L HCl solution to maintain pH 6.9, performing enzymolysis for 8 hr, immediately inactivating enzyme with boiling water for 10min, cooling, centrifuging (8000 rpm,15 min), and lyophilizing supernatant to obtain sesame protein hydrolysate.
(4) Preparation of lutein-loaded nano delivery system:
preparing sesame protein hydrolysate dispersion liquid with the concentration of 0.25% (w/v), regulating the pH value to 7.0, adding a certain amount of lutein dichloromethane solution (1% w/v) into the sesame protein hydrolysate dispersion liquid to ensure that the mass ratio of the sesame protein hydrolysate to lutein is 12.5:1, magnetically stirring and uniformly mixing, performing ice bath ultrasonic treatment (150 w, working for 2s and gaps of 2s and 35 min), and volatilizing in a water bath to remove dichloromethane to obtain the sesame protein hydrolysate.
Examples 4 to 6
This example provides three carotenoid-loaded nanodelivery systems, and the specific operation differs from example 1 only in that the sesame protein hydrolysate concentration in step (4) was 0.5, 1.0 and 2.5%, respectively, and the other operating conditions remained unchanged.
Examples 7 to 10
The present example provides four carotenoid-loaded nanodelivery systems, the specific operation differs from example 1 only in that the mass ratio of sesame protein hydrolysate to β -carotene in step (4) is 1:1, 2:1, 6.25:1 and 25:1, respectively, and the other operating conditions remain unchanged.
Examples 11 to 14
This example provides four carotenoid-loaded nanodelivery systems, the specific operation differing from example 1 only in that the ultrasound time in step (4) was 5, 15, 25 and 45min, respectively, with the other operating conditions remaining unchanged.
Examples 15 to 18
This example provides four carotenoid-loaded nanodelivery systems, the specific operation differing from example 1 only in that the pH of the system in step (4) was 4, 5, 6 and 8, respectively, and the other operating conditions remained unchanged.
Comparative example 1
The comparative example provides a nano delivery system for supporting carotenoid, wherein the steps (1) and (2) are the same as the example 1, except that the step (3) in the example 1 is omitted, namely, sesame protein is not subjected to enzymolysis, then sesame protein dispersion liquid with the concentration of 0.25% (w/v) is prepared, the pH value is adjusted to 7.0, a certain amount of beta-carotene petroleum ether solution (1% w/v) is added, the mass ratio of the sesame protein to the beta-carotene is 12.5:1, magnetic stirring and mixing are carried out uniformly, then ice bath ultrasonic treatment (150 w, working 2s, gap 2s and 35 min) is carried out, and petroleum ether is volatilized and removed in a water bath at 60 ℃ for 1h, thus obtaining the nano delivery system.
Comparative example 2
This comparative example provides a carotenoid-loaded nanodelivery system, which differs from example 1 only in that soybean meal is used instead of sesame meal, and other operating conditions are referred to in example 1.
Comparative example 3
The specific operation of the nano delivery system for supporting carotenoid is different from that of the embodiment 1 only in that the steps (1) and (2) in the embodiment 1 are omitted, casein is directly selected to prepare casein zymolyte according to the parameter condition of the step (3), then casein zymolyte dispersion liquid with the concentration of 0.25% (w/v) is prepared, the pH value is regulated to 7.0, a certain amount of beta-carotene petroleum ether solution (1% w/v) is added into the casein zymolyte dispersion liquid, the mass ratio of the casein zymolyte to the beta-carotene is 12.5:1, magnetic stirring and mixing are carried out uniformly, then ice bath ultrasonic treatment (150 w, working 2s, gap 2s and 35 min) is carried out, and petroleum ether is volatilized and removed in a water bath at 60 ℃ for 1h, thus obtaining the nano delivery system.
Test example 1
This test example characterizes the average particle size (nm), PDI and Zeta potential (mV) of the carotenoid-loaded nanodelivery systems prepared in examples 1-18, and was run in parallel 3 times, with the results shown in Table 1:
TABLE 1
As can be seen from the results in Table 1, the particle size distribution of the carotenoid-loaded nano delivery system prepared by the preparation method provided by the invention is uniform, the sesame protein hydrolysate concentration, the mass ratio of the sesame protein hydrolysate to beta-carotene, the ultrasonic time and the pH value of the system influence the particle size distribution and the Zeta potential of the product to a certain extent, wherein the smaller the PDI, the more uniform the particle size distribution of the system, the smaller the particle size and the more stable the Zeta potential absolute value.
Test example 2
The carotenoid-loaded nano delivery system prepared in example 1 was characterized by the entrapment rate and drug loading rate, fourier transform infrared spectroscopy (FTIR), transmission Electron Microscopy (TEM), X-ray diffraction (XRD), and Circular Dichroism (CD), and was measured in parallel for 3 times, specifically as follows:
(1) Determination of the entrapment and drug loading
1mg of beta-carotene is accurately weighed and dissolved in 10mL of normal hexane to prepare 0.1mg/mL of beta-carotene mother liquor, then petroleum ether is respectively diluted into beta-carotene solutions with a certain concentration gradient, petroleum ether is used as a blank control, absorbance is measured at 450nm, and a standard curve is drawn. 1mL of the product of example 1 is added with 4mL of normal hexane, vortex for 1min, centrifuge for 3min at 5000r/min, supernatant is taken, normal hexane is taken as a blank control, absorbance is measured at 450nm, free beta-carotene content is calculated by substituting into a standard curve, and the embedding rate and drug loading rate are calculated according to the following formula.
The embedding rate and drug loading rate of 3 groups of samples were measured, and the results are shown in table 2:
TABLE 2
Sample of Embedding ratio (%) Drug loading rate (%)
1 74.31 11.01
2 76.15 11.28
3 75.23 11.14
As can be seen from the data in table 2, the nano-delivery system according to the present invention has high embedding rate and high loading rate of beta-carotene.
(2) Fourier transform infrared spectroscopy (FTIR)
Taking a small amount of lyophilized sample powder (beta-carotene, sesame protein hydrolysate prepared in step (3) of example 1, end product prepared in step (4) of example 1, sesame protein hydrolysate prepared in step (3) of example 1 and beta-carrotSimple mixture of elements), tabletting with KBr, and scanning wave band range of 400-1000 cm -1 Resolution is 8cm -1 The number of scans was 32, and the infrared spectrum was measured by a fourier infrared spectrometer.
The results are shown in FIG. 2. Beta-carotene is 965cm in infrared spectrum -1 Has strong characteristic absorption peak, is attributed to trans-common olefin-CH=CH-out-of-plane bending vibration, and is 1360cm -1 And 1448cm -1 Absorption peaks of 1556cm for flexural vibrations of olefins C-H radicals symmetrical and antisymmetric -1 Corresponding to the vibration of the aromatic ring skeleton, 2852cm -1 And 2916cm -1 Absorption peak of the symmetric and antisymmetric stretching vibration of methylene C-H is 2949cm -1 Is a methyl C-H antisymmetric telescopic vibration absorption peak of 3030cm -1 Telescoping vibration attributed to = C-H, indicating that the β -carotene is an all-trans isomer. Compared with the characteristic peak of sesame protein zymolyte, the sesame protein zymolyte-beta-carotene nano micelle shows a typical absorption peak of beta-carotene, which proves that the beta-carotene nano micelle is successfully prepared, and meanwhile, 1082cm -1 The strong absorption peak appears, presumably caused by the expansion vibration generated by the reaction of embedding beta-carotene which is involved in the self-assembly of C-O originally contained in the polypeptide in the sesame protein hydrolysate.
(3) Transmission Electron Microscope (TEM)
Samples (beta-carotene, sesame protein hydrolysate prepared in step (3) of example 1, end product prepared in step (4) of example 1, and simple mixture of sesame protein hydrolysate prepared in step (3) of example 1) and beta-carotene) were placed on a copper mesh, and then dyed with phosphotungstic acid. The samples were morphologically observed using TEM at an accelerating voltage of 100 kV.
The results are shown in FIG. 3. It can be seen that the particle size of the nano micelle after loading beta-carotene is increased compared with the nano micelle of the empty sesame protein hydrolysate. Aggregation occurs in the mixture of pure beta-carotene and sesame protein hydrolysate. The sesame protein zymolyte self-assembled nano micelle loaded with the beta-carotene presents a regular round nano particle structure and is uniformly dispersed, which proves that embedding the beta-carotene through the polypeptide self-assembly effect is beneficial to reducing the particle size, thereby being beneficial to the absorption and utilization of the organisms to the beta-carotene.
(4) X-ray diffraction (XRD)
A small amount of lyophilized sample powder (β -carotene, sesame protein hydrolysate prepared in step (3) of example 1, end product prepared in step (4) of example 1, simple mixture of sesame protein hydrolysate prepared in step (3) of example 1 and β -carotene) was taken, and XRD was used for testing, and the test conditions were: the scanning angle range is 3-80 degrees, the scanning speed is 6 degrees/min, the tube current is 40mA, the tube voltage is 40kV, and the Cu target wavelength is 1.5406 angstroms.
The results are shown in FIG. 4. The sesame protein zymolyte has a stronger diffraction peak at 21 degrees and corresponds to the alpha-helical structure of protein, the main diffraction peak type of the sesame protein zymolyte and beta-carotene mixture is not changed, the sesame protein zymolyte and beta-carotene mixture exists in an amorphous form, and only one discrete Long Feng appears in a diffraction pattern, so that the pure mixing of the sesame protein zymolyte and the beta-carotene has no influence on the crystal structure of sesame polypeptide. In contrast, sesame protein hydrolysate-beta-carotene nano-micelles showed sharper characteristic diffraction peaks at 23 ° and 32 °, indicating that after embedding beta-carotene, the crystallization characteristics of sesame protein hydrolysate were changed, probably due to the formation of crystallization regions by hydrogen bonds formed in or between molecules.
(5) Round two Chromatograph (CD)
Samples (sesame protein, sesame protein hydrolysate prepared in step (3) of example 1, end product prepared in step (4) of example 1) were dissolved in 0.01mol/L phosphate buffer solution at pH 7.0 to prepare a protein solution of 0.1mg/mL at the starting wavelength: 190nm, termination wavelength: circular dichromatic scanning is carried out in the range of 260nm, and the step length is as follows: 1nm, 3 times, 1mm optical path length of the sample cell, phosphate buffer as a blank.
The results are shown in FIG. 5. The data obtained were analyzed by the circular dichroism web site dichlorweb (http:// dichlorweb. Cryst. Bbk. Ac. Uk). The changes in the secondary structure of the protein are shown in table 3:
TABLE 3 Table 3
As can be seen from the data in Table 3, sesame protein is alpha+beta type protein, enzymolysis results in increased alpha-helix and random coil structure in the protein, and reduced beta-sheet and beta-corner, but after loading beta-carotene, the proportion of alpha-helix is greatly reduced, when the alpha-helix content in protein molecule is lower, the beta-sheet and random coil content is higher, the disorder of protein molecule is increased, the secondary structure is more loose, and the micelle structure is changed compared with the protein zymolyte. There is a significant linear negative correlation between the α -helix content and the surface hydrophobicity, and therefore most likely due to self-assembly of the proteolytic enzyme into a nanoparticle structure due to hydrophobic interactions.
Test example 3
The in vitro slow release effect of the carotenoid-loaded nano delivery system prepared in example 1 was evaluated in this test example, and the specific procedure is as follows:
10mL of the product prepared in example 1 was injected into 10mL of simulated gastric fluid (containing 3.2mg/mL pepsin and 2.0mg/mL sodium chloride, pH 2.5) and placed in a 100mL Erlenmeyer flask, and then the mixture was continuously shaken at 37℃and 150rpm for 2 hours, and samples were taken every 30 minutes for analysis. After gastric digestion, the pH was adjusted to 7.0 with a pre-formulated 1.0mol/L NaOH solution, followed by injection of 20mL of simulated intestinal fluid (containing 4.1mg/mL bile salts, 6.0mg/mL trypsin, 6.0mg/mL pancreatic lipase, 7.28mg/mL calcium chloride dihydrate, 8.8mg/mL sodium chloride, pH 7.0) and continued shaking at 37℃for 2 hours at 150rpm, sampling every 30min for analysis. And (3) taking the free beta-carotene solution as a control, measuring the content of the free beta-carotene by a spectrophotometry method, and calculating the release rate of the beta-carotene.
As shown in fig. 6, it can be seen that compared with free beta-carotene, the self-assembled and embedded beta-carotene of sesame protein hydrolysate can be released more slowly in the simulated gastrointestinal environment, which is more beneficial for the absorption, transformation and utilization of beta-carotene by organisms after passing through the gastrointestinal environment.
Test example 4
The pharmacokinetics of the carotenoid-loaded nanodelivery system prepared in example 1 was evaluated in this test example, as follows:
healthy SD male rats are selected for 12, 9-10 weeks of week old, and the weight is 500-600 g. Rats were fasted without water withdrawal 18h before blood collection and the experiment was divided into a β -carotene aqueous dispersion group, a β -carotene oil solution group and a β -carotene nanomicelle group (product of example 1), 4 per group, each at 50mg β -carotene/kg intragastric. After gastric lavage, each rat was at different time points: the orbital vein blood collection is carried out for 0.5, 1, 2, 3, 6, 9 and 12 hours and stored in a heparin tube, the blood collection is immediately carried out, the room temperature centrifugation is carried out for 10 minutes at 4500rpm, the blood plasma is sucked into a 1.5mL centrifuge tube, and the blood plasma is stored in a refrigerator at the temperature of minus 80 ℃ to be tested.
The content of beta-carotene in the plasma is detected by Agilent 1290-6460LC/MS liquid chromatography/four-stage rod tandem mass spectrometry. Detection conditions: the chromatographic column is Agilent Eclipse Plus C 18 column (50 mm. Times.2.1 mm,1.8 μm), mobile phase 100% acetonitrile, elution time 18min, flow rate 0.5mL/min, and sample injection amount 1. Mu.L. Mass spectrometry scan mode: multiple Reaction Monitoring (MRM), parent ion (m/z): 536.3, daughter ion (m/z): 444 collision energy: 10V. The test data were analyzed using agilent software Qualitative Analysis b.06.00.
As a result, as shown in FIG. 7, it can be seen that the time points at which the maximum plasma concentrations of the aqueous beta-carotene dispersion, the beta-carotene oil solution and the beta-carotene nano-micelles occurred were 8 hours, 6 hours and 8 hours, respectively, and the peak concentrations were 23.78ng/mL, 55.49ng/mL and 64.98ng/mL, respectively. Under the same stomach-filling dosage, compared with beta-carotene aqueous dispersion liquid and beta-carotene oil solution, the sesame protein hydrolysate-beta-carotene nano micelle has higher peak reaching concentration in plasma, can obviously promote the oral absorption of beta-carotene in rats, and improves the oral bioavailability of beta-carotene.
Test example 5
The present test example evaluates the protective effect of the carotenoid-loaded nano-delivery system prepared in example 2 on ulcerative colitis, and is specifically as follows:
SPF-class female C57BL/6 mice (8-10 weeks) are adopted in the experiment, animals are fed for 1 week and are randomly divided into a control group, a model group, an astaxanthin aqueous dispersion liquid group, a sesame protein hydrolysate empty nano micelle group and a sesame protein hydrolysate-astaxanthin nano micelle group provided in example 2 after being adapted to the environment, and each group of mice is fed in separate cages, and 8 mice are fed in each group. The acute ulcerative colitis model was established by the method disclosed in the reference (Cooper H S et al, clinicopathologic study of dextran sulfate sodium experimental murine colitis, laboratory Investigation,1993, vol.2, no. 69, 238-249), with the model and experimental groups freely drinking 3% dextran sodium sulfate solution (DSS solution, formulated with distilled water) for 5 consecutive days, with fresh DSS solution changed every 1 day, and the control group drinking distilled water. The experimental group started to perform gastric lavage on the sesame protein hydrolysate-astaxanthin nano-micelle provided in example 23 days before modeling of ulcerative colitis, and the gastric lavage dose was calculated as 50mg astaxanthin/kg; the gastric lavage dose of the astaxanthin aqueous dispersion liquid group and the sesame protein zymolyte empty nano micelle group is equal to the dose of astaxanthin and sesame protein zymolyte in the sesame protein zymolyte-astaxanthin nano micelle. The control and model groups replaced the samples with distilled water for up to the last 1 day of molding. Subsequently, each group of mice was dissected, the entire intestinal section from the anus to the distal cecum was rapidly removed, the length was weighed and measured, and the weight-to-length ratio of the colon was calculated.
Mice in each group were scored for mouse Disease Activity (DAI), which is the average of the sum of weight loss score, stool trait score, and hematocrit score, by the method disclosed in the reference (Wirtz S et al Chemically induced mouse models of intestinal inflammation, nature Protocols,2007, volume 2, phase 3, 541). Mice were observed daily for mental state, activity, hair gloss, stool characteristics (including whether bloody stool and diarrhea), etc., body weight was recorded on day 5 and scores were recorded, and DAI scoring criteria are shown in table 4.
TABLE 4 Table 4
Scoring of Weight loss (%) Stool characteristics Hematochezia blood
0 <1 Normal state Negative of
1 1～5 Loosening Weak positive
2 5～10 Semi-forming thin Positive and negative
3 10～20 Non-forming thin film Strong positive
4 >20 Water sample diarrhea Blood stool with naked eyes
In Table 4, the method for determining the occult blood of the mouse comprises the following steps: daily application of mouse feces on clean filterDropwise adding 2-3 drops of o-tolidine on paper, and then dropwise adding 3% H 2 O 2 Several drops, the color change of the feces was observed. Judging the occult blood result: (1) negative: no change in color within 2 min; (2) weak positive: gradually changing from light blue to blue after 10 s; (3) positive: initially a light blue brown color, slowly changing to a deep blue brown color; (4) strong positive: immediately appear dark blue brown.
The control, model and experimental mice were tested for their colon weight to length ratio and DAI score as described above and the results are shown in table 5.
TABLE 5
In table 5, the different letters a to d represent significant differences between groups in the same column (P < 0.05).
As can be seen from the data in table 5, the ratio of the weight and the length of the colon of the mice is the ratio of the weight and the length of the colon, reflects the edema degree of the colon per unit length, and the index of the weight/the length of the colon of the model group is remarkably increased compared with that of the control group, so that the sesame protein hydrolysate-astaxanthin nano-micelle prepared in example 2 can remarkably reduce the edema degree of the colon and restore the colon to a normal state. The weight length ratio of the colon of the mice treated by the empty nano-micelle of the astaxanthin aqueous dispersion liquid and the sesame protein hydrolysate is not obviously different from that of the model group, which shows that the protection effect of the astaxanthin on the colon can be enhanced after the astaxanthin is embedded in the sesame protein hydrolysate to form the nano-micelle.
The DAI index is an index for comprehensively evaluating the mental state, activity condition, hair glossiness and stool property of the mice, and is also an index for reflecting the severity of inflammation, the DAI index of a model group is obviously increased relative to a control group, and each experimental group is improved to different degrees relative to the model group. Compared with the empty nano micelle treatment group of the astaxanthin aqueous dispersion liquid and the sesame protein zymolyte, the sesame protein zymolyte-astaxanthin nano micelle prepared by the method of the embodiment 2 has better improvement effect on the intestinal inflammation injury of mice, which is related to the improvement of the bioavailability of the astaxanthin by the formation of the nano micelle and the synergistic effect of the astaxanthin and the sesame protein zymolyte in the nano micelle when the ulcerative colitis symptom is improved.
Test example 6
The present test example evaluates the protective effect of the carotenoid-loaded nano delivery system prepared in example 3 on retinal photodamage, and is specifically as follows:
the blue-violet rabbits are adaptively raised for one week, freely picked up with water and eaten, and then randomly divided into 5 groups according to body weight, namely a control group, a model group, a lutein water dispersion group, a sesame protein hydrolysate empty nano micelle group and a sesame protein hydrolysate-lutein nano micelle group provided in the example 3, wherein each group comprises 5 experimental animals. The control group and the model group are respectively provided with corresponding samples after being provided with stomach, the other groups are respectively provided with stomach-filling drinking water, the stomach is filled for 2 weeks, the dark adaptation is carried out for 24 hours, the photo damage modeling is carried out, the compound topiramate eye drops are used for mydriasis on two eyes, the eyes are placed in an illumination box after 20 minutes, illumination is started, the illumination intensity is 15000+/-1000 lux, the time is 2 hours, and the administration is continued for 1 week after illumination. After the end of the experiment, the eye function was evaluated by performing ERG examination on each group of bluish-violet rabbits, recording the dark vision, bright vision and maximum response ERG, and analyzing the b-wave amplitude. After ERG recording, the air embolism method exempts the eugenolysis from blue and violet, extracts the eyeball, and after fixation, carries out paraffin section, H & E staining, and analyzes the retinal ONL thickness.
The b-wave changes and retinal ONL thickness of the control, model and experimental groups were tested as described above and the results are shown in table 6.
TABLE 6
In table 6, the different letters a to d represent significant differences between groups in the same column (P < 0.05).
From the results in table 6, the model group was seen to have significantly reduced scotopic, photopic and maximum response b-wave amplitudes and retinal ONL thickness relative to the control group. After the lutein aqueous dispersion liquid, the sesame protein hydrolysate empty nano micelle and the sesame protein hydrolysate-lutein nano micelle provided in the embodiment 3 are ingested, b wave amplitude and retina ONL thickness can be improved to different degrees, wherein the effect strength sequence is as follows: example 3> lutein aqueous dispersion > sesame protein hydrolysate empty nano-micelle. This shows that all three samples can repair the damage of the visual pole cells and the visual cone cells of the blue-violet rabbit, improve the scotopic vision capability and have the optimal effect in the embodiment 3. The sesame protein zymolyte-lutein nano micelle provided in the oral administration example 3 can restore functions of the video rod cells and the video cone cells to normal level, can effectively inhibit apoptosis of photoreceptor cells, and can improve retinal structural damage caused by illumination. The results show that the sesame protein zymolyte-lutein nano micelle can improve the protection effect on visual health by enhancing the bioavailability of lutein and the synergistic effect in the components.
Test example 7
This test example evaluates the recovery of DSS-damaged Caco-2 cells with the carotenoid-loaded nanodelivery systems prepared in example 1, comparative examples 1-3, as follows:
caco-2 cells were grown at 2X 10 4 The density of individuals/wells was inoculated into 96-well culture plates. After 12h incubation, the original medium was removed, 100. Mu.L of each group of samples (200. Mu.g/mL) and DSS (10 mg/mL) were added to each well, and after 24h incubation, the supernatant was discarded, cell viability was measured by CCK-8 method, and cell membrane integrity was measured by LDH kit. The results are shown in Table 7.
TABLE 7
Sample of Cell viability (%) Cell membrane integrity (%)
DSS injury group 79.6 50.1
Example 1 110.5 87.9
Comparative example 1 91.7 69.2
Comparative example 2 99.4 82.3
Comparative example 3 105.2 84.5
As can be seen from the data in Table 7, the beta-carotene-loaded nano-micelle prepared by the method of example 1 can better improve the survival rate and the integrity of cell membranes of Caco-2 cells, thereby more effectively protecting the intestinal barrier function, compared with comparative examples 1 to 3. Therefore, in the process of preparing the nano-micelle of β -carotene, a step of performing enzymolysis on sesame protein is indispensable. In addition, the result proves that the sesame protein is selected to replace soybean protein or casein to prepare zymolyte, and the zymolyte is used for embedding beta-carotene through polypeptide self-assembly, so that the physiological activity of the beta-carotene is improved, and the method has more advantages in the aspect of repairing intestinal barrier injury.
The applicant states that the present invention is illustrated by the above examples as a carotenoid-loaded nanodelivery system, and methods of making and using the same, but the present invention is not limited to, i.e., does not mean that the present invention must be practiced in dependence upon the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.

Claims (10)

1. A nano-delivery system for supporting carotenoid, which is characterized by comprising a carrier material sesame protein hydrolysate and the carotenoid coated in the sesame protein hydrolysate.
2. The carotenoid-loaded nano-delivery system according to claim 1, wherein the sesame protein hydrolysate is prepared by a preparation method comprising the steps of:
mixing sesame protein and protease for enzymolysis reaction, inactivating enzyme, centrifuging, and freeze-drying supernatant to obtain the sesame protein hydrolysate.
3. The carotenoid-loaded nano-delivery system according to claim 2, wherein the protease comprises any one or a combination of at least two of pepsin, trypsin, cathepsin, papain, subtilisin, neutral protease or alkaline protease;
preferably, the mass ratio of the protease to the sesame protein is 1 (50-200);
preferably, the pH of the enzymolysis reaction system is 1.5-8.5, and the temperature is 30-55 ℃;
preferably, the enzymolysis reaction time is 2-10h.
4. The carotenoid-loaded nano-delivery system according to claim 2, wherein the sesame protein is obtained by an extraction method comprising the steps of:
removing grease in the sesame raw material by adopting a solvent leaching method, and extracting sesame protein in the defatted sesame raw material by adopting an alkali extraction and acid precipitation method;
preferably, the sesame raw material is sesame seed meal;
preferably, the alkali extraction and acid precipitation method comprises the following steps: dissolving protein at pH 9.0-11.0 and 50-55deg.C, centrifuging, adjusting pH of supernatant to 3.5-5.5, and standing to precipitate protein.
5. The carotenoid-loaded nano-delivery system according to any of claims 1-4, wherein the carotenoid comprises any one or a combination of at least two of a-carotene, β -carotene, γ -carotene, lycopene, lutein, astaxanthin, capsanthin or zeaxanthin.
6. The method of preparing a carotenoid loaded nano-delivery system according to any one of claims 1-5, wherein the method of preparing comprises:
and mixing the sesame protein hydrolysate dispersion liquid with carotenoid solution, and carrying out ultrasonic treatment to obtain the carotenoid-loaded nano delivery system.
7. The method of preparing a carotenoid-loaded nano-delivery system according to claim 6, wherein the concentration of the sesame protein hydrolysate dispersion is 0.25-2.5%, preferably 0.25-0.45%;
preferably, the concentration of the carotenoid solution is 0.5-1.5%;
preferably, the mass ratio of the sesame protein hydrolysate to the carotenoid is 10:1-20:1;
preferably, the solvent of the carotenoid solution is selected from any one or a combination of at least two of petroleum ether, n-hexane, dichloromethane or acetone;
preferably, the working parameters of the ultrasonic treatment are 50-250w and 30-45min.
8. The method of preparing a carotenoid-loaded nano-delivery system according to claim 6 or 7, wherein the sesame protein hydrolysate dispersion is further pH-adjusted to 4.0-8.0, preferably 6.0-8.0, before mixing with the carotenoid solution;
preferably, the ultrasonic treatment is carried out after the end of the ultrasonic treatment, and the treatment mode comprises the steps of placing in a water bath at 50-70 ℃ for 0.5-2h.
9. Use of a carotenoid-loaded nano-delivery system according to any one of claims 1-5 in the manufacture of a medicament for the prevention, amelioration or treatment of ulcerative colitis.
10. Use of the carotenoid-loaded nano-delivery system according to any one of claims 1-5 for the preparation of a food, pharmaceutical or health care product for maintaining visual health.
CN202311318535.9A 2023-10-12 2023-10-12 Carotenoid-loaded nano delivery system and preparation method and application thereof Pending CN117338944A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311318535.9A CN117338944A (en) 2023-10-12 2023-10-12 Carotenoid-loaded nano delivery system and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311318535.9A CN117338944A (en) 2023-10-12 2023-10-12 Carotenoid-loaded nano delivery system and preparation method and application thereof

Publications (1)

Publication Number Publication Date
CN117338944A true CN117338944A (en) 2024-01-05

Family

ID=89360773

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311318535.9A Pending CN117338944A (en) 2023-10-12 2023-10-12 Carotenoid-loaded nano delivery system and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN117338944A (en)

Similar Documents

Publication Publication Date Title
US4680314A (en) Process for producing a naturally-derived carotene/oil composition by direct extraction from algae
Fernández-Sevilla et al. Biotechnological production of lutein and its applications
US10834949B2 (en) Method of microalgal biomass processing for high-value chemicals production, the resulting composition of butyrogenic algal slowly fermenting dietary fiber, and a way to improve colon health using a slowly fermenting butyrogenic algal dietary fiber
US8048652B2 (en) Biomass hydrolysate and uses and production thereof
JP5795572B2 (en) Method for separating carotenoid crystals from various plants
US20090247633A1 (en) Nutrient extracts derived from green plant materials
Rodríguez-Sifuentes et al. Importance of downstream processing of natural astaxanthin for pharmaceutical application
EP0608172A1 (en) Phaffia rhodozyma mutants, process for the production of beta-caroten and use of a beta-caroten rich biomass
CN113925157B (en) Beta-carotene emulsion and preparation method thereof
WO2020011176A1 (en) Method for extracting and isolating lutein crystal from vegetable oil resin containing lutein diester
Akepach et al. Bioaccessibility of carotenoids (β-carotene and lutein) from intact and disrupted microalgae (Chlamydomonas reinhardtii)
CN1493622A (en) Preparation method of lycopene
Murthy et al. Comparative evaluation of hepatoprotective activity of carotenoids of microalgae
US8481769B2 (en) Isolation and purification of cartenoids from marigold flowers
CN117338944A (en) Carotenoid-loaded nano delivery system and preparation method and application thereof
CN107897647B (en) Green preparation method of water-soluble lutein
JP5767716B2 (en) Method for extracting lutein-containing silk extract
Xiaofei et al. Effects of heat pump drying temperature and dietary fat on carrot β-carotene bioaccessibility
AU595824B2 (en) Narurally-derived carotene/oil composition and the process for the production thereof by direct extraction of carotene from algae
CN1301262C (en) Method for producing glucolipid substance using ganglioside as main component
JP2007319015A (en) Method for producing carotenoid
US20040176475A1 (en) Isolation of lutein from alfalfa
CA2815271A1 (en) Extraction of oil-soluble pigment from micro-organisms
RU2166868C1 (en) Method for obtaining biologically active product
BR102022010286A2 (en) COMPOUND OF PROTEIN ISOLATED FROM MACAÚBA ALMOND, PROCESSES FOR OBTAINING THE PROTEIN ISOLATE AND USES

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination