CN116098289A - Preparation method of DHA-containing rice bran-based carrier - Google Patents
Preparation method of DHA-containing rice bran-based carrier Download PDFInfo
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- CN116098289A CN116098289A CN202211722366.0A CN202211722366A CN116098289A CN 116098289 A CN116098289 A CN 116098289A CN 202211722366 A CN202211722366 A CN 202211722366A CN 116098289 A CN116098289 A CN 116098289A
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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
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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
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
- A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
- A23L7/107—Addition or treatment with enzymes not combined with fermentation with microorganisms
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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
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
- A23L7/115—Cereal fibre products, e.g. bran, husk
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
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Abstract
The invention discloses a preparation method of a rice bran-based carrier containing DHA, which comprises the following steps: s10, grinding and sieving rice bran, and soaking to obtain rice bran mixed liquor. S20, pulping and homogenizing the rice bran mixed solution, and performing suction filtration to obtain filtrate; s30, adding cellulase and pectase into the filtrate for enzymolysis, and centrifuging to obtain an upper cream pasty substance; s40, washing and freeze-drying the upper cream paste substance to obtain rice bran oil base; s50, dissolving the rice bran oil base in water to form a water phase, dissolving the fish oil containing DHA in the oil to form an oil phase, and uniformly mixing the water phase and the oil phase to obtain colostrum; s60, performing rotary steaming, homogenizing and standing on the colostrum to obtain the DHA-containing rice bran-based carrier. The natural components in the rice bran are used as the stabilizer of the carrier, so that the use of the synthetic emulsifier is avoided, and the application range of rice bran resources is widened. The preparation process is not complicated, the organic solvent residue is less, and the preparation method is natural and environment-friendly.
Description
Technical Field
The invention relates to the technical field of foods, in particular to a preparation method of a rice bran-based carrier containing DHA.
Background
Docosahexaenoic acid (Docosahexaenoic Acid, DHA) is a polyunsaturated fatty acid of the omega-3 type extracted mainly from fish oils. DHA is mainly present in glycerophospholipids of animals, is mostly present in the retina and cerebral cortex in humans, and can be produced from alpha-linolenic acid during in vivo metabolism, but the production is low, mainly through food supplementation. Studies show that DHA has a plurality of probiotic functions, including promoting the development of brain nerve cells, reducing blood fat, delaying aging, enhancing memory function and the like, and has attracted a great deal of attention of a plurality of scholars and researchers. However, DHA is an unsaturated fatty acid, and has 6 double bonds on the group, so that DHA is unstable and is extremely easily oxidized. Meanwhile, factors such as light, heat, pH and the like also influence the property of DHA, so that the physiological activity of DHA is reduced. These defects greatly limit the use of DHA.
Disclosure of Invention
The invention mainly aims to provide a preparation method of a rice bran-based carrier containing DHA, aiming at preparing the rice bran-based carrier containing DHA and improving the bioavailability of DHA.
In order to achieve the above object, the present invention provides a method for preparing a rice bran-based carrier containing DHA, comprising the following steps:
s10, grinding and sieving rice bran, and soaking to obtain rice bran mixed liquor.
S20, pulping and homogenizing the rice bran mixed solution, and performing suction filtration to obtain filtrate;
s30, adding cellulase and pectase into the filtrate for enzymolysis, and centrifuging to obtain an upper cream pasty substance;
s40, washing and freeze-drying the upper cream paste substance to obtain rice bran oil base;
s50, dissolving the rice bran oil base in water to form a water phase, dissolving the fish oil containing DHA in the oil to form an oil phase, and uniformly mixing the water phase and the oil phase to obtain colostrum;
s60, performing rotary steaming, homogenizing and standing on the colostrum to obtain the DHA-containing rice bran-based carrier.
Optionally, step S10 includes:
grinding and sieving rice bran, and adding phosphate buffer solution for soaking to obtain rice bran mixed solution.
Optionally, in step S20, the homogenized rotation speed is 3500-10500 rpm.
Optionally, in step S30, the sum of the masses of the cellulase and pectase is 1% -3% of the mass of the filtrate.
Optionally, in step S30, the enzymolysis time is 2-6 hours.
Optionally, in step S30, the temperature of the enzymolysis is 40-60 ℃.
Optionally, step S40 includes:
s41, washing the upper-layer cream paste substance with a washing liquid, and centrifugally collecting the upper-layer substance, wherein the washing liquid comprises medium-chain triglyceride, glucose and potassium chloride;
s42, freeze-drying the upper layer substance to obtain rice bran oil base.
Optionally, in step S40, the temperature of the lyophilization is-60 ℃ to-40 ℃.
Optionally, in step S50, the mass ratio of the aqueous phase to the oil phase is 90:10 to 95:5.
Alternatively, in step S60,
the homogeneous power is 240-600 w; and/or the number of the groups of groups,
the homogenizing time is 5-10 min.
In the technical scheme provided by the invention, the rice bran-based carrier containing DHA is prepared, and the bioavailability of DHA is improved. The natural components in rice bran are used for replacing the traditional industrial emulsifier as an emulsion stabilizer, so that carcinogenicity and anaphylactic reaction possibly brought by the industrial emulsifier can be avoided. The part of oily solid particles in rice bran can be adsorbed and accumulated on the interface between two non-phase solutions (generally expressed by oil phase and water phase) to stabilize liquid drops and prevent the liquid drops from coalescing, and the emulsion has more stable performance and lower toxicity, so that the emulsion is safer in vivo, the sustained and controlled release effect on the embedded nutrient is achieved, and the bioavailability of the embedded substance is improved. The natural components in the rice bran are used as the stabilizer of the carrier, so that the use of the synthetic emulsifier is avoided, and the application range of rice bran resources is widened. The preparation process is not complicated, the organic solvent residue is less, and the preparation method is natural and environment-friendly. Develops a nanoscale DHA-type carrier, and provides basic basis for application of DHA fish oil in the field of health-care foods and research on DHA absorption mechanism.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other related drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flowchart of a method for preparing a DHA-containing rice bran-based carrier according to an embodiment of the present invention;
FIG. 2 is a graph showing the particle size distribution of a DHA-based carrier prepared in example 1 according to the present invention;
FIG. 3 is a morphology diagram of a DHA-based rice bran-based carrier prepared in example 1 of the present invention;
FIG. 4 is a schematic diagram showing the stability of the DHA-based carrier prepared in example 1 according to the present invention;
FIG. 5 is a schematic view showing the brightness of DHA-based carrier prepared in example 1 according to the present invention;
FIG. 6 is a graph showing cytotoxicity test results of the DHA-based rice bran carrier prepared in example 1 of the present invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Docosahexaenoic acid (Docosahexaenoic Acid, DHA) is a polyunsaturated fatty acid of the omega-3 type extracted mainly from fish oils. DHA is mainly present in glycerophospholipids of animals, is mostly present in the retina and cerebral cortex in humans, and can be produced from alpha-linolenic acid during in vivo metabolism, but the production is low, mainly through food supplementation. Studies show that DHA has a plurality of probiotic functions, including promoting the development of brain nerve cells, reducing blood fat, delaying aging, enhancing memory function and the like, and has attracted a great deal of attention of a plurality of scholars and researchers. However, DHA is an unsaturated fatty acid, and has 6 double bonds on the group, so that DHA is unstable and is extremely easily oxidized. Meanwhile, factors such as light, heat, pH and the like also influence the property of DHA, so that the physiological activity of DHA is reduced. These defects greatly limit the use of DHA.
In view of the above, the invention provides a preparation method of a rice bran-based carrier containing DHA, which aims to prepare the rice bran-based carrier containing DHA and improve the bioavailability of DHA.
Referring to fig. 1, the preparation method of the rice bran-based carrier containing DHA provided by the invention comprises the following steps:
s10, grinding and sieving rice bran, and soaking to obtain rice bran mixed liquor.
Preferably, step S10 includes:
grinding and sieving rice bran, and adding phosphate buffer solution for soaking to obtain rice bran mixed solution.
During specific operation, grinding: the rice bran is milled by a mill, so that the particles are smaller and the particle size is uniform.
Sieving: sieving the milled rice bran with 60 mesh sieve (pore diameter of 0.15 mm) to remove impurities and broken rice in the rice bran.
Soaking: phosphate buffer (phosphate buffered saline, PBS, ph=7.0) is added to the sieved rice bran according to a feed-liquid ratio of 1:10, the pH stability of the components in the rice bran system is maintained, and the rice bran is soaked overnight after being uniformly mixed.
S20, pulping and homogenizing the rice bran mixed solution, and performing suction filtration to obtain filtrate.
In the embodiment of the invention, during specific operation, pulping: pouring the rice bran mixed liquor into a beater for beating, and destroying cell walls and fiber layers of rice bran to release endogenous substances. The power of the beater was set at 1500w and the working time was 1.5min.
Homogenizing: transferring the rice bran solution after pulping into a 500mL beaker, homogenizing the rice bran solution by using a homogenizer, further destroying plant tissues of the rice bran, and fully releasing endogenous components. The working speed of the homogenizer is 3500-10500 rpm, the optimal scheme is 7000rpm, and the working time is 5min.
And (3) suction filtration: and (3) carrying out suction filtration on the homogenized rice bran solution by using a suction filter, wherein the specification of qualitative filter paper is 12.5cm, and collecting the lower filtrate for later use.
S30, adding cellulase and pectase into the filtrate for enzymolysis, and centrifuging to obtain the upper cream paste substance.
In the embodiment of the invention, the enzymolysis is carried out during the specific operation: transferring the collected filtrate into a 500mL beaker, adjusting the pH of the components to 4, adding cellulase and pectase (the mass ratio of the cellulase to the pectase is 1:1, the sum of the masses of the cellulase and the pectase is 1% -3% of the mass of the filtrate, preferably 2%), and carrying out enzymolysis for 2-6 hours, preferably 4 hours, wherein the enzymolysis temperature is 40-60 ℃. Preferably 55 ℃.
And (3) centrifuging: centrifuging the rice bran solution subjected to enzymolysis by using a refrigerated centrifuge at a centrifugal temperature of 4 ℃ for 30min with a centrifugal power of 10000rpm. And collecting cream paste substances on the upper layer of the centrifuge tube after centrifugation.
And S40, washing and freeze-drying the upper cream paste substance to obtain rice bran oil base.
Preferably, step S40 includes:
s41, washing the upper-layer cream paste substance with a washing liquid, and centrifugally collecting the upper-layer substance, wherein the washing liquid comprises medium-chain triglyceride, glucose and potassium chloride;
s42, freeze-drying the upper layer substance to obtain rice bran oil base.
Preferably, in step S40, the temperature of the lyophilization is-60 ℃ to-40 ℃.
During specific operation, washing: PBS (containing 0.1% medium chain triglyceride, 0.6mol/L glucose, 0.5mol/L potassium chloride, pH=7.0) is added to the collected cream-like substance according to a feed liquid ratio of 1:1, and the mixture is washed thoroughly. And then, continuing to freeze and centrifuge, and collecting cream paste substances on the upper layer of the centrifuge tube. The centrifugal washing step was repeated 3 times, after which the cream-like substance after washing was collected, to which 0.1% NaN was added 3 Preventing bacteria contamination.
And (3) freeze-drying: freeze-drying the cream paste material in a freeze dryer at-60 to-40 ℃ for 3 days. And collecting oily viscous solid particles after freeze-drying, namely rice bran oil base.
S50, dissolving the rice bran oil base in water to form a water phase, dissolving the fish oil containing DHA in the oil to form an oil phase, and uniformly mixing the water phase and the oil phase to obtain the colostrum.
Preferably, in step S50, the mass ratio of the aqueous phase to the oil phase is 90:10 to 95:5.
Specifically, the oil phase is dissolved: fish oil (containing 12% DHA) was dissolved in n-hexane and mixed well to form an oil phase.
Preparation of colostrum: dissolving rice bran oil base with water, diluting into 2% solution, mixing in high-speed disperser with power of 3500rpm/min and working time of 5min to form water phase. The aqueous phase is placed in an ultrasonic extractor, and the DHA-containing oil phase (water-oil mass ratio is 90:10-95:5, preferably water: oil=92:8) is slowly added dropwise into the aqueous phase, and the mixture is uniformly mixed under the condition of ultrasonic treatment to form the colostrum.
S60, performing rotary steaming, homogenizing and standing on the colostrum to obtain the DHA-containing rice bran-based carrier.
Preferably, in step S60, the process of the present invention,
the homogeneous power is 240-600 w; and/or the number of the groups of groups,
the homogenizing time is 5-10 min.
Specifically, rotary steaming: transferring the prepared colostrum into a rotary evaporator evaporation bottle, removing n-hexane in the system through rotary evaporation operation, and further concentrating the carrier emulsion. The spin-steaming temperature was 60℃and the rotational speed was 100rpm.
Homogenizing: placing the concentrated carrier in a cell disruption instrument for further homogenizing and shearing. The homogenization shearing operation is performed under ice water bath conditions to ensure stability of the DHA. The power of the crusher is preferably 480w (the power range is 240-600 w), the working time is preferably 7min (the working time is 5-10 min), the probe model is 12, the sample processing amount is more than 12mL, and the working interval time is set to be 1s. The working stage of the crusher is to pay attention to the temperature of the outer ice water bath, and if the temperature is higher than 40 ℃, the ice water bath is replaced.
Cooling and standing: and standing the emulsified carrier emulsion at 25 ℃ in a water bath condition, and cooling to room temperature.
And (3) a finished product: the prepared carrier is uniform and stable, has certain viscosity, and the emulsion is milky and contains a certain DHA smell, and has little or no foam.
The rice bran is a milled product obtained by primarily processing rice to obtain brown rice and then performing a series of whitening operations. Rice bran is usually composed of seed coats, pericarps, aleurone layers, endosperm, etc., and is often mixed with rice germ and broken rice, a major byproduct of rice processing. The rice bran yield is about 6% of the total rice, and the nutritional ingredients may be about 60% of the rice nutrition, including proteins, dietary fibers, carbohydrates, fats, etc. However, rice bran is not suitable for direct use as a food material because a small amount of rice husk and a certain amount of dust and microorganisms are mixed in the rice bran during the process. For a long time, the rice bran is mainly used as livestock and poultry feed in China for raising chickens, pigs, ducks, geese, aquatic products and the like, and the development of the rice bran in other industries is limited.
Nanocarriers are delivery systems that deliver nutrients or drugs into the body's environment or specific target tissues, and are one of the hot spots of current interest. Many fat-soluble substances are low in stability and bad in smell, resulting in difficulty in full use of nutrients. The nano emulsion is one of nano carriers, and the emulsion can stabilize an oil-water interface through a small molecular emulsifier and a plurality of macromolecular substances, so that the nano emulsion is often applied to various fields of biopharmaceuticals, drug delivery, food industry, fine chemical industry, cosmetics and the like, can stabilize the property of the nano emulsion while coating nutrient substances, and better plays the nutrition and physiological characteristics.
Part of the plant and animal ingredients are the basic sources of natural emulsion stabilizers because of their nature of being naturally degradable, good ecological benefits, availability in nature and harmlessness to health, etc. make them of great advantage as natural emulsion stabilizers for food products.
In the technical scheme provided by the invention, the rice bran-based carrier containing DHA is prepared, and the bioavailability of DHA is improved. The natural components in rice bran are used for replacing the traditional industrial emulsifier as an emulsion stabilizer, so that carcinogenicity and anaphylactic reaction possibly brought by the industrial emulsifier can be avoided. The part of oily solid particles in rice bran can be adsorbed and accumulated on the interface between two non-phase solutions (generally expressed by oil phase and water phase) to stabilize liquid drops and prevent the liquid drops from coalescing, and the emulsion has more stable performance and lower toxicity, so that the emulsion is safer in vivo, the sustained and controlled release effect on the embedded nutrient is achieved, and the bioavailability of the embedded substance is improved. The natural components in the rice bran are used as the stabilizer of the carrier, so that the use of the synthetic emulsifier is avoided, and the application range of rice bran resources is widened. The preparation process is not complicated, the organic solvent residue is less, and the preparation method is natural and environment-friendly. Develops a nanoscale DHA-type carrier, and provides basic basis for application of DHA fish oil in the field of health-care foods and research on DHA absorption mechanism.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
Example 1
Grinding: the rice bran is milled by a mill, so that the particles are smaller and the particle size is uniform.
Sieving: sieving the milled rice bran with 60 mesh sieve (pore diameter of 0.15 mm) to remove impurities and broken rice in the rice bran.
Soaking: phosphate buffer (phosphate buffered saline, PBS, ph=7.0) is added to the sieved rice bran according to a feed-liquid ratio of 1:10, the pH stability of the components in the rice bran system is maintained, and the rice bran is soaked overnight after being uniformly mixed.
Pulping: pouring the rice bran mixed liquor into a beater for beating, and destroying cell walls and fiber layers of rice bran to release endogenous substances. The power of the beater was set at 1500w and the working time was 1.5min.
Homogenizing: transferring the rice bran solution after pulping into a 500mL beaker, homogenizing the rice bran solution by using a homogenizer, further destroying plant tissues of the rice bran, and fully releasing endogenous components. The working speed of the homogenizer was 7000rpm, and the working time was 5min.
And (3) suction filtration: and (3) carrying out suction filtration on the homogenized rice bran solution by using a suction filter, wherein the specification of qualitative filter paper is 12.5cm, and collecting the lower filtrate for later use.
Enzymolysis: transferring the collected filtrate into a 500mL beaker, adjusting the pH of the components to 4, and adding cellulase and pectase (the mass ratio of the cellulase to the pectase is 1:1, the sum of the masses of the cellulase and the pectase is 2% of the mass of the filtrate) into the beaker, wherein the enzymolysis time is 4 hours, and the enzymolysis temperature is 55 ℃.
And (3) centrifuging: centrifuging the rice bran solution subjected to enzymolysis by using a refrigerated centrifuge at a centrifugal temperature of 4 ℃ for 30min with a centrifugal power of 10000rpm. And collecting cream paste substances on the upper layer of the centrifuge tube after centrifugation.
Washing: PBS (containing 0.1% medium chain triglyceride, 0.6mol/L glucose, 0.5mol/L potassium chloride, pH=7.0) is added to the collected cream-like substance according to a feed liquid ratio of 1:1, and the mixture is washed thoroughly. And then, continuing to freeze and centrifuge, and collecting cream paste substances on the upper layer of the centrifuge tube. The centrifugal washing step was repeated 3 times, after which the cream-like substance after washing was collected, to which 0.1% NaN was added 3 Preventing bacteria contamination.
And (3) freeze-drying: freeze-drying the cream paste in a freeze dryer at-50deg.C for 3 days. And collecting oily viscous solid particles after freeze-drying, namely rice bran oil base.
Oil phase dissolution: fish oil (containing 12% DHA) was dissolved in n-hexane and mixed well to form an oil phase.
Preparation of colostrum: dissolving rice bran oil base with water, diluting into 2% solution, mixing in high-speed disperser with power of 3500rpm/min and working time of 5min to form water phase. The aqueous phase was placed in an ultrasonic extractor and the DHA-containing oil phase (water: oil=92:8 mass ratio) was slowly added dropwise to the aqueous phase, and mixed well under ultrasonic treatment to form colostrum.
Rotary steaming: transferring the prepared colostrum into a rotary evaporator evaporation bottle, removing n-hexane in the system through rotary evaporation operation, and further concentrating the carrier emulsion. The spin-steaming temperature was 60℃and the rotational speed was 100rpm.
Homogenizing: placing the concentrated carrier in a cell disruption instrument for further homogenizing and shearing. The homogenization shearing operation is performed under ice water bath conditions to ensure stability of the DHA. The power of the crusher is 480w, the working time is 7min, the probe type is 12, the sample processing amount is 12mL, and the working interval time is set to be 1s. The working stage of the crusher is to pay attention to the temperature of the outer ice water bath, and if the temperature is higher than 40 ℃, the ice water bath is replaced.
Cooling and standing: and standing the emulsified carrier emulsion at 25 ℃ in a water bath condition, and cooling to room temperature.
And (3) a finished product: the prepared carrier is uniform and stable, has certain viscosity, and the emulsion is milky and contains a certain DHA smell, and has little or no foam.
Example 2
Grinding: the rice bran is milled by a mill, so that the particles are smaller and the particle size is uniform.
Sieving: sieving the milled rice bran with 60 mesh sieve (pore diameter of 0.15 mm) to remove impurities and broken rice in the rice bran.
Soaking: phosphate buffer (phosphate buffered saline, PBS, ph=7.0) is added to the sieved rice bran according to a feed-liquid ratio of 1:10, the pH stability of the components in the rice bran system is maintained, and the rice bran is soaked overnight after being uniformly mixed.
Pulping: pouring the rice bran mixed liquor into a beater for beating, and destroying cell walls and fiber layers of rice bran to release endogenous substances. The power of the beater was set at 1500w and the working time was 1.5min.
Homogenizing: transferring the rice bran solution after pulping into a 500mL beaker, homogenizing the rice bran solution by using a homogenizer, further destroying plant tissues of the rice bran, and fully releasing endogenous components. The working speed of the homogenizer is 3500rpm, and the working time is 5min.
And (3) suction filtration: and (3) carrying out suction filtration on the homogenized rice bran solution by using a suction filter, wherein the specification of qualitative filter paper is 12.5cm, and collecting the lower filtrate for later use.
Enzymolysis: transferring the collected filtrate into a 500mL beaker, adjusting the pH of the component to 4, adding cellulase and pectase (the mass ratio of the cellulase to the pectase is 1:1, and the sum of the masses of the cellulase and the pectase is 1% of the mass of the filtrate) into the beaker, and carrying out enzymolysis for 2 hours at the enzymolysis temperature of 40 ℃.
And (3) centrifuging: centrifuging the rice bran solution subjected to enzymolysis by using a refrigerated centrifuge at a centrifugal temperature of 4 ℃ for 30min with a centrifugal power of 10000rpm. And collecting cream paste substances on the upper layer of the centrifuge tube after centrifugation.
Washing: PBS (containing 0.1% medium chain triglyceride, 0.6mol/L glucose, 0.5mol/L potassium chloride, pH=7.0) is added to the collected cream-like substance according to a feed liquid ratio of 1:1, and the mixture is washed thoroughly. After thatAnd (5) continuing to freeze and centrifuge, and collecting cream paste substances on the upper layer of the centrifuge tube. The centrifugal washing step was repeated 3 times, after which the cream-like substance after washing was collected, to which 0.1% NaN was added 3 Preventing bacteria contamination.
And (3) freeze-drying: freeze-drying the cream paste in a freeze dryer at-60deg.C for 3 days. And collecting oily viscous solid particles after freeze-drying, namely rice bran oil base.
Oil phase dissolution: fish oil (containing 12% DHA) was dissolved in n-hexane and mixed well to form an oil phase.
Preparation of colostrum: dissolving rice bran oil base with water, diluting into 2% solution, mixing in high-speed disperser with power of 3500rpm/min and working time of 5min to form water phase. Placing the water phase in an ultrasonic extractor, slowly adding the DHA-containing oil phase (the water-oil mass ratio is 90:10) dropwise into the water phase, and uniformly mixing under the ultrasonic treatment condition to form the colostrum.
Rotary steaming: transferring the prepared colostrum into a rotary evaporator evaporation bottle, removing n-hexane in the system through rotary evaporation operation, and further concentrating the carrier emulsion. The spin-steaming temperature was 60℃and the rotational speed was 100rpm.
Homogenizing: placing the concentrated carrier in a cell disruption instrument for further homogenizing and shearing. The homogenization shearing operation is performed under ice water bath conditions to ensure stability of the DHA. The power of the crusher is 240w, the working time is 5min, the probe type is 12, the sample processing amount is 12mL, and the working interval time is set to be 1s. The working stage of the crusher is to pay attention to the temperature of the outer ice water bath, and if the temperature is higher than 40 ℃, the ice water bath is replaced.
Cooling and standing: and standing the emulsified carrier emulsion at 25 ℃ in a water bath condition, and cooling to room temperature.
And (3) a finished product: the prepared carrier is uniform and stable, has certain viscosity, and the emulsion is milky and contains a certain DHA smell, and has little or no foam.
Example 3
Grinding: the rice bran is milled by a mill, so that the particles are smaller and the particle size is uniform.
Sieving: sieving the milled rice bran with 60 mesh sieve (pore diameter of 0.15 mm) to remove impurities and broken rice in the rice bran.
Soaking: phosphate buffer (phosphate buffered saline, PBS, ph=7.0) is added to the sieved rice bran according to a feed-liquid ratio of 1:10, the pH stability of the components in the rice bran system is maintained, and the rice bran is soaked overnight after being uniformly mixed.
Pulping: pouring the rice bran mixed liquor into a beater for beating, and destroying cell walls and fiber layers of rice bran to release endogenous substances. The power of the beater was set at 1500w and the working time was 1.5min.
Homogenizing: transferring the rice bran solution after pulping into a 500mL beaker, homogenizing the rice bran solution by using a homogenizer, further destroying plant tissues of the rice bran, and fully releasing endogenous components. The working speed of the homogenizer was 10500rpm, and the working time was 5min.
And (3) suction filtration: and (3) carrying out suction filtration on the homogenized rice bran solution by using a suction filter, wherein the specification of qualitative filter paper is 12.5cm, and collecting the lower filtrate for later use.
Enzymolysis: transferring the collected filtrate into a 500mL beaker, adjusting the pH of the component to 4, adding cellulase and pectase (the mass ratio of the cellulase to the pectase is 1:1, and the sum of the masses of the cellulase and the pectase is 3% of the mass of the filtrate) into the beaker, and carrying out enzymolysis for 6 hours at the enzymolysis temperature of 60 ℃.
And (3) centrifuging: centrifuging the rice bran solution subjected to enzymolysis by using a refrigerated centrifuge at a centrifugal temperature of 4 ℃ for 30min with a centrifugal power of 10000rpm. And collecting cream paste substances on the upper layer of the centrifuge tube after centrifugation.
Washing: PBS (containing 0.1% medium chain triglyceride, 0.6mol/L glucose, 0.5mol/L potassium chloride, pH=7.0) is added to the collected cream-like substance according to a feed liquid ratio of 1:1, and the mixture is washed thoroughly. And then, continuing to freeze and centrifuge, and collecting cream paste substances on the upper layer of the centrifuge tube. The centrifugal washing step was repeated 3 times, after which the cream-like substance after washing was collected, to which 0.1% NaN was added 3 Preventing bacteria contamination.
And (3) freeze-drying: freeze-drying the cream paste in a freeze dryer at-40deg.C for 3 days. And collecting oily viscous solid particles after freeze-drying, namely rice bran oil base.
Oil phase dissolution: fish oil (containing 12% DHA) was dissolved in n-hexane and mixed well to form an oil phase.
Preparation of colostrum: dissolving rice bran oil base with water, diluting into 2% solution, mixing in high-speed disperser with power of 3500rpm/min and working time of 5min to form water phase. Placing the water phase in an ultrasonic extractor, slowly adding the DHA-containing oil phase (the water-oil mass ratio is 95:5) dropwise into the water phase, and uniformly mixing under the ultrasonic treatment condition to form the colostrum.
Rotary steaming: transferring the prepared colostrum into a rotary evaporator evaporation bottle, removing n-hexane in the system through rotary evaporation operation, and further concentrating the carrier emulsion. The spin-steaming temperature was 60℃and the rotational speed was 100rpm.
Homogenizing: placing the concentrated carrier in a cell disruption instrument for further homogenizing and shearing. The homogenization shearing operation is performed under ice water bath conditions to ensure stability of the DHA. The power of the crusher is 600w, the working time is 10min, the probe model is 12, the sample processing amount is 12mL, and the working interval time is set to be 1s. The working stage of the crusher is to pay attention to the temperature of the outer ice water bath, and if the temperature is higher than 40 ℃, the ice water bath is replaced.
Cooling and standing: and standing the emulsified carrier emulsion at 25 ℃ in a water bath condition, and cooling to room temperature.
And (3) a finished product: the prepared carrier is uniform and stable, has certain viscosity, and the emulsion is milky and contains a certain DHA smell, and has little or no foam.
Example 4
Grinding: the rice bran is milled by a mill, so that the particles are smaller and the particle size is uniform.
Sieving: sieving the milled rice bran with 60 mesh sieve (pore diameter of 0.15 mm) to remove impurities and broken rice in the rice bran.
Soaking: phosphate buffer (phosphate buffered saline, PBS, ph=7.0) is added to the sieved rice bran according to a feed-liquid ratio of 1:10, the pH stability of the components in the rice bran system is maintained, and the rice bran is soaked overnight after being uniformly mixed.
Pulping: pouring the rice bran mixed liquor into a beater for beating, and destroying cell walls and fiber layers of rice bran to release endogenous substances. The power of the beater was set at 1500w and the working time was 1.5min.
Homogenizing: transferring the rice bran solution after pulping into a 500mL beaker, homogenizing the rice bran solution by using a homogenizer, further destroying plant tissues of the rice bran, and fully releasing endogenous components. The working speed of the homogenizer is 5000rpm, and the working time is 5min.
And (3) suction filtration: and (3) carrying out suction filtration on the homogenized rice bran solution by using a suction filter, wherein the specification of qualitative filter paper is 12.5cm, and collecting the lower filtrate for later use.
Enzymolysis: transferring the collected filtrate into a 500mL beaker, adjusting the pH of the components to 4, and adding cellulase and pectase (the mass ratio of the cellulase to the pectase is 1:1, the sum of the masses of the cellulase and the pectase is 2% of the mass of the filtrate) into the beaker, wherein the enzymolysis time is 2-6 h, preferably 4h, and the enzymolysis temperature is 50 ℃.
And (3) centrifuging: centrifuging the rice bran solution subjected to enzymolysis by using a refrigerated centrifuge at a centrifugal temperature of 4 ℃ for 30min with a centrifugal power of 10000rpm. And collecting cream paste substances on the upper layer of the centrifuge tube after centrifugation.
Washing: PBS (containing 0.1% medium chain triglyceride, 0.6mol/L glucose, 0.5mol/L potassium chloride, pH=7.0) is added to the collected cream-like substance according to a feed liquid ratio of 1:1, and the mixture is washed thoroughly. And then, continuing to freeze and centrifuge, and collecting cream paste substances on the upper layer of the centrifuge tube. The centrifugal washing step was repeated 3 times, after which the cream-like substance after washing was collected, to which 0.1% NaN was added 3 Preventing bacteria contamination.
And (3) freeze-drying: freeze-drying the cream paste in a freeze dryer at-40deg.C for 3 days. And collecting oily viscous solid particles after freeze-drying, namely rice bran oil base.
Oil phase dissolution: fish oil (containing 12% DHA) was dissolved in n-hexane and mixed well to form an oil phase.
Preparation of colostrum: dissolving rice bran oil base with water, diluting into 2% solution, mixing in high-speed disperser with power of 3500rpm/min and working time of 5min to form water phase. Placing the water phase in an ultrasonic extractor, slowly adding the DHA-containing oil phase (the water-oil mass ratio is 90:10) dropwise into the water phase, and uniformly mixing under the ultrasonic treatment condition to form the colostrum.
Rotary steaming: transferring the prepared colostrum into a rotary evaporator evaporation bottle, removing n-hexane in the system through rotary evaporation operation, and further concentrating the carrier emulsion. The spin-steaming temperature was 60℃and the rotational speed was 100rpm.
Homogenizing: placing the concentrated carrier in a cell disruption instrument for further homogenizing and shearing. The homogenization shearing operation is performed under ice water bath conditions to ensure stability of the DHA. The power of the crusher is 500w, the working time is preferably 8min, the probe model is 12, the sample processing amount is more than 12mL, and the working interval time is set to be 1s. The working stage of the crusher is to pay attention to the temperature of the outer ice water bath, and if the temperature is higher than 40 ℃, the ice water bath is replaced.
Cooling and standing: and standing the emulsified carrier emulsion at 25 ℃ in a water bath condition, and cooling to room temperature.
And (3) a finished product: the prepared carrier is uniform and stable, has certain viscosity, and the emulsion is milky and contains a certain DHA smell, and has little or no foam.
Taking example 1 as an example for illustrating the beneficial effects of the DHA-containing rice bran-based carrier prepared by the invention, the DHA-containing rice bran-based carrier prepared in example 1 is subjected to the following operations:
1. the particle size of the support was characterized using a malvern laser particle sizer and the results are shown in figure 2.
As can be seen from FIG. 1, the particle size distribution of the DHA-containing rice bran-based carrier has only one single peak, about 300nm, and no other peak appears, which indicates that the particle size of the prepared DHA-containing rice bran-based carrier is smaller and the DHA-containing rice bran-based carrier is uniformly distributed in an oil-water system.
2. The morphology of the carrier was observed by an optical microscope, and the results are shown in FIG. 3.
As shown in FIG. 3, the DHA-containing rice bran-based carrier emulsion was stable and uniform and milky. Under an optical microscope, the rice bran-based carrier containing DHA is round, and the droplet sizes in the system are basically consistent, which shows that the prepared rice bran-based carrier containing DHA is uniformly distributed in the emulsion, and the particle size is consistent with the particle size result.
3. The stability of the carrier was analyzed by a stability analyzer, and the results are shown in fig. 4.
The stability of the DHA-containing rice bran-based carrier is expressed in terms of the Turbocan stability index (Turbiscan stability index, TSI), with a higher TSI value representing a more unstable carrier emulsion system. In fig. 4, the change of TSI value of the DHA-containing rice bran-based carrier after three hours of standing at room temperature is shown, and it can be seen that the TSI value of the DHA-containing rice bran-based carrier emulsion after three hours of standing is 1.98, which indicates that the DHA-containing rice bran-based carrier has good stability.
4. The color difference of the emulsion was measured by using a color difference meter, and the result is shown in FIG. 5.
The measurement result of the color difference meter is represented by L, a and b values, and the measurement result represents the chromaticity value of the color of the object, namely the color space coordinate of the color. The L value represents darkness (black and white), the a value represents red-green, and the b value represents yellow-blue. Wherein L is a positive value, indicating that the sample is brighter than the standard plate; if the value is negative, the description is darker; the value a indicates that the template is redder than normal if it shows a positive value and that it indicates that it is greener if it shows a negative value; the b value is positive, indicating that the template is more yellow than normal, and negative, indicating that it is blue. As can be seen from fig. 5, the prepared DHA-containing rice bran-based carrier has higher brightness, and the color tends to be green and blue compared to the standard test conditions of the color difference meter. Since fish oil is yellow, the results indicate that DHA has been successfully loaded into the emulsion; the prepared rice bran-based carrier containing DHA is white and has better organoleptic properties.
5. The newly prepared DHA-containing rice bran-based vector was diluted to different concentrations and added to HepG-2 liver cancer cells in the logarithmic phase, and the effect of DHA-containing rice bran-based vector on cytotoxicity was observed, and the results are shown in FIG. 6.
As shown in FIG. 6, the survival rate of HepG-2 cells showed a decrease trend with increasing concentration of the DHA-containing rice bran-based vector. Omega-3 fatty acids and derivatives thereof stimulate tumor cells, induce oxidative stress in tumor cells, and induce apoptosis. The conclusion shows that the DHA is embedded by taking rice bran oil base as a carrier, so that the anticancer effect of the DHA can be fully exerted, and the bioavailability of the DHA is improved.
In conclusion, the rice bran-based carrier containing DHA prepared by the invention improves the bioavailability of DHA. The natural components in rice bran are used for replacing the traditional industrial emulsifier as an emulsion stabilizer, so that carcinogenicity and anaphylactic reaction possibly brought by the industrial emulsifier can be avoided. The part of oily solid particles in rice bran can be adsorbed and accumulated on the interface between two non-phase solutions (generally expressed by oil phase and water phase) to stabilize liquid drops and prevent the liquid drops from coalescing, and the emulsion has more stable performance and lower toxicity, so that the emulsion is safer in vivo, the sustained and controlled release effect on the embedded nutrient is achieved, and the bioavailability of the embedded substance is improved. The natural components in the rice bran are used as the stabilizer of the carrier, so that the use of the synthetic emulsifier is avoided, and the application range of rice bran resources is widened. The preparation process is not complicated, the organic solvent residue is less, and the preparation method is natural and environment-friendly. Develops a nanoscale DHA-type carrier, and provides basic basis for application of DHA fish oil in the field of health-care foods and research on DHA absorption mechanism.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for preparing a rice bran-based carrier containing DHA, which is characterized by comprising the following steps:
s10, grinding and sieving rice bran, and soaking to obtain rice bran mixed liquor;
s20, pulping and homogenizing the rice bran mixed solution, and performing suction filtration to obtain filtrate;
s30, adding cellulase and pectase into the filtrate for enzymolysis, and centrifuging to obtain an upper cream pasty substance;
s40, washing and freeze-drying the upper cream paste substance to obtain rice bran oil base;
s50, dissolving the rice bran oil base in water to form a water phase, dissolving the fish oil containing DHA in the oil to form an oil phase, and uniformly mixing the water phase and the oil phase to obtain colostrum;
s60, performing rotary steaming, homogenizing and standing on the colostrum to obtain the DHA-containing rice bran-based carrier.
2. The method for preparing a DHA-containing rice bran-based carrier according to claim 1, wherein step S10 comprises:
grinding and sieving rice bran, and adding phosphate buffer solution for soaking to obtain rice bran mixed solution.
3. The method for preparing a DHA-based carrier according to claim 1, wherein the homogenizing speed is 3500-10500 rpm in step S20.
4. The method of claim 1, wherein in step S30, the sum of the masses of the cellulase and pectase is 1% to 3% of the mass of the filtrate.
5. The method for preparing a DHA-based carrier according to claim 1, wherein the enzymolysis time is 2-6 h in the step S30.
6. The method for preparing a DHA-based carrier according to claim 1, wherein the enzymolysis temperature is 40-60 ℃ in step S30.
7. The method of preparing a DHA-containing rice bran-based carrier according to claim 1, wherein step S40 comprises:
s41, washing the upper-layer cream paste substance with a washing liquid, and centrifugally collecting the upper-layer substance, wherein the washing liquid comprises medium-chain triglyceride, glucose and potassium chloride;
s42, freeze-drying the upper layer substance to obtain rice bran oil base.
8. The method of claim 1, wherein in step S40, the lyophilization temperature is-60 ℃ to-40 ℃.
9. The method of preparing a DHA-containing rice bran-based carrier according to claim 1, wherein the mass ratio of the aqueous phase to the oil phase in step S50 is 90:10-95:5.
10. The method for preparing a DHA-based carrier according to claim 1, wherein, in the step S60,
the homogeneous power is 240-600 w; and/or the number of the groups of groups,
the homogenizing time is 5-10 min.
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