Preparation method of astaxanthin nanoemulsion

Abstract

The invention discloses a preparation method of astaxanthin nanoemulsion, and belongs to the field of astaxanthin, the preparation method comprises the following steps: S1, heating 120-150 parts by mass of haematococcus pluvialis oil to 28-30 DEG C, keeping the temperature, and continuously stirring for later use to obtain a component A; s2, dissolving 4-7 parts by mass of a polygonatum odoratum extract and 3-5 parts by mass of a black pepper extract in 5-20 parts by mass of purified water, and heating to 28-30 DEG C to obtain a component B; s3, emulsification treatment: mixing the component A and the component B according to a mass ratio of 1: (1-2), adding a compound emulsifier which is 1-3 times of the mass of the mixture, uniformly stirring, treating by using a high-speed dispersion homogenizer, and then treating by using an ultrasonic cell disruptor; when a high-speed dispersion homogenizer is used for treatment, high-speed shearing dispersion is carried out for 9-15 min at the speed of 10000 r / min to 12000 r / min; when an ultrasonic cell disruptor is used for treatment, ultrasonic dispersion is carried out for 2 min at the power of 100 W, then ultrasonic dispersion is carried out for 4 min at the power of 200 W, the mode of working for 1 s and intermittent for 1 s is selected, and the astaxanthin nanoemulsion is obtained. The nano-emulsion prepared by the invention is good in stability and high in bioavailability.

Description

Technical Field

[0001] The present invention belongs to the field of astaxanthin, and specifically relates to a preparation method of astaxanthin nanoemulsion. Background Art

[0002] Extracts of Haematococcus pluvialis are the main source of natural astaxanthin. Astaxanthin has extremely strong antioxidant properties and good coloring ability, and has extensive applications in the fields of food, aquaculture, cosmetics, and medicine. However, due to properties such as the low water solubility and chemical instability of astaxanthin, common astaxanthin products on the market are mostly oil-based. In order to facilitate carrying, consumption, and protection, astaxanthin oil is often encapsulated into capsules, resulting in a lack of rich dosage forms of astaxanthin products on the market and making it difficult to meet the needs of diversified applications. Summary of the Invention

[0003] In order to overcome the problems existing in the prior art, the present invention proposes a preparation method of astaxanthin nanoemulsion. The nanoemulsion prepared by the present invention has advantages such as strong stability and high bioavailability.

[0004] To achieve the above object, the first aspect of the present invention provides a preparation method of astaxanthin nanoemulsion, comprising the following steps: S1, heating 120 - 150 parts by mass of Haematococcus pluvialis oil containing 5% - 15% astaxanthin to 28 - 30 °C, keeping warm and continuously stirring for standby to obtain component A; S2, dissolving 4 - 7 parts by mass of Polygonatum odoratum extract and 3 - 5 parts by mass of Piper nigrum extract in 5 - 20 parts by mass of purified water, and heating to 28 - 30 °C to obtain component B; S3, emulsification treatment: after mixing component A and component B in a mass ratio of 1:1 - 2, adding a compound emulsifier 1 - 3 times the mass of the mixture, stirring evenly, first using a high-speed dispersion homogenizer for treatment, and then using an ultrasonic cell disrupter for treatment; when using the high-speed dispersion homogenizer for treatment: under 10000 r / min - 12000 r / min, high-speed shear dispersion for 9 - 15 min; when using the ultrasonic cell disrupter for treatment: at a power of 100 W, ultrasonic dispersion for 2 min, and then at a power of 200 W, ultrasonic dispersion for 4 min, selecting a mode of working for 1 s and intermittent for 1 s, then the astaxanthin nanoemulsion is obtained.

[0005] Further, the compound emulsifier in step S3 comprises Tween - 80, sodium caseinate, and pea protein in a mass ratio of 3 - 5:1 - 2:1 - 2. During emulsification treatment, first add Tween - 80, stir evenly and then perform high-speed shear dispersion for 1 / 3 of the time; then add sodium caseinate, stir evenly and then perform high-speed shear dispersion for 1 / 3 of the time; finally add pea protein, stir evenly and then perform high-speed shear dispersion for 1 / 3 of the time.

[0006] The second aspect of the present invention provides a nanoemulsion prepared by the preparation method provided in the first aspect.

[0007] The third aspect of the present invention provides the use of the nanoemulsion described in the second aspect in the preparation of gel candies, soft capsules, and beverages.

[0008] Through the above technical solutions, the present invention can at least achieve the following beneficial effects: The nanoemulsion prepared by the present invention has good stability and high bioavailability. Detailed implementation manners

[0009] Unless otherwise specified, the materials and reagents used in the present invention are commercially available.

[0010] In the present invention: The Haematococcus pluvialis oil containing 5%-15% astaxanthin is extracted by our company (Yunnan Green A Bio-Engineering Co., Ltd.) itself.

[0011] The pea protein is purchased from Xi'an Shizeyuan Biotechnology Co., Ltd., and the effective substance content is 80%; Sodium caseinate CAS number: 9005-46-3, the effective ingredient content is 98%, purchased from Hubei Furunde Food Ingredients Co., Ltd. The polygonatum odoratum extract and black pepper extract are purchased from Shanyang Lianfeng Biotechnology Co., Ltd. (both are water extracts, and the specifications are both 20:1); Glycerol is purchased from Jiangxi Yipsen Pharmaceutical Co., Ltd.; Purified water is prepared by our company (Yunnan Green A Bio-Engineering Co., Ltd.) itself. Example 1

[0012] A preparation method of astaxanthin nanoemulsion comprises the following steps: S1, heating 130 parts by mass of the Haematococcus pluvialis oil containing 10% astaxanthin to 29 °C, keeping warm and continuously stirring for standby to obtain component A; S2, dissolving 5 parts by mass of the polygonatum odoratum extract and 4 parts by mass of the black pepper extract in 13 parts by mass of purified water, and heating to 29 °C to obtain component B; S3, emulsification treatment: after mixing component A and component B according to a mass ratio of 1:1.5, adding a compound emulsifier twice the mass of the mixture, the compound emulsifier contains Tween-80, sodium caseinate and pea protein with a mass ratio of 4:1.5:1.5, stirring evenly, first using a high-speed dispersion homogenizer for treatment, and then using an ultrasonic cell crusher for treatment; The steps of treatment with a high-speed dispersion homogenizer are as follows: When using a high-speed dispersion homogenizer for treatment, control the temperature of the mixed solution below 35 °C. The specific addition method of the composite emulsifier is as follows: First, add Tween-80 and stir evenly. Then, use a high-speed dispersion homogenizer to perform high-speed shear dispersion at 11000 r / min for 4 min. Next, add sodium caseinate, stir evenly, and then use a high-speed dispersion homogenizer to perform high-speed shear dispersion at 11000 r / min for 4 min. Finally, add pea protein, stir evenly, and then use a high-speed dispersion homogenizer to perform high-speed shear dispersion at 11000 r / min for 4 min. When using an ultrasonic cell disruptor for treatment: Disperse ultrasonically at a power of 100 W for 2 min, and then disperse ultrasonically at a power of 200 W for 4 min. Select the mode of working for 1 s and intermittent for 1 s to obtain astaxanthin nanoemulsion. Example 2

[0013] A preparation method of astaxanthin nanoemulsion comprises the following steps: S1. Heat 120 parts by mass of Haematococcus pluvialis oil containing 5% astaxanthin to 28 °C, keep it warm and stir continuously for standby to obtain component A; S2. Dissolve 4 parts by mass of Polygonatum odoratum extract and 3 parts by mass of Piper nigrum extract in 5 parts by mass of purified water, and heat to 28 °C to obtain component B; S3. Emulsification treatment: After mixing component A and component B in a mass ratio of 1:1, add a composite emulsifier that is 1 time the mass of the mixture. The composite emulsifier contains Tween-80, sodium caseinate, and pea protein in a mass ratio of 3:1:1. After stirring evenly, first use a high-speed dispersion homogenizer for treatment, and then use an ultrasonic cell disruptor for treatment; The steps of treatment with a high-speed dispersion homogenizer are as follows: When using a high-speed dispersion homogenizer for treatment, control the temperature of the mixed solution below 35 °C. The specific addition method of the composite emulsifier is as follows: First, add Tween-80 and stir evenly. Then, use a high-speed dispersion homogenizer to perform high-speed shear dispersion at 10000 r / min for 3 min. Next, add sodium caseinate, stir evenly, and then use a high-speed dispersion homogenizer to perform high-speed shear dispersion at 10000 r / min for 3 min. Finally, add pea protein, stir evenly, and then use a high-speed dispersion homogenizer to perform high-speed shear dispersion at 10000 r / min for 3 min. When using an ultrasonic cell disruptor for treatment: Disperse ultrasonically at a power of 100 W for 2 min, and then disperse ultrasonically at a power of 200 W for 4 min. Select the mode of working for 1 s and intermittent for 1 s to obtain astaxanthin nanoemulsion. Example 3

[0014] A preparation method of astaxanthin nanoemulsion comprises the following steps: S1. Heat 150 parts by mass of Haematococcus pluvialis oil containing 15% astaxanthin to 30 °C, keep it warm and stir continuously for standby to obtain Component A; S2. Dissolve 7 parts by mass of Polygonatum odoratum extract and 5 parts by mass of black pepper extract in 20 parts by mass of purified water, and heat to 30 °C to obtain Component B; S3. Emulsification treatment: After mixing Component A and Component B in a mass ratio of 1:2, add a compound emulsifier that is 3 times the mass of the mixture. The compound emulsifier contains Tween-80, sodium caseinate, and pea protein in a mass ratio of 5:2:2. After stirring evenly, first use a high-speed dispersion homogenizer to process, and then use an ultrasonic cell disrupter to process; The steps of the high-speed dispersion homogenizer treatment are as follows: When using the high-speed dispersion homogenizer for treatment, control the temperature of the mixed solution below 35 °C. The specific addition method of the compound emulsifier is as follows: First add Tween-80, stir evenly, and then use a high-speed dispersion homogenizer to perform high-speed shear dispersion at 12000 r / min for 5 min; then add sodium caseinate, stir evenly, and use a high-speed dispersion homogenizer to perform high-speed shear dispersion at 12000 r / min for 5 min; finally, add pea protein and stir evenly, and then use a high-speed dispersion homogenizer to perform high-speed shear dispersion at 12000 r / min for 5 min; When using the ultrasonic cell disrupter for treatment: Disperse ultrasonically at a power of 100 W for 2 min, and then disperse ultrasonically at a power of 200 W for 4 min. Select the mode of working for 1 s and intermittent for 1 s to obtain astaxanthin nanoemulsion.

[0015] Example 4 Preparation of Astaxanthin Gel Candies Prepare astaxanthin gel candies according to the formula recorded in Table 1 below: Table 1 Astaxanthin Gel Candy Formula Table

[0016] Among them, linseed oil is purchased from Heze Zhonghe Jianyuan Biotechnology Co., Ltd., γ-aminobutyric acid is purchased from Bloomage Biotechnology Co., Ltd., beeswax is purchased from Cangzhou Dongfang Beeswax Glue Industry Co., Ltd., gelatin is purchased from Kunming Sigui Economic and Trade Co., Ltd., and glycerol is purchased from Jiangxi Yipsen Pharmaceutical Co., Ltd.

[0017] The specific preparation method is as follows: Content preparation 1.1 Add the weighed linseed oil (take 60 wt%) and beeswax to the batching tank, stir and heat to melt (temperature does not exceed 60 °C). Cool to about 30 °C, and then add the remaining linseed oil, astaxanthin nanoemulsion, and γ-aminobutyric acid in sequence, and stir until basically evenly dispersed.

[0018] 1.2 Pass the obtained suspension through a colloid mill (7 - 10 microns) three times, and then transfer it to a batching tank for vacuum degassing to obtain a uniform, dark red - black viscous oily suspension liquid, which is the content.

[0019] Sol 2.1 Add pure water to the sol tank and heat it to 80°C. Add glycerol, mix well, then heat to 80°C again. Open the vacuum pump, evacuate the air, start stirring at 140 - 150 rpm, and suck gelatin into the sol tank through the feed pipe. After completion, close the feed valve and the vacuum pump, and continue stirring for 20 minutes. Open the vacuum pump for vacuum degassing and continue stirring (140 - 150 rpm). When the temperature drops below 60°C, stop the vacuum.

[0020] 2.2 Conduct vacuum degassing. During vacuum degassing, observe the state and weight of the glue solution at any time. The weight loss caused by the evaporation of water in the glue solution should be controlled within the range of 5 - 7% of the total input amount. When there are only fine bubbles churning on the liquid surface and the bubbles are like many small bright spots, it indicates that the degassing is complete.

[0021] 2.3 Stop the vacuum pump, break the vacuum. After sampling, observing and measuring that the viscosity is qualified (range: 8000 - 12000 cps), keep it warm at 60 - 65°C for standby to obtain the capsule wall material.

[0022] 3. Capsule making Make capsules from the content and the capsule wall material through a capsule making machine. The prepared capsules weigh about 500 mg each and contain about 12 mg of astaxanthin per capsule.

[0023] Example 5 Preparation of astaxanthin beverage Prepare an astaxanthin beverage according to the formula recorded in Table 2 below: Table 2 Astaxanthin beverage formula table

[0024] Among them, fish collagen peptide is purchased from Dezhou Lanli Biotechnology Co., Ltd., fructooligosaccharide is purchased from Quantum Hi - Tech (Guangdong) Biotechnology Co., Ltd., passion fruit concentrated juice is purchased from Gansu Yishengxiang Biotechnology Co., Ltd., white tomato extract, green tea extract, citric acid, sucralose, potassium sorbate are purchased from Guangzhou Jingfang Medical Technology Development Co., Ltd., sodium hyaluronate is purchased from Bloomage Biotechnology Co., Ltd., arabic gum is purchased from Suzhou Purecare Food Co., Ltd., and DL - malic acid is purchased from Anhui Xuelang Biotechnology Co., Ltd.

[0025] The specific preparation method is as follows: Weighing: Weigh each material according to the proportion in the formula table.

[0026] Mixing: Add water (purified water) accounting for 60 wt% of the total water amount into the batching tank and heat it to 65°C ± 5°C. Add passion fruit concentrated juice and fructooligosaccharide into the batching tank, start stirring until the added materials are completely dissolved, and the stirring time is 2 - 5 min; First, mix fish collagen, arabic gum, citric acid, DL-malic acid, sucralose, green tea extract, sodium hyaluronate, white tomato extract, and potassium sorbate evenly, dissolve them completely with the remaining 35 wt% of water, and add them into the batching tank. Dissolve the astaxanthin nano-aqueous solution completely with the remaining water, add it into the batching tank, and the stirring time is 1 min - 2 min. After the stirring ends, the mixing is completed.

[0027] Quantitative measurement: Take a small amount of the liquid material, cool it to room temperature (20 - 25°C) for measurement. Control Brix: 9.8 ± 1; pH value: 4.08 ± 0.2.

[0028] Filling: After the mixing is completed and the physical and chemical indexes of the liquid material are detected to be qualified, transfer the liquid material into the hopper, adjust the filling volume of the filling machine, with a filling volume of 50 ml, and the control range is 50 - 54 ml.

[0029] Capping: Seal with a capping machine.

[0030] Sterilization: After the filling is completed, neatly stack the sealed beverages in the turnover baskets (stainless steel), place them in a water bath, and sterilize them at a constant temperature of 85°C for 40 min.

[0031] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that it does not contain polygonatum extract.

[0032] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that it does not contain black pepper extract.

[0033] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that it does not contain black pepper extract and polygonatum extract.

[0034] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that sodium caseinate is replaced with Tween - 80.

[0035] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that pea protein is replaced with Tween - 80.

[0036] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that sodium caseinate and pea protein are replaced with Tween - 80.

[0037] Stability test Test protocol design: According to the Guidelines for Stability Testing of Drug Substances and Preparations (Part IV of Chinese Pharmacopoeia 2020 Edition, 9001), 100 g of the astaxanthin oil as the raw material of the soft capsules of the present invention and the nanoemulsions prepared in Examples 1 to 3 and Comparative Examples 1 to 6 were taken respectively. They were placed in clean brown glass bottles and stored for 6 months under the conditions of temperature 40°C ± 2°C and relative humidity 75% ± 5%. Samples were taken at 0 month, 1 month, 2 months, 3 months, and 6 months respectively. The test results are shown in Table 3 below: Table 3 Results of Product Stability Investigation

[0038] As can be seen from Table 1 above, after six months of accelerated test, the attenuation rate of the astaxanthin raw material was 37.20%; the attenuation rate of astaxanthin in the nanoemulsion of Example 1 was 4.09%, the attenuation rate of astaxanthin in the nanoemulsion of Example 2 was 4.85%, and the attenuation rate of astaxanthin in the nanoemulsion of Example 3 was 3.64%; the attenuation rate of astaxanthin in the nanoemulsion of Comparative Example 1 was 7.53%, the attenuation rate of astaxanthin in the nanoemulsion of Comparative Example 2 was 9.06%, and the attenuation rate of astaxanthin in the nanoemulsion of Comparative Example 3 was 14.43%; the attenuation rate of astaxanthin in the nanoemulsion of Comparative Example 4 was 15.61%, the attenuation rate of astaxanthin in the nanoemulsion of Comparative Example 5 was 8.18%, and the attenuation rate of astaxanthin in the nanoemulsion of Comparative Example 6 was 18.96%. Thus, it can be seen that the nanoemulsion prepared by the present invention has high stability.

[0039] Detection of Average Particle Size of Nanoemulsion The products prepared in Examples 1 to 3 and Comparative Examples 1 to 6 were diluted 100 times with distilled water and then filled into the sample cell. The average particle size of the nanoemulsion prepared in each example and comparative example was measured using a nano particle size and zeta potential analyzer. The results are statistically shown in Table 4 below: Table 4 Statistical Table of Average Particle Size of Products Diluted 100 Times

[0040] As can be seen from Table 2 above, in the comparative examples without adding sodium caseinate, pea protein, or neither of them, the average particle size of the emulsion was significantly higher than that of Example 1.

[0041] Absorption and Utilization Test SPF-grade male Wistar rats were used. After 1 week of adaptive feeding, they were randomly divided into 10 groups: astaxanthin raw material group, Examples 1 to 3, and Comparative Examples 1 to 6, with 30 rats in each group. According to the equivalent conversion of the daily dietary intake of adults (60 kg) (astaxanthin 12 mg / d) into the rat dose, the astaxanthin raw material group (astaxanthin content 10%), the products of Examples 1 to 3, and the products of Comparative Examples 1 to 6 were administered by gavage. 12 h before the experiment, the rats were fasted but not water-deprived. At 0, 0.5, 1, 2, 4, 8, 10, 12, 16, and 24 h after gavage, they were anesthetized with 3% sodium pentobarbital, sacrificed after blood collection from the abdominal aorta, and the serum was separated. The serum was quickly frozen in liquid ammonia and then placed in a -80 °C refrigerator for standby.

[0042] Determination of astaxanthin content Extract astaxanthin from biological samples, and try to perform the experiment under light avoidance as much as possible. Pretreatment of serum samples: Take an appropriate amount of serum, add 5 times the volume of chloroform-methanol solution (2:1, v / v), vortex for 1 min, let stand for 5 min, centrifuge at 8000 r / min at 4 °C for 5 min, collect the lower layer solution, repeat the extraction 3 times, combine the extracts, filter through a 0.45 μm filter membrane, and then dry under nitrogen. Re-dissolve with methanol-methyl tert-butyl ether solution (1:1, v / v) for testing.

[0043] Calculation of bioavailability According to the change curve of serum astaxanthin content, the trapezoidal area method was used to calculate the area under the curve (Area under the curve, AUC 0-t ), and the bioavailability of astaxanthin in different experimental groups was measured by the size of AUC 0-t . AUC 0-t Calculation formula: AUC 0-t =0.25A + 0.5B + 0.75C + 1.5D + 3E + 3F + 2G + 3H + 6I + 4J In the formula, A, B, C, D, E, F, G, H, I, and J represent the serum astaxanthin concentration levels at the time nodes of 0, 0.5, 1, 2, 4, 8, 10, 12, 16, and 24 h, respectively.

[0044] Table 5 Change curves of serum astaxanthin content in rats of different experimental groups and AUC 0-t value

[0045] As can be seen from Table 5 above, after preparing astaxanthin oil into nanoemulsion and applying it to the production of gel candies and beverages, the bioavailability of astaxanthin can be significantly improved in the present invention.

[0046] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in terms of form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing an astaxanthin nanoemulsion, characterized in that: The following steps are included: S1, heating 120-150 parts by weight of Haematococcus pluvialis oil containing 5%-15% astaxanthin to 28-30° C., keeping the temperature and stirring continuously for later use, to obtain component A; S2, dissolving 4-7 parts by weight of polygonatum odoratum extract and 3-5 parts by weight of black pepper extract in 5-20 parts by weight of purified water, and heating to 28-30° C. to obtain component B; S3, emulsification treatment: after mixing component A and component B in a mass ratio of 1:1-2, add a composite emulsifier in an amount of 1-3 times the mass of the mixture, stir evenly, first use a high-speed dispersing homogenizer for treatment, and then use an ultrasonic cell crusher for treatment; when using a high-speed dispersing homogenizer for treatment: at 10000r / min-12000r / min, high-speed shear dispersion for 9-15min; when using an ultrasonic cell crusher for treatment: at a power of 100W, ultrasonic dispersion for 2min, then at a power of 200W, ultrasonic dispersion for 4min, select a mode of working for 1s and resting for 1s, and astaxanthin nanoemulsion is obtained.

2. The method for preparing an astaxanthin nanoemulsion according to claim 1, characterized in that: In the step S3, the composite emulsifier comprises Tween-80, sodium caseinate and pea protein in a mass ratio of 3-5:1-2:1-2. During the emulsification process, Tween-80 is added first, stirred evenly and then dispersed by high-speed shear for 1 / 3 of the time; then sodium caseinate is added, stirred evenly and then dispersed by high-speed shear for 1 / 3 of the time; finally, pea protein is added, stirred evenly and then dispersed by high-speed shear for 1 / 3 of the time.

3. The astaxanthin nanoemulsion obtained by the preparation method of the astaxanthin nanoemulsion according to any one of claims 1 to 2.

4. Use of the astaxanthin nanoemulsion as claimed in claim 3 in preparing gel candies, soft capsules and beverages.

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