Preparation method of astaxanthin nanoemulsion

Abstract

The present invention discloses a preparation method of astaxanthin nanoemulsion, belonging to the field of astaxanthin, which comprises the following steps: S1, heating 120-150 parts by mass of Haematococcus pluvialis oil 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 black pepper 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 B according to a mass ratio of 1:1-2, adding a compound emulsifier with a mass 1-3 times that of the mixture, stirring evenly, first using a high-speed dispersion homogenizer for treatment, and then using an ultrasonic cell disruptor 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 disruptor for treatment: with a power of 100 W, ultrasonic dispersion for 2 min, and then with a power of 200 W, ultrasonic dispersion for 4 min, selecting the mode of working for 1 s and intermittent for 1 s, namely obtaining the astaxanthin nanoemulsion. The nanoemulsion prepared by the present invention has good stability and high 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 wide applications in the fields of food, aquaculture, cosmetics, and medicine. However, due to the properties of 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 mostly encapsulated into capsules, resulting in a lack of rich dosage forms of astaxanthin products on the market and being 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 provides 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:

[0005] 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;

[0006] 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;

[0007] 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 disruptor for treatment; when using the high-speed dispersion homogenizer for treatment: at 10000 r / min - 12000 r / min, high-speed shear dispersion for 9 - 15 min; when using the ultrasonic cell disruptor 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.

[0008] Further, in the step S3, the composite emulsifier comprises Tween-80, sodium caseinate and pea protein with a mass ratio of 3-5:1-2:1-2. During the emulsification treatment, first add Tween-80, stir evenly and then perform high-speed shearing and dispersion for 1 / 3 of the time; then add sodium caseinate, stir evenly and perform high-speed shearing and dispersion for 1 / 3 of the time; finally add pea protein, stir evenly and perform high-speed shearing and dispersion for 1 / 3 of the time.

[0009] The second aspect of the present invention provides the nanoemulsion prepared by the preparation method provided in the first aspect.

[0010] The third aspect of the present invention provides the application of the nanoemulsion described in the second aspect in the preparation of gel candies, soft capsules and beverages.

[0011] 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

[0012] Unless otherwise specified, the materials and reagents used in the present invention are commercially available.

[0013] In the present invention:

[0014] The Haematococcus pluvialis oil containing 5%-15% astaxanthin is extracted by our company (Yunnan Green A Biotechnology Co., Ltd.) itself.

[0015] Pea protein is purchased from Xi'an Shizeyuan Biotechnology Co., Ltd., and the effective substance content is 80%;

[0016] The CAS number of sodium caseinate is 9005-46-3, the effective ingredient content is 98%, and it is purchased from Hubei Furunde Food Raw Material Co., Ltd.

[0017] The polygonatum odoratum extract and black pepper extract are purchased from Shanyang Lianfeng Biotechnology Co., Ltd. (water extracts, and the specifications are both 20:1);

[0018] Glycerol is purchased from Jiangxi Yipusheng Pharmaceutical Co., Ltd.;

[0019] Purified water is prepared by our company (Yunnan Green A Biotechnology Co., Ltd.) itself. Example 1

[0020] A preparation method of astaxanthin nanoemulsion comprises the following steps:

[0021] S1, heat 130 parts by mass of the Haematococcus pluvialis oil containing 10% astaxanthin to 29 °C, keep warm and stir continuously for standby to obtain component A;

[0022] S2. Dissolve 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 heat to 29 °C to obtain Component B;

[0023] S3. Emulsification treatment: After mixing Component A and Component B in a mass ratio of 1:1.5, add a compound emulsifier twice the mass of the mixture. The compound emulsifier contains Tween-80, sodium caseinate, and pea protein in a mass ratio of 4:1.5:1.5. After stirring evenly, first use a high-speed dispersion homogenizer for treatment, and then use an ultrasonic cell disruptor for treatment;

[0024] The steps for treatment with the high-speed dispersion homogenizer are: 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: First add Tween-80, stir evenly, and then use the high-speed dispersion homogenizer to perform high-speed shear dispersion for 4 min under the condition of 11,000 r / min; then add sodium caseinate, stir evenly, and use the high-speed dispersion homogenizer to perform high-speed shear dispersion for 4 min under the condition of 11,000 r / min; finally, add pea protein and stir evenly, and then use the high-speed dispersion homogenizer to perform high-speed shear dispersion for 4 min under the condition of 11,000 r / min;

[0025] When using the ultrasonic cell disruptor for treatment: Perform ultrasonic dispersion for 2 min at a power of 100 W, and then perform ultrasonic dispersion for 4 min at a power of 200 W. Select the mode of working for 1 s and intermittent for 1 s, and then astaxanthin nanoemulsion is obtained. Example Two

[0026] A preparation method of astaxanthin nanoemulsion, comprising the following steps:

[0027] 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;

[0028] S2. Dissolve 4 parts by mass of the polygonatum odoratum extract and 3 parts by mass of the black pepper extract in 5 parts by mass of purified water, and heat to 28 °C to obtain Component B;

[0029] S3. Emulsification treatment: After mixing Component A and Component B in a mass ratio of 1:1, add a compound emulsifier once the mass of the mixture. The compound 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;

[0030] 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 for 3 minutes under the condition of 10,000 r / min; then add sodium caseinate, stir evenly, and use a high-speed dispersion homogenizer to perform high-speed shear dispersion for 3 minutes under the condition of 10,000 r / min; finally, add pea protein, stir evenly, and use a high-speed dispersion homogenizer to perform high-speed shear dispersion for 3 minutes under the condition of 10,000 r / min;

[0031] When using an ultrasonic cell disruptor for treatment: Disperse ultrasonically at a power of 100 W for 2 minutes, and then disperse ultrasonically at a power of 200 W for 4 minutes. Select the mode of working for 1 s and intermittent for 1 s to obtain astaxanthin nanoemulsion. Example Three

[0032] A preparation method of astaxanthin nanoemulsion comprises the following steps:

[0033] S1, Heat 150 parts by mass of Haematococcus pluvialis oil containing 15% astaxanthin to 30°C, keep warm and stir continuously for standby to obtain component A;

[0034] 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;

[0035] S3, Emulsification treatment: After mixing component A and component B according to a mass ratio of 1:2, add a composite emulsifier 3 times the mass of the mixture. The composite emulsifier contains Tween-80, sodium caseinate and pea protein with a mass ratio of 5:2:2. After stirring evenly, first use a high-speed dispersion homogenizer for treatment, and then use an ultrasonic cell disruptor for treatment;

[0036] 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 for 5 minutes under the condition of 12,000 r / min; then add sodium caseinate, stir evenly, and use a high-speed dispersion homogenizer to perform high-speed shear dispersion for 5 minutes under the condition of 12,000 r / min; finally, add pea protein, stir evenly, and use a high-speed dispersion homogenizer to perform high-speed shear dispersion for 5 minutes under the condition of 12,000 r / min;

[0037] When using an ultrasonic cell disruptor for treatment: Disperse ultrasonically at a power of 100 W for 2 minutes, and then disperse ultrasonically at a power of 200 W for 4 minutes. Select the mode of working for 1 s and intermittent for 1 s to obtain astaxanthin nanoemulsion.

[0038] Example 4 Preparation of Astaxanthin Gel Candy

[0039] Prepare astaxanthin gel candy according to the formula recorded in Table 1 below:

[0040] Table 1 Astaxanthin Gel Candy Formula Table

[0041]

[0042] Among them, linseed oil was purchased from Heze Zhonghe Jianyuan Biotechnology Co., Ltd., γ-aminobutyric acid was purchased from Bloomage Biotechnology Co., Ltd., beeswax was purchased from Cangzhou Dongfang Beeswax Glue Industry Co., Ltd., gelatin was purchased from Kunming Sigui Economic and Trade Co., Ltd., and glycerol was purchased from Jiangxi Yipsen Pharmaceutical Co., Ltd.

[0043] The specific preparation method is as follows:

[0044] Content Preparation

[0045] 1.1 Add the weighed linseed oil (take 60wt%) and beeswax into the mixing 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.

[0046] 1.2 Pass the obtained suspension through a colloid mill (7 - 10 microns) three times, and then transfer it to a mixing tank for vacuum defoaming to obtain a uniform, dark red and black viscous oily suspension liquid, that is, the content.

[0047] Sol Preparation

[0048] 2.1 Add pure water to the sol tank and heat to 80°C. Add glycerol, mix well and then heat to 80°C again. Open the vacuum pump, evacuate, stir at 140 - 150 rpm, and suck gelatin into the sol tank with a feed pipe. After completion, close the feed valve and the vacuum pump, and continue to stir for 20 minutes. Open the vacuum pump, evacuate and defoam, and continue to stir (140 - 150 rpm). When the temperature drops below 60°C, stop the vacuum.

[0049] 2.2 Evacuate and defoam. When evacuating, 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 is controlled within the range of 5 - 7% of the total feed amount. When there are only small bubbles churning on the liquid surface and the bubbles are like many small bright spots, it means that the defoaming is complete.

[0050] 2.3 Stop the vacuum pump, break the vacuum, take a sample to observe and measure the viscosity to be qualified (range: 8000 - 12000 cps), and keep it warm at 60 - 65°C for standby to obtain the capsule wall material.

[0051] 3. Capsule Making

[0052] The content and the capsule wall material are made into capsules by a capsule-making machine. The prepared capsules weigh about 500 mg each and contain about 12 mg of astaxanthin per capsule.

[0053] Example 5 Preparation of an astaxanthin beverage

[0054] Prepare an astaxanthin beverage according to the formula recorded in Table 2 below:

[0055] Table 2 Astaxanthin beverage formula table

[0056]

[0057] 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.

[0058] The specific preparation method is as follows:

[0059] Weighing: Weigh each material according to the proportion in the formula table.

[0060] Blending: Add 60 wt% of the total water volume (purified water) to the mixing tank and heat it to 65°C ± 5°C. Add passion fruit concentrated juice and fructooligosaccharide to the mixing tank and start stirring until the added materials are completely dissolved. Stirring time: 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, and dissolve them completely with the remaining 35 wt% of water, then add them to the mixing tank. Dissolve the astaxanthin nano-aqueous solution completely with the remaining water and add it to the mixing tank. Stirring time: 1 min - 2 min. After stirring ends, the blending is completed.

[0061] 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.

[0062] Filling: After the blending is completed and the physical and chemical indexes of the liquid material are detected to be qualified, transfer the liquid material to the hopper, adjust the filling volume of the filling machine to 50 ml, and the control range is 50 - 54 ml.

[0063] Sealing: Seal with a capping machine.

[0064] Sterilization: After filling is completed, the sealed beverages are neatly stacked in turnover baskets (stainless steel), placed in a water bath, and sterilized at a constant temperature of 85°C for 40 minutes.

[0065] Comparative Example 1

[0066] The difference between Comparative Example 1 and Example 1 is that it does not contain polygonatum extract.

[0067] Comparative Example 2

[0068] The difference between Comparative Example 2 and Example 1 is that it does not contain black pepper extract.

[0069] Comparative Example 3

[0070] The difference between Comparative Example 3 and Example 1 is that it does not contain black pepper extract and polygonatum extract.

[0071] Comparative Example 4

[0072] The difference between Comparative Example 4 and Example 1 is that sodium caseinate is replaced with Tween-80.

[0073] Comparative Example 5

[0074] The difference between Comparative Example 5 and Example 1 is that pea protein is replaced with Tween-80.

[0075] Comparative Example 6

[0076] The difference between Comparative Example 6 and Example 1 is that sodium caseinate and pea protein are replaced with Tween-80.

[0077] Stability Test

[0078] Test Scheme Design: According to the guiding principles for the stability test of raw drugs and preparations (Part IV of the Chinese Pharmacopoeia 2020 Edition, 9001), 100 g of astaxanthin oil, 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 are respectively taken. Placed in a clean brown glass bottle, placed for 6 months under the conditions of a temperature of 40°C ± 2°C and a relative humidity of 75% ± 5%, and samples are taken at 0 month, 1 month, 2 months, 3 months, and 6 months respectively. The test results are shown in Table 3 below:

[0079] Table 3 Results of Product Stability Investigation

[0080]

[0081] As can be seen from Table 1 above, after six months of accelerated testing, 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.

[0082] Detection of the average particle size of nanoemulsion

[0083] 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 a sample cell, and a nano particle size and zeta potential analyzer was used to measure the average particle size of the nanoemulsion prepared in each example and comparative example. The results are statistically shown in Table 4 below:

[0084] Table 4 Statistical table of the average particle size of the product diluted 100 times

[0085]

[0086] As can be seen from Table 2 above, in the comparative examples without adding sodium caseinate, pea protein, and without adding both, the average particle size of the emulsion was significantly higher than that of Example 1.

[0087] Absorption and utilization test

[0088] 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, 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, the rats were anesthetized with 3% sodium pentobarbital, sacrificed by taking blood 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.

[0089] Determination of astaxanthin content

[0090] Extract astaxanthin from biological samples, and try to operate in the dark as much as possible during the experiment. 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 blow dry with nitrogen, and redissolve with methanol-methyl tert-butyl ether solution (1:1, v / v) for testing.

[0091] Calculation of bioavailability

[0092] According to the change curve of serum astaxanthin content, the trapezoidal area method is used to calculate the area under the curve (Area under the curve, AUC 0-t ), and the bioavailability of astaxanthin in different experimental groups is 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

[0093] In the formula, A, B, C, D, E, F, G, H, I, and J respectively represent the serum astaxanthin concentration levels at the time nodes of 0, 0.5, 1, 2, 4, 8, 10, 12, 16, and 24 h.

[0094] Table 5 Change curves of serum astaxanthin content in rats in different experimental groups AUC 0-t value

[0095]

[0096] It can be seen from Table 5 above that the present invention can significantly improve the bioavailability of astaxanthin after preparing astaxanthin oil into nanoemulsion and applying it to the production of gel candies and beverages.

[0097] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not 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 form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A preparation method of astaxanthin nanoemulsion, characterized in that: It includes the following steps: S1. Heat 120 - 150 parts by mass of Haematococcus pluvialis oil containing 5% - 15% astaxanthin to 28 - 30 °C, keep warm and continuously stir for standby to obtain component A; S2. Dissolve 4 - 7 parts by mass of the aqueous extract of Polygonatum odoratum and 3 - 5 parts by mass of the aqueous extract of Piper nigrum in 5 - 20 parts by mass of purified water, and heat 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 compound emulsifier with a mass 1 - 3 times that of the mixture, stir evenly, first use a high-speed dispersion homogenizer for treatment, and then use an ultrasonic cell disrupter for treatment; When using the high-speed dispersion homogenizer for treatment: at 10,000 r / min - 12,000 r / min, carry out high-speed shearing dispersion for 9 - 15 min; When using the ultrasonic cell disrupter for treatment: at a power of 100 W, carry out ultrasonic dispersion for 2 min, and then at a power of 200 W, carry out ultrasonic dispersion for 4 min, select the mode of working for 1 s and intermittent for 1 s, then the astaxanthin nanoemulsion is obtained; Among them, the compound emulsifier contains Tween - 80, sodium caseinate and pea protein with a mass ratio of 3 - 5:1 - 2:1 - 2. During the emulsification treatment, first add Tween - 80, stir evenly and carry out high-speed shearing dispersion for 1 / 3 of the time; then add sodium caseinate, stir evenly and carry out high-speed shearing dispersion for 1 / 3 of the time; finally add pea protein, stir evenly and carry out high-speed shearing dispersion for 1 / 3 of the time.

2. The astaxanthin nanoemulsion prepared by the method for preparing astaxanthin nanoemulsion according to claim 1.

3. The application of the astaxanthin nanoemulsion according to claim 2 in the preparation of gel candies, soft capsules, and beverages.

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