A highly stable DHA algal oil nanoemulsion, its preparation method and application

Abstract

This invention belongs to the field of health food and pharmaceutical technology, specifically relating to a highly stable DHA algal oil nanoemulsion, its preparation method, and its application. The raw materials include DHA algal oil, emulsifier, and water; the emulsifier includes plant-based protein isolate and natural polysaccharides, wherein the natural polysaccharides are selected from Poria cocos polysaccharides and Tremella fuciformis polysaccharides. This invention provides a method for improving the stability of DHA algal oil nanoemulsions. The prepared DHA algal oil nanoemulsion has a stable particle size and excellent zeta potential. Furthermore, the use of plant protein base and polysaccharide complex synergistically enhances the stability of the nanoemulsion and can also synergistically enhance the body's memory function with DHA, providing new ideas for the further development and application research of DHA algal oil nanoemulsions.

Description

Technical Field

[0001] This invention belongs to the field of health food and pharmaceutical technology, specifically relating to a highly stable DHA algal oil nanoemulsion, its preparation method, and its application. Background Technology

[0002] DHA has various benefits, including promoting brain and eye nerve development, anti-inflammatory and anti-tumor effects, enhancing immunity, regulating lipid metabolism, promoting gut health, and improving cardiovascular health.

[0003] Because DHA and other ω-3 polyunsaturated fatty acids have a large number of double bonds in their molecules, they are easily oxidized during processing, transportation and storage. In particular, fatty acid oxidation caused by heating produces hydroperoxides, which are then further oxidized to produce small molecule compounds such as aldehydes, ketones and acids, resulting in a rancid taste and seriously affecting the quality of the oil.

[0004] Furthermore, DHA oxidation reduces its content, affecting its efficacy; and the oxidation products are harmful to health, causing lipid peroxidation upon ingestion, inducing various physiological abnormalities and leading to diseases. To control DHA oxidation, improve its stability, and facilitate its application in food and health supplements, the main methods currently employed are adding antioxidants and encapsulation technology using algal oil to slow down and control DHA algal oil oxidation. Adding antioxidants effectively slows down lipid oxidation and is a common practice in the food industry. Butyl hydroxyisool (BHA) and tert-butylhydroquinone (TBHQ) are commonly used synthetic antioxidants to improve the stability of lipid oxidation, but these synthetic antioxidants are gradually being phased out due to their potential toxicity.

[0005] Compared to additives, encapsulation technology is gaining increasing attention. Emulsion systems, as a superior encapsulation modality, can address issues such as low stability and bioavailability in the application of active substances. Emulsion-encapsulated algal oil not only exhibits good antioxidant effects but also effectively solves problems related to poor water solubility and low bioavailability of algal oil. Emulsion systems are crucial for improving the oxidative stability of DHA and enabling it to exert its functional effects.

[0006] Chinese invention patent application CN103549442A discloses a method for preparing high-oil-loading DHA algal oil microcapsule powder. The microcapsule powder, by its total mass, consists of 48-65% outer wall material, 33-50% core material, 1.2-1.5% emulsifier, and 0.5% antioxidant. The preparation process is as follows: First, the outer wall material and emulsifier are dissolved in water and mixed evenly at 50-60℃. The mixture is then emulsified and sheared to obtain a pre-emulsion. The core material and antioxidant are added, and emulsification and shearing continue. The mixture is then homogenized twice using a high-pressure homogenizer at 40-60 MPa to obtain a final emulsion. Finally, it is spray-dried to obtain high-oil-loading DHA algal oil microcapsule powder. The spray drying inlet air temperature is 180-210℃, and the outlet air temperature is 80-100℃. This invention can protect the original properties of DHA, improve its stability, and extend its shelf life. However, its preparation is relatively complex, and the high-temperature spray drying process easily causes oxidation of DHA.

[0007] Another Chinese invention patent application, CN104719913A, discloses a method for preparing algal oil DHA nano-suspension. The method involves adding lecithin emulsifier to alkaline water, shearing and mixing, heating to 50-70°C, adding algal oil DHA, shearing and mixing again, adjusting the pH of the system, and maintaining the system temperature for gradient homogenization. This preparation method uses a small amount of emulsifier, does not add other excipients, especially antioxidants, and is safe for consumption. The preparation method is simple, but the drug loading is still relatively low, requiring large doses, and the homogenization method is relatively complex. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a highly stable DHA algal oil nanoemulsion, its preparation method, and its applications.

[0009] To achieve the objectives of this invention, the following technical solution is adopted: A highly stable DHA algal oil nanoemulsion, comprising DHA algal oil, emulsifier, and water; wherein the emulsifier comprises plant protein isolate and natural polysaccharide; wherein the plant protein isolate is selected from whey protein isolate or pea protein isolate, and the natural polysaccharide is Poria cocos polysaccharide and Tremella fuciformis polysaccharide.

[0010] Furthermore, the plant protein isolate is selected from pea protein isolate.

[0011] Furthermore, the mass ratio of the plant protein isolate, Poria cocos polysaccharide, and Tremella fuciformis polysaccharide is 0.1-2:0.1-0.5:0.5-1.

[0012] Furthermore, the mass ratio of the DHA algal oil, emulsifier, and water is 35-45:1-5:50-65.

[0013] Proteins are amphiphilic biopolymers because they contain both hydrophobic and hydrophilic groups. The diverse properties and types of proteins available in nature make it possible to produce emulsions with different characteristics. Besides their excellent interfacial adsorption and emulsifying properties, proteins also possess high nutritional value, making them excellent raw materials for stable nanoemulsions. Sodium caseinate exhibits good surface activity, stability, gelling properties, hydrophilicity, and self-assembly characteristics. As a redundant layer absorber at the oil-water interface, it reduces the tension between phases. The binding layer between oil droplets and casein generates sufficient space and electrostatic repulsion to form a stable emulsion. Therefore, it is a good material for preparing nanocarriers.

[0014] Pea protein molecules contain both hydrophilic and hydrophobic amino acid regions. The hydrophilic portions (such as charged glutamic acid and lysine) tend to face the aqueous phase, while the hydrophobic portions (such as leucine and valine) are anchored in the oil phase. This structure allows them to effectively adsorb at the oil-water interface to form a stable film. However, there is currently no research on the physicochemical properties of combining pea protein with other emulsifiers to improve the particle size and zeta potential of nanoemulsions.

[0015] Polysaccharides are natural polymers composed of monosaccharides linked by glycosidic bonds. Some polysaccharides possess amphoteric properties, containing both nonpolar and polar groups, enabling them to adsorb onto oil droplet surfaces and stabilize emulsion formation. To date, gum arabic is the most widely used natural polysaccharide emulsifier in the food industry. Gum arabic is a colloidal substance with emulsifying capabilities, providing stable emulsification of oils over a wide pH range. During emulsification, gum arabic forms a stable elastic film, reducing the surface tension at the oil-water interface, providing strong support for the preparation of various oil-in-water food emulsion systems. Its amphiphilic structure gives it superior adsorption performance at the oil-water interface. During adsorption at the interface, hydrophobic chains tend towards the oil phase, while hydrophilic chains suspend in the water, entangled and covering the oil droplet surface, providing strong steric repulsion to resist droplet aggregation, thereby maintaining emulsion stability. However, there is very little research on the emulsifying properties of natural plant polysaccharides. The excellent natural polysaccharides obtained in this invention, namely Poria cocos polysaccharide and Tremella fuciformis polysaccharide, which are compounded with proteins, not only have the function of emulsifier bioactivity, but also greatly improve the physicochemical properties of nanoemulsions while regulating human functions, thus providing a new approach for the development of nanoemulsions.

[0016] Preferably, the Poria cocos polysaccharide can be a commercially available product, and more preferably a self-made product, prepared as follows: Poria cocos was extracted with water, filtered, and concentrated to obtain a concentrated solution. The concentrated solution was then precipitated with alcohol to obtain a precipitate, which was then dried to obtain the final product.

[0017] Preferably, the alcohol precipitation is specifically performed as follows: 95% ethanol is added to the concentrate until the final concentration of the concentrate is 50-60% ethanol, and precipitate 1 and supernatant 1 are obtained. Then, ethanol is added to supernatant 1 until the final concentration is 70-80% ethanol, and precipitate 2 and supernatant 2 are obtained. Then, 95% ethanol is added to supernatant 2 until the final concentration is 80-90% ethanol, and precipitate 3 is obtained. Precipitates 1, 2 and 3 are combined and dried to obtain Poria cocos polysaccharide.

[0018] All ethanol concentrations mentioned above are volume concentrations.

[0019] Poria cocos is the dried sclerotium of the fungus *Poria cocos*. The β-(1→3)D-glucan backbone and numerous hydrophilic hydroxyl groups of Poria cocos polysaccharides endow them with strong water absorption, high viscosity, and film-forming properties. These characteristics enable them to stabilize the water-oil interface and form a homogeneous emulsion system. Polysaccharides from *Tremella fuciformis*, *Lentinula edodes*, and *Ganoderma lucidum* also exhibit certain similar properties.

[0020] However, this invention has found that Poria cocos polysaccharides exhibit superior emulsifying properties due to their unique helical conformation and hydrophilic group distribution. Especially when combined with Tremella fuciformis polysaccharides, they demonstrate significant emulsifying stability. Furthermore, Poria cocos possesses various effects, including promoting diuresis and dampness elimination, strengthening the spleen and calming the mind, regulating immunity, improving memory, anti-inflammatory and analgesic properties, regulating blood sugar, and delaying aging. As the core active ingredient of Poria cocos, Poria cocos polysaccharides play an indispensable role in its spleen-strengthening and mind-calming effects through free radical scavenging and anti-inflammatory properties. Therefore, Poria cocos polysaccharides, possessing both emulsifying properties and biological activity, warrant further investigation.

[0021] However, the molecular weight of Poria cocos polysaccharides varies considerably, and different extraction and purification methods significantly affect their structure and physiological activity. This study, through comparison, yielded polysaccharides with superior emulsification and biological activity, demonstrating promising applications in the pharmaceutical, food, and excipient fields.

[0022] The second objective of this invention is to provide a method for preparing the above-mentioned DHA algal oil nanoemulsion, comprising the following steps: (1) Prepare a composite dispersion by adding water to the emulsifier; (2) DHA algal oil was added to the composite dispersion and sheared to obtain a crude emulsion; (3) The crude emulsion is obtained by sonication.

[0023] Further, in step (1), the plant protein isolate is mixed with water, the pH is adjusted to 6.5-7.0, and stirred at 300-500 r / min and 45-55℃ for 0.5-2 h to make an emulsion; then natural polysaccharides are added, and stirred at 400-600 r / min and 55-65℃ for 1-3 h to obtain a composite dispersion.

[0024] Furthermore, the shearing described in step (2) is shearing at 10000-15000 rpm for 3-10 minutes.

[0025] Furthermore, the ultrasonic power in step (3) is 200-250W, and the ultrasonic emulsification time is 10-15min.

[0026] Another objective of this invention is to provide the application of the above-mentioned highly stable DHA algal oil nanoemulsion or the DHA algal oil nanoemulsion prepared by the above-described preparation method in the preparation of DHA algal oil products.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention finds a method to improve the stability of DHA algal oil nanoemulsion, prepares stable particle size and excellent zeta potential, and uses Tremella fuciformis polysaccharide with good biocompatibility and biological efficacy to not only promote the stability of nanoemulsion, but also to synergistically enhance the body's memory with DHA. Further research found that adding natural polysaccharide Poria cocos polysaccharide to Tremella fuciformis polysaccharide can synergistically promote the preparation of more stable DHA algal oil nanoemulsion, providing a new idea for the research on the formulation development and application of DHA nanoemulsion.

[0028] (2) The DHA algal oil nanoemulsion prepared by the method of the present invention has excellent particle size and zeta potential, and exhibits good stability within 6 months of accelerated testing. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. All the following raw materials are commercially available conventional raw materials. Specifically, the supplier of Tremella fuciformis polysaccharide is Shanxi Shengda Yue Biotechnology Co., Ltd., specification: 90% / 1kg. The supplier of Lentinus edodes polysaccharide is Shaanxi Dennis Biotechnology Co., Ltd., specification: 50%. The supplier of soybean lecithin is Shanxi Shengda Yue Biotechnology Co., Ltd., specification: 98% / 1kg. The supplier of pea protein isolate is Xi'an Bainiankang Biotechnology Co., Ltd. The suppliers of soy protein isolate and whey protein isolate are Xi'an Lvteng Biotechnology Co., Ltd.

[0030] Example 1 The raw materials for the DHA algal oil nanoemulsion in this embodiment are: DHA algal oil, emulsifier and water in a mass ratio of 40:4:56; The emulsifiers are pea protein isolate, poria cocos polysaccharide, and tremella polysaccharide in a mass ratio of 0.1:0.1:0.5.

[0031] The preparation method of Poria cocos polysaccharide is as follows: Poria cocos is extracted twice with water at a volume of 8 times that of the medicinal material, each extraction lasting 0.5 hours. The mixture is then filtered and concentrated to 1 / 3 of the original filtrate volume to obtain a concentrated solution. 95% ethanol is added to the concentrated solution until the volume concentration of the concentrated solution is 55% ethanol. Alcohol precipitation is performed to obtain precipitate 1 and supernatant 1. 95% ethanol is added to supernatant 1 again until the volume concentration of ethanol is 75% ethanol. Alcohol precipitation is performed to obtain precipitate 2 and supernatant 2. 95% ethanol is added to supernatant 2 again until the final concentration is 85% ethanol. Alcohol precipitation is performed to obtain precipitate 3 and supernatant 3. The precipitates are combined and freeze-dried to obtain Poria cocos polysaccharide.

[0032] The preparation method of DHA algal oil nanoemulsion in this embodiment is as follows: (1) Take pea protein isolate, add it to deionized water, adjust the pH to 6.5, stir at 400 r / min and 50℃ for 0.5 h; then add Poria cocos polysaccharide and Tremella fuciformis polysaccharide, stir at 500 r / min and 60℃ for 1 h to obtain a composite dispersion. (2) Add DHA algal oil to the composite dispersion and shear at 12000 rpm for 8 min to obtain crude emulsion; (3) The crude emulsion is obtained by ultrasonic emulsification at 230W for 12 minutes.

[0033] Example 2 The raw materials for the DHA algal oil nanoemulsion in this embodiment are: DHA algal oil, emulsifier and water in a mass ratio of 35:1:64; wherein, the emulsifier is plant protein isolate, Poria cocos polysaccharide and Tremella fuciformis polysaccharide in a mass ratio of 2:0.5:1, and the plant protein is selected from pea protein isolate.

[0034] The preparation method of Poria cocos polysaccharide is as follows: Poria cocos is extracted twice with water at a volume of 8 times that of the medicinal material, each extraction lasting 0.5 hours. The mixture is then filtered and concentrated to 1 / 3 of the original filtrate volume to obtain a concentrated solution. 95% ethanol is added to the concentrated solution until the volume concentration of the concentrated solution is 50% ethanol. Alcohol precipitation is performed to obtain precipitate 1 and supernatant 1. 95% ethanol is added to supernatant 1 again until the volume concentration of ethanol is 80% ethanol. Alcohol precipitation is performed to obtain precipitate 2 and supernatant 2. 95% ethanol is added to supernatant 2 again until the final concentration is 90% ethanol. Alcohol precipitation is performed to obtain precipitate 3 and supernatant 3. The precipitates are combined and freeze-dried to obtain Poria cocos polysaccharide.

[0035] The preparation method of DHA algal oil nanoemulsion in this embodiment is as follows: (1) Take pea protein isolate, add it to deionized water, adjust the pH to 7, stir at 500 r / min and 55℃ for 1 h; then add Poria cocos polysaccharide and Tremella fuciformis polysaccharide, stir at 400 r / min and 55℃ for 1 h to obtain a composite dispersion. (2) Add DHA algal oil to the composite dispersion and shear at 10,000 rpm for 10 min to obtain a crude emulsion; (3) The crude emulsion is obtained by ultrasonic emulsification at 200W for 15 minutes.

[0036] Example 3 The raw materials for the DHA algal oil nanoemulsion in this embodiment are: DHA algal oil, emulsifier and water in a mass ratio of 45:5:50; wherein, the emulsifier is plant protein isolate, Poria cocos polysaccharide and Tremella fuciformis polysaccharide in a mass ratio of 0.5:0.5:1, and the plant protein is selected from whey protein isolate.

[0037] The preparation method of Poria cocos polysaccharide is as follows: Poria cocos is extracted twice with water at a volume of 8 times that of the medicinal material, each extraction lasting 0.5 hours. The mixture is then filtered and concentrated to 1 / 3 of the original filtrate volume to obtain a concentrated solution. 95% ethanol is added to the concentrated solution until the volume concentration of the concentrated solution is 60% ethanol. Alcohol precipitation is performed to obtain precipitate 1 and supernatant 1. 95% ethanol is added to supernatant 1 again until the volume concentration of ethanol is 70% ethanol. Alcohol precipitation is performed to obtain precipitate 2 and supernatant 2. 95% ethanol is added to supernatant 2 again until the final concentration is 80% ethanol. Alcohol precipitation is performed to obtain precipitate 3 and supernatant 3. The precipitates are combined and freeze-dried to obtain Poria cocos polysaccharide.

[0038] The preparation method of DHA algal oil nanoemulsion in this embodiment is as follows: (1) Take the isolated whey protein, add it to deionized water, adjust the pH to 7, stir at 300 r / min and 45℃ for 1 h; then add Poria cocos polysaccharide and Tremella fuciformis polysaccharide, stir at 600 r / min and 65℃ for 1 h to obtain a composite dispersion. (2) Add DHA algal oil to the composite dispersion and shear at 15000 rpm for 3 min to obtain crude emulsion; (3) The crude emulsion is obtained by ultrasonic emulsification at 250W for 10 minutes.

[0039] Comparative Example 1 The difference between this comparative example and Example 1 lies in the emulsifier, as detailed below: The raw materials for DHA algal oil nanoemulsion are: DHA algal oil, emulsifier and water in a mass ratio of 40:4:56; wherein the emulsifier is plant protein isolate, shiitake mushroom polysaccharide and soybean lecithin in a mass ratio of 0.1:0.1:0.5, and the plant protein is selected from pea protein isolate.

[0040] The preparation method of DHA algal oil nanoemulsion is as follows: (1) Take pea protein isolate and soybean lecithin and add them to deionized water, adjust the pH to 6.5, stir at 400 r / min and 50℃ for 0.5 h; then add lentinan and stir at 500 r / min and 60℃ for 1 h to obtain a composite dispersion. (2) Add DHA algal oil to the composite dispersion and shear at 12000 rpm for 8 min to obtain crude emulsion; (3) The crude emulsion is obtained by ultrasonic emulsification at 230W for 12 minutes.

[0041] Comparative Example 2 The difference between this comparative example and Example 1 lies in the emulsifier. Specifically: The raw materials for DHA algal oil nanoemulsion are: DHA algal oil, emulsifier and water in a mass ratio of 40:4:56; wherein the emulsifier is pea protein isolate and poria cocos polysaccharide in a mass ratio of 0.1:0.6.

[0042] The preparation method of Poria cocos polysaccharide is as follows: Poria cocos is extracted twice with water at a volume of 8 times that of the medicinal material, each extraction lasting 0.5 hours. The mixture is then filtered and concentrated to 1 / 3 of the original filtrate volume to obtain a concentrated solution. 95% ethanol is added to the concentrated solution until the volume concentration of the concentrated solution is 55% ethanol. Alcohol precipitation is performed to obtain precipitate 1 and supernatant 1. 95% ethanol is added to supernatant 1 again until the volume concentration of ethanol is 75% ethanol. Alcohol precipitation is performed to obtain precipitate 2 and supernatant 2. 95% ethanol is added to supernatant 2 again until the final concentration is 85% ethanol. Alcohol precipitation is performed to obtain precipitate 3 and supernatant 3. The precipitates are combined and freeze-dried to obtain Poria cocos polysaccharide.

[0043] The preparation method of DHA algal oil nanoemulsion is as follows: (1) Take pea protein isolate, add it to deionized water, adjust the pH to 6.5, stir at 400 r / min and 50℃ for 0.5 h; then add Poria cocos polysaccharide, stir at 500 r / min and 60℃ for 1 h to obtain a composite dispersion. (2) Add DHA algal oil to the composite dispersion and shear at 12000 rpm for 8 min to obtain crude emulsion; (3) The crude emulsion is obtained by ultrasonic emulsification at 230W for 12 minutes.

[0044] Comparative Example 3 The difference between this comparative example and Example 1 is that the emulsifier is different, as detailed below.

[0045] The raw materials for DHA algal oil nanoemulsion are: DHA algal oil, emulsifier and water in a mass ratio of 40:4:56; wherein the emulsifier is pea protein isolate and tremella polysaccharide in a mass ratio of 0.1:0.6.

[0046] The preparation method of DHA algal oil nanoemulsion is as follows: (1) Take pea protein isolate, add it to deionized water, adjust the pH to 6.5, stir at 400 r / min and 50℃ for 0.5 h; then add tremella polysaccharide, stir at 500 r / min and 60℃ for 1 h to obtain a composite dispersion. (2) Add DHA algal oil to the composite dispersion and shear at 12000 rpm for 8 min to obtain crude emulsion; (3) The crude emulsion is obtained by ultrasonic emulsification at 230W for 12 minutes.

[0047] Comparative Example 4 The difference between this comparative example and Example 1 is that the emulsifier is different, as detailed below.

[0048] The raw materials for DHA algal oil nanoemulsion are: DHA algal oil, emulsifier and water in a mass ratio of 40:4:56; wherein the emulsifier is soy protein isolate, gum arabic and xanthan gum in a mass ratio of 0.1:0.1:0.5.

[0049] The preparation method of DHA algal oil nanoemulsion is as follows: (1) Take soy protein isolate, add it to deionized water, adjust the pH to 6.5, stir at 400 r / min and 50℃ for 0.5 h; then add gum arabic and xanthan gum, stir at 500 r / min and 60℃ for 1 h to obtain a composite dispersion. (2) Add DHA algal oil to the composite dispersion and shear at 12000 rpm for 8 min to obtain crude emulsion; (3) The crude emulsion is obtained by ultrasonic emulsification at 230W for 12 minutes.

[0050] Comparative Example 5 The difference between this comparative example and Example 1 lies in the preparation method of Poria cocos polysaccharide and the ultrasonic emulsification parameters, as detailed below.

[0051] The raw materials for DHA algal oil nanoemulsion are: DHA algal oil, emulsifier and water in a mass ratio of 40:4:56; wherein the emulsifier is pea protein isolate, poria cocos polysaccharide and tremella polysaccharide in a mass ratio of 0.1:0.1:0.5.

[0052] The preparation method of Poria cocos polysaccharide is as follows: Poria cocos is extracted twice with 8 times the weight of the medicinal material in water, each extraction lasting 0.5 hours. After filtration, the extract is concentrated to 1 / 3 of the original filtrate volume to obtain a concentrated solution. 95% ethanol is added to the concentrated solution until the volume concentration of the concentrated solution is 45% ethanol. Alcohol precipitation is performed to obtain precipitate 1 and supernatant 1. 95% ethanol is added to supernatant 1 again until the volume concentration of ethanol is 65%. Alcohol precipitation is performed to obtain precipitate 2 and supernatant 2. 95% ethanol is added to supernatant 2 again until the final concentration is 75% ethanol. Alcohol precipitation is performed to obtain precipitate 3 and supernatant 3. The precipitates are combined and freeze-dried to obtain Poria cocos polysaccharide.

[0053] The preparation method of DHA algal oil nanoemulsion is as follows: (1) Take pea protein isolate, add it to deionized water, adjust the pH to 6.5, stir at 400 r / min and 50℃ for 0.5 h; then add Poria cocos polysaccharide and Tremella fuciformis polysaccharide, stir at 500 r / min and 60℃ for 1 h to obtain a composite dispersion. (2) Add DHA algal oil to the composite dispersion and shear at 12000 rpm for 8 min to obtain crude emulsion; (3) The crude emulsion is obtained by sonicating at 375W for 8 minutes.

[0054] Test Example 1: Storage Stability of DHA Nanoemulsion Storage experiments were conducted on the DHA algal oil nanoemulsions prepared in Examples 1-3 and Comparative Examples 1-5 for 0 months, 3 months, and 6 months. The particle size and zeta potential of the algal oil nanoemulsions were measured using a nanoparticle size analyzer and a zeta potential analyzer. At the same time, the changes in the emulsion morphology were observed. The storage conditions were a temperature of 40°C and a relative humidity of 75%.

[0055] Table 1

[0056] Table 2

[0057] Table 3

[0058] Particle size and potential are important characterization methods for emulsions. The smaller the droplet size, the weaker the Brownian motion of the emulsion, and therefore the more stable it is. The greater the electrostatic repulsion between droplets, the less likely they are to flocculate into large droplets. Generally, the larger the absolute value of the Zeta potential of the droplets in an emulsion, the better its stability.

[0059] As shown in Tables 1-3, the DHA nanoemulsions prepared in October were uniform with particle sizes ranging from 268 to 351 nm. Examples 1-3 showed better absolute zeta potential values. During accelerated processing at 3 and 6 months, the nanoemulsions exhibited some instability, which was more pronounced in Comparative Examples 1-4. Comparative Example 5 only showed slight stratification at 6 months of accelerated processing, but its zeta potential decreased significantly. Examples 1-3 were superior, while Example 1 showed the best overall stability.

[0060] Test Example 2: Experimental Study on the Improvement of Memory by the DHA Nanoemulsion of the Present Invention One hundred SPF-grade male Kunming mice were randomly divided into 10 groups: control group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, and Comparative Example 5 group. Except for the control group and model group, the other groups were administered DHA nanoemulsion prepared in their respective groups by gavage at 1 g / kg. The control group and model group were administered an equal volume of physiological saline once a day for 10 consecutive days. Training began on the morning of the 9th day. Twenty minutes before training, each group of mice was injected intraperitoneally with scopolamine hydrobromide injection at 2 mg / kg to induce a memory deficit model. The mice were then placed in a bright box for 5 minutes of training. Immediately after entering a dark room, the mice were given an electric shock (2-3 seconds) to form an avoidance memory of the dark room. After the electric shock, the mice remained in the dark room for 30 seconds to reinforce the stimulus association. One hour after the last oral administration, the mice were placed back into the open box. The latency period from when the mice were placed in the open box to when they crawled into the dark box and the number of errors in 5 minutes were recorded. The results are shown in Table 4.

[0061] The Dark Avoidance Method: The passive avoidance conditioning chamber measures 36×12×12cm and consists of two chambers, one bright and one dark. A lamp is placed on the top of the bright chamber, and a copper grid is installed at the bottom of the dark chamber, supplying a 36-volt current. A 3cm diameter opening connects the two chambers. A timer is connected to the chamber to record the latency of the animal entering the dark chamber through the opening from the bright chamber. During the experiment, a mouse is placed in the bright chamber with its head facing away from the opening. Upon entering the dark chamber, the mouse immediately receives an electric shock, and the timer stops simultaneously. The mouse is then removed, and the average latency for each group of animals to enter the dark chamber and the number of times they enter the dark chamber (an incorrect response) within 5 minutes are recorded.

[0062] Table 4

[0063] Note: Comparison between the model group and the blank group. ### p < 0.001, comparison between each experimental group and the model group, &&& p < 0.001, compared with Example 1 group, △ p < 0.05 △△ p < 0.01, △△△ p < 0.001. Compared with Example 2 group, ▽ p < 0.05 ▽▽ p < 0.01, ▽▽▽ p < 0.001. Compared with Example 3 group, ◇ p < 0.05 ◇◇ p < 0.01.

[0064] As shown in Table 4, the latency of the model mice was significantly longer and the number of errors within 5 minutes was significantly higher than that of the control group, with a statistically significant difference between the two groups, indicating successful modeling. After each group of mice was administered the prepared DHA nanoemulsion by gavage for 10 days, compared with the model group, the latency of Examples 1-3 and Comparative Examples 1-5 was significantly prolonged and the number of errors was significantly reduced, showing highly statistically significant differences. &&& (p < 0.001). Comparative Examples 1-5 used different additives with emulsifying and biological activity and different methods for preparing Poria cocos polysaccharides. The DHA algal oil nanoemulsions prepared by these examples shortened the latency period for mice to enter the dark room to varying degrees, and the number of errors increased accordingly. It can be seen that the products prepared by the raw materials and preparation methods of this invention not only have good stability, but also have a certain promoting effect on the in vivo absorption and improvement of the biological activity of the drug. The composition of this invention has good application prospects.

[0065] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A highly stable DHA algal oil nanoemulsion, characterized in that, The raw materials include DHA algal oil, emulsifiers, and water; the emulsifiers include plant protein isolate and natural polysaccharides; the plant protein isolate is selected from whey protein isolate or pea protein isolate, and the natural polysaccharides are Poria cocos polysaccharide and Tremella fuciformis polysaccharide.

2. The highly stable DHA algal oil nanoemulsion according to claim 1, characterized in that, The plant-based protein isolate is selected from pea protein isolate.

3. The highly stable DHA algal oil nanoemulsion according to claim 1, characterized in that, The mass ratio of the plant protein isolate, Poria cocos polysaccharide, and Tremella fuciformis polysaccharide is 0.1-2:0.1-0.5:0.5-1.

4. The highly stable DHA algal oil nanoemulsion according to claim 1, characterized in that, The mass ratio of DHA algal oil, emulsifier and water is 35-45:1-5:50-65.

5. The highly stable DHA algal oil nanoemulsion according to claim 1, characterized in that, The preparation method of the Poria cocos polysaccharide includes the following steps: Poria cocos was extracted with water, filtered, and concentrated to obtain a concentrated solution. The concentrated solution was then precipitated with alcohol to obtain a precipitate, which was then dried to obtain the final product.

6. The highly stable DHA algal oil nanoemulsion according to claim 5, characterized in that, The alcohol precipitation process involves adding 95% ethanol to the concentrate until the final concentration is 50-60% ethanol, resulting in precipitate 1 and supernatant 1. Then, 95% ethanol is added to supernatant 1 until the final concentration is 70-80% ethanol, resulting in precipitate 2 and supernatant 2. Finally, 95% ethanol is added to supernatant 2 until the final concentration is 80-90% ethanol, resulting in precipitate 3. The precipitates 1, 2, and 3 are combined and dried to obtain Poria cocos polysaccharide.

7. A method for preparing a highly stable DHA algal oil nanoemulsion according to any one of claims 1-6, characterized in that, Includes the following steps: (1) Prepare a composite dispersion by adding water to the emulsifier; (2) Add DHA algal oil to the composite dispersion and shear to obtain a crude emulsion; (3) The crude emulsion is obtained by sonication.

8. The preparation method according to claim 7, characterized in that, In step (1), plant protein isolate is mixed with water, pH is adjusted to 6.5-7.0, and stirred at 300-500 r / min and 45-55℃ for 0.5-2 h to form an emulsion; then natural polysaccharides are added, and stirred at 400-600 r / min and 55-65℃ for 1-3 h to obtain a composite dispersion.

9. The preparation method according to claim 7, characterized in that, The shearing in step (2) is performed at 10,000-15,000 rpm for 3-10 minutes; the ultrasonic power in step (3) is 200-250W, and the ultrasonic emulsification time is 10-15 minutes.

10. The application of the highly stable DHA algal oil nanoemulsion according to any one of claims 1-6 or the DHA algal oil nanoemulsion prepared by the preparation method according to any one of claims 7-9 in the preparation of DHA algal oil products.

Images