Nanometer ultramicro liquid DHA composition and preparation method thereof

Abstract

The invention discloses a nano ultramicro liquid DHA composition and a preparation method thereof, and belongs to the technical field of functional foods. The composition is prepared from the following raw materials: DHA algae oil, phosphatidylserine, walnut peptide, cerebroprotein peptide, curcumin, piperine, a herba cistanche extract, a hericium erinaceus extract, vitamin B6, vitamin B12, magnesium citrate, maltitol liquid, concentrated apple clear juice, yellow lemon concentrated liquid, green lemon concentrated juice, sodium citrate, edible essence, polyglycerol fatty acid ester and soybean powder phospholipid. Sodium ascorbate, glycerol and water; the preparation method comprises the following steps: (1) weighing raw materials; (2) premixing the component 1; (3) premixing a component 2; (4) pre-dissolving the component 3; (5) preparing a coarse emulsion; (6) performing superfine emulsification; (7) blending and mixing; (8) homogenizing and emulsifying; (9) filling and sealing; and (10) sterilizing. The composition is suitable for heart and intelligence nutrition supplement of children and special crowds, and has good taste, physical stability and oral absorption performance.

Description

Technical Field

[0001] This invention relates to the field of functional food technology, and more specifically to a nano-ultrafine liquid DHA composition and its preparation method. Background Technology

[0002] Docosahexaenoic acid (DHA) is an important member of the omega-3 polyunsaturated fatty acid family, widely found in neural tissues such as the brain and retina, and plays a crucial role in children's cognitive and visual development and in maintaining adult brain function. In recent years, with the increasing public awareness of health, DHA has become one of the fastest-growing core ingredients in the children's nutritional supplement market.

[0003] Currently, commercially available DHA products are mainly available in the form of soft capsules, chewable tablets, or drops, with the carrier mostly being an oil-based matrix. However, these traditional dosage forms share the following common problems: 1. Poor sensory experience: DHA algal oil itself has a distinct fishy or algal odor, which may cause children to refuse to take it; 2. Poor stability: DHA is easily oxidized, leading to the degradation of active ingredients, affecting efficacy and producing unpleasant odors; 3. Low absorption efficiency: DHA is a fat-soluble component. Traditional oil-based DHA is not fully emulsified in the gastrointestinal tract, and its absorption depends on bile secretion, resulting in a limited absorption rate, especially in children whose digestive functions are not yet mature. 4. Inconvenient dosage form: Soft capsules are difficult to swallow and are not suitable for young children; liquid products are prone to separation and precipitation, and have poor storage stability.

[0004] Therefore, how to develop a novel DHA supplement is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a nano-ultrafine liquid DHA composition and its preparation method, so as to solve the shortcomings of existing DHA supplements such as strong fishy smell, low absorption rate, poor stability and unpleasant taste.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A nano-ultrafine liquid DHA composition comprising the following raw materials in parts by weight: 200-600 parts DHA algal oil, 1-20 parts phosphatidylserine, 5-50 parts walnut peptide, 1-20 parts brain protein peptide, 5-20 parts curcumin, 0.01-0.1 parts piperine, 5-50 parts Cistanche deserticola extract, 5-150 parts Hericium erinaceus extract, 0.2-2 parts vitamin B6, and vitamin B12. 0.04-0.4 parts, magnesium citrate 350-600 parts, maltitol solution 1000-2000 parts, concentrated apple juice 1200-2000 parts, concentrated yellow lemon juice 100-400 parts, concentrated green lemon juice 100-150 parts, sodium citrate 20-50 parts, edible flavoring 10-50 parts, polyglycerol fatty acid ester 20-200 parts, soybean powder phospholipid 10-100 parts, sodium ascorbate 10-200 parts, glycerin 20-200 parts, and water 10000-13000 parts.

[0007] The preferred composition is: 500 parts DHA algal oil, 10 parts phosphatidylserine, 10 parts walnut peptide, 5 parts brain protein peptide, 5 parts curcumin, 0.05 parts piperine, 10 parts Cistanche deserticola extract, 10 parts Hericium erinaceus extract, 1.5 parts vitamin B6, 0.2 parts vitamin B12, 400 parts magnesium citrate, 1500 parts maltitol solution, 1500 parts concentrated apple juice, 280 parts concentrated yellow lemon juice, 120 parts concentrated green lemon juice, 35 parts sodium citrate, 20 parts edible flavoring, 50 parts polyglycerol fatty acid ester, 20 parts soybean powder phospholipid, 50 parts sodium ascorbate, 50 parts glycerin, and 11200 parts water.

[0008] The functions of each raw material in the nano-ultrafine liquid DHA composition of this invention are described below: DHA algal oil: The main active ingredient, providing DHA.

[0009] Phosphatidylserine: a neurotrophic factor that improves memory.

[0010] Walnut peptides: plant-derived neuroactive peptides.

[0011] Brain protein peptides: promote nerve cell repair.

[0012] Curcumin: Anti-inflammatory and antioxidant, synergistic protection.

[0013] Piperine: an absorption enhancer that improves the utilization rate of curcumin and DHA.

[0014] Cistanche deserticola extract: Nourishes the kidneys and improves intelligence, aiding cognition.

[0015] Hericium erinaceus extract: stimulates the production of nerve growth factor (NGF) and protects the brain.

[0016] Vitamin B6: Participates in the synthesis of neurotransmitters.

[0017] Vitamin B12: Supports myelin formation.

[0018] Magnesium citrate: Magnesium supplementation helps with sleep and regulates nerve excitability.

[0019] Maltitol solution: humectant, sweetener, osmotic pressure regulator.

[0020] Concentrated apple juice: a natural juice carrier that improves taste.

[0021] Yellow lemon concentrate: provides natural acidity and flavor.

[0022] Lime concentrate: Masks flavor and adjusts pH.

[0023] Sodium citrate: pH buffer.

[0024] Food flavorings: enhance flavor and improve taste.

[0025] Polyglycerol fatty acid esters: the main emulsifier.

[0026] Soybean powder phospholipids: aid in emulsification and stabilize the interfacial film.

[0027] Sodium ascorbate: an antioxidant that prevents DHA oxidation.

[0028] Glycerin: a solubilizer and humectant.

[0029] Water: solvent matrix.

[0030] The specific objectives of the nano-ultrafine liquid DHA composition of this invention are as follows: 1. To provide a nanoscale liquid DHA formulation based on LipoMatrix™ ultra-microemulsification technology, achieving efficient emulsification and steady-state delivery of DHA; 2. A uniform droplet system with an average particle size of less than 500 nm (D50) was constructed, which significantly improved the dispersibility and lymphatic absorption efficiency of DHA in the intestine; 3. By scientifically compounding a variety of neuroactive components (such as phosphatidylserine, walnut peptides, and brain protein peptides), multiple pathways are synergistically promoted to enhance brain development and cognitive function; 4. By introducing flavor control technology and agile masking components, a sensory experience of "refreshing and non-fishy, ​​with a pleasant flavor" is achieved; 5. Provide a standardized manufacturing process that can be scaled up industrially to ensure consistent product quality and the feasibility of large-scale production; 6. Achieve DHA bioavailability by more than 300% compared to traditional soft capsule oil formulations, achieving a nutrient delivery effect of "high load, high absorption, and low dosage".

[0031] The nanoemulsion system of the nano-ultrafine liquid DHA composition of the present invention has the following characteristics: 1. Average particle size (D50): 437 nm, PDI < 0.2, uniform distribution; 2. System type: O / W type nanoemulsion (oil-in-water); 3. Stabilization mechanism: The synergistic effect of the two emulsifiers (polyglycerol fatty acid esters + soybean lecithin) forms a dense interfacial film, which resists aggregation; 4. Antioxidant system: Sodium ascorbate and sodium citrate work synergistically to combat oxidation and extend shelf life to 18 months.

[0032] The inventive points and innovations of the nano-ultrafine liquid DHA composition of this invention are summarized as follows: 1. The first-ever "nano-ultra-micro liquid DHA composition" dosage form breaks through the limitations of traditional oil and capsule formulations, achieving a three-in-one combination of high absorption, good taste, and easy administration; 2. By applying LipoMatrix™ microemulsification technology, a DHA droplet size of ≤500nm was achieved, thus constructing a stable nanoemulsion system; 3. The formula features a synergistic combination of multiple active ingredients, including DHA, neuropeptides, plant extracts, vitamins, and minerals, forming a "golden matrix" of brain nutrition. 4. The integration of flavor control and taste masking technologies addresses the sensory challenges of high-nutrient-density liquid products; 5. The preparation process can be scaled up industrially, covering standardized operations across the entire chain of premixing, two-stage emulsification, formulation, and filling.

[0033] A method for preparing a nano-ultrafine liquid DHA composition specifically includes the following steps: (1) Weigh the raw materials Weigh each raw material according to the above-mentioned parts by weight of nano-ultrafine liquid DHA composition; (2) Premixed component 1 Polyglycerol fatty acid esters, soybean powder phospholipids, and sodium ascorbate were mixed to obtain component 1; (3) Premixed component 2 Component 2 is obtained by mixing brain protein peptides, curcumin, piperine, vitamin B6 and vitamin B12. (4) Pre-dissolved component 3 Sodium citrate and magnesium citrate were dissolved in water to obtain component 3; (5) Preparation of crude emulsion Component 1 and glycerol were added to water, dispersed by high-speed shearing, kept warm, DHA algal oil was added, and shearing was continued to obtain a crude emulsion; (6) Ultra-microemulsification The crude emulsion was subjected to ultra-micro emulsification to obtain a nano-emulsion; (7) Mixing Maltitol solution, component 3, lime concentrate, phosphatidylserine, walnut peptide, cistanche extract, hericium erinaceus extract, concentrated apple juice, yellow lemon concentrate, component 2 and edible flavoring are dissolved in water in sequence, stirred evenly, and then nanoemulsion and rinsing water are added and the volume is adjusted to obtain the preparation solution. (8) Homogenization and emulsification The prepared solution was filtered and homogenized to obtain a homogenized emulsion. (9) Filling After the homogenized emulsion passes the tests for pH, flavor, and appearance, it is quantitatively filled into strip bags to obtain the filled product. (10) Sterilization The filled product is sterilized, cooled, packaged, and stored to obtain a nano-ultrafine liquid DHA composition.

[0034] Furthermore, in step (2) above, the mixing time is 2-3 minutes.

[0035] Furthermore, in step (5) above, the high-speed shear dispersion time is 2 min; the heat preservation temperature is 50℃; and the continuous shearing time is 4 min.

[0036] Furthermore, in step (6) above, the equipment for ultra-micro emulsification is a micro-jet homogenizer, with a set pressure of 15000-20000 PSI, a temperature of 20℃, and cyclic homogenization twice; the particle size of the nano-emulsion is ≤500nm.

[0037] Furthermore, in step (8) above, the sieve mesh size for filtration is 100 mesh; the equipment for homogenization and emulsification is a high-pressure homogenizer with a pressure of 30 MPa and a flow rate of 0.2 kg / s.

[0038] Furthermore, in step (10) above, the sterilization method is water bath pasteurization, the temperature is 90-94℃, and the time is 15min.

[0039] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows: 1. Significantly improved absorption efficiency Using LipoMatrix™ microemulsification technology, the DHA droplet size is controlled at 437nm, which greatly increases the specific surface area and improves the efficiency of intestinal contact and chylomicron encapsulation. Bioavailability tests show that the DHA absorption rate of the composition of this invention is 300% higher than that of traditional soft capsule oil, achieving "4 times higher absorption". Piperine synergistically enhances the transmembrane transport of curcumin and DHA.

[0040] 2. Sensory experience significantly improved Combining an agile flavor-masking module with a systematic taste bud testing design, it achieves a "refreshing, fishy, ​​and pleasantly flavorful" drinking experience; multiple natural fruit juices (apple juice, lemon juice) and edible flavorings work together to effectively mask the off-flavors of DHA and plant extracts.

[0041] 3. Synergistic effect of the formula A scientifically formulated blend of various neuroactive ingredients (phosphatidylserine, walnut peptides, brain protein peptides, Hericium erinaceus, etc.) provides "DHA+" multidimensional brain nutrition support; vitamins B6 / B12 and magnesium work synergistically to regulate neurotransmitter metabolism, relieve anxiety, and improve sleep quality.

[0042] 4. High stability and security The dual emulsification system (polyglycerol fatty acid ester + soybean lecithin) constructs a stable interfacial film to prevent emulsion droplet aggregation; the sodium ascorbate + sodium citrate antioxidant system effectively delays DHA oxidation; and there are no organic solvent residues throughout the process, meeting food safety standards.

[0043] 5. The process has strong scalability. The process parameters are clearly defined, the equipment is universal, and it is suitable for continuous industrial production; the two-stage emulsification of micro-jet homogenization and high-pressure homogenization ensures batch consistency.

[0044] 6. The dosage form has significant advantages. The liquid form is easy for children to take, requiring no swallowing; each packet is individually packaged for easy portability and precise dosage control.

[0045] In summary, this invention, based on nano-microemulsification technology, constructs a DHA liquid formulation with low odor, high stability, and high absorption rate. It possesses characteristics such as nanoscale particle size, high bioavailability, good flavor, stable storage, and a dosage form suitable for children. It is suitable for the mental and intellectual nutritional supplementation of children and special populations, has good taste, physical stability, and oral absorption performance, and has achieved stable industrial production through innovative processes. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] In the following examples, DHA algal oil (containing ≥50% DHA) was commercially available; other excipients all met the Chinese Pharmacopoeia or food-grade standards; and all equipment used was general-purpose equipment for the food / pharmaceutical industry with no special customization requirements.

[0048] Example 1 The nano-micro liquid DHA composition comprises the following ingredients by weight: 200 mg DHA algal oil, 1 mg phosphatidylserine, 5 mg walnut peptide, 1 mg brain protein peptide, 5 mg curcumin, 0.01 mg piperine, 5 mg Cistanche deserticola extract, 5 mg Hericium erinaceus extract, 0.2 mg vitamin B6, 0.04 mg vitamin B12, 350 mg magnesium citrate, 1000 mg maltitol solution, 1200 mg concentrated apple juice, 100 mg concentrated yellow lemon juice, 100 mg concentrated green lemon juice, 20 mg sodium citrate, 10 mg edible flavoring, 20 mg polyglycerol fatty acid ester, 10 mg soybean powder phospholipid, 10 mg sodium ascorbate, 20 mg glycerin, and 10000 mg water. The preparation method of the above-mentioned nano-ultrafine liquid DHA composition specifically includes the following steps: (1) Weigh the raw materials Weigh each raw material according to the weight of the above-mentioned nano-ultrafine liquid DHA composition; (2) Premixed component 1 Add polyglycerol fatty acid esters, soybean powder phospholipids and sodium ascorbate into a transparent mixing bag and mix manually for 3 minutes to obtain component 1; (3) Premixed component 2 Brain protein peptides, curcumin, piperine, vitamin B6 and vitamin B12 were mixed in another mixing bag and mixed evenly to obtain component 2; (4) Pre-dissolved component 3 Add 10 kg of 60°C hot water to a stainless steel bucket, then add sodium citrate and magnesium citrate, and stir until completely dissolved to obtain component 3; (5) Preparation of crude emulsion Add 60°C hot water to the mobile tank, then add component 1 and glycerol in sequence, disperse by high-speed shearing for 2 min, slowly add DHA algal oil while keeping warm at 50°C, and continue shearing for 4 min to obtain crude emulsion; (6) Ultra-microemulsification The crude emulsion was introduced into a microfluidics homogenizer (Model: M110EH) for ultra-microemulsification. The pressure was set at 15000-20000 PSI and the temperature at 20℃. The homogenization was repeated twice to obtain a nanoemulsion with a particle size ≤500nm. (7) Mixing Add 2 / 3 of the dissolving water (40℃) to the premix tank, then add maltitol solution, component 3, lime concentrate, phosphatidylserine, walnut peptide, cistanche extract, hericium erinaceus extract, concentrated apple juice, yellow lemon concentrate, component 2 and edible flavoring in sequence. After stirring evenly, add nanoemulsion and rinsing water, and bring the volume to the target volume to obtain the preparation solution. (8) Homogenization and emulsification After filtering the prepared solution through a 100-mesh sieve, it was homogenized and emulsified by a high-pressure homogenizer at a pressure of 30 MPa and a flow rate of 0.2 kg / s to obtain a homogenized emulsion. (9) Filling After the homogenized emulsion passes the pH, flavor and appearance tests, it is quantitatively filled into strip bags (aluminum-plastic composite strip bags, 10mL per bag) to obtain the filled product. (10) Sterilization The filled product is pasteurized in a water bath at 92°C for 15 minutes. After cooling, it is packaged and stored to obtain the nano-ultrafine liquid DHA composition.

[0049] Example 2 The nano-micro liquid DHA composition comprises the following ingredients by weight: 500 mg DHA algal oil, 10 mg phosphatidylserine, 10 mg walnut peptide, 5 mg brain protein peptide, 5 mg curcumin, 0.05 mg piperine, 10 mg Cistanche deserticola extract, 10 mg Hericium erinaceus extract, 1.5 mg vitamin B6, 0.2 mg vitamin B12, 400 mg magnesium citrate, 1500 mg maltitol solution, 1500 mg concentrated apple juice, 280 mg concentrated yellow lemon juice, 120 mg concentrated green lemon juice, 35 mg sodium citrate, 20 mg edible flavoring, 50 mg polyglycerol fatty acid ester, 20 mg soybean powder phospholipid, 50 mg sodium ascorbate, 50 mg glycerin, and 11200 mg water. The preparation method of the above-mentioned nano-ultrafine liquid DHA composition specifically includes the following steps: (1) Weigh the raw materials Weigh each raw material according to the weight of the above-mentioned nano-ultrafine liquid DHA composition; (2) Premixed component 1 Add polyglycerol fatty acid esters, soybean powder phospholipids and sodium ascorbate into a transparent mixing bag and mix manually for 3 minutes to obtain component 1; (3) Premixed component 2 Brain protein peptides, curcumin, piperine, vitamin B6 and vitamin B12 were mixed in another mixing bag and mixed evenly to obtain component 2; (4) Pre-dissolved component 3 Add 10 kg of 60°C hot water to a stainless steel bucket, then add sodium citrate and magnesium citrate, and stir until completely dissolved to obtain component 3; (5) Preparation of crude emulsion Add 60°C hot water to the mobile tank, then add component 1 and glycerol in sequence, disperse by high-speed shearing for 2 min, slowly add DHA algal oil while keeping warm at 50°C, and continue shearing for 4 min to obtain crude emulsion; (6) Ultra-microemulsification The crude emulsion was introduced into a microfluidics homogenizer (Model: M110EH) for ultra-microemulsification. The pressure was set at 15000-20000 PSI and the temperature at 20℃. The homogenization was repeated twice to obtain a nanoemulsion with a particle size ≤500nm. (7) Mixing Add 2 / 3 of the dissolving water (40℃) to the premix tank, then add maltitol solution, component 3, lime concentrate, phosphatidylserine, walnut peptide, cistanche extract, hericium erinaceus extract, concentrated apple juice, yellow lemon concentrate, component 2 and edible flavoring in sequence. After stirring evenly, add nanoemulsion and rinsing water, and bring the volume to the target volume to obtain the preparation solution. (8) Homogenization and emulsification After filtering the prepared solution through a 100-mesh sieve, it was homogenized and emulsified by a high-pressure homogenizer at a pressure of 30 MPa and a flow rate of 0.2 kg / s to obtain a homogenized emulsion. (9) Filling After the homogenized emulsion passes the pH, flavor and appearance tests, it is quantitatively filled into strip bags (aluminum-plastic composite strip bags, 10mL per bag) to obtain the filled product. (10) Sterilization The filled product is pasteurized in a water bath at 92°C for 15 minutes. After cooling, it is packaged and stored to obtain the nano-ultrafine liquid DHA composition.

[0050] Example 3 The nano-micro liquid DHA composition comprises the following ingredients by weight: DHA algal oil 600mg, phosphatidylserine 20mg, walnut peptide 50mg, brain protein peptide 20mg, curcumin 20mg, piperine 0.1mg, Cistanche deserticola extract 50mg, Hericium erinaceus extract 150mg, vitamin B6 2mg, vitamin B12 0.4mg, magnesium citrate 600mg, maltitol solution 2000mg, concentrated apple juice 2000mg, yellow lemon concentrate 400mg, green lemon concentrate 150mg, sodium citrate 50mg, edible flavor 50mg, polyglycerol fatty acid ester 200mg, soybean powder phospholipid 100mg, sodium ascorbate 200mg, glycerin 200mg, and water 13000mg. The preparation method of the above-mentioned nano-ultrafine liquid DHA composition specifically includes the following steps: (1) Weigh the raw materials Weigh each raw material according to the weight of the above-mentioned nano-ultrafine liquid DHA composition; (2) Premixed component 1 Add polyglycerol fatty acid esters, soybean powder phospholipids and sodium ascorbate into a transparent mixing bag and mix manually for 3 minutes to obtain component 1; (3) Premixed component 2 Brain protein peptides, curcumin, piperine, vitamin B6 and vitamin B12 were mixed in another mixing bag and mixed evenly to obtain component 2; (4) Pre-dissolved component 3 Add 10 kg of 60°C hot water to a stainless steel bucket, then add sodium citrate and magnesium citrate, and stir until completely dissolved to obtain component 3; (5) Preparation of crude emulsion Add 60°C hot water to the mobile tank, then add component 1 and glycerol in sequence, disperse by high-speed shearing for 2 min, slowly add DHA algal oil while keeping warm at 50°C, and continue shearing for 4 min to obtain crude emulsion; (6) Ultra-microemulsification The crude emulsion was introduced into a microfluidics homogenizer (Model: M110EH) for ultra-microemulsification. The pressure was set at 15000-20000 PSI and the temperature at 20℃. The homogenization was repeated twice to obtain a nanoemulsion with a particle size ≤500nm. (7) Mixing Add 2 / 3 of the dissolving water (40℃) to the premix tank, then add maltitol solution, component 3, lime concentrate, phosphatidylserine, walnut peptide, cistanche extract, hericium erinaceus extract, concentrated apple juice, yellow lemon concentrate, component 2 and edible flavoring in sequence. After stirring evenly, add nanoemulsion and rinsing water, and bring the volume to the target volume to obtain the preparation solution. (8) Homogenization and emulsification After filtering the prepared solution through a 100-mesh sieve, it was homogenized and emulsified by a high-pressure homogenizer at a pressure of 30 MPa and a flow rate of 0.2 kg / s to obtain a homogenized emulsion. (9) Filling After the homogenized emulsion passes the pH, flavor and appearance tests, it is quantitatively filled into strip bags (aluminum-plastic composite strip bags, 10mL per bag) to obtain the filled product. (10) Sterilization The filled product is pasteurized in a water bath at 92°C for 15 minutes. After cooling, it is packaged and stored to obtain the nano-ultrafine liquid DHA composition.

[0051] Performance testing The nano-micro liquid DHA composition prepared in Example 2 was used to test its particle size distribution, quality detection and DHA bioavailability.

[0052] I. Particle Size Distribution Test sample: Nano-micro liquid DHA composition prepared in Example 2 Sample description: Liquid Test item: Particle size distribution Test method: GB / T 19627-2005 Particle size analysis - Photon correlation spectroscopy Test results are shown in Table 1.

[0053] Table 1. Particle size distribution of the nano-micro liquid DHA composition in Example 2.

[0054] II. Quality Inspection Test sample: Nano-micro liquid DHA composition prepared in Example 2 Sample description: Liquid Test item: Quality inspection Test results are shown in Table 2.

[0055] Table 2 Quality testing of the nano-ultrafine liquid DHA composition in Example 2

[0056] III. DHA Bioavailability Testing 1. Experimental Objective The bioavailability of different DHA formulations in cells was detected using CACO-2 and statistical analysis was performed.

[0057] 2. Experimental Materials 2.1 Main Experimental Reagents Table 3 Main Experimental Reagents

[0058] 2.2 Main Experimental Instruments Table 4 Main Experimental Instruments

[0059] 3. Experimental Procedure 3.1 Sample Information Sample information is shown in Table 5. All samples were directly used for cell experiments after being vortexed and suspended in DMEM medium.

[0060] Table 5 Sample Information

[0061] 3.2 Caco-2 cell culture and passage 3.2.1 Cell resuscitation 1) Remove the cells from the liquid nitrogen tank and quickly place them in a 37°C water bath. Gently shake the cells to allow them to thaw quickly. Avoid prolonged water bathing or excessively high temperatures to prevent cell damage. 2) Transfer the thawed cell suspension to a centrifuge tube, add an appropriate amount of culture medium, and centrifuge to remove the cryopreservation solution; 3) After centrifugation, discard the supernatant, resuspend the cells in fresh culture medium, seed them into pre-prepared culture dishes or flasks, and incubate them in an incubator; 4) It usually takes 24-48 hours for the revived cells to fully adhere to the wall. During this period, interference with the cells should be minimized.

[0062] 3.2.2 Cell passage 1) Remove the old culture medium and gently wash the cells with PBS to remove residual culture medium and dead cells; 2) Add an appropriate amount of trypsin for digestion. The digestion time depends on the tightness of cell adhesion and the concentration of trypsin. 3) After digestion, add serum-containing culture medium to stop digestion, and gently pipette to detach the cells; 4) Transfer the cell suspension to a centrifuge tube and centrifuge to collect the cells; 5) After centrifugation, discard the supernatant, resuspend the cells in fresh culture medium, and perform cell counting.

[0063] 3.2.3 Cell Culture 1) Caco-2 cells were inoculated at a rate of 5 × 10⁻⁶. 4 cells / cm 2 12-well Transwell chambers inoculated with permeable polycarbonate membrane; 2) Add 0.5 mL of cell suspension to the top side of the chamber and 1.5 mL of complete culture medium to the bottom side. Change the culture medium on the top and bottom sides every other day. After one week, change it every day and culture for 14 days. 3) Transmembrane resistance was assessed using a transepithelial resistivity meter to verify the integrity and compactness of the Caco-2 cell monolayer. A transepithelial resistivity > 800 Ω / cm was considered adequate. 2 At that time, the Caco-2 cell monolayer model was successfully established; 4) Discard the upper and lower layers of culture medium in the chamber, and rinse the upper and lower layers of the chamber with PBS to thoroughly wash away any residual culture medium; 5) Add 0.5 mL of DMEM cell culture medium diluted with various preparations to the top side to release the final products (the effective DHA content added to each well on the top side is 50 μg), add 1.5 mL of blank DEME culture medium to the base side, place in an incubator to simulate small intestinal epithelial absorption and permeation for 4 h, collect 1.5 mL of solution from the base side chamber, combine three parallel wells together, and detect the DHA content on the base side. 6) Bioavailability calculation formula: Papp / cm / s=(dQ / dT) / (A×C0).

[0064] 3.3 DHA Extraction 1) Take 4 mL of sample, add undecylate triglyceride internal standard solution, add 5 mL of anhydrous ethanol, mix well and transfer to a separatory funnel, extract with 25 mL of diethyl ether-petroleum ether mixture, shake for 2 min, let stand for 5 min, and extract 3 times. 2) The upper layer solution was collected in a flask and concentrated to dryness using a rotary evaporator at 40°C; 3) Add 8 mL of 2% sodium hydroxide methanol solution to the flask, connect the reflux condenser, and reflux in an 80°C water bath for 20 min; 4) Add 7 mL of 14% boron trifluoride methanol solution from the top of the reflux condenser, continue refluxing in an 80°C water bath for 2 min, rinse the reflux condenser with a small amount of water, stop heating, and quickly cool the flask to room temperature. 5) Accurately add 6 mL of n-heptane, shake for 2 min, add 5 mL of saturated sodium chloride aqueous solution, let stand to separate the layers, and transfer about 5 mL of the upper n-heptane extract solution to a 10 mL test tube. Add about 2 g of anhydrous sodium sulfate, shake for 1 min, let stand for 2 min, and transfer 1 mL of the upper solution to a sample vial for testing.

[0065] 3.4 Gas chromatography-mass spectrometry (GC-MS) detection FFAP (30m×0.25mm×0.5μm) was used, with high-purity helium (99.999%) as the carrier gas. The flow rate was 1.0mL / min, the injection volume was 10.0μL, the injection port temperature was 250℃, and the temperature was programmed to increase from an initial temperature of 80℃ to 230℃ at a rate of 10℃ / min and hold for 15min.

[0066] The ionization method used was electron bombardment with an energy of 70 eV, an ion source temperature of 230℃, a transfer line temperature of 270℃, a solvent delay of 3 min, and full ion scanning.

[0067] 4. Experimental Results 4.1 In vitro bioavailability of DHA formulations in each group When undigested samples were added to DMEM cell culture medium, obvious oil floated in Yingxinsheng DHA Algae oil Softgel, and precipitated at the bottom when QINBAOBAO PS+DHA algal oil beverage dissolved in DMEM. Enbele DHA phosphatidylserine liquid beverage was able to form an emulsion suspension with DMEM, showing the best dispersibility.

[0068] When the three DHA preparations were shaken and suspended in DMEM solution, QINBAOBAO PS+DHA algal oil drink dissolved in the cell culture medium with obvious particle texture, Yingxinsheng DHA Algae oil Softgel remained suspended above the DMEM culture medium, and Enbele DHA phosphatidylserine liquid drink remained a miscible emulsion, also showing the best dispersibility.

[0069] During cell culture, a small number of cells aggregated and floated in all three different formulations, as shown in Tables 6 and 7. The undigested DHA osmotic enbele DHA phosphatidylserine liquid drink was the best, followed by QINBAOBAO PS+DHA algal oil drink, and Yingxinsheng DHA Algae oil Softgel was the worst under the same treatment conditions.

[0070] Table 6. Total Lower Chamber DHA (throughput) for Each Group

[0071] Table 7 Bioavailability Results for Each Group

[0072] 4.2 Comparison of in vitro results of DHA preparations in different groups The initial total amount for all groups was 50 μg. Undigested samples of each formulation were added to the upper chamber of a Transwell apparatus and absorbed through Caco-2 cells for 4 hours. As shown in Table 8, the absolute intake of Enbelo DHA phosphatidylserine liquid drink was 2.598%, followed by QINBAOBAO PS+DHA algal oil drink at 1.908%, and the lowest was Yingxinsheng DHA Algae oil Softgel at 0.6498%. Compared to Yingxinsheng DHA Algae oil Softgel, the bioavailability of Enbelo DHA phosphatidylserine liquid drink was increased by 300%, and that of QINBAOBAO PS+DHA algal oil drink by 200%.

[0073] Table 8 Comparison of in vitro results of lower chamber DHA formulations in different groups

[0074] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nano-ultrafine liquid DHA composition, characterized in that, The ingredients include the following parts by weight: DHA algal oil 200-600 parts, phosphatidylserine 1-20 parts, walnut peptide 5-50 parts, brain protein peptide 1-20 parts, curcumin 5-20 parts, piperine 0.01-0.1 parts, cistanche extract 5-50 parts, hericium erinaceus extract 5-150 parts, vitamin B6 0.2-2 parts, vitamin B12 0.04-0.4 parts, magnesium citrate 350-600 parts, maltitol solution 1000-2000 parts, concentrated apple juice 1200-2000 parts, concentrated yellow lemon juice 100-400 parts, concentrated green lemon juice 100-150 parts, sodium citrate 20-50 parts, edible flavoring 10-50 parts, polyglycerol fatty acid ester 20-200 parts, soybean powder phospholipid 10-100 parts, sodium ascorbate 10-200 parts, glycerin 20-200 parts, and water 10000-13000 parts.

2. The nano-micro liquid DHA composition according to claim 1, characterized in that, The ingredients include the following parts by weight: 500 parts DHA algal oil, 10 parts phosphatidylserine, 10 parts walnut peptide, 5 parts brain protein peptide, 5 parts curcumin, 0.05 parts piperine, 10 parts Cistanche deserticola extract, 10 parts Hericium erinaceus extract, 1.5 parts vitamin B6, 0.2 parts vitamin B12, 400 parts magnesium citrate, 1500 parts maltitol solution, 1500 parts concentrated apple juice, 280 parts concentrated yellow lemon juice, 120 parts concentrated green lemon juice, 35 parts sodium citrate, 20 parts edible flavoring, 50 parts polyglycerol fatty acid ester, 20 parts soybean powder phospholipid, 50 parts sodium ascorbate, 50 parts glycerin, and 11200 parts water.

3. A method for preparing a nano-ultrafine liquid DHA composition, characterized in that, Specifically, the following steps are included: (1) Weigh the raw materials Weigh each raw material according to the weight proportions of the nano-ultrafine liquid DHA composition according to claim 1 or 2; (2) Premixed component 1 Polyglycerol fatty acid esters, soybean powder phospholipids, and sodium ascorbate were mixed to obtain component 1; (3) Premixed component 2 Component 2 is obtained by mixing brain protein peptides, curcumin, piperine, vitamin B6 and vitamin B12. (4) Pre-dissolved component 3 Sodium citrate and magnesium citrate were dissolved in water to obtain component 3; (5) Preparation of crude emulsion Component 1 and glycerol were added to water, dispersed by high-speed shearing, kept warm, DHA algal oil was added, and shearing was continued to obtain a crude emulsion; (6) Ultra-microemulsification The crude emulsion was subjected to ultra-micro emulsification to obtain a nano-emulsion; (7) Mixing Maltitol solution, component 3, lime concentrate, phosphatidylserine, walnut peptide, cistanche extract, hericium erinaceus extract, concentrated apple juice, yellow lemon concentrate, component 2 and edible flavoring are dissolved in water in sequence, stirred evenly, and then nanoemulsion and rinsing water are added and the volume is adjusted to obtain the preparation solution. (8) Homogenization emulsification The prepared solution was filtered and homogenized to obtain a homogenized emulsion. (9) Filling After the homogenized emulsion passes the tests for pH, flavor, and appearance, it is quantitatively filled into strip bags to obtain the filled product. (10) Sterilization The filled product is sterilized, cooled, packaged, and stored to obtain the nano-ultrafine liquid DHA composition.

4. The method for preparing a nano-ultrafine liquid DHA composition according to claim 3, characterized in that, In step (2), the mixing time is 2-3 minutes.

5. The method for preparing a nano-ultrafine liquid DHA composition according to claim 3, characterized in that, In step (5), the high-speed shear dispersion time is 2 min; the heat preservation temperature is 50℃; and the continuous shearing time is 4 min.

6. The method for preparing a nano-ultrafine liquid DHA composition according to claim 3, characterized in that, In step (6), the ultra-micro emulsification equipment is a micro-jet homogenizer, with a set pressure of 15000-20000 PSI, a temperature of 20℃, and a cycle homogenization of 2 times; the particle size of the nano-emulsion is ≤500nm.

7. The method for preparing a nano-ultrafine liquid DHA composition according to claim 3, characterized in that, In step (8), the sieve used for filtration has a mesh size of 100; the homogenizing and emulsifying equipment is a high-pressure homogenizer with a pressure of 30 MPa and a flow rate of 0.2 kg / s.

8. The method for preparing a nano-ultrafine liquid DHA composition according to claim 3, characterized in that, In step (10), the sterilization method is water bath pasteurization, the temperature is 90-94℃, and the time is 15min.

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