Highly stable emulsions comprising a polypeptide and methods of making the same

Abstract

Provided herein are high-stability emulsions comprising polypeptides and methods for preparing the same. An emulsion composition is provided, comprising 0.01-1 wt% of polypeptides, 0.2-0.8 wt% of hydrophilic colloids, 5-25 wt% of fillers, 5-25 wt% of co-emulsifiers, ≤60 wt% of oil and fat, and water; wherein the polypeptides as a single emulsifier are one or more of walnut polypeptides, corn polypeptides, wheat polypeptides and soybean polypeptides, and the proportion of polypeptides with a molecular weight below 1000 Da is 50-90%. The emulsion composition comprises a fat-soluble functional ingredient. The composition of the present application can effectively improve the stability of the emulsion and reduce the phenomenon of oil separation or stratification. The use of the composition of the present application can significantly improve the bioavailability of the fat-soluble functional ingredient.

Description

Technical Field

[0001] This invention relates to emulsions, and more particularly to highly stable, bioacceptable emulsions containing polypeptides and methods for their preparation. Background Technology

[0002] An emulsion is a heterogeneous liquid dispersion system in which one liquid is dispersed in the form of tiny droplets within another liquid phase. Oil-in-water emulsions are highly efficient carriers for delivering hydrophobic bioactive substances. Oil-in-water emulsions improve the water dispersibility of hydrophobic bioactive substances while effectively reducing the impact of environmental factors such as pH and enzymes on these substances, thus holding significant application potential in the food and pharmaceutical fields.

[0003] Despite this, emulsions often exhibit instability during production and storage, such as flocculation, coagulation, suspension, and degradation, which significantly shortens their shelf life. Studies have shown that the stability of emulsion systems is mainly affected by the composition of the oil phase and the aqueous phase. In aqueous systems, the choice of emulsifier and its combination with other hydrophilic colloids are crucial. Currently, commonly used emulsifiers are divided into two categories: synthetic small-molecule surfactants such as Tween and Span, and large-molecule natural emulsifiers such as polysaccharides and proteins. Among these, proteins are favored due to their amphiphilic properties.

[0004] In recent years, peptides, the enzymatic hydrolysis products of proteins, have been used for interfacial stabilization of emulsions due to their good molecular extensibility. There have also been some reports in this field regarding the application of peptides as emulsifiers. Chinese patent application CN103202340A discloses an A3 subunit protein peptide nutritional emulsifier and its preparation method, which discloses that the A3 subunit protein peptide is obtained by enzymatic hydrolysis with pepsin to obtain small molecule peptides with a molecular weight of 1000-5000 Daltons. However, Chinese patent application CN103202340A does not verify the emulsifying ability of the A3 subunit protein peptide, nor does it mention whether it can prepare emulsion products with a high proportion of oil, nor does it examine the stability of the emulsion.

[0005] There is a need in the art for peptide emulsifiers, methods for preparing emulsion products containing a high proportion of oils, and emulsion products. Summary of the Invention

[0006] The inventors discovered the following common problems in the application of peptide emulsions: (1) The amount of peptide added is large, and the content of the emulsifiable oil phase is low, resulting in high production costs; (2) Peptides need to be used in combination with synthetic emulsifiers (such as Tween, Span, etc.) to achieve better results, which greatly reduces their safety; (3) The emulsion has poor stability, and most of the emulsions prepared in the study show stratification after being stored at 4°C or 25°C for a period of time (7-30 days); (4) The preparation environment of peptide emulsions is mostly near neutral (pH 6.5-7.0), under which microorganisms are more likely to multiply, which is not conducive to long-term storage; (5) The content of hydrophobic bioactive substances is low, and their in vitro bioacceptability is low. Therefore, it is of great significance to develop a highly stable emulsion system containing peptides.

[0007] To address the problems of existing technologies, the inventors, through research, discovered an emulsion composition containing peptides as a single or sole emulsifier, wherein the peptides have a molecular weight <1000 Da and constitute 50-90% of the composition. The emulsion prepared from the emulsion composition of this invention has an oil content ≤60%, and the resulting emulsion exhibits uniform particle size, good stability, and maintains excellent stability even under acidic conditions of pH 2.5-4.5. Under these low pH conditions, the risk of excessive microbial contamination during transportation and storage can be reduced.

[0008] The emulsion composition of the present invention comprises peptides, hydrocolloids, fillers, co-emulsifiers, oils, and water. The present invention can improve the stability of emulsions using peptides as emulsifiers and increase the bioacceptability of poorly absorbed, fat-soluble functional ingredients such as curcumin.

[0009] This invention provides a highly stable emulsion composition. The composition comprises peptides, hydrocolloids, fillers, co-emulsifiers, oils, and water; wherein the peptides, with a molecular weight <1000 Da, account for 50-90%, and the peptides constitute 0.01-1% of the composition, playing a crucial role in the emulsification and stabilization of the oils; the hydrocolloids constitute 0.2-0.8% of the composition, significantly influencing the inhibition of oil precipitation and emulsion separation. The composition of this invention can be consumed directly or applied to beverages, gummies, soft capsules, and foods for special medical purposes (e.g., meal replacement shakes). The dosage of this product can be adjusted according to the type of oil or the daily intake of functional ingredients.

[0010] In one aspect, the present invention provides an emulsion composition comprising 0.01-1 wt% of a polypeptide, 0.2-0.8 wt% of a hydrocolloid, 5-25 wt% of a filler, 5-25 wt% of a co-emulsifier, ≤60 wt% of an oil, and water. Water may be used to bring the amount of the emulsion composition to 100 wt%.

[0011] In one embodiment, the peptide is a single or sole emulsifier. In one embodiment, the peptide is one or more of walnut peptides, corn peptides, wheat peptides, and soybean peptides. In one embodiment, the proportion of peptides with a molecular weight <1000 Da is 50-90%. In one embodiment, the emulsion composition contains a fat-soluble functional ingredient. In one embodiment, the oil content is 30wt%-60wt%.

[0012] In one embodiment, the hydrocolloid is one or more of xanthan gum, pectin, and locust bean gum.

[0013] In one embodiment, the filler is one or more of fructooligosaccharides, sucrose, erythritol, xylitol, maltitol, isomaltitol, lactitol, maltodextrin, and xylooligosaccharides.

[0014] In one embodiment, the co-emulsifier is one or more of glycerol and propylene glycol.

[0015] In one embodiment, the oil is one or more of fish oil, algal oil, soybean oil, corn oil, sunflower oil, flaxseed oil, and medium-chain triglycerides.

[0016] In one implementation, the fat-soluble functional ingredient is one or more of curcumin, coenzyme Q10, lutein esters, and astaxanthin.

[0017] In one embodiment, the content of the fat-soluble functional ingredient is 0.03-1 wt%.

[0018] In one embodiment, the pH of the emulsion composition is 2.5-4.5.

[0019] In one embodiment, the emulsion composition is preservative-free.

[0020] In one aspect, the present invention provides a method for preparing an emulsion using the emulsion composition described herein, the method comprising:

[0021] (1) Prepare an aqueous phase comprising a co-emulsifier, a hydrophilic colloid, a filler, a polypeptide and water. Preferably, the co-emulsifier is dissolved in water, followed by the hydrophilic colloid and the filler, and then the polypeptide is added and dissolved further. The pH of the solution is then adjusted using an acidity regulator to obtain the aqueous phase for later use.

[0022] (2) Pour the oil into the aqueous phase, or add the fat-soluble functional component into the oil to dissolve and obtain the oil phase, and then pour the oil phase into the aqueous phase; to obtain the emulsion. The emulsion can be an emulsion with no oil on the surface and no layering.

[0023] In one implementation, step (2) is performed by shearing at 6000-12000 rpm for 2-10 minutes using a high-speed shearing machine.

[0024] In one embodiment, an acidity regulator is used to adjust the pH of the emulsion to 2.5-4.5. In one embodiment, the acidity regulator is citric acid.

[0025] In one aspect, the present invention provides an emulsion prepared by the method described herein.

[0026] In one aspect, the present invention provides the use of the emulsion compositions or emulsions described herein in food, health food, pharmaceuticals and cosmetics.

[0027] In one aspect, the present invention provides the use of polypeptides as a single emulsifier or the sole emulsifier in the preparation of the emulsion compositions or emulsions described herein.

[0028] In another aspect, the present invention provides products comprising the emulsion described herein. The products are one or more of beverages, gummies, soft capsules, and foods for special medical purposes, such as meal replacement shakes.

[0029] In this document, the polypeptide can be a walnut polypeptide, with 50-90% having a molecular weight <1000 Da. In one embodiment, the walnut polypeptide can be prepared as follows: walnut protein is enzymatically hydrolyzed using an alkaline protease, the pH of the hydrolysate is adjusted to 8.0-10.0, the protease is inactivated by boiling in a water bath, centrifuged, and spray-dried. In one embodiment, a walnut protein:purified water ratio of 1:15-1:8 (w / v) is used. In one embodiment, the mass of the alkaline protease is 0.5-1.2% of the walnut protein. In one embodiment, enzymatic hydrolysis is performed at 45-55°C. In one embodiment, the pH of the hydrolysate is adjusted to 8.0-10.0 every 30 minutes during enzymatic hydrolysis. In one embodiment, the protease is inactivated by boiling in a water bath for 10 minutes at the predetermined hydrolysis time. In one embodiment, the hydrolysate is centrifuged at 5000-10000g for 30 minutes at 4°C and spray-dried to obtain the walnut polypeptide. In one embodiment, the walnut polypeptide can be prepared as follows: A material-to-liquid ratio of walnut protein to purified water of 1:15-1:8 (w / v) and an enzyme-to-substrate ratio of 0.5-1.2% are used. Enzymatic hydrolysis is carried out at 45-55°C, adjusting the pH of the hydrolysate to 8.0-10.0 every 30 minutes. At the predetermined hydrolysis time, the protein is inactivated by heating in a boiling water bath for 10 minutes. The hydrolysate is centrifuged at 5000-10000g for 30 minutes at 4°C and then spray-dried to obtain the walnut polypeptide. In one embodiment, the hydrolysis time is 2, 3, 4, 5, 6, 7, or 8 hours.

[0030] The advantages of this invention include:

[0031] (1) The proportion of stable oil in the emulsion of the prior art is 20%, while the emulsion composition or the proportion of oil in the emulsion of the present invention can still maintain the stability of the emulsion when it reaches 60% of the formulation ratio.

[0032] (2) When fat-soluble functional ingredients are added to the composition or emulsion involved in this invention, the bioacceptability rate is close to 40% in in vitro digestion experiments, which is much higher than that of the prior art.

[0033] (3) The composition of the present invention can be applied to products with low pH, and the composition can remain stable under pH < 4.5 conditions.

[0034] (4) The compositions or emulsions involved in this invention may not contain preservatives.

[0035] (5) The compositions or emulsions of the present invention may contain only polypeptides as emulsifiers. Attached Figure Description

[0036] Figure 1 Reference diagram for evaluating the appearance of emulsions. From left to right, the scores are 5, 4, 3, 2, and 1. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0038] In one aspect, the present invention provides an emulsion composition comprising a polypeptide, a hydrocolloid, a filler, a co-emulsifier, an oil, and water.

[0039] In this paper, peptides may be used as emulsifiers, either as a single emulsifier or as the sole emulsifier. The peptides may be one or more of walnut peptides, corn peptides, wheat peptides, and soybean peptides. The peptide content may be 0.01-1 wt%, for example, 0.05 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.7 wt%, 0.8 wt%, or 0.9 wt%. In one embodiment, the proportion of peptides with a molecular weight <1000 Da is 50-90%, for example 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%. For example, walnut peptides can be prepared as follows: walnut protein is hydrolyzed using an alkaline protease, the pH of the hydrolysate is adjusted to 8.0-10.0, the protease is inactivated by heating in a boiling water bath, centrifugation is performed, and the supernatant is spray-dried.

[0040] In this study, hydrocolloids can inhibit lipid precipitation and emulsion separation. The hydrocolloid can be one or more of xanthan gum, pectin, and locust bean gum. The content of the hydrocolloid can be 0.2-0.8 wt%, for example, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, or 0.7 wt%.

[0041] In this document, the filler may be one or more of fructooligosaccharides, sucrose, erythritol, xylitol, maltitol, isomaltitol, lactitol, maltodextrin, and xylooligosaccharides. The filler content may be 5-25 wt%, for example, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, or 24 wt%.

[0042] In this document, the co-emulsifier may be one or more of glycerol and propylene glycol. The content of the co-emulsifier may be 5-25 wt%, for example 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, or 24 wt%.

[0043] In this article, oils can be one or more of fish oil, algal oil, soybean oil, corn oil, sunflower seed oil, flaxseed oil, and medium-chain triglycerides. The oil content can be ≤60wt%, for example, 1wt%, 5wt%, 10wt%, 15wt%, 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt%, 30wt%, 31wt%, 32wt%, 33wt%, 34wt%, 35wt%, 36wt%, 37wt%, 38wt%, 39wt%, 40wt%, 41wt%, 42wt%, 43wt%, 44wt%, 45wt%, 46wt%, 47wt%, 48wt%, 49wt%, 50wt%, 51wt%, 52wt%, 53wt%, 54wt%, 55wt%, 56wt%, 57wt%, 58wt%, or 59wt%.

[0044] In this document, the emulsion composition may contain a fat-soluble functional ingredient. The fat-soluble functional ingredient may be one or more of curcumin, coenzyme Q10, lutein esters, and astaxanthin. The content of the fat-soluble functional ingredient may be 0.03-1 wt%, for example, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, or 0.9%.

[0045] In this paper, the pH of the emulsion composition can be 2.5-4.5, for example 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3 or 4.4.

[0046] In this document, the emulsion composition may contain water. The water content may be 10wt%-70wt%, for example 11wt%, 12wt%, 13wt%, 14wt%, 15wt%, 16wt%, 17wt%, 18wt%, 19wt%, 20wt%, 21wt%, 22wt%, 23wt%, 24wt%, 25wt%, 26wt%, 27wt%, 28wt%, 29wt%, 30wt%, 31wt%, 32wt%, 33wt%, 34wt%, 35wt%, 36wt%, 37wt%, 38wt%. %, 39wt%, 40wt%, 41wt%, 42wt%, 43wt%, 44wt%, 45wt%, 46wt%, 47wt%, 48wt%, 49wt%, 50wt%, 51wt%, 52wt%, 53wt%, 54wt%, 55wt%, 56wt%, 57wt%, 58wt%, 59wt%, 60wt%, 61wt%, 62wt%, 63wt%, 64wt%, 65wt%, 66wt%, 67wt%, 68wt%, or 69wt%.

[0047] This document also covers methods for preparing emulsions using the emulsion compositions described herein, the methods comprising:

[0048] (1) Dissolve the emulsifier in water, then dissolve the hydrophilic colloid and filler in water sequentially, add the peptide and continue dissolving, adjust the pH of the solution using an acidity regulator to obtain an aqueous phase for later use; and

[0049] (2) Pour the oil into the aqueous phase, or add the fat-soluble functional component into the oil to dissolve and obtain the oil phase, and then pour the oil phase into the aqueous phase to obtain the emulsion.

[0050] An emulsion can be an emulsion with no oil on its surface and no layering.

[0051] In the method described in this paper, step (2) can be performed by shearing at 6000-12000 rpm for 2-10 minutes using a high-speed shearing machine.

[0052] In the method described herein, an acidity regulator is used to adjust the pH of the emulsion to 2.5–4.5, for example, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, or 4.4. In one embodiment, the acidity regulator is citric acid.

[0053] This article also covers emulsions prepared by the methods described herein. The characteristics described above for emulsion compositions also apply to emulsions. For example, the emulsions described herein have one or more of the following characteristics: (1) an oil content ≤ 60 wt%; (2) good stability and overall emulsion quality at pH 2.5–4.5; (3) a non-oil-free surface and no stratification; and (4) improved bioacceptability of fat-soluble functional ingredients when the emulsion contains such ingredients.

[0054] This document also covers the use of the emulsion compositions or emulsions described herein in food, health food, pharmaceutical, and cosmetic applications. This document also covers the use of the peptides described herein as a single or sole emulsifier in the preparation of the emulsion compositions or emulsions described herein.

[0055] This invention also covers products comprising the emulsion described herein. Products may be one or more of beverages, gummies, soft capsules, and foods for special medical purposes, such as meal replacement shakes.

[0056] The emulsion composition of the present invention

[0057] This document provides exemplary examples of emulsion compositions of the present invention, which include:

[0058] Polypeptides: 0.01-1 wt%, with 50-90% having a molecular weight <1000 Da, preferably one or a combination of walnut polypeptides, corn polypeptides, wheat polypeptides and soybean polypeptides;

[0059] Hydrophilic colloid: 0.2-0.8 wt%, preferably one or a combination of xanthan gum, pectin and locust bean gum;

[0060] Filler: 5-25 wt%, preferably one or a combination of fructooligosaccharides, sucrose, erythritol, xylitol, maltitol, isomaltitol, lactitol, maltodextrin, and xylooligosaccharides;

[0061] Co-emulsifier: 5-25 wt%, preferably one or a combination of glycerol and propylene glycol;

[0062] Oils: ≤60wt%, preferably one or a combination of fish oil, algae oil, soybean oil, corn oil, sunflower seed oil, flaxseed oil, and medium-chain triglycerides;

[0063] The remainder is water.

[0064] If the above components are not within the specified range, the emulsion may experience oil precipitation or stratification, which will affect the stability of the emulsion.

[0065] The method of the present invention

[0066] This document provides a method for preparing the emulsion composition of the present invention, which includes:

[0067] (1) Dissolve the emulsifier in purified water, then dissolve the hydrophilic colloid and filler in it in sequence. After the other components are completely dissolved, add the peptide and continue to dissolve. Adjust the pH value of the solution with an acidity regulator to obtain an aqueous phase for later use.

[0068] (2) Pour the oil into the aqueous phase, or dissolve the fat-soluble functional ingredient in the formulated amount of oil to obtain an oil phase, and then pour the oil phase into the aqueous phase; shear with a high-speed shearing machine at 6000-12000 rpm for 2-10 min to obtain an emulsion. The emulsion can be an emulsion without oil on the surface and without layering. In one embodiment, peptides, hydrocolloids, fillers, co-emulsifiers, and water in a certain proportion range can improve the stability of high-oil-content emulsions and significantly improve the bioacceptability of the fat-soluble functional ingredients added thereto. The proportion of peptides with a molecular weight distribution <1000 Da can be between 50-90%.

[0069] Example

[0070] The following embodiments are provided to illustrate the present invention. Those skilled in the art should understand that the embodiments are merely illustrative and not restrictive. The invention is limited only by the scope of the appended claims.

[0071] 1. Materials and Sources

[0072] Xanthan gum, fructooligosaccharides, glycerin, and fish oil are all sourced from commercially available ingredients.

[0073] Preparation method of walnut polypeptides: Walnut protein (supplier: Hebei Lehuo): purified water at a material-to-liquid ratio of 1:15-1:8 (w / v), with an enzyme-to-substrate ratio of 0.5-1.2%, is hydrolyzed at 45-55℃. The pH of the hydrolysate is adjusted to 8.0-10.0 every 30 minutes. At the predetermined hydrolysis time, the protein is inactivated by heating in a boiling water bath for 10 minutes. The hydrolysate is centrifuged at 5000-10000g for 30 minutes at 4℃ and spray-dried to obtain walnut polypeptides. The percentages of walnut polypeptides with molecular weights <1000Da (1-5) at hydrolysis times of 0.5h, 2h, 4h, 8h, and 14h were 34.4%, 52.6%, 76.9%, 90.2%, and 97.3%, respectively. The molecular weight determination method for walnut polypeptides refers to GB / T 22492 (Appendix A), and the determination equipment is an Agilent HPLC 1260.

[0074] 2. Preparation method

[0075] (1) Dissolve glycerol in purified water, then dissolve xanthan gum and fructooligosaccharide in it in sequence. Stir thoroughly until completely dissolved, then add walnut polypeptide and continue to dissolve. Adjust the pH of the solution with citric acid to obtain an aqueous phase for later use.

[0076] (2) Pour the fish oil into the aqueous phase and shear it for 2-10 minutes using a high-speed shearing machine at 6000-12000 rpm to obtain the composition.

[0077] 3. Testing Methods

[0078] Instruments used: Malvern MS3000 laser particle size analyzer, Lumisizer 611 full-function stability analyzer

[0079] Test steps:

[0080] (1) Appearance evaluation: Observe whether the prepared emulsion is uniform, whether it is layered, and whether there is oil floating on the surface.

[0081] (2) Evaluation of droplet size: The droplet size of the emulsion was measured using a Malvern MS3000 laser particle size analyzer when the shading degree reached 5-20%. The surface area average particle size D[3,2] and volume average particle size D[4,3] were obtained to characterize the droplet size of the emulsion.

[0082] (3) Instability index: The sample was placed in a Lumisizer 611 full-function stability analyzer and centrifuged at 4000 rpm for 4.5 h at 25 °C to characterize the instability index of the sample. The lower the instability index, the less likely the sample is to separate into layers.

[0083] 4. Evaluation Criteria:

[0084] Table 1: Emulsion Stability Evaluation Indicators

[0085]

[0086] Example 1: Determining the suitable molecular weight range of peptides for emulsion preparation

[0087] The emulsion was prepared according to the formulation in Table 2, and the pH was adjusted to 3.5 using a pH adjuster (15% citric acid solution). The D[3,2] / μm, D[4,3] / μm, instability index, and emulsion stability evaluation score were then evaluated.

[0088] Table 2: Experimental Study on Molecular Weight Distribution of Walnut Polypeptides

[0089]

[0090]

[0091] Formulas 1-5 were tested using walnut peptides with different molecular weight distributions. Formulas 2-4 showed that when the proportion of walnut peptides with a molecular weight <1000Da was 50-90%, an emulsion with excellent stability could be prepared, with an emulsion stability evaluation score of 4.5 or higher. However, Formulas 1 and 5 showed that when the proportion of peptides with a molecular weight <1000Da was less than 50% (34.4%) or more than 90% (97.3%), the particle size and instability index of the emulsion increased, and the stability was significantly reduced. The appearance was slightly layered or with obvious oil floating on the surface.

[0092] Determine the content of the formulation composition.

[0093] The emulsion was prepared according to the formulation in Table 3, and the pH was adjusted to 3.5 using a pH adjuster (15% citric acid solution). The emulsion's D[3,2] / μm, D[4,3] / μm, instability index, emulsion stability score, and appearance were evaluated.

[0094] Table 3: Experiments on the range of components in the composition

[0095]

[0096]

[0097] Table 3 examines the quality of emulsions prepared with walnut polypeptides of the same molecular weight distribution (76.9% of which have a molecular weight <1000) under different emulsion formulation ratios.

[0098] Surprisingly, formulations 3, 7, and 8 showed high emulsion stability when the proportion of walnut peptides in the formulation was between 0.01% and 1%, with stability evaluation scores of 4 points for each. However, formulations 6 and 9 showed that when the proportion of walnut peptides in the formulation was less than 0.01% or more than 1%, the stability evaluation scores of the emulsions were unqualified, with scores of 1.75 and 1 points, respectively.

[0099] Formulations 10-14 show that xanthan gum has a significant impact on emulsion stability. When xanthan gum content is between 0.2% and 0.8%, it has a positive effect on inhibiting droplet migration and improving emulsion stability. When xanthan gum content is below 0.2%, the amount is insufficient to stabilize the emulsion, resulting in a stability evaluation score of 2. When xanthan gum content is above 0.8%, flocculation occurs in the emulsion, particle size increases, and stability decreases, resulting in a stability evaluation score of 3.5.

[0100] Formulas 15, 16, and 17 show that when the content of fructooligosaccharides and glycerol is 5-25%, the emulsion has good stability, with stability evaluation scores all above 4. Formula 18, by increasing the proportion of fish oil to 60%, does not significantly affect the stability of the emulsion, and the emulsion stability evaluation score is as high as 4.5.

[0101] Example 3: Experiment investigating alternative composition components

[0102] The emulsion was prepared according to the formulation in Table 4, and the pH was adjusted to 3.5 using a pH adjuster (15% citric acid solution). The D[3,2] / μm, D[4,3] / μm, instability index, and emulsion stability evaluation score were then evaluated.

[0103] Among them, the proportions of corn peptides, wheat peptides, soybean peptides and collagen peptides with a molecular weight <1000Da were 86.03%, 79.26%, 81.32% and 87.49%, respectively.

[0104] Table 4: Experiments on the Investigation of Component Substitution Schemes in the Composition

[0105]

[0106] In formulations 19-22, replacing walnut peptides with corn peptides, wheat peptides, and soybean peptides respectively resulted in emulsions with essentially no difference in stability compared to the walnut peptide emulsion, with stability scores all above 4. However, replacing walnut peptides with collagen peptides of similar molecular weight distribution resulted in an emulsion quality score of only 3, and issues such as oil floating and unsatisfactory sample appearance arose. These results indicate that not all peptides exhibit excellent emulsifying and oil-stabilizing effects at this specific specification (similar molecular weight distribution ratio). In formulations 23-24, replacing xanthan gum with pectin and locust bean gum respectively still yielded emulsions of relatively good overall quality, with stability and appearance scores both around 4.

[0107] Example 4: The effect of the formulation composition on improving the bioacceptability of fat-soluble functional ingredients

[0108] The emulsion was prepared according to the formulation in Table 5, and the pH was adjusted to 3.5 using a pH adjuster (15% citric acid solution). The emulsion's D[3,2] / μm, D[4,3] / μm, instability index, emulsion stability score, and bioacceptability were evaluated.

[0109] Bioacceptability determination method:

[0110] Take an appropriate amount of sample into a digestion flask, add purified water to 7.5 ml, and add 7.5 ml of oral digestion solution containing 3 mg / ml oral mucin (Sigma-Aldrich M2378) (the oral digestion solution should be prepared fresh and contains 159.4 mg sodium chloride, 32.8 mg ammonium nitrate, 20.2 mg potassium chloride, 61.6 mg potassium citrate, 127 mg potassium dihydrogen phosphate, 14.6 mg sodium lactate, 19.8 mg urea, and 2.1 mg sodium dihydrogen urate per 100 ml of water). Stir well, adjust the pH to 6.8, and digest at 37°C for 10 min. Add 15 ml of gastric digestion solution containing 3.2 mg / ml pepsin (Sigma-Aldrich P7125) to the oral digested sample (the gastric digestion solution should be prepared fresh and contains 200 mg sodium chloride per 100 ml of water; adjust the pH to 1.2 with concentrated hydrochloric acid), adjust the pH to 2.0, and digest at 37°C for 2 h. Add 1.5 ml of enteric digestion solution (freshly prepared enteric digestion solution containing 21.87 g sodium chloride and 3.67 g calcium chloride monohydrate per 100 ml of water), 2.5 ml of PBS solution containing 24 mg / ml lipase (Sigma-Aldrich L3126), 2.5 ml of PBS solution containing 24 mg / ml trypsin (Sigma-Aldrich P7545), and 3.5 ml of PBS solution containing 54 mg / ml bile salts to the gastric digested sample. Adjust the pH to 7.0 and digest at 37°C for 2 hours. During digestion, titrate the digestion solution with sodium hydroxide solution to neutralize the free fatty acids produced. After enteric digestion, take 2 ml of the digested sample and centrifuge at 15000 rpm for 30 min at 4°C. Collect the clear intermediate micelle layer and determine the content of functional materials in the undigested sample and the micelle layer.

[0111] The testing methods for functional materials are as follows:

[0112] Total curcumin: High-performance liquid chromatography (HPLC) was used. An appropriate amount of sample was dissolved and diluted to the mark with methanol via ultrasonication. The solution was then diluted again and diluted to the mark with methanol to obtain the test solution. Curcumin, demethoxycurcumin, and bisdemethoxycurcumin were used as reference standards. Acetonitrile-glacial acetic acid solution was used as the mobile phase. Detection was performed at a wavelength of 430 nm. The total curcumin content in the product was calculated using the external standard method.

[0113] Coenzyme Q10: Assay according to the "Content Determination" section under Coenzyme Q10 in Part II of the Chinese Pharmacopoeia;

[0114] Lutein esters: Using ultraviolet spectrophotometry, a suitable amount of sample was extracted three times with a small amount of water and an appropriate amount of hexane-acetone-toluene-anhydrous ethanol solution. After centrifugation, the supernatant was collected and concentrated to dryness. A quantitative amount of hexane-ethyl acetate solution was added to dissolve the supernatant as the test solution. Using hexane as a blank, the concentration was measured at 445 nm. The content of lutein esters in the product was calculated using the absorption coefficient of 1394 at 445 nm.

[0115] Astaxanthin: Ultraviolet spectrophotometry was used. An appropriate amount of sample was dissolved in acetic acid-dimethyl sulfoxide solution at a warm temperature, brought to a final volume, and filtered. The solution was diluted with acetic acid-dimethyl sulfoxide solution to prepare the test solution. Acetic acid-dimethyl sulfoxide solution was used as a blank control. The absorbance value A was measured at a wavelength of 489 nm. The astaxanthin content in the product was calculated using the absorption coefficient of 1908 at 489 nm.

[0116] Calculate the bioacceptability of the sample based on the measured content of functional materials:

[0117] Bioacceptability = (Content of functional components in the micelle layer / Content of functional components before digestion) * 100%

[0118] Table 5. Experiments on Bioacceptability Assessment

[0119]

[0120]

[0121] As seen in formulations 25-31, the molecular weight distribution of walnut peptides and the proportion of their combined content in the emulsion both affect the bioacceptability of curcumin. In formulations 25 and 27, the proportions of walnut peptides with a molecular weight <1000 Da are less than 50% and greater than 90%, respectively, resulting in a significant decrease in the bioacceptability of curcumin, to 16% and 18%, respectively.

[0122] In formulations 28 and 29, the proportion of walnut peptides was 0% and above 1%, respectively, and the bioacceptability of curcumin also decreased significantly. In formulations 30 and 31, the proportion of xanthan gum added was below 0.2% and above 0.8%, respectively, and the bioacceptability of curcumin (9% and 8%) was significantly lower than that of formulation 26 (51%). As can be seen from formulations 32-34, when curcumin was replaced with coenzyme Q10, lutein esters and astaxanthin, it still had a high bioacceptability, with corresponding bioacceptability rates of 56%, 37% and 42%, respectively.

[0123] Example 5: Application effect of the formulation composition under different pH conditions

[0124] The initial pH of the formulation composition was 6.8. The emulsion was adjusted to pH 2.3-10.4 using pH adjusters (15% citric acid solution or 15% potassium hydroxide solution), as shown in Table 6. The D[3,2] / μm, D[4,3] / μm, instability index, and emulsion stability evaluation score of the emulsion were evaluated.

[0125] Table 6 Application of formulation combinations under low pH conditions

[0126]

[0127]

[0128] As shown in formulations 35-39, different pH values ​​also affect the stability of the emulsion. Formulation 35 shows that when the pH is below 2.5, the emulsion stability is compromised, with a stability evaluation score of only 1.5. Formulation 38 shows that without adjusting the pH of the composition, a stable emulsion cannot be prepared, and the stability evaluation score of the prepared emulsion is also only 1.5. Formulation 39 shows that when the emulsion is alkaline, the stability evaluation score is 1.75, indicating that the emulsion stability is also compromised. In summary, the optimal pH for the emulsion is 2.5-4.5.

[0129] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An emulsion composition comprising 0.01-1 wt% of a polypeptide, 0.2-0.8 wt% of a hydrocolloid, 5-25 wt% of a filler, 5-25 wt% of a co-emulsifier, ≤60 wt% of an oil, and water; wherein the polypeptide is one or more of walnut polypeptide, corn polypeptide, wheat polypeptide, and soybean polypeptide, and the proportion of polypeptides with a molecular weight <1000 Da is 50-90%; the pH of the emulsion composition is 2.5-4.

5.

2. The emulsion composition according to claim 1, wherein the emulsion composition comprises a fat-soluble functional ingredient.

3. The emulsion composition according to claim 1, wherein the polypeptide is a single emulsifier.

4. The emulsion composition according to claim 1, wherein the oil content is 30wt%-60wt%.

5. The emulsion composition according to claim 1, wherein the hydrocolloid is one or more of xanthan gum, pectin and locust bean gum.

6. The emulsion composition according to any one of claims 1-5, wherein the filler is one or more of fructooligosaccharides, sucrose, erythritol, xylitol, maltitol, isomaltitol, lactitol, maltodextrin, and xylooligosaccharides.

7. The emulsion composition according to any one of claims 1-5, wherein the co-emulsifier is one or more of glycerol and propylene glycol.

8. The emulsion composition according to any one of claims 1-5, wherein the oil is one or more selected from fish oil, algal oil, soybean oil, corn oil, sunflower seed oil, flaxseed oil, and medium-chain triglycerides.

9. The emulsion composition according to claim 2, wherein the fat-soluble functional component is one or more of curcumin, coenzyme Q10, lutein ester and astaxanthin.

10. The emulsion composition according to claim 9, wherein the content of the fat-soluble functional ingredient is 0.03-1 wt%.

11. The emulsion composition according to any one of claims 1-5, wherein the emulsion composition is free of preservatives.

12. A method for preparing an emulsion using an emulsion composition according to any one of claims 1-11, the method comprising: (1) Prepare an aqueous phase comprising a co-emulsifier, a hydrophilic colloid, a filler, a polypeptide and water; and (2) Pour the oil into the aqueous phase, or add the fat-soluble functional ingredients into the oil to dissolve and obtain the oil phase, and then pour the oil phase into the aqueous phase; to obtain an emulsion, wherein the pH of the emulsion is adjusted to 2.5-4.5 using an acidity regulator.

13. The method according to claim 12, further comprising dissolving a co-emulsifier in water, and then sequentially dissolving the hydrophilic colloid and the filler therein. Add the polypeptide to continue dissolving, adjust the pH of the solution using an acidity regulator, and prepare an aqueous phase for later use.

14. The method according to claim 12, wherein the emulsion is an emulsion with no grease on its surface and without layering.

15. The method according to claim 12, wherein the aqueous phase is prepared by shearing at 6000-12000 rpm for 2-10 min using a high-speed shearing machine.

16. The method of claim 12, wherein the acidity regulator is citric acid.

17. An emulsion prepared by the method according to any one of claims 12-16.

18. Use of the emulsion composition according to any one of claims 1-11 or the emulsion according to claim 17 in the preparation of food, health food, pharmaceuticals and cosmetics.

19. Use of polypeptides as emulsifiers or as a single emulsifier in the preparation of emulsion compositions according to any one of claims 1-11 or in emulsions according to claim 17.

20. A product comprising the emulsion according to claim 17.

21. The product according to claim 20 is one or more of beverages, gummies, soft capsules, and foods for special medical purposes.

22. The product according to claim 21 is a meal replacement shake.

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