A Pickering emulsion based on almond protein isolate and its preparation method and application

Abstract

The present invention discloses a Pickering emulsion based on almond protein isolate and its preparation method and application. The preparation method of the emulsion comprises the following steps: (1) pH offset treatment is performed on almond protein to obtain modified almond protein; (2) modified almond protein is dissolved in water, and sugar substances (glucose, lactose and maltodextrin) are added for glycosylation modification to obtain almond protein glycosylation products; (3) almond protein glycosylation products are dissolved in water as aqueous phase, and β-carotene is added to corn oil as oil phase, and then the two are mixed and homogenized to obtain the Pickering emulsion based on almond protein isolate. The inventive method can improve the emulsification performance of almond protein, can effectively improve the stability of β-carotene, and realize the sustained release delivery of β-carotene.

Description

Technical Field

[0001] The present invention relates to the technical field of almond protein processing, and in particular to a Pickering emulsion based on almond protein isolate, and a preparation method and application thereof. Background Art

[0002] In recent years, people have paid more and more attention to healthy lifestyles, and active ingredients such as curcumin, β-carotene, and astaxanthin have attracted much attention due to their excellent health benefits. Among them, β-carotene is a plant pigment found in many fruits and plants. Its strong free radical scavenging ability gives it a variety of biological effects, such as improving vision, enhancing immunity, anti-inflammatory, lowering blood pressure, and anti-cancer. However, during food processing and storage, various conditions such as heat treatment, ultraviolet radiation, and oxygen will affect its activity. In addition, problems such as low water solubility and low oral bioavailability pose challenges to its application in food.

[0003] Pickering emulsion is a reliable means to achieve the stabilization of bioactive ingredients and is widely used in the food, cosmetics, pharmaceutical and cleaning industries. As a thermodynamically unstable system, Pickering emulsion requires the use of stabilizers to achieve long-term stability. In current research, inorganic particles (silicon dioxide, calcium carbonate and graphene, etc.) and organic particles (soy protein, zein, cellulose nanocrystals and chitosan, etc.) are commonly used as stabilizers. Compared with inorganic particles, protein as a natural amphiphilic polymer can not only stabilize the emulsion, but also has the advantages of being green and safe, widely available, having good biocompatibility, biodegradability, high nutritional value and high acceptability as a food ingredient. In addition, due to people's concerns about the safety of animal protein, plant protein has become the preferred alternative to animal protein.

[0004] Almond is a nutritious food rich in lipids and proteins. As an important raw material for food processing and production, almond has the effects of regulating blood lipids, reducing cholesterol absorption, reducing the occurrence of cardiovascular and cerebrovascular diseases, anti-oxidation and controlling weight. Almond meal is a by-product of the oil extraction industry, containing more than 50% protein, and is a potential source of high-quality plant protein. Almond protein is mainly composed of globulin and albumin, while gluten and alcohol-soluble proteins only account for a small part. In addition, almond protein has good emulsification, foaming and oil retention properties, and can be used as an animal protein substitute. It has high development value in the fields of food supplements and functional foods. However, at present, almond meal is mostly used as feed, fertilizer or directly discarded, causing great waste of resources and ecological pollution. In addition, due to the poor solubility of almond protein in acidic environment, this limits its application in the food industry.

[0005] pH shift is one of the commonly used food processing methods in food industry production and daily life. It puts the protein in an extreme acid-base environment, and through the two processes of unfolding and refolding, the protein is in a "molten state", which significantly improves the solubility and emulsification of the protein. It has the advantages of simple operation, cost-effectiveness and environmental protection. Glycosylation is a widely used protein modification method, but traditional glycosylation modification has problems such as long reaction time and low degree of grafting. The application effect of a single modification method is limited. At present, there are no reports on the synergistic modification of almond protein by combining pH shift with glycosylation. Summary of the invention

[0006] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing a Pickering emulsion based on almond protein isolate.

[0007] Another object of the present invention is to provide a Pickering emulsion based on almond protein isolate prepared by the method.

[0008] Another object of the present invention is to provide an application of the Pickering emulsion based on almond protein isolate.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A method for preparing a Pickering emulsion based on almond protein isolate comprises the following steps:

[0011] (1) pH shift treatment of almond protein isolate: add almond protein to water, stir to dissolve, adjust the pH value to 10.0-12.0, and then stand at room temperature for pH shift treatment. After the treatment is completed, adjust the pH back to neutral, add a NaCl solution with a final concentration of 50 mmol / L, and freeze-dry to obtain modified almond protein;

[0012] (2) Glycosylation modification: dissolving the modified almond protein obtained in step (1) in water, then adding a carbohydrate, stirring and mixing, placing in a 4°C refrigerator overnight for hydration reaction, then adjusting the pH value to 8.0 and sealing and placing at 80±5°C for glycosylation reaction, terminating the reaction in an ice bath after the reaction is completed, and freeze-drying to obtain an almond protein glycosylation product; wherein the carbohydrate is at least one of glucose, lactose and maltodextrin;

[0013] (3) Preparing a Pickering emulsion loaded with β-carotene: dissolving the almond protein glycosylation product obtained in step (2) in water, adjusting the pH value to 7.0, stirring and carrying out a hydration reaction overnight to obtain an aqueous phase; adding β-carotene to corn oil, stirring and mixing in a light-proof water bath at 60±5°C to obtain an oil phase; then mixing the aqueous phase and the oil phase and homogenizing them to obtain a Pickering emulsion loaded with β-carotene, i.e., the Pickering emulsion based on almond protein isolate.

[0014] The almond protein described in step (1) is preferably prepared by the following method: adding defatted almond powder to water, stirring and extracting under the conditions of pH 9.0 and 45±5°C, centrifuging to obtain the supernatant after extraction and adjusting the pH value to 4.5, then centrifuging to obtain the precipitate, and then redissolving the precipitate in water and adjusting the pH value to neutral, dialyzing and freeze-drying to obtain almond protein freeze-dried powder.

[0015] The solid-to-liquid ratio of the defatted almond powder to water is preferably 1:10 (g / mL).

[0016] The water is preferably deionized water.

[0017] The stirring extraction time is 1 to 3 hours, preferably 2 hours.

[0018] The centrifugal conditions are: centrifugation at 8000 rpm for 20 min.

[0019] The dialysis is performed using a dialysis bag with a molecular weight cutoff of 3500Da; preferably, the dialysis is performed for 2 days using a dialysis bag with a molecular weight cutoff of 3500Da.

[0020] The water described in steps (1), (2) and (3) is preferably deionized water.

[0021] The stirring and dissolving time in step (1) is preferably 2 hours.

[0022] The pH value in step (1) is adjusted by using a NaOH solution; preferably, a 2 mol / L NaOH solution is used for adjustment.

[0023] The pH value adjusted in step (1) is preferably adjusted to 12.

[0024] The standing time at room temperature in step (1) is preferably 1 hour.

[0025] In step (1), the concentration of the NaCl solution used is preferably 1 mol / L.

[0026] In step (1), in the system formed after adding the NaCl solution, the final concentration of the modified almond protein is 2% by mass.

[0027] The mass ratio of the modified almond protein and the carbohydrate substance described in step (2) is 1:1.

[0028] The amount of water described in step (2) is calculated based on 25-30 ml of water per gram of almond protein; preferably, it is calculated based on 25 ml of water per gram of almond protein.

[0029] The stirring time in step (2) is preferably 3 h.

[0030] The glycosylation reaction time in step (2) is 0 to 2 h (excluding 0), preferably 0.5 h.

[0031] The concentration of the almond protein glycosylation product in the aqueous phase described in step (3) is 2% by mass.

[0032] The stirring time in step (3) is 2 h.

[0033] The concentration of β-carotene in the oil phase in step (3) is 0.1% by mass.

[0034] The volume ratio of the water phase to the oil phase in step (3) is 1:1.

[0035] The homogenization treatment described in step (3) includes pre-homogenization and high-pressure homogenization; wherein, the condition of the pre-homogenization is: shearing at 20,000 rpm for 1 min; the condition of the high-pressure homogenization is: circulating twice at 60 MPa.

[0036] The pre-homogenization is preferably performed by using a handheld homogenizer at high speed.

[0037] The high-pressure homogenization is preferably performed by using a high-pressure microfluidizer.

[0038] A Pickering emulsion based on almond protein isolate is prepared by any of the methods described above.

[0039] The application of the Pickering emulsion based on almond protein isolate in the food industry.

[0040] Compared with the prior art, the present invention has the following advantages and effects:

[0041] (1) The present invention uses defatted almond protein as a raw material, and synergistically modifies almond protein isolate by pH shift treatment combined with glycosylation; then, β-carotene is added to corn oil, and stirred in a light-proof water bath at 60°C for 2 hours to fully dissolve it; finally, the oil phase containing β-carotene and the water phase containing the modified almond protein isolate are evenly mixed, and a uniform and stable emulsion is obtained by high-speed shearing and high-pressure homogenization; this method improves the emulsification properties of almond protein, and enhances its biological activity by encapsulating β-carotene, and the prepared β-carotene-loaded emulsion has high encapsulation rate and bioavailability, and has excellent stability and sustained-release effect.

[0042] (2) The present invention synergistically modifies almond protein by combining pH shift with glycosylation. Compared with untreated protein and single modified almond protein, the emulsion stabilized by the synergistically modified almond protein has a smaller emulsion droplet size, higher emulsification stability and antioxidant properties.

[0043] (3) The present invention provides an embedding delivery carrier for β-carotene, which has good encapsulation effect and high bioavailability, can effectively improve the stability of β-carotene, and realize the sustained-release delivery of β-carotene.

[0044] (4) The raw protein used in the present invention has high nutritional value, is safe and non-toxic, and has a simple, economical, green and safe preparation process, and is suitable for application in the food industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is an evaluation result diagram of the glycosylation reaction degree of almond protein / modified almond protein; wherein A is the grafting degree; B is the intermediate product content; and C is the browning degree.

[0046] Figure 2 It is the CLSM picture of the emulsion prepared with almond protein / modified almond protein.

[0047] Figure 3 These are the appearance pictures of the emulsion prepared with almond protein / modified almond protein after storage for 0d and 30d.

[0048] Figure 4 It is a graph showing the changes in lipid hydroperoxides (LH) and thiobarbituric acid reactants (TBARs) during storage; wherein A is the change in lipid hydroperoxides; and B is the change in thiobarbituric acid reactants.

[0049] Figure 5 This is a graph showing the release of β-carotene in different emulsions.

[0050] Figure 6 These are optical microscope photos of HepG2 cells in different concentrations of β-carotene emulsions.

[0051] Figure 7 This is a statistical graph of the retention rate of β-carotene after the emulsion was treated at different temperatures.

[0052] Figure 8 This is a statistical graph showing the retention rate of β-carotene in the emulsion at different pH values.

[0053] Fig. 9 This is a statistical graph showing the retention rate of β-carotene in the emulsion at different salt ion concentrations.

[0054] Fig.10 This is a statistical graph showing the retention rate of β-carotene in the emulsion at different illumination times under ultraviolet light.

[0055] Fig.11 This is a statistical graph of the retention rate of β-carotene after the emulsion was stored at room temperature for 30 days. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually in accordance with conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0057] Unless otherwise specified, the test results in the embodiments of the present invention are all obtained by repeating the test more than three times.

[0058] The preparation process of the almond protein isolate involved in the present invention is as follows:

[0059] Defatted almond powder (purchased from Guangdong Nanxing Tianhong Nut Products Co., Ltd.) was added to deionized water at a solid-liquid ratio of 1:10 (g / mL), and the pH was adjusted to 9.0 with a 2mol / L NaOH solution, then stirred in a 45°C water bath for 2h for extraction. After the alkaline extraction, it was centrifuged at 8000rpm for 20min, and the supernatant was taken for use. The pH value of the supernatant was adjusted to 4.5 with a 2mol / L HCl solution, allowed to stand for 30min, centrifuged at 8000rpm for 20min, and the precipitate was collected. The precipitate was redissolved in deionized water, the pH was adjusted to neutral, and dialyzed with a 3500Da dialysis bag for 2d. Finally, it was freeze-dried to obtain almond protein powder (API).

[0060] Example 1 Preparation of pH Shift Processed API

[0061] Weigh 2g of freeze-dried API and add it to 95mL of deionized water, stirring for 2h to fully dissolve it. Then adjust the pH of the solution to 12.0 with 2mol / LNaOH, let it stand at room temperature for 1h, and then adjust the pH of the solution back to neutral to terminate the reaction. Finally, adjust the NaCl concentration of the reaction solution to 50mmol / L with 1mol / L NaCl solution, and make the solution volume to 100mL. A portion of the reactant solution was placed in a -4℃ refrigerator for subsequent testing, and the rest was freeze-dried to obtain modified almond protein PA12.

[0062] Example 2 Preparation of Glycosylated Modified API

[0063] Weigh 4g of API and dissolve it in 100mL of deionized water, then add 4g of different types of sugars (glucose GLC, lactose LAC and maltodextrin MAL (DE: 15-20%; purchased from Shanghai McLean Biotechnology Co., Ltd.)), stir magnetically for 3h and place in a 4℃ refrigerator overnight for hydration. Then adjust the pH of the solution to 8.0 and seal it, place it in a 80℃ constant temperature water bath to react for 0-2h (0.5h in this experiment), and terminate the reaction with an ice bath. A portion of the reactant solution was placed in a -4℃ refrigerator for subsequent testing, and the rest was freeze-dried to obtain almond protein glycosylation product powders, named A-GLC, A-LAC, and A-MAL respectively.

[0064] Example 3 Preparation of pH shift combined with glycosylation modified API

[0065] (1) Weigh 2 g of freeze-dried API and add it to 95 mL of deionized water. Stir for 2 h to fully dissolve it. Then adjust the pH of the solution to 12.0 with 2 mol / L NaOH, let it stand at room temperature for 1 h, and then adjust the pH of the solution back to neutral to terminate the reaction. Finally, adjust the NaCl concentration of the reaction solution to 50 mmol / L with 1 mol / L NaCl solution, and make the solution dilute to 100 mL. Freeze-dry to obtain modified almond protein PA12.

[0066] (2) Weigh 4 g of PA12 and dissolve it in 100 mL of deionized water. Then add 4 g of different types of sugars (glucose GLC, lactose LAC and maltodextrin MAL) respectively. After magnetic stirring for 3 h, place it in a 4 °C refrigerator for hydration overnight. Then adjust the pH of the solution to 8.0 and seal it. Place it in a constant temperature water bath at 80 °C for 0-2 h (0.5 h in this experiment). After the reaction is completed, use an ice bath to terminate the reaction. A portion of the reaction solution is placed in a -4 °C refrigerator for subsequent testing. The rest is freeze-dried to obtain almond protein glycosylation product powders, which are named PA-GLC, PA-LAC, and PA-MAL.

[0067] Example 4 Preparation of simple blend of API and sugar

[0068] 4 g of API was weighed and dissolved in 100 mL of deionized water, followed by the addition of 4 g of different types of sugars (glucose GLC, lactose LAC, and maltodextrin MAL), followed by magnetic stirring for 3 h and then placed in a 4 °C refrigerator overnight for hydration. A portion of the reaction solution was placed in a -4 °C refrigerator for subsequent testing, and the rest was freeze-dried to obtain a simple blend of API and sugar, named A / GLC, A / LAC, and A / MAL, respectively.

[0069] Example 5 Preparation of a simple blend of PA12 and sugar

[0070] (1) Weigh 2 g of freeze-dried API and add it to 95 mL of deionized water. Stir for 2 h to fully dissolve it. Then adjust the pH of the solution to 12.0 with 2 mol / L NaOH, let it stand at room temperature for 1 h, and then adjust the pH of the solution back to neutral to terminate the reaction. Finally, adjust the NaCl concentration of the reaction solution to 50 mmol / L with 1 mol / L NaCl solution, and make the solution dilute to 100 mL. Freeze-dry to obtain modified almond protein PA12.

[0071] (2) 4 g of PA12 was weighed and dissolved in 100 mL of deionized water, and then 4 g of different types of sugars (glucose GLC, lactose LAC and maltodextrin MAL) were added respectively. After magnetic stirring for 3 h, the mixture was placed in a 4 °C refrigerator overnight for hydration. A portion of the reaction solution was placed in a -4 °C refrigerator for subsequent testing, and the rest was freeze-dried to obtain a simple blend of PA12 and sugar, which was named PA / GLC, PA / LAC, and PA / MAL, respectively.

[0072] Example 6 Evaluation of the degree of glycosylation reaction

[0073] (1) Determination of grafting degree: Weigh 80 mg of o-phthalaldehyde (OPA) and dissolve it in 2 mL of methanol, add 50 mL of 0.1 M sodium tetraborate buffer (pH 9.5), 5 mL of 20% (w / v) sodium dodecyl sulfate (SDS) solution and 200 μL of β-mercaptoethanol, and then dilute to 100 mL with deionized water to obtain OPA reagent. Dilute the sample solution to be tested (A-GLC, A-LAC, A-MAL, PA-GLC, PA-LAC, PA-MAL) to a protein concentration (based on unmodified API, the same below) of 5 mg / mL, take 200 μL of the dilution and 4 mL of OPA reagent and mix them evenly, incubate at 35°C for 2 minutes, and measure its absorbance at 340 nm using a UV-visible spectrophotometer. Deionized water is used as a blank control. The grafting degree of the sample is calculated according to the following formula:

[0074]

[0075] In the formula, A his the absorbance of the heated protein, A s is the absorbance of the sample.

[0076] (2) Determination of intermediate products: dilute the sample solution to be tested with deionized water to a protein concentration of 0.5% (w / v), measure the absorbance at 294 nm, and use deionized water as a blank control.

[0077] (3) Determination of browning degree: The sample solution to be tested was diluted to a protein concentration of 1% (w / v), and the absorbance was measured at 420 nm, with deionized water used as a blank control.

[0078] The results are as follows Figure 1 As shown in the figure: 30 minutes before the glycosylation reaction, the grafting degree of all glycosylation products increased rapidly, but within 30-120 minutes, the grafting degree of all glycosylation products did not increase significantly, among which A-GLC, A-LAC, and A-MAL reached the maximum grafting degree at 120 minutes, 90 minutes, and 90 minutes, which were 15.15%, 7.99%, and 6.55%, respectively; PA-GLC, PA-LAC, and PA-MAL all reached the maximum grafting degree at the 30th minute, which were 23.44%, 22.21%, and 21.68%, respectively. This shows that pH shift treatment helps to promote the glycosylation reaction ( Figure 1 A). The intermediate product and browning degree have the same change trend as the grafting degree. With the increase of reaction time, the intermediate product and browning degree first increase significantly within 0-30min, and then increase slowly. Considering the grafting degree, intermediate reaction product and browning degree, the glycosylation reaction time is determined to be 30min ( Figure 1 B and Figure 1 C).

[0079] Example 7

[0080] (1) Determination of free thiol content

[0081] The sample solutions prepared in Examples 1 to 5 were diluted to a protein concentration of 10 mg / mL (with API as a control), and the pH was adjusted to 7.0. Then, 0.5 mL of the 10 mg / mL sample solution was mixed with 2.0 mL of Tris-glycine buffer (86 mmol / LTris, 90 mmol / L glycine, 4 mmol / L ethylenediaminetetraacetic acid (EDTA), pH 8.0), vortexed for 10 seconds, and 50 μL of Ellman's reagent (5,5-dithio-bis-(2-nitrobenzoic acid)) was added. After mixing evenly, the mixture was placed in a 25°C constant temperature water bath for incubation for 30 minutes, and then centrifuged at 10000 rpm for 10 minutes. The supernatant was taken to measure its absorbance at 412 nm, and the absorbance at 13600 M was measured. - 1 cm-1 The free thiol content was calculated as the molar extinction coefficient. The sample solution without Ellman's reagent was used as a blank control. The free thiol content was calculated as follows:

[0082] Free thiol content (μmol / g) = 73.53*A S1 *D / C;

[0083] In the formula, A S1 is the absorbance of the sample at 412 nm, D is the dilution factor, and C is the API concentration (mg / mL).

[0084] Results analysis: The free thiol content of the samples measured by the above method is shown in Table 1. It can be seen from Table 1 that compared with the free thiol content of the untreated API, both single modification (pH shift treatment, glycosylation modification) and synergistic modification (pH shift combined with glycosylation modification) increased the free thiol content of the API. In addition, the glycosylation reaction reduced the content of free thiol groups in the complex, which may be because the thiol groups were oxidized to form disulfide bonds during the reaction.

[0085] (2) Determination of surface hydrophobicity

[0086] The sample solutions prepared in Examples 1 to 5 were diluted to different protein concentrations (0.005, 0.05, 0.10, 0.25 and 0.50 mg / mL) (with API as control) using phosphate buffer (0.1 mol / L, pH 7.0), and then 2.0 mL of sample solutions of different concentrations were respectively drawn, 20 μL of 8 mM 8-aniline-1-naphthalenesulfonic acid (ANS) solution was added, vortexed and mixed, and reacted in the dark for 15 min. The excitation wavelength and emission wavelength of the instrument were set to 390 nm and 468 nm, respectively, the slit width was 5 nm, and the scanning rate was 10 nm / s. The initial slope of the fluorescence intensity and protein concentration obtained by scanning is the surface hydrophobicity of the protein.

[0087] Result analysis: The measurement results are shown in Table 1. It can be seen from Table 1 that compared with the surface hydrophobicity of untreated API, both single modification and synergistic modification increased the surface hydrophobicity of API, and the surface hydrophobicity of API after synergistic modification was the largest.

[0088] (3) Determination of solubility

[0089] The sample solutions prepared in Examples 1 to 5 were diluted with deionized water to a protein concentration of 0.2 mg / mL (with API as a control), and the pH of the dispersion was adjusted to 7.0. After constant stirring for 2 h with a magnetic stirrer, the mixture was centrifuged at 8000 rpm for 15 min. The supernatant was mixed with the Coomassie Brilliant Blue solution, and the absorbance was measured at 595 nm. With bovine serum albumin as a standard, the solubility of the protein sample was calculated according to the following formula:

[0090]

[0091] Where m1 is the protein content of the supernatant and m2 is the total protein content.

[0092] Result analysis: The solubility of the samples measured by the above method is shown in Table 1. It can be seen from Table 1 that compared with the solubility of the untreated API, both single modification and synergistic modification increased the solubility of the API. In addition, the solubility of the synergistically modified glycosylation product is lower than that of the traditional glycosylation product and the simple blend of PA12 and sugar. This may be due to the high surface hydrophobicity of the synergistically modified glycosylation product, which leads to a decrease in its solubility.

[0093] (4) Determination of emulsification properties

[0094] The sample solutions prepared in Examples 1 to 5 were diluted with deionized water to a protein concentration of 1% (w / v) (with API as a control), and the pH was adjusted to 7.0 to obtain a protein solution; then 7.5 mL of the protein solution and 2.5 mL of corn oil were taken and homogenized at 15000 rpm for 1 min using a handheld homogenizer. Immediately after homogenization, 50 μL of fresh emulsion was drawn from the bottom, 0.1% (w / v) sodium dodecyl sulfate (SDS) solution was added and diluted 100 times, and the absorbance of the solution was measured at 500 nm using a UV-visible spectrophotometer, with 0.1% (w / v) SDS solution as a blank control. The emulsifying activity (EAI) and emulsifying stability (ESI) of almond protein were calculated according to the following formulas:

[0095]

[0096]

[0097] In the formula, D represents the dilution factor of the solution (100), C represents the sample concentration before forming the emulsion, represents the volume fraction of the oil phase (0.25), A0 represents the absorbance of the emulsion at 0 min, A 10 Represents the absorbance value of the emulsion after 10 minutes.

[0098] Results: The EAI and ESI of the samples are shown in Table 1. Compared with the EAI and ESI of the untreated API, both single modification and synergistic modification increased the EAI and ESI of the API, and the EAI and ESI of the API after synergistic modification were the largest. This indicates that both pH shift treatment and glycosylation modification can improve the emulsification of the API, and the combination of pH shift and glycosylation can more effectively improve the emulsification activity and emulsification stability of the API.

[0099] Table 1

[0100]

[0101]

[0102] Example 8 Preparation and characterization of stable emulsions of natural almond protein and modified almond protein

[0103] The natural almond protein powder (API) and the modified almond protein prepared in Examples 1 to 5 were dissolved in deionized water (1%, w / v), the pH was adjusted to 7.0, 5% (v / v) corn oil was added as the oil phase, the two phases were mixed evenly, and the crude emulsion was obtained by pre-homogenization at high speed (10000rpm) by a handheld homogenizer for 1min, and then the fine emulsion was obtained by secondary homogenization at 500W power (20kHz) for 6min, and finally a final concentration of 0.02% (w / v) sodium azide solution was added to prevent microbial growth. The microscopic morphology of the emulsion was observed using a laser confocal scanning microscope (CLSM) for sampling, and the fresh emulsion was placed in a sealed screw-cap glass bottle and stored at room temperature for 30d to observe the appearance changes of the emulsion before and after storage.

[0104] The results are as follows Figure 2 and Figure 3 Shown: From Figure 2 It can be seen that compared with the emulsion prepared with untreated protein, the emulsions prepared with single modified and synergistic modified proteins have smaller emulsion droplet size; among them, the emulsions stabilized by PA-GLC, PA-LAC and PA-MAL have the smallest emulsion droplet size, which are 254.53±4.38nm, 256.40±2.25nm and 257.60±1.56nm, respectively; in addition, the emulsions stabilized by PA-GLC, PA-LAC and PA-MAL have the smallest Zeta potential, which are -44.23±0.70mV, -45.54±0.25mV and -41.97±0.21mV, respectively. The above results indicate that the combination of pH shift and glycosylation can more effectively improve the emulsification activity and emulsification stability of API. Figure 3 It can be seen that after 30 days of storage, the emulsion prepared by API had serious stratification, and the emulsions prepared by PA12, A / GLC, A / LAC, A / MAL, PA / GLC, PA / LAC, PA / MAL, A-GLC, A-LAC, and A-MAL had lower stratification than API. However, the emulsions prepared by PA-GLC, PA-LAC, and PA-MAL did not show stratification after 30 days of storage, showing good storage stability.

[0105] Example 9 Determination of oil oxidation products in natural almond protein and modified almond protein stabilized emulsions

[0106] The fresh emulsions prepared in Example 8 were placed in sealed screw-capped glass tubes and stored at room temperature for 30 days, and then the lipid hydroperoxides and thiobarbituric acid reactants were determined in the following steps:

[0107] (1) Lipid hydroperoxide (LH): 0.5 mL of the emulsion sample was added to 1.5 mL of mixture A (isooctane: 2-propanol = 3:1, v / v), and then vortexed 3 times (20 s each time). After centrifugation at 3000 g for 5 min, 0.2 mL of the supernatant was collected and added to 2.8 mL of mixture B (methanol: 1-butanol = 2:1, v / v). Next, 15 uL of ammonium thiocyanate solution (3.94 M) and 15 uL of ferrous ion solution (0.288 M ferrous sulfate solution and 0.264 M barium chloride solution were mixed in equal volumes, and the supernatant was taken after centrifugation, which was the ferrous ion solution). After incubation at 25 ° C for 20 min, the absorbance was measured at 510 nm. The LH content was calculated based on the H2O2 standard curve.

[0108] (2) Thiobarbituric acid reactants (TBARS): 0.2 mL of the emulsion sample was mixed with 1.8 mL of distilled water and 4 mL of thiobarbituric acid (TBA), and the mixture was placed in boiling water for 15 min. After centrifugation at 10,000 rpm for 5 min, the supernatant was taken and the absorbance at 532 nm was measured. The level of TBARS was obtained from the standard curve of 1,1,3,3-tetraethoxypropane.

[0109] Changes in LH and TBARs content of different emulsions after storage at room temperature for 30 days Figure 4 As shown: During the entire storage process, the LH content of all samples gradually increased, and the change curve of TBARs content showed the same trend as LH, among which the LA and TBARs content of PA-GLC, PA-MAL and PA-LAC increased the slowest. The above results show that compared with unmodified API, single modified API and simple mixture, the synergistic modified API has a better ability to inhibit lipid oxidation.

[0110] In summary, compared with unmodified API, single modified API and simple mixture, synergistic modified API has better emulsification, emulsion stability and ability to inhibit lipid oxidation. Therefore, PA-GLC, PA-MAL and PA-LAC were selected to prepare Pickering emulsions loaded with β-carotene, and Tween-20 was used as a control to further explore its release characteristics.

[0111] Example 10 Preparation of Pickering emulsion loaded with β-carotene

[0112] PA-GLC, PA-MAL and PA-LAC were dissolved in deionized water (2%, w / v), adjusted to pH 7.0, stirred for 2h and hydrated overnight to obtain an aqueous phase. 0.1% (w / v) β-carotene (BC) was added to corn oil and stirred in a dark water bath at 60°C for 2h to obtain an oil phase. After the two phases were mixed evenly in a ratio of 1:1 (v / v), a crude emulsion was obtained by high-speed pre-homogenization with a handheld homogenizer (10000rpm, 1min), and then a high-pressure microfluidizer was used for high-pressure homogenization (60MPa, two cycles) to obtain a Pickering emulsion loaded with β-carotene. At the same time, Tween-20 was used as a control, that is, Tween-20 was added to deionized water to prepare a 2% (w / v) Tween-20 solution as the aqueous phase, and the remaining steps were the same as the preparation steps of the Pickering emulsion loaded with β-carotene.

[0113] Example 11 Determination of β-carotene embedding rate

[0114] Take 1 mL of the Pickering emulsion loaded with β-carotene prepared in Example 10, add 3 mL of n-hexane, vortex vigorously, centrifuge at 10000g for 5 min, collect the n-hexane phase, and continue to extract and precipitate with n-hexane until the sample is clear, and finally use a multifunctional microplate reader to measure the absorbance of the sample at 450nm. The emulsion loaded with β-carotene in Tween-20 is used as a control. The calculation formula of β-carotene (BC) embedding rate is as follows:

[0115]

[0116] Results: The embedding efficiency of β-carotene in the emulsions prepared by Tween-20, PA-GLC, PA-LAC and PA-MAL were 43.17±2.94%, 83.65±1.03%, 87.62±0.40% and 81.41±0.45%, respectively. Compared with Tween-20, the emulsions prepared by PA-GLC, PA-LAC and PA-MAL had higher embedding efficiency.

[0117] Example 12 Release of β-carotene

[0118] (1) Construction of an in vitro simulated gastrointestinal digestion model: ① Take 10 mL of the prepared simulated gastric fluid SGF (containing 2 g / L sodium chloride, 3.2 g / L pepsin and 7 mL / L concentrated hydrochloric acid (12 M)). Adjust the pH to 2.0 with 0.5 M sodium hydroxide solution and incubate at 37° C. for 5 min. Add the β-carotene-loaded Pickering emulsion sample prepared in Example 10 to the simulated gastric fluid SGF at a ratio of 1:1 (v / v), and vibrate the mixture in a water bath for 2 h (37° C., 250 rpm). Use the Tween-20-loaded β-carotene emulsion as a control. After gastric digestion is completed (2 h), take a sample for subsequent analysis. ② Prepare 20 mL of simulated intestinal fluid SIF (containing 8 mg / mL bile salts, 1 mg / mL pancreatic enzymes and 5 mM calcium chloride; adjust the pH of the phosphate buffer to 7.0) and incubate at 37° C. for 5 min. The samples after gastric digestion were added to simulated intestinal fluid (SIF), and the pH of the digestion solution was immediately adjusted to 7.0 with 1.0 M sodium hydroxide solution, followed by digestion in a water bath shaker for 2 h.

[0119] (2) In the in vitro digestion test, 0.5 mL of sample was collected every 20 min, and then 0.5 mL of deionized water, 2 mL of anhydrous ethanol and 3 mL of n-hexane were added. After vortexing for 10 s, the sample was allowed to stand for 10 min. The n-hexane phase was then removed and the remaining sample was extracted twice with n-hexane until the n-hexane phase was colorless. The n-hexane phases were combined and the volume was adjusted to 10 mL. Finally, the absorbance was measured at 450 nm. The release rate of β-carotene was calculated according to the following formula:

[0120]

[0121] Where: C1 represents the concentration of β-carotene released after simulated gastrointestinal digestion, and C0 represents the concentration of β-carotene in the original emulsion.

[0122] The release of β-carotene from different emulsions during in vitro digestion Figure 5 As shown: the release of β-carotene from Tween-20, PA-GLC, PA-LAC and PA-MAL emulsions increased with the increase of digestion time. Compared with Tween-20, the emulsions prepared by PA-GLC, PA-LAC and PA-MAL had a better sustained release effect.

[0123] Example 13 Bioaccessibility of β-carotene

[0124] The sample after in vitro digestion in Example 12 (4 hours in total) was centrifuged at 15,000 rpm for 30 minutes to obtain three layers of separation. They are the undigested oil upper layer, the difficult-to-digest or undigested solid lower layer, and the micelle middle layer representing the digestion process. The middle micelle phase was aspirated using a syringe, the content of β-carotene was determined (the method is the same as in Example 12), and the bioaccessibility of β-carotene was calculated; wherein the bioaccessibility of β-carotene was calculated according to the following formula:

[0125]

[0126] Where C1 represents the concentration of β-carotene in micelles, and C0 represents the concentration of β-carotene in the original emulsion.

[0127] Results: The bioaccessibility of β-carotene in the emulsions prepared by Tween-20, PA-GLC, PA-LAC and PA-MAL was 20.21±1.06%, 51.26±0.91%, 56.04±0.70% and 48.41±0.14%. Compared with Tween-20, the emulsions loaded with β-carotene prepared by PA-GLC, PA-LAC and PA-MAL had stronger bioaccessibility.

[0128] Example 14 Effects of different β-carotene emulsions on cell activity

[0129] HepG2 cells (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were added to DMEM complete medium containing 1% (w / v) penicillin, 1% (w / v) streptomycin and 10% (v / v) fetal bovine serum and cultured in a cell culture incubator at 37°C and 5% CO2. 0.25% trypsin-EDTA was used to detach cells from the culture flask. HepG2 cells in the logarithmic growth phase were plated at 1×10 4 The cells were inoculated at a density of 10 cells / well in a 96-well plate, 5 replicate wells were set up, and cultured in an incubator for 24 hours. The PA-GLC, PA-LAC and PA-MAL stabilized emulsion samples prepared in Example 10 were added at different concentrations (50, 100, 250, 500, 1000 μg / mL) and cultured for 24 hours. Then 10 μL of CCK-8 solution was added to each well of the plate and placed in a 37°C incubator and shaken for 3 hours. The absorbance was then detected using a multifunctional microplate reader with a wavelength set to 450nm. Cells treated with pure culture medium were used as negative controls. The cell survival rate was calculated according to the following formula:

[0130]

[0131] Result analysis: Figure 6Optical microscope photos of HepG2 cells in different concentrations of β-carotene emulsions. Figure 6 It can be seen that most of the cells in the blank control group were spindle-shaped or triangular-shaped, and there were no large numbers of dead cells suspended in the culture medium. The cells treated with the five concentrations of emulsions were spindle-shaped or triangular-shaped, and no obvious shrinkage occurred. Moreover, the cell viability of the cells treated with the three emulsions in the concentration range of 50-1000 μg / mL was above 90%. The above results show that the β-carotene emulsions prepared by 50-1000 μg / mL PA-GLC, PA-LAC and PA-MAL have no toxic effect on cells.

[0132] Example 15 Processing stability of β-carotene emulsion

[0133] (1) Thermal stability: 2 mL of the β-carotene-loaded Pickering emulsion prepared in Example 10 was transferred to glass bottles, and then placed in water baths at 25, 50, 70, and 90° C., respectively. After 30 min, the emulsion was taken out and cooled in an ice bath, and the β-carotene retention rate was measured. The β-carotene-loaded emulsion prepared with Tween-20 was used as a control.

[0134] (2) pH stability: 2 mL of the β-carotene-loaded Pickering emulsion prepared in Example 10 was transferred to glass bottles, and the pH thereof was adjusted to 3, 5, 7, 9, and 11, respectively, and the β-carotene retention rate was measured. The β-carotene-loaded emulsion prepared with Tween-20 was used as a control.

[0135] (3) Ionic stability: NaCl was added to the β-carotene-loaded Pickering emulsion prepared in Example 10 to prepare β-carotene emulsions containing different salt concentrations (NaCl concentrations were 0, 100, 200, 300, and 400 mmol / L), and the β-carotene retention rate was measured. The β-carotene-loaded emulsion prepared with Tween-20 was used as a control.

[0136] (4) Light stability: 2 mL of the β-carotene-loaded Pickering emulsion prepared in Example 10 was transferred to glass bottles and exposed to ultraviolet light (365-375 nm) for 6 h. The β-carotene content was measured every 2 h, and the β-carotene retention rate was measured after the light exposure. The β-carotene-loaded emulsion prepared with Tween-20 was used as a control.

[0137] (5) Storage stability: The β-carotene-loaded Pickering emulsions prepared in Example 10 were placed at room temperature (25° C.) in the dark and stored for 30 days, and samples were taken regularly every week to determine the β-carotene retention rate. The β-carotene-loaded emulsion prepared with Tween-20 was used as a control.

[0138] The calculation formula of the above β-carotene retention rate is as follows:

[0139]

[0140] Where C1 is the concentration of β-carotene in the sample, and C2 is the original concentration of β-carotene in the emulsion.

[0141] Result analysis: Figures 7 to 11 As shown, compared with Tween-20, the β-carotene-loaded emulsions prepared by PA-GLC, PA-LAC and PA-MAL have better processing stability.

[0142] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a Pickering emulsion based on almond protein isolate, characterized in that: The steps include: (1) pH shift treatment of almond protein isolate: add almond protein to water, stir to dissolve, adjust the pH value to 12.0, and then let it stand at room temperature for pH shift treatment. After the treatment is completed, adjust the pH back to neutral, add a NaCl solution with a final concentration of 50 mmol / L, and freeze-dry to obtain modified almond protein; (2) Glycosylation modification: dissolving the modified almond protein obtained in step (1) in water, then adding a sugar substance, stirring and mixing evenly, placing in a refrigerator at 4°C overnight for hydration reaction, then adjusting the pH value to 8.0 and sealing and placing at 80±5°C for glycosylation reaction, after the reaction is completed, terminating the reaction in an ice bath, freeze-drying, and obtaining an almond protein glycosylation product; wherein the sugar substance is at least one of glucose, lactose and maltodextrin; (3) Preparing a Pickering emulsion loaded with β-carotene: dissolving the almond protein glycosylation product obtained in step (2) in water, adjusting the pH value to 7.0, stirring and carrying out a hydration reaction overnight to obtain an aqueous phase; adding β-carotene to corn oil, stirring and mixing in a light-proof water bath at 60±5°C to obtain an oil phase; then mixing the aqueous phase and the oil phase and homogenizing them to obtain a Pickering emulsion loaded with β-carotene, i.e., the Pickering emulsion based on almond protein isolate; The almond protein described in step (1) is prepared by the following method: adding defatted almond powder to water, stirring and extracting at pH 9.0 and 45±5°C, centrifuging to obtain the supernatant after extraction and adjusting the pH value to 4.5, then centrifuging to obtain the precipitate, then redissolving the precipitate in water and adjusting the pH value to neutral, dialyzing and freeze-drying to obtain almond protein freeze-dried powder; The mass ratio of the modified almond protein and the carbohydrate substance described in step (2) is 1:1; The concentration of the almond protein glycosylation product in the aqueous phase described in step (3) is 2% by mass; The concentration of β-carotene in the oil phase in step (3) is 0.1% by mass; The volume ratio of the water phase to the oil phase in step (3) is 1:

1.

2. The method for preparing a Pickering emulsion based on almond protein isolate according to claim 1, characterized in that: The solid-to-liquid ratio of the defatted almond powder to water is 1 g / 10 mL; The stirring extraction time is 1 to 3 hours; The dialysis is performed using a dialysis bag with a molecular weight cut-off of 3500Da.

3. The method for preparing a Pickering emulsion based on almond protein isolate according to claim 1, characterized in that: The homogenization treatment described in step (3) includes pre-homogenization and high-pressure homogenization; wherein the condition of the pre-homogenization is: shearing at 20,000 rpm for 1 min; the condition of the high-pressure homogenization is: circulating twice at 60 MPa.

4. The method for preparing a Pickering emulsion based on almond protein isolate according to claim 1, characterized in that: The stirring and dissolving time in step (1) is 2 h; The pH value in step (1) is adjusted by using a NaOH solution; The standing time at room temperature in step (1) is 1 h; The glycosylation reaction time in step (2) is 0 to 2 hours, excluding 0; The stirring time in step (2) is 3 h; The stirring time in step (3) is 2 h.

5. A Pickering emulsion based on almond protein isolate, characterized in that: It is prepared by the method according to any one of claims 1 to 4.

6. Application of the Pickering emulsion based on almond protein isolate according to claim 5 in the food industry.

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