Preparation method and application of ethanol modified soybean protein isolate-chitosan co-stabilized high internal phase emulsion

Abstract

The invention provides a preparation method and application of an ethanol-modified soybean protein isolate-chitosan co-stable high internal phase emulsion, natural soybean protein isolate is modified by adding ethanol to obtain soybean protein isolate particles, then chitosan is added as an emulsifier, the high internal phase emulsion is prepared by using an ultrasonic-centrifugal means, and the high internal phase emulsion is prepared by using an ethanol-modified soybean protein isolate-chitosan co-stable high internal phase emulsion. Therefore, an adjustable high-internal-phase emulsion with excellent viscoelasticity and higher stability is prepared, and the encapsulation efficiency of astaxanthin and the stability in a complex environment can be effectively improved. The preparation process is completely established on the basis of natural'green ', does not use any synthetic particles or surfactants, does not generate harmful substances, is short in production cycle, rich in nutrition, high in economic utilization value and convenient to store, and improves the application potential in the fields of foods, packaging materials, medicines and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of high internal phase emulsion preparation, and specifically relates to a preparation method and application of an ethanol-modified soybean protein isolate-chitosan co-stabilized high internal phase emulsion. Background Art

[0002] High Internal Phase Emulsions (HIPEs) refer to emulsions with a dispersed phase volume fraction greater than or equal to 74%. Due to their superior stability, semisolid texture, and adjustable viscoelasticity, HIPEs have become a research hotspot in the field of food colloids. HIPEs can be prepared by one-step, two-step, phase-inversion, and mold-curing methods, each of which has its own advantages and limitations for specific applications. Various bioactive substances can be encapsulated in HIPEs to improve their stability and bioavailability, and new preparation methods and natural amphiphilic polymers (such as proteins and polysaccharides) stabilizers are still needed to prepare HIPEs with enhanced stability.

[0003] Soy protein isolate contains 20 essential amino acids and is widely used in the food industry due to its nutritional value and versatility. The amphiphilicity of soy protein isolate can achieve diffusion and adsorption behavior at the interface of oil droplets during the emulsification process, thereby stabilizing the emulsion interface. Although the emulsification of soy protein isolate has been widely recognized in a large number of studies, it still does not meet the requirements of high emulsification performance proteins in the actual production of the food industry, so its application range is very limited. Therefore, when soy protein isolate is used alone as an emulsifier, it is generally necessary to improve its functional properties by modification, addition of polysaccharides, etc. Among them, ethanol has a smaller polarity and a lower dielectric constant than water, which can easily destroy the non-covalent interaction of proteins and effectively improve the emulsification of proteins; chitosan is non-toxic and healthy, has excellent hydrophilic behavior and thickening properties, and the presence of hydroxyl and amino groups provides the potential to enhance the stability of emulsions. The combination of the two modification methods can greatly improve the emulsification ability of soy protein isolate.

[0004] Astaxanthin is a carotenoid and is the best natural additive with antioxidant effect found in nature so far. However, astaxanthin has poor water solubility and is unstable in high temperature, oxygen and light environment, and is easily destroyed, thereby reducing its biological activity. Therefore, using HIPEs to protect astaxanthin from damage is of practical significance to the development of astaxanthin in the food industry. Summary of the invention

[0005] The main purpose of the present invention is to propose a preparation method and application of an ethanol-modified soy protein isolate-chitosan co-stabilized high internal phase emulsion, aiming to provide a preparation method that effectively maintains the stability of HIPE and apply it to the encapsulation of astaxanthin.

[0006] To achieve the above objectives, the present invention provides a method for preparing an ethanol-modified soy protein isolate-chitosan co-stabilized high internal phase emulsion, wherein soy protein isolate particles are prepared using ethanol-modified soy protein isolate, chitosan is added as an emulsifier, and an O / W high internal phase emulsion is prepared using ultrasound-centrifugation means to encapsulate astaxanthin and improve the encapsulation rate of astaxanthin.

[0007] The present invention provides a preparation method and application of an ethanol-modified soy protein isolate-chitosan co-stabilized high internal phase emulsion, which is characterized by comprising the following steps: Step 1, dissolving soy protein isolate in an ethanol solution, sealing the solution with a plastic wrap and stirring at a low speed for 4 hours, removing the ethanol by a rotary evaporator, and freeze-drying to obtain modified soy protein isolate particles; Step 2, adjusting the pH values ​​of the modified soy protein isolate solution and the chitosan solution to 2 respectively, mixing the two in a volume ratio of 1:1 to obtain a modified soy protein isolate-chitosan mixed solution as an aqueous phase; Step 3, soybean oil is used as the oil phase, the water phase and the oil phase are mixed in a volume ratio of 7:3, and the mixture is processed to obtain a pre-emulsion; Step 4, adjusting the pH value of the pre-emulsion and centrifuging to obtain HIPEs; Optionally, in step 1, the ethanol treatment concentration is 0%, 40% (v / v); Optionally, in step 2, the mass fraction of the modified soybean protein is 5%, and the mass fraction of chitosan is 0% or 4%; Preferably, in step 2, the pH values ​​of the modified soy protein isolate solution and the chitosan solution are adjusted to 2 respectively; Optionally, in step 3, the mixture is treated by homogenization (10,000 rpm) or ultrasound (250 W cm -2 ); Preferably, in step 4, the pH value of the pre-emulsion is adjusted to 5; Preferably, in step 4, the centrifugal force is 10000 g and the centrifugation time is 5 min; The present invention also provides a method for preparing an ethanol-modified soybean protein isolate-chitosan co-stabilized high internal phase emulsion, which is applied in embedding astaxanthin, a hydrophobic active substance.

[0008] The effective effects of the present invention are:

[0009] (1) The present invention prepares a high internal phase emulsion, wherein natural soy protein isolate is modified by adding ethanol to obtain soy protein isolate particles, and chitosan is added as an emulsifier to prepare the high internal phase emulsion by ultrasound-centrifugation, thereby preparing a high internal phase emulsion with excellent viscoelastic properties, stronger stability and adjustable properties.

[0010] (2) The high internal phase emulsion prepared by the present invention is completely based on natural "green" principles, does not use any synthetic particles or surfactants, does not produce harmful substances, has a short production cycle, is rich in nutrition, has high economic value, is easy to store, has good self-sustaining properties, and can improve its application potential in the fields of food, packaging materials, and medicine.

[0011] (3) The high internal phase emulsion prepared by the present invention can be used to encapsulate astaxanthin, and the astaxanthin encapsulation rate is as high as 96.08%. It has good thermal stability. After heating at 90°C for 1 hour, the astaxanthin retention rate is as high as 98.69%, which greatly retains the astaxanthin. It also has good storage stability and can retain astaxanthin for at least two months without leakage. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0013] Figure 1 Comparison of astaxanthin encapsulation efficiency in Examples 1 to 3 of the present invention and Comparative Example 2

[0014] Figure 2 The astaxanthin retention rate of HIPEs after heating at 60 and 90°C in Examples 1 to 3 of the present invention and Comparative Examples 1 to 2 is shown in FIG.

[0015] Figure 3 The retention rate of astaxanthin after freezing and thawing of HIPEs in Examples 1 to 3 of the present invention and Comparative Examples 1 to 2 is shown in FIG.

[0016] Figure 4 The retention rate of astaxanthin in HIPEs of Examples 1 to 3 of the present invention and Comparative Examples 1 to 2 after storage for 56 days DETAILED DESCRIPTION

[0018] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below. If the specific conditions are not specified in the embodiments, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. The technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0019] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0020] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0021] In the following examples, the defatted soybean meal was purchased from Zhaoyuan Wenji Food Co., Ltd., on which soybean protein isolate was extracted, and the chitosan was purchased from Beijing Solable Technology Co., Ltd.

[0022] Example 1

[0023] (1) Soybean protein isolate was extracted from defatted soybean meal using the “alkali extraction and acid precipitation” method.

[0024] (2) SPI (1%) was dissolved in 40% (v / v) ethanol, and the solution was stirred at a low speed for 4 hours in a glass beaker covered with a sealing film (to avoid ethanol evaporation). The ethanol was then removed by a rotary evaporator (40°C), and the sample was freeze-dried to obtain ethanol-modified soy protein isolate particles.

[0025] (3) Dissolve ethanol-modified soy protein isolate (5 mg / mL) in deionized water and stir for 2 h to obtain an ethanol-modified soy protein isolate solution as the aqueous phase; dissolve astaxanthin in soybean oil (0.2 mg / mL) to obtain an oil phase.

[0026] (4) The water phase and the oil phase were mixed in a ratio of 7:3 and shear homogenized at 10,000 rpm to prepare an O / W pre-emulsion.

[0027] (5) Centrifuge the O / W pre-emulsion at 10,000 g for 5 min to obtain astaxanthin-loaded HIPE.

[0028] Example 2

[0029] (1) Soybean protein isolate was extracted from defatted soybean meal using the “alkali extraction and acid precipitation” method.

[0030] (2) SPI (1%) was dissolved in 40% (v / v) ethanol, and the solution was stirred at a low speed for 4 hours in a glass beaker covered with a sealing film (to avoid ethanol evaporation). The ethanol was then removed by a rotary evaporator (40°C), and the sample was freeze-dried to obtain ethanol-modified soy protein isolate particles.

[0031] (3) Dissolve the ethanol-modified soy protein isolate in deionized water (5 mg / mL) and stir for 2 h to obtain an ethanol-modified soy protein isolate solution as the aqueous phase; dissolve astaxanthin in soybean oil (0.2 mg / mL) to obtain an oil phase.

[0032] (4) The water phase and the oil phase were mixed in a volume ratio of 7:3 and heated at 250 W cm -2 The O / W pre-emulsion was prepared by ultrasonication at an ultrasonic power of 10 min (5 s on, 2 s off).

[0033] (5) Centrifuge the O / W pre-emulsion at 10,000 g for 5 min to obtain astaxanthin-loaded HIPE.

[0034] Example 3

[0035] (1) Soybean protein isolate was extracted from defatted soybean meal using the “alkali extraction and acid precipitation” method.

[0036] (2) SPI (1%) was dissolved in 40% (v / v) ethanol, and the solution was stirred at a low speed for 4 hours in a glass beaker covered with a sealing film (to avoid ethanol evaporation). The ethanol was then removed by a rotary evaporator (40°C), and the sample was freeze-dried to obtain ethanol-modified soy protein isolate particles.

[0037] (3) Ethanol-modified soy protein isolate was dissolved in deionized water (5 mg / mL) and stirred for 2 h to obtain an ethanol-modified soy protein isolate solution. Chitosan (4 mg / mL) was dissolved in acetic acid solution (0.1%, v / v) to prepare a chitosan solution. Astaxanthin was dissolved in soybean oil (0.2 mg / mL) to obtain an oil phase.

[0038] (4) The pH values ​​of the modified soy protein isolate solution and the chitosan solution were adjusted to 2 respectively, and the two were mixed in equal volumes at a ratio of 1:1 to obtain a mixed solution of ethanol-modified soy protein isolate and chitosan as the aqueous phase.

[0039] (5) The water phase and the oil phase were mixed in a volume ratio of 7:3 and heated at 250 W cm -2 The O / W pre-emulsion was prepared by ultrasonication at an ultrasonic power of 10 min (5 s on, 2 s off).

[0040] (6) The pH value of the O / W pre-emulsion was adjusted to 5 and centrifuged at 10,000 g for 5 min to obtain the astaxanthin-loaded HIPE.

[0041] Comparative Example 1

[0042] Astaxanthin was dissolved in soybean oil (0.2 mg / mL) and stirred overnight to obtain an emulsion encapsulating astaxanthin.

[0043] Comparative Example 2

[0044] (1) Dissolve soy protein isolate in deionized water (5 mg / mL) and stir for 2 h to obtain a soy protein isolate solution as the aqueous phase; dissolve astaxanthin in soybean oil (0.2 mg / mL) to obtain an oil phase.

[0045] (2) The soy protein isolate solution and astaxanthin-loaded soybean oil were mixed in a volume ratio of 7:3 and sheared and homogenized at 10,000 rpm to prepare an O / W pre-emulsion.

[0046] (2) Centrifuge the O / W pre-emulsion at 10,000 g for 5 min to obtain astaxanthin-loaded HIPE.

[0047] Test methods and results

[0048] (1) Method for evaluating astaxanthin encapsulation efficiency

[0049] 0.2 g of the astaxanthin-loaded high internal phase emulsion was mixed with 4.8 ml of a mixture of methanol and dichloromethane (1:2, v / v), shaken for 3 min and centrifuged at 6000 rpm for 10 min. The absorbance of the supernatant was measured at 480 nm using a Lambda 35 UV spectrophotometer.

[0050] Astaxanthin encapsulation rate

[0051] Among them, m 1 is the encapsulated amount of astaxanthin; m 0 is the amount of astaxanthin added, the standard curve of astaxanthin: y=5.964x+0.0386 (R 2 =0.9981), the embedding efficiency of astaxanthin is as follows Figure 1 As shown, the astaxanthin embedding rate in Example 3 is the highest, which is 96.07%, and the astaxanthin embedding rate in Comparative Example 2 is the lowest, which is 55.48%.

[0052] (2) Product thermal stability analysis and testing methods

[0053] HIPEs were placed in a sample bottle and heated at 60 and 90 °C for 60 min in the dark, and then immediately cooled to room temperature. The astaxanthin concentration was measured using test method (1) and the astaxanthin retention rate was calculated according to the following formula:

[0054] Astaxanthin retention rate (%)

[0055] Among them, C 0 and C t are the initial and heated astaxanthin concentrations, respectively. The thermal stability of HIPEs is shown in Figure 2 As shown, the examples are higher than the control examples, and the astaxanthin retention rate of Example 3 is the largest, which is 98.60%. At this time, the astaxanthin will not be destroyed at a high temperature of 90°C, indicating that the HIPE structure is well preserved at this time.

[0056] (3) Product freeze-thaw analysis and testing methods

[0057] Place HIPEs in a sample bottle and place it in the dark at -20°C for 24 h. Then take it out and place it for 2 h. Measure the astaxanthin concentration using test method (1) and calculate the astaxanthin retention rate.

[0058] Astaxanthin retention rate (%)

[0059] Among them, C 0 and C 1 are the initial and post-freeze-thaw astaxanthin concentrations, respectively. The retention rate of astaxanthin is as follows Figure 3 As shown, under freezing conditions, astaxanthin itself is not easily destroyed, but HIPEs are very unstable. Among all the examples, the astaxanthin retention rate of Example 3 is the largest, which is 97.54%, indicating that the HIPE at this time can encapsulate astaxanthin intactly and has the best freeze-thaw stability.

[0060] (4) Product storage stability and testing methods

[0061] The HIPEs were placed in sample bottles and stored at 4°C in the dark for 7, 14, 21, 28, and 56 days. The astaxanthin concentration was measured using the test method (1) and the astaxanthin retention rate was calculated.

[0062] Astaxanthin retention rate (%)

[0063] Among them, C 0 and C t are the concentrations of astaxanthin at the initial stage and on the tth day, respectively. The retention rate of astaxanthin is as follows: Figure 4As shown, at the same time, all the examples were higher than the comparative examples, among which Example 3 had the highest astaxanthin retention rate of 94.79%, which was much higher than other examples and comparative examples, indicating that after two months of storage, there was no change in the appearance of HIPEs.

[0064] In summary, the present invention is based on ethanol-modified soy protein isolate-chitosan to prepare a high internal phase emulsion, which has a prolonged storage period and good freeze-thaw stability. It is used to load astaxanthin and can resist changes in the external environment to achieve the purpose of slow decomposition of astaxanthin.

[0065] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A preparation method and application of an ethanol-modified soybean protein isolate-chitosan co-stabilized high internal phase emulsion, characterized in that: The following steps are involved: Step 1, dissolving soy protein isolate in an ethanol solution, stirring the solution at a low speed for 4 hours in a beaker covered with a sealing film, removing the ethanol by a rotary evaporator, and obtaining modified soy protein isolate particles after freeze-drying; Step 2, respectively adjusting the pH values ​​of the modified soy protein isolate solution and the chitosan solution, mixing the two in a volume ratio of 1:1 to obtain a modified soy protein isolate-chitosan mixed solution as an aqueous phase; Step 3, soybean oil is used as the oil phase, the water phase and the oil phase are mixed in a volume ratio of 7:3, and the mixture is processed to obtain a pre-emulsion; Step 4, adjusting the pH value of the pre-emulsion and centrifuging to obtain HIPEs; 2. The preparation method and application of an ethanol-modified soybean protein isolate-chitosan co-stabilized high internal phase emulsion according to claim 1, characterized in that: In step 1, The ethanol treatment concentration is 0% (v / v) or 40% (v / v); 3. The preparation method and application of an ethanol-modified soybean protein isolate-chitosan co-stabilized high internal phase emulsion according to claim 1, characterized in that: In step 2, The mass fraction of modified soy protein isolate is 5%, and the mass fraction of chitosan is 0% or 4%; 4. The preparation method and application of an ethanol-modified soybean protein isolate-chitosan co-stabilized high internal phase emulsion according to claim 1, characterized in that: In step 2, The pH values ​​of the modified soy protein isolate solution and chitosan solution were adjusted to 2 respectively; 5. The preparation method and application of an ethanol-modified soybean protein isolate-chitosan co-stabilized high internal phase emulsion according to claim 1, characterized in that: In step 3, The mixture was treated by homogenization (10,000 rpm) or ultrasound (250 W cm -2 ); 6. The preparation method and application of an ethanol-modified soybean protein isolate-chitosan co-stabilized high internal phase emulsion according to claim 1, characterized in that: In step 4, The pH value of the pre-emulsion was adjusted to 5; 7. The preparation method and application of an ethanol-modified soybean protein isolate-chitosan co-stabilized high internal phase emulsion according to claim 1, characterized in that: In step 4, The centrifugal force was 10000 g and the centrifugation time was 5 min; 8. A preparation method and application of an ethanol-modified soybean protein isolate-chitosan co-stabilized high internal phase emulsion, characterized in that: Prepared according to the preparation method according to any one of claims 2 to 7; 9. A method for preparing an ethanol-modified soy protein isolate-chitosan co-stabilized high internal phase emulsion according to claims 2-8 and its application in encapsulating astaxanthin, a hydrophobic active substance.

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