Isoelectric point determination method of gel composition

Abstract

The invention provides an isoelectric point determination method of a gel composition. The isoelectric point determination method comprises the following steps: S1, preparing a colloidal solution from the gel composition taking gelatin as a colloid component and water; s2, sequentially adding ethanol and diethyl ether into the colloidal solution to obtain a first mixed solution, performing oscillation and standing layering treatment, removing supernatant, adding diethyl ether, and repeating the operation to obtain a second mixed solution; s3, adding ethanol into the second mixed solution to obtain a colloidal precipitate, and drying the colloidal precipitate to obtain a colloidal sample; and S4, preparing a colloidal solution from the colloidal sample and water, carrying out deionization treatment to obtain a solution to be detected, and measuring the pH value of the solution to be detected, namely the isoelectric point of the gel composition. According to the method, other components in the gel composition are removed through an ethanol-diethyl ether extraction system, a high-purity colloid sample is obtained, charge interference of other components is eliminated, it is ensured that a detected object is a gelatin component in a gel skeleton, and the accuracy of a detection result of the isoelectric point of the gel composition is improved.

Description

Technical Field

[0001] The invention relates to the technical field of isoelectric point testing, and in particular to a method for determining the isoelectric point of a gel composition. Background Art

[0002] Gel is a special dispersion system in which colloidal particles or polymers in a sol or solution connect with each other under certain conditions to form a spatial network structure. The gaps in the structure are filled with a liquid (which can also be a gas in a dry gel, also called an aerogel) serving as a dispersion medium. The dispersion system thus formed is a gel.

[0003] A gel composition is a material system composed of specific components in a certain ratio that forms a gel. As a type of soft material system with a three-dimensional network structure, gel compositions are widely used in drug delivery, cosmetics, food industry, and tissue engineering.

[0004] Gelatin is a protein derived from the partial hydrolysis of collagen in animal connective tissue or epidermal tissue. It is a colorless, odorless, non-volatile, transparent, hard, amorphous substance. When mixed with other chemicals and subjected to certain conditions (such as heating), gelatin can form a gel composition.

[0005] Currently, the commonly used method for testing the isoelectric point of a gel composition is to directly mix the gel composition with an anion exchange resin and a cation exchange resin to remove free ions in the system. The pH value of the system is then measured using a pH meter. The measured pH value is the isoelectric point of the gel composition. However, the isoelectric point of the gel composition measured using this method is inaccurate. Summary of the Invention

[0006] In order to solve the problem of inaccurate detection results in existing isoelectric point testing methods when testing the isoelectric point of a gel composition, the present invention provides a method for determining the isoelectric point of a gel composition.

[0007] According to a first aspect of the present invention, there is provided a method for determining the isoelectric point of a gel composition, comprising the following steps: S1. A colloidal solution is prepared by preparing a gel composition with water, wherein the gel composition contains a colloid, which is gelatin; S2. Ethanol and diethyl ether were sequentially added to the colloidal solution to obtain a first mixed solution, the first mixed solution was shaken, and the mixture was allowed to stand to separate the first mixed solution after shaking. After removing the supernatant, diethyl ether was added to the system and the shaking treatment, standing and removing the supernatant were repeated to obtain a second mixed solution; S3. Ethanol was added to the second mixed solution to obtain a colloidal precipitate, and the colloidal precipitate was dried to obtain a colloidal sample; S4. Prepare a colloidal solution by mixing the colloidal sample with water, deionize the colloidal solution to obtain a test solution, and measure the pH value of the test solution. The measured pH value is the isoelectric point of the gel composition.

[0008] Existing gel compositions with gelatin as the colloidal component typically contain, in addition to gelatin, a variety of additives (such as the fat-soluble pigment lutein ester, emulsifiers, plasticizers, oils, etc.). These components will interact with the charge of gelatin during the isoelectric point detection of the gel composition, thereby seriously interfering with the charge signal, affecting the isoelectric point detection results of the gel composition, and further resulting in inaccurate isoelectric point detection results of the gel composition.

[0009] In gel compositions containing gelatin as the colloidal component, gelatin serves as the three-dimensional network backbone of the gel. Its surface charge distribution (particularly the ionization states of amino and carboxyl groups) directly determines the point at which the net charge of the gel as a whole returns to zero (i.e., the isoelectric point). Key properties of gel compositions containing gelatin as the colloidal component, such as swelling behavior, ion responsiveness, and drug release kinetics, undergo abrupt changes near the isoelectric point, primarily driven by the charge characteristics of gelatin. Therefore, measuring the isoelectric point of gel compositions containing gelatin as the colloidal component essentially measures the intrinsic isoelectric point of the gelatin component within the gel composition.

[0010] The isoelectric point determination method provided by the present invention is proposed for a gel composition with gelatin as a colloid component. The method is suitable for isoelectric point detection of a gel composition with gelatin as a colloid component. First, a gel composition with gelatin as a colloid component is prepared with water to form a colloid solution. Ethanol and ether are added to the colloid solution in sequence. The density of ether is lower than that of ethanol and ether is difficult to dissolve the colloid components in the gel composition. After oscillation treatment and standing, the colloid (i.e., gelatin) in the gel composition is precipitated to the lower layer under the action of ethanol, and other components in the gel composition (such as oil, emulsifier, lutein ester, etc.) are extracted to the upper layer by forming a supernatant organic phase with ether. The aqueous layer is located between the supernatant organic phase and the colloidal precipitate in the lower layer. After removing the supernatant, the lower layer material is subjected to ether filtration. Repeated extraction operations are performed to obtain a second mixed liquid containing a colloidal component, the colloid in the second mixed liquid is precipitated with ethanol and dried to obtain a colloidal sample, the colloidal sample is mixed with water to obtain a colloidal solution, and the pH value of the test solution obtained by mixing the colloidal solution with anions and cations is measured to obtain the isoelectric point of the gel composition. The method provided by the present invention can selectively remove components such as fat-soluble additives and water-soluble impurities in the gel composition from the gel composition through an ethanol-ether extraction system to obtain a high-purity colloidal sample, eliminate the charge interference of other components in the gel composition on the isoelectric point determination to the greatest extent, ensure that the detection object is the colloidal component in the gel skeleton, and thus improve the accuracy of the detection result of the isoelectric point of the gel composition.

[0011] Furthermore, the method provided by the present invention does not require expensive equipment such as a chromatograph and a laser scattering instrument. Instead, the method can accurately measure the isoelectric point of the gel composition using only conventional solvents such as ethanol and ether and a pH meter, thereby reducing detection costs.

[0012] Preferably, the above S1 includes the following operations: after mixing the gel composition with water, heating the system to 30-40°C and keeping it warm for 5-10 min while shaking in a water bath, then heating the system to T1 at a rate of 0.3-0.7°C / min and keeping it warm for 5-10 min to obtain a colloidal solution, wherein the denaturation temperature of the gel composition is T2, T1 and T2 satisfy, T1<T2.

[0013] During the preparation of the colloidal solution, the gel composition is mixed with water and the system is heated to 30-40°C and kept warm for 5-10 minutes under water bath oscillation. The temperature is then raised to T1 at a specific heating rate and kept warm for 5-10 minutes, wherein T1 is less than the denaturation temperature T2 of the gel composition. By heating under water bath oscillation conditions and regulating the temperature and heating rate within the above range, the gel composition can be better dissolved in water to form a colloidal solution, and the oil-soluble substances in the gel composition can be suspended in the form of particles or oil droplets, so that the droplet size in the colloidal solution is more consistent and the dispersion is more uniform, thereby improving the accuracy of the isoelectric point determination result of the final gel composition.

[0014] Preferably, in S1, the volume ratio of the gel composition to water in the colloidal solution is (1-3):(2-5).

[0015] Preferably, in S2, the volume ratio of the colloidal solution, ethanol, and diethyl ether in the first mixed solution is (2-4):(1-2):(3-5).

[0016] By regulating the volume ratio of the colloidal solution containing the gel composition to ethanol and ether to meet the aforementioned range, the colloidal components in the gel composition can form a dense, flocculent colloidal precipitate with minimal water content under the action of ethanol, while also ensuring that other components in the gel composition are fully extracted into the supernatant, thereby reducing residual lipids. These two effects enable efficient separation and purification of the colloidal components in the gel composition, improving the efficiency of impurity removal and the recovery / extraction rate of the colloid in the gel composition, thereby further improving the accuracy of the isoelectric point test results of the gel composition and bringing the isoelectric point of the gel composition closer to the intrinsic isoelectric point of the colloidal components (i.e., gelatin) contained therein.

[0017] Preferably, in S2, oscillating the first mixed solution comprises the following operation: oscillating the first mixed solution at a temperature of T1 at a frequency of no more than 30 times / min for 10 to 40 seconds.

[0018] The operation of oscillating the first mixed solution containing the colloidal solution, ethanol, and diethyl ether using a constant temperature oscillator is performed at temperature T1, and the oscillation frequency and oscillation time are controlled within the above-mentioned range. First, performing the oscillation operation at a low temperature (T1 temperature) can prevent the volatilization of diethyl ether and the denaturation of the colloid in the colloidal solution. Second, oscillating the first mixed solution at a frequency of no more than 30 times / min for 10 to 40 seconds can reduce the risk of excessive emulsification of the colloidal solution, resulting in a reduced colloid extraction rate, and the risk of incomplete impurity removal due to the colloid encapsulating other components, thereby affecting the accuracy of the final isoelectric point determination result.

[0019] Preferably, in S1, the gel composition further contains carotenoids, and the carotenoids include at least one of lutein esters, zeaxanthin, β-carotene, lycopene, astaxanthin, fucoxanthin, and canthaxanthin.

[0020] Preferably, S3 includes the following operations: preparing a mixed solvent using a non-polar solvent, a strong polar solvent, and a weak polar solvent, adding the mixed solvent to the second mixed liquid and shaking it, standing to allow the shaken second mixed liquid containing the mixed solvent to separate, removing the supernatant to obtain a colloidal precipitate, and drying the colloidal precipitate to obtain a colloidal sample; the volume ratio of the non-polar solvent, the strong polar solvent, and the weak polar solvent in the mixed solvent is (5~15):(8~18):(5~10); the non-polar solvent includes at least one of cyclohexane, n-hexane, pentane, and heptane; the strong polar solvent includes ethanol, and the strong polar solvent also includes at least one of acetone, ethyl acetate, and tetrahydrofuran; the weak polar solvent includes at least one of toluene and ethylbenzene.

[0021] When a gel composition contains both a colloid (gelatin) and carotenoids, if the colloid component is separated and purified from the gel composition using only an ether-ethanol extraction system, the presence of carotenoids in the gel composition will result in a decrease in the purity of the separated colloid and affect the accuracy of the final isoelectric point test result.

[0022] Among the carotenoids such as lutein ester, zeaxanthin, β-carotene, lycopene, astaxanthin, fucoxanthin and canthaxanthin, although zeaxanthin, astaxanthin and fucoxanthin contain hydroxyl (-OH) or keto group (C=O), these two groups only bring slight polarity, and their long carbon chain structure dominates the non-polar characteristics. Therefore, the above-mentioned carotenoids are generally non-polar substances. Based on the non-polar properties of the above-mentioned carotenoids and the above-mentioned problems, this solution mixes the non-polar solvent, the strong polar solvent and the weak polar solvent according to the above-mentioned ratio to obtain a mixed solvent, and adds it to the second mixed solution for oscillation treatment. During the standing and supernatant removal operations, the strongly polar solvent in the mixed solvent is used to precipitate the colloidal components in the second mixed liquid and balance the non-polar second mixed liquid with the weakly polar solvent (the second mixed liquid is non-polar due to the presence of non-polar carotenoids). The non-polar solvent is then used to extract the colloidal components in the second mixed liquid. Thus, through the synergistic effect of the non-polar solvent, the strongly polar solvent, and the weakly polar solvent in the mixed solvent, the carotenoids in the gel composition can be efficiently removed and the colloid is precipitated in the lower layer, so that a high-purity colloid can be obtained from the gel composition, further improving the accuracy of the isoelectric point test results of the gel composition.

[0023] Preferably, the non-polar solvent is n-hexane; the strong polar solvent includes ethanol, and the strong polar solvent also includes acetone or ethyl acetate; the weak polar solvent is toluene.

[0024] Preferably, the mixed solvent is formed by mixing n-hexane, acetone, toluene and ethanol in a volume ratio of (5-15):(5-10):(5-10):(3-8).

[0025] The mixed solvent used in this solution is composed of n-hexane, acetone, toluene, and ethanol mixed in the above-mentioned volume ratio. The synergistic effect of the components in the mixed solvent is optimized, which is conducive to further improving the removal efficiency of carotenoids and the purity of the colloid, as well as the accuracy of the isoelectric point test results of the gel composition.

[0026] Preferably, in S3, the shaking treatment after adding the mixed solvent to the second mixed liquid comprises the following operations: adding the mixed solvent to the second mixed liquid and shaking at a frequency of 15 to 25 times / min for 10 to 40 seconds.

[0027] After adding the mixed solvent to the second mixed liquid, an oscillation treatment is performed and the oscillation frequency and oscillation time are adjusted to be within the above-mentioned range. This can not only ensure that the second mixed liquid does not separate into layers, but also reduce the risk of emulsification of droplets in the second mixed liquid due to excessively high oscillation frequency when the second mixed liquid and the mixed solvent are evenly mixed. This leads to a better demulsification effect (demulsification refers to the complete destruction of the emulsion into two immiscible phases), and better separation of the oil phase and the water phase, which is beneficial to improving the separation effect of the colloid and its accuracy in the subsequent isoelectric point determination process.

[0028] Preferably, in S3, before drying the colloidal precipitate, the process further includes homogenizing the colloidal precipitate using ethanol.

[0029] Homogenizing the colloidal precipitate with ethanol before drying it can improve the dispersion effect of the colloid and further remove water-soluble impurities in the colloidal precipitate, thereby improving the purity of the obtained colloidal sample and the accuracy of its isoelectric point detection results.

[0030] Preferably, in S3, the mass fraction of ethanol used in the process of homogenizing the colloidal precipitate with ethanol is 80%.

[0031] Preferably, the drying temperature used in the drying process of the colloidal precipitate is 60-105° C., and the drying time is 30-80 min.

[0032] During the drying process of the colloidal precipitate separated from the gel composition, the drying temperature and drying time are controlled within the above ranges, which can ensure that the colloidal precipitate is fully dried, so that the results obtained when the dried colloidal precipitate is subsequently used to prepare a test solution and perform isoelectric point determination are more accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a diagram showing the liquid state in the system after the first mixed liquid is shaken and allowed to stand for stratification, ether is added, the shaking is repeated, and the mixture is allowed to stand in step S2 of Example 1.

[0034] Figure 2 This is a colloidal precipitation image obtained after adding ethanol to the second mixed solution for repeated homogenization and centrifugation in step S3 of Example 1.

[0035] Figure 3 This is a diagram showing the demulsification effect during the determination of the isoelectric point of the gel composition using the methods provided in Examples 9 and 19.

[0036] Figure 4 The figure shows the removal effect of lutein ester during the detection of the isoelectric point of the gel composition using the methods provided in Examples 7, 9, 14, and 15. DETAILED DESCRIPTION

[0037] The following is a further clear and complete description of the technical features of the technical solution provided by the present invention in conjunction with specific embodiments. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts are within the scope of protection of the present invention.

[0038] Example 1 A method for determining the isoelectric point of a gel composition comprises the following steps: S1. The gel composition was mixed with water in a volume ratio of 1:2. The temperature of the system was raised to 35°C with shaking in a water bath and kept at this temperature for 8 min. The temperature was then raised to 50°C at a rate of 0.5°C / min and kept at this temperature for 8 min to obtain a colloidal solution. Among them, the above-mentioned gel composition is prepared with reference to Chinese patent CN119302418B, and the specific preparation steps are as follows: a. The mass percentages of the raw materials in the formula of the gel composition are as follows: gelatin 6%, xylitol 20%, maltitol 18%, DHA algae oil 35%, citric acid monohydrate 0.65%, sodium citrate dihydrate 0.2%, glycerin 2%, orange essence 2%, and the remainder is water; b. Weigh the formulated amount of water, glycerin and maltitol solution in a beaker and mix them evenly, then add gelatin, heat at 70°C until the colloid is completely dissolved, then add xylitol, citric acid monohydrate and sodium citrate dihydrate and stir until completely dissolved, keep warm for use, and obtain an aqueous phase; c. Weigh the formulated amount of DHA algae oil in a beaker, add the formulated amount of orange essence to obtain an oil phase; d. Place the aqueous phase in a homogenizer for shearing, slowly add the oil phase to the aqueous phase for shear homogenization, and obtain a gel composition; S2. Ethanol and diethyl ether were sequentially added to the colloidal solution in a volume ratio of 3:1:4 to obtain a first mixed solution. The first mixed solution was oscillated at 25 times / min at 50°C for 25 seconds using a thermostatic oscillator. The first mixed solution was allowed to stand to separate the layers. The supernatant was removed, and diethyl ether was added to the system. The shaking, standing, and supernatant removal steps were repeated 3-5 times to obtain a second mixed solution. S3. Add 80% ethanol by mass to the second mixed solution and homogenize using a homogenizer. Repeat this process 3–8 times. Centrifuge at 7000 rpm for 5 min to obtain a colloidal precipitate. Dry the colloidal precipitate at 105°C for 50 min to obtain a colloidal sample. S4. Weigh 0.2 g of the colloidal sample, mix it with 40 mL of water, and heat it in a 50°C water bath to dissolve it to prepare a colloidal solution. After cooling, the colloidal solution is mixed with 0.6 g of anion exchange resin and 0.6 g of cation exchange resin in a 30°C constant temperature oscillator and automatically oscillated for 1 h to obtain a test solution. Measure the pH value of the test solution. The measured pH value is the isoelectric point of the gel composition.

[0039] Example 2 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the differences in the structure are: (1) the conditions involved in the preparation process of the colloidal solution in step S1 are different. Specifically, the gel composition and water are mixed in a volume ratio of 2:3, and the system is heated to 30°C and kept warm for 10 minutes under water bath oscillation. Then, the system is heated to 50°C at a rate of 0.3°C / min and kept warm for 10 minutes to obtain a colloidal solution; (2) in step S2, the volume ratio of the colloidal solution, ethanol, and ether in the first mixed solution is 2:1.5:5, and the oscillation frequency used for the oscillation treatment of the first mixed solution is 30 times / min and the oscillation time is 10 s; (3) in step S3, the drying temperature used in the drying treatment of the colloidal precipitate is 60°C and the drying time is 90 minutes.

[0040] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0041] Example 3 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the differences in the structure are: (1) the conditions involved in the preparation process of the colloidal solution in step S1 are different. Specifically, the gel composition and water are mixed in a volume ratio of 3:5, and the system is heated to 40°C and kept warm for 5 minutes under water bath oscillation. Then, the system is heated to 50°C at a rate of 0.7°C / min and kept warm for 5 minutes to obtain a colloidal solution; (2) in step S2, the volume ratio of the colloidal solution, ethanol, and ether in the first mixed solution is 1:2:2, and the oscillation frequency used for the oscillation treatment of the first mixed solution is 20 times / min and the oscillation time is 40 s; (3) in step S3, the drying temperature used in the drying treatment of the colloidal precipitate is 150°C and the drying time is 30 minutes.

[0042] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0043] Example 4 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the difference in structure is that the heating rate used in the preparation process of the colloidal solution in step S1 is 0.1° C. / min.

[0044] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0045] Example 5 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the difference in structure is that the heating rate used in the preparation process of the colloidal solution in step S1 is 0.9° C. / min.

[0046] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0047] Example 6 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the difference in structure is that in step S2, the oscillation frequency used for oscillating the first mixed solution is 35 times / min.

[0048] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0049] Example 7 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the difference in structure is that in step S1, an equal amount of lutein ester is used instead of DHA algae oil during the preparation of the gel composition.

[0050] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0051] Example 8 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the difference in structure is that the operation steps of step S3 are different. Specifically, step S3 includes the following operations: n-hexane, acetone, toluene, and ethanol are mixed in a volume ratio of 10:7:7:6 to prepare a mixed solvent; the mixed solvent is added to a second mixed solution and the solution is shaken at a frequency of 20 times / min for 25 seconds; the solution is allowed to stand to allow the shaken second mixed solution containing the mixed solvent to separate into layers; the supernatant is removed; the addition of the mixed solvent, shaking, standing, and supernatant removal are repeated 2 to 8 times to obtain a colloidal precipitate; the colloidal precipitate is dried at 105°C for 50 minutes to obtain a colloidal sample.

[0052] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0053] Example 9 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the differences in the structure are: (1) in step S1, an equal amount of lutein ester is used instead of DHA algae oil in the preparation process of the gel composition; (2) the operation steps of step S3 are different. Specifically, step S3 includes the following operations: n-hexane, acetone, toluene, and ethanol are mixed in a volume ratio of 10:7:7:6 to prepare a mixed solvent, the mixed solvent is added to the second mixed solution and the solution is shaken at a frequency of 20 times / min for 25 s, the solution is allowed to stand to allow the shaken second mixed solution containing the mixed solvent to separate into layers, the supernatant is removed, and the operation of adding the mixed solvent, shaking, standing, and removing the supernatant is repeated 2 to 8 times to obtain a colloidal precipitate, and the colloidal precipitate is dried at 105°C for 50 min to obtain a colloidal sample.

[0054] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those in Example 1.

[0055] Example 10 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 9, the difference in structure is that the mixed solvent used in step S3 is a mixture of n-hexane, acetone, toluene, and ethanol in a volume ratio of 5:10:5:8.

[0056] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those of Example 9.

[0057] Example 11 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 9, the difference in structure is that the mixed solvent used in step S3 is a mixture of n-hexane, acetone, toluene, and ethanol in a volume ratio of 10:5:10:3.

[0058] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those of Example 9.

[0059] Example 12 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 9, the difference in structure is that in step S3, an equal amount of cyclohexane is used instead of n-hexane.

[0060] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those of Example 9.

[0061] Example 13 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 9, the difference in structure is that in step S3, an equal amount of heptane is used instead of n-hexane, and an equal amount of tetrahydrofuran is used instead of acetone.

[0062] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those of Example 9.

[0063] Example 14 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 9, the difference in structure is that in step S3, an equal amount of pentane is used instead of n-hexane, an equal amount of ethyl acetate is used instead of acetone, and an equal amount of ethylbenzene is used instead of toluene.

[0064] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those of Example 9.

[0065] Example 15 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 9, the difference in structure is that the mixed solvent used in step S3 is a mixture of n-hexane, acetone, toluene, and ethanol in a volume ratio of 3:12:7:6.

[0066] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those of Example 9.

[0067] Example 16 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 9, the difference in structure is that the mixed solvent used in step S3 is a mixture of n-hexane, acetone, toluene, and ethanol in a volume ratio of 10:3:3:10.

[0068] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those of Example 9.

[0069] Example 17 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 9, the difference in structure is that the mixed solvent used in step S3 is a mixture of n-hexane, acetone, toluene, and ethanol in a volume ratio of 12:7:12:2.

[0070] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those of Example 9.

[0071] Example 18 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 9, the difference in structure is that in step S3, the frequency of oscillation used in the oscillation treatment operation after adding the mixed solvent to the second mixed solution is 10 times / min.

[0072] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those of Example 9.

[0073] Example 19 This embodiment provides a method for determining the isoelectric point of a gel composition. Compared with Example 9, the difference in structure is that in step S3, the frequency of oscillation used in the oscillation treatment operation after adding the mixed solvent to the second mixed liquid is 30 times / min.

[0074] Except for the above differences, the materials, formula ratios and preparation operations used in this embodiment are strictly consistent with those of Example 9.

[0075] Comparative Example 1 This comparative example provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the difference in structure is that in step S2, the order of adding ethanol and diethyl ether in the preparation step of the first mixed solution is different. Specifically, diethyl ether and ethanol are added sequentially to the colloidal solution in a volume ratio of 3:1:4 of colloidal solution, ethanol, and diethyl ether to obtain the first mixed solution.

[0076] Except for the above differences, the materials, formula ratios and preparation operations used in this comparative example are strictly consistent with those in Example 1.

[0077] Comparative Example 2 This comparative example provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the difference in structure is that in step S2, the preparation steps of the first mixed solution are different. Specifically, ethanol and diethyl ether are mixed in a volume ratio of 1:4 and then added to the colloidal solution to obtain the first mixed solution. The volume ratio of the colloidal solution, ethanol, and diethyl ether is 3:1:4.

[0078] Except for the above differences, the materials, formula ratios and preparation operations used in this comparative example are strictly consistent with those in Example 1.

[0079] Comparative Example 3 This comparative example provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the difference in structure is that in step S2, an equal amount of petroleum ether is used instead of ether.

[0080] Except for the above differences, the materials, formula ratios and preparation operations used in this comparative example are strictly consistent with those in Example 1.

[0081] Comparative Example 4 This comparative example provides a method for determining the isoelectric point of a gel composition. Compared with Example 1, the method differs in that, in step S2, after the first mixed solution that has been shaken is allowed to stand to separate into layers and the supernatant is removed, the steps of adding ether to the system and repeating the shaking, standing, and supernatant removal are omitted.

[0082] Except for the above differences, the materials, formula ratios and preparation operations used in this comparative example are strictly consistent with those in Example 1.

[0083] Test Example 1 Colloid Extraction Efficiency In this test example, the isoelectric point of the gel composition was detected using the method provided in Examples 1-19 and Comparative Examples 1-4. During the entire detection process, the extraction rate of the colloid component (i.e., gelatin) in the gel composition was calculated as one of the indicators for evaluating the accuracy of the isoelectric point determination results of the gel composition using the determination methods provided in Examples 1-19 and Comparative Examples 1-4. The colloid extraction rate was calculated using the following formula: colloid extraction rate (%) = mass of the colloid sample obtained in step S3 (g) / mass of the colloid in the gel composition in step S1 (g) × 100%.

[0084] Table 1 Colloid extraction rate

[0085] The colloid extraction rate results of the gel composition during the determination of the isoelectric point of the gel composition using the determination methods provided in Examples 1 to 19 and Comparative Examples 1 to 4 are shown in Table 1.

[0086] In the process of detecting the isoelectric point of the gel composition with gelatin as the colloid component according to the method provided in Examples 1 to 3, the colloid extraction rate is as high as more than 70%. Moreover, by comparing the colloid extraction rates of Examples 1 to 3 with those of Comparative Examples 1 to 4, it can be seen that in the process of determining the isoelectric point of the gel composition, the type of ether reagent used, the order of adding ethanol and diethyl ether, and the number of times diethyl ether is added will affect the colloid extraction rate in the gel composition. Regardless of whether diethyl ether is added first and then ethanol in Comparative Example 1 or ethanol and diethyl ether are mixed and then added in Comparative Example 2, the colloid extraction rate in the gel composition is higher than that in the method provided in Examples 1 to 3. When the colloid solution is added, emulsification occurs easily, which increases the difficulty of subsequent colloid extraction. Therefore, in the process of determining the isoelectric point of the gel composition, ethanol and ether are sequentially added to the colloidal solution to obtain a first mixed solution, which is then shaken, allowed to stand for stratification, and the supernatant is removed. Then, ether is added to the system and the above-mentioned shaking, standing, and supernatant removal operations are repeated. This can improve the colloid extraction rate, which is conducive to improving the accuracy of the final isoelectric point determination result and making the measured isoelectric point of the gel composition closer to the intrinsic isoelectric point of the colloidal component therein.

[0087] By comparing the colloid extraction rate results of Examples 1, 4, and 5, it can be seen that in the preparation process of the colloid solution, after the gel composition is mixed with water, the system is heated under water bath oscillation. The heating rate involved in the heating process will affect the subsequent colloid extraction rate. The isoelectric point determination method of the gel composition provided in Example 1 is to heat the system under water bath oscillation after the gel composition is mixed with water and the heating rate is controlled within the range of 0.3-0.7°C / min. This can not only make the gel composition better dissolved in water to form a colloid solution, but also ensure that the oil-soluble substances in the gel composition are suspended in the form of particles or oil droplets, so that the emulsion in the colloid solution is The droplet size consistency is better and the dispersion is more uniform, thereby improving the colloid extraction rate, which will be beneficial to improving the accuracy of the isoelectric point determination results of the gel composition. However, the heating rate used in the preparation process of the colloid solution provided by the method provided in Example 4 is too low, which easily leads to the droplets being too large and unevenly dispersed. The heating rate used in the preparation process of the colloid solution provided by Example 5 is too high, which easily destroys the formation of the droplets. Both too low and too high heating rates used in the preparation process of the colloid solution will affect the formation of droplets in the colloid solution, thereby affecting the subsequent colloid extraction rate and the accuracy of the final isoelectric point determination results of the gel composition.

[0088] Comparison of the colloid extraction rates of Examples 1 and 6 reveals that the oscillation frequency of the first mixed solution prepared from the colloidal solution, ethanol, and diethyl ether during the oscillation process affects the colloid extraction rate. This is primarily because the oscillation frequency of the first mixed solution is higher than 35 times / min, which easily leads to emulsification and makes it difficult to separate the oil phase and the aqueous phase. This significantly reduces the colloid extraction rate and, in turn, affects the isoelectric point detection results of the gel composition.

[0089] Compared to Example 1, the gel composition used in the isoelectric point determination method provided in Example 7 contains lutein esters in addition to gelatin, and only an ethanol-ether extraction system is used to separate and purify the colloidal components from the gel composition. In contrast, in the methods provided in Examples 9 to 11, in addition to using the ethanol-ether extraction system, a mixed solvent prepared from n-hexane, acetone, toluene, and ethanol in specific proportions is used to extract the colloidal components in the second mixed solution during isoelectric point determination of the gel composition containing both gelatin and lutein esters. Comparing the colloid extraction rate data of Examples 1, 7, 8, and 9, it can be seen that the presence of lutein esters reduces the yield and purity of the colloid and affects the accuracy of the final isoelectric point test results. The synergistic effect of the non-polar solvent, the strongly polar solvent, and the weakly polar solvent in the mixed solvent can efficiently remove the lutein esters from the gel composition and precipitate the colloid in the lower layer, thereby improving the colloid extraction rate, thereby obtaining a high-purity colloid from the gel composition and further improving the accuracy of the isoelectric point test results of the gel composition.

[0090] By comparing the colloid extraction rate data of Examples 9, 12, 13, 14, 15, 16, and 17, it can be seen that the components and their ratios in the mixed solvent will affect the colloid extraction rate.

[0091] Comparison of the colloid extraction rate data of Examples 9, 18, and 19 shows that the oscillation frequency used in the oscillation treatment process after adding the mixed solvent to the second mixed liquid affects the colloid extraction rate. This is mainly because too low an oscillation frequency can reduce the size consistency and dispersion uniformity of the emulsion droplets and even cause stratification, while too high an oscillation frequency can easily cause emulsion demulsification and lead to separation of the oil phase and the aqueous phase. Both too low and too high an oscillation frequency can affect the removal effect of lutein esters, thereby reducing the colloid extraction rate and further affecting the isoelectric point detection results of the gel composition.

[0092] Test Example 2 Demulsification Effect In this test example, the isoelectric point of the gel composition was detected using the methods provided in Examples 1-19 and Comparative Examples 1-4. During the isoelectric point detection of the gel composition using the methods provided in Examples 1-8 and Comparative Examples 1-4, the state of the liquid in the system after the first mixed liquid was shaken and allowed to stand for stratification, the addition of ether, repeated shaking, and the standing operation in step S2 was observed. During the isoelectric point detection of the gel composition using the methods provided in Examples 9-19, the state of the liquid in the system after the second mixed liquid was added with the mixed solvent, shaken and allowed to stand for stratification, the addition of ether, repeated shaking, and the standing operation in step S3 was observed. The demulsification effect was evaluated based on the state of the liquid in the system, and the demulsification effect was used to indirectly evaluate the effectiveness of the isoelectric point determination method for the gel composition provided in Examples 1-19 and Comparative Examples 1-4. The better the demulsification effect, the more effective it is in preventing liquid emulsification, the more obvious the stratification, the easier it is to obtain colloid precipitation, the higher the yield of colloid, and the more accurate the isoelectric point of the gel composition finally measured.

[0093] Table 2 Demulsification effect

[0094] The demulsification effects during the determination of the isoelectric point of the gel composition using the methods provided in Examples 1 to 19 and Comparative Examples 1 to 4 are shown in Table 2. In Table 2, the demulsification effects were evaluated by visual observation and scoring. If obvious stratification appeared in the liquid in the system after standing, the demulsification effect was good. If an emulsified layer and only partial stratification appeared in the liquid in the system after standing, the demulsification effect was average. If the liquid in the system was evenly dispersed and almost no stratification existed, the demulsification effect was poor. Scores from 1 to 10 indicate that the demulsification effect ranged from good to poor, with a score of 10 indicating the best demulsification effect and a score of 1 indicating the worst demulsification effect.

[0095] In step S2 of Example 1, the first mixed solution is shaken and allowed to stand for stratification, ether is added and the shaken process is repeated, and the liquid state of the system after the standing operation is as follows: Figure 1 As shown; in step S3 of Example 1, ethanol is added to the second mixed solution for repeated homogenization and the colloidal precipitate obtained after centrifugation is as shown Figure 2 As shown; the demulsification effect of the isoelectric point of the gel composition was measured using the method provided in Examples 9 and 19. Figure 3 As shown, Figure 3 A is a diagram showing the state of the liquid in the system after adding a mixed solvent to the second mixed liquid, shaking and standing for stratification, adding ether, repeating the shaking, and standing for stratification in step S3 of Example 9. Figure 3 B is a diagram showing the state of the liquid in the system after adding a mixed solvent to the second mixed liquid for shaking and standing to separate layers, adding ether and repeating the shaking, and standing to separate layers in step S3 of Example 19.

[0096] It can be seen from the demulsification effects of Example 1 and Comparative Examples 1 to 4 that, in the process of determining the isoelectric point of the gel composition, the type of ether reagent used, the order of adding ethanol and diethyl ether, and the number of times diethyl ether is added will affect the demulsification effect; the demulsification effects of Examples 1, 4, and 5 can illustrate that in the preparation process of the colloidal solution, the gel composition is mixed with water and the system is heated under oscillation in a water bath. If the heating rate involved in the heating process is too high or too low, the demulsification effect will be reduced; the demulsification effects of Examples 1 and 6 and Examples 9, 18, and 19 can demonstrate the influence of the oscillation frequency on the demulsification effect; the demulsification effects of Examples 9, 12 to 17 illustrate that the components and their ratios in the mixed solvent will also affect the demulsification effect to a certain extent.

[0097] Test Example 3 Lutein ester removal effect In this test example, the isoelectric point of the gel composition was detected using the methods provided in Examples 7 and 9 to 19. During the detection of the isoelectric point of the gel composition using the methods provided in Examples 9 to 19, the color of the supernatant after adding the mixed solvent to the second mixed solution and shaking and stratifying in step S3 was observed. During the detection of the isoelectric point of the gel composition using the method provided in Example 7, the color of the supernatant after shaking and stratifying the first mixed solution, adding ether, and repeating the shaking and stratifying in step S2 was observed. The color of the supernatant was used to evaluate the removal effect of lutein esters in the gel composition, and the removal effect of lutein esters was used to indirectly evaluate the removal effect of the lutein esters in Examples 7 and 9 to 19. 19 provides the effect of the isoelectric point determination method of the gel composition. Lutein ester itself is dark reddish brown. When lutein ester is prepared into a gel composition with other components such as gelatin and the lutein ester in the gel composition is extracted by referring to the methods provided in Examples 7, 9 to 19, the lutein ester is yellow in the extraction system. The darker the color of the supernatant, the more lutein ester is extracted into the supernatant, the better the lutein ester removal effect, the higher the purity of the extracted colloidal component, and the higher the accuracy of the isoelectric point of the gel composition finally measured. The lighter the color of the supernatant, the less lutein ester is extracted into the supernatant, the worse the lutein ester removal effect, the lower the purity of the extracted colloidal component, and the lower the accuracy of the isoelectric point of the gel composition finally measured. In addition, in the isoelectric point determination method of the gel composition provided in Examples 7, 9 to 19, other carotenoids (zeaxanthin, β-carotene, lycopene, astaxanthin) were used instead of lutein esters in the preparation step of the gel composition, and the removal effects of these carotenoids, zeaxanthin, β-carotene, lycopene, and astaxanthin, were evaluated according to the above method. The darker the color of the supernatant, the more carotenoids extracted into the supernatant, and the better the carotenoid removal effect. The lighter the color of the supernatant, the less carotenoids extracted into the supernatant, and the worse the carotenoid removal effect.

[0098] Table 3 Carotenoid removal effect

[0099] In the process of detecting the isoelectric point of the gel composition using the method provided in Examples 7, 9 to 19, the carotenoid removal effect is shown in Table 3. In Table 3, the carotenoid removal effect was evaluated by visual observation and scoring. If the supernatant color is darker, it means that the carotenoid removal effect is good, and if the supernatant color is lighter, it means that the carotenoid removal effect is poor. The scores from 1 to 10 represent the carotenoid removal effect from good to poor, with a score of 10 indicating the best carotenoid removal effect and a score of 1 indicating the worst carotenoid removal effect.

[0100] The removal effect of lutein esters during the detection of the isoelectric point of the gel composition using the methods provided in Examples 7, 9, 14, and 15 is as follows: Figure 4 As shown, Figure 4 A is a color result of the supernatant after the first mixed solution is shaken and allowed to stand for stratification, ether is added, and the shaking is repeated, and the stratification is performed in step S2 during the detection of the isoelectric point of the gel composition using the method provided in Example 7. Figure 4 B, C, and D are color results of the supernatant after adding the mixed solvent to the second mixed solution for shaking and standing for stratification in step S3 during the detection of the isoelectric point of the gel composition using the methods provided in Examples 9, 14, and 15.

[0101] The gel composition used in the isoelectric point determination method provided in Example 7 contains carotenoids in addition to gelatin. Using only the ethanol-ether extraction system to separate and purify the colloidal components from the gel composition results in poor carotenoid removal.

[0102] Compared with Example 7, in the method provided in Examples 9 to 11, in addition to using the ethanol-ether extraction system, a mixed solvent prepared by preparing n-hexane, acetone, toluene, and ethanol in specific proportions is used to extract the colloidal components in the second mixed solution during the determination of the isoelectric point of the gel composition containing both gelatin and carotenoids, thereby significantly improving the removal effect of carotenoids.

[0103] By comparing the carotenoid removal effects of Examples 9, 12, 13, 14, 15, 16, and 17, it can be seen that the components and their ratios in the mixed solvent will affect the carotenoid removal effect of the gel composition.

[0104] By comparing the lutein ester removal effects of Examples 9, 18, and 19, it can be seen that the oscillation frequency during the oscillation treatment after adding the mixed solvent to the second mixed solution will affect the carotenoid removal effect in the gel composition.

[0105] Test Example 4 In this test example, the isoelectric point of the gel composition was determined using the methods provided in Examples 1-19 and Comparative Examples 1-4. In the preparation of the gel compositions involved in Examples 1-19 and Comparative Examples 1-4, gelatin 180 freeze-acid method (purchased from Fujian Funingpu Biotechnology Co., Ltd.) was used as the control group, and its isoelectric point was tested in accordance with GB 6783-94. The preparation method of the test solution (containing gelatin) for the isoelectric point test was the same as step S4 of Example 1. Each experiment was repeated twice, and the final results were averaged. The isoelectric point test results are shown in Table 4. The difference between the isoelectric point of the gel composition (containing gelatin 180 freeze-acid method) measured by the methods provided in Examples 1-19 and Comparative Examples 1-4 and the isoelectric point of the gel composition (containing gelatin 180 freeze-acid method) measured in accordance with GB 6783-94 was calculated.

[0106] The freezing number (180 freezing) in "Gelatin 180 freezing-acid method" refers to the gel strength of gelatin, also known as Bloom's strength. Gelatin is divided into alkaline gelatin (also known as type B gelatin), acid gelatin (also known as type A gelatin) and enzymatic gelatin according to the production method. "Gelatin 180 freezing-acid method" refers to gelatin with a gel strength of 180 and produced by the alkaline method.

[0107] Table 4 Isoelectric point test results of different gel compositions using the methods provided in Examples 1 to 19 and Comparative Examples 1 to 4

[0108] The results of measuring the isoelectric points of the gel compositions using the methods provided in Examples 1 to 19 and Comparative Examples 1 to 4 are shown in Table 4.

[0109] Compared with Comparative Examples 1 to 4, the isoelectric point detection results (5.75-5.76) of the gel composition with gelatin as the colloid component using the method provided in Examples 1 to 3 are very close to the isoelectric point detection result (5.89) of gelatin according to GB 6783-94. This indicates that in the process of determining the isoelectric point of the gel composition, adding ethanol and ether to the colloid solution in sequence to obtain a first mixed solution, shaking the mixed solution, standing to separate the layers, and removing the supernatant, and then adding ether to the system and repeating the above shaking, standing, and removing the supernatant operations can improve the colloid extraction rate, which is conducive to improving the accuracy of the final isoelectric point determination result, making the measured isoelectric point of the gel composition closer to the intrinsic isoelectric point of the colloid component therein.

[0110] By comparing the isoelectric point detection results of Examples 1, 4, and 5 with the control group, it can be seen that during the preparation of the colloidal solution, the gel composition is mixed with water and then the system is heated under oscillation in a water bath. The heating rate involved in this heating process will affect the subsequent colloid extraction rate and thus affect the accuracy of the final isoelectric point detection result. The isoelectric point determination method of the gel composition provided in Example 1, in which the gel composition is mixed with water and then the system is heated under oscillation in a water bath and the heating rate is controlled within the range of 0.3-0.7°C / min, can improve the accuracy of the isoelectric point determination result of the gel composition.

[0111] By comparing the isoelectric point detection results of Example 1, Example 6 and the control group, it can be seen that the oscillation frequency of the first mixed solution prepared from the colloidal solution, ethanol and diethyl ether during the oscillation process will affect the colloid extraction rate and thus affect the final isoelectric point detection result. Controlling the oscillation frequency of the first mixed solution to no more than 35 times / min can improve the accuracy of the isoelectric point detection result of the gel composition.

[0112] Compared to Example 1, the gel composition used in the isoelectric point determination method provided in Example 7 contains lutein esters in addition to gelatin, and only an ethanol-ether extraction system is used to separate and purify the colloidal components from the gel composition. In contrast, in the methods provided in Examples 9 to 11, in addition to using the ethanol-ether extraction system, a mixed solvent prepared by mixing n-hexane, acetone, toluene, and ethanol in specific proportions is used to extract the colloidal components in the second mixed solution during the isoelectric point determination process of the gel composition containing both gelatin and carotenoids. Among the isoelectric point detection results of Examples 1, 7, 8, 9, and the control group, the isoelectric point of Example 9 is closest to that of the control group. This indicates that the synergistic effect of the non-polar solvent, the strongly polar solvent, and the weakly polar solvent in the mixed solvent can efficiently remove carotenoids from the gel composition and increase the extraction rate of gelatin, further improving the accuracy of the isoelectric point test results of the gel composition.

[0113] By comparing the isoelectric point detection results of Examples 9, 12, 13, 14, 15, 16, and 17, it can be seen that the components and their ratios in the mixed solvent will affect the accuracy of the isoelectric point detection results.

[0114] By comparing the isoelectric point detection results of Examples 9, 18, and 19, it can be shown that if the oscillation frequency used in the oscillation treatment after adding the mixed solvent to the second mixed liquid is too low, the size uniformity and dispersion uniformity of the emulsion droplets will be reduced, and even stratification may occur. If the oscillation frequency is too high, emulsion demulsification may easily occur, resulting in separation of the oil phase and the water phase. If the oscillation frequency is too low or too high, the extraction rate of gelatin will be affected, and thus the accuracy of the isoelectric point detection result of the gel composition will be affected.

[0115] Test Example 5 In this test example, the isoelectric point of a gel product (i.e., a gel composition) made of different gelatin materials is detected using the isoelectric point determination method of the gel composition provided in Example 9. Each group of experiments is repeated twice, and the final results are averaged. The isoelectric point detection results of Example 9 are compared with the isoelectric point detection results of different gelatin materials with reference to GB 6783-94, and the deviation between the isoelectric points obtained by detecting the gel product made of different gelatin materials using the method provided in Example 9 and the isoelectric points obtained by detecting the isoelectric points of different gelatin materials with reference to GB 6783-94 is calculated according to the following formula: For the gel product, isoelectric point deviation = |isoelectric point of the gel product measured in Example 9 - isoelectric point of the pure gelatin material measured with reference to GB 6783-94| / isoelectric point of the gel product made of different gelatin materials with reference to GB The isoelectric point of the pure gelatin material measured by 6783-94 was × 100%. The preparation method of the test solution (containing gelatin) for the isoelectric point test was referred to step S4 of Example 1, and the gel product was prepared according to the preparation method of the gel composition in step S1 of Example 1. The results are shown in Table 5.

[0116] The gelatin materials used in this test example include gelatin 180 freeze-acid method, gelatin 180 freeze-alkaline method, gelatin 220 freeze-acid method, gelatin 220 freeze-alkaline method, gelatin 250 freeze-acid method, and gelatin 250 freeze-alkaline method, among which: 180 freeze-alkaline method and gelatin 250 freeze-alkaline method were purchased from Rousselot Biochemical Technology Co., Ltd., 180 freeze-acid method, gelatin 220 freeze-acid method, gelatin 220 freeze-alkaline method, and gelatin 250 freeze-acid method were purchased from Fujian Funingpu Biotechnology Co., Ltd. The freeze numbers (180 freeze, 220 freeze, 250 freeze) in the above-mentioned gelatin materials refer to the gel strength of gelatin, also known as Bloom's strength. Gelatin is divided into alkaline gelatin (also known as type B gelatin), acid gelatin (also known as type A gelatin) and enzymatic gelatin according to the production method. The above-mentioned 180 freeze-acid method, gelatin 220 freeze-acid method, and gelatin 250 freeze-acid method are all produced by the acid method, and gelatin 180 freeze-alkaline method, gelatin 220 freeze-alkaline method, and gelatin 250 freeze-alkaline method are all produced by the alkaline method.

[0117] The gel products (i.e., gel compositions) used in this test example were all prepared with reference to the gel composition preparation method in step S1 of Example 1, wherein the gelatin used in the 180 freeze-acid method yogurt was the 180 freeze-acid method, the gelatin used in the 180 freeze-alkaline method yogurt was gelatin 180 freeze-alkaline method, the gelatin used in the 220 freeze-acid method yogurt was gelatin 220 freeze-acid method, the gelatin used in the 220 freeze-alkaline method yogurt was gelatin 220 freeze-alkaline method, the gelatin used in the 250 freeze-acid method yogurt was gelatin 250 freeze-acid method, and the gelatin used in the 250 freeze-alkaline method yogurt was gelatin 250 freeze-alkaline method.

[0118] Table 5 Comparison of isoelectric point test results of gel products made from different gelatin materials using the method provided in Example 9 and the isoelectric point test results of different gelatin materials according to GB 6783-94

[0119] The isoelectric point test results of gel products made from different gelatin materials using the isoelectric point determination method for gel compositions provided in Example 9 and the isoelectric point test results of pure gelatin materials used in gel products according to GB 6783-94 are shown in Table 5.

[0120] As shown in Table 5, when the method provided in Example 9 was used to test a gel product (i.e., a gel composition) containing gelatin as the colloidal component, the deviation between the measured isoelectric point and the isoelectric point of pure gelatin material measured in accordance with GB 6783-94 was extremely small, falling within 4%. These results demonstrate that the method provided in Example 9, using an ethanol-ether extraction system combined with a mixed solvent composed of n-hexane, acetone, toluene, and ethanol, can selectively remove components such as fat-soluble additives, water-soluble impurities, and carotenoids from a gel composition containing both gelatin and carotenoids, thereby obtaining a high-purity colloidal sample. This method also minimizes charge interference from other components in the gel composition on isoelectric point determination, ensuring that the target of detection is the gelatin component in the gel matrix. This improves the accuracy of the isoelectric point test results for the gel composition, making the final measured isoelectric point closer to the intrinsic isoelectric point of gelatin.

[0121] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents, but these modifications or replacements are all within the scope of protection of the present invention.

Claims

1. A method for determining the isoelectric point of a gel composition, characterized in that: The following steps are involved: S1. A colloidal solution is prepared by preparing a gel composition with water, wherein the gel composition contains a colloid, and the colloid is gelatin; S2. Ethanol and diethyl ether are sequentially added to the colloidal solution to obtain a first mixed solution, the first mixed solution is shaken, and the first mixed solution is allowed to stand to separate after the shaking. After removing the supernatant, diethyl ether is added to the system and the shaking, standing, and supernatant removal operations are repeated to obtain a second mixed solution; S3. adding ethanol to the second mixed solution to obtain a colloidal precipitate, and drying the colloidal precipitate to obtain a colloidal sample; S4. Prepare a colloidal solution by mixing the colloidal sample with water, deionize the colloidal solution to obtain a test solution, and measure the pH value of the test solution. The measured pH value is the isoelectric point of the gel composition.

2. The method for determining the isoelectric point of a gel composition according to claim 1, wherein: The S1 comprises the following operations: mixing the gel composition with water, heating the system to 30-40°C and keeping the temperature for 5-10 min while shaking in a water bath, then heating the system to T1 at a rate of 0.3-0.7°C / min and keeping the temperature for 5-10 min to obtain a colloidal solution, wherein the denaturation temperature of the gel composition is T2, and T1 and T2 satisfy T1<T2.

3. The method for determining the isoelectric point of a gel composition according to claim 1, wherein: In S1, the volume ratio of the gel composition and the water in the colloidal solution is (1-3):(2-5), and / or, In S2, the volume ratio of the colloidal solution, the ethanol, and the diethyl ether in the first mixed solution is (2-4):(1-2):(3-5).

4. The method for determining the isoelectric point of a gel composition according to claim 2, wherein: In S2, oscillating the first mixed solution includes the following operations: oscillating the first mixed solution at a temperature of T1 at a frequency of no more than 30 times / min for 10 to 40 seconds.

5. The method for determining the isoelectric point of a gel composition according to claim 1, wherein: In the above S1, the gel composition further contains carotenoids, and the carotenoids include at least one of lutein ester, zeaxanthin, β-carotene, lycopene, astaxanthin, fucoxanthin, and canthaxanthin.

6. The method for determining the isoelectric point of a gel composition according to claim 5, wherein: The step S3 comprises the following steps: preparing a mixed solvent using a non-polar solvent, a strongly polar solvent, and a weakly polar solvent; adding the mixed solvent to the second mixed solution and shaking the solution; standing the solution to allow the shaken second mixed solution containing the mixed solvent to separate into layers; removing the supernatant to obtain a colloidal precipitate; and drying the colloidal precipitate to obtain a colloidal sample. The volume ratio of the non-polar solvent, the strong polar solvent, and the weak polar solvent in the mixed solvent is (5-15):(8-18):(5-10); The non-polar solvent includes at least one of cyclohexane, n-hexane, pentane, and heptane; The highly polar solvent includes ethanol, and the highly polar solvent also includes at least one of acetone, ethyl acetate, and tetrahydrofuran; The weakly polar solvent includes at least one of toluene and ethylbenzene.

7. The method for determining the isoelectric point of a gel composition according to claim 6, wherein: The non-polar solvent is n-hexane; The strong polar solvent includes ethanol, and the strong polar solvent also includes acetone or ethyl acetate; The weakly polar solvent is toluene.

8. The method for determining the isoelectric point of a gel composition according to claim 7, wherein: The mixed solvent is prepared by mixing n-hexane, acetone, toluene and ethanol in a volume ratio of (5-15):(5-10):(5-10):(3-8).

9. The method for determining the isoelectric point of a gel composition according to claim 6, wherein: In S3, the shaking treatment after adding the mixed solvent to the second mixed liquid includes the following operations: adding the mixed solvent to the second mixed liquid and shaking at a frequency of 15 to 25 times / min for 10 to 40 seconds.

10. The method for determining the isoelectric point of a gel composition according to claim 1, wherein: In said S3, before drying the colloidal precipitate, the process further includes homogenizing the colloidal precipitate with ethanol. and / or, In the step S3 , the drying temperature used in the drying process of the colloidal precipitate is 60-105° C., and the drying time is 30-80 min.

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