Method for determination of isoelectric point of a gel composition

Abstract

The application provides a method for measuring the isoelectric point of a gel composition, comprising the following steps: S1. preparing a colloidal solution by using a gel composition with gelatin as a colloidal component and water; S2. sequentially adding ethanol and diethyl ether into the colloidal solution to obtain a first mixed solution, and performing oscillation and static layering treatment, adding diethyl ether after removing supernatant, and repeating the above operation to obtain a second mixed solution; S3. adding ethanol into the second mixed solution to obtain a colloidal precipitate and performing drying treatment to obtain a colloidal sample; S4. preparing a colloidal solution by using the colloidal sample and water, and performing deionization treatment to obtain a to-be-measured solution, and measuring the pH value of the to-be-measured solution as the isoelectric point of the gel composition. The ethanol-diethyl ether extraction system is used to remove other components in the gel composition, a high-purity colloidal sample is obtained, the charge interference of other components is eliminated, the gelatin component in the gel skeleton is ensured as the detection object, and the accuracy of the detection result of the isoelectric point of the gel composition is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of isoelectric point testing, and particularly relates to a method for determining the isoelectric point of a gel composition. BACKGROUND

[0002] Gel is a special dispersion system, in which colloidal particles or polymers in sol or solution are connected to each other under certain conditions to form a spatial network structure, and the structure gap is filled with liquid (or gas in dry gel, also known as aerogel) as a dispersion medium, and thus a dispersion system is formed.

[0003] Gel composition refers to a substance system formed by specific components in a certain ratio and forming a gel state. As a kind of soft matter system with three-dimensional network structure, gel composition is widely used in drug delivery, cosmetics, food industry and tissue engineering fields.

[0004] Gelatin is a protein obtained by partial hydrolysis of collagen in animal connective tissue or epidermal tissue, which is a colorless, odorless, non-volatile, transparent and hard amorphous substance. Gelatin can form a gel composition after being mixed with other chemical substances under certain conditions (such as heating).

[0005] At present, the commonly used isoelectric point testing method of gel composition is to mix the gel composition with anion exchange resin and cation exchange resin uniformly to remove free ions in the system, and then use a pH meter to measure the pH value of the system, and the measured pH value is the isoelectric point of the gel composition. However, the isoelectric point of the gel composition measured by the above method is not accurate. SUMMARY

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

[0007] According to a first aspect of the present application, a method for determining the isoelectric point of a gel composition is provided, comprising the following steps:

[0008] S1. A colloidal solution is prepared by using a gel composition and water, wherein the gel composition contains a gelatin as a gel;

[0009] S2. Ethanol and diethyl ether are sequentially added to the colloidal solution to obtain a first mixed solution, the first mixed solution is subjected to oscillation treatment, and is left to stand to separate the oscillation-treated first mixed solution into layers, after removing the supernatant, diethyl ether is added to the system and the oscillation treatment, standing and removing the supernatant are repeated to obtain a second mixed solution;

[0010] S3. Adding ethanol to the second mixed solution to obtain a colloidal precipitate, and drying the colloidal precipitate to obtain a colloidal sample;

[0011] S4. Preparing a colloidal solution by using the colloidal sample and water, and deionizing the colloidal solution to obtain a to-be-tested solution, measuring the pH value of the to-be-tested solution, and the measured pH value is the isoelectric point of the gel composition.

[0012] The existing gel composition with gelatin as the colloidal component usually contains various additives (such as fat-soluble pigment lutein ester, emulsifier, plasticizer, oil, etc.) in addition to gelatin. These components will interact with the charge of gelatin at the isoelectric point detection of the gel composition, thereby seriously interfering with the charge signal, affecting the isoelectric point detection result of the gel composition, and further leading to inaccurate detection of the isoelectric point of the gel composition.

[0013] In the gel composition with gelatin as the colloidal component, gelatin serves as the three-dimensional network skeleton of the gel, and the surface charge distribution (especially the ionization state of the amino and carboxyl groups) directly determines the net charge zero point (i.e. the isoelectric point) of the whole gel. The swelling behavior, ion responsiveness, drug release kinetics and other key properties of the gel composition with gelatin as the colloidal component mutate near the isoelectric point, and these phenomena are mainly driven by the charge characteristics of gelatin. Therefore, the detection of the isoelectric point of the gel composition with gelatin as the colloidal component essentially measures the intrinsic isoelectric point of the gelatin component in the gel composition.

[0014] The isoelectric point determination method provided by the present application is proposed for the gel composition with gelatin as the colloid component, and is suitable for the isoelectric point detection of the gel composition with gelatin as the colloid component. The gel composition with gelatin as the colloid component is first mixed with water to prepare a colloid solution, and then ethanol and diethyl ether are sequentially added to the colloid solution. The density of diethyl ether is less than that of ethanol, and diethyl ether is difficult to dissolve the colloid component 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 (such as oil, emulsifier, lutein ester, etc.) in the gel composition are extracted to the upper layer with diethyl ether to form a supernatant organic phase. The water layer is located between the supernatant organic phase and the lower layer of colloid precipitate. After removing the supernatant, the lower layer material is repeatedly extracted with diethyl ether to obtain a second mixed solution containing the colloid component. The colloid in the second mixed solution is precipitated by ethanol and dried to obtain a colloid sample. The colloid sample is mixed with water to obtain a colloid solution, and the pH value of the test solution obtained by mixing the colloid solution with anions and cations is determined to obtain the isoelectric point of the gel composition. The method provided by the present application can selectively remove fat-soluble additives and water-soluble impurities and other components in the gel composition through the ethanol-diethyl ether extraction system, obtain a high-purity colloid sample, and maximize the elimination of the charge interference of other components in the gel composition on the isoelectric point determination, so as to ensure that the detection object is the colloid component in the gel skeleton, thereby improving the accuracy of the detection result of the isoelectric point of the gel composition.

[0015] In addition, the method provided by the present application does not need to rely on expensive equipment such as chromatograph and laser scattering instrument, and only needs to use conventional solvents such as ethanol and diethyl ether and a pH meter to realize the purpose of accurately determining the isoelectric point of the gel composition, and the detection cost is also reduced.

[0016] Preferably, the S1 includes the following operation: after the gel composition is mixed with water, the system is warmed to 30-40℃ under water bath oscillation and incubated for 5-10 min, and then the system is warmed to T1 at a rate of 0.3-0.7℃ / min and incubated for 5-10 min to obtain a colloid solution, wherein the denaturation temperature of the gel composition is T2, T1 and T2 satisfy T1

[0017] In the preparation of the colloidal solution, after the gel composition is mixed with water, the system is warmed to 30-40°C under water bath oscillation for 5-10 min, and then warmed to T1 at a specific warming rate and kept for 5-10 min, wherein T1 < the denaturation temperature T2 of the gel composition. By warming under water bath oscillation and adjusting the temperature and warming rate to 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 size uniformity of the emulsion droplets in the colloidal solution is better and the dispersion is more uniform, thereby improving the accuracy of the isoelectric point determination result of the final gel composition.

[0018] Preferably, in S1, the volume ratio of the gel composition in the colloidal solution to water is (1-3):(2-5).

[0019] 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).

[0020] By adjusting the volume ratio of the colloidal solution containing the gel composition, ethanol and diethyl ether to meet the above range, it can be ensured that the colloidal components in the gel composition can form dense and low-moisture flocculent colloidal precipitate under the action of ethanol, and other components in the gel composition can be fully extracted into the supernatant, reducing lipid residues. Through the above two aspects, efficient separation and purification of the colloidal components in the gel composition are realized, the impurity removal efficiency of the gel composition and the recovery rate / extraction rate of the colloidal are improved, thereby further improving the accuracy of the isoelectric point test result of the gel composition, and the isoelectric point of the gel composition is closer to the intrinsic isoelectric point of the colloidal component (i.e. gelatin) contained therein.

[0021] Preferably, in S2, the oscillation treatment of the first mixed solution includes the following operation: oscillating the first mixed solution at a frequency of not more than 30 times / min for 10-40 s at T1.

[0022] The oscillation treatment of the first mixed solution containing the colloidal solution, ethanol and diethyl ether using the constant temperature oscillator is carried out at T1, and the oscillation frequency and oscillation time are adjusted to the above range. Firstly, the oscillation operation at low temperature (T1) can prevent the volatilization of diethyl ether and the denaturation of colloids in the colloidal solution. Secondly, oscillating the first mixed solution at a frequency of not more than 30 times / min for 10-40 s can reduce the risk of excessive emulsification of the colloidal solution, which reduces the extraction rate of the colloids and causes incomplete impurity removal due to the colloids wrapping other components, thereby affecting the accuracy of the final isoelectric point determination result.

[0023] Preferably, in S1, the gel composition further contains carotenoids, the carotenoids including at least one of lutein ester, zeaxanthin, β-carotene, lycopene, astaxanthin, fucoxanthin, canthaxanthin.

[0024] Preferably, S3 includes the following operations: a mixed solvent is prepared by using a non-polar solvent, a strong polar solvent, and a weak polar solvent; the mixed solvent is added to the second mixed solution and subjected to oscillation treatment; standing is performed to make the second mixed solution containing the mixed solvent subjected to the oscillation treatment stratify; supernatant is removed to obtain a colloidal precipitate; the colloidal precipitate is subjected to drying treatment 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 further includes at least one of acetone, ethyl acetate, and tetrahydrofuran; the weak polar solvent includes at least one of toluene and ethylbenzene.

[0025] When the gel composition contains both a colloid (gelatin) and carotenoids, if the colloid component is separated and purified from the gel composition only by an ether-ethanol extraction system, the presence of the 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.

[0026] Among the lutein ester, zeaxanthin, β-carotene, lycopene, astaxanthin, fucoxanthin, and canthaxanthin, although the zeaxanthin, astaxanthin, and fucoxanthin contain hydroxyl groups (—OH) or ketone groups (C=O), these two groups only bring slight polarity, and the long carbon chain structure dominates the non-polar property, so the above-mentioned carotenoids belong to non-polar substances in general. Based on the non-polar property of the above-mentioned carotenoids and the above-mentioned problems, the non-polar solvent, the strong polar solvent, and the weak polar solvent are mixed according to the above-mentioned ratio to prepare a mixed solvent, which is added to the second mixed solution for oscillation treatment, standing, and supernatant removal. The strong polar solvent in the mixed solvent is used to precipitate the colloid component in the second mixed solution and balance the non-polarity of the second mixed solution (the second mixed solution exhibits non-polarity due to the presence of non-polar carotenoids) with the weak polar solvent, and the non-polar solvent is used to extract the colloid component in the second mixed solution. Therefore, through the synergistic effect of the non-polar solvent, the strong polar solvent, and the weak 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 is obtained from the gel composition, and the accuracy of the isoelectric point test result of the gel composition is further improved.

[0027] Preferably, the non-polar solvent is n-hexane; the strong polar solvent includes ethanol, and the strong polar solvent further includes acetone or ethyl acetate; and the weak polar solvent is toluene.

[0028] Preferably, 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).

[0029] The mixed solvent used in the present scheme is prepared by mixing n-hexane, acetone, toluene and ethanol in the above-mentioned volume ratio. The synergistic effect of the components in the mixed solvent is optimal, which is conducive to further improving the removal efficiency of carotenoids, the purity of the colloid and the accuracy of the isoelectric point test results of the gel composition.

[0030] Preferably, in S3, the oscillation treatment after adding the mixed solvent to the second mixed solution includes the following operation: adding the mixed solvent to the second mixed solution and oscillating at a frequency of 15-25 times / min for 10-40 s.

[0031] The oscillation treatment after adding the mixed solvent to the second mixed solution and the oscillation frequency and oscillation time are controlled within the above-mentioned ranges, which can not only ensure that the second mixed solution is not stratified, but also reduce the risk of emulsification of the emulsion droplets in the second mixed solution due to too high oscillation frequency under the condition that the second mixed solution is uniformly mixed with the mixed solvent, and the demulsification effect is better (demulsification refers to the complete destruction of the emulsion into two immiscible phases), and the oil phase and the water phase are better separated, which is conducive to improving the separation effect of the colloid and its accuracy in the subsequent isoelectric point determination process.

[0032] Preferably, in S3, before the drying treatment of the colloid precipitate, it further includes the operation of homogenizing the colloid precipitate with ethanol.

[0033] The homogenization of the colloid precipitate with ethanol before the drying treatment of the colloid precipitate can improve the dispersion effect of the colloid, and further remove water-soluble impurities in the colloid precipitate, thereby improving the purity of the obtained colloid sample and the accuracy of the isoelectric point test results.

[0034] Preferably, in S3, the mass fraction of ethanol used in the homogenization of the colloid precipitate with ethanol is 80%.

[0035] Preferably, the drying temperature used in the drying treatment of the colloid precipitate is 60-105℃, and the drying time is 30-80 min.

[0036] Controlling the drying temperature and drying time within the above-mentioned ranges during the drying treatment of the colloid precipitate separated from the gel composition can ensure that the colloid precipitate is fully dried, so that the results measured when the dried colloid precipitate is used to prepare a test solution and perform isoelectric point determination are more accurate. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 The liquid state result diagram of the system after the oscillation treatment, standing and delamination, adding ether and repeating the oscillation treatment and standing operation of the first mixed solution in step S2 of Example 1.

[0038] Figure 2 The gel precipitate diagram obtained after repeating the homogenization treatment and centrifugation of the second mixed solution by adding ethanol in step S3 of Example 1.

[0039] Figure 3 The demulsification effect diagram during the measurement of the isoelectric point of the gel composition by the method provided in Examples 9 and 19.

[0040] Figure 4 The removal effect diagram of the lutein ester during the measurement of the isoelectric point of the gel composition by the method provided in Examples 7, 9, 14 and 15. DETAILED DESCRIPTION

[0041] The technical features in the technical solutions provided by the present application will be further described clearly and completely in combination with the specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0042] Example 1

[0043] A method for measuring the isoelectric point of a gel composition, comprising the following steps:

[0044] S1. mixing the gel composition with water at a volume ratio of 1:2, then heating the system to 35℃ in a water bath oscillator and keeping for 8 min, then heating the system to 50℃ at a rate of 0.5℃ / min and keeping for 8 min to obtain a gel solution;

[0045] The gel composition is prepared according to the Chinese patent CN119302418B, and the specific preparation steps are as follows: a. The mass percentage of each raw material in the formula of the gel composition is as follows, gelatin 6%, xylitol 20%, maltitol 18%, DHA algal oil 35%, citric acid monohydrate 0.65%, sodium citrate dihydrate 0.2%, glycerol 2%, orange flavor 2%, and the remaining part is water; b. The formula amount of water, glycerol and maltitol liquid is weighed and mixed uniformly in a beaker, then gelatin is added, heated to 70℃ until the colloid is completely dissolved, then xylitol, citric acid monohydrate and sodium citrate dihydrate are added and stirred until completely dissolved, and kept for standby, to obtain an aqueous phase; c. The formula amount of DHA algal oil is weighed in a beaker, and the formula amount of orange flavor is added to obtain an oil phase; d. The aqueous phase is placed in a homogenizing shear machine for shearing, and the oil phase is slowly added to the aqueous phase for shearing homogenization to obtain a gel composition;

[0046] S2. Add ethanol and diethyl ether to the colloid solution in the order of ethanol, diethyl ether, according to the volume ratio of 3:1:4, to obtain a first mixed solution, use a constant temperature oscillator to oscillate the first mixed solution at 50℃ with a frequency of 25 times / min for 25s, stand to stratify the first mixed solution after oscillation treatment, remove the supernatant, add diethyl ether to the system and repeat the above oscillation treatment, standing and removing the supernatant operation (repeat 3-5 times) to obtain a second mixed solution;

[0047] S3. Add 80% ethanol by mass fraction to the second mixed solution and homogenize with a homogenizer, repeat 3-8 times, centrifuge at 7000 rpm / min for 5 min to obtain a colloid precipitate, dry the colloid precipitate at 105℃ for 50 min to obtain a colloid sample;

[0048] S4. Weigh 0.2 g of the colloid sample and mix with 40 mL of water, then heat and dissolve in a 50℃ water bath to prepare a colloid solution, after cooling, mix the colloid solution with 0.6 g of anion exchange resin and 0.6 g of cation exchange resin in a 30℃ constant temperature oscillator for automatic oscillation for 1 h to obtain a test solution, measure the pH value of the test solution, and the measured pH value is the isoelectric point of the gel composition.

[0049] Example 2

[0050] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the method is different from that in Embodiment 1 in that (1) conditions involved in the preparation process of the colloidal solution in step S1 are different, specifically, the gel composition and water are mixed according to a volume ratio of 2:3, then the system is heated to 30 DEG C and kept for 10 min under water bath oscillation, then the system is heated to 50 DEG C at a rate of 0.3 DEG C / min and kept for 10 min, and finally the colloidal solution is obtained; (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 for the oscillation treatment of the first mixed solution is 30 times / min and the oscillation time is 10 s; and (3) in step S3, the drying temperature for the drying treatment of the colloidal precipitate is 60 DEG C and the drying time is 90 min.

[0051] Except for the above differences, the materials, formula ratio and preparation operation in the embodiment are strictly kept consistent with those in Embodiment 1.

[0052] Embodiment 3

[0053] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the method is different from that in Embodiment 1 in that (1) conditions involved in the preparation process of the colloidal solution in step S1 are different, specifically, the gel composition and water are mixed according to a volume ratio of 3:5, then the system is heated to 40 DEG C and kept for 5 min under water bath oscillation, then the system is heated to 50 DEG C at a rate of 0.7 DEG C / min and kept for 5 min, and finally the colloidal solution is obtained; (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 for the oscillation treatment of the first mixed solution is 20 times / min and the oscillation time is 40 s; and (3) in step S3, the drying temperature for the drying treatment of the colloidal precipitate is 150 DEG C and the drying time is 30 min.

[0054] Except for the above differences, the materials, formula ratio and preparation operation in the embodiment are strictly kept consistent with those in Embodiment 1.

[0055] Embodiment 4

[0056] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the method is different from that in Embodiment 1 in that the heating rate in the preparation process of the colloidal solution in step S1 is 0.1 DEG C / min.

[0057] Except for the above differences, the materials, formula ratio and preparation operation in the embodiment are strictly kept consistent with those in Embodiment 1.

[0058] Embodiment 5

[0059] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the difference from the embodiment 1 is that the heating rate is 0.9 DEG C / min in the preparation process of the colloidal solution in the step S1.

[0060] Except for the above difference, the materials, the formula ratio and the preparation operation of the embodiment are consistent with the embodiment 1.

[0061] Embodiment 6

[0062] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the difference from the embodiment 1 is that the oscillation frequency is 35 times / min in the oscillation treatment of the first mixed solution in the step S2.

[0063] Except for the above difference, the materials, the formula ratio and the preparation operation of the embodiment are consistent with the embodiment 1.

[0064] Embodiment 7

[0065] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the difference from the embodiment 1 is that the equal amount of lutein ester is used to replace the DHA algal oil in the preparation process of the gel composition in the step S1.

[0066] Except for the above difference, the materials, the formula ratio and the preparation operation of the embodiment are consistent with the embodiment 1.

[0067] Embodiment 8

[0068] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the difference from the embodiment 1 is that the operation steps of the step S3 are different, and specifically, the step S3 comprises the following operation: the mixed solvent is prepared by mixing n-hexane, acetone, toluene and ethanol according to the volume ratio of 10:7:7:6, the mixed solvent is added into the second mixed solution and oscillated at the frequency of 20 times / min for 25 s, the second mixed solution containing the mixed solvent after the oscillation treatment is layered by standing, the supernatant is removed, and the operation of adding the mixed solvent, oscillation treatment, standing and removing the supernatant is repeated for 2-8 times to obtain the colloidal precipitate, and the colloidal precipitate is dried at 105 DEG C for 50 min to obtain the colloidal sample.

[0069] Except for the above difference, the materials, the formula ratio and the preparation operation of the embodiment are consistent with the embodiment 1.

[0070] Embodiment 9

[0071] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the method is different from that in the embodiment 1 in that: (1) in the step S1, equal amount of leaf yellow ester is used to replace the DHA algal oil in the preparation process of the gel composition; and (2) the operation steps in the step S3 are different, and specifically, the step S3 comprises the following operations: after mixing n-hexane, acetone, toluene and ethanol according to a volume ratio of 10:7:7:6, a mixed solvent is prepared, the mixed solvent is added into the second mixed solution and oscillated at a frequency of 20 times / min for 25 s, and the second mixed solution containing the mixed solvent after the oscillation treatment is allowed to stand to be layered, supernatant is removed, and the oscillation treatment, standing and supernatant removal operations are repeated 2-8 times, a colloid precipitate is obtained, and the colloid precipitate is dried at 105 DEG C for 50 min to obtain a colloid sample.

[0072] In addition to the above difference, the materials, formula ratio and preparation operation adopted in the embodiment are strictly consistent with those in the embodiment 1.

[0073] Embodiment 10

[0074] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the method is different from that in the embodiment 9 in that: the mixed solvent used in the step S3 is mixed by n-hexane, acetone, toluene and ethanol according to a volume ratio of 5:10:5:8.

[0075] In addition to the above difference, the materials, formula ratio and preparation operation adopted in the embodiment are strictly consistent with those in the embodiment 9.

[0076] Embodiment 11

[0077] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the method is different from that in the embodiment 9 in that: the mixed solvent used in the step S3 is mixed by n-hexane, acetone, toluene and ethanol according to a volume ratio of 10:5:10:3.

[0078] In addition to the above difference, the materials, formula ratio and preparation operation adopted in the embodiment are strictly consistent with those in the embodiment 9.

[0079] Embodiment 12

[0080] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the method is different from that in the embodiment 9 in that: in the step S3, equal amount of cyclohexane is used to replace n-hexane.

[0081] In addition to the above difference, the materials, formula ratio and preparation operation adopted in the embodiment are strictly consistent with those in the embodiment 9.

[0082] Embodiment 13

[0083] The present example provides a method for determining the isoelectric point of a gel composition, which differs from example 9 in 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.

[0084] In addition to the above differences, the materials used, the formulation ratios, and the preparation operations of the present example are strictly consistent with those of example 9.

[0085] Example 14

[0086] The present example provides a method for determining the isoelectric point of a gel composition, which differs from example 9 in 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.

[0087] In addition to the above differences, the materials used, the formulation ratios, and the preparation operations of the present example are strictly consistent with those of example 9.

[0088] Example 15

[0089] The present example provides a method for determining the isoelectric point of a gel composition, which differs from example 9 in that the mixed solvent used in step S3 is composed of n-hexane, acetone, toluene, and ethanol in a volume ratio of 3:12:7:6.

[0090] In addition to the above differences, the materials used, the formulation ratios, and the preparation operations of the present example are strictly consistent with those of example 9.

[0091] Example 16

[0092] The present example provides a method for determining the isoelectric point of a gel composition, which differs from example 9 in that the mixed solvent used in step S3 is composed of n-hexane, acetone, toluene, and ethanol in a volume ratio of 10:3:3:10.

[0093] In addition to the above differences, the materials used, the formulation ratios, and the preparation operations of the present example are strictly consistent with those of example 9.

[0094] Example 17

[0095] The present example provides a method for determining the isoelectric point of a gel composition, which differs from example 9 in that the mixed solvent used in step S3 is composed of n-hexane, acetone, toluene, and ethanol in a volume ratio of 12:7:12:2.

[0096] In addition to the above differences, the materials used, the formulation ratios, and the preparation operations of the present example are strictly consistent with those of example 9.

[0097] Example 18

[0098] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the difference from the embodiment 9 is that in the step S3, the frequency of the oscillation in the oscillation treatment operation after the mixed solvent is added into the second mixed solution is 10 times / min.

[0099] In addition to the above difference, the materials, the formula ratio and the preparation operation of the embodiment are strictly consistent with the embodiment 9.

[0100] Embodiment 19

[0101] The embodiment provides a method for measuring the isoelectric point of a gel composition, and the difference from the embodiment 9 is that in the step S3, the frequency of the oscillation in the oscillation treatment operation after the mixed solvent is added into the second mixed solution is 30 times / min.

[0102] In addition to the above difference, the materials, the formula ratio and the preparation operation of the embodiment are strictly consistent with the embodiment 9.

[0103] Comparative Example 1

[0104] The comparative example provides a method for measuring the isoelectric point of a gel composition, and the difference from the embodiment 1 is that in the step S2, the adding sequence of the ethanol and the ether in the preparation step of the first mixed solution is different, and specifically, the ether and the ethanol are sequentially added into the colloidal solution according to the volume ratio of 3:1:4 to obtain the first mixed solution.

[0105] In addition to the above difference, the materials, the formula ratio and the preparation operation of the comparative example are strictly consistent with the embodiment 1.

[0106] Comparative Example 2

[0107] The comparative example provides a method for measuring the isoelectric point of a gel composition, and the difference from the embodiment 1 is that in the step S2, the preparation step of the first mixed solution is different, and specifically, the ethanol and the ether are mixed according to the volume ratio of 1:4 and then added into the colloidal solution to obtain the first mixed solution, and the volume ratio of the colloidal solution, the ethanol and the ether is 3:1:4.

[0108] In addition to the above difference, the materials, the formula ratio and the preparation operation of the comparative example are strictly consistent with the embodiment 1.

[0109] Comparative Example 3

[0110] The comparative example provides a method for measuring the isoelectric point of a gel composition, and the difference from the embodiment 1 is that in the step S2, the same amount of petroleum ether is used to replace the ether.

[0111] Except for the above-mentioned difference, the materials, formulation, and preparation operation used in this comparative example are strictly consistent with those of Example 1.

[0112] Comparative Example 4

[0113] This comparative example provides a method for measuring the isoelectric point of a gel composition, which is different from Example 1 in that, in step S2, after the first mixture subjected to the oscillation treatment is allowed to stand to separate the supernatant, the operation of adding diethyl ether to the system and repeating the oscillation treatment, standing, and removing the supernatant is omitted.

[0114] Except for the above-mentioned difference, the materials, formulation, and preparation operation used in this comparative example are strictly consistent with those of Example 1.

[0115] Test Example 1: Colloid extraction efficiency

[0116] This test example uses the methods provided in Examples 1-19 and Comparative Examples 1-4 to detect the isoelectric point of a gel composition. During the entire detection process, the extraction rate of the colloid component (i.e., gelatin) in the gel composition is calculated as one of the indicators for evaluating the accuracy of the isoelectric point measurement results of the gel composition obtained by the measurement methods provided in Examples 1-19 and Comparative Examples 1-4. The extraction rate of the colloid is calculated by 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) x 100%.

[0117] Table 1: Colloid extraction rate

[0118]

[0119] The results of the colloid extraction rate in the gel composition during the measurement of the isoelectric point of the gel composition using the measurement methods provided in Examples 1-19 and Comparative Examples 1-4 are shown in Table 1.

[0120] The method provided in Examples 1-3 has a colloid extraction rate of more than 70% in the detection of the isoelectric point of the gel composition with gelatin as the colloid component. By comparing the colloid extraction rates of Examples 1-3 and Comparative Examples 1-4, it can be seen that in the determination of the isoelectric point of the gel composition, the type of ether reagent, the order of addition of ethanol and diethyl ether, and the number of times of adding diethyl ether all affect the colloid extraction rate in the gel composition. Whether the diethyl ether is added first and then the ethanol is added in Comparative Example 1 or the ethanol and diethyl ether are mixed and then added to the colloid solution in Comparative Example 2, emulsification is easy to occur, which increases the difficulty of subsequent colloid extraction. Therefore, in the determination of the isoelectric point of the gel composition, after adding the ethanol and diethyl ether to the colloid solution in sequence to obtain a first mixed solution and performing oscillation treatment, standing and layering, and removing the supernatant, adding diethyl ether to the system and repeating the oscillation treatment, standing and removing the supernatant operation can improve the colloid extraction rate, which is beneficial 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 colloid component.

[0121] By comparing the colloid extraction rates of Examples 1, 4 and 5, it can be seen that in the preparation of the colloid solution, after the gel composition is mixed with water, the system is warmed under water bath oscillation, and the warming rate involved in this warming process affects the subsequent colloid extraction rate. The isoelectric point determination method of the gel composition provided in Example 1 controls the warming rate to be within the range of 0.3-0.7℃ / min after the gel composition is mixed with water and the system is warmed under water bath oscillation, which 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 size consistency of the emulsion droplets in the colloid solution is better and the dispersion is more uniform, thereby improving the colloid extraction rate, which is beneficial to improving the accuracy of the isoelectric point determination result of the gel composition. The warming rate used in the preparation of the colloid solution in Example 4 is too low, which is easy to cause the emulsion droplets to be too large and not uniformly dispersed. The warming rate used in the preparation of the colloid solution in Example 5 is too high, which is easy to destroy the formation of the emulsion droplets. Too low or too high warming rate in the preparation of the colloid solution will affect the formation of the emulsion droplets in the colloid solution, thereby affecting the subsequent colloid extraction rate and the accuracy of the final isoelectric point determination result of the gel composition.

[0122] By comparing the colloid extraction rates of Example 1 and Example 6, it can be seen that the oscillation frequency of the first mixed solution prepared from the colloid solution, ethanol and diethyl ether in the oscillation treatment process affects the colloid extraction rate. This is mainly because when the oscillation frequency of the first mixed solution is higher than 35 times / min, emulsification is easy to occur, which makes it difficult to separate the oil phase and the water phase, which greatly reduces the colloid extraction rate and further affects the isoelectric point detection result of the gel composition.

[0123] Compared with Example 1, the gel composition used in the isoelectric point determination method provided by Example 7 contains not only gelatin but also lutein ester, and only the ethanol-ethyl ether extraction system is used to separate and purify the colloid component from the gel composition, while the methods provided by Examples 9-11 use not only the ethanol-ethyl ether extraction system but also a mixed solvent prepared from n-hexane, acetone, toluene and ethanol in a specific ratio to extract the colloid component in the second mixed solution during the determination of the isoelectric point of the gel composition containing both gelatin and lutein ester. By comparing the colloid extraction rate data of Examples 1, 7, 8, 9, it can be seen that the presence of lutein ester can reduce the yield and purity of the colloid and affect the accuracy of the final isoelectric point test results. The synergistic effect of the non-polar solvent, strong polar solvent and weak polar solvent in the mixed solvent can efficiently remove lutein ester from the gel composition and make the colloid precipitate in the lower layer, thereby improving the extraction rate of the colloid and obtaining high-purity colloid from the gel composition, and further improving the accuracy of the isoelectric point test results of the gel composition.

[0124] 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 can affect the extraction rate of the colloid.

[0125] By comparing the colloid extraction rate data of Examples 9, 18 and 19, it can be seen that the oscillation frequency used during the oscillation process after adding the mixed solvent to the second mixed solution can affect the extraction rate of the colloid. This is mainly because too low oscillation frequency can reduce the consistency and uniformity of the emulsion droplets and even cause stratification, and too high oscillation frequency can easily cause demulsification and lead to the separation of the oil phase and the water phase. Too low or too high oscillation frequency can affect the removal effect of lutein ester, thereby reducing the extraction rate of the colloid and further affecting the isoelectric point detection results of the gel composition.

[0126] Test Example 2 Demulsification Effect

[0127] This test example uses the methods provided in Examples 1-19 and Comparative Examples 1-4 to detect the isoelectric point of the gel composition. During the detection of the isoelectric point 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 shaking and allowing the first mixture to separate into layers, adding ether and repeating the shaking and allowing the mixture to stand in step S2 is observed. During the detection of the isoelectric point of the gel composition using the methods provided in Examples 9-19, the state of the liquid in the system after adding a mixed solvent to the second mixture, shaking and allowing it to separate into layers, adding ether and repeating the shaking and allowing the mixture to stand in step S3 is observed. The state of the liquid in the system is used to evaluate the demulsification effect. The demulsification effect is used to indirectly evaluate the effectiveness of the isoelectric point determination methods for the gel compositions provided in Examples 1-19 and Comparative Examples 1-4. A better demulsification effect prevents liquid emulsification, results in more obvious layering, makes it easier to obtain colloidal precipitation, and increases the colloid yield, ultimately leading to a more accurate measured isoelectric point of the gel composition.

[0128] Table 2. Demulsification Effect

[0129]

[0130] The demulsification effect during the determination of the isoelectric point of the gel composition using the methods provided in Examples 1-19 and Comparative Examples 1-4 is shown in Table 2. In Table 2, the demulsification effect was evaluated by visual observation and scoring. If the liquid in the system showed obvious stratification after standing, it indicated a good demulsification effect. If the liquid in the system had an emulsion layer after standing and only showed partial stratification, it indicated a moderate demulsification effect. If the liquid in the system was evenly dispersed and there was almost no stratification, it indicated a poor demulsification effect. The score from 1 to 10 represents the demulsification effect from good to bad, with a score of 10 indicating the best demulsification effect and a score of 1 indicating the worst demulsification effect.

[0131] In step S2 of Example 1, the first mixture is subjected to shaking treatment and static separation, followed by the addition of diethyl ether and repeated shaking treatment. After static separation, the liquid state of the system is as follows: Figure 1 As shown; in step S3 of Example 1, ethanol is added to the second mixture for repeated homogenization, and the resulting colloidal precipitate after centrifugation is as follows: Figure 2 As shown; the demulsification effect during the determination of the isoelectric point of the gel composition using the methods provided in Examples 9 and 19 is as follows. Figure 3 As shown, where, Figure 3 Figure A is a diagram showing the state of the liquid in the system after step S3 of Example 9, in which a mixed solvent is added to the second mixture, followed by shaking and settling, addition of ether, repeated shaking, and settling. Figure 3The state of the liquid in the system after the addition of the mixed solvent to the second mixed solution, the oscillation treatment, the standing and delamination, the addition of diethyl ether, and the repeated oscillation treatment and standing operation in Example 19, Step S3.

[0132] From the demulsification effects of Examples 1 and Comparative Examples 1 to 4, it can be seen that the type of ether reagent used, the order of addition of ethanol and diethyl ether, and the number of times of addition of diethyl ether all affect the demulsification effect during the determination of the isoelectric point of the gel composition. The demulsification effects of Examples 1, 4 and 5 show that the preparation of the colloidal solution by mixing the gel composition with water and then heating the system in a water bath under oscillation, and the heating rate involved in the heating process, both affect the demulsification effect. The demulsification effects of Examples 1, 6, and 9, 18 and 19 show the effect of the oscillation frequency on the demulsification effect. The demulsification effects of Examples 9 and 12 to 17 show that the components and their proportions in the mixed solvent also affect the demulsification effect to some extent.

[0133] Test Example 3: Lutein ester removal effect

[0134] The test example utilizes the method provided in Examples 7, 9-19 to detect the isoelectric point of the gel composition. In the process of detecting the isoelectric point of the gel composition using the method provided in Examples 9-19, the color of the supernatant after the oscillation treatment and static stratification operation of adding the mixed solvent to the second mixed solution in step S3 is observed. In the process of detecting the isoelectric point of the gel composition using the method provided in Example 7, the color of the supernatant after the oscillation treatment and static stratification operation of the first mixed solution in step S2, the repeated oscillation treatment and static stratification operation of adding diethyl ether is observed. The removal effect of lutein ester in the gel composition is evaluated by the color of the supernatant. The removal effect of lutein ester is used to indirectly evaluate the effect of the isoelectric point determination method of the gel composition provided in Examples 7, 9-19. Lutein ester itself is deep red brown. During the preparation of the gel composition with other components such as gelatin and the extraction of lutein ester in the gel composition according to the method provided in Examples 7, 9-19, lutein ester is yellow in the extraction system. The deeper the color of the supernatant, the more lutein ester is extracted into the supernatant, the better the removal effect of lutein ester, the higher the purity of the extracted colloidal component, and the higher the accuracy of the finally measured isoelectric point of the gel composition. The lighter the color of the supernatant, the less lutein ester is extracted into the supernatant, the worse the removal effect of lutein ester, the lower the purity of the extracted colloidal component, and the lower the accuracy of the finally measured isoelectric point of the gel composition. In addition, in the isoelectric point determination method of the gel composition provided in Examples 7, 9-19, other carotenoids (zeaxanthin, β-carotene, lycopene, astaxanthin) are used to replace lutein ester in the preparation step of the gel composition, and the removal effect of zeaxanthin, β-carotene, lycopene, and astaxanthin is evaluated according to the above method. The deeper the color of the supernatant, the more carotenoids are extracted into the supernatant, the better the removal effect of carotenoids, the lighter the color of the supernatant, the less carotenoids are extracted into the supernatant, the worse the removal effect of carotenoids.

[0135] Table 3 Carotenoid removal effect

[0136]

[0137] In the process of detecting the isoelectric point of the gel composition using the method provided in Examples 7, 9-19, the removal effect of carotenoids is shown in Table 3. In Table 3, the removal effect of carotenoids is evaluated by visual observation and scoring. If the supernatant color is darker, it means that the removal effect of carotenoids is better. If the supernatant color is lighter, it means that the removal effect of carotenoids is worse. The score from 1 to 10 represents the removal effect of carotenoids from good to bad. Score 10 represents the best removal effect of carotenoids, and score 1 represents the worst removal effect of carotenoids.

[0138] The removal effect of lutein ester in the process of detecting the isoelectric point of gel composition by the method provided in Examples 7, 9, 14, and 15 is shown in the following table, wherein, Figure 4 Figure 4 A in the table is the color result of the supernatant after the oscillation treatment and standing and layering of the first mixed solution in step S2, the repeated oscillation treatment, and the standing and layering operation of adding diethyl ether in the process of detecting the isoelectric point of gel composition by the method provided in Example 7, Figure 4 B, C, and D in the table are the color result of the supernatant after the oscillation treatment and standing and layering operation of adding mixed solvents to the second mixed solution in step S3 in the process of detecting the isoelectric point of gel composition by the methods provided in Examples 9, 14, and 15, respectively.

[0139] The gel composition used in the isoelectric point determination method provided in Example 7 contains carotenoids in addition to gelatin, and only the ethanol-diethyl ether extraction system is used to separate and purify the colloidal component from the gel composition, and the removal effect of carotenoids is poor.

[0140] Compared with Example 7, the method provided in Examples 9-11 uses a mixed solvent prepared from n-hexane, acetone, toluene, and ethanol in a specific ratio to extract the colloidal component in the second mixed solution in addition to the ethanol-diethyl ether extraction system in the process of determining the isoelectric point of the gel composition containing both gelatin and carotenoids, and the removal effect of carotenoids is significantly improved.

[0141] 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 removal effect of carotenoids in the gel composition.

[0142] By comparing the lutein ester removal effects of Examples 9, 18, and 19, it can be seen that the oscillation frequency in the oscillation treatment process after adding the mixed solvent to the second mixed solution will affect the removal effect of carotenoids in the gel composition.

[0143] Test Example 4

[0144] ​The isoelectric point of the gel composition was determined by the method provided in Examples 1-19 and Comparative Examples 1-4. In the preparation of the gel composition involved in Examples 1-19 and Comparative Examples 1-4, the gelatin used was Gelatin 180 Freeze-Acid Method (purchased from Fujian Funing Pu Biotechnology Co., Ltd.), and the above-mentioned Gelatin 180 Freeze-Acid Method was used as a control group and its isoelectric point was detected according to GB 6783-94. The preparation method of the test solution (containing gelatin) for isoelectric point testing was according to the step S4 of Example 1. Each group of experiments was repeated twice, and the final result was the average value. 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 method provided in Examples 1-19 and Comparative Examples 1-4 and the isoelectric point of the gelatin 180 Freeze-Acid Method measured according to GB 6783-94 was calculated.

[0145] The freeze number (180 freeze) in "Gelatin 180 Freeze-Acid Method" refers to the gel strength of gelatin, also known as bloom strength. Gelatin is divided into alkali gelatin (also known as type B gelatin), acid gelatin (also known as type A gelatin), and enzyme gelatin according to the production method. "Gelatin 180 Freeze-Acid Method" refers to gelatin with a gel strength of 180 bloom and produced by alkali method.

[0146] Table 4 Isoelectric point test results of different gel compositions by the method provided in Examples 1-19 and Comparative Examples 1-4

[0147]

[0148] The results of the isoelectric point determination of the gel composition by the method provided in Examples 1-19 and Comparative Examples 1-4 are shown in Table 4.

[0149] Compared with Comparative Examples 1-4, the detection results of the isoelectric point of the gel composition with gelatin as the colloidal component by the method provided in Examples 1-3 (5.75-5.76) were very close to the detection results of the isoelectric point of gelatin according to GB 6783-94 (5.89), which indicated that during the isoelectric point determination of the gel composition, after adding ethanol and diethyl ether to the colloidal solution to obtain a first mixture and subjecting it to oscillation treatment, standing and layering, and removing the supernatant, adding diethyl ether to the system and repeating the above oscillation treatment, standing and removing the supernatant operation could improve the extraction rate of the colloid, which was beneficial to improve the accuracy of the final isoelectric point determination result, and the measured isoelectric point of the gel composition was closer to the intrinsic isoelectric point of the colloidal component.

[0150] By comparing the isoelectric point detection results of Example 1, 4, 5 and the control group, it can be known that in the preparation process of the colloidal solution, the temperature of the system is raised under water bath oscillation after the gel composition is mixed with water, and the temperature raising rate involved in the process will affect the extraction rate of the gel and thus the accuracy of the final isoelectric point detection result. The isoelectric point determination method of the gel composition provided in Example 1 can improve the accuracy of the isoelectric point determination result of the gel composition by controlling the temperature raising rate within the range of 0.3-0.7 ℃ / min after the gel composition is mixed with water and the system is raised in temperature under water bath oscillation.

[0151] By comparing the isoelectric point detection results of Example 1, 6 and the control group, it can be known 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 extraction rate of the gel and thus the final isoelectric point detection result. Controlling the oscillation frequency of the first mixed solution to be not more than 35 times / min can improve the accuracy of the isoelectric point detection result of the gel composition.

[0152] Compared with Example 1, the gel composition used in the isoelectric point determination method provided in Example 7 contains not only gelatin but also lutein ester, and only an ethanol-diethyl ether extraction system is used to separate and purify the gel component from the gel composition. In the methods provided in Examples 9-11, in addition to the ethanol-diethyl ether extraction system, a mixed solvent prepared from n-hexane, acetone, toluene and ethanol in a specific ratio is also used to extract the gel component in the second mixed solution during the determination of the isoelectric point of the gel composition containing both gelatin and carotenoids. Among the isoelectric point detection results of Example 1, 7, 8, 9 and the control group, the isoelectric point of Example 9 is closest to that of the control group, which indicates that the synergistic effect of the non-polar solvent, strong polar solvent and weak polar solvent in the mixed solvent can efficiently remove carotenoids in the gel composition and improve the extraction rate of gelatin, further improving the accuracy of the isoelectric point test result of the gel composition.

[0153] By comparing the isoelectric point detection results of Example 9, 12, 13, 14, 15, 16 and 17, it can be known that the components in the mixed solvent and their ratios will affect the accuracy of the isoelectric point detection result.

[0154] By comparing the isoelectric point detection results of Example 9, 18 and 19, it can be known that too low oscillation frequency in the oscillation process after the mixed solvent is added to the second mixed solution will reduce the uniformity and dispersion uniformity of the emulsion droplets, and even cause stratification. Too high oscillation frequency is prone to cause demulsification and thus cause the separation of the oil phase and the water phase. Too low or too high oscillation frequency will affect the extraction rate of gelatin, and thus affect the accuracy of the isoelectric point detection result of the gel composition.

[0155] Test Example 5

[0156] The isoelectric point of the gel products (i.e. gel compositions) made from different gelatin materials was detected by the isoelectric point detection method provided in Example 9, each group of experiments was repeated twice, the final result was averaged, the isoelectric point detection result of Example 9 was compared with the isoelectric point detection result of different gelatin materials according to GB 6783-94, and the deviation between the isoelectric point detected by the method provided in Example 9 for the gel products made from different gelatin materials and the isoelectric point detected by GB 6783-94 for different gelatin materials was calculated according to the following formula: for the gel products, the isoelectric point deviation = |the isoelectric point of the gel product measured by Example 9 - the isoelectric point of the pure gelatin material measured by GB 6783-94| / the isoelectric point of the pure gelatin material measured by GB 6783-94 x 100%, the preparation method of the test solution (containing gelatin) for isoelectric point test was referred to the step S4 of Example 1, the gel products were prepared according to the gel composition preparation method in step S1 of Example 1, and the results are shown in Table 5.

[0157] The gelatin materials used in this test example include 180 freeze-acid method, gelatin 180 freeze-alkali method, gelatin 220 freeze-acid method, gelatin 220 freeze-alkali method, gelatin 250 freeze-acid method, and gelatin 250 freeze-alkali method, wherein:

[0158] The 180 freeze-alkali method and the gelatin 250 freeze-alkali method were purchased from Rosebio Biotech Co., Ltd., and the 180 freeze-acid method, the gelatin 220 freeze-acid method, the gelatin 220 freeze-alkali method, and the gelatin 250 freeze-acid method were purchased from Fujian Funingpu Biotech Co., Ltd.

[0159] The freeze number (180 freeze, 220 freeze, 250 freeze) in the above gelatin materials refers to the gel strength of the gelatin, also known as bloom strength. Gelatin is divided into alkali gelatin (also known as B-type gelatin), acid gelatin (also known as A-type gelatin), and enzyme gelatin according to the production method. The above 180 freeze-acid method, gelatin 220 freeze-acid method, and gelatin 250 freeze-acid method are produced by acid method, and the gelatin 180 freeze-alkali method, gelatin 220 freeze-alkali method, and gelatin 250 freeze-alkali method are produced by alkali method.

[0160] The gel products (i.e. gel compositions) used in the present test example were prepared according to the gel composition preparation method of step S1 of Example 1, wherein the gelatin used in the 180 freeze-acid extracted cheese was gelatin 180 freeze-acid, the gelatin used in the 180 freeze-alkali extracted cheese was gelatin 180 freeze-alkali, the gelatin used in the 220 freeze-acid extracted cheese was gelatin 220 freeze-acid, the gelatin used in the 220 freeze-alkali extracted cheese was gelatin 220 freeze-alkali, the gelatin used in the 250 freeze-acid extracted cheese was gelatin 250 freeze-acid, and the gelatin used in the 250 freeze-alkali extracted cheese was gelatin 250 freeze-alkali.

[0161] Table 5. Comparison of the isoelectric point test results of the 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

[0162]

[0163] The isoelectric point test results of the gel products made from different gelatin materials using the isoelectric point determination method of the gel composition provided in Example 9 and the isoelectric point test results of the pure gelatin materials used in the gel products according to GB 6783-94 are shown in Table 5.

[0164] As can be seen from Table 5, when the gel products (i.e. gel compositions) using gelatin as the colloid component are tested using the method provided in Example 9, the deviation between the measured isoelectric point and the isoelectric point of the pure gelatin material measured according to GB 6783-94 is very small, all within 4%. The above results can prove that the method provided in Example 9 can selectively remove the fat-soluble additives, water-soluble impurities and carotenoids and other components in the gel composition from the gel composition containing both gelatin and carotenoids by using the ethanol-ethyl ether extraction system combined with the mixed solvent composed of n-hexane, acetone, toluene and ethanol, obtain a high-purity colloid sample, and eliminate the charge interference of other components in the gel composition on the isoelectric point determination to the greatest extent, thereby ensuring that the test object is the gelatin component in the gel skeleton, and thus improving the accuracy of the isoelectric point detection results of the gel composition and making the finally measured isoelectric point closer to the intrinsic isoelectric point of the gelatin.

[0165] The above examples are only used to illustrate the technical solutions of the present application and not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the above examples, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents, and these modifications or replacements are within the protection scope of the present application.

Claims

1. A method for determining the isoelectric point of a gel composition, characterized in that, Includes the following steps: S1. The gel composition is mixed with water at a volume ratio of (1~3):(2~5), and the system is heated to 30~40℃ and kept at that temperature for 5~10 min under water bath shaking. Then, the system is heated to T1 at a rate of 0.3~0.7℃ / min and kept at that temperature for 5~10 min to obtain a colloidal solution. The gel composition contains a colloid and carotenoids. The colloid is gelatin. The carotenoids include at least one of lutein ester, zeaxanthin, β-carotene, lycopene, astaxanthin, fucoxanthin, and canthaxanthin. The denaturation temperature of the gel composition is T2. T1 and T2 satisfy the condition that T1 < T2. S2. Ethanol and diethyl ether are added sequentially to the colloidal solution to obtain a first mixture. The first mixture is oscillated at a frequency not exceeding 30 times / min for 10-40 s at temperature T1. After standing, the first mixture after oscillation is separated into layers. After removing the supernatant, diethyl ether is added to the system and the above oscillation, standing and supernatant removal operations are repeated to obtain a second mixture. The volume ratio of the colloidal solution, the ethanol and the diethyl ether in the first mixture is (2-4):(1-2):(3-5). S3. Prepare a mixed solvent using a non-polar solvent, a strong polar solvent, and a weak polar solvent. Add the mixed solvent to the second mixture and shake it. Let it stand so that the shaken second mixture containing the mixed solvent separates into layers. Remove the supernatant to obtain a colloidal precipitate. Dry the colloidal precipitate to obtain a colloidal sample. The volume ratio of the nonpolar solvent, the strongly polar solvent, and the weakly polar solvent in the mixed solvent is (5~15):(8~18):(5~10); The nonpolar solvent includes at least one of cyclohexane, n-hexane, pentane, and heptane; The strongly polar solvent includes ethanol, and the strongly 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; S4. Prepare a colloidal solution using the colloidal sample and 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 the gel composition as described in claim 1, characterized in that: The nonpolar solvent is n-hexane; The strongly polar solvent includes ethanol, and the strongly polar solvent also includes acetone or ethyl acetate; The weakly polar solvent is toluene.

3. The method for determining the isoelectric point of the gel composition as described in claim 2, characterized in that: The mixed solvent is composed of n-hexane, acetone, toluene, and ethanol in a volume ratio of (5~15):(5~10):(5~10):(3~8).

4. The method for determining the isoelectric point of the gel composition as described in claim 1, characterized in that, In step S3, the oscillation process after adding the mixed solvent to the second mixture includes the following operation: adding the mixed solvent to the second mixture and oscillating at a frequency of 15 to 25 times / min for 10 to 40 seconds.

5. The method for determining the isoelectric point of the gel composition as described in claim 1, characterized in that: In step S3, before drying the colloidal precipitate, a homogenization process using ethanol is further included. And / or, In step S3, the drying temperature used in the drying process of the colloidal precipitate is 60~105℃, and the drying time is 30~80 min.

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