Identification method for adulteration of sodium alginate gel in instant bird's nest products

Abstract

The present invention provides a method for identifying sodium alginate gel adulteration in instant bird's nest products, comprising: S1, crushing the bird's nest, adding an acid solution to heat the product in a water bath, cooling and centrifuging, removing a first filtrate, rinsing the filter residue, adding an alkaline solution, shaking and diluting with water, and filtering to obtain a second filtrate as a test sample; S2, subjecting the test sample to high performance liquid chromatography-evaporative light scattering detector / differential refractive index detector detection and analysis, and determining whether the test sample contains sodium alginate gel by comparing the spectra of the test sample with those of a standard substance. The present invention adopts a sample pretreatment method of adding acid, washing with water, and dissolving with alkali, which can remove other interfering components in the bird's nest on the one hand, and can also fully dissolve the gel-like sodium alginate gel, and then use a high performance liquid chromatography-evaporative light scattering detector / differential refractive index detector determination method to identify the sodium alginate gel adulteration in the bird's nest.

Description

Technical Field

[0001] The invention relates to a method for identifying sodium alginate gel adulteration in instant bird's nest products. Background Art

[0002] Bird's nests are nests built by the saliva secreted by swiftlets during the breeding season, or by the condensation of saliva and feathers. Bird's nests have long been considered a health food and medicine, nourishing the skin and moistening the lungs. Used both as food and medicine, bird's nests are rich in protein, amino acids, sialic acid, and inorganic elements. Research has shown that bird's nests have antioxidant, immune-enhancing, anti-aging, antiviral, blood clotting inhibitory, and intestinal flora regulation benefits.

[0003] Current research on bird's nest authentication focuses on identifying bird's nests and adulterants, identifying the origin and production methods of bird's nests, and identifying the species of bird's nests. These methods rely on the unique properties of the bird's nest itself or the differences between adulterants and bird's nests, such as molecular biological testing. These methods are generally complex, expensive, and unable to quantify adulterants. Sodium alginate is a polysaccharide biopolymer extracted from brown algae, composed of α-1-guluronic acid and β-d-mannuronic acid. It is non-toxic, hydrophilic, soluble, and thickening, and is widely used in various fields. If sodium alginate gel is used to counterfeit bird's nests, while current detection methods can identify them, they are complex and costly.

[0004] Sodium alginate and calcium ions can undergo ion exchange to produce calcium alginate gel. From a morphological perspective, it is difficult to distinguish calcium alginate from bird's nest. The present invention utilizes the principle of ion exchange. Calcium alginate gel reacts with hydrochloric acid or sulfuric acid at 100°C to displace calcium ions. Sodium hydroxide solution is then added to form a sodium alginate solution. High-performance liquid chromatography is then used for detection and analysis. By comparing the spectra of the sample to be tested with the standard substance, it is determined whether the sample to be tested contains sodium alginate gel. Accurate quantification is then performed using a standard curve established using the standard substance. This method can quickly and accurately detect the sodium alginate content corresponding to sodium alginate gel doping in bird's nests and similar foods, with good repeatability and recovery, making it suitable for analysis of large quantities of samples. Summary of the Invention

[0005] The present invention provides a method for identifying sodium alginate gel adulteration in instant bird's nest products. The method provided by the present invention has simple and quick pretreatment operations, fully stable chemical reactions, high accuracy, small deviation between measurement results and actual results, good reproducibility, and can meet the laboratory's needs for identifying sodium alginate gel adulteration in bird's nests and similar foods and determining the corresponding sodium alginate content.

[0006] The present invention is achieved in that:

[0007] A method for identifying sodium alginate gel adulteration in instant bird's nest products, comprising:

[0008] S1, crush and homogenize the bird's nest sample containing sodium alginate gel, add acid solution, heat in a water bath at 80℃-100℃ for 5-60min, cool and centrifuge, discard the supernatant, wash and centrifuge with water several times, retain the insoluble matter, add alkaline solution, shake well, dilute to volume with water, and filter to obtain the sample to be tested;

[0009] S2. Performing high performance liquid chromatography-evaporative light scattering detector / differential refractive index detector detection and analysis on the sample to be tested, and determining whether the sample to be tested contains sodium alginate gel by comparing the spectra of the sample to be tested with those of the standard substance.

[0010] The present invention further provides a method for detecting sodium alginate gel adulteration in gel foods, comprising:

[0011] S1. Grind and homogenize a gel food sample containing sodium alginate gel, add an acid solution, heat in a water bath at 80°C-100°C for 5-60 minutes, cool and centrifuge, discard the supernatant, wash with water and centrifuge several times, retain the insoluble matter, add an alkaline solution, shake well, dilute to volume with water, and filter to obtain the sample to be tested;

[0012] S2. Performing high performance liquid chromatography-evaporative light scattering detector / differential refractive index detector detection and analysis on the sample to be tested, and determining whether the sample to be tested contains sodium alginate gel by comparing the spectra of the sample to be tested with those of the standard substance.

[0013] The beneficial effects of the present invention are as follows: by adopting a sample pretreatment method of adding acid, washing with water, and dissolving with alkali, the present invention can, on the one hand, remove other interfering components in the bird's nest, and can also fully dissolve the gel-like sodium alginate gel. The sodium alginate gel in the bird's nest can be identified by a high-performance liquid chromatography-evaporative light scattering detector / differential refractive index detector determination method, and the identification and analysis of the sodium alginate gel in the bird's nest can be completed within 4 minutes. The method of the present invention can further detect sodium alginate gel in samples with an impure solid content of more than 1wt%, with good linearity in the concentration range of 0.10-0.50mg / mL, and both precision and accuracy meet the analytical requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 1 is a graph showing sodium alginate concentration and peak area according to an embodiment of the present invention.

[0016] Figure 2 、 Figure 3 Spectrum of sodium alginate standard solution according to an embodiment of the present invention.

[0017] Figure 4-Figure 6 This is a spectrum of sodium alginate gel in the bird's nest sample according to the embodiment of the present invention.

[0018] Figure 7 This is a graph showing the effect of acid on sodium alginate content in accordance with an embodiment of the present invention.

[0019] Figure 8 3 is a graph showing the effect of water bath time on sodium alginate content in accordance with an embodiment of the present invention.

[0020] Figure 9 1 is a graph showing the effect of pH on sodium alginate content in accordance with an embodiment of the present invention.

[0021] Figure 10 This is the sodium alginate chromatogram of the amino chromatographic column of the present invention.

[0022] Figure 11 This is the sodium alginate chromatogram of the strong cation chromatographic column of the present invention.

[0023] Figure 12 This is the chromatogram of sodium alginate on a phenyl column of the present invention.

[0024] Figure 13 It is the chromatogram of the hydrophilic C18 column sodium alginate of the present invention.

[0025] Figure 14 The chromatogram of the hydrophilic C18 column sodium alginate on a differential refractive index detector of the present invention is shown. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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 ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention for which protection is sought, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] In the description of the present invention, the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, a feature specified as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

[0028] The embodiment of the present invention provides a method for identifying sodium alginate gel adulteration in an instant bird's nest product, comprising:

[0029] S1, crush and homogenize the bird's nest sample containing sodium alginate gel, add acid solution, heat in a water bath at 80℃-100℃ for 5-60min, cool and centrifuge, discard the supernatant, wash and centrifuge with water several times, retain the insoluble matter, add alkaline solution, shake well, dilute to volume with water, and filter to obtain the sample to be tested;

[0030] S2. Performing high performance liquid chromatography-evaporative light scattering detector / differential refractive index detector detection and analysis on the sample to be tested, and determining whether the sample to be tested contains sodium alginate gel by comparing the spectra of the sample to be tested with those of the standard substance.

[0031] In step S1, as a further improvement, the step of adding the acid solution and heating in a water bath comprises:

[0032] A sulfuric acid solution with a concentration of 0.5-1.5 mol / L is added and heated in an 80-100°C water bath for 5-60 minutes. The main purpose of this step is to remove the cross-linking state in the sodium alginate gel and allow it to fully dissolve in water. The concentration of sulfuric acid cannot be too high, as it will oxidize the bird's nest and other substances and dissolve them, affecting subsequent reactions. Preferably, a sulfuric acid solution with a concentration of 0.7-1.0 mol / L is added and heated in a 95-100°C water bath for 25-45 minutes. In one embodiment, a sulfuric acid solution with a concentration of 0.9 mol / L is added and heated in a 100°C water bath for 30 minutes. The ratio of the weighed bird's nest sample to the acid solution is (0.5-5.0) g: (1-15) ml. In one embodiment, the ratio of the bird's nest to the sulfuric acid solution is approximately 1 g: 2 ml. Experiments have shown that the above method can fully dissolve more than 99 wt% of the sodium alginate gel in the bird's nest without damaging other substances.

[0033] As a further improvement, the step of adding the alkaline solution comprises:

[0034] Sodium hydroxide solution is added to adjust the pH to 8-9. The added alkaline solution is a 0.5-1.5.0 mol / L sodium hydroxide solution. Adding sodium hydroxide to adjust the pH serves two purposes: neutralizing the sulfuric acid in the solution to prevent damage to the chromatographic column and converting the product into a sodium alginate solution, which facilitates subsequent testing.

[0035] As a further improvement, the steps of shaking, diluting with water, and filtering to obtain the sample to be tested as the sample to be tested include:

[0036] Shake well and dilute to volume with water. Pass the mixture through a 0.22 μm aqueous filter membrane to obtain the second filtrate as the sample to be tested. Excessive particles may clog the chromatographic column.

[0037] In step S2, as a further improvement, the optimal liquid chromatography conditions are: a hydrophilic C18 column; mobile phase A is acetonitrile, and mobile phase B is first-grade water. The ratio of mobile phase A to mobile phase B is 20:80; the flow rate is 1.0 mL / min; the column temperature is 40°C; and the injection volume is 5 μL. Experiments have shown that this column can effectively separate sodium alginate. This prevents the influence of mannuronic acid in bird's nest on the detection of sodium alginate. Other control experiments revealed that other types of columns and mobile phases were unable to effectively separate sodium alginate and mannuronic acid.

[0038] As a further improvement, in another embodiment, the detector conditions include evaporation temperature: 60° C., atomization temperature: 60° C., evaporation gas volume: 1.65 SLM, and LED light intensity: 100%.

[0039] As a further improvement, the method further comprises:

[0040] S3, drawing a standard quantitative curve and calculating the sodium alginate gel doping content in the bird's nest according to the standard quantitative curve.

[0041] The above detection method is not limited to the detection of sodium alginate gel adulteration in bird's nests, but can also be used for some gel foods, such as shark fin, peach gum, pectin, ice jelly and other foods.

[0042] The embodiment of the present invention further provides a method for detecting sodium alginate gel adulteration in gel foods, comprising:

[0043] S1. Grind and homogenize a gel food sample containing sodium alginate gel, add an acid solution, heat in a water bath at 80°C-100°C for 5-60 minutes, cool and centrifuge, discard the supernatant, wash with water and centrifuge several times, retain the insoluble matter, add an alkaline solution, shake well, dilute to volume with water, and filter to obtain the sample to be tested;

[0044] S2. Performing high performance liquid chromatography-evaporative light scattering detector / differential refractive index detector detection and analysis on the sample to be tested, and determining whether the sample to be tested contains sodium alginate gel by comparing the spectra of the sample to be tested with those of the standard substance.

[0045] The detection method is basically the same as the detection method for sodium alginate gel doping in bird's nests, and will not be repeated here.

[0046] In order to further demonstrate the beneficial effects of the present invention and to better understand the present invention, the following determination test is further used to illustrate the properties and application performance of the high performance liquid chromatography-evaporative light scattering detector / differential refractive index detector determination method of sodium alginate in bird's nest of the present invention, but it should not be understood as a limitation of the present invention. For those skilled in the art, the properties of the method obtained by other determination experiments based on the above invention content and the application based on the above properties are also considered to fall within the scope of protection of the present invention.

[0047] 1. Instruments and reagents

[0048] Instruments: Agilent 1260 high performance liquid chromatograph with evaporative light scattering detector (ELSD) (Agilent, USA); electronic balance (Mettler-Toledo).

[0049] Reagents: sodium alginate standard (Xinyang Zhongjian Metrology Biotechnology Co., Ltd.); acetonitrile (chromatographic grade, Anhui Tiandi High Purity Solvent Co., Ltd.); methanol (chromatographic grade, Anhui Tiandi High Purity Solvent Co., Ltd.); ultrapure water.

[0050] 2. Standard solution and configuration

[0051] Accurately pipette 0.10, 0.20, 0.30, 0.40, and 0.50 mL of 10 mg / mL sodium alginate standard stock solution into a 10 mL volumetric flask, add water to the scale to make sodium alginate standard solutions with concentrations of 0.10, 0.20, 0.30, 0.40, and 0.50 mg / mL. Take 5 μL of the sample and plot the peak area-concentration of sodium alginate to obtain the regression equation of the standard curve. The results are shown in the figure. Figure 1 .

[0052] 3. Instrument analysis conditions

[0053] Liquid chromatography conditions

[0054] Chromatographic column: hydrophilic C18 column; mobile phase A is acetonitrile, mobile phase B is first-grade water, the ratio is 20:80; flow rate is 1.0 mL / min; column temperature is 40°C; injection volume is 5 μL.

[0055] 3.2. Evaporative light scattering detector conditions

[0056] Evaporation temperature: 60℃, atomization temperature: 60℃, evaporation gas volume: 1.65SLM, LED light intensity: 100%.

[0057] 4. Experimental methods and results

[0058] 4.1 Sample processing

[0059] Crush and homogenize the bird's nest, weigh 5 g, add 10 mL of 0.9 mol / L sulfuric acid solution, incubate in a 100°C water bath for 30 min, cool to room temperature, centrifuge at 10,000 r / min for 30 min, discard the supernatant, wash and centrifuge several times, add 1.0 mol / L sodium hydroxide solution to dissolve until the pH is 8-9, dilute to 100 mL with water, filter through a 0.22 μm aqueous filter membrane, and use the filtrate as the sample to be tested.

[0060] 4.1.1 Single Factor Investigation

[0061] The instant bird's nest samples used were all prepared by mixing a 0.5% sodium alginate solution with a 2.0% calcium lactate solution. Other pretreatment conditions and detector conditions were the same. The effects of hydrochloric acid and sulfuric acid, the amount of acid added, the water bath time, and the pH value of the alkaline solution added to the solution on the sodium alginate content were investigated.

[0062] 4.1.2 Investigation of acid hydrolysis conditions

[0063] Since both hydrochloric acid solution and sulfuric acid solution can react with calcium alginate gel and replace calcium ions, a single factor experiment was conducted to investigate the effects of hydrochloric acid and sulfuric acid as well as the amount of acid added on the sodium alginate content. The amount of acid added was within the range of 1ml-15ml. As the amount of acid added increased, the sodium alginate content increased and eventually stabilized. Compared with hydrochloric acid, 10mL, 0.9mol / L sulfuric acid had the best effect. The results are shown in the figure. Figure 7 .

[0064] 4.1.3 Investigation of water bath time

[0065] Under the condition that the amount of 0.9 mol / L sulfuric acid added is 10 ml, the effect of 100 ℃ water bath time on the sodium alginate content is investigated. The water bath time is within the investigation range of 5-50 min. As the reaction time prolongs, the sodium alginate content gradually increases and reaches the maximum when the water bath time is 30 min. Since too short a reaction time will lead to incomplete calcium ion replacement, the sodium alginate content decreases. Finally, 30 min is selected as the water bath time. The results are shown in Figure 2. Figure 8 .

[0066] 4.1.4 Investigation of the amount of sodium hydroxide solution added

[0067] Sodium alginate can be better dissolved under alkaline conditions, but the pH has an effect on the chromatographic column, so it is necessary to determine the amount of sodium hydroxide solution to be added by determining the pH of the sample solution. Weigh 5g of the sample, add 10mL, 0.9mol / L sulfuric acid solution in a 100℃ water bath for 30min, cool to room temperature, centrifuge at 10000r / min for 30min, discard the supernatant, wash and centrifuge several times, add sodium hydroxide solution to dissolve and adjust the pH to 4-5, 6-7, 8-9, 10-11, 11-12, 12-13 respectively, make up to 100mL with water, filter through a 0.22μm aqueous phase membrane, and put on the machine. As the pH gradually increases, the sodium alginate content gradually increases. When the pH range is 8-9, it reaches the maximum, so the pH range is determined to be 8-9. See the results. Figure 9 .

[0068] 4.2 Optimization of chromatographic conditions

[0069] 4.2.1 Examination of chromatographic columns

[0070] The separation effects of amino column, strong cation column, phenyl column and hydrophilic C18 column on sodium alginate were investigated respectively. The analytical chromatograms of sodium alginate by different columns are shown in Figure 10-13 , the results show that Figure 10-12 , the peak shape is poor and the response value is low; Figure 13 The peak shape, response value and separation are relatively good, so the final chromatographic column is: hydrophilic C18 chromatographic column.

[0071] 4.2.2 Investigation of mobile phase ratio

[0072] The invention determines that the mobile phase A is acetonitrile and the mobile phase B is first-grade water. The mobile phase ratios are investigated respectively, and the mobile phase ratios are V(A):V(B)=10:90, V(A):V(B)=20:80, V(A):V(B)=30:70, and V(A):V(B)=40:60, respectively, for on-machine determination. Because the peak time has a certain influence on the result accuracy of the test sample, through test analysis, when the V(A):V(B) ratio of the mobile phase acetonitrile to water is 20:80, the retention time of the sample chromatographic peak is stable, the separation is optimal, and the peak shape is sharp, so the mobile phase V(A):V(B)=20:80 is finally determined.

[0073] 4.3 Linear range and detection limit

[0074] Under the set experimental conditions, sodium alginate: 0.10-0.50mg / mL. Take 5μL of sample, use the peak area-concentration of sodium alginate to plot, get the standard curve regression equation, record the spectrum to get the linear equation and correlation coefficient, the linear regression equation is shown in Figure 1, and its linear correlation coefficient is greater than 0.999. The detection limit (LOD) and quantification limit (LOQ) of the instrument are determined by the stepwise dilution method of the standard solution, and the target concentration corresponding to the signal-to-noise ratio S / N ≥ 3 and S / N ≥ 10 are used as the instrument detection limit and quantification limit, respectively. Based on a sample weight of 5g and a fixed volume of 25mL, the detection limit is 0.25g / kg and the quantification limit is 0.75g / kg. The standard sodium alginate was tested, and the test results are as follows Figure 2-Figure 3 .

[0075] 4.4 Precision

[0076] Since sodium alginate gel cannot be added to the bird's nest for verification, different sampling amounts were used to investigate its accuracy. Six parallel precision tests were conducted with gradient sampling amounts of 1g, 2g, and 5g, and the precision was calculated. The RSD was between 1.5% and 2.0%. The specific results are shown in Table 1.

[0077] Table 1 Precision of sodium alginate gel in bird's nest (n=6)

[0078]

[0079]

[0080] 4.5 Detection of adulterated bird’s nest samples

[0081] The bird's nests mixed with sodium alginate gel in different proportions were tested, and 6 parallel samples were tested in each batch. Figure 4 、 Figure 5 、 Figure 6 and Figure 14 By comparing the spectrum obtained with the standard under the same conditions, it can be seen that the bird's nest sample contains sodium alginate gel. According to the established standard curve, the sodium alginate content is calculated. The results are shown in Table 2. The relative standard deviation is between 9.5 and 11.5, and the minimum detection doping value is 10% of the sodium alginate gel solids.

[0082] Table 2 Detection of sodium alginate adulteration in bird's nest (n=6)

[0083]

[0084] The embodiment of the present invention adopts a sample pretreatment method of adding acid, washing with water, and dissolving with alkali, and a determination method using high performance liquid chromatography-evaporative light scattering detector to analyze the doping of sodium alginate gel in bird's nest. The entire experimental method is simple to operate, fast and efficient, accurate, sensitive and reproducible, and is suitable for rapid detection and analysis of sodium alginate gel doping in bird's nest. The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for identifying sodium alginate gel adulteration in instant bird's nest products, characterized in that: include: S1: Grind and homogenize the bird's nest sample containing sodium alginate gel, add a sulfuric acid solution with a concentration of 0.5-1.5 mol / L, heat in a water bath at 80-100°C for 5-60 minutes, cool and centrifuge, discard the supernatant, wash and centrifuge with water several times, retain the insoluble matter, add alkaline solution, shake well, dilute to volume with water, and filter to obtain the sample to be tested; S2. Perform high performance liquid chromatography-evaporative light scattering detector / differential refractive index detector (DRD) detection and analysis on the sample to be tested. By comparing the spectra of the sample to be tested with those of the standard substance, determine whether the sample to be tested contains sodium alginate gel. The liquid chromatography conditions are as follows: a hydrophilic C18 column; mobile phase A is acetonitrile, mobile phase B is first-grade water; and the ratio of mobile phase A to mobile phase B is 20:

80.

2. The method for identifying sodium alginate gel adulteration in instant bird's nest products according to claim 1, characterized in that: Further including: A standard quantitative curve is drawn and the sodium alginate gel doping content in the bird's nest is calculated based on the standard quantitative curve.

3. The method for identifying sodium alginate gel adulteration in instant bird's nest products according to claim 1, characterized in that: The step of adding sulfuric acid solution comprises: The concentration of the sulfuric acid solution is 0.9 mol / L; the ratio of the bird's nest sample weight to the sulfuric acid solution is (0.5-5.0) g: (1-15) ml.

4. The method for identifying sodium alginate gel adulteration in instant bird's nest products according to claim 1, characterized in that: The step of adding alkaline solution comprises: Sodium hydroxide solution is added to make its pH reach 8-9; the added alkaline solution is 1.0 mol / L sodium hydroxide solution.

5. The method for identifying sodium alginate gel adulteration in instant bird's nest products according to claim 1, characterized in that: The steps of shaking, diluting with water, and filtering to obtain the sample to be tested include: The mixture was shaken and diluted to volume with water, and passed through a 0.22 μm aqueous filter membrane to obtain the second filtrate as the sample to be tested.

6. The method for identifying sodium alginate gel adulteration in instant bird's nest products according to claim 1, characterized in that: The flow rate was 1.0 mL / min; the column temperature was 40°C; and the injection volume was 5 μL.

7. The method for identifying sodium alginate gel adulteration in instant bird's nest products according to claim 1, characterized in that: The detector conditions include evaporation temperature: 60°C, atomization temperature: 60°C, evaporation gas volume: 1.65SLM, LED light intensity: 100%.

8. The method for identifying sodium alginate gel adulteration in instant bird's nest products according to claim 1, characterized in that: The centrifugal separation in step S1 is performed at a speed of 8000-10000 rad / min for 15-30 min.

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