Method for measuring glycerol content in collagen gel

Abstract

This invention proposes a method for determining the glycerol content in collagen colloids. It utilizes the oxidation of free glycerol by periodic acid to formaldehyde, which then undergoes a Hantzsch reaction with two molecules of acetylacetone in the presence of ammonium acetate. This is the first time this method has been applied to the determination of glycerol content in collagen colloids, and it is automated using an automated analyzer. This method can be used to detect collagen colloidal materials. The sample pretreatment primarily utilizes the isoelectric point precipitation property of collagen. The pH of the collagen solution sample is pre-adjusted to approximately 5.0, close to the isoelectric point of collagen colloids. The precipitation of proteins clarifies the solution and facilitates the release of glycerol into the solution, resulting in more accurate measurement results. This invention offers advantages such as simplicity, speed, accuracy, and safety in determining the glycerol content in collagen colloids.

Description

Technical Field

[0001] This invention relates to a method for determining the glycerol content in collagen colloids, belonging to the field of food quality testing technology. Background Technology

[0002] Natural sausage casings are thin film products made from the large or small intestines of livestock through processes such as salting, washing, soaking, scraping, and drying. They possess excellent tissue structure, good water retention and air permeability, and a rich collagen content. They are primarily used as the outer layer of sausage products and can also be made into sutures for use as bowstrings and surgical sutures. With the expanding market demand for sausage casings, natural casings, due to their high production costs, low efficiency, and difficulty in quality control, can no longer meet the requirements of modern sausage casing processing. Therefore, artificial casings, as a substitute for natural casings, have gradually gained widespread use. Collagen casings, in particular, are mainly made from collagen fibers from pig or cow hides. After adding auxiliary materials, they are chemically and mechanically processed into collagen clumps, which are then extruded, inflated, dried, and heat-set. They offer advantages such as a smooth and crisp texture, high air permeability, and resistance to high-temperature cooking. Furthermore, due to their thinness, high tensile strength, high compressive strength, and high elongation, they are more suitable for mechanized production and can maximize cost savings.

[0003] In the production of collagen casings, precise control of various indicators and parameters of the raw material collagen is necessary to ensure the stable quality of the final casing product. Currently, there is no unified measurement standard or specific method for the glycerol content in collagen. Current methods for determining glycerol in food samples mainly include: 1) Chemical method: Glycerol reacts with potassium periodate in an aqueous solution to produce formic acid, which is then neutralized with standard sodium hydroxide to obtain the free glycerol content. While this method is low-cost, it is cumbersome, time-consuming, and sometimes prone to significant random errors; 2) Enzymatic assay: This method specifically reacts with glycerol in solution, offering high specificity, but requires expensive reagents, making it unsuitable for industrial applications and large-scale sample testing; 3) Gas chromatography: This method offers high accuracy and sensitivity, but its complex procedures and high testing cost. Therefore, developing a low-cost, rapid, accurate, and safe detection method for the colloidal glycerol content of collagen is essential for ensuring the quality control of the collagen casing process. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects of existing general methods for detecting glycerol in food, and to propose a low-cost, easy-to-operate, highly accurate and safe method for determining the glycerol content in collagen colloids.

[0005] The technical solution of this invention: a method for determining the glycerol content in collagen colloids, specifically including the following steps:

[0006] 1) Sample pretreatment

[0007] Weigh approximately 10.0 g of the collagen colloidal sample to be tested and place it in a homogenizer bag, making two copies. First, add 190 mL of deionized water and homogenize for 10 minutes. Then, add 10 mL of 0.1 mol / L NaOH to adjust the pH of the solution to approximately 5.0 and continue homogenizing for 10 minutes. Filter the sample using a Buchner funnel and pipette 10 mL of the filtrate into a 100 mL volumetric flask. Dilute to the mark with deionized water.

[0008] 2) Determination of the glycerol standard curve

[0009] Prepare standard solutions of glycerol with different concentration gradients, dilute and bring to volume, add sodium periodate solution, shake and then add acetylacetone solution, heat and shake to react, cool rapidly after the reaction is complete, and measure its absorbance at 410 nm using an automatic analyzer, and plot the standard curve of glycerol concentration-absorbance.

[0010] 3) Automatic detection and analysis

[0011] ① Set up the automatic analyzer with deionized water, sodium periodate and acetylacetone, turn on the switch, start the tension and pressure system, and run the system for 15 minutes;

[0012] ② Place the standard solution sample cups for the standard curve in the syringe in order of increasing concentration. Start working vertically from the top left side and insert 4 cups containing samples. Analyze after all the standard samples and samples are in place.

[0013] ③ Enter the sample weight and sample dilution factor to start the analysis. Plot a graph with peak height as the Y-axis and concentration as the X-axis, and read the glycerol concentration of the sample from the graph.

[0014] 4) Result Calculation

[0015] The formula for calculating the glycerol content in collagen colloidal samples is as follows:

[0016] Glycerin content (%) = (C × 10) -6 ×V ×100×ρ) / W

[0017] Where C is the glycerol concentration of the sample to be tested (ppm), V is the total volume of the diluent (2000 mL), W is the sample mass (10.0 g), and ρ is 1 g / cm³.

[0018] Compared with the prior art, the advantages of the present invention are as follows:

[0019] 1) This invention utilizes the oxidation of free glycerol by periodic acid to formaldehyde. Formaldehyde then undergoes a Hantzsch reaction with two molecules of acetylacetone in the presence of ammonium acetate to produce a yellow product, 3,5-diacetyl-1,4-dihydrodimethylpyridine, which has the highest absorbance at a wavelength of 410 nm, thus aiding in the accurate determination of glycerol. This invention is the first to apply the above method to the determination of glycerol content in collagen colloids and uses an automated analyzer for automatic analysis, which can be used to detect collagen raw materials.

[0020] 2) Sample pretreatment is crucial for the accuracy and rapid analysis of results. The pH value of the collagen colloid to be tested is around 2.0. If the sample is directly dissolved in water and tapped, the mixture is found to be turbid, and small particles are still present after filtration. However, this invention utilizes the property of collagen isoelectric point precipitation. The pH value of the collagen solution is adjusted to around 5.0 in advance, making it close to the isoelectric point of the collagen colloid, and then tapped. The protein precipitation effect clarifies the solution and also facilitates the release of glycerol into the solution, making the solution filtration more efficient and the measurement results more accurate. Attached Figure Description

[0021] Appendix Figure 1 This is a standard curve of glycerol concentration-absorbance measured in an embodiment of the present invention.

[0022] Appendix Figure 2 This is a graph showing the detection results of glycerol content in collagen colloidal samples obtained in an embodiment of the present invention. Detailed Implementation

[0023] The technical solution of the present invention will be further described below with reference to embodiments. In the description of this specification, the content of each embodiment means that the specific technical features described therein are included in at least one embodiment of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same implementation or example. Moreover, the specific technical features described may be combined in any suitable manner in one or more implementations or examples.

[0024] The specific equipment and materials used in this embodiment are as follows:

[0025] Equipment: Analytical balance, 100 mL conical flask, 50 mL deionized water separator, mechanical vibrator, 2 mL ball pipette, 50 mL volumetric flask with stopper, homogenizer bag, beat homogenizer, Buchner funnel, 20 mL ball pipette, automatic analyzer, disposable sample cups.

[0026] The automatic analyzer is a spectrophotometer that automatically injects and measures samples. Its main unit model is SEAL Analytical Pump 4, and its injector model is SEAL Analytical XY-2 Sampler.

[0027] Materials: Sodium periodate solution, acetylacetone solution, ammonium acetate, and glycerol standard solution.

[0028] The preparation process of the glycerol reference solution is as follows:

[0029] Weigh approximately 5g of glycerol into a 100ml volumetric flask and dilute to the mark with distilled water to obtain a 5% glycerol solution. Accurately transfer 5ml of the 5% glycerol solution into a 500ml volumetric flask and dilute to the mark with distilled water to obtain a 0.05% glycerol reference solution.

[0030] The preparation process of the glycerol standard solution is as follows:

[0031] Use a pipette to transfer 10, 20, 30, 40, 50, and 60 ml of 0.05% glycerol reference solution into six 200 ml volumetric flasks, respectively, and dilute to the mark with distilled water. Shake well and set aside to obtain glycerol standard solutions of 25, 50, 75, 100, 125, and 150 ppm.

[0032] The preparation process of acetylacetone solution is as follows:

[0033] Mix equal volumes of 0.8 mol / L (4.8 g / 100 ml) acetic acid solution and 2.0 mol / L (15.4 g / 100 ml) ammonium acetate solution to obtain a buffer solution with a pH of 5.5; pipette 15 ml of acetylacetone (analytical grade) into a 2000 ml volumetric flask, add acetic acid and ammonium acetate buffer solution to dilute to the mark, and shake well for later use.

[0034] The preparation process of sodium periodate solution is as follows:

[0035] Weigh approximately 2.57 g of sodium periodate (analytical grade) into a 2000 ml volumetric flask, and dilute to the mark with ammonium acetate buffer solution to obtain a 6 mmol / L sodium periodate solution.

[0036] The method for determining the glycerol content in collagen colloids proposed in this embodiment includes the following specific steps:

[0037] 1) Sample pretreatment

[0038] Weigh approximately 10.0 g of the collagen colloidal sample to be tested and place it in a homogenizer bag, making two copies. First, add 190 mL of deionized water and homogenize for 10 minutes. Then, add 10 mL of 0.1 mol / L NaOH to adjust the pH of the solution to approximately 5.0 and continue homogenizing for 10 minutes. Filter the sample using a Buchner funnel and pipette 10 mL of the filtrate into a 100 mL volumetric flask. Dilute to the mark with deionized water.

[0039] 2) Determination of the glycerol standard curve

[0040] Glyceryl standard solutions of different concentration gradients were prepared, diluted to a final volume, and then sodium periodate solution was added. After shaking, acetylacetone solution was added, and the reaction was heated and shaken. After the reaction was completed, the solution was rapidly cooled, and the absorbance was measured at 410 nm using an automatic analyzer. A standard curve of glyceryl concentration versus absorbance was plotted. The standard curve of glyceryl concentration versus absorbance obtained in this example is shown below. Figure 1 As shown.

[0041] 3) Automatic detection and analysis

[0042] Set up the automatic analyzer with deionized water, sodium periodate, and acetylacetone to eliminate interference from these substances on the absorbance of the measurement system. Turn on the switch and start the tension and pressure system, allowing it to run for 15 minutes. Before analysis, a constant curve (5% absorbance) should be visible on the computer screen. Place the glycerol standard solutions in the injector in ascending order of concentration, and then add the sample to be tested. Perform the analysis once all the glycerol standard solutions and samples are in place.

[0043] 4) Computer settings

[0044] On the computer screen of the automated analyzer, click the "load form" button, click the file named 1-glycerol.frm, enter your own filename in the format (e-xxx), and save the file. On the computer screen, click "tray protocol," which will open a list of all sample cups. Enter the sample weight and sample dilution factor, and click "OK." The program is ready to run; click "Start Analysis." Plot the peak height (Y-axis) against the concentration (X-axis) and read the glycerol concentration of the sample from the graph.

[0045] 5) Result Calculation

[0046] The formula for calculating the glycerol content in collagen colloidal samples is as follows:

[0047] Glycerin content (%) = (C × 10) -6 ×V ×100×ρ) / W

[0048] Where C is the glycerol concentration of the sample to be tested (ppm), V is the total volume of the diluent (2000 mL), W is the sample mass (10.0 g), and ρ is 1 g / cm³.

[0049] The detection results of glycerol content in collagen colloidal samples measured in this embodiment are as follows: Figure 2 As shown.

[0050] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for measuring the content of glycerol in collagen gel, characterized by, The method specifically comprises the following steps: 1) sample pretreatment; 2) determination of glycerol standard curve; 3) automatic detection analysis; 4) result calculation; The step 1) sample pretreatment specifically comprises the following contents: 10.0 g of the collagen colloid sample to be measured is weighed, put into a homogenizer to beat the bag, and duplicated; first, 190 mL of deionized water is added, and beating is performed for 10 minutes; then, 10 mL of 0.1 mol / L NaOH is added to adjust the solution pH value to 5.0, and beating is continuously performed for 10 minutes; the sample is filtered through a Buchner funnel, 10 mL of the filtrate is sucked into a 100 mL volumetric flask by using a pipette, and deionized water is used to dilute to the scale; The step 2) determination of glycerol standard curve specifically comprises the following contents: Glycerol standard solutions with different concentration gradients are prepared, after dilution and constant volume, sodium periodate solution is added, after shaking, acetylacetone solution is added, heating and oscillation reaction is performed, after the reaction is completed, rapid cooling is performed, and the absorbance is measured at 410 nm by using an automatic analyzer, and a standard curve of glycerol concentration-absorbance is drawn; The step 3) automatic detection analysis specifically comprises the following contents: ① The automatic analyzer is set with deionized water, sodium periodate and acetylacetone, the switch is turned on, the tension and pressure system is started, and the system is run for 15 minutes; ② The standard solution sample cups for establishing the standard curve are placed in the sample injector in the order of increasing concentration, vertical work is started from the top left hand position, 4 sample cups are inserted, after all the standard samples and samples are in place, analysis is performed; ③ The sample weight and sample dilution multiple are input, analysis is started, the peak height is taken as the Y axis, and the concentration is taken as the X axis to draw a graph, and the glycerol concentration of the sample to be measured is read from the graph; The step 4) result calculation specifically comprises the following contents: The calculation formula of the glycerol content in the collagen colloid sample is as follows: Glycerol content (%) = (C x 10 -6 x V x 100 x p) / W Wherein C is the glycerol concentration of the sample to be measured (ppm), V is the total volume of the diluent (2000 mL), W is the sample weight (10.0 g), and ρ is 1 g / cm³.

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