Method for determining D-psicose in processed food and application of method

Abstract

The invention provides a method for determining D-psicose in processed food and application of the method. The method specifically comprises the following steps: extracting a sample with warm water at 50-60 DEG C, precipitating protein by adopting a Carrez reagent, and removing interference impurities; chromatographic conditions are defined as follows: a calcium type strong cation exchange column is adopted, pure water containing 50 mg / L of EDTA-Ca is adopted as a mobile phase, the flow rate is 0.4-0.6 mL / min, and the column temperature is 80 DEG C. The result shows that the linear relation of the D-psicose is good in the concentration range of 1.0 mg / mL to 20.0 mg / mL, and the correlation coefficient is 0.999999; according to the method, the LOD is 0.002 g / 100g, and the LOQ is 0.005 g / 100g. Under the experimental conditions of standard addition before simulation processing, the average recovery rate of 18 matrixes including dairy products, bakery products and the like is 81.5-128.1%, and the RSD is less than 3.0%. The determination method provided by the invention is simple in pretreatment, high in sensitivity, strong in interference resistance and good in accuracy, effectively solves the ubiquitous problems of matrix wrapping, matrix interference, difficulty in isomer separation and the like when the D-psicose in the processed food is detected, can realize rapid and accurate detection of the D-psicose in all dosage forms of processed food, and has a wide prospect.

Description

Technical Field

[0001] This invention belongs to the field of high performance liquid chromatography detection technology, specifically relating to a method for determining D-allulose in processed foods and its application. Background Technology

[0002] D-Allulose is the C-3 epimer of D-fructose. Its sweetness is about 70% that of sucrose, but its calorie content is extremely low (approximately 0.4 kcal / g), only 0.3% of that of sucrose. Numerous studies have shown that D-Allulose also possesses a variety of unique physiological functions, including inhibiting postprandial blood glucose elevation, improving insulin resistance, reducing visceral fat accumulation, and exhibiting antioxidant effects. Therefore, it has broad application prospects in the food industry.

[0003] In 2014, the U.S. Food and Drug Administration (FDA) granted D-allulose GRAS (Generally Recognized As Safe) certification, and in 2019, it was allowed to be excluded from the "total sugar" and "added sugar" categories on nutrition labels. In my country, the National Health Commission issued Announcement No. 4 in 2025, officially approving D-allulose as a new food ingredient, marking the beginning of standardized development for its application in my country's food industry.

[0004] With the official approval of D-allulose as a new food ingredient, its application has expanded from single-component liquid beverages to a wide range of processed foods, including but not limited to flavored syrups, jams, flavored wines, liquid beverages, flavored green tea powder, table sweeteners, yogurt, ice cream, egg yolk powder, cakes, glutinous rice balls, custard buns, jellies, fish balls, spicy shredded chicken, compound seasonings, chocolate, salad dressings, and many other food categories. The matrix composition of these processed foods is far more complex than that of liquid beverages. Classifying matrix types according to the GB2760-2024 food classification standard covers baked and flour products, dairy products, egg products, aquatic and meat products, seasonings, candies and jellies, and solid and liquid beverages. When testing D-allulose in these complex matrix foods, various technical challenges are intertwined, seriously affecting the accuracy, stability, and reliability of the test results. Specifically:

[0005] First, the matrix encapsulation effect is prevalent and has a significant impact in complex food systems. For example, during high-temperature processing of baked goods such as cakes and custard buns, the starch in the raw materials undergoes retrogradation, and the proteins undergo denaturation and cross-linking, forming a dense microstructure. D-allulose molecules are easily embedded or adsorbed by this dense structure. Similarly, during the processing and refrigeration of glutinous rice balls, the starch gelatinizes and then cools, resulting in retrogradation and the formation of a dense matrix that encapsulates D-allulose. In chocolate preparation, the network structure formed by cocoa butter crystallization and the three-dimensional network structure formed by jelly gel both encapsulate D-allulose to varying degrees. Conventional extraction processes cannot effectively disrupt the dense microstructure of these foods, preventing the complete release of encapsulated D-allulose into the extract. This leads to generally low detection results, making it difficult to objectively reflect the actual D-allulose content in the food. Based on the inventors' prior research, the encapsulation effect is particularly pronounced in flour products such as cakes, custard buns, and glutinous rice balls, as well as in chocolate and jelly, and has the most significant impact on the detection results.

[0006] Secondly, complex food systems can lead to severe matrix interference. For example, egg yolk powder, as a dehydrated egg product, is rich in high-protein components, and these proteins easily dissolve into the extract during the extraction process. Spicy shredded chicken and fish balls, belonging to braised meat and frozen seafood products respectively, both contain large amounts of high-protein and high-fat substances. Fat easily emulsifies to form a colloidal system, while protein easily denatures to form large molecular impurities. Chocolate and salad dressings are rich in oils, while dairy products such as ice cream and yogurt contain milk proteins and milk fats. Compound seasonings and flavored wines contain various large molecular flavoring components and colloidal substances. Jams and flavored syrups contain pectin, polysaccharides, and other large molecular substances, all of which can become interfering components. Without targeted purification techniques, these impurities will enter the chromatographic detection system along with D-allulose, resulting in irreversible adsorption. This leads to decreased column efficiency, increased column pressure, and retention time drift, not only reducing the accuracy of the detection results but also compromising the stability of the detection process. In severe cases, it can prevent the detection work from being carried out normally. Among them, egg yolk powder, spicy shredded chicken, fish balls, chocolate, and salad dressing are the most serious matrix interferences.

[0007] Finally, there is the common problem of isomer separation difficulties in the food detection process of D-allulose. Currently, the standard method for detecting carbohydrates in food in my country is the amino column method recommended by the national standard GB 5009.8. However, this method has significant limitations when applied to complex processed food systems for D-allulose detection: flavoring syrups, jams, table sweeteners, liquid beverages, and flavored green tea powder inherently contain a certain amount of D-fructose; starch hydrolysis in cakes, custard buns, and glutinous rice balls produces D-fructose during processing; and fermentation in dairy products such as yogurt and ice cream also generates D-fructose. As C-3 epimers, D-allulose and D-fructose have highly similar chemical structures, making baseline separation difficult using the amino column method. The resolution (Rs) is typically below 1.0, failing to effectively distinguish the two isomers and leading to overestimation of the results. Meanwhile, the amino groups on the surface of the amino column are prone to undergo Maillard reactions with reducing sugars (including D-allulose, D-fructose and other reducing sugars) in various food samples, generating Schiff base products. This leads to irreversible degradation of the column performance, shortens the column's lifespan, and increases maintenance and detection costs. This problem is particularly prominent when used for the detection of foods with high sugar content, such as jams, syrups, and sweeteners.

[0008] In summary, existing detection technologies cannot simultaneously solve the technical problems of matrix encapsulation, matrix interference, and isomer separation difficulties in the detection of D-allulose in complex processed foods, nor can they guarantee the long-term stability of chromatographic columns, thus failing to meet the actual detection needs of the industry.

[0009] Therefore, developing a universal D-allulose detection method that can simultaneously solve the aforementioned technical challenges and cover all categories of products specified in GB 2760 has become a critical technical bottleneck that urgently needs to be overcome in the current food testing field. In view of this, this invention is proposed. Summary of the Invention

[0010] To address the shortcomings of existing technologies in the detection of D-allulose in processed foods, such as matrix encapsulation, matrix interference, difficulties in isomer separation, short column life, and high detection costs, this invention provides a highly sensitive, interference-resistant, and accurate method for detecting D-allulose in processed foods with complex matrices. This method enables rapid and accurate detection of D-allulose in all dosage forms (liquid, solid, and semi-solid) of processed foods.

[0011] To achieve the above objectives, the present invention provides a method for determining D-allulose in processed foods, comprising the following steps:

[0012] Preparation of D-allulose standard working solution: Accurately weigh D-allulose standard and prepare a 100 mg / mL standard stock solution with water; dilute the prepared standard stock solution stepwise with water to prepare a series of standard working solutions with concentrations of 0.1-20.0 mg / mL;

[0013] Preparation of test solution: Add 50-60℃ warm water to the processed food sample for extraction, then add Carrez reagent, dilute to volume with water, centrifuge, take the supernatant and filter the membrane to obtain the test solution;

[0014] Chromatographic detection: A calcium cation exchange column was used at a column temperature of 80℃. Ultrapure water containing 50 mg / L EDTA-Ca was used as the mobile phase. The peak areas of a series of standard working solutions were detected by a differential refractive index detector, and a standard curve was plotted to obtain the regression equation. Then, the peak area of ​​the test sample solution was detected under the same chromatographic conditions, and its concentration was calculated by substituting it into the regression equation. The D-allulose content in the processed food was then obtained.

[0015] In a preferred embodiment, the linear correlation coefficient R of the standard curve 2 ≥0.99999.

[0016] In a preferred embodiment, the detection limit of the determination method for D-allulose in processed foods is 0.002 g / 100g.

[0017] In a preferred embodiment, the limit of quantification for D-allulose in processed foods by the determination method is 0.005 g / 100g.

[0018] In a preferred embodiment, in the step of preparing the D-allulose standard working solution, the concentrations of the standard working solution are 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 5.0 mg / mL, 10.0 mg / mL, and 20.0 mg / mL, respectively.

[0019] In a preferred embodiment, in the step of preparing the test solution, the mass-to-volume ratio of the processed food to warm water is (1-2.5) g / 15 mL.

[0020] In a preferred embodiment, in the step of preparing the test sample solution, the processed food includes one or more of the following categories as classified by the GB2760-2024 food classification standard: baked goods, dairy products, egg products, aquatic and meat products, condiments, candies and jellies, and solid and liquid beverages; preferably, the processed food includes one or more of the following: syrup, jam, flavored wine, liquid beverage, flavored green tea powder, table sweeteners, yogurt, ice cream, egg yolk powder, cake, glutinous rice balls, custard buns, jelly, fish balls, spicy shredded chicken, compound seasonings, chocolate, and salad dressing.

[0021] In a preferred embodiment, when the processed food sample is solid or semi-solid in the step of preparing the test solution, the specific operation is as follows: after crushing and mixing the sample, add warm water at 50-60℃, and extract with ultrasound at 100-500 W for 10-30 minutes to promote the full release of sugar.

[0022] When the food sample is in liquid state, the specific operation is as follows: add 50-60℃ warm water to the uniform sample and shake until uniform.

[0023] In a preferred embodiment, in the step of preparing the test solution, the volume ratio of the 50-60°C warm water to the Carrez reagent is (10-20):1; the Carrez reagent consists of Carrez I solution (zinc acetate solution) and Carrez II solution (potassium ferrocyanide solution), and the volume ratio of the two is 1:1.

[0024] In the step of preparing the test solution, the centrifugation conditions can be conventional parameters known to those skilled in the art, such as centrifuging at 10000 r / min for 10 min.

[0025] In the step of preparing the test solution, the filter membrane is a 0.22 μm aqueous microporous filter membrane.

[0026] In a preferred embodiment, in the chromatographic detection step, the calcium-type strong cation exchange column is a Waters Sugar-Pak I.

[0027] In a preferred embodiment, the specific chromatographic conditions in the chromatographic detection step include: flow rate: 0.4-0.6 mL / min; detection cell temperature: 35-45 ℃; injection volume: 10-20 μL; preferably, the chromatographic conditions include: flow rate: 0.5 mL / min; detection cell temperature: 40 ℃; injection volume: 10 μL.

[0028] Another object of the present invention is to provide an application of any of the above methods in determining the D-allulose content in processed foods, including but not limited to: using the determination method for quality control and finished product compliance testing during the production process of processed foods.

[0029] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0030] In this invention, processing foods are extracted using warm water at 50-60℃, which promotes the release of sugars embedded in starch / protein while avoiding the degradation of allulose or the Maillard reaction caused by high temperatures. The Carrez reagent, composed of zinc acetate solution and potassium ferrocyanide solution, effectively precipitates proteins in processed foods, resolving the matrix effect problem. Adding 50 mg / L EDTA-Ca to pure water effectively prevents metal ions in the mobile phase from replacing calcium ions on the column, extending column life. By limiting the temperature to 80℃ using a high-temperature calcium-type column, the optimal temperature for separating allulose and fructose (Rs>1.5) can be achieved, thus solving the isomer separation problem; below this temperature, separation is poor, and above this temperature, the column pressure is too high.

[0031] By synergistically applying the aforementioned technical means, the detection method provided by this invention achieves extremely high resolution (Rs>1.5) and extremely low limit of detection (LOD) of 0.002, fully meeting the trace analysis requirements for low-content samples. Validation with spiked loading before simulated processing showed that the recovery rate of this method remained stable between 81.5% and 128.1% in 18 processed foods containing high levels of fat and protein, with RSDs less than 3.0% for all samples, demonstrating that the method maintains good precision and extraction stability in various complex systems. Furthermore, this method is not limited to food dosage forms and can detect liquid, solid, and semi-solid processed foods, exhibiting good versatility and broad application prospects. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 This is the standard curve drawn in Embodiment 1 of the present invention;

[0034] Figure 2 This is the HPLC chromatogram (200 mg / L) of the D-allulose standard solution in Example 1 of the present invention.

[0035] Figure 3 The LOD chromatogram of D-allulose in Example 1 of this invention (standard solution concentration 2.0 mg / L);

[0036] Figure 4The LOQ chromatogram of D-allulose in Example 1 of this invention (standard solution concentration 5.0 mg / L);

[0037] Figure 5 The image shows the HPLC chromatogram of the cake sample from Example 3 of this invention (detected content 4.8 g / 100g). Detailed Implementation

[0038] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0039] Unless otherwise specified, the technical means used in this invention are conventional means well known to those skilled in the art, and all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0040] In this embodiment of the invention, the D-allulose standard (purity ≥ 99.0%) used was purchased from Sigma-Aldrich. In this embodiment of the invention, the Carrez I solution (zinc acetate solution) was prepared as follows: 21.9 g of zinc acetate was weighed, 3 mL of glacial acetic acid was added, dissolved in water, and the volume was adjusted to 100 mL; the Carrez II solution (potassium ferrocyanide solution) was prepared as follows: 10.6 g of potassium ferrocyanide was weighed, dissolved in water, and the volume was adjusted to 100 mL; zinc acetate and potassium ferrocyanide (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. In this embodiment of the invention, the EDTA-Ca used was calcium disodium ethylenediaminetetraacetate, with the chemical formula C. 10 H 12 CaN2Na2O8, analytical grade. The water used in the experiment was ultrapure water (resistivity 18.2 MΩ·cm).

[0041] In this embodiment of the invention, the instruments and equipment used include: an Agilent 1260 Infinity II high-performance liquid chromatograph equipped with a G7111A quaternary pump, a G7129A autosampler, and a G7116A column oven; an Agilent 1260 Infinity II differential refractive index detector (RID): model G7162A, Agilent Technologies, USA; a Waters Sugar-Pak I column: a calcium cation exchange column, 6.5 mm × 300 mm, 10 μm, Waters Corporation, USA; a VGT-2227QT mechanically controlled ultrasonic cleaner: Guangdong Gote Ultrasonic Co., Ltd.; and an H1750R high-speed refrigerated benchtop centrifuge: Hunan Xiangyi Laboratory Instrument Development Co., Ltd.

[0042] Example 1

[0043] S1 Preparation of D-allulose standard stock solution: Accurately weigh 1.0 g of D-allulose standard (accurate to 0.0001 g), and prepare a 100 mg / mL standard stock solution with deionized water. Store at 4 ℃.

[0044] S2 Preparation of D-aloxone standard working solution: The prepared standard stock solution was diluted stepwise with deionized water to prepare a series of standard working solutions with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 5.0 mg / mL, 10.0 mg / mL and 20.0 mg / mL.

[0045] S3. Plotting the Standard Curve: Inject the prepared series of standard working solutions sequentially into the high-performance liquid chromatograph (HPLC) in ascending order of concentration. Measure the solutions under the set chromatographic conditions and record the peak area (Y, unit: nRIU*s) for each concentration. Each concentration is injected in triplicate, and the average peak area from the three injections is used for linear regression analysis. Plot the D-allulose concentration (X, unit: mg / mL) on the x-axis and the corresponding average peak area (Y) on the y-axis. Perform linear regression analysis using Excel software to obtain the regression equation, correlation coefficient (R²), and linear range. The chromatographic conditions included: a Waters Sugar-Pak I calcium cation exchange column (6.5 mm × 300 mm, 10 μm); mobile phase: ultrapure water containing 50 mg / L EDTA-Ca; flow rate: 0.5 mL / min; column temperature: 80 ℃; detector: Agilent 1260 Infinity II RID (G7162A); detector cell temperature: 40 ℃; and injection volume: 10 μL.

[0046] S4. Limit of Detection (LOD) and Limit of Quantification (LOQ): Accurately pipette 0.1 mg / mL of the standard working solution and serially dilute with water to obtain dilutions with concentrations of 0.0002 mg / mL (0.2 mg / L), 0.0005 mg / mL (0.5 mg / L), 0.001 mg / mL (1 mg / L), 0.002 mg / mL (2 mg / L), 0.005 mg / mL (5 mg / L), and 0.01 mg / mL (10 mg / L). Inject the solutions according to the chromatographic conditions in step S4 and record the chromatogram and signal-to-noise ratio (S / N) for each dilution. The concentration corresponding to a S / N of approximately 3 is taken as the limit of detection (LOD), and the concentration corresponding to an S / N of approximately 10 is taken as the limit of quantification (LOQ). Using the actual sample pretreatment conditions (5 g sample, 50 mL final volume), calculate the LOD and LOQ of D-allulose in the sample using the following formula:

[0047] LOD in sample (g / 100g) = (LOD concentration of standard solution (mg / L) × final volume (mL)) / (sample amount (g) × 1000) × 100;

[0048] LOQ in the sample (g / 100g) = (LOQ concentration of standard solution (mg / L) × final volume (mL)) / (sample amount (g) × 1000) × 100.

[0049] Results and Discussion:

[0050] (1) The results of the standard curve are as follows Figure 1 As shown, D-allulose exhibited good linearity in the range of 1.0 mg / mL–20.0 mg / mL, with a regression equation of Y = 239134.198X – 3857.9366 and a correlation coefficient R0. 2 = 0.99999.

[0051] (3) Results of limit of detection and limit of quantitation are as follows Figure 3 and Figure 4 As shown, when the concentration of the standard solution is 2 mg / L, the signal-to-noise ratio (S / N) of the D-allulose chromatographic peak is approximately 3, and the limit of detection (LOD) of D-allulose in the sample is calculated to be 0.002 g / 100g according to the formula; when the concentration of the standard solution is 5 mg / L, the signal-to-noise ratio (S / N) of the chromatographic peak is approximately 10, and the limit of quantitation (LOQ) is calculated to be 0.005 g / 100g.

[0052] In this embodiment, a series of standard working solutions with concentration gradients from 1.0 mg / mL to 20.0 mg / mL were successfully prepared using D-allulose standard. The established standard curves showed excellent linearity (R0). 2=0.99999). The method provided by this invention has a detection limit of 0.002 g / 100g and a quantitation limit of 0.005 g / 100g, which fully meets the trace analysis requirements for low-content samples and is significantly superior to the sensitivity reported by conventional amino column methods.

[0053] Example 2

[0054] (1) Sample preparation

[0055] Preparation of blank matrix: 18 kinds of solid, semi-solid or liquid processed foods were prepared according to the matrix types in Table 1. During the preparation, D-allulose was not added, which served as the blank matrix group without D-allulose.

[0056] Preparation of spiked samples: Take the same raw materials used to prepare the blank matrix, add the theoretical amount of D-allulose standard, and prepare spiked samples of 18 kinds of solid, semi-solid or liquid processed foods; three copies of each processed food are prepared simultaneously as parallel test samples.

[0057] (2) Preparation of test solution: Among the 18 spiked samples, solid or semi-solid processed food was taken, the sample was crushed evenly, and 5g (accurate to 0.001 g) was weighed into a beaker, 30 mL of 60℃ warm water was added, and ultrasonic extraction was performed at 100 W for 20 min to ensure that the sugars wrapped in starch or matrix were completely released. After cooling, the samples were transferred to a 50 mL volumetric flask. Liquid processed food was taken, shaken evenly, and 5g (accurate to 0.001 g) was weighed into a 50 mL volumetric flask, 30 mL of 60℃ warm water was added, and the mixture was shaken until it was evenly dissolved.

[0058] Add 1 mL of Carrez I solution and 1 mL of Carrez II solution to the volumetric flasks mentioned above, respectively, and shake well after each addition of Carrez solution to precipitate the protein. Dilute to the mark with water, mix well, and let stand for 10 min. Take the supernatant and centrifuge at 10000 r / min for 10 min. Filter the supernatant through a 0.22 μm aqueous microporous membrane and collect the filtrate as the test solution.

[0059] (3) Preparation of blank control solution: Take a blank matrix without D-allulose and prepare a blank control solution in the same way as the test solution.

[0060] (4) Chromatographic detection: For each processed food, the blank control solution and three parallel test solutions were injected sequentially into the high-performance liquid chromatograph and measured under the same chromatographic conditions as the standard curve established in Example 1. The chromatograms of the blank control solution and the peak areas corresponding to D-allulose in the test solutions were recorded. After confirming that there were no interfering peaks in the blank control solution, the peak areas of the test solutions were substituted into the D-allulose standard curve established in Example 1 to calculate the measured D-allulose content of each sample.

[0061] Table 1

[0062]

[0063] Results and Discussion: The spiked recovery and precision results of 18 processed food samples are shown in Table 2. Table 2 shows that the average recovery rate of the method provided by this invention in different matrices ranged from 81.49% to 128.14%. Although the experiment used a pre-processing spiked mode, the recovery rate of some high-protein or high-fat matrices (such as egg yolk powder and cake) fluctuated due to matrix effects or physical adsorption during processing. However, overall, the RSD of all samples was less than 3.0%, demonstrating that the method of this invention maintains good precision and extraction stability in various complex systems and can meet the quantitative detection needs of most processed foods.

[0064] Table 2

[0065]

[0066] Example 3

[0067] Preparation of the test solution: After uniformly pulverizing a commercially available low-sugar cake, weigh 5g (accurate to 0.001g) into a beaker, add 30 mL of 60℃ warm water, and extract using ultrasound at 100 W for 20 min to ensure complete release of sugars encapsulated in starch or matrix. After cooling, transfer to a 50 mL volumetric flask. Add 1 mL of Carrez I solution and 1 mL of Carrez II solution to the volumetric flask sequentially, shaking thoroughly after each addition to precipitate proteins. Dilute to the mark with water, mix well, and let stand for 10 min. Centrifuge the supernatant at 10000 r / min for 10 min, filter the supernatant through a 0.22 μm aqueous microporous membrane, and collect the filtrate as the test solution.

[0068] Chromatographic detection: The test solution was injected into a high-performance liquid chromatograph and measured under the same chromatographic conditions as the standard curve established in Example 1. The peak area corresponding to D-allulose in the test solution was recorded. The peak area of ​​D-allulose in the test solution was substituted into the regression equation of the established D-allulose standard curve to calculate the concentration of D-allulose in the test solution.

[0069] Results and Discussion: The content detected in the low-sugar cake was 4.8 g / 100g, and its chromatogram is shown below. Figure 5 As shown in the figure, after precipitating protein-bound material with Carrez reagent and filtering at 0.22 μm, the baseline of the cake sample remained stable. This is consistent with the chromatogram of the aforementioned standard sample. Figure 3 , Figure 4 In comparison, the retention time of D-allulose in the samples was consistent, and the peak shape was intact. Although there were trace matrix-associated peaks (shoulder peaks) near the main peak, thanks to the optimization of the 80 ℃ high-temperature elution conditions, the target peak and the interfering peaks were effectively separated, without interfering with the quantitative integration.

[0070] Example 4

[0071] Preparation of the test solution: After shaking a certain brand of sparkling water, weigh 5g (accurate to 0.001g) into a 50mL volumetric flask, add 30mL of 60℃ warm water, and shake until uniformly dissolved. Add 1mL of Carrez I solution and 1mL of Carrez II solution to the volumetric flask in sequence, shaking thoroughly after each addition to precipitate the protein; dilute to the mark with water, mix well, and let stand for 10min; take the supernatant and centrifuge at 10000 r / min for 10min, filter the supernatant through a 0.22 μm aqueous microporous membrane, and collect the filtrate as the test solution;

[0072] Chromatographic detection: The test solution was injected into a high-performance liquid chromatograph and measured under the same chromatographic conditions as the standard curve established in Example 1. The peak area corresponding to D-allulose in the test solution was recorded. The peak area of ​​D-allulose in the test solution was substituted into the regression equation of the established D-allulose standard curve to calculate the concentration of D-allulose in the test solution.

[0073] Results and Discussion: The content of the substance was found to be 2.5 g / 100mL in a certain brand of sparkling water.

[0074] Comparative Example

[0075] Using the cake from Example 2 as a sample (the matrix contains high starch, high protein, and fat), the theoretical addition amount of D-allulose is 6.12 g / 100g. The difference between the measured and theoretical addition amounts of D-allulose was tested under different treatment parameters.

[0076] Using the complete scheme of this invention (60℃ warm water ultrasonic extraction + Carrez reagent purification + EDTA-Ca mobile phase + 80℃ column temperature), the measured content of D-allulose was 7.33 g / 100g (recovery rate 119.78%) after treatment. The obtained HPLC chromatogram showed that the chromatographic peaks were sharp and symmetrical, and the separation degree (Rs) from D-fructose was > 1.5.

[0077] Comparative Example 1

[0078] The only difference from Example 2 is that in step (2), the 60°C warm water is replaced with 25°C room temperature water. The rest of the methods and steps are completely the same as in Example 2.

[0079] Results and Discussion: The D-allulose content was measured to be only 4.00 g / 100g, with a recovery rate reduced to 65.4%. The experimental results indicate that room temperature water cannot effectively break the physical encapsulation of sugars by aged starch and denatured proteins. The 50-60℃ warm water combined with ultrasound, specifically designed for this study, is the key condition for disrupting matrix encapsulation and achieving complete release of the target substance.

[0080] Comparative Example 2

[0081] The only difference from Example 2 is that in step (2), the addition of Carrez I solution and Carrez II solution is omitted; the rest of the methods and steps are completely consistent with Example 2. That is, the liquid that was sonicated in 60°C warm water in Example 2 is diluted with water, centrifuged, and filtered to obtain the test solution.

[0082] Results and Discussion: The baseline noise of the obtained chromatograms increased significantly, and a broad impurity interference packet appeared at the front of the target peak, making accurate integration impossible; moreover, after 10 consecutive injections, the column pressure increased by 30 bar. This indicates that Carrez reagent plays an irreplaceable role in removing interference from high-protein / high-lipid matrices and protecting the chromatographic column, and is a prerequisite for stable detection of complex matrices.

[0083] Comparative Example 3

[0084] The only difference from Example 2 is that in step (4), the mobile phase is pure water and does not contain EDTA-Ca. The rest of the methods and steps are completely consistent with Example 2.

[0085] Results and Discussion: After 100 hours of continuous operation, the retention time (Rt) of D-allulose shifted forward by 0.8 min, and the theoretical plate number decreased by approximately 15%. This demonstrates that calcium ions are easily lost from calcium-type columns under high-temperature conditions of 80°C. Adding EDTA-Ca to the mobile phase can dynamically replenish the lost calcium ions, maintaining the separation performance and long-term stability of the column. This is the key advantage of the method in this invention over conventional industrial chromatographic conditions.

[0086] Comparative Example 4

[0087] The only difference from Example 2 is that in step (4), the column temperature is adjusted from 80°C to 60°C. The rest of the methods and steps are completely the same as in Example 2.

[0088] Results and Discussion: In the obtained chromatograms, the peaks of D-allulose and the coexisting D-fructose overlapped significantly, and the resolution (Rs) dropped to 0.9, making baseline separation and accurate quantification impossible. This indicates that a column temperature of 80℃ is the critical condition for achieving baseline separation of allulose and fructose (Rs>1.5). This high-temperature condition is not an obvious choice, but rather an innovative setting based on specific separation requirements.

[0089] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for determining D-allulose in processed foods, characterized in that, Includes the following steps: Preparation of D-allulose standard working solution: Accurately weigh D-allulose standard and prepare a 100 mg / mL standard stock solution with water; dilute the prepared standard stock solution stepwise with water to prepare a series of standard working solutions with concentrations of 0.1-20.0 mg / mL; Preparation of test solution: Add 50-60℃ warm water to the processed food sample for extraction, then add Carrez reagent, dilute to volume with water, centrifuge, take the supernatant and filter the membrane to obtain the test solution; Chromatographic detection: A calcium cation exchange column was used at a column temperature of 80℃. Ultrapure water containing 50 mg / L EDTA-Ca was used as the mobile phase. The peak areas of a series of standard working solutions were detected by a differential refractive index detector, and a standard curve was plotted to obtain a regression equation. Then, the peak area of ​​the test sample solution was detected under the same chromatographic conditions, and its concentration was calculated by substituting it into the regression equation. The D-allulose content in the processed food was then obtained.

2. The method for determining D-allulose in processed foods as described in claim 1, characterized in that, The linear correlation coefficient R of the standard curve 2 ≥0.99999.

3. The method for determining D-allulose in processed foods as described in claim 1, characterized in that, The detection limit for D-allulose in processed foods by the aforementioned assay method is 0.002 g / 100g.

4. The method for determining D-allulose in processed foods as described in claim 1, characterized in that, The limit of quantification for D-allulose in processed foods by the aforementioned determination method is 0.005 g / 100g.

5. The method for determining D-allulose in processed foods as described in claim 1, characterized in that, In the step of preparing the test solution, the mass-to-volume ratio of the processed food to warm water is (1-2.5) g / 15 mL.

6. The method for determining D-allulose in processed foods as described in claim 5, characterized in that, In the step of preparing the test sample solution, the processed food includes one or more of the following categories as classified according to the GB 2760-2024 food classification standard: baked goods, dairy products, egg products, aquatic and meat products, condiments, confectionery and jelly, and solid and liquid beverages.

7. The method for determining D-allulose in processed foods as described in claim 6, characterized in that, In the step of preparing the test solution, when the processed food sample is solid or semi-solid, the specific operation is as follows: after crushing and mixing the sample, add warm water at 50-60℃, and extract by ultrasound for 10-30 min to promote the full release of sugar. When the processed food sample is in liquid state, the specific operation is as follows: add 50-60℃ warm water to the well-mixed sample and shake until uniform.

8. The method for determining D-allulose in processed foods as described in claim 1, characterized in that, In the step of preparing the test solution, the volume ratio of the 50-60℃ warm water to the Carrez reagent is (10-20):1; the Carrez reagent consists of Carrez I solution and Carrez II solution, and the volume ratio of the two is 1:

1.

9. The method for determining D-allulose in processed foods as described in claim 1, characterized in that, In the chromatographic detection step, the specific chromatographic conditions include: flow rate: 0.4-0.6 mL / min; detection cell temperature: 35-45 ℃; injection volume: 10-20 μL.

10. The application of the method according to any one of claims 1-9 in determining the D-allulose content in processed foods, characterized in that, The applications include using the assay method for quality control and finished product compliance testing during food processing.

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