Methods for rapid detection of preservatives in processed foods

Abstract

This invention provides a rapid method for detecting preservatives in processed foods. The method involves first pre-treating the processed food using solid-phase extraction (SPE) technology, followed by measurement using surface-enhanced Raman scattering (SERS) technology to obtain the Raman spectrum of the processed food. By analyzing the Raman spectrum of the processed food, the presence and content of preservatives can be determined. This rapid method for detecting preservatives in processed foods increases the accuracy of detection and reduces testing costs, thereby improving food safety.

Description

Technical Field

[0001] This invention relates to food safety, and in particular to a method for rapidly detecting preservatives in processed foods. Prior Technology

[0002] Commonly used antibacterial preservatives in food additives include compounds such as benzoic acid (BA), sorbic acid (SA), and dehydroacetic acid (DHA). Their main function is to extend the shelf life of food and prevent microbial growth that leads to spoilage. To increase the solubility of these compounds in water, they are often present in salt form, such as sodium benzoate, sodium sorbate, potassium benzoate, potassium sorbate, calcium sorbate, and sodium dehydroacetate. These antibacterial preservatives or their salts are widely used in protein-rich foods, such as dairy products and seafood.

[0003] Because excessive intake of antimicrobial preservatives can damage the liver and kidneys, governments worldwide have established maximum residue limits (MRLs) for these preservatives in food. Currently, the most common methods for detecting antimicrobial preservative content in food are quantitative analysis using HPLC (high-performance liquid chromatography) and photodiode array detectors. However, these methods suffer from drawbacks such as expensive equipment, long processing times, and complex operation. Therefore, they cannot provide immediate and rapid detection of antimicrobial preservative content at the point of sale or manufacture, increasing the difficulty of verifying whether food additives exceed permitted levels. Other research indicates that surface-enhanced Raman scattering (SERS) combined with a substrate containing nanoscale metal thin films can serve as a tool for detecting food additives, offering advantages such as short detection time and high sensitivity. However, the SERS signal is affected by the components in the processed sample, such as fats, proteins, and organic matter, leading to a decrease in detection accuracy. Summary of the Invention

[0004] The main objective of this invention is to provide a rapid method for detecting preservatives in processed foods, which can suppress the interference of components in processed foods on SERS signals, thereby improving the accuracy and sensitivity of detecting preservatives in processed foods.

[0005] Another objective of this invention is to provide a rapid method for detecting preservatives in processed foods. The detection results are consistent with those of the standard methods currently used in Taiwan, and the detection time and steps are shortened. Therefore, the rapid method for detecting preservatives in processed foods disclosed in this invention can reduce detection costs and improve detection convenience compared with existing standard methods.

[0006] Therefore, in order to achieve the above objectives, this invention discloses a method for rapid detection of preservatives in processed foods, which mainly includes a pretreatment step, a measurement step, and an analysis step, wherein:

[0007] This pretreatment step involves using solid-phase extraction (SPE) technology to pretreat a processed food product to obtain a test solution. Specifically, this pretreatment step further includes the following steps:

[0008] Step a-1: Perform an extraction process on the processed food to obtain a supernatant.

[0009] Step a-2: Pass the supernatant through an extraction column.

[0010] Step a-3: Rinse the extraction column.

[0011] Step a-4: Rinse the column to obtain an extract, and then mix the extract with water to obtain the test solution.

[0012] The measurement step uses surface-enhanced Raman scattering (SERS) technology to obtain the Raman spectrum of the processed food. Specifically, the measurement step involves placing a droplet of the test solution onto a SERS substrate and then performing SERS measurement to produce the SERS spectrum of the processed food.

[0013] The analytical step involves analyzing the Raman spectrum of the processed food, such as comparing the Raman spectra of various preservatives or compound standards, to obtain information about the preservatives in the processed food, such as whether it contains preservatives and the amount of preservatives.

[0014] In one embodiment of the present invention, the SERS measurement is performed by irradiating the center of a SERS substrate with a laser light.

[0015] In another embodiment of the present invention, the droplet and the SERS substrate have a contact angle between 30° and 120°, with 85° to 95° being preferred.

[0016] In a further embodiment of the present invention, the volume of the droplet is 1.5-7.5µL, wherein a volume of 4.5µL is preferred. Simple Explanation of the Diagram

[0017] Figure 1 shows the results of analyzing the Raman band intensity of preservative molecules in the rinsing solution at different stages. Figure 2 shows the effect of different contact angles between food sample droplets and the SERS substrate on the SERS measurement results. Figure 3 shows the effect of different extraction solutions on the Raman band of preservative molecules. Figure 4 shows the linear relationship between BA concentration and Raman band intensity (Log(y) = 0.9278 Log(x) + 0.6985, R² = 0.9762). Figure 5 shows the linear relationship between SA concentration and Raman band intensity (Log(y) = 0.86 Log(x) + 1.1007, R² = 0.9556). Figure 6 shows the linear relationship between DHA concentration and Raman band intensity (Log(y) = 0.8847 Log(x) + 0.5639, R² = 0.9817). Figure 7 shows the SERS measurement results of squid pills with 0.2 g / kg BA and 0.2 g / kg SA added. Figure 8 shows the SERS measurement results of squid pills with 0.3 g / kg BA and 0.2 g / kg SA added. Figure 9 shows the SERS measurement results of squid pills with added 0.3 g / kg BA and 0.3 g / kg SA. Figure 10 shows the SERS measurement results of squid pills with 0.05 g / kg BA added. Figure 11 shows the SERS measurement results of squid pills with 0.05 g / kg SA added. Figure 12 shows the SERS measurement results of squid pills with added 0.05 g / kg DHA. Implementation

[0018] This invention provides a rapid method for detecting preservatives in processed foods. The method mainly comprises two stages: the first stage involves pretreating the processed food using solid-phase extraction (SPE) to obtain a test solution; the second stage involves measuring the test solution using surface-enhanced Raman scattering (SERS) to obtain the Raman spectrum of the processed food. By analyzing the Raman spectrum of the processed food, the presence and content of preservatives can be determined. This rapid method for detecting preservatives in processed foods reduces interference from components within the processed food, such as phospholipids, proteins, peptides, and free amino acids, on the SERS signal. It also simplifies the detection steps and reduces the time required. Therefore, this method can increase the accuracy of detection and reduce the cost, making it widely applicable in the field of food testing, thereby improving food safety and protecting public health.

[0019] In one embodiment of the present invention, the preprocessing procedure of the first stage includes the following steps:

[0020] Step 1-1: Extract the processed food with a first-grade alcohol aqueous solution to obtain a sample supernatant;

[0021] Steps 1-2: Pass the sample supernatant through an extraction column;

[0022] Steps 1-3: Rinse the extraction column with a second lower alcohol aqueous solution, wherein the concentration of the second lower alcohol aqueous solution is lower than the concentration of the first lower alcohol aqueous solution;

[0023] Steps 1-4: Elute the column with a third lower alcohol aqueous solution and collect the eluent. Then mix the eluent with water to obtain the test solution, wherein the concentration of the third lower alcohol aqueous solution is higher than the concentration of the first lower alcohol aqueous solution.

[0024] The first, second, and third lower alcohol aqueous solutions are prepared from an alcohol and deionized water, respectively, and the alcohol may be methanol, ethanol, propanol, etc.

[0025] For example, the first lower alcohol aqueous solution is a 50% (v / v) methanol aqueous solution, the second lower alcohol aqueous solution is a 20% (v / v) methanol aqueous solution, and the third lower alcohol aqueous solution is an 80% (v / v) methanol aqueous solution.

[0026] In step 1-2, the extraction column system is a SEP column.

[0027] In step 1-2, the purification column is first cleaned and activated with the first low-grade alcohol aqueous solution before the sample supernatant is added.

[0028] In steps 1-2, the sample supernatant passes through an extraction column at a rate of 1 drop per second.

[0029] Specifically, the second phase of the measurement procedure includes the following steps:

[0030] Step 2-1: Place a droplet of the solution to be tested onto a SERS substrate, with a contact angle between the droplet and the SERS substrate, the contact angle being between 30° and 120°;

[0031] Step 2-2: Perform SERS measurement on the droplets on the SERS substrate.

[0032] In step 2-1, the volume of the droplet is 1.5-7.5µL.

[0033] In step 2-2, the conditions for SERS measurement include using a laser light source with a wavelength of 785nm.

[0034] In embodiments of the present invention, the preservative contains benzoic acid (BA), sorbic acid (SA), dehydroacetic acid (DHA), or other compounds with antibacterial and preservative effects.

[0035] The term "processed foods" refers to agricultural, livestock, or aquatic products that have been processed through food processing methods. These processing methods refer to physical, chemical, or microbiological approaches used to alter the form of food to improve its preservation, nutritional value, palatability, or flavor, such as freezing, sterilization, and the addition of food additives.

[0036] The term "surface-enhanced Raman scattering" is a method based on Raman technology, which is currently used as an analytical tool to identify various food additives.

[0037] The term "BA" is an abbreviation for benzoic acid.

[0038] The term "SA" is an abbreviation for sorbic acid, which is also known as hexadienoic acid.

[0039] The term "DHA" is an abbreviation for dehydrated acetic acid, which is also known as salicylic acid.

[0040] The term "SERS" is an abbreviation for Surface Enhanced Raman Scattering.

[0041] The term "SEP" is an abbreviation for solid-phase extraction.

[0042] To illustrate the technical features and effects of this invention, several examples will be provided below, along with accompanying drawings, for detailed explanation.

[0043] In the following examples, unless otherwise specified, the SERS measurement method used is as follows: after a food sample droplet is placed on a SERS substrate, the measurement is performed using a portable Raman spectrometer (RMS-1000, Oceanhood Inc., Zhongli, TW) under a 785 nm light source, with a measurement power of 50 mW and an integration time of 2 seconds; and the SERS substrate used is a silver nanoarray with a predetermined size, such as 2.2 mm x 2.2 mm.

[0044] Example 1: Preparation of Reagents

[0045] 10 mg of sodium benzoate and potassium sorbate were dissolved in 2 mL of deionized water and ultrasonicated for 5 minutes to obtain benzoic acid solvent and sorbic acid solution, respectively. 10 mg of dehydrated sodium acetate was dissolved in 2 mL of deionized water and ultrasonicated for 5 minutes to obtain dehydrated acetic acid solution. These solutions were then diluted with a deionized water-methanol solution (1:1, V / V, DI H₂O / MeOH) to prepare mobile phase standard samples of the required concentrations for subsequent applications.

[0046] Additionally, 50 mg of R6G (rhodamine 6 G) standard was dissolved in 0.5 mL of methanol, ultrasonicated for 5 minutes, and then diluted to the required concentration by adding deionized water-methanol solution (1:1, V / V, DI H₂O / MeOH) for use in subsequent examples.

[0047] Example 2: Preparation of Food Samples

[0048] Add a predetermined concentration of preservatives, such as SA, BA, or DHA, to a commercially available food product as a food sample. For example, add SA to squid balls at a concentration of 1.0 g / kg.

[0049] Example 3: Pre-processing procedures for food samples

[0050] Place 2 grams of homogenized food sample and 5 mL of deionized water-methanol solution (1:1, V / V, DI H₂O / MeOH) in a 15 mL centrifuge tube, mix thoroughly, such as by shaking the test tube for 1 minute, then let stand for 3 minutes, and then obtain the sample supernatant.

[0051] 200 μL of sample supernatant was passed through an SPE column (PT-001 / 3 mL; GETECH, Taiwan) at a rate of 1 drop per second. The SPE column was pre-purified with 2 mL of 50% deionized water-methanol solution (1:1, V / V, DI H₂O / MeOH). After passing through the SPE column, the column was flushed with 600 μL of deionized water-methanol solution (8:2, V / V, DI H₂O / MeOH) at a flow rate of 1 drop per second. Finally, 400 μL of deionized water-methanol solution (2:8, V / V, DI H₂O / MeOH) was used as the elution solution for subsequent SERS analysis.

[0052] Example 4: Detection of the intensity of the main characteristic peaks of various preservative molecules

[0053] In this example, squid balls containing 0.5 g / kg of BA, SA and DHA were used as food samples.

[0054] Each food sample underwent the pretreatment procedure as described in Example 3, and extracts from different stages (three stages in total) were collected. Each stage's extract was mixed with 200 μL of deionized water at a 1:1 ratio to obtain the test solution for each stage. Each stage's test solution was dropped onto a SERS substrate at a volume of 4.5 μL, and then SERS measurements were performed. The results are shown in Figure 1.

[0055] As shown in Figure 2, the main Raman band intensities of BA, SA, and DHA in the first extract are higher than those in the second or third extract, indicating that most of the preservative molecules, BA, SA, and DHA, are extracted in the first extract. Therefore, the first extract was selected as the detection solution in subsequent examples.

[0056] Example 5: Analysis of the Influence of Droplet Contact Angle on SERS Measurement in Food Samples

[0057] In this example, a squid ball with a concentration of 1.0 g / kg SA was used as the food sample, and the pretreatment was carried out in accordance with the steps of Example 3 to obtain an SA squid ball test solution.

[0058] SA (Squid Oleifera) test solution was dropped onto a SERS substrate at different droplet volumes: 1.5, 3.0, 4.5, and 7.5 μL. Different contact angles (θc) were generated between the droplets and the SERS substrate, namely 37°, 63°, 89°, and 105°, respectively, before SERS measurements were performed. Additionally, a 4.5 μL volume of SA (Squid Oleifera) test solution was dropped onto the SERS substrate and allowed to air dry as a control group with a contact angle of 0°, and SERS measurements were also performed.

[0059] Figure 2 shows the SERS measurement results of SA squid pellet test solutions under different conditions. As can be seen from Figure 2, the SA squid pellet test solution dried in air (contact angle 0°) showed almost no signal in the SERS measurement. This result indicates that when the test solution is directly dried on the surface of the SERS substrate, the preservative molecules cannot be adsorbed onto the SERS substrate surface, and the SERS substrate generates fluorescence interference, thus causing Raman signal suppression.

[0060] When the contact angle between the food sample droplet and the SERS substrate is greater than 0°, a distinct Raman characteristic peak of SA can be measured. This result shows that the contact angle between the food sample droplet and the SERS substrate affects the intensity of the SA Raman characteristic peak. Furthermore, as shown in Figure 2, the intensity of the SA Raman characteristic peak is highest when the contact angle between the food sample droplet and the SERS substrate is approximately 90° (i.e., the food sample droplet volume is 4.5 μL). When the contact angle is less than 90°, the increase in SA molecular signal decreases as the contact angle decreases, and when the contact angle is greater than 90°, the increase in SA molecular signal decreases as the contact angle increases.

[0061] Example 5: The impact of tubing pretreatment on SERS measurements

[0062] In this example, squid pills with added 0.5 g / kg of BA, SA, and DHA were used as food samples. Each food sample was divided into two groups. One group underwent the same pretreatment procedure as in Example 3; the other group used pure methanol as the extraction solvent. After completing their respective pretreatment procedures, each food sample was dropped onto a SERS substrate (droplet volume 4.5 μL) for SERS measurement. The results are shown in Figure 3.

[0063] The results in Figure 3 show that when the extract does not contain water, the SERS system cannot detect obvious characteristic peaks of SA, BA, or DHA. However, when the extract contains water, the SERS spectrum does not show obvious characteristic bands of SA, BA, or DHA.

[0064] Example 6: Correlation Analysis between Preservative Concentration and Raman Band Intensity

[0065] In this example, squid pills containing different concentrations of BA, SA, and DHA were used as food samples, with preservative concentrations of 0.1, 0.5, 1, and 2 g / kg, respectively. The food samples were processed according to the pretreatment procedure in Example 3. The first-stage extract of each food sample was mixed with deionized water at a 1:1 ratio to obtain the detection solution for each food sample, and then SERS measurements were performed. The laser irradiation power before measurement was 30 mW, the irradiation time was 15 seconds, and the titration volume was 4.5 µL. The relationship between different concentrations of BA, SA, and DHA and Raman band intensity was analyzed, and the results are shown in Figures 4 to 6.

[0066] As shown in Figures 4 to 6, there is a linear relationship between the concentration of preservatives and the intensity of the Raman band. The main characteristic peak of BA is 1002 cm⁻¹; the main characteristic peak of SA is 1645 cm⁻¹; and the main characteristic peak of DHA is 631 cm⁻¹.

[0067] Example 7: Sensitivity Analysis

[0068] The food samples used in this example include: squid balls with 0.2 g / kg BA and 0.2 g / kg SA, squid balls with 0.3 g / kg BA and 0.2 g / kg SA, squid balls with 0.3 g / kg BA and 0.3 g / kg SA, squid balls with 0.05 g / kg BA, squid balls with 0.05 g / kg SA, and cheese with 0.01 g / kg DHA. Referring to Example 3, the pretreatment procedures for each food sample were performed, and SERS measurements were conducted. The results are shown in Figures 7 to 12.

[0069] The SERS spectrum shown in Figure 7 includes multiple characteristic bands, such as 845 cm⁻¹, 1002 cm⁻¹, 1154 cm⁻¹, 1595 cm⁻¹, and 1645 cm⁻¹, indicating that BA and SA can be detected and identified simultaneously. Comparing Figure 7 with Figures 8 and 9, it can be seen that when the concentration of BA and / or SA in the food sample increases, the intensity of the corresponding Raman bands increases significantly. Specifically, Figure 8 shows a significant increase in the intensity of the two Raman bands at 845 cm⁻¹ and 1002 cm⁻¹; the SERS spectrum in Figure 9 shows an increase in the intensity at 845 cm⁻¹, 1002 cm⁻¹, 1154 cm⁻¹, 1595 cm⁻¹, and 1645 cm⁻¹, respectively.

[0070] A distinct BA characteristic peak appears at 845 cm⁻¹, 1002 cm⁻¹, and 1595 cm⁻¹ in the SERS spectrum shown in Figure 10; a distinct SA characteristic peak appears at 1154 cm⁻¹ and 1645 cm⁻¹ in the SERS spectrum shown in Figure 11; and a distinct DHA characteristic peak appears at 631 cm⁻¹, 705 cm⁻¹, 940 cm⁻¹, and 1595 cm⁻¹ in the SERS spectrum shown in Figure 12. These results demonstrate that the detection method disclosed in this invention has extremely high sensitivity and can detect preservatives in very low concentrations in food samples.

[0071] The results above show that the signal-to-noise ratios for BA, SA, and DHA are 3.62, 4.14, and 3.25, respectively. The detection limits (LODs) of the method disclosed in this invention for BA and SA are 0.05 g / kg, while the detection limit for DHA is 0.10 g / kg. Further calculations show that the limits of quantification (LOQs) for BA, SA, and DHA are 0.183 g / kg, 0.144 g / kg, and 0.201 g / kg, respectively.

[0072] Since the detection limits of BA, SA and DHA disclosed in this invention are significantly lower than the residue limits of BA, SA and / or DHA specified by the Taiwan Food and Drug Administration for processed fish paste, seafood products and dairy products, it shows that the rapid detection method for preservatives in processed foods disclosed in this invention can meet the needs of practical testing.

[0073] Example 10: Accuracy Analysis

[0074] Thirty samples of minced fish products, processed seafood, and cheese products were purchased from the supermarket for testing. The minced fish products included fish sauce, fish dumplings, crab sticks, tempura, fish cake, milkfish balls, cod balls, fish balls, marlin balls, salmon balls, caviar rolls, and caviar balls. The seafood products included cuttlefish balls, cuttlefish balls, cuttlefish and shrimp balls, octopus fish cake, shrimp balls, shrimp balls, squid balls, and squid paste. The cheese products included cheddar cheese, mozzarella cheese, gouda cheese, truffle cheese, Emmental cheese, sweet potato cheese, Parmesan cheese, and processed cheese.

[0075] Referring to the content of Example 3, the above-mentioned test samples were subjected to preprocessing procedures, followed by SERS measurement. The results of SERS spectrum analysis of each test sample were used to determine whether BA, SA and / or DHA were detected in the test sample, the location of characteristic peaks of BA, SA and / or DHA, and their intensity.

[0076] The preservative content of the above-mentioned samples was determined using the standard method (MOHWA0020.03) announced by the Taiwan Food and Drug Administration. This result was taken as the true result and compared with the SERS measurement results. Statistical analysis was performed to determine the specificity, sensitivity, false positives, and false negatives of the detection method disclosed in this invention for processed fish, seafood products, and dairy products. The results are shown in Table 1 below.

[0077] Table 1: Accuracy analysis of the detection method disclosed in this invention for processed foods. Processed foods Specificity (%) Sensitivity (%) False positives (%) False negative (%) Processed fish and seafood products 100% 88.88% 0% 11.11%. dairy products 100% 100% 0% 0%

[0078] The results above show that the method for rapid detection of preservatives in processed foods disclosed in this invention can indeed detect preservatives in processed foods quickly and accurately, and the detection sensitivity and specificity are excellent.

[0079] Furthermore, conventional techniques require significant time and manpower, and are costly to analyze. For example, the method for detecting preservatives in cheese, as announced by the Republic of China, involves steps such as liquid sample dilution, extraction, distillation, and HPLC / UV analysis. Because the dilution process lacks purification, subsequent HPLC / UV analysis is prone to matrix interference, leading to inaccurate results, and the entire process takes more than one hour. The rapid detection method for preservatives in processed foods disclosed in this invention includes a rapid purification pretreatment procedure, and the overall detection time is only 10-15 minutes. Therefore, compared to conventional techniques, the rapid detection method for preservatives in processed foods disclosed in this invention significantly improves the practicality and accuracy of detection, contributing to improved food safety and the efficiency of immediate verification.

[0080] none

[0081] none

Claims

1. A method for rapid detection of preservatives in processed foods, comprising the following main steps: Step a: performing a pretreatment procedure on a processed food using solid-phase extraction technology to obtain a test solution, wherein, The pretreatment procedure includes the following steps: a-1: Extracting the processed food with a first low-alcohol aqueous solution to obtain a supernatant; a-2: Passing the supernatant through an extraction column; a-3: Rinsing the extraction column with a second low-alcohol aqueous solution, wherein the concentration of the second low-alcohol aqueous solution is lower than the concentration of the first low-alcohol aqueous solution; a-4: Evaporating the column with a third low-alcohol aqueous solution to obtain an extract, and then mixing the extract with water to obtain the test solution, wherein the concentration of the third low-alcohol aqueous solution is higher than the concentration of the first low-alcohol aqueous solution; Step b: Placing a droplet of the test solution on a SERS substrate and performing a SERS measurement procedure to obtain the SERS spectrum of the processed food, wherein the droplet and the SERS substrate have a contact angle between 30° and 120°, and the volume of the droplet is 1.5-7.5µL; Step c: Analyze the position and / or intensity of characteristic bands or peaks in the SERS spectrum of the processed food to obtain information on the types or contents of preservatives in the processed food; wherein, the types of preservatives in the processed food are benzoic acid, sorbic acid, and dehydrated acetic acid.

2. The method for rapid detection of preservatives in processed foods as described in claim 1, wherein, The first, second, and third lower alcohol aqueous solutions are prepared from an alcohol and deionized water, respectively.

3. The method for rapid detection of preservatives in processed foods as described in claim 1, wherein, The first, second, and third lower alcohol aqueous solutions are all methanol aqueous solutions.

4. The method for rapid detection of preservatives in processed foods as described in claim 1, wherein, The contact angle is 85-95°.

5. The method for rapid detection of preservatives in processed foods as described in claim 1, wherein, The volume of the droplet is 4.5 µL.

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