Histamine is a biogenic amine, produced in spoiled fish and some fermented products, which causes a foodborne disease similar to an allergic reaction. Because regulatory levels on histamine in food have been set by many countries or organizations, a quick and accurate analysis of histamine is of great interest. An enzymatic histamine determination method on the basis of a colorimetric assay has been used to detect histamine for raw and canned tuna due to its simplicity and rapidity. However, note that some compounds in fermented foods interfere with assay results. In this study, the pretreatments and conditions of the assay for fermented foods were evaluated. Lowering the reaction temperature from 37 to 23°C was considerably effective in reducing the interference. As a result, histamine in salami and sauerkraut (≥5 to 10 mg/kg) could be determined with a 25-fold dilution, as in the manufacturer's instructions. Histamine in soy sauce (≥10 to 20 mg/L) could also be determined with a 100-fold dilution. Removing fat and protein in cheese samples by using perchloric acid with a resultant 25-fold dilution and removing polyphenol with polyvinylpolypyrrolidone for red wine with a fivefold dilution were feasible; the limits of quantification were 5 mg/kg and 1 mg/L, respectively. Good recovery rates, precision repeatability, and correlations with a high-performance liquid chromatography method were confirmed. These protocols are expected to be applicable for histamine determination in various foods and useful for preventing histamine food poisoning.
Histamine in fermented foods could be determined by an enzymatic assay.
Lowering assay temperature and diluting were effective in reducing the interference.
The removal of interfering substances in cheese and red wine were essential.
Method validation studies characterized the recoveries and limit of quantification.
Histamine is one of the most toxic biogenic amines produced by bacterial decarboxylation of histidine in foods (4, 7, 22). Cooking can inactivate both the enzyme and the microorganisms for histamine production, but the histamine that has been formed cannot be eliminated because it is heat stable (26). Histamine intoxication that leads to allergic-like reactions is caused by the intake of high levels of histamine in foods (4, 7, 22). Particularly, the consumption of contaminated fish belonging to the family Scombridae, such as tuna and mackerel, presents a high risk of harm from histamine because of the high content of free histidine in the muscle tissues (7). Currently, the U.S. Food and Drug Administration and the European Union have set a limit of 50 and 100 mg/kg for histamine content in fish meat, respectively (7, 13, 19). The European Union also adopted a limit of 200 and 400 mg/kg for histamine content in enzymatically matured fishery products and fish sauce, respectively (7, 13, 19). Besides fishery products, histamine can also be found in fermented foods such as wine, dry sausage, sauerkraut, cheese, and soy sauce (1, 4, 6, 9, 13, 17, 19, 22–24). Because the consumption of a large amount of these foods with a high content of histamine can lead to serious health consequences, interest in the control of histamine in fermented foods has emerged.
To determine histamine levels in foods, the AOAC International Official Methods of Analysis 977.13 fluorescent method, high-performance liquid chromatography (HPLC) methods, enzyme immunoassay methods, and enzymatic assay methods are used (7). Specifically, the enzymatic assay provides an advantage over other assay methods due to easy extraction, ease of use, and a short time to results (20). The Histamine Test (Kikkoman Biochemifa, Tokyo, Japan) is a commercially available test kit that is a colorimetric enzymatic assay on the basis of a histamine dehydrogenase reaction (Fig. 1) (20). This reaction, in the presence of 1-methoxy-5-methylphenazinium methyl sulfate, produces a colored tetrazolium salt that can be measured by absorption at 470 nm (21). Because the selectivity of the enzyme is good, false positives are not observed for other biogenic amines, except for agmatine and putrescine (21). The enzymatic assay can determine levels of histamine >10 mg/kg for raw and canned tuna and 80 mg/kg for anchovy fish sauce and is approved for AOAC Research Institute Performance Tested Method certification (21). It has been used for quality control of fishery products.
However, note that some compounds in fermented foods interfere with the enzymatic assay results (9). In this method, the tetrazolium salt develops color due to electron transfer, which is caused by histamine decomposition by the enzyme (Fig. 1). If there is any oxidation or reduction substance in the reaction, interference with the enhancement of color development is possible, resulting in inaccurate histamine levels (3). Sample dilution is an effective method to reduce this interference. Regarding raw or canned fish, because the interfering substances are not abundant, a 25-fold sample dilution was enough to avoid the interference (21). In fact, the histamine in fish sauce can be determined when a sample is diluted at least 200 times (21). Though the resultant limit of quantification (LOQ) is 80 mg/kg, it is an acceptable sensitivity because the strictest regulation of histamine levels for fish sauce is 200 mg/kg in Canada (2). However, further dilution is undesirable because detection sensitivities decrease.
To avoid interference by oxidation or reduction substances, the pretreatments and conditions of the enzymatic histamine assay were optimized for fermented meat, dairy, vegetable, beverages, and seasoning. Method validation studies were conducted to characterize the recoveries and LOQ. The effect of the change in conditions on the selectivity for other biogenic amines was also characterized.
MATERIALS AND METHODS
Commercially available dry salami (Italy), sauerkraut (Germany), Cheddar cheese (New Zealand), Brie cheese (France), red wine (70% Merlot, 20% Cabernet Franc, and 10% Cabernet Sauvignon, France), and regular soy sauce (Japan) were used for a spike test. Seven red wines for a correlation study were from France (Carignan, Grenache Noir, and Merlot and as described previously), the United States (Cabernet Sauvignon), Italy (Montepulciano), Chile (Cabernet Sauvignon and Syrah), and South Africa (Pinotage). The EDTA disodium salt sample treatment buffer was from Dojindo Laboratories (Kumamoto, Japan). Perchloric acid (9.2 N) and polyvinylpolypyrrolidone (PVPP) were from Nacalai Tesque (Kyoto, Japan). Nuclease-free water was used for sample preparations, enzymatic assays, and HPLC assays.
Histamine solution (1,000 ppm) for spike tests, selectivity tests, and an HPLC standard was prepared by first drying histamine dihydrochloride (Sigma-Aldrich, Tokyo, Japan) in a desiccator at 23°C for 2 h. Then, the dried histamine dihydrochloride (167 mg) was weighed and dissolved in 0.1 N HCl and adjusted to 100 mL with a 100-mL plastic graduated cylinder. Agmatine and putrescine solutions (1,000 ppm) for selectivity tests were also prepared in the same manner. Dried agmatine sulfate (175 mg; Sigma-Aldrich) and putrescine dihydrochloride (183 mg; Sigma-Aldrich) were dissolved in 0.1 N HCl, and 100-mL solutions were prepared in the same manner, respectively. These solutions were subsequently stored at 4°C.
Preparation of histamine-spiked samples for enzymatic histamine assays
For soy sauce, five replicates for each histamine concentration (0, 10, 20, 40, 100, 200, and 400 mg/L) were prepared for the spike test. The sample (0.5 mL) was transferred to 35 heat-resistant 50-mL test tubes with caps. Then, 0, 5, 10, 20, 50, 100, or 200 μL of 1,000 ppm of histamine solution was added to five test tubes and mixed, respectively. Successively, 49.5 mL of 0.1 M EDTA (pH 8.0) was added to each tube and mixed to achieve a 100-fold dilution. All tubes were boiled for 20 min in a water bath and cooled by placing them in an ice bath.
For salami and sauerkraut, five replicates for each histamine concentration (0, 5, 10, 20, 40, 100, and 150 mg/kg) were prepared for the spike test. The sample preparation procedures were the same as the manufacturer's instruction for raw and canned tuna (21). The samples were homogenized with a blender. The homogenized sample (1 g) was transferred to 35 heat-resistant 50-mL test tubes with caps. Then, 0, 5, 10, 20, 50, 100, or 150 μL of 1,000 ppm of histamine solution was added to five test tubes and mixed, respectively. Successively, 24 mL of 0.1 M EDTA (pH 8.0) was added to each tube and mixed to achieve a 25-fold dilution. All tubes were boiled for 20 min in a water bath and cooled by placing them in an ice bath. Successively, the tubes were centrifuged (10,000 × g for 5 min), and the supernatant was recovered.
For cheese, five replicates for each histamine concentration (0, 5, 10, 20, 50, 100, and 150 mg/kg) were prepared for the spike test. The samples were homogenized with a blender. The homogenized sample (1 g) was transferred to 35 heat-resistant 50-mL test tubes with caps. Then, 0, 5, 10, 20, 50, 100, or 150 μL of 1,000 ppm of histamine solution was added to five test tubes and mixed, respectively. Successively, 18 mL of 0.1 M EDTA (pH 8.0) was added to each tube and mixed. All tubes were boiled for 20 min in a water bath and cooled by placing them in an ice bath. Then, 3 mL of 1 N perchloric acid was added, and the mixture was left for 10 min in an ice bath to precipitate fat and protein. After centrifugation (10,000 × g for 5 min), the supernatant (11 mL) was collected, and 1.5 mL of 1 M potassium hydroxide was added and mixed for neutralization to obtain 25-fold diluted samples.
For red wine, five replicates for each histamine concentration (0, 1, 2, 4, 10, and 20 mg/L) were prepared for the spike test. The sample (20 mL) was transferred to six heat-resistant 50-mL test tubes with caps. Then, 0, 20, 40, 80, 200, or 400 μL of 1,000 mg/kg histamine solution was added to the tubes and mixed, respectively. The samples in 50-mL test tubes with caps were boiled for 20 min in a water bath. Each heat-treated sample (2 mL) was dispensed into five 50-mL test tubes with caps. Successively, 6 mL of water and 1 g of PVPP were mixed, and the solution was left at 23°C for 1 h to absorb pigments to the resin. After centrifugation (10,000 × g for 5 min), the supernatant (4 mL) was collected in a tube, and 1 mL of 0.5 M EDTA (pH 8.0) was added and mixed to obtain fivefold diluted samples with 0.1 M EDTA. Regarding the correlation study using seven wines, the assays were performed in duplicate, and the mean values were calculated.
Enzymatic assay of histamine
The Histamine Test (Kikkoman Biochemifa) was used for the enzymatic assay of histamine (20, 21). The assay procedures were based on the manufacturer's instructions. To assay n samples, 2n + 2 plastic test tubes were prepared. When there were many samples to assay, simultaneous assays up to n = 10 were carried out. First, 0.5 mL of the included histamine standard (Estd) solution (4 mg/kg) and 0.5 mL of the enzyme solution were added to a test tube for the assay of the absorbance of the Estd. Second, for the assay of the absorbance of the reagent blank (Ec), 0.5 mL of nuclease-free water and 0.5 mL of the included buffer (Tris HCl) were added to another test tube. Third, 0.5 mL of the extracted sample solution was added to two other plastic test tubes. To carry out the assay of the absorbance of the sample (Es), 0.5 mL of the enzyme solution was added to the one tube with the sample. Instead of the enzyme solution, 0.5 mL of the included buffer (Tris HCl) was added to the other tube with the sample for the assay of the absorbance of the sample blank (Eb), with caution to avoid the contamination of the enzyme solution. Finally, 0.5 mL of the colorimetric solution was added to all test tubes, and the tubes were mixed. Although 37°C for 15 min was recommended as the incubation condition in the manual, incubation at 23°C in a water bath for 15 min was performed in this study to reduce false coloring. During the incubation, the sample tubes were covered with aluminum foil to protect them from light.
The value 4 in the formula means that the histamine concentration of the standard solution was 4 ppm.
Determination of LOQ
Selectivity study for the enzymatic assay
Solutions of 1,000 ppm of histamine, agmatine, and putrescine were diluted to 5 ppm with 0.1 M EDTA (pH 8.0). Each 5 ppm of solution of histamine, agmatine, and putrescine was assayed in the same manner, as previously described. The incubation was performed for 15 min at 23 or 37°C. The assays were performed in duplicate, and the mean (Es − Eb) values of agmatine and putrescine were expressed as relative values at each temperature, while the mean value for histamine was considered to be 100%.
HPLC histamine assay
Endogenous histamine of all matrices was determined by the HPLC method on the basis of the conditions as described by Izquierdo-Pulido et al. (8). A Shimadzu HPLC instrument model prominence (Kyoto, Japan) equipped with a controller (CBM-20A), a tertiary gradient pump (LC-20AD), an injector fitted with 100-μL sample loop, an autoinjector SIL-20AC, an online degasser DGU-20A5R, and a column oven CTO-20AC was used. The system also included a fluorescence detector (RF-20A XS). An L-Column ODS (4.6 by 150 mm inside diameter, 3 μm; Chemicals Evaluation and Research Institute, Tokyo, Japan) was used for chromatographic separation. Each measurement was performed once. Data were acquired and processed by using LabSolutions software (Shimadzu).
Some components in soy sauce interfere with the assay results (9). As preliminary tests, the conditions of the enzymatic histamine assay of regular soy sauce were investigated by using the sample that contains 3.8 mg/L endogenous histamine (Table 1). Even the Eb value with a 50-fold dilution, without the enzyme solution, and under the reaction at 37°C for 15 min was “scale over”; the portable absorptiometer RGB can measure up to 1.5 at 470 nm. The absorbance derived from the sample color, where water was used in place of the colorimetric solution, was only 0.154. Accordingly, it was thought that the interference issue in the histamine assay of soy sauce was not due to the sample color but due to the nonspecific and nonenzymatic coloring reaction of the redox materials (3). To decrease this nonspecific reaction, the reaction temperature was lowered to 23°C that is known as one of representative room temperature. Then, the Eb decreased to 0.85. Generally, the absorbance of Estd at 4 ppm shows ca. 0.8 at 37°C, and the equivalent Estd value was confirmed, even at 23°C, due to excess enzyme. To keep the sum of Eb and Estd less than 1.5 for the spike test, a sample with a 100-fold dilution was evaluated, and the Eb successfully reduced to 0.474. Thus, the dilution factor (100-fold) and the reaction temperature (23°C) were determined for the histamine assay of soy sauce.
The data from the histamine spike test, including the means, sr, RSDr, and recovery rates at each concentration are summarized in Table 1. All plots of determined values versus concentrations are shown in Figure 2A. The recoveries of all spiked matrices ranged from 81.4 to 122.5% (data not shown), and the mean was 92.5%. The RSDr at all spike levels was <14% though nonspiked samples showed RSDr values of 17.3% (Table 1), resulting from the fact that the native concentration was below the LOQ. The results showed good linearity and the overall goodness-of-fit R2 was 0.9995 (Fig. 2A).
To calculate LOQ, sr was plotted against concentration, and regression analysis was performed (Supplemental Fig. S1A). As shown in Table 2, the estimated LOQ for regular soy sauce is 13.3 mg/L. The recovery and RSDr values with 13.8 mg/L histamine (endogenous + spiking) were 106.9 and 13.8%, respectively (Table 1). Thus, the LOQ was validated.
Because lowering the reaction temperature to 23°C was quite effective in reducing nonspecific coloring, the enzymatic histamine assays for other fermented food matrices were also conducted at this temperature.
Salami and sauerkraut
Endogenous histamines for salami and sauerkraut were not detected and 5.0 mg/kg, respectively (Table 1). These fermented foods also caused nonenzymatic false coloring, as expected (data not shown). Fortunately, the histamine spike test was successful just by lowering the reaction temperature to 23°C and without changing the dilution factor (25-fold) set for raw and canned tuna. The data from the histamine spike test, including the means, sr, RSDr, and recovery rates at each concentration are summarized in Table 1. All plots of determined values versus concentrations are shown in Figure 2B and 2C. The recoveries of all spiked matrices ranged from 93.0 to 145.0% (data not shown), and the mean was 105.5% for salami. For sauerkraut, the recoveries of all matrices, including nonspiked samples, ranged from 90.5 to 159.1% (data not shown), and the mean was 110.1%. The RSDr for all samples of both matrices, including nonspiked samples, was <9% (Table 1). The results showed good linearity, and both matrices showed an overall goodness-of-fit R2 exceeding 0.9993 (Fig. 2B and 2C).
According to the regression analyses (Fig. S1B and S1C), the estimated LOQs for salami and sauerkraut are 9.2 and 3.0 mg/kg, as shown in Table 2, respectively. The recovery and RSDr values at 5 mg/kg histamine (spiking) for salami were 134.4 and 5.6%, respectively (Table 1). The recovery and RSDr values at 5 mg/kg histamine (endogenous) for sauerkraut were 152.3 and 3.6%, respectively (Table 1). Thus, the LOQs were validated.
Because a sauerkraut sample naturally contaminated with histamine was available, its histamine content was determined. The HPLC method and the enzymatic method showed that the histamine content was 49.0 and 44.1 mg/kg, respectively. The relative recovery of the enzymatic method compared with the HPLC method was 90.0%.
Cheddar and Brie were chosen as the representative test samples of hard and soft cheeses, respectively. The turbidity derived from the fat and protein in the cheese sample made the histamine assay difficult. Therefore, the fat and protein in the cheese were removed by perchloric acid, as is widely done for extractions of biogenic amines in foods (12), and resultant 25-fold diluted samples were obtained. Endogenous histamines for both Cheddar and Brie were not detected (Table 1). The data from the histamine spike test, including the means, sr, RSDr, and recovery rates at each concentration are summarized in Table 1. All plots of determined values versus concentrations are shown in Figure 2D and 2E. The recoveries of all spiked matrices ranged from 86.8 to 117.8% (data not shown), and the mean was 99.5% for Cheddar. For Brie, the recoveries of all spiked matrices ranged from 96.1 to 107.6% (data not shown), and the mean was 101.4%. The RSDr at all spike levels was <5.7%. Nonspiked samples showed RSDr values of 10 to 15%, resulting from the fact that the native concentration was below the LOQ. The results showed good linearity, and both matrices showed an overall goodness-of-fit R2 exceeding 0.9993 (Fig. 2D and 2E).
According to the regression analyses (Fig. S1D and S1E), the estimated LOQ values for Cheddar and Brie were 4.5 and 4.1 mg/kg, as shown in Table 2, respectively. The recovery and RSDr values at 5 mg/kg histamine (spiking) for cheddar were 110.1 and 5.7%, respectively (Table 1). The recovery and RSDr values at 5 mg/kg histamine (spiking) for Brie were 102.9 and 4.1%, respectively (Table 1). Thus, the LOQs were validated.
The regulatory limits for histamine in wines have not yet been established; however, several countries recommend upper limits for histamine (10 mg/L in Switzerland and Australia, 3.5 mg/L in Netherlands, 2 mg/L in Germany, 5 mg/L in Finland, 8 mg/L in France, and 6 mg/L in Belgium) (6, 18). It has been suggested that 8 to 20 mg/L of histamine may cause negative physiological effects if consumed in large quantities (6, 22). Furthermore, 2 mg/L of histamine in alcoholic beverages was suggested as an upper limit for human consumption (13). To measure histamine at several parts per million, a fivefold dilution of the samples was desirable, though the standard dilution factor was set at 25-fold for raw and canned tuna. However, interference by the nonenzymatic color reaction from redox materials occurred severely in red wine, probably due to the presence of polyphenol (data not shown). Because PVPP removes polyphenol in wine (11), the optimal pretreatment conditions (the amount of PVPP, the solvent for the absorption, and the time) was established, as described in the “Materials and Methods” (data not shown).
The endogenous level of histamine for red wine used for the histamine spiking test was 1.7 mg/L (Table 1). The data from the histamine spike test, including the means, sr, RSDr, and recovery rates at each concentration are summarized in Table 1. All plots of determined values versus concentrations are shown in Figure 2F. All recoveries of all matrices, including nonspiked samples, ranged from 71.1 to 83.3% (data not shown), and the mean was 79.4%. The RSDr of all samples, including nonspiked samples, was <7.1%. The results showed good linearity and the overall goodness-of-fit R2 was 0.9996 (Fig. 2F).
According to the regression analyses (Fig. S1F), the estimated LOQ for red wine was 0.9 ppm, as shown in Table 2. The recovery and RSDr values with 1.7 mg/L histamine (endogenous) were 80.6 and 7.1%, respectively (Table 1). Thus, the LOQs were validated.
In addition to the sample that was used for the spike test, six red wine samples were analyzed. According to the HPLC method, the endogenous histamine levels were 1.0 to 8.3 mg/L. The enzymatic assay showed a good correlation with the HPLC assay (R2 = 0.9972; Fig. 3). The relative recoveries of the enzymatic method compared with the HPLC method were 89.8 to 102.2%.
Selectivity for biogenic amines
The previous validation study under the incubation condition at 37°C showed that putrescine may cause a slight positive interference and agmatine leads to an increase in the apparent histamine concentration (21). The effect on the selectivity when the reaction temperature was lowered to 23°C was evaluated by using 5 ppm of histamine, agmatine, and putrescine. At 37°C, the relative measurement values for agmatine and putrescine toward histamine were 45.2 and 6.2%, respectively. Lowering the temperature to 23°C reduced these relative measurement values to 35.6 and 2.1%.
To use the enzymatic histamine assay kit for soy sauce, elimination of interfering compounds with a weak cation exchange column has been recommended (9). The procedures are as follows. Soy sauce is diluted 100-fold with a phosphate buffer. This solution is applied to a weak cation exchange column, and then the column is washed with a phosphate buffer. Finally, histamine is eluted with an equal volume of sodium chloride solution to the applied sample volume (9). All of the eluate is collected and mixed, resulting in an assay sample with 100-fold dilution. This protocol can be used for other matrices if the extraction is performed by using a 25-fold dilution in phosphate buffer. Although this method is effective for accurate determination of histamine, the preparation of the buffer and the procedures of the treatment are laborious, and they are impractical for a multisample assay. Accordingly, a more simple and rapid method is required.
In this study, it was found that lowering the reaction temperature from 37 to 23°C was considerably effective in reducing the nonspecific color development by redox materials. As a result, histamine in salami and sauerkraut could be determined by using a 25-fold dilution, and these LOQs were 5 to 10 mg/kg as in the manufacturer's instructions (Table 2). Histamine in soy sauce could also be determined by the combination of an additional fourfold dilution without the column pretreatment. Consequently, the LOQ for soy sauce was 10 to 20 mg/L (Table 2). Regarding cheese and wine, further pretreatments were necessary, that is, removing the fat and protein in cheese with perchloric acid and removing polyphenol in wine with PVPP. Accordingly, a reliable determination of histamine in these foods could be achieved. The LOQ for cheese was 5 mg/kg (Table 2). Specifically, the dilution of wine can be reduced to fivefold, and the LOQ level (1 mg/L; Table 2) covers the specific histamine limits (2 mg/L) in several European countries (6).
The Estd was constant (ca. 0.8), regardless of whether the reaction was performed at 37 or 23°C. This enzymatic histamine kit is based on an end point assay, and sufficient amounts of enzyme to enable the completion of the color reaction within 15 min, even at 23°C. This kit has a shelf life of 42 months when stored at 2 to 8°C (21). According to two-way analysis of variance, no statistically significant differences were observed for Estd between the new kit and the kit after storage at 4°C for 46 months when the reaction was carried out at 20 and 37°C, respectively (data not shown). Hence, there is no need to change the shelf life, and the robustness of an incubation temperature down to 20°C is indicated.
Lowering the reaction temperature from 37 to 23°C was also preferable from the viewpoint of selectivity for histamine. As a result, the positive interference by putrescine becomes practically negligible (only 2% toward histamine). However, the positive interference by agmatine was still approximately 35%. Regarding the histamine assay in tuna, it is assumed that agmatine interference can also be negligible because the amount of arginine, a precursor of agmatine is remarkably lower than that of histidine, a precursor of histamine in tuna (14, 21). However, the possibility that agmatine development can occur in fermented foods should be considered due to the generation of arginine by proteolytic digestion in the fermentation process. Therefore, note that the presence of agmatine would cause false-positive results for the enzymatic histamine assay of fermented foods.
For example, it is reasonable that the actual histamine contents in red wine, whose recovery rate was ca. 80% (Table 1) are about 1.25 times as much as the corresponding measurement values.
There are many other fermented foods with concerns about histamine content, such as miso (13), beer (15), white wine (6), yogurt (1), dried bonito (17), kimchi (25), and salt-ripened anchovy (16). Furthermore, the histamine content of dried fish (4), dressed fried fish (27), and processed fish seasoned with soy sauce (10), miso (10), or tomato (5) have also raised concerns. The combination of lowering the reaction temperature, dilution, pretreatment for removing fat, protein, and polyphenol that were examined in this study seems to be successful for the modification of the histamine assay protocol for these foods. In these cases, a validation study to determine the recovery and the LOQ is essential.
When the measurement values are compared with the regulatory values, the units should be considered. The units used in regulations for fishery products and fish sauce are milligrams per kilogram (7, 13, 19). However, the unit of specific limits for wine is milligram per liter (6). In this study, the units of the measurements were milligrams per kilogram for solid foods and milligrams per liter for liquid foods because the solid and liquid samples were measured by weight and volume, respectively. If the unit of milligram per liter needs to be converted to milligram per kilogram, the value (milligram per liter) should be divided by the density (gram per milliliter).
The test kit was originally designed for use by personnel with basic laboratory skills in a laboratory environment (21). The features of the kit are its simplicity due to its easy pretreatment, single calibration measurement without a complex calculation to create multipoint calibration curves, and its accuracy due to the subtraction of the blank coloring of each sample. Moreover, the constant Estd is useful as an internal quality control to determine the skill of the person performing the assay and the quality of reagents. Although a portable absorptiometer with a 2-cm optical path length was used in this study, a general spectrophotometer with a 1-cm optical path length is also available (21). Then, it is necessary to consider that the LOQ values obtained by the 1-cm cuvette are theoretically twice as large as the values obtained by the 2-cm cuvette. If a centrifuge for pretreatment is not available, filtration through folded filter paper with a plastic funnel into a clean plastic tube is an alternate method for the separation, according to the previous robustness study (21). The developed protocols are versatile and can be used for histamine determination in various foods and offer a convenient strategy for the prevention of histamine food poisoning.
In summary, this study demonstrated that the enzymatic histamine assay could be applicable for the determination of histamine in soy sauce, salami, sauerkraut, cheese, and wine if the reactions are performed at 23°C. An additional dilution is necessary for soy sauce. Regarding cheese and wine, extra pretreatments for the removal of interfering substances are essential. Measurements of spiked samples and naturally contaminated samples showed excellent correlation. Good recovery rates and precision repeatability were also shown for all spiked matrices. The arranged protocols seem to be useful as a basis for the application of the enzymatic histamine assay to other fermented foods and complexly processed or seasoned fishery products.
Supplemental material associated with this article can be found online at: https://doi.org/10.4315/JFP-20-082.s1