A Comprehensive Analysis of ATP Tests: Practical Use and Recent Progress in the Total Adenylate Test for the Effective Monitoring of Hygiene.

ABSTRACT: Rapid hygiene monitoring tests based on the presence of ATP have been widely used in the food industry to ensure that adequate cleanliness is maintained. In this study, the practical applications and limitations of these tests and recent technological progress for facilitating more accurate control were evaluated. The presence of ATP on a surface indicates improper cleaning and the presence of contaminants, including organic debris and bacteria. Food residues are indicators of insufficient cleaning and are direct hazards because they may provide safe harbors for bacteria, provide sources of nutrients for bacterial growth, interfere with the antimicrobial activity of disinfectants, and support the formation of biofilms. Residues of allergenic foods on a surface may increase the risk of allergen cross-contact. However, ATP tests cannot detect bacteria or allergenic proteins directly. To ensure efficient use of commercially available ATP tests, in-depth knowledge is needed regarding their practical applications, methods for determining pass-fail limits, and differences in performance. Conventional ATP tests have limitations due to possible hydrolysis of ATP to ADP and AMP, which further hinders the identification of food residues. To overcome this problem, a total adenylate test was developed that could detect ATP+ADP+AMP (A3 test). The A3 test is suitable for the detection of adenylates from food residues and useful for verification of hygiene levels. The A3 test in conjunction with other methods, such as microorganism culture and food allergen tests, may be a useful strategy for identifying contamination sources and facilitating effective hygiene management.

Validation Study of LuciPacTM A3 Surface for Hygiene Monitoring through Detection of ATP, ADP, and AMP from Stainless Steel Surfaces: AOAC Performance Tested MethodSM 051901.

BACKGROUND: The LuciPac A3 Surface Hygiene Monitoring System based on the detection of total adenylate, ATP+ADP+AMP (A3), has been developed by Kikkoman Biochemifa. OBJECTIVE: This A3 swabbing assay kit was validated for Performance Tested MethodsSM (PTM) certification. METHODS: The LuciPac A3 Surface Hygiene Monitoring System was evaluated for limit of detection (LOD) for each adenylate with pure analyte solutions, detection of food residues and microbial residues on stainless steel surfaces, interference by disinfectants, and selectivity of the method response. RESULTS: Pure analyte studies performed by the method developer and the independent laboratory showed good linearity (R2 > 0.9854) and repeatability precision (RSDr < 20% for ≥2.5 fmol/assay). The LOD values for each adenylate were around 10 relative light units or 2.5 fmol/assay. The repeatability precision in the method developer laboratory for the matrix study (raw chicken breast, sliced deli ham, orange juice, yogurt, and apple pie) and the microbial study (Cronobacter sakazakii, Lactobacillus acidophilus, and Saccharomyces cerevisiae) were 8-30% and 10-35%, respectively. The repeatability precision of independent laboratory testing was 13-27% for orange juice and 16-43% for ham. Interference by several disinfectants indicate that rinsing is recommended to be performed after the use of sanitizing agents and before testing with LuciPac A3 Surface. Selectivity testing revealed that no positive interference and no inhibition were caused by adenylate analogues. Instrument variation, lot-to-lot consistency, accelerated stability (30°C, 5 weeks) were confirmed, and the method was shown to be robust against shaking time. CONCLUSIONS: The LuciPac A3 Surface has been successfully validated. HIGHLIGHTS: This A3 swabbing assay kit was qualified for PTM certification No. 051901.

Modified Enzymatic Assays for the Determination of Histamine in Fermented Foods.

ABSTRACT: 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.

Comparison of Detection Limits for Allergenic Foods between Total Adenylate (ATP+ADP+AMP) Hygiene Monitoring Test and Several Hygiene Monitoring Approaches.

ABSTRACT: Validation and verification of cleaning and inspection methods are essential to prevent the spread of allergens via cross-contact. Among the hygiene monitoring tests used on-site, the ATP test is rapid and provides quantifiable results. Nevertheless, because a wide variety of foods contain significant amount of ADP and/or AMP due to the degradation of ATP, the ATP+ADP+AMP (A3) test is preferred for detecting food debris. Hence, the A3 test may be valuable in screening food debris that may contain residual allergens. In this study, the detection limits of the A3 test for 40 foods that are regulated in several countries as allergenic were compared with those of the other hygiene monitoring tests used on-site: the conventional ATP test with similar sensitivity for ATP, the protein swab test that detects as little as 50 µg of protein, and the lateral flow immunoassay (LFI). The A3 test demonstrated lower detection limits than did the ATP test. The detection sensitivity of the A3 test was greater than that of the protein swab test except for its use on gelatin (extracted protein). The cleaning validation performance using a stainless steel model in fish and meat revealed that the A3 test is efficient in verifying the levels of remaining food debris. Although LFI displayed the best sensitivities for 10 of 14 foods, it is not commercially available for some specific allergens; however, the A3 test can detect such food debris. Moreover, the detection limits of the A3 test were preferable or comparable to those of LFI for crustacean shellfish and for processed grains, with the exception of wheat flour and buckwheat. A field study in a food processing plant demonstrated that the amount of both A3 and milk protein (enzyme-linked immunosorbent assay) considerably decreased as the cleaning steps progressed. Therefore, the A3 test is effective in detecting the risk for allergen cross-contact after inadequate cleaning.

Validation Study of Histamine Test for the Determination of Histamine in Selected Fish Products.

Background: Since prescribed limits for histamine in fish have been set by various regulatory bodies around the world, the rapid, specific and easy determination of histamine is in high demand. Objective: The enzymatic histamine assay developed by Kikkoman Biochemifa Co. was validated. Methods: Fresh and frozen raw tuna, canned tuna in oil and water, and anchovy fish sauce were used for the validation study under the specific guidelines of the AOAC Research Institute Performance Tested MethodSM (PTM) program. Results: Good linearity (R² > 0.9997) for histamine standard solutions, recovery rates (90.3-125.2%), and repeatability precisions (RSDr < 10%) were shown for all spiked tested matrixes. The claimed LOQ values, 20 and 10 mg/kg for solid fish and 160 and 80 mg/kg for fish sauce using a 1 and 2 cm optical path length spectrophotometer, respectively, were validated. The cross-reactivity test showed no positive interference of other biogenic amines, except for agmatine and putrescine. Moreover, no inhibition was confirmed among the 12 biogenic amines. Stability data supported the shelf life (42 months at 4°C), lot-to-lot consistency was demonstrated, and the method was shown to be robust. Independent laboratory testing using canned tuna in oil demonstrated that the recovery ranged from 93.5 to 124.7%, and the RSDr at all spike levels was <20%. Conclusions: The simple and rapid enzymatic histamine assay method has been successfully validated. Highlights: This histamine quantitative assay kit was qualified for PTM certification No. 041802.

Development of a Novel Hygiene Monitoring System Based on the Detection of Total Adenylate (ATP+ADP+AMP).

ATP is the universal energy molecule found in animals, plants, and microorganisms. ATP rapid hygiene monitoring tests have been employed in the food industry to ensure that adequate cleanliness is being maintained. However, because ATP is hydrolyzed to ADP and AMP by metabolic processes, by heat treatment, or under acidic or alkaline conditions, total adenylate (ATP+ADP+AMP [A3]) could be a more reliable sanitation indicator of food residues that may cause biofilm formation and allergen contamination. Therefore, a novel hygiene monitoring system to measure A3 was developed based on the luciferin-luciferase assay with the combination of two enzymes, pyruvate kinase and pyruvate phosphate dikinase, that can convert ADP into ATP and recycle AMP into ATP, respectively. The newly developed A3 assay system afforded stable bioluminescence signals and equivalent linear calibration curves between relative light units (RLU) and the amounts of ATP, ADP, and AMP, respectively. To verify the significance of the A3 method, the ratios of ATP, ADP, and AMP in various food samples were determined; large amounts of ADP and AMP were found in a variety of foods, such as meat, seafood, dairy, nuts, fruits, vegetables, and fermented foods. Sanitation monitoring of stainless steel exposed to raw meat was also examined, and the A3 method achieved a 200-RLU level, the typical benchmark value, after complete washing with detergent and rinsing. In contrast, a conventional ATP method showed less than 200 RLU after only a light cold and hot water rinse. In conclusion, the A3 assay appeared to be suitable for detection of adenylates from food residues that are not detected by the conventional ATP assay.

Detection of Gluten during the Fermentation Process To Produce Soy Sauce.

Advances have been made to provide people with celiac disease (CD) access to a diverse diet through an increase in the availability of gluten-free food products and regulations designed to increase label reliability. Despite advances in our knowledge regarding CD and analytical methods to detect gluten, little is known about the effects of fermentation on gluten detection. The enzyme-linked immunosorbent assay (ELISA) and lateral flow devices routinely used by analytical laboratories and regulatory agencies to test for the presence of gluten in food were examined for their ability to detect gluten during the fermentation processes leading to the production of soy sauce, as well as in finished products. Similar results were observed irrespective of whether the soy sauce was produced using pilot-plant facilities or according to a homemade protocol. In both cases, gluten was not detected after moromi (brine-based) fermentation, which is the second stage of fermentation. The inability to detect gluten after moromi fermentation was irrespective of whether the assay used a sandwich configuration that required two epitopes or a competitive configuration that required only one epitope. Consistent with these results was the observation that ELISA, lateral flow devices, and Western immunoblot analyses were unable to detect gluten in commercial soy sauce, teriyaki sauce, and Worcestershire sauce. Although reports are lacking on problems associated with the consumption of fermented soy-containing sauces by consumers with CD, additional research is needed to determine whether all immunopathogenic elements in gluten are hydrolyzed during soy sauce production.

Identification, characterization, and molecular cloning of a novel hyaluronidase, a member of glycosyl hydrolase family 16, from Penicillium spp.

Hyaluronidase (HAase) activity was detected in the culture supernatants of Penicillium purpurogenum and Penicillium funiculosum. The HAase from Penicillium spp. (HAase-P) was a hyaluronate 4-glycanohydrolase, which catalyzed the endolytic hydrolysis of the ß-1,4 glycosidic linkage, as do vertebrate HAases. The gene encoding HAase-P was cloned and expressed in Escherichia coli. According to homology analyses of the deduced amino acid sequences, HAase-P is not classified into any of the known HAase groups, but belongs to glycoside hydrolase family 16, which includes endo-ß-1,3(4)-glucanase. Regarding the substrate specificities, no chondroitinase and glucanase activities were detected. Judging from homology analyses and enzymatic properties, HAase-P seems to be a new type of HAase.

N1,N12-diacetylspermine oxidase from Debaryomyces hansenii T-42: purification, characterization, molecular cloning and gene expression.

An FAD-dependent N(1),N(12)-diacetylspermine oxidase (DASpmOX), which seems suitable for enzymatic determination of the tumor marker N(1),N(12)-diacetylspermine (DASpm), was isolated from Debaryomyces hansenii T-42. DASpmOX exhibited the most excellent specificity toward DASpm among all polyamine oxidases found to date, and the specificity for DASpm could be raised by adjusting the pH of the buffer and adding TritonX-100. In potassium phosphate (pH 7.0) with 0.3% TritonX-100, this enzyme did not have any detectable activity toward free polyamines, and the reaction rate of N(1),N(8)-diacetylspermidine, N(1)-acetylspermine, N(1)-acetylspermidine, and N(8)-acetylspermidine was only 19%, 7.8%, 7.8%, and 1.0% of that of DASpm, respectively. The gene encoding DASpmOX was cloned and expressed in Escherichia coli. The apparent k(cat) and K(m) values of recombinant enzyme for DASpm were found to be 158 s(-1) and 3.1 x 10(-4) M under the conditions described above, respectively.

N-ethylmaleimide-resistant acyl-coenzyme A oxidase from Arthrobacter ureafaciens NBRC 12140: molecular cloning, gene expression and characterization of the recombinant enzyme.

N-ethylmaleimide (NEM)-resistant acyl-coenzyme A oxidase (ACO) has been desired for the determination of free fatty acids (FFAs). In order to meet this demand, we prepared recombinant ACO from Arthrobacter ureafaciens NBRC 12140. The coding region of the gene was 2109, encoding a protein of 703 amino acids with a predicted molecular mass of 76.5 kDa. The heterologous expression level in Escherichia coli was 520-fold higher than that in the native strain. The purified enzyme retained more than 60% activity after incubation in the presence of 10 mM NEM at 37 degrees C for 4 h, while other commercially available ACOs showed only less than 10% activities after the same NEM treatment. We presume that this is due to the presence of only three cysteines in ACO from A. ureafaciens. Site-directed mutagenesis studies and close scrutiny of the three-dimensional structures of other related ACOs suggested that these cysteines were buried in the protein and unreactive to NEM. The recombinant enzyme was used for the colorimetric determination of free fatty acid, which gave a linear calibration.

Thermostabilization of porcine kidney D-amino acid oxidase by a single amino acid substitution.

D-amino acid oxidase (DAO) is of considerable practical importance, such as bioconversion and enzymatic assay. In this study, we succeeded in obtaining a thermostable mutant DAO from porcine kidney by a single amino acid substitution. This mutant enzyme, F42C, was stable at 55 degrees C, while the wild-type enzyme was stable only up to 45 degrees C. The Km values of F42C for D-amino acids was about half of those of the wild-type enzyme. This mutant DAO with improved stability and affinity for its substrates is advantageous for the determination of D-amino acids.

Histamine dehydrogenase from Rhizobium sp.: gene cloning, expression in Escherichia coli, characterization and application to histamine determination.

The gene encoding histamine dehydrogenase in Rhizobium sp. 4--9 has been cloned and overexpressed in Escherichia coli. The coding region of the gene was 2,079 bp and encoded a protein of 693 amino acids with a calculated molecular mass of 76,732 Da. This histamine dehydrogenase was related to histamine dehydrogenase from Nocardioides simplex (54.5% identical), trimethylamine dehydrogenase from Methylophilus methylotrophus (39.3% identical) and dimethylamine dehydrogenase from Hyphomicrobium X (38.1% identical), which have a covalent 6-S-cysteinyl flavin mononucleotide and a [4Fe--4S] cluster as redox cofactors. Sequence alignment and a UV-visible absorption spectrum supported the presence of these cofactors in this histamine dehydrogenase. The investigation of the enzymatic properties suggested that this enzyme exhibited the most excellent substrate specificity toward histamine among all amine oxidases or dehydrogenases found to date. The recombinant enzyme was able to be used for the colorimetric determination of histamine, which gave a linear calibration curve and identical data with conventional methods.

Improvement in thermal stability and substrate binding of pig kidney D-amino acid oxidase by chemical modification.

Chemical modification was evaluated to stabilize pig kidney D-amino acid oxidase (pkDAAO), which is required for analytical determination of D-amino acids. Optimization of modification conditions was performed to obtain high recovery yield and stability, and chemical modification at 30 degrees C for 12 h with a highly concentrated enzyme solution gave dextran-conjugated pkDAAO with a 70% yield of activity. pkDAAO was stable at less than 55 degrees C at pH 6.0, while the conjugated enzyme was stable even at 70 degrees C. In addition, the conjugated enzyme showed decreased Km values for D-amino acids. Because of these outstanding characteristics, this new material is expected to be available for use as a liquid assay reagent.

Distribution and properties of novel deglycating enzymes for fructosyl peptide in fungi.

Our fungal culture collection was screened for fructosyl peptide oxidase, an enzyme that could be used for the determination of glycated hemoglobin in diabetic subjects with hyperglycemia. Fructosyl peptide oxidases were found in strains of eight genera: Achaetomiella, Achaetomium, Chaetomium, Coniochaeta, Eupenicillium, Gelasinospora, Microascus and Thielavia. By their substrate specificity toward N(alpha)-fructosyl valyl-histidine (alpha-keto-amine) and N(epsilon)-fructosyl lysine (epsilon-keto-amine), fructosyl peptide oxidases could be categorized into two groups: (1) enzymes that oxidize both alpha-keto-amine and epsilon-keto-amine, and (2) enzymes that preferably oxidize alpha-keto-amine. A fructosyl peptide oxidase from Achaetomiella virescens ATCC 32393, active toward both N(alpha)-fructosyl valyl-histidine and N(epsilon)-fructosyl lysine, was purified to homogeneity and characterized. The enzyme was monomeric ( M(r)=50,000), was most active at 40 degrees C and pH 8.0, and had a covalently bound flavin as a prosthetic group. Apparent K(m) values for N(alpha)-fructosyl valyl-histidine and N(epsilon)-fructosyl lysine were 2.30 and 1.69 mM, respectively. N(alpha)-fructosyl valyl-histidine was consumed and the same molar amount of valyl-histidine was produced by the fructosyl peptide oxidase reaction. This enzyme could be useful for the measurement of hemoglobin A(1C), the N-terminal valine residue of the beta-subunit of which is glycated.

Lipase-Catalyzed Enantiomeric Resolution of Ceramides.

Lipase-catalyzed enantiomeric kinetic resolution of ceramides related to C(16)-sphinganine and C(18)-sphingenine is described. Two hydroxy groups in readily available racemic N-stearoyl-erythro-C(16)-sphinganine were acetylated, and several kinds of lipases were screened for the hydrolysis of this substrate. Among them, a Burkholderia cepacia lipase (SC lipase A, Sumitomo Chemical Co., Ltd.) showed the highest reactivity and enantioselectivity. The rate of hydrolysis and selectivity were greatly affected by some additives. Especially, the combined use of a detergent, Triton X-100, and the solid support, Florisil, for immobilization showed the highest enantioselectivity (E = ca. 170), although the reaction rate turned low. Introduction of a double bond into the substrate (N-stearoyl-erythro-C(18)-sphingenine) also retarded the hydrolysis. By utilizing the preferential hydrolysis of the acetate on the primary hydroxy group, another advantageous feature of this enzyme-catalyzed reaction, the resulting product could directly be used as the glycosyl acceptor for cerebroside synthesis.