Quantification of Total Sulfite in Shrimps by BIOFISH 300 SUL Method: First Action 2021.09.

BACKGROUND: Sulfite is the oldest and most widely used additive in our food supply with antioxidant and preservative properties. Due to its allergenic-like reactions and other adverse health effects, its use is regulated by international regulatory bodies. Therefore, food industries as well as regulatory laboratories must ensure that the maximum concentration of sulfite permitted is not exceeded. The AOAC INTERNATIONAL-approved official method for the quantification of sulfites is the Optimized Monier-Williams Method (AOAC Official Method 990.28), which consists of a time-consuming titration. OBJECTIVE: The present study aims to demonstrate the reliability of the BIOFISH 300 SUL, a simple, fast, and accurate method, as an alternative, for the quantification of total sulfites in shrimp, with a lower LOQ than that of the OMA 990.28, set at 10 mg/Kg. METHODS: The BIOFISH 300 SUL method is a highly specific biosensor based on its proprietary enzyme-based electrode, for the rapid quantification of total sulfite. The test kit consists of an electrochemical reader (biosensor BIOFISH 300) and disposable electrodes (Biotest) that are capable of providing an electrical signal proportional to the amount of sulfite in the sample analyzed. The method mainly consists of the extraction of sulfite from the solid matrix in an aqueous solution, and its subsequent quantification by the device in less than 3 min. RESULTS: Comparative studies between BIOFISH 300 SUL and OMA 990.28 were conducted for naturally contaminated and spiked samples of raw and boiled shrimp with sulfite levels covering the 7-150 mg/kg range in order to determine linearity, recovery, repeatability, intermediate reproducibility, and accuracy. CONCLUSION: The BIOFISH 300 SUL method demonstrated high accuracy and precision for the whole range of quantification (7-150 mg/kg). Its ease of use and fast response make it the ideal technology to be implemented by the industry. HIGHLIGHTS: BIOFISH 300 SUL was adopted as a First Action Official MethodSM by the AOAC Expert Review Panel for Sulfites in Seafood Methods in February 2021 after rigorous review.

Quantification of Lactose in Lactose-Free and Low-Lactose Milk and Milk Products by BIOMILK 300/3000 Lac, Collaborative Study: Final Action 2020.09.

BACKGROUND: In April 2020, the BIOMILK 300 LAC method for the quantification of lactose in lactose-free and low-lactose dairy products was adopted as First Action Official Method of AnalysisSM2020.09 by AOAC INTERNATIONAL. To test the reproducibility of the method, as the last step toward the Final Action status, a collaborative study was organized by BIOLAN Microbiosensores. OBJECTIVE: Fifteen collaborators within the European Union took part in this study, where nine different samples were sent as duplicate blind test portions for lactose determination. The data obtained were used to determine method repeatability and reproducibility and also to validate the new version of the biosensor. METHOD: The test method is based on the direct enzymatic recognition-electrochemical detection of trace levels of lactose over a wide range of dairy samples by means of the BIOMILK 300 biosensor, in less than 5 min, and without intricate sample pretreatments. RESULTS: All samples resulted in a repeatability relative standard deviation (RSDr) of <8.1% and a reproducibility relative standard deviation (RSDR) of 14% maximum, meeting requirements from Standard Method Performance Requirement (SMPR®) 2018.09. CONCLUSIONS: The method has proved to give reproducible results for the quantification of lactose in lactose-free and low-lactose dairy products. HIGHLIGHTS: On the basis of these results, the enzymatic amperometric biosensor method developed by BIOLAN Microbiosensores was adopted as a Final Action Official Method in July 2022.

Modification of BIOFISH 300 HIS for Determination of Histamine in Fish and Fish Products: AOAC Performance Tested MethodSM 051604.

BACKGROUND: The BIOFISH 300 HIS method for the quantification of histamine in fish and fish products was validated by the AOAC Research Institute and granted Performance Tested MethodSM certification in 2016. The method is based on the use of an electrochemical reader (BIOFISH 300 device) together with disposable gold electrodes (Biotest) modified with a specific enzyme that oxidizes the histamine molecules and produces a detectable and quantifiable electric current signal. It was validated for raw fish (tuna, mackerel, sardine, and anchovy), cooked fish (tuna), canned fish (tuna in water, tuna in oil, mackerel in tomato sauce, and pickled sardine), and salted fish (canned salted anchovy). The validated ranges were 30-150 mg/kg histamine for canned salted anchovy and 10-200 mg/kg histamine for all other matrices. OBJECTIVE: The objective of the present report is to validate some method modifications, namely the use of a new reader (BIOFISH 3000), the inclusion of new quantification ranges, new matrixes in the certified claims, a histamine reference solution (Verifying Solution) that will enable the user to perform a system verification, and also the inclusion of the kit ASC1-10 for the elimination of ascorbic acid from sample extract. METHODS: New quantification ranges can be obtained by diluting the sample appropriately into the measurement cuvette, thereby adjusting the concentration to the internal calibration range of the device. New matrixes such as fish meal, preserved salted anchovy, and fish with added ascorbic acid were validated. The new digitalized reader enables new functionalities such as the use of an intuitive app and cloud storage of the results, with no changes in the data acquisition and processing. As the use of the new reader does not imply any change at the electrochemical readout level, all assays were performed using the BIOFISH 3000 device. RESULTS: The method was shown to be linear in all the quantitation ranges configured. Accuracy and recovery studies over a wide range of matrixes showed that the method yields comparable results to the reference method used. Stability and product consistency testing of kit components demonstrated that the system produces accurate results when expiration dates from the manufacturer are met. CONCLUSIONS: All modifications made to the BIOFISH 300 HIS method, PTM 051604, have been validated satisfactorily, under criteria of the AOAC Research Institute. HIGHLIGHTS: The use of the BIOFISH 300/3000 HIS method is ideal as a routine histamine control method for the fish industry. After a simple aqueous extraction of the sample, results are obtained in less than 3 minutes. All data generated by the new BIO3000 biosensor are sent to a web platform, allowing new functionalities to the user. All these technical capabilities represent a competitive advantage over other existing technologies.

Quantification of Lactose in Lactose-Free and Low-Lactose Milk and Milk Products by BIOMILK 300 Lac Biosensor: First Action 2020.09.

BACKGROUND: In 2018, the AOAC Stakeholder Panel on Strategic Food Analytical Methods approved Standard Method Performance Requirement (SMPR®) 2018.009, for lactose in low-lactose or lactose-free milk, milk products, and products containing dairy ingredients, establishing the minimum recommended performance characteristics to be addressed during the evaluation of methods. Subsequently, AOAC INTERNATIONAL opened a call for methods under the Official Method of AnalysisSM program with the aim of finding a candidate method for confirming compliance with regulatory standards and dispute resolution. OBJECTIVE: A biosensor-based analytical method, BIOMILK 300 LAC, was developed by BIOLAN Microbiosensores S.L. (www.biolanmb.com) to rapidly, easily, and accurately quantify lactose in free or low-lactose dairy products. In response to the AOAC call for methods, BIOLAN performed a single laboratory validation of this method against SMPR 2018.009. Several different matrixes were tested, including: milk, sugary plain yogurt, fruit plain yogurt, flavored liquid yogurt, Greek yogurt, cream, soft cheese, infant formula, café latte, chocolate milk, and high-protein milk shake. Evaluated method parameters included: linearity, selectivity, matrix effect, recovery, accuracy, repeatability, intermediate reproducibility, robustness, reagent lot-to-lot consistency, and stability. METHODS: The method is based on the use of the Biotest gold electrode together with the BIOMILK 300 biosensor reader, for the quantification of residual levels of lactose in dairy samples via an enzymatic recognition/electrochemical transduction system. RESULTS: Assay linearity, applicability to different matrixes, recovery, and precision demonstrated that the method is fit for purpose. The method proved to be robust, consistent, and stable, under conditions detailed in the Instructions For Use guide. CONCLUSION: The overall results were within requirements stated by SMPR 2018.009 for low-lactose and lactose-free milk, milk products and products containing dairy ingredients. HIGHLIGHTS: The BIOMILK 300 LAC method enables the quantification of reduced levels of lactose in less than 5 min, without the requirement for expert technicians, toxic solvents or intricate procedures, maintaining a high degree of precision and accuracy in the results. BIOMILK 300 LAC was adopted as a First Action Official MethodSM by the Expert Review Panel of Low-lactose Methods in April 2020 after rigorous review.

Measurement Time Reduction by Means of Mathematical Modeling of Enzyme Mediated RedOx Reaction in Food Samples Biosensors.

The possibility of measuring in real time the different types of analytes present in food is becoming a requirement in food industry. In this context, biosensors are presented as an alternative to traditional analytical methodologies due to their specificity, high sensitivity and ability to work in real time. It has been observed that the behavior of the analysis curves of the biosensors follow a trend that is reproducible among all the measurements and that is specific to the reaction that occurs in the electrochemical cell and the analyte being analyzed. Kinetic reaction modeling is a widely used method to model processes that occur within the sensors, and this leads to the idea that a mathematical approximation can mimic the electrochemical reaction that takes place while the analysis of the sample is ongoing. For this purpose, a novel mathematical model is proposed to approximate the enzymatic reaction within the biosensor in real time, so the output of the measurement can be estimated in advance. The proposed model is based on adjusting an exponential decay model to the response of the biosensors using a nonlinear least-square method to minimize the error. The obtained results show that our proposed approach is capable of reducing about 40% the required measurement time in the sample analysis phase, while keeping the error rate low enough to meet the accuracy standards of the food industry.

Validation of the Biofish-300 SUL Enzymatic Biosensor for the Detection of Sulfite in Crustacean.

Background: Sulfites are some of the oldest and most widespread preservatives in our food supply. They are food additives that have antioxidant properties, but they are also recorded as allergens by the main international regulatory bodies on food safety because of their adverse health effect. Hence, sulfites maximum concentration in foodstuff is regulated and they must be ensured by the agro-food processing industries. The most widely used technique for the quantification of sulfites is the Modified Monier-Williams (AOAC Official Method 990.28). Objective: In this method, SO2 is released from sulfites and some bound compounds when the sample is mixed with an acid (normally hydrochloric acid, but sometimes phosphoric acid) and heated. The SO2 is distilled using a stream of nitrogen gas, which carries the gaseous SO2 into an absorbing solution of hydrogen peroxide (H2O2) where it is oxidised to sulphuric acid. The amount of SO2 distilled into the H2O2 is determined by titration with 0.1M sodium hydroxide. Apart from being time consuming (at least 2 h) and the usage of toxic solvents, the method presents some other disadvantages that make it inappropriate as a routine-control technique for the agro-food industry. Hence, the industry demands simple, fast and accurate methods for sulfite level monitoring. Methods: BIOLAN is a SME that develops and commercializes biosensors for quantitative analysis of food quality and safety parameters, based on its proprietary enzyme-based electrochemical biosensor technology platform. This technology enables high accurate and robust analysis with a compact device that help the users to control the quality in an easy and safety manner. Biofish-300 SUL method is a highly specific enzimatic biosensor for the rapid quantification of sulfite, measured as SO2 content, in crustaceans. It consists on the extraction of sulfite in an aqueous based solution, by the aid of an Ultra-turrax or similar, and its subsequent quantification by the biosensor after previous calibration (3 min). Results: Sulfite in raw shrimp head-on, raw shrimp head-off, and boiled shrimp was analyzed, and performance was examined using naturally contaminated and spiked samples by comparisons with AOAC Official Methods of AnalysisSM (OMA) 990.28. Linearity, selectivity, matrix, consistency, and robustness were evaluated. All results were within acceptable ranges except robustness, which reflected deviation in the sample volume and ultraturrax time compared with the standard assay procedures described in the Biofish 300 SUL Instruction Manual. Accuracy, assessed as a comparison of the Biofish results with the OMA results, ranged from 82 to 115% in all samples except for fortified raw shrimp head-on, in which the low level yielded an accuracy of 138%. The method bias was in general negative in both incurred and fortified high levels, and slightly positive in incurred low levels. Repeatability was very good as shown by the low RSDr values, demonstrating acceptable repeatability precision with results <10% in most of the evaluated values. Regression analyses showed a good correlation between the Biofish and OMA methods with R² = 0.99 in all cases. Conclusions: As a whole, accuracy, recovery and bias within range results indicate that the kit provides accurate and precise sulfite quantification for all the evaluated matrices, confirming that sample preparation and assay procedures produce acceptable results. Biofish 300 SUL has proved to be a suitable tool for monitoring sulfite levels in quality control routines due to its high accuracy, precision, rapid response and ease of use. Highlights: With a simple sample preparation, results are obtained in approximately 3 min, making a big difference with other technologies that require specific skills or tedious sample pretreatments and analysis procedures.

Validation of the Biofish-300 HIS Enzymatic Biosensor for the Detection of Histamine in Fishery Products.

The Biofish-300 HIS method is a simple, reliable, and specific enzymatic biosensor for the detection of histamine. This technology is highly specific and selective and allows quantification of histamine in fishery products (fresh/frozen and processed) in a short time frame (2-3 min). Histamine in raw tuna, raw mackerel, raw sardine, raw anchovy, boiled tuna, canned tuna in water, canned tuna in oil, canned mackerel in tomato sauce, canned pickled sardine, and canned salted anchovy was analyzed using a water-based extract. Matrix-specific assay procedures and calibration curves were used to enable analyses to be carried out across multiple sample types. The performance of this assay was examined using samples that were naturally contaminated (reference materials and interlaboratory studies) and spiked with histamine. All data were judged against previously established acceptance criteria. Performance measures were evaluated for linearity, selectivity, matrix, lot consistency, and robustness. Results produced in all performance measures, except robustness, were within acceptable ranges. Out-of-range robustness results reflected deviation in sample volume compared to the standard assay procedures. Positive interferences from the presence of agmatine were shown.

Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1.

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His(6)PheA1 and His(6)PheA2 were purified and its catalytic activity characterized. His(6)PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His(6)PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His(6)PheA1 and His(6)PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.

Three-dimensional growth as multicellular spheroid activates the proangiogenic phenotype of colorectal carcinoma cells via LFA-1-dependent VEGF: implications on hepatic micrometastasis.

BACKGROUND: The recruitment of vascular stromal and endothelial cells is an early event occurring during cancer cell growth at premetastatic niches, but how the microenvironment created by the initial three-dimensional (3D) growth of cancer cells affects their angiogenesis-stimulating potential is unclear. METHODS: The proangiogenic profile of CT26 murine colorectal carcinoma cells was studied in seven-day cultured 3D-spheroids of <300 mum in diameter, produced by the hanging-drop method to mimic the microenvironment of avascular micrometastases prior to hypoxia occurrence. RESULTS: Spheroid-derived CT26 cells increased vascular endothelial growth factor (VEGF) secretion by 70%, which in turn increased the in vitro migration of primary cultured hepatic sinusoidal endothelium (HSE) cells by 2-fold. More importantly, spheroid-derived CT26 cells increased lymphocyte function associated antigen (LFA)-1-expressing cell fraction by 3-fold; and soluble intercellular adhesion molecule (ICAM)-1, given to spheroid-cultured CT26 cells, further increased VEGF secretion by 90%, via cyclooxygenase (COX)-2-dependent mechanism. Consistent with these findings, CT26 cancer cells significantly increased LFA-1 expression in non-hypoxic avascular micrometastases at their earliest inception within hepatic lobules in vivo; and angiogenesis also markedly increased in both subcutaneous tumors and hepatic metastases produced by spheroid-derived CT26 cells. CONCLUSION: 3D-growth per se enriched the proangiogenic phenotype of cancer cells growing as multicellular spheroids or as subclinical hepatic micrometastases. The contribution of integrin LFA-1 to VEGF secretion via COX-2 was a micro environmental-related mechanism leading to the pro-angiogenic activation of soluble ICAM-1-activated colorectal carcinoma cells. This mechanism may represent a new target for specific therapeutic strategies designed to block colorectal cancer cell growth at a subclinical micrometastatic stage within the liver.

Purification, characterization and cloning of aldehyde dehydrogenase from Rhodococcus erythropolis UPV-1.

The enzyme responsible for formaldehyde removal in industrial wastewaters by cells of Rhodococcus erythropolis UPV-1 was identified as a broad-specific aldehyde dehydrogenase (EC 1.2.1.3). The enzyme was purified to electrophoretic homogeneity from ethanol-grown cells with a specific activity of 19.5 U mg-1 protein and an activity recovery of 56%. The enzyme showed an isoelectric point (pI) of 5.3 and was a trimer of 162 kDa consisting of three identical 54-kDa subunits. It was specific for NAD+ and showed hyperbolic kinetics for this coenzyme (Km=90 microM), but sigmoidal kinetics for the aliphatic aldehydes used as substrates. The enzyme affinity for aldehydes increased with their hydrocarbon chain length, ranging from 333 microM for formaldehyde to 85 nM for n-octanal. The corresponding calculated Hill coefficients were in the 1.55-2.77 range. With n-propanal as substrate, the optimum pH and temperature for activity were 9.5-10.0 and 47.5 degrees C, respectively, with an Ea for catalysis of 28.6 kJ mol-1. NAD+ protected the enzyme against thermal inactivation, but aldehydes were ineffective. The activity was severely inhibited by p-hydroxymercuribenzoate, indicating that a thiol was essential for catalysis. The 1,524-bp aldhR gene encoding a 507-amino-acid protein was expressed in cells of Escherichia coli M15 as a hexahistidine-tagged protein.