A green-footprint approach for parallel multiclass analysis of contaminants in roasted coffee via LC-HRMS.

The development and validation of a simple, comprehensive, and environment-friendly procedure to determine pesticide residues, naturally occurring and processing contaminants in roasted coffee is presented. A solid-liquid extraction of pesticides and mycotoxins with ethyl acetate and the concurrent partition of acrylamide to an aqueous phase follows a parallel analytical strategy that requires a single analytical portion to determine contaminants that are typically analyzed by dedicated single residue methods. The partition rules the lipids out of the aqueous extract before an "in-tube" dispersive solid phase microextraction (dSPME) for acrylamide retention. This is followed by the elution with buffer prior to injection. This extract is independently introduced into the system front end followed by the injection of the compounds from the organic phase, yet all spotted in the same run. A novel liquid chromatography high-resolution mass spectrometry (LC-HRMS) method setup enables the quantification of 186 compounds at 10 µg/kg, 226 at 5 µg/kg, and the acrylamide at 200 µg/kg for a total of 414 molecules, with acceptable recoveries (70-120%) and precision (RSD < 20%) making this strategy significantly faster and cost-effective than the dedicated single residue methods. Even though the presence of chlorpyrifos, acrylamide, and ochratoxin A was confirmed on samples of different origins, the findings were below the limit of quantification. During the storage of raw coffee, no proof of masking of OTA was found; however, condensation with glucose was evidenced during thermal processing experiments with sucrose by using stable isotope labeling (SIL). No detected conjugates were found in roasted nor in commercial sugar-added torrefacto samples, an industrial processing usually carried out above the decomposition temperature of the disaccharide.

Advancements in Chemical and Biosensors for Point-of-Care Detection of Acrylamide.

Acrylamide (AA), an odorless and colorless organic small-molecule compound found generally in thermally processed foods, possesses potential carcinogenic, neurotoxic, reproductive, and developmental toxicity. Compared with conventional methods for AA detection, bio/chemical sensors have attracted much interest in recent years owing to their reliability, sensitivity, selectivity, convenience, and low cost. This paper provides a comprehensive review of bio/chemical sensors utilized for the detection of AA over the past decade. Specifically, the content is concluded and systematically organized from the perspective of the sensing mechanism, state of selectivity, linear range, detection limits, and robustness. Subsequently, an analysis of the strengths and limitations of diverse analytical technologies ensues, contributing to a thorough discussion about the potential developments in point-of-care (POC) for AA detection in thermally processed foods at the conclusion of this review.

A Mass Spectrometry Strategy for Protein Quantification Based on the Differential Alkylation of Cysteines Using Iodoacetamide and Acrylamide.

Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.

Adding pulse flours to cereal-based snacks and bakery products: An overview of free asparagine quantification methods and mitigation strategies of acrylamide formation in foods.

Thermal processing techniques can lead to the formation of heat-induced toxic substances. Acrylamide is one contaminant that has received much scientific attention in recent years, and it is formed essentially during the Maillard reaction when foods rich in carbohydrates, particularly reducing sugars (glucose, fructose), and certain free amino acids, especially asparagine (ASN), are processed at high temperatures (>120°C). The highly variable free ASN concentration in raw materials makes it challenging for food businesses to keep acrylamide content below the European Commission benchmark levels, while avoiding flavor, color, and texture impacts on their products. Free ASN concentrations in crops are affected by environment, genotype, and soil fertilization, which can also influence protein content and amino acid composition. This review aims to provide an overview of free ASN and acrylamide quantification methods and mitigation strategies for acrylamide formation in foods, focusing on adding pulse flours to cereal-based snacks and bakery products. Overall, this review emphasizes the importance of these mitigation strategies in minimizing acrylamide formation in plant-based products and ensuring safer and healthier food options.

Evaluation of the efficiency of thermostable L-asparaginase from B. licheniformis UDS-5 for acrylamide mitigation during preparation of French fries.

A thermostable L-asparaginase was produced from Bacillus licheniformis UDS-5 (GenBank accession number, OP117154). The production conditions were optimized by the Plackett Burman method, followed by the Box Behnken method, where the enzyme production was enhanced up to fourfold. It secreted L-asparaginase optimally in the medium, pH 7, containing 0.5% (w/v) peptone, 1% (w/v) sodium chloride, 0.15% (w/v) beef extract, 0.15% (w/v) yeast extract, 3% (w/v) L-asparagine at 50 °C for 96 h. The enzyme, with a molecular weight of 85 kDa, was purified by ion exchange chromatography and size exclusion chromatography with better purification fold and percent yield. It displayed optimal catalysis at 70 °C in 20 mM Tris-Cl buffer, pH 8. The purified enzyme also exhibited significant salt tolerance too, making it a suitable candidate for the food application. The L-asparaginase was employed at different doses to evaluate its ability to mitigate acrylamide, while preparing French fries without any prior treatment. The salient attributes of B. licheniformis UDS-5 L-asparaginase, such as greater thermal stability, salt stability and acrylamide reduction in starchy foods, highlights its possible application in the food industry.

Efficient optimization and development of two methods for the determination of acrylamide in deep-frying oil by liquid chromatography-tandem mass spectrometry: Application of multifactor analysis assessment strategy.

A new multifactor analysis assessment strategy was developed for evaluating, optimizing, and comparing analytical techniques for acrylamide in frying oils. Based on five indices (absolute recovery, absolute matrix effect, the intensity of the full ion scan, and the precursor ion scan to m/z 184 and m/z 241), the proposed strategy was performed with radar analysis, relative contribution analysis, and the entropy-weighted technique for order performance by similarity to ideal solution analysis. Two novel methods based on quick, easy, cheap, effective, rugged, and safe extraction methodology and gel permeation chromatography-liquid-liquid extraction followed by liquid chromatography-tandem mass spectrometry have been developed for the analysis of acrylamide in frying oils. Two methods were suitable for rapid and sensitive analysis of acrylamide in oils in different laboratories, with a limit of quantitation at 2 µg/kg, and the average recovery ranging from 92.5% to 107.8%, with relative standard deviations below 10%. When considering automation efficiency and matrix effects, gel permeation chromatography is the most efficient method, whereas the other method has an advantage when analyzing large samples. The developed methods were used in a pilot study to analyze frying oils with acrylamide content below 9.82 µg/kg, showing that the repeated frying process did not produce significant content of acrylamide in oils.

Biochemical Characterization of Thermostable Acrylamide Amidohydrolase from Aspergillus fumigatus with Potential Activity for Acrylamide Degradation in Various Food Products.

Acrylamide is the major by-product of the Maillard reactions in foods with the overheating processes of L-asparagine-rich foods with reducing sugars that usually allied with neurotoxicity and carcinogenicity. Several approaches have been used to prevent the formation of acrylamide, however, degrading the already formed acrylamide in foods remains unequivocal. Acrylamide hydrolyzing enzyme "amidohydrolase" is one of the most promising enzymes for acrylamide degradation in foods. So, amidohydrolase "amidase" from thermotolerant Aspergillus fumigatus EFBL was purified to their electrophoretic homogeneity by gel-filtration and ion-exchange chromatography, with overall purification folds 2.8 and yield 9.43%. The apparent molecular subunit structure of the purified A. fumigatus amidase was 50 kDa, with highest activity at reaction temperature of 40 °C and pH of 7.5 The enzyme displayed a significant thermal stability as revealed from the value of T1/2 (13.37 h), and thermal denaturation rate (Kr 0.832 × 10-3 min) at 50 °C, with metalloproteinic identity. The purified enzyme had a significant activity for acrylamide degradation in various food products such as meat, cookies, potato chips, and bread as revealed from the HPLC analysis and LC-MS analysis. So, with the purified amidase, the acrylamide in the food products was degraded by about 95% to acrylic acid, ensuring the possibility of using this enzyme in abolishing the toxic acrylamide in the foods products. This is the first report exploring the potency of A. fumigatus amidase for an actual degradation of acrylamide in foods efficiently. Further biochemical analyses are ongoing to assess the affinity of this enzyme for selective hydrolyses of acrylamide in foods, without affecting the beneficial stereochemical related compounds.

Production and Inhibition of Acrylamide during Coffee Processing: A Literature Review.

Coffee is the third-largest beverage with wide-scale production. It is consumed by a large number of people worldwide. However, acrylamide (AA) is produced during coffee processing, which seriously affects its quality and safety. Coffee beans are rich in asparagine and carbohydrates, which are precursors of the Maillard reaction and AA. AA produced during coffee processing increases the risk of damage to the nervous system, immune system, and genetic makeup of humans. Here, we briefly introduce the formation and harmful effects of AA during coffee processing, with a focus on the research progress of technologies to control or reduce AA generation at different processing stages. Our study aims to provide different strategies for inhibiting AA formation during coffee processing and investigate related inhibition mechanisms.

Screening of Acrylamide of Par-Fried Frozen French Fries Using Portable FT-IR Spectroscopy.

Current assays for acrylamide screening rely heavily on LC-MS/MS or GC-MS, techniques that are not suitable to support point of manufacturing verification because it can take several weeks to receive results from a laboratory. A portable sensor that can detect acrylamide levels in real-time would enable in-house testing to safeguard both the safety of the consumer and the economic security of the agricultural supplier. Our objective was to develop a rapid, accurate, and real-time screening technique to detect the acrylamide content in par-fried frozen French fries based on a portable infrared device. Par-fried French fries (n = 70) were manufactured at times ranging from 1 to 5.5 min at 180 °C to yield a wide range of acrylamide levels. Spectra of samples were collected using a portable FT-IR device operating from 4000 to 700 cm-1. Acrylamide was extracted using QuEChERS and quantified using uHPLC-MS/MS. Predictive algorithms were generated using partial least squares regression (PLSR). Acrylamide levels in French fries ranged from 52.0 to 812.8 µg/kg. The best performance of the prediction algorithms required transformation of the acrylamide levels using a logarithm function with models giving a coefficient of correlation (Rcv) of 0.93 and RPD as 3.8, which means the mid-IR model can be used for process control applications. Our data corroborate the potential of portable infrared devices for acrylamide screening of high-risk foods.

Chemical Contamination in Bread from Food Processing and Its Environmental Origin.

Acrylamide (AA), furan and furan derivatives, polycyclic aromatic amines (PAHs), monochloropropanediols (MCPDs), glycidol, and their esters are carcinogens that are being formed in starchy and high-protein foodstuffs, including bread, through baking, roasting, steaming, and frying due to the Maillard reaction. The Maillard reaction mechanism has also been described as the source of food processing contaminants. The above-mentioned carcinogens, especially AA and furan compounds, are crucial substances responsible for the aroma of bread. The other groups of bread contaminants are mycotoxins (MTs), toxic metals (TMs), and pesticides. All these contaminants can be differentiated depending on many factors such as source, the concentration of toxicant in the different wheat types, formation mechanism, metabolism in the human body, and hazardous exposure effects to humans. The following paper characterizes the most often occurring contaminants in the bread from each group. The human exposure to bread contaminants and their safe ranges, along with the International Agency for Research on Cancer (IARC) classification (if available), also have been analyzed.

Antioxidative Properties and Acrylamide Content of Functional Wheat-Flour Cookies Enriched with Wild-Grown Fruits.

This study aimed to evaluate the impact of widely grown fruits (wild roses, elderberries, sea buckthorns, rowans, chokeberries, and hawthorns) as a functional ingredient in wheat-flour cookie formulation on antioxidative properties with a simultaneous reduction of the carcinogen-like compound acrylamide. The organoleptic features of the cookies were assessed by a panel of consumers. The following parameters were measured: chemical composition, total polyphenols, polyphenolic profile, antioxidant activity, and acrylamide content. The overall ratings of the tested cookies with the addition of chokeberries, hawthorns, sea buckthorns, and elderberries were more than satisfactory, while wild rose and rowan cookies were the most widely accepted and best rated by the panelists. The antioxidant activity of the tested cookies was 1.1−15.22 µmol trolox·g−1 dm and 2.46−26.12 µmol Fe (II)·g−1 dm as measured by the ABTS and FRAP methods, respectively. All the fruit-enriched cookies had significantly higher antioxidative properties (p < 0.05) in comparison to the control cookies, but among the fruit-enriched cookies, there were differences in the quality and quantity of particular polyphenols. The acrylamide content was significantly decreased by 59% (hawthorn), 71% (rowan), 87% (wild rose), 89% (sea buckthorn), 91% (elderberry), and 94% (chokeberry) compared with the control cookies (p < 0.05). Cookies enriched with wild-grown fruits could constitute a promising novel snack food.

Relevance of dietary exposure to acrylamide formed in heat-processed agri-food products.

OBJECTIVES: Acrylamide (AA) is considered one of the contaminants that occur in heat-processed agri-food products, which through diet can increase the risk of developing cancer for consumers of all age groups. METHODS: This review analysed the level of acrylamide of the most important heat-processed agri-food products that contribute to the dietary exposure of the population of different European countries and the assessment of health risks related to the presence of AA in food. RESULTS: The results of monitoring AA concentrations in agri-food products, reported individually by researchers or projects such as CONTAM in 2015 and the UK Food Standard Agency in 2017, show that some products exceeding the recently set European reference level are reported as such for specific values - mean UB/RLs in µg.kg-1: French fries (550/500), coffee dry (523/400), coffee substitutes (1,499/500, 400), processed cereal-based baby foods (76/40), potato crisps and snacks (2,214/750), breakfast cereals (744/300), biscuits and crackers (637/350, 400), and coffee substitutes (1,897/500). Average values (µg/kg body weight per day) of exposure to AA from food for different age groups (EFSA) are estimated at 0.4-1.9, but in different European countries, as reported by several studies (including Romania), are between 1.4 and 3.4. CONCLUSION: Starting from the genotoxic and carcinogenic potential of acrylamide, it is important to regularly monitor the presence of acrylamide and its levels in food and to investigate the food pattern of the population to detect the share of foods at risk of exposure.

Acrylamide in human breast milk - the current state of knowledge.

Human milk is a first choice in infant nutrition. It not only provides all the nutrients necessary for the proper infant`s development but also contains bioactive factors that provide natural protection against infections. Unfortunately, chemical contaminants can pass to breast milk and pose a health risk for the breastfed infant's health. Acrylamide is a typical process contaminant and in food it is formed as a result of the Maillard reaction. Numerous studies have shown that acrylamide is a neurotoxic and carcinogenic compound. So far there have been published only three studies on the acrylamide content in human milk. In two of them, the acrylamide level in most of the tested samples did not exceed the value of 0.5 µg/L. In the third study, the authors assessed the circulation of acrylamide in the body of two breastfeeding women after consuming products with high acrylamide content. Depending on the time elapsed after the meal, the acrylamide content ranged from 3.17 µg/L to 18.8 µg /L. These studies show that the breastfeeding mothers' diet may have a significant influence on the level of acrylamide in their milk. However, it seems that the acrylamide content in breast milk is also influenced by the time of breast milk collection, including the time elapsed after the mother's meal. To assess the exposure of breastfed infants to acrylamide in human milk, more data is needed on the acrylamide content in human milk at different stages of lactation and using standard protocols for human milk sampling.

Characterization of Penicillium crustosum L-asparaginase and its acrylamide alleviation efficiency in roasted coffee beans at non-cytotoxic levels.

This work aims at isolating a fungal source for L-asparaginase production to be applied in reducing acrylamide levels in coffee beans at non-cytotoxic levels. An L-asparaginase-producing fungus was isolated from an agricultural soil sample and identified as Penicillium crustosum NMKA 511. A maximum L-asparaginase activity of 19.10 U/mL was obtained by the above-mentioned fungus when grown under optimum conditions (i.e. 16.96 g/L sucrose as carbon source, 1.92 g/L peptone as nitrogen source, pH 7.7 and 33.5 °C). Further, the produced L-asparaginase was purified and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that P. crustosum L-asparaginase was a heterodimer enzyme with molecular weights of approximately 41.3 and 44.4 kDa. Also, the purified P. crustosum L-asparaginase was specific towards L-asparagine and showed negligible and no effects towards L-glutamine and D-asparagine, respectively. Additionally, the purified L-asparaginase reduced the acrylamide levels by 80.7% and 75.8% in light and dark roasted coffee beans, respectively. The amount of L-asparaginase used to reduce acrylamide was considered safe when cell viability reached 94.6%.

An eco-friendly solvent-free reaction based on peptide probes: design an extraction-free method for analysis of acrylamide under microliter volume.

Acrylamide is a group 2A carcinogen and potential endocrine disruptor that can enter the ecosystem by various routes and has recently become a dangerous pollutant. This widely used chemical can enter the human body via air inhalation, food or water consumption, or skin contact. In this study, we developed a peptide probe for the detection of acrylamide by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) after its micro-tagging with a peptide. Direct detection of acrylamide by MALDI-TOF MS is not feasible due to its poor ionization in the MALDI interface, which hinders its analysis by the technique. After microwave irradiation for 2 min, the formed acrylamide-peptide derivative was detected easily by MALDI-TOF MS without the need for extraction procedures. The procedure does not involve organic solvents and a water-soluble peptide that allows detection of acrylamide in small sample volumes with a limit of detection (LOD) of 0.05 ng/µL. The relative standard deviation (RSD) and relative error (RE) of the measurements were < 6.7% for intra- and inter-day assays. Gel-washing solutions from a polyacrylamide gel experiment were used as a model to study the efficiency of the developed method. Finally, we used the proposed method for the detection of free acrylamide in small volumes of lung epithelial cells (a model to test the air inhalation of acrylamide under a tiny volume of sample) and human urine. The developed method will enable rapid acrylamide detection in environmental and biological samples via a green approach based on microwave-assisted derivatization in water alongside the use of a less toxic derivatization reagent, reusable target plate, and miniaturization protocols.

Acrylamide in coffee: formation and possible mitigation strategies - a review.

It is widely known that acrylamide, present in some different heat-treated foods, is an important toxic compound to humans. Coffee beverage is one of the most important sources of acrylamide, because the raw bean contains the reaction substrates and it is processed at very high temperature during roasting. Due to its high consumption all over the world, it is necessary to find applicable solutions to decrease the concentration of this undesired Maillard reaction product.The present review summarizes the advance made in understanding the acrylamide formation and describes the potential acrylamide reduction strategies along all coffee production steps, from raw material to coffee brew preparation with a dominant focus on roasting stage.Currently, it is quite established that the selection of the highest quality Arabica green coffee variety, high roasting thermal input and shortest brewing techniques lead to low final acrylamide levels. There are also few innovative interventions proposed for acrylamide control in coffee such as enzymatic treatments of raw material, vacuum or steam roasting, roasted beans supercritical fluid extraction, final beverage treatments like yeast fermentation and amino acids/additive additions. However, for these strategies the impact on the desired sensorial and nutritional coffee brew properties must be evaluated and some proposed procedures are still difficult to be applied at real industrial scale. Furthermore, in-depth studies are needed in order to find appropriate and practical solutions for acrylamide mitigation in coffee with a holistic risk/benefit approach.

Development of certified reference materials for the determination of cadmium and acrylamide in cocoa.

Since 1 January 2019 a maximum content of 0.6 mg kg-1 cadmium (Cd) in cocoa powder sold to the final consumer or as an ingredient in sweetened cocoa powder sold to the final consumer (drinking chocolate) is set by the Commission Regulation (EU) No. 488/2014. Monitoring compliance with the specified limit value requires analytical measuring methods and reference materials for quality control. However, suitable certified reference materials intended for quality assurance and quality control purposes are still lacking. Therefore, three cocoa reference materials (ERM®-BD513, ERM®-514 and ERM®-515) were developed according to the requirements of ISO 17034 and the recommendations of ISO Guide 35. The whole process of reference material development, including material preparation, assessment of homogeneity and stability, characterisation and value assignment is presented. The assignment of the certified mass fractions was based upon an interlaboratory comparison study involving 19 expert laboratories for Cd and 12 laboratories for acrylamide. The certified mass fractions and expanded uncertainties (k = 2) of the reference materials were (0.181 ± 0.009) mg kg-1 Cd (ERM®-BD513), (0.541 ± 0.024) mg kg-1 Cd (ERM®-BD514) and (0.690 ± 0.029) mg kg-1 Cd (ERM®-BD515). Acrylamide contents are given for information.

A Review on Acrylamide in Food: Occurrence, Toxicity, and Mitigation Strategies.

Acrylamide (AA) is a food contaminant present in a wide range of frequently consumed foods, which makes human exposure to this toxicant unfortunately unavoidable. However, efforts to reduce the formation of AA in food have resulted in some success. This review aims to summarize the occurrence of AA and the potential mitigation strategies of its formation in foods. Formation of AA in foods is mainly linked to Maillard reaction, which is the first feasible route that can be manipulated to reduce AA formation. Furthermore, manipulating processing conditions such as time and temperature of the heating process, and including certain preheating treatments such as soaking and blanching, can further reduce AA formation. Due to the high exposure to AA, recognition of its toxic effect is necessary, especially in developing countries where awareness about AA health risks is still very low. Therefore, this review also focuses on the different toxic effects of AA exposure, including neurotoxicity, genotoxicity, carcinogenicity, reproductive toxicity, hepatotoxicity, and immunotoxicity.

Estimation of acrylamide exposure in Italian schoolchildren consuming a canteen menu: health concern in three age groups.

Acrylamide (AA) is a food process contaminant with carcinogenic and genotoxic properties that is formed in thermally treated foods, especially carbohydrate-rich. Dietary exposure to AA, due to school canteen foods, was estimated in schoolchildren aged 3-13 years by combining the AA concentration in foods with the amount and frequency of food consumption. Potato products and bakery products presented the highest mean levels of AA (841 and 244 µg/kg, respectively) followed by meat (222 µg/kg) and egg products (151 µg/kg). The mean total AA intake was estimated at 2.16 µg/kg bw/d, with the highest percentages provided by potato products (34.5%), meat products (26.1%) and bread (24.5%). The calculated margins of exposure (MOEs) for the average AA exposure (84 and 212 for the benchmark dose lower confidence limits (BMDL) of 0.17 and 0.43 mg/kg bw/d) suggest that there is a health concern with respect to students eating in school canteens.

Occurrence of Acrylamide in Italian Baked Products and Dietary Exposure Assessment.

Acrylamide (AA) is a neo-formed toxic compound that develops in foods during cooking at temperatures above 120 °C. AA shows in vivo neurotoxic and carcinogenic effects, and it is potentially carcinogenic for humans. Its occurrence is common in baked food, such as bread and similar products. This study set out to analyze bread and sweets from the Italian market to evaluate the effects of the benchmark thresholds set by EU Regulation 2017/2158 and to ascertain the exposure of the Italian population to AA, across three age groups, through the consumption of baked products, according to the margin of exposure (MOE) approach. Two hundred samples were tested, and the content of AA ranged from 31 to 454 µg/kg for bread and products thereof and from 204 to 400 µg/kg for the sweets category. The exposure data did not show any neurotoxic health concern, whereas the MOE related to the carcinogenic endpoint is well below the minimum safety value of 10,000.