A machine learning-based electronic nose system using numerous low-cost gas sensors for real-time alcoholic beverage classification.

This study introduces numerous low-cost gas sensors and a real-time alcoholic beverage classification system based on machine learning. Dogs possess a superior sense of smell compared to humans due to having 30 times more olfactory receptors and three times more olfactory receptor types than humans. Thus, in odor classification, the number of olfactory receptors is a more influential factor than the number of receptor types. From this perspective, this study proposes a system that utilizes distinctive data patterns resulting from heterogeneous responses among numerous low-cost homogeneous MOS-based sensors with poor gas selectivity. To evaluate the performance of the proposed system, learning data were gathered using three alcoholic beverage groups including different aged whiskeys, Korean soju with 99% same compositions, and white wines made from the Sauvignon blanc variety, sourced from various countries. The electronic nose system was developed to classify alcoholic samples measured using 30 gas sensors in real time. The samples were injected into a gas chamber for 60 seconds, followed by a 60-second injection of clean air. After preprocessing the time-series data into four distinct datasets, the data were analyzed using a machine learning algorithm, and the classification results were compared. The results showed a high classification accuracy of over 99%, and it was observed that classification performance varied depending on data preprocessing. As the number of gas sensors increased, the prediction accuracy improved, reaching up to 99.83 ± 0.21%. These experimental results indicated that the proposed electronic nose system's classification performance was comparable to that of commercial electronic nose systems. Additionally, the implementation of an alcoholic beverage classification system based on a pretrained LDA model demonstrated the feasibility of real-time classification using the proposed system.

Effects of Active Ingredients in Alcoholic Beverages and Their De-Alcoholized Counterparts on High-Fat Diet Bees: A Comparative Study.

The mechanisms by which alcohol, alcoholic beverages, and their de-alcoholized derivatives affect animal physiology, metabolism, and gut microbiota have not yet been clarified. The polyphenol, monosaccharide, amino acid, and organic acid contents of four common alcoholic beverages (Chinese Baijiu, beer, Chinese Huangjiu, and wine) and their de-alcoholized counterparts were analyzed. The research further explored how these alcoholic beverages and their non-alcoholic versions affect obesity and gut microbiota, using a high-fat diet bee model created with 2% palm oil (PO). The results showed that wine, possessing the highest polyphenol content, and its de-alcoholized form, particularly when diluted five-fold (WDX5), markedly improved the health markers of PO-fed bees, including weight, triglycerides, and total cholesterol levels in blood lymphocytes. WDX5 treatment notably increased the presence of beneficial microbes such as Bartonella, Gilliamella, and Bifidobacterium, while decreasing Bombilactobacillus abundance. Moreover, WDX5 was found to closely resemble sucrose water (SUC) in terms of gut microbial function, significantly boosting short-chain fatty acids, lipopolysaccharide metabolism, and associated enzymatic pathways, thereby favorably affecting metabolic regulation and gut microbiota stability in bees.

Effect of S-adenosyl-methionine accumulation on hineka odor in sake brewed with a non-Kyokai yeast.

Hineka is a type of off-flavor of sake and is attributed to the presence of several compounds, including a major one called dimethyl trisulfide (DMTS). The production of the main precursor of DMTS involves yeast methionine salvage pathway. The DMTS-producing potential (DMTS-pp) of sake brewed using the Km67 strain, a non-Kyokai sake yeast, is lower than that of sake brewed using Kyokai yeast; however, the detailed mechanism is unclear. We focused on S-adenosyl-methionine (SAM) and aimed to elucidate the mechanism that prevents DMTS production in sake brewed using the Km67 strain. We revealed that SAM is involved in DMTS production in sake, and that the conversion of SAM to the DMTS precursor occurs through an enzymatic reaction rather than a chemical reaction. Based on previous reports on ADO1 and MDE1 genes, sake brewing tests were performed using the Km67 Δmde1, Δado1, and Δmde1Δado1 strains. A comparison of the SAM content of pressed sake cakes and DMTS-pp of sake produced using the Km67 Δado1 strain showed an increase in both SAM content and DMTS-pp compared to those produced using the parent strain. However, the Km67 Δmde1Δado1 strain showed little increase in DMTS-pp compared to the Km67 Δmde1 strain, despite an increase in SAM content. These results suggest that SAM accumulation in yeast plays a role in the production of DMTS in sake through the methionine salvage pathway. Moreover, the low SAM-accumulation characteristic of the Km67 strain contributes to low DMTS production in sake.

Distribution and Sensory Impact of (2E,4E,6Z)-nonatrienal and Trans-4,5-epoxy-(E)-2-decenal in Wines and Spirits.

This study reports the distribution of (2E,4E,6Z)-nonatrienal (1) and trans-4,5-epoxy-(E)-2-decenal (2) in wines and spirits. We validated a quantification method using solid-phase extraction (SPE) and negative chemical ionization (NCI, NH3) gas chromatography-mass spectrometry (GC-MS) analysis. Both were identified for the first time in wines and spirits from different grape varieties and raw materials. Their olfactory detection thresholds (ODTs) were 16 and 60 ng/L, respectively. Analysis of 66 wines showed that the highest levels of (1) (441.3 ng/L) and (2) (386.5 ng/L) were found in red and white wines, respectively. At these levels, they modify the balance of the fruity expression of red (fresh to cooked fruits) and white (vegetal/green hazelnut nuance) wines. Similar quantitative and sensory analyses were conducted in spirits. With ODT estimated at 500 and 400 ng/L and concentrations ranging from trace amounts to 1.1 and 2.4 µg/L respectively, (1) and (2) can contribute directly to the aroma of spirits.

A State-of-the-Science Review of Alcoholic Beverages and Polycyclic Aromatic Hydrocarbons.

BACKGROUND: The association between alcohol and certain cancers is well established, yet beyond ethanol and its metabolite acetaldehyde, little is known about the presence of other carcinogenic compounds in alcoholic beverages, including polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (a Group I carcinogen). OBJECTIVES: We summarized the published literature on PAH levels in alcoholic beverages to identify potential gaps in knowledge to inform future research. METHODS: Medline and Scopus were searched for primary research published from January 1966 to November 2023 that quantified PAH levels among various types of alcoholic beverages, including whisky, rum, brandy, gin, vodka, wine, and beer. Studies that were not primary literature were excluded; only studies that quantified PAH content in the specified alcoholic beverages were included. RESULTS: Ten studies published from 1966 to 2019 met the criteria for review. Other than beverage type, no publication reported selection criteria for their samples of tested alcohol products. Studies used a variety of analytical methods to detect PAHs. Of the 10 studies, 7 were published after 2000, and 6 assessed <20 products. Of the studies, 7 examined spirits; 3, beer; and 4, wines. Benzo[a]pyrene was most prevalent among spirit products, particularly whisky, with values generally exceeding acceptable levels for drinking water. Some beer and wine products also contained PAHs, albeit at lower levels and less frequently than spirit products. DISCUSSION: PAHs are found in some alcohol products and appear to vary by beverage type. However, there is an incomplete understanding of their presence and levels among large, representative samples from the range of currently available alcohol products. Addressing this gap could improve understanding of alcohol-cancer relationships and may have important implications for public health and the regulation of alcohol products. In addition, novel methods, such as direct mass spectroscopy, may facilitate more thorough testing of samples to further investigate this relationship. https://doi.org/10.1289/EHP13506.

Comparison of two data processing approaches for aroma marker identification in different distilled liquors using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry dataset.

Whisky, brandy, rum, and Chinese baijiu are popular distilled spirits globally, and the volatile components play an essential role in the aroma of these distilled liquors. Volatile compounds in whisky, brandy, rum, and three main aroma types of Chinese baijiu (strong, light, and sauce) were investigated using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS). Two significant variable detection approaches, the variable importance in the projection (VIP) and nonparametric test (Mann-Whitney U test), were compared to determine the volatile markers among these samples. It was found that the VIP model was more efficient in screening significant variants than the U test. A total of 117 common markers were selected by both the VIP and U test methods with potential aroma contributions. Esters and acids were the main aroma markers for baijiu, diethyl esters were the main aroma markers for brandy, whereras pyrazines, lactones, and furans were the main aroma markers for whisky. Based on the chosen markers, different unknown distilled liquors were successfully classified in the model validation. This study provided a feasible methodology for spirit sample speculation based on volatile composition obtained by GC×GC-TOFMS.

Correlation of the presence of acrolein with higher alcohols, glycerol, and acidity in cachaças.

Acrolein is a toxic aldehyde that can be present in various beverages, such as cachaça and other distilled spirits from sugarcane. The objective of this work was to detect and quantify acrolein in samples of cachaça produced by different processes in all regions of Brazil and to evaluate the possible routes of formation of this contaminant from the correlation with other secondary compounds present in the beverage using principal component analysis. Approximately 27.0% of the samples analyzed were outside the limit established by Brazilian legislation for this contaminant, with an average acrolein concentration of 14.01 mg 100 mL-1 anhydrous alcohol (aa). In the other samples, the average concentration was 0.97 mg 100 mL-1 aa. After selecting the variables that most closely correlated with the presence of acrolein in beverages, a positive correlation was found with the presence of butan-2-ol, propan-1-ol and volatile acids, and a slight correlation with the presence of phenolic compounds. Therefore, the presence of acrolein in cachaça can be associated with contamination of the fermentation must by bacteria of the genus Lactobacillus, as a result of the chemical degradation and enzymatic conversion of the glycerol produced during fermentation.

Research progress on the application of different controlling strategies to minimizing ethyl carbamate in grape wine.

Ethyl carbamate (EC) is a probable carcinogenic compound commonly found in fermented foods and alcoholic beverages and has been classified as a category 2A carcinogen by the International Agency for Research on Cancer (IARC). Alcoholic beverages are one of the main sources of EC intake by humans. Therefore, many countries have introduced a standard EC limit in alcoholic beverages. Wine is the second largest alcoholic beverage in the world after beer and is loved by consumers for its rich taste. However, different survey results showed that the detection rate of EC in wine was almost 100%, while the maximum content was as high as 100 µg/L, necessitating EC content regulation in wine. The existing methods for controlling the EC level in wine mainly include optimizing raw fermentation materials and processes, using genetically engineered strains, and enzymatic methods (urease or urethanase). This review focused on introducing and comparing the advantages, disadvantages, and applicability of methods for controlling EC, and proposes two possible new techniques, that is, changing the fermentation strain and exogenously adding phenolic compounds. In the future, it is hoped that the feasibility of this prospect will be verified by pilot-scale or large-scale application to provide new insight into the regulation of EC during wine production. The formation mechanism and influencing factors of EC in wine were also introduced and the analytical methods of EC were summarized.

A review of the polyphenols extraction from apple pomace: novel technologies and techniques of cell disintegration.

Apple pomace, a solid waste produced during industrial processing of apple juice or cider, is a rich source of high value-added compounds such as polyphenols. This review summarizes present studies on the qualitative and quantitative methods, including Folin-Ciocalteu colorimetric, high pressure liquid chromatography (HPLC) and fluorescence spectrum, as well as enhanced extraction methods of polyphenols in apple pomace by different traditional and novel technologies, including ultrasounds (US), microwave (MW), pulsed electric fields (PEF), high voltage electrical discharges (HVED) and enzyme. The principles and characteristics of different effective enhanced extraction technologies of polyphenols in apple pomace were compared. In addition, the different cell disruption analysis methods, such as destructive detection method (electrical conductivity disintegration index, Zc), image analysis method (including scanning electron microscopy, SEM, and confocal laser scanning microscopy, CLSM), and nondestructive method (such as magnetic resonance imaging, MRI) are presented in this review. The study proved that there was a correlation between destructive detection method and image analysis method. However, each of the technologies reviewed in this study has some disadvantages to overcome, and some mechanisms need to be further substantiated. Therefore, more competitive techniques for polyphenols extraction and analysis of cell disintegration are needed to emerge in the future.

Production of caproic acid by Rummeliibacillus suwonensis 3B-1 isolated from the pit mud of strong-flavor baijiu.

Caproic acid is the precursor of ethyl caproate, the main representative flavor substance of strong-flavor baijiu (SFB). Caproic acid-producing bacteria are considered to be the most important type of acid-producing microorganisms in the pit mud of the SFB ecosystem. In this study, the Rummeliibacillus suwonensis 3B-1 with a high yield of caproic acid (4.064 g/L) was screened from SFB pit mud. The genome of the R. suwonensis 3B-1 was sequenced, the total size was found to be 4117,671 bp and a calculated GC content of 35.86%. The caproic acid biosynthesis pathway was identified and analyzed, and it showed that 3B-1 could not only use ethanol, but it could also use glucose and other carbon sources as substrates to produce caproic acid. According to the genome analysis and with an optimized medium, the optimal conditions for caproic acid production were yeast powder at 3 g/L, sodium acetate at 15 g/L, and 1% biotin at 8 mL/100 mL. The yield of caproic acid reached 4.627 g/L, an increase of 13.9%, which was higher than that of general caproic acid bacteria. This is the first report of the synthesis of caproic acid by R. suwonensis. This strain could be used to produce caproic acid, an artificial pit mud preparation, and/or an enhanced inoculum in the production of SFB.

Exploring the volatile profile of whiskey samples using solid-phase microextraction Arrow and comprehensive two-dimensional gas chromatography-mass spectrometry.

We present a novel sample preparation method for the extraction and preconcentration of volatile organic compounds from whiskey samples prior to their determination by comprehensive two-dimensional gas chromatography (GC × GC) coupled to mass spectrometry (MS). Sample preparation of the volatile compounds, important for the organoleptic characteristics of different whiskeys and their acceptance and liking by the consumers, is based on the use of the solid-phase microextraction (SPME) Arrow. After optimization, the proposed method was compared with conventional SPME regarding the analysis of different types of whiskey (i.e., Irish whiskey, single malt Scotch whiskey and blended Scotch whiskey) and was shown to exhibit an up to a factor of six higher sensitivity and better repeatability by a factor of up to five, depending on the compound class. A total of 167 volatile organic compounds, including terpenes, alcohols, esters, carboxylic acids, ketones, were tentatively-identified using the SPME Arrow technique, while a significantly lower number of compounds (126) were determined by means of conventional SPME. SPME Arrow combined with GC × GC-MS was demonstrated to be a powerful analytical tool for the exploration of the volatile profile of complex samples, allowing to identify differences in important flavour compounds for the three different types of whiskey investigated.

Exploring the response patterns of strong-flavor baijiu brewing microecosystem to fortified Daqu under different pit ages.

The fermentation of strong-flavor baijiu depends largely on the evolution of the microbial community that originated from Daqu and pit mud (PM). Applying fortified Daqu (FD) has been proven to be an effective strategy to improve the quality and yield of baijiu. However, it is unclear what the effects of FD on the liquor brewing microecosystem under different pit ages because of the temporal heterogeneity of the PM community. Taking 2-year (new) and 40-year (aged) pits as the objects, the influence of FD on the metabolic profile, physicochemical parameters, and community diversity in Zaopei and PM was investigated by polyphase detecting approaches. Present results showed that the metabolic profiles of Zaopei were significantly improved by FD, whereas those of PM were mainly dependent on pit age. Aspergillus, Caproiciproducens, and Methanosarcina were more abundant in the aged pit, while Kazachstania, Lactobacillus, and Sphingomonas dominated in the new pit, whether in Zaopei or in PM. The interaction relationships among the communities were also altered by FD, and the co-occurrence network, especially the increased links between archaea and bacteria in the new pit. Notably, this interaction in the aged pit distinctly affected the hexanoic acid content based on the Mantel test. The results of PICRUSt2 analysis inferred that FD perhaps improved the interspecies hydrogen transfer in the new pit and increased the carbon flow of hexanoic acid production during chain elongation in the aged pit. These results provide new insights into the production of high-quality strong-flavor baijiu and the aging of PM.

Fabrication of a fluorescence probe via molecularly imprinted polymers on carbazole-based covalent organic frameworks for optosensing of ethyl carbamate in fermented alcoholic beverages.

Ethyl carbamate (EC), which is a group 2A carcinogen, is a byproduct formed in the alcohol fermentation process that can accumulate with heating, transportation, and storage. In this study, molecularly imprinted polymers (MIPs) on carbazole-based covalent organic frameworks (COFs) were prepared as a fluorescence probe for the optosensing of EC in fermented alcoholic beverages. The excellent optical properties of carbazole-based COFs coupled with the good adsorption and selectivity of MIPs provided fast and efficient recognition of EC. MIPs on carbazole-based COFs exhibited advantages of high efficiency, a good separation effect, fluorescence dependence, and reproducibility. A good linear relationship was obtained over the concentration range of 1-200 µg L-1, with a low limit of detection (LOD) of 0.607 µg L-1. The RSD precision and five-cycle reproducibility were lower than 4.91% and 6.38%, respectively, and the recoveries were 85.30%-109.49%. This optosensor was applied to quantify EC contents in several fermented alcoholic beverages, all of which were less than LOD. The results of the optosensors based on MIPs on carbazole-based COFs were then validated using standard gas chromatography-mass spectrometry (GC-MS), which gave results consistent with the proposed method.

A high-throughput, cheap, and green method for determination of ethanol in cachaça and vodka using 96-well-plate images.

A high-throughput method for the determination of ethanol in vodka and cachaça using 96-well-plate digital images was proposed and validated. The standard method consists of beverage distillation, measuring its density using a pycnometer, and converting it into ethanol content. It is simple, but it is time-consuming and susceptive to error. The proposed method exploits the suppression of phenolphthalein ionization by ethanol in an alkaline solution and the fading of the pink solution was converted into ethanol content. It consists in mixing 1 mL of sample with 0.1 mL of an alkaline phenolphthalein solution. 96-well-plate images were acquired using a desktop scanner. Red, green, and blue (RGB) values from the 96 wells were automatically extracted using ReadPlate (ImageJ plugin). Then, RGB values were exported to a spreadsheet that converted these values into analytical signals and calculated the ethanol content in beverages. The ethanol content of cachaças and vodkas was 33-45% (v/v) and it was also the linear range of the proposed method. The method's precision was evaluated using relative standard deviation (RSD). Five cachaças and three vodkas were analyzed. Each beverage was analyzed six times on the same day (intra-day repeatability) and three consecutive days (inter-day repeatability) by three different analysts (inter-analyst repeatability). The intra-day repeatability average was 1.7% (1.2-2.2% range), the intra-day repeatability average was 2.6% (1.9-3.5% range), and the inter-analyst repeatability average was 4% (2.6-6.2% range). Accuracy was evaluated by comparing the proposed method with the standard method using a percent error and a paired t-test. The average percent error was 1.9%, in the paired t-test, the p-value average value was 0.25. The proposed method can analyze 12 samples in 30 min, whereas the standard method spends around 1 h on each sample. Thus, the proposed method provides high-throughput compared with the standard method.

Moderate Wine Consumption and Health: A Narrative Review.

Although it is clearly established that the abuse of alcohol is seriously harmful to health, much epidemiological and clinical evidence seem to underline the protective role of moderate quantities of alcohol and in particular of wine on health. This narrative review aims to re-evaluate the relationship between the type and dose of alcoholic drink and reduced or increased risk of various diseases, in the light of the most current scientific evidence. In particular, in vitro studies on the modulation of biochemical pathways and gene expression of wine bioactive components were evaluated. Twenty-four studies were selected after PubMed, Scopus and Google Scholar searches for the evaluation of moderate alcohol/wine consumption and health effects: eight studies concerned cardiovascular diseases, three concerned type 2 diabetes, four concerned neurodegenerative diseases, five concerned cancer and four were related to longevity. A brief discussion on viticultural and enological practices potentially affecting the content of bioactive components in wine is included. The analysis clearly indicates that wine differs from other alcoholic beverages and its moderate consumption not only does not increase the risk of chronic degenerative diseases but is also associated with health benefits particularly when included in a Mediterranean diet model. Obviously, every effort must be made to promote behavioral education to prevent abuse, especially among young people.

Comparative 13 C-metabolic flux analysis indicates elevation of ATP regeneration, carbon dioxide, and heat production in industrial Saccharomyces cerevisiae strains.

BACKGROUND: Various industrial Saccharomyces cerevisiae strains are used for specific processes, such as sake, wine brewing and bread making. Understanding mechanisms underlying the fermentation performance of these strains would be useful for further engineering of the S. cerevisiae metabolism. However, the relationship between the fermentation performance, intra-cellular metabolic states, and other phenotypic characteristics of industrial yeasts is still unclear. In this study, 13 C-metabolic flux analysis of four diploid yeast strains-laboratory, sake, bread, and wine yeasts-was conducted. RESULTS: While the Crabtree effect was observed for all strains, the metabolic flux level of glycolysis was elevated in bread and sake yeast. Furthermore, increased flux levels of the TCA cycle were commonly observed in the three industrial strains. The specific rates of CO2 production, net ATP regeneration, and metabolic heat generation estimated from the metabolic flux distribution were two to three times greater than those of the laboratory strain. The elevation in metabolic heat generation was correlated with the tolerance to low-temperature stress. CONCLUSION: These results indicate that the metabolic flux distribution of sake and bread yeast strains contributes to faster production of ethanol and CO2 . It is also suggested that the generation of metabolic heat is preferable under the actual industrial fermentation conditions.

Optimization and Validation of a Headspace Solid-Phase Microextraction with Comprehensive Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometric Detection for Quantification of Trace Aroma Compounds in Chinese Liquor (Baijiu).

The detection of trace aroma compounds in samples with complex matrices such as Chinese liquor (Baijiu) requires a combination of several methods, which makes the analysis process very complicated. Therefore, a headspace solid-phase microextraction (HS-SPME) method coupled with two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS) was developed for the quantitation of a large number of trace compounds in Baijiu. Optimization of extraction conditions via a series of experiments revealed that dilution of the alcohol content of 8 mL of Baijiu to 5%, followed by the addition of 3.0 g of NaCl and subsequent SPME extraction with DVB/CAR/PDMS fiber coating over 45 min at 45 °C was the most suitable. To check the matrix effects, various model Baijiu matrices were investigated in detail. The quantitative method was established through an optimized model synthetic solution, which can identify 119 aroma compounds (esters, alcohols, fatty acids, aldehydes and ketones, furans, pyrazines, sulfur compounds, phenols, terpenes, and lactones) in the Baijiu sample. The developed procedure provided high recovery (86.79-117.94%), good repeatability (relative standard deviation < 9.93%), high linearity (R2 > 0.99), and lower detection limits than reported methods. The method was successfully applied to study the composition of volatile compounds in different types of Baijiu. This research indicated that the optimized HS-SPME-GC×GC-TOFMS method was a valid and accurate procedure for the simultaneous determination of different types of trace compounds in Baijiu. This developed method will allow an improved analysis of other samples with complex matrices.

Margin of Exposure Analyses and Overall Toxic Effects of Alcohol with Special Consideration of Carcinogenicity.

Quantitative assessments of the health risk of the constituents of alcoholic beverages including ethanol are reported in the literature, generally with hepatotoxic effects considered as the endpoint. Risk assessment studies on minor compounds such as mycotoxins, metals, and other contaminants are also available on carcinogenicity as the endpoint. This review seeks to highlight population cancer risks due to alcohol consumption using the margin of exposure methodology. The individual and cumulative health risk contribution of each component in alcoholic beverages is highlighted. Overall, the results obtained consistently show that the ethanol contributes the bulk of harmful effects of alcoholic beverages, while all other compounds only contribute in a minor fashion (less than 1% compared to ethanol). Our data provide compelling evidence that policy should be focused on reducing total alcohol intake (recorded and unrecorded), while measures on other compounds should be only secondary to this goal.

Occurrence of Ethyl Carbamate in Foods and Beverages: Review of the Formation Mechanisms, Advances in Analytical Methods, and Mitigation Strategies.

ABSTRACT: Ethyl carbamate (EC) is a process contaminant that can be formed as a by-product during fermentation and processing of foods and beverages. Elevated EC concentrations are primarily associated with distilled spirits, but this compound has also been found at lower concentrations in foods and beverages, including breads, soy sauce, and wine. Evidence from animal studies suggests that EC is a probable human carcinogen. Consequently, several governmental institutions have established allowable limits for EC in the food supply. This review includes EC formation mechanisms, occurrence of EC in the food supply, and EC dietary exposure assessments. Current analytical methods used to detect EC will be covered, in addition to emerging technologies, such as nanosensors and surface-enhanced Raman spectroscopy. Various mitigation methods have been used to maintain EC concentrations below allowable limits, including distillation, enzymatic treatments, and genetic engineering of yeast. More research in this field is needed to refine mitigation strategies and develop methods to rapidly detect EC in the food supply.