Sensitive detection of viable Cronobacter sakazakii by bioluminescent reporter phage emitting stable signals with truncated holin.

As outbreaks of foodborne illness caused by the opportunistic pathogen Cronobacter sakazakii (Cs) continue to occur, particularly in infants consuming powdered infant formula (PIF), the need for sensitive, rapid, and easy-to-use detection of Cs from food and food processing environments is increasing. Here, we developed bioluminescent reporter bacteriophages for viable Cs-specific, substrate-free, rapid detection by introducing luciferase and its corresponding substrate-providing enzyme complex into the virulent phage ΦC01. Although the reporter phage ΦC01_lux, constructed by replacing non-essential genes for phage infectivity with a luxCDABE reporter operon, produced bioluminescence upon Cs infection, the emitted signal was quickly decayed due to the superior bacteriolytic activity of ΦC01. By truncating the membrane pore-forming protein holin and thus limiting its function, the bacterial lysis was delayed and the resultant engineered reporter phage ΦC01_lux_Δhol could produce a more stable and reliable bioluminescent signal. Accordingly, ΦC01_lux_Δhol was able to detect at least an average of 2 CFU/ml of Cs artificially contaminated PIF and Sunsik and food contact surface models within a total of 7 h of assays, including 5 h of pre-enrichment for Cs amplification. The sensitive, easy-to-use, and specific detection of live Cs with the developed reporter phage could be applied as a novel complementary tool for monitoring Cs in food and food-related environments for food safety and public health.

Análise microbiológica de sushi comercializado em Rio Branco, Acre / Microbiological analysis of sushi marketed in Rio Branco, Acre / Análisis microbiológico de sushi comercializado en Rio Branco, Acre

O Brasil é um dos países mais diversificados no ramo gastronômico oferecendo vários alimentos diferentes aos seus consumidores, com base nos próprios pratos típicos ou provenientes de outras culturas. O pescado trata-se de um alimento perecível que necessita de atenções especiais em seu processamento. Falhas nas condições higiênico-sanitárias, associadas com a não cocção do alimento, podem ocasionar em uma contaminação e proliferação de bactérias, o que leva à uma grande preocupação a nível de saúde pública. O estudo analisou os aspectos microbiológicos de sushi comercializado na cidade de Rio Branco ­ Acre verificando os parâmetros de qualidade e as condições higiênicas sanitárias, comparando os resultados obtidos com a legislação vigente estabelecida pela ANVISA. Foram escolhidos 5 estabelecimentos aleatoriamente, sendo escolhidas 3 amostras de sushis do tipo niguiri de cada. As análises microbiológicas incluíram coliformes totais e coliformes termotolerantes utilizando a técnica dos tubos multiplos e a técnica de semeadura por profundidade para mesófilos e Salmonella. Constatou-se que todas as amostras tiveram um crescimento bacteriano e presença sugestiva de Salmonella, tornando o alimento impróprio para o consumo e mostrando uma falha nas condições higiênico- sanitária ao qual o sushi é processado e armazenado. É necessário maior fiscalização dos órgãos responsáveis e cuidado dos estabelecimentos que vendem sushi na cidade de Rio Branco, para que o produto vendido seja de boa qualidade e não cause malefícios a saúde de quem o consome.(AU)

Brazil is one of the most diversified countries in the gastronomic field, offering several different foods to its consumers, based on typical dishes or from other cultures. Fish is a perishable food that requires special attention in its processing. Failures in hygienic-sanitary conditions, coupled with the consumption of undercooked food, can lead to contamination and the proliferation of bacteria, which raises significant concerns regarding public health. The study analyzed the microbiological aspects of sushi sold in the city of Rio Branco - Acre, verifying the quality parameters and the hygienic sanitary conditions, comparing the obtained results with the current legislation established by ANVISA. Five establishments were randomly selected, and three samples of nigiri sushi were chosen from each establishment. The microbiological analysis included total coliforms and thermotolerant coliforms using the multiple tube technique, as well as depth seeding technique for mesophiles and Salmonella. It was found that all samples exhibited bacterial growth and suggested the presence of Salmonella, rendering the food unsuitable for consumption and indicating a failure in the hygienic-sanitary conditions under which the sushi was processed and stored. Greater inspection by the responsible authorities and improved care by establishments selling sushi in the city of Rio Branco are necessary to ensure that the product sold is of good quality and does not pose harm to the health of consumers.(AU)

Brasil es uno de los países más diversificados en el campo gastronómico, ofreciendo muchos alimentos diferentes a sus consumidores, basados en platos típicos ode otras culturas El pescado es un alimento perecedero que necesita especial atención en su elaboración. Las fallas en las condiciones, higiénico-sanitarias asociadas a la no cocción de los alimentos, pueden conducir a la contaminación y proliferación de bacterias, lo que genera una gran preocupación en términos de salud pública. El estudio analizó los aspectos microbiológicos del sushi comercializado en la ciudad de Rio Branco - Acre, verificando los parámetros de calidad y las condiciones higiénicas sanitarias, comparando los resultados obtenidos con la legislación vigente establecida por la ANVISA. Se eligieron 5 establecimientos al azar, y de cada uno se escogieron 3 muestras de sushi niguiri. Los análisis microbiológicos incluyeron coliformes totales y coliformes termotolerantes mediante la técnica de tubos múltiples y la técnica de siembra profunda para mesófilos y Salmonella. Se encontró que todas las muestras presentaban crecimiento bacteriano y la sugestiva presencia de Salmonella, lo que hace que el alimento no sea apto para el consumo y presenta una falla en las condiciones higiénico-sanitarias en las que se procesa y almacena el sushi. Se necesita mayor fiscalización por parte de los órganos responsables y cuidado de los establecimientos que venden sushi en la ciudad de Rio Branco, para que el producto vendido sea de buena calidad y no cause daño a la salud de quien lo consume.(AU)

16S ribosomal RNA-depletion PCR and its application in cause analysis of yogurt package shrinkage.

Fermentative bacteria, the main microbiota in yogurt, interfere with the detection of unintended bacterial contaminants. The removal of fermentative bacteria and enrichment of unintended bacterial contaminants is a challenging task in bacterial detection. The present study developed a new 16S rRNA-depletion PCR for such enrichment and detection. Specifically, a single-guide RNA was designed and synthesized based on the 16S rRNA sequence of Streptococcus thermophilus, with the highest DNA abundance in the yogurt. The CRISPR-Cas9 system was used to specifically cleave and remove the genomic DNA of the fermentative bacteria, followed by PCR amplification. This method improved the detection sensitivity, simplified the operation steps, and reduced the detection cost of PCR analysis. We also used the 16S rRNA-depletion PCR to amplify and detect the unintended bacterial contaminants in yogurts with shrunken packages and analyzed the underlying reasons to prevent this issue of product quality.

Development of nanobody-horseradish peroxidase-based sandwich ELISA to detect Salmonella Enteritidis in milk and in vivo colonization in chicken.

BACKGROUND: Salmonella Enteritidis (S. Enteritidis) being one of the most prevalent foodborne pathogens worldwide poses a serious threat to public safety. Prevention of zoonotic infectious disease and controlling the risk of transmission of S. Enteriditidis critically requires the evolution of rapid and sensitive detection methods. The detection methods based on nucleic acid and conventional antibodies are fraught with limitations. Many of these limitations of the conventional antibodies can be circumvented using natural nanobodies which are endowed with characteristics, such as high affinity, thermal stability, easy production, especially higher diversity. This study aimed to select the special nanobodies against S. Enteriditidis for developing an improved nanobody-horseradish peroxidase-based sandwich ELISA to detect S. Enteritidis in the practical sample. The nanobody-horseradish peroxidase fusions can help in eliminating the use of secondary antibodies labeled with horseradish peroxidase, which can reduce the time of the experiment. Moreover, the novel sandwich ELISA developed in this study can be used to detect S. Enteriditidis specifically and rapidly with improved sensitivity. RESULTS: This study screened four nanobodies from an immunized nanobody library, after four rounds of screening, using the phage display technology. Subsequently, the screened nanobodies were successfully expressed with the prokaryotic and eukaryotic expression systems, respectively. A sandwich ELISA employing the SE-Nb9 and horseradish peroxidase-Nb1 pair to capture and to detect S. Enteritidis, respectively, was developed and found to possess a detection limit of 5 × 104 colony forming units (CFU)/mL. In the established immunoassay, the 8 h-enrichment enabled the detection of up to approximately 10 CFU/mL of S. Enteriditidis in milk samples. Furthermore, we investigated the colonization distribution of S. Enteriditidis in infected chicken using the established assay, showing that the S. Enteriditidis could subsist in almost all parts of the intestinal tract. These results were in agreement with the results obtained from the real-time PCR and plate culture. The liver was specifically identified to be colonized with quite a several S. Enteriditidis, indicating the risk of S. Enteriditidis infection outside of intestinal tract. CONCLUSIONS: This newly developed a sandwich ELISA that used the SE-Nb9 as capture antibody and horseradish peroxidase-Nb1 to detect S. Enteriditidis in the spike milk sample and to analyze the colonization distribution of S. Enteriditidis in the infected chicken. These results demonstrated that the developed assay is to be applicable for detecting S. Enteriditidis in the spiked milk in the rapid, specific, and sensitive way. Meanwhile, the developed assay can analyze the colonization distribution of S. Enteriditidis in the challenged chicken to indicate it as a promising tool for monitoring S. Enteriditidis in poultry products. Importantly, the SE-Nb1-vHRP as detection antibody can directly bind S. Enteritidis captured by SE-Nb9, reducing the use of commercial secondary antibodies and shortening the detection time. In short, the developed sandwich ELISA ushers great prospects for monitoring S. Enteritidis in food safety control and further commercial production.

The Use of Unique, Environmental Lactic Acid Bacteria Strains in the Traditional Production of Organic Cheeses from Unpasteurized Cow's Milk.

The aim of this study was to use local LAB cultures for the production of organic acid-rennet cheeses from unpasteurized cow's milk. Under industrial conditions, three types of cheese were produced, i.e., traditionally with acid whey (AW), with starter culture L. brevis B1, or with starter culture L. plantarum Os2. Strains were previously isolated from traditional Polish cheeses. Chemical composition, physico-chemical, microbiological, and sensory studies during 2 months of storage were carried out. As a result of this research, it was found that the basic composition was typical for semi-hard, partially skimmed cheeses. Mainly saturated fatty acids were detected. The cheeses were rich in omega-3, -6, and -9 fatty acids and conjugated linoleic acid (CLA), and were characterized by good lipid quality indices (LQI). All of the cheeses were characterized by a high number of lactic acid bacteria, with Enterobacteriaceae, yeast, molds, and staphylococci contaminants, which is typical microbiota for unpasteurized milk products. Water activity, pH, and total acidity were typical. A lower oxidation-reduction potential (ORP) of cheeses with the addition of strains and stability of the products during storage were observed. The B1 and Os2 cheeses were lighter, less yellow, had a more intense milk and creamy aroma, were softer, moister, and more elastic than AW cheese. The research results indicate the possibility of using environmental LAB strains in the production of high-quality acid-rennet cheeses, but special attention should be paid to the production process due to the microbiological quality of the cheeses.

[Management of Food Hygiene and Safety by Hazard Analysis and Critical Control Point (HACCAP)].

Advances in food production technology and newer distribution systems have made it easier to obtain fresh ingredients from both within and outside of Japan. Although convenient, mass distribution of food over wide areas involves the risk of expanding the health damage caused by foods. Comprehensive management from production to consumption, using systems, such as hazard analysis and critical control point (HACCP), is required to ensure the safety of foods. Improved inspection methods are also required to detect the effects of environmental changes on food. In this paper, we demonstrate the use of "Methods of Analysis in Health Science 2020" for food hygiene and safety management.

To culture or not to culture: careful assessment of metabarcoding data is necessary when evaluating the microbiota of a modified-atmosphere-packaged vegetarian meat alternative throughout its shelf-life period.

BACKGROUND: As the increased consumption of ready-to-eat meat alternatives is a fairly recent trend, little is known about the composition and dynamics of the microbiota present on such products. Such information is nonetheless valuable in view of spoilage and food safety prevention. Even though refrigeration and modified-atmosphere-packaging (MAP) can extend the shelf-life period, microbial spoilage can still occur in these products. In the present study, the microbiota of a vegetarian alternative to poultry-based charcuterie was investigated during storage, contrasting the use of a culture-dependent method to a culture-independent metagenetic method. RESULTS: The former revealed that lactic acid bacteria (LAB) were the most abundant microbial group, specifically at the end of the shelf-life period, whereby Latilactobacillus sakei was the most abundant species. Metabarcoding analysis, in contrast, revealed that DNA of Xanthomonas was most prominently present, which likely was an artifact due to the presence of xanthan gum as an ingredient, followed by Streptococcus and Weissella. CONCLUSIONS: Taken together, these results indicated that Lb. sakei was likely the most prominent specific spoilage organisms (SSO) and, additionally, that the use of metagenetic analysis needs to be interpreted with care in this specific type of product. In order to improve the performance of metagenetics in food samples with a high DNA matrix but a low bacterial DNA load, selective depletion techniques for matrix DNA could be explored.

Taqman qPCR Quantification and Fusarium Community Analysis to Evaluate Toxigenic Fungi in Cereals.

Fusarium head blight (FHB) is an economically important plant disease. Some Fusarium species produce mycotoxins that cause food safety concerns for both humans and animals. One especially important mycotoxin-producing fungus causing FHB is Fusarium graminearum. However, Fusarium species form a disease complex where different Fusarium species co-occur in the infected cereals. Effective management strategies for FHB are needed. Development of the management tools requires information about the diversity and abundance of the whole Fusarium community. Molecular quantification assays for detecting individual Fusarium species and subgroups exist, but a method for the detection and quantification of the whole Fusarium group is still lacking. In this study, a new TaqMan-based qPCR method (FusE) targeting the Fusarium-specific elongation factor region (EF1α) was developed for the detection and quantification of Fusarium spp. The FusE method was proven as a sensitive method with a detection limit of 1 pg of Fusarium DNA. Fusarium abundance results from oat samples correlated significantly with deoxynivalenol (DON) toxin content. In addition, the whole Fusarium community in Finnish oat samples was characterized with a new metabarcoding method. A shift from F. culmorum to F. graminearum in FHB-infected oats has been detected in Europe, and the results of this study confirm that. These new molecular methods can be applied in the assessment of the Fusarium community and mycotoxin risk in cereals. Knowledge gained from the Fusarium community analyses can be applied in developing and selecting effective management strategies for FHB.

Is skipping the definition of primary and secondary models possible? Prediction of Escherichia coli O157 growth by machine learning.

To predict bacterial population behavior in food, statistical models with specific function form have been applied in the field of predictive microbiology. Modelers need to consider the linear or non-linear relationship between the response and explanatory variables in the statistical modeling approach. In the present study, we focused on machine learning methods to skip definition of primary and secondary structure model. Support vector regression, extremely randomized trees regression, and Gaussian process regression were used to predict population growth of Escherichia coli O157 at 15 and 25 °C without defining the primary and secondary models. Furthermore, the support vector regression model was applied to predict small population of bacteria cells with probability theory. The model performance of the machine learning models were nearly equal to that of the current statistical models. Machine learning models have a potential for predicting bacterial population behavior.

Multiplex PCR method for the detection of human norovirus, Salmonella spp., Shigella spp., and shiga toxin producing Escherichia coli in blackberry, coriander, lettuce and strawberry.

A multiplex PCR method was developed for the simultaneous detection of murine norovirus (MNV-1) as a surrogate for human norovirus (HuNoV) GI and GII, Salmonella spp., Shigella spp., and Shiga toxin producing Escherichia coli (STEC) in fresh produce. The toxicity of the glycine buffer on bacterial pathogens viability was evaluated. The growth of each of the three pathogens (previously stressed) was evaluated at 35 and 41.5 °C in modified buffered peptone water (mBPW) and trypticase soy broth (TSB), supplemented with vancomycin, novobiocin and brilliant green at two concentration levels. The selected conditions for simultaneous enrichment were: 41.5 °C/mBPW/supplemented with 8 ppm vancomycin, 0.6 ppm novobiocin and 0.2 ppm brilliant green. The pathogens and aerobic plate count (APC) growth was evaluated in the enrichment of lettuce, coriander, strawberry and blackberry under the best enrichment conditions. Starting from 1 to 10 CFU/mL, Salmonella reached from 7.63 to 8.91, Shigella 6.81 to 7.76 and STEC 7.43 to 9.27 log CFU/mL. The population reached for the APC was 5.11-6.56 log CFU/mL. Simultaneous detection by PCR was done using designed primers targeting invA, ipaH, stx1 and stx2 genes, and MNV-1. The detection sensitivity was 10-100 PFU for the MNV-1 and 1-10 CFU for each pathogenic bacteria. This protocol takes 6 h for MNV-1 and 24 h for Salmonella spp., Shigella spp., and STEC detection from the same food portion. In total, 200 samples were analyzed from retail markets from Queretaro, Mexico. Two strawberry samples were positive for HuNoV GI and one lettuce sample was positive for STEC. In conclusion, the method developed in this study is capable of detecting HuNoV GI and GII, Salmonella spp., Shigella spp and STEC from the same fresh produce sample.

Accurate classification of Listeria species by MALDI-TOF mass spectrometry incorporating denoising autoencoder and machine learning.

Listeria monocytogenes belongs to the category of facultative anaerobic bacteria, and is the pathogen of listeriosis, potentially lethal disease for humans. There are many similarities between L. monocytogenes and other non-pathogenic Listeria species, which causes great difficulties for their correct identification. The level of L. monocytogenes contamination in food remains high according to statistics from the Food and Drug Administration. This situation leads to food recall and destruction, which has caused huge economic losses to the food industry. Therefore, the identification of Listeria species is very important for clinical treatment and food safety. This work aims to explore an efficient classification algorithm which could easily and reliably distinguish Listeria species. We attempted to classify Listeria species by incorporating denoising autoencoder (DAE) and machine learning algorithms in matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). In addition, convolutional neural networks were used to map the high dimensional original mass spectrometry data to low dimensional core features. By analyzing MALDI-TOF MS data via incorporating DAE and support vector machine (SVM), the identification accuracy of Listeria species was 100%. The proposed classification algorithm is fast (range of seconds), easy to handle, and, more importantly, this method also allows for extending the identification scope of bacteria. The DAE model used in our research is an effective tool for the extraction of MALDI-TOF mass spectrometry features. Despite the fact that the MALDI-TOF MS dataset examined in our research had high dimensionality, the DAE + SVM algorithm was still able to exploit the hidden information embedded in the original MALDI-TOF mass spectra. The experimental results in our work demonstrated that MALDI-TOF mass spectrum combined with DAE + SVM could easily and reliably distinguish Listeria species.

Bacteriophage-Mediated Modulation of Bacterial Competition during Selective Enrichment of Campylobacter.

Selective media using antimicrobial supplements generate unique microbial ecology to facilitate bacterial isolation. However, antibiotic-resistant bacteria indigenous to samples can interfere with the isolation process using selective media. Recent studies showed that extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is highly prevalent on retail raw chicken and compromises the efficacy of Campylobacter isolation because ESBL-producing E. coli are resistant to antimicrobial supplements in Campylobacter-selective media and outgrows Campylobacter. The objective of this study was to improve Campylobacter isolation by inhibiting the growth of ESBL-producing E. coli using bacteriophages (phages). The supplementation of Campylobacter-selective media with E. coli phages reduced the level of ESBL-producing E. coli during the enrichment step. When E. coli phages were combined with the antimicrobial supplements of Campylobacter-selective media, antimicrobial synergy was observed, particularly with rifampicin, an antibiotic used in Preston medium. Although the same materials (i.e., phages and selective media) were used, the sequence of combining the materials markedly influenced the inhibition of ESBL-producing E. coli and the isolation of Campylobacter. These findings indicated that the modulation of microbial competition at the enrichment step was critical to the successful isolation of fastidious bacteria and that phages can be utilized to facilitate the selective enrichment of target bacteria by inhibiting their competitive bacteria. IMPORTANCE Phages are promising antimicrobial alternatives. In this study, we first demonstrated that phages can be used to facilitate selective isolation of fastidious bacteria that are prone to be outgrown by bacterial competitors during isolation. The effectiveness of a phage-based isolation method was primarily dependent on the antimicrobial synergy between phages and antibiotics used in selective media. The same approach could be applied to the development of isolation methods for other fastidious bacteria.

Antibacterial Properties of Melanoidins Produced from Various Combinations of Maillard Reaction against Pathogenic Bacteria.

Novel melanoidins are produced by the Maillard reaction. Here, melanoidins with high antibacterial activity were tested by examining various combinations of reducing sugars and amino acids as reaction substrates. Twenty-two types of melanoidins were examined by combining two reducing sugars (glucose and xylose) and eleven l-isomers of amino acids (alanine, arginine, glutamine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine) to confirm the effects of these melanoidins on the growth of Listeria monocytogenes at 25°C. The melanoidins produced from the combination of d-xylose with either l-phenylalanine (Xyl-Phe) or l-proline (Xyl-Pro), for which absorbance at 420 nm was 3.5 ± 0.2, completely inhibited the growth of L. monocytogenes at 25°C for 48 h. Both of the melanoidins exhibited growth inhibition of L. monocytogenes which was equivalent to the effect of nisin (350 IU/mL). The antimicrobial spectrum of both melanoidins was also investigated for 10 different species of bacteria, including both Gram-positive and Gram-negative species. While Xyl-Phe-based melanoidin successfully inhibited the growth of Bacillus cereus and Brevibacillus brevis, Xyl-Pro-based melanoidin inhibited the growth of Salmonella enterica Typhimurium. However, no clear trend in the antimicrobial spectrum of the melanoidins against different bacterial species was observed. The findings in the present study suggest that melanoidins generated from xylose with phenylalanine and/or proline could be used as potential novel alternative food preservatives derived from food ingredients to control pathogenic bacteria. IMPORTANCE Although the antimicrobial effect of melanoidins has been reported in some foods, there have been few comprehensive investigations on the antimicrobial activity of combinations of reaction substrates of the Maillard reaction. The present study comprehensively investigated the potential of various combinations of reducing sugars and amino acids. Because the melanoidins examined in this study were produced simply by heating in an autoclave at 121°C for 60 min, the targeted melanoidins can be easily produced. The melanoidins produced from combinations of xylose with either phenylalanine or proline exhibited a wide spectrum of antibiotic effects against various pathogens, including Listeria monocytogenes, Bacillus cereus, and Salmonella enterica Typhimurium. Since the antibacterial effect of the melanoidins on L. monocytogenes was equivalent to that of a nisin solution (350 IU/mL), we might expect a practical application of melanoidins as novel food preservatives.

Partial Purification and Characterization of Bacteriocin-Like Inhibitory Substances Produced by Streptomyces sp. Isolated from the Gut of Chanos chanos.

Bacteriocin-like inhibitory substances (BLIS) have sparked great interest because of their promising use in food as natural antimicrobial agents. In this work, six Streptomyces isolates obtained from the gut of Chanos chanos demonstrated their ability to produce extracellular metabolites with inhibitory activity against Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus. Exposure of the extracellular metabolites to proteolytic enzymes (i.e., proteinase-K, trypsin, and pepsin) revealed high sensitivity and confirmed their proteinaceous nature. The metabolites were stable at high temperatures (up to 100°C for 30 min) and a wide range of pH (pH 2.0-7.0). Fractionation of the crude BLIS by filtration yielded three fractions based on molecular weight: <3 kDa, 3-10 kDa, and >10 kDa. Analysis of the antibacterial activity of these fractions showed increased specific activity, especially in the fraction with a molecular weight (MW) of <3 kDa, relative to the crude sample. The fraction with MW < 3 kDa had minimum inhibitory and bactericidal concentrations in ranges 0.04-0.62 mg·mL-1 and 0.08-1.25 mg·mL-1, respectively. This fraction also showed better temperature and pH stability compared with crude BLIS. Brine shrimp toxicity assay revealed that this fraction has moderate toxicity with a 50% lethal concentration of 226.975 µg·mL-1 (i.e., moderate toxicity) to Artemia salina. Identification of the peptide sequences of this fraction by liquid chromatography-tandem mass spectrometry yielded 130 proteins with retention times of 15.21-19.57 min. Eleven proteins with MWs of 1345.66-2908.35 Da and composed of less than 30 amino acid residues with high hydrophobicity (15.34-26.22 kcal·mol-1) appeared to be responsible for the antibacterial activity of the fraction. This study revealed the potential application of BLIS from Streptomyces, especially BLIS SCA-8, as antibacterial agents.

Evaluating the Performance of Lateral Flow Devices for Total Aflatoxins with Special Emphasis on Their Robustness under Sub-Saharan Conditions.

As aflatoxins are a global risk for humans and animals, testing methods for rapid on-site screening are increasingly needed alongside the standard analytical laboratory tools. In the presented study, lateral flow devices (LFDs) for rapid total aflatoxin screening were thoroughly investigated with respect to their matrix effects, cross-reactivity, their performance under harsh conditions in Sub-Saharan Africa (SSA), and their stability, as well as when compared with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To analyze the matrix effects, qualitative test kits offering a certain cutoff level were used to screen different nut samples. In addition, these tests were challenged on their cross-reactivity with 230 fungal toxins and metabolites. Furthermore, the resulting measurements performed under harsh tropical conditions (up to 38.4 °C and 91% relative humidity) in SSA, specifically Burkina Faso and Mozambique, were compared with the results from a well-established and validated LC-MS/MS-based reference method. The comparison of the on-site LFD results with the reference method showed a good agreement: 86.4% agreement, 11.8% non-agreement, and 1.8% invalid test results. To test the robustness of the cutoff tests, short- and long-term stability testing was carried out in Mozambique and Nigeria. For both experiments, no loss of test performance could be determined. Finally, a subset of African corn samples was shipped to Austria and analyzed under laboratory conditions using semiquantitative aflatoxin tests. A good correlation was found between the rapid strip tests and the LC-MS/MS reference method. Overall, the evaluated LFDs showed satisfying results regarding their cross-reactivity, matrix effects, stability, and robustness.

Validation of New ELISA Technique for Detection of Aflatoxin B1 Contamination in Food Products versus HPLC and VICAM.

Toxin-contaminated foods and beverages are a major source of illness, may cause death, and have a significant negative economic impact worldwide. Aflatoxin B1 (AFB1) is a potent toxin that may induce cancer after chronic low-level exposure. This study developed a quantitative recombinant AflR gene antiserum ELISA technique for aflatoxin B1 detection in contaminated food products. Aflatoxin B1 residuals from 36 food samples were analyzed with HPLC and VICAM. DNA was extracted from aflatoxin-contaminated samples and the AflR gene amplified using PCR. PCR products were purified and ligated into the pGEM-T vector. Recombinant plasmids were sequenced and transformed into competent E. coli (BL21). Molecular size and B-cell epitope prediction for the recombinant protein were assessed. The purified protein was used to induce the production of IgG antibodies in rabbits. Serum IgG was purified and labeled with alkaline phosphatase. Finally, indirect-ELISA was used to test the effectiveness of polyclonal antibodies for detection of aflatoxin B1 in food samples.

Optimization of the QuEChERS-Based Analytical Method for Investigation of 11 Mycotoxin Residues in Feed Ingredients and Compound Feeds.

Mycotoxins are toxic substances naturally produced by various fungi, and these compounds not only inflict economic damage, but also pose risks to human and animal health. The goal of the present study was to optimize the QuEChERS-based extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of 11 mycotoxins, such as aflatoxins (AFs), ochratoxin A (OTA), fumonisins (FBs), T-2 toxin, HT-2 toxin, zearalenone (ZEN), and deoxynivalenol (DON), commonly found in feed. The QuEChERS method, characterized by being "quick, easy, cheap, effective, rugged, and safe", has become one of the most common extractions and clean-up procedures for mycotoxin analyses in food. Therefore, in this experiment, an optimal method for the analysis of 11 mycotoxins in feed was established by modifying the general QuEChERS method. In this process, it was confirmed that even if feed samples of different weights were extracted, the quantitative value of mycotoxins in the feed was not affected. To reduce matrix effects, 13C-labeled compounds and deuterium were used as internal standards. This optimized method was then applied in the determination of 11 mycotoxins in 736 feed ingredients and compound feeds obtained from South Korea. The results showed that the occurrence rates of FBs, ZEN, and DON were 59.4%, 38.0%, and 32.1%, respectively, and OTA, AFs, and T-2 toxin and HT-2 toxin were found in fewer than 1% of the 736 feeds. The mean concentration ranges of FBs, ZEN, and DON were 757-2387, 44-4552, and 248-9680 µg/kg, respectively. Among the samples in which DON and ZEN were detected, 10 and 12 samples exceeded the management recommendation standards presented by the Ministry of Agriculture, Food and Rural Affairs (MAFRA). However, when the detected concentrations of DON and ZEN were compared with guideline levels in foreign countries, such as the US, Japan, China, and the EU, the number of positive samples changed. In addition, the co-occurrence of mycotoxins in the feed was analyzed, and the results showed that 43.8% of the samples were contaminated with two or three mycotoxins, among which the co-occurrence rate of FBs, ZEN, and DON was the highest. In conclusion, these results suggest the need for stricter management standards for FBs, DON, and ZEN in South Korea, and emphasize the importance of the continuous monitoring of feeds.

Evaluation of the Antimicrobial Efficacy of N-Acetyl-l-Cysteine, Rhamnolipids, and Usnic Acid-Novel Approaches to Fight Food-Borne Pathogens.

In the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-l-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.

A Systematic Review on Nanoencapsulation Natural Antimicrobials in Foods: In Vitro versus In Situ Evaluation, Mechanisms of Action and Implications on Physical-Chemical Quality.

Natural antimicrobials (NA) have stood out in the last decade due to the growing demand for reducing chemical preservatives in food. Once solubility, stability, and changes in sensory attributes could limit their applications in foods, several studies were published suggesting micro-/nanoencapsulation to overcome such challenges. Thus, for our systematic review the Science Direct, Web of Science, Scopus, and Pub Med databases were chosen to recover papers published from 2010 to 2020. After reviewing all titles/abstracts and keywords for the full-text papers, key data were extracted and synthesized. The systematic review proposed to compare the antimicrobial efficacy between nanoencapsulated NA (nNA) and its free form in vitro and in situ studies, since although in vitro studies are often used in studies, they present characteristics and properties that are different from those found in foods; providing a comprehensive understanding of primary mechanisms of action of the nNA in foods; and analyzing the effects on quality parameters of foods. Essential oils and nanoemulsions (10.9-100 nm) have received significant attention and showed higher antimicrobial efficacy without sensory impairments compared to free NA. Regarding nNA mechanisms: (i) nanoencapsulation provides a slow-prolonged release to promote antimicrobial action over time, and (ii) prevents interactions with food constituents that in turn impair antimicrobial action. Besides in vitro antifungal and antibacterial, nNA also demonstrated antioxidant activity-potential to shelf life extension in food. However, of the studies involving nanoencapsulated natural antimicrobials used in this review, little attention was placed on proximate composition, sensory, and rheological evaluation. We encourage further in situ studies once data differ from in vitro assay, suggesting food matrix greatly influences NA mechanisms.