Quantitative detection and survival analysis of VBNC Salmonella Typhimurium in flour using droplet digital PCR and DNA-intercalating dyes.

The difficulty in detecting viable but non-culturable (VBNC) Salmonella by culture-dependent methods poses a risk to food safety. In our study, we applied a viability test to Salmonella following a lethal treatment and to flour samples inoculated with Salmonella to evaluate the effectiveness of viability polymerase chain reaction (PCR). Our findings revealed that the combination of both ddPCR and qPCR with those DNA-intercalating dyes could quantify viable cells at low concentrations when the plate counting method failed to detect them post-inactivation. Prolonged UV exposure did not induce cell membrane disruption, as confirmed with PMA-ddPCR, with insignificant differences in gene copies. However, samples exposed to DyeTox13 and DyeTox13 + EMA showed lower gene copy numbers, implying that enzymatic activity was decreased by UV exposure duration. In addition, temperature-dependent survival in flour revealed uniform decay rates and D values (time required for a 1 log reduction) of DNA in untreated samples across various temperatures. By contrast, different decay rates were observed with DNA-intercalating dyes (DyeTox13 and DyeTox13 + EMA), showing faster metabolic activity loss at higher temperatures in flour. The decay rates and D values, determined through plate counting and those DNA-intercalating dyes, indicated the potential presence of VBNC Salmonella. A strong correlation between DyeTox13 dyes and the plate counting method suggested DyeTox13 as a rapid alternative for detecting Salmonella in flour. The ddPCR with DNA-intercalating dyes could effectively evaluate Salmonella viability, facilitating more precise monitoring of VBNC in food. IMPORTANCE: Salmonella, a major foodborne pathogen, poses significant risks, particularly to vulnerable groups like infants, older people, and the immunocompromised. Accurate detection is vital for public health and food safety, given its potential to cause severe and life-threatening symptoms. Our study demonstrated digital polymerase chain reaction (ddPCR) with DNA-intercalating dyes for identifying the different physiological statuses of Salmonella. Also, the application of ddPCR with DNA-intercalating dyes offers quantification of viable cells post-disinfection as an alternative method in food. Utilizing ddPCR and DNA-intercalating dyes, we enhanced the detection of VBNC Salmonella, a form often undetectable by conventional methods. This innovative approach could significantly improve the precision and efficiency of detection for viable Salmonella. By providing deeper insights into its transmission potential, our method is a critical tool in preventing outbreaks and ensuring the safety of food products. This research contributes substantially to global efforts in controlling foodborne illnesses and safeguarding public health.

The microbiological quality of flour products in the UK with respect to Salmonella and Shiga-toxin-producing Escherichia coli.

AIM: To investigate the possible contamination of raw flour and raw flour-based products, such as pancake/batter mixes, with Salmonella, generic Escherichia coli, and Shiga-toxin-producing E. coli (STEC). Samples included flours available for sale in the UK over a period of four months (January to April 2020). The Bread and Flour regulations, 1998 state the permitted ingredients in flour and bread but it does not specify the regular monitoring of the microbiological quality of flour and flour-based products. METHODS AND RESULTS: Samples of raw flour were collected by local authority sampling officers in accordance with current guidance on microbiological food sampling then transported to the laboratory for examination. Microbiological testing was performed to detect Salmonella spp., generic E. coli, and STEC characterized for the presence of STEC virulence genes: stx1, stx2, and subtypes, eae, ipah, aggR, lt, sth, and stp, using molecular methods Polymerase Chain Reaction (PCR). Of the 882 flours sampled, the incidence of Salmonella was 0.1% (a single positive sample that contained multiple ingredients such as flour, dried egg, and dried milk, milled in the UK), and 68 samples (7.7%) contained generic E. coli at a level of >20 CFU/g. Molecular characterization of flour samples revealed the presence of the Shiga-toxin (stx) gene in 10 samples (5 imported and 5 from the UK) (1.1%), from which STEC was isolated from 7 samples (0.8%). Salmonella and STEC isolates were sequenced to provide further characterization of genotypes and to compare to sequences of human clinical isolates held in the UKHSA archive. Using our interpretive criteria based on genetic similarity, none of the STEC flour isolates correlated with previously observed human cases, while the singular Salmonella serotype Newport isolate from the mixed ingredient product was similar to a human case in 2019, from the UK, of S. Newport. Although there have been no reported human cases of STEC matching the isolates from these flour samples, some of the same serotypes and stx subtypes detected are known to have caused illness in other contexts. CONCLUSION: Results indicate that while the incidence was low, there is a potential for the presence of Salmonella and STEC in flour, and a genetic link was demonstrated between a Salmonella isolate from a flour-based product and a human case of salmonellosis.

Sourdough fermentation for the valorization of sorghum flour: Microbiota characterization and metabolome profiling.

Due to a large adaptability to different cultivation conditions and limited input compared to other cereals, sorghum is considered an emerging crop. Its antioxidant properties, high fiber content and low glycemic index also make it a valuable addition to a healthy diet, nevertheless, the presence of antinutritional factors and the lack of gluten, hamper its use as food ingredient. This study investigated the impact of sourdough fermentation on sorghum nutritional quality. Lactic acid bacteria dominating sorghum flour and sourdough were identified by culture-dependent analysis revealing Lactiplantibacillus plantarum as the dominant species found in the mature sourdough, whereas Weissella cibaria and Weissella paramesenteroides were the species isolated the most after the first refreshment. Among yeasts, Saccharomyces cerevisiae was the most prevalent. Lactic acid bacteria pro-technological and functional performances as starter were evaluated in sorghum type-II sourdoughs through an integrated characterization combining chromatographic and NMR spectroscopic techniques. The metabolic profile of the strains mainly grouped together W. cibaria strains and W. paramesenteroides AI7 which distinguished for the intense proteolysis but also for the presence of compounds particularly interesting from a physiological perspective (allantoin, glutathione, γ-aminobutyric acid and 2-hydroxy-3-methylbutyric acid), whose concentration increased during fermentation in a species or strain specific matter.

Insights into the starch and proteins molecular structure changes of foxtail millet sourdough: Effect of fermentation from grains of cereal to pre-meal.

This study investigated the effects of foxtail millet sourdough fermentation time (0, 8, 16, and 24 h) on the protein structural properties, thermomechanical, fermentation, dynamic rheological, starch granules crystalline regions molecular mobility, and starch microstructural characteristics. The fermentation led to a significant increase in the concentration of free amino acids from protein hydrolysis. Fourier transform infrared spectroscopy (FTIR) revealed changes in protein secondary structure and the presence of functional groups of different bioactive compounds. The result of thermomechanical properties showed a significant increase in the stability (0.70-0.79 min) and anti-retrogradation ability (2.29-3.14 Nm) of lactic acid bacteria (LAB) sourdough compared to the control dough, showing a wider processing applicability with radar profiler index. In contrast, sourdoughs with lower tan δ values had higher elasticity and strength. Scanning electron microscopy showed that the surface of the starch appeared from smooth to uneven with patchy shapes and cavities, which declined the crystallinity from 34.00 % to 21.57 %, 23.64 %, 25.09 %, and 26.34 % respectively. Fermentation changed the To, Tp, Tc, and ΔH of the starch. The results of the study will have great potential for application in the whole grain sourdough industry.

Validation of a Simulated Commercial English Muffin Baking Process to control Salmonella Contamination.

A validation study was conducted to investigate the effect of the English muffin baking process to control Salmonella contamination and to study the thermal inactivation kinetic parameters (D- and z-values) of Salmonella in English muffin dough. The unbleached bread flour was inoculated with 3 serovar Salmonella cocktail (Salmonella serovars viz., Newport, Typhimurium, and Senftenberg), and dried back to its preinoculated water activity levels with 7.46 ± 0.12 log CFU/g of Salmonella concentration. The Salmonella inoculated flour was used to prepare English muffin batter and baked at 204.4°C (400°F) for 18 min and allowed to cool at ambient air for 15 min. The English muffins reached 99 ± 0°C (211.96 ± 0.37°F) as their maximum mean internal temperature during baking. The pH and aw of English muffin dough were 5.01 ± 0.01 and 0.947 ± 0.003, respectively. At the end of the 18-min baking period, the Salmonella inoculated English muffins recorded a more than 5 log CFU/g reduction on the injury-recovery media. The D-values of 3 serovar cocktails of Salmonella at 55, 58.5, and 62°C were 42.0 ± 5.68, 15.6 ± 0.73, and 3.0 ± 0.32 min, respectively; and the z-value was 6.2 ± 0.59°C. The water activity (aw) of the English muffin crumb (0.947 ± 0.003 to 0.9557 ± 0.001) remained statistically unchanged during baking, whereas the aw of the muffin crust decreased significantly (0.947 ± 0.003 to 0.918 ± 0.002) by the end of 18 min of baking. This study validates and documents the first scientific evidence that baking English muffins at 204.4°C (400°F) for 18 min acts as an effective kill step by controlling Salmonella population by >5 log CFU/g.

Exploring the flavor changes in mung bean flour through Lactobacillus fermentation: insights from volatile compounds and non-targeted metabolomics analysis.

BACKGROUND: Mung beans are highly nutritious but their leguminous flavor limits their development. Lactic acid bacteria (LAB) fermentation can decrease unwanted bean flavors in legumes and enhance their flavor. This study examined the influence of Lactobacillus fermentation on the flavor characteristics of mung bean flour (MBF) using volatile compounds and non-targeted metabolomics. RESULTS: Lactobacillus plantarum LP90, Lactobacillus casei LC89, and Lactobacillus acidophilus LA85 eliminated 61.37%, 48.29%, and 43.73%, respectively, of the primary bean odor aldehydes from MBF. The relative odor activity value (ROAV) results showed that fermented mung bean flour (FMBF) included volatile chemicals that contributed to fruity, flowery, and milky aromas. These compounds included ethyl acetate, hexyl formate, 3-hydroxy-2-butanone, and 2,3-butanedione. The levels of amino acids with a fresh sweet flavor increased significantly by 93.89, 49.40, and 35.27% in LP90, LC89, and LA85, respectively. A total of 49 up-regulated and 13 down-regulated significantly differential metabolites were annotated, and ten metabolic pathways were screened for contributing to the flavor. The correlation between important volatile compounds and non-volatile substances relies on two primary metabolic pathways: the citric acid cycle pathway and the amino acid metabolic system. CONCLUSION: The flavor of MBF was enhanced strongly by the process of Lactobacillus fermentation, with LP90 having the most notable impact. These results serve as a reference for identifying the flavor of FMBF. © 2024 Society of Chemical Industry.

Farofas de mandioca temperadas: avaliação microbiológica e da atividade de água / Cassava flour seasoned: microbiological evaluation and water activity

Produzida artesanalmente em casas de farinha, pode ser comercializada em feiras livres e supermercados. Este trabalho foi realizado com o objetivo de avaliar microbiologicamente duas amostras de farofas de mandioca temperadas e verificar o comportamento da atividade de água ao longo de um período de trinta e cinco dias de armazenamento sob temperatura constante de 30 ºC. A análise quanto a atividade de água foi realizada logo após o processamento e durante todo o período de armazenamento, com intervalos de sete dias. Com relação à análise microbiológica as amostras estavam dentro dos padrões estabelecidos pela legislação. A atividade de água diminuiu com a temperatura e com o tempo de armazenamento, porém, com valores inferiores a 0,6.

Efeito da farinha de banana verde no crescimento de bactérias láticas contidas nos grãos de kefir / Effect of green banana flour on the growth of lactic acid bacteria contained in kefir grains

Kefir é um produto fermentado, ácido, levemente alcoólico, produzido a partir de grãos que apresentam uma população microbiana simbiótica considerada estável, imersos em uma matriz constituída de polissacarídeos e proteínas. Está relacionado à reparação da mucosa intestinal, redução dos sintomas de intolerância à lactose, estimulação do sistema imunitário, redução do colesterol além de propriedades tumorais. O Kefir pode ser consumido com diferentes produtos, dentre eles a farinha de banana verde, que vem sendo amplamente pesquisada pelo seu potencial prebiótico. Diante do exposto, o objetivo do presente estudo foi verificar o efeito prebiótico de farinha de banana verde no crescimento de bactérias láticas contidas nos grãos de Kefir. Foram realizados dois tratamentos, sendo um apenas com leite fermentado por grãos de Kefir e outro onde após a preparação da bebida foram adicionados 2% de farinha de banana verde. Para avaliação da viabilidade foi realizado plaqueamento em ágar deMan Rogosa Sharpe (MRS) e M17 para a contagem de lactobacilos e cocos Gram positivos respectivamente. A contagem de bactéria lática variou de 8,31 a 9,45 log UFC/g, indicando que a adição de 2% de farinha de banana não interferiu na viabilidade de bactérias láticas presentes no kefir. Entretanto, apesar da farinha de banana verde não apresentar efeito prebiótico, a mesma pode ser considerada uma boa opção para ser adicionada à essa bebida fermentada, com intuito de agregar valor nutricional ao produto.

Kefir is fermented milk, acidic, lightly alcoholic, produced in a handmade way from grains that present a stable symbiotic microbial population, immersed in a matrix consisting of polysaccharides and proteins. It is related to repair of the intestinal mucosa, reduction of symptoms of lactose intolerance, stimulation of the immune system, reduction of cholesterol in addition to tumor properties. Kefir can be consumed with different products, among them green banana flour that has been widely researched for its prebiotic potential. In view of the above, the objective of the present study was to verify the prebiotic effect of green banana flour on the growth of lactic bacteria contained in Kefir grains. Two treatments were performed, one with only fermented milk by grains of Kefir and another where after preparation of the beverage was added 2% of green banana flour. To assess viability plating was performed on deMan Rogosa Sharpe agar (MRS) and M17 for the counts of lactobacilli and Gram positive cocci respectively. The lactic acid bacteria count ranged from 8.31 to 9.45 log CFU / g, indicating that the addition of 2% of banana flour did not interfere in the viability of lactic acid bacteria present in kefir. However, although green banana flour does not present a prebiotic effect, it can be considered as a good option to be added to this fermented beverage, in order to add nutritional value to the product.

Qualidade microbiológica da farinha multimistura distribuída no município de Campo Grande, MS / Microbiological quality of flour multi-mixture distributed in the city of Campo Grande, MS

A multimistura vem sendo utilizada em ações na área de atenção básica à saúde, destinada prioritariamente a famílias de baixa renda que apresentem crianças em risco nutricional. A farinha de multimistura é obtida através da secagem, torragem, moagem e mistura de farelos e pó de folhas escuras. É produzida em nível comunitário, podendo ter condições higiênico-sanitárias inadequadas, oferecendo um risco de contaminação elevado, o que poderá acarretar em doença às crianças que fazem uso deste alimento. Este estudo teve como objetivo avaliar as condições higiênico-sanitárias da farinha de multimistura distribuída em Campo Grande-MS. Das sete amostras de farinha analisadas microbiologicamente, 71,4% apresentaram índices elevados de contaminação por coliformes totais e 42,8% das amostras apresentaram-se contaminadas com coliformes termotolerantes. Não foram detectados em nenhuma amostra analisada a presença de Salmonella sp. e Bacillus cereus. Devido à presença de contaminantes de origem fecal, o seu consumo torna-se inapropriado em especial quando consideramos que o mesmo é empregado em programas de alimentação infantil e combate a carências nutricionais, o que poderia debilitar ainda mais o estado nutricional dos consumidores deste alimento.

Impact of solar dehydration on composition and antioxidant properties of acai (Euterpe oleracea Mart.)

Commercial products derived from the acai fruit (Euterpe oleracea Mart.) are available in Brazil, but in Venezuela, it is only known by ethnic indigenous groups of the Amazon. In this study, acai flour was made by solar dehydration and the effect of processing on the composition, microbiological quality, and antioxidant properties of such flour were evaluated. The fruit was purchased in Puerto Ayacucho, Venezuela, and a portion was manually pulped. Microbiological quality, proximal composition, minerals, polyphenols, tannins, anthocyanins, and antioxidant capacity were evaluated. The remaining portion of fruit was blanched in a solution of ascorbic acid and citric acid at 98ºC for 1min in the same manner, manually pulped, dried by solar dehydration and the acai flour was also analysed. From the composition of the acai flour, its high content of fat (22.9%), protein (13.7%), dietary fibre (20.5%), total polyphenols (1.60 g/kg) and antioxidant capacity (79.97%) stood out. The blanching of the fruit and the solar dehydrating of the acai pulp did not modify the composition, but they improved its microbiological quality and reduced phenolic compounds and antioxidant capacity. The flour obtained is stable and innocuous and could be used to diversify the diet of the indigenous people of the Amazon region.

Los productos comerciales derivados del fruto del acai (Euterpe oleracea Mart) están disponibles en Brasil, no así en Venezuela donde sólo los grupos indígenas del Amazonas lo conocen y consumen. En este estudio se elaboró una harina de acai mediante deshidratación solar y se evaluó el efecto del procesamiento en la composición, calidad microbiológica y propiedades antioxidantes de dicha harina. Los frutos fueron adquiridos en Puerto Ayacucho, Venezuela y una porción de ellos se despulpó manualmente y se le determinó calidad microbiológica, composición proximal, contenido de minerales, contenido de polifenoles, taninos, antocianinas y la capacidad antioxidante. La otra porción del fruto se escaldó en una solución de ácido ascórbico y ácido cítrico a 98ºC por 1min, se despulpó manualmente y se eliminó su humedad por deshidratación solar; a la harina de acai obtenida se le hicieron los mismos análisis que a la pulpa. De la composición del acai resaltó su alto contenido de grasa (22.9%), proteína (13.7%), fibra dietética (20.5%), polifenoles totales (1.60 g/kg-1) y su capacidad antioxidante (79.97%). El lavado y escaldado de los frutos y la posterior deshidratación solar de la pulpa del acai no modificó significativamente su composición, pero si mejoró la calidad microbiológica y redujo los compuestos fenólicos y la capacidad antioxidante. La harina obtenida es estable e inocua y se podría utilizar para diversificar la dieta de los indígenas de la región del Amazonas.

Perfil electroforético y calidad microbiológica de la harina de lombriz Eisenia fetida / Electrophoretic profile and microbiological quality flour from the worm Eisenia fetida

An electrophoretic and microbiological study more probable number (NMP), total (CT) and fecal coliforms (CF); aerobic-mesophils bacteria (BAM), molds and yeasts in worm's flour of Eisenia fetida was carried out. In the electrophoretic profiles using gels of standard concentration, different protein fractions with molecular weights between 39.6 and 43.5 kDa were separated. When gels with growing gradient of concentration were used, proteins with higher molecular weights (124.4 and 106.8 kDa) were separated with more detail. The protein characterization using different electrophoretic systems used in this study allows studying different protein fractions that could have a nutritional importance. The counts of BAM, CT, CF, molds and yeasts in this flour were low, indicating an innocuous product. With the use of this non-conventional resource, an interesting field of investigation begins involving food enrichment, representing an alternative at the industrial level.

Se realizó un estudio electroforético y microbio-lógico [número más probable (NMP), coliformes totales (CT) y fecales (CF); bacterias aerobias-mesófilas (BAM), mohos y levaduras] en harina de lombriz Eisenia fétida. En los perfiles electroforéticos con geles de concentración estándar se separaron diferentes fracciones proteicas con pesos moleculares comprendidos entre 39,6 y 43,5 kDa, valores similares a los reportados por otros investigadores. Cuando se usaron geles con gradiente creciente de concentración, se lograron separar en forma mas detallada proteínas con pesos moleculares más altos (124,4 y 106,8 kDa). En este estudio, la caracterización de proteínas utilizando distintos sistemas electroforéticos permitió analizar las diferentes fracciones proteicas que pudiesen tener una importancia nutricional. Los recuentos de BAM, CT, CF, mohos y levaduras en esta harina fueron bajos, lográndose un producto inocuo. Es importante destacar, que con la utilización de este recurso no convencional, se inicia un interesante campo de investigación en lo que se refiere al enriquecimiento de alimentos, representando una alternativa a nivel industrial.

Higiene sanitizaçäo e controle da qualidade no processamento de massas alimentícias / Higienic sanitition and quality control on processing of past alimentary

Verificou-se as condiçöes de produçäo de massas alimentícias em dez micro e pequenas empresas do Estado do Rio Grande do Sul. A partir dos resultados obtidos, foram elaborados e entregues, às empresas, pareceres individuais contendo informaçöes sobre a situaçäo encontrada e sugestöes para melhoria de instalaçöes e equipamentos. Paralelamente, através dos pareceres, elaborou-se diagnóstico do setor e um manual de higiene, sanitizaçäo e controle da qualidade. Concluída esta etapa, repassou-se as informaçöes do manual aos funcionários de vinte empresas através de palestras, distribuiçäo de cartilhas e cartazes, com o objetivo de colocar, em prática, as medidas recomendadas. Considera-se fundamental a implementaçäo de programas de controle da qualidade nas empresas, a fim de se obter significativa melhoria da qualidade higiênico-sanitária dos produtos comercializados