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The production of whole-liquid eggs is of significant economic and nutritional importance. This study aimed to assess the phenotypic and genotypic diversity of mesophilic aerobic spore-forming bacteria (n = 200) isolated from pasteurized whole liquid egg and liquid egg yolk. The majority of the isolates were identified as belonging to the genera Bacillus (86 %), followed by Brevibacillus (10 %) and Lysinibacillus (4 %). For the phenotypic characterization, isolates were subjected to various heat shocks, with the most significant reductions observed at 80 °C/30 min and 90 °C/10 min for isolates recovered from raw materials. On the other hand, the decrease was similar for isolates recovered from raw material and final product at 100 °C/5 min and 110 °C/5 min. Genotypic genes related to heat resistance (cdnL, spoVAD, dacB, clpC, dnaK, and yitF/Tn1546) were examined for genotypic characterization. The dnaK gene showed a positive correlation with the highest thermal condition tested (110 °C/5 min), while 100 °C/5 min had the highest number of positively correlated genes (clpC, cdnL, yitF/Tn1546, and spoVAD). Whole Genome Sequencing of four strains revealed genes related to sporulation, structure formation, initiation and regulation, stress response, and DNA repair in vegetative cells. The findings of this study indicate that these mesophilic aerobic spore-forming bacteria may adopt several strategies to persist through the process and reach the final product. As the inactivation of these microorganisms during egg processing is challenging, preventing raw materials contamination and their establishment in processing premises must be reinforced.

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Escherichia coli harboring a transmissible locus of stress tolerance (tLST) and the ability to form biofilms represent a serious risk in dairy production. Thus, we aimed to evaluate the microbiological quality of pasteurized milk from two dairy producers in Mato Grosso, Brazil, with a focus on determining the possible presence of E. coli with heat resistance (60 °C/6 min), biofilm-forming potential phenotypes and genotypes, and antimicrobial susceptibility. For this, fifty pasteurized milk samples from producers named A and B were obtained for 5 weeks to investigate the presence of Enterobacteriaceae members, coliforms, and E. coli. For heat resistance, E. coli isolates were exposed to a water bath at 60 °C for 0 and 6 min. In antibiogram analysis, eight antibiotics belonging to six antimicrobial classes were analyzed. The potential to form biofilms was quantified at 570 nm, and curli expression by Congo Red was analyzed. To determine the genotypic profile, we performed PCR for the tLST and rpoS genes, and pulsed-field gel electrophoresis (PFGE) was used to investigate the clonal profile of the isolates. Thus, producer A presented unsatisfactory microbiological conditions regarding Enterobacteriaceae and coliforms for weeks 4 and 5, while all samples analyzed for producer B were contaminated at above-the-limit levels established by national and international legislation. These unsatisfactory conditions enabled us to isolate 31 E. coli from both producers (7 isolates from producer A and 24 isolates from producer B). In this way, 6 E. coli isolates (5 from producer A and 1 from producer B) were highly heat resistant. However, although only 6 E. coli showed a highly heat-resistant profile, 97% (30/31) of all E. coli were tLST-positive. In contrast, all isolates were sensitive to all antimicrobials tested. In addition, moderate or weak biofilm potential was verified in 51.6% (16/31), and the expression of curli and presence of rpoS was not always related to this biofilm potential. Therefore, the results emphasize the spreading of heat-resistant E. coli with tLST in both producers and indicate the biofilm as a possible source of contamination during milk pasteurization. However, the possibility of E. coli producing biofilm and surviving pasteurization temperatures cannot be ruled out, and this should be investigated.

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Current technology that enables bioethanol production from agricultural biomass imposes harsh conditions for Saccharomyces cerevisiae's metabolism. In this work, the genetic architecture of industrial bioethanol yeast strain SA-1 was evaluated. SA-1 segregant FMY097 was previously described as highly aldehyde resistant and here also as thermotolerant: two important traits for the second-generation industry. A Quantitative Trait Loci (QTL) mapping of 5-hydroxymethylfurfural (HMF) -resistant segregants of hybrid FMY097/BY4742 disclosed a region in chromosome II bearing alleles with uncommon non-synonymous (NS) single nucleotide polymorphisms (SNPs) in FMY097: MIX23, PKC1, SEA4, and SRO77. Allele swap to susceptible laboratory strain BY4742 revealed that SEA4FMY097 enhances robustness towards HMF, but the industrial fitness could not be fully recovered. The genetic network arising from the causative genes in the QTL window suggests that intracellular signaling TOR (Target of Rapamycin) and CWI (Cell Wall Integrity) pathways are regulators of this phenotype in FMY097. Because the QTL mapping did not result in one major allelic contribution to the evaluated trait, a background effect in FMY097's HMF resistance is expected. Quantification of NADPH - cofactor implied in endogenous aldehyde detoxification reactions - supports the former hypothesis, given its high availability in FMY097. Regarding thermotolerance, SEA4FMY097 grants BY4742 ability to grow in temperatures as high as 38 ºC in liquid, while allele PKC1FMY097 allows growth up to 40 ºC in solid medium. Both SEA4FMY097 and PKC1FMY097 encode rare NS SNPs, not found in other > 1013S. cerevisiae. Altogether, these findings point towards crucial membrane and stress mediators for yeast robustness.

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In our study, we analyzed 30years of climatological data revealing the bean production risks for Western Amazonia. Climatological profiling showed high daytime and nighttime temperatures combined with high relative humidity and low vapor pressure deficit. Our understanding of the target environment allows us to select trait combinations for reaching higher yields in Amazonian acid soils. Our research was conducted using 64 bean lines with different genetic backgrounds. In high temperatures, we identified three water use efficiency typologies in beans based on detailed data analysis on gasometric exchange. Profligate water spenders and not water conservative accessions showed leaf cooling, and effective photosynthate partitioning to seeds, and these attributes were found to be related to higher photosynthetic efficiency. Thus, water spenders and not savers were recognized as heat resistant in acid soil conditions in Western Amazonia. Genotypes such as BFS 10, SEN 52, SER 323, different SEFs (SEF 73, SEF 10, SEF 40, SEF 70), SCR 56, SMR 173, and SMN 99 presented less negative effects of heat stress on yield. These genotypes could be suitable as parental lines for improving dry seed production. The improved knowledge on water-use efficiency typologies can be used for bean crop improvement efforts as well as further studies aimed at a better understanding of the intrinsic mechanisms of heat resistance in legumes.

RESUMO

This study found variability in the time required for tubes of media in heating block wells to reach 60 °C, resulting in significant effects on heat resistance measurements. To determine the extent that methodology changed heat resistance measurements, we compared the heat resistance of Shiga-toxin producing Escherichia coli (STEC) strains with and without the locus of heat resistance (LHR) using both heating block and water bath methods. A total of 34 strains of STEC were used along with a generic E. coli which has been identified as heat-resistant and used as a positive control. The E. coli strains were incubated in a water bath and a heating block set at 60 °C to determine come up time to 60 °C (T0) and for 6 additional minutes (T6) to calculate the D60 value. After incubation, the colony forming units (CFU) were enumerated and mean log CFU/mL from biological replicates was calculated. To compare reductions from T0 to T6, standard deviations among replicates within heating method and correlation of the D60 values generated across methods were determined using Mixed model and Correlation analyses. Our findings indicate that the method chosen to evaluate heat resistance of E. coli can dramatically influence results as there was not a significant correlation between D60 values for the same isolate determined by water bath and heating block methods. The water bath method generates more reliable and consistent heat resistance data and should be used in future evaluations of heat resistance in E. coli. Moreover, PCR screening for the LHR would only be moderately useful for predicting phenotypic heat-resistance of E. coli. Considering water bath data only, LHR-positive STEC isolates were either moderately heat-resistant (1 to 5 log reduction) or heat-sensitive (> 5 log reduction). As LHR-negative STEC were also moderately heat-resistant, prediction of phenotypic heat resistance from genotype requires further refinement.

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AIMS: First, two inactivation models were compared for different phenotypic profiles of Escherichia coli O26 using ultraviolet-C light (UV-C) and thermal treatment (T), by means of Central Composite Rotatable Design of Experiment (CCRD). Second, we aimed to evaluate the subsequent survival and persistence of cells in simulated gastric fluid (SGF). METHODS AND RESULTS: Two strains of E. coli O26, a wild-type strain and a clinical ATCC strain were used in both steps. A CCRD was used in a 22 arrangement in random order. The goodness-of-fit of the models was determined. The lack of fit, and the normality of residual data were checked with the Shapiro-Wilk test, and the model accuracy factor, bias factor and the model mean square error (MSE) were measured. Subsequently, the resistance capacity of the strains was evaluated after exposure to simulated gastric acid. The CCRD results obtained indicate that the mild heat (<70°C) has a recovery effect. In addition, for the clinical strain, the UV-C and heat (above 70°C) has an additive inactivation effect. Moreover, temperature (65°C) induced SGF resistance by the wild-type and clinical strain. For the clinical strain, cells exposed to UV-C were more sensitive to SGF. In contrast to clinical strain, exposing cells of the wild-type strain to UV-C increased the survival capacity in the SGF. CONCLUSION: Response surface analyses showed that the wild-type O26 strain has higher persistence under unfavourable conditions than the clinical strain, and the stresses caused by applied microbial control technologies can increase the survival capacity in the SGF. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shed light on different phenotypic responses in the same bacterium serogroup. Moreover, the impact of the study was that strain selection criteria must be adequate to develop effective models of inactivation.

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Lentinula edodes (Berk.) Pegler (shiitake) é o segundo cogumelo mais consumido no mundo e sua produção pode ser feita em toras de madeira ou em cultivo axênico utilizando resíduos agroindustriais como substrato. O cultivo do shiitake no Brasil tem sido gravemente prejudicado pela alta incidência de fungos contaminantes, como Trichoderma sp., e pela ausência de linhagens mais adaptadas às temperaturas tropicais. Duas linhagens resistentes ao Trichoderma sp. foram obtidas por meio de seleção quando crescidas em contato com o fungo contaminante e, por meio de fragmentação micelial, foram obtidas linhagens resistentes a temperaturas elevadas, capazes de crescer a 30º C em meio de cultura.

Lentinula edodes (Berk.) Pegler (shiitake) is the second most consumed mushroom in the world and its production can be made in natural logs or in axenic culture using agroindustrial residues as substrate. The cultivation of shiitake in Brazil has been seriously harmed by the high incidence of contaminant fungi such as Trichoderma sp. and by the absence of strains adapted to tropical temperatures. Two strains resistant to Trichoderma sp. were obtained by selection when grown in contact with the contaminant fungus and, by mycelial fragmentation, strains were obtained that can resist high temperatures and grow at 30º C in a culture medium.

RESUMO

The objective of this study was to isolate lactic acid bacteria (LAB) from vacuum packaged beef and to investigate their antagonist activity. LAB mean counts of 5.19 log cfu/cm² were obtained from five samples of vacuum packaged beef. Two hundred isolates were selected and screened for the inhibitory effect on five ATCC reference Lactobacillus strains. Thirty six isolates showed activity in the agar spot test against at least two of the indicator strains. However, only six cell free supernatants (CFS) from these isolates exhibited activity against the indicator strains using the well-diffusion test and conditions that eliminated the effects of organic acids and hydrogen peroxide. L. acidophilus was the most sensitive indicator tested, whereas L. plantarum and L. fermentum were the most resistant ones. Identification by MIDI system indicated that these LAB isolates were Lactococcus lactis subsp. cremoris, Pediococcus acidilactici, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus casei GC subgroup A. The antagonistic factors produced by most of these LAB against L. acidophilus were resistant to heat treatment (100ºC for 10 min) and stable over a wide pH range (4.0 to 9.0). These data suggest that these isolates could be used as promising hurdles aiming increased safety and extended shelf life of meat products.

Este trabalho teve como objetivo isolar bactérias ácido láticas (BAL) de carne bovina embalada a vácuo e investigar sua atividade antimicrobiana. Uma média de 5,19 log ufc/cm² BAL foi obtida de cinco amostras. Duzentos isolados foram selecionados e o efeito inibitório sobre cinco cepas ATCC referência de Lactobacillus foi pesquisado. Trinta e seis isolados apresentaram atividade inibitória pelo teste 'agar spot' de pelo menos duas das cepas indicadoras. Sobrenadantes isentos de células (SIC) destes isolados foram obtidos. Apenas seis SIC apresentaram atividade contra as cepas indicadoras usando o método de difusão em poços e condições que eliminaram a influência do peróxido de hidrogênio e de ácidos orgânicos. L. acidophilus foi a cepa indicadora mais sensível, enquanto L. plantarum e L. fermentum foram as mais resistentes. Identificação pelo sistema MIDI indicaram que as BAL isoladas eram Lactococcus lactis subsp. cremoris, Pediococcus acidilactici, Lactobacillus delbrueckii subsp. bulgaricus e Lactobacillus casei GC subgrupo A. O efeito antagonista ao L. acidophilus dos SIC destas BAL foi resistente ao tratamento térmico (100ºC/10 min) e estável em ampla faixa de pH (4,0 a 9,0). Estes dados sugerem que os isolados obtidos poderiam ser utilizados como barreiras promissoras no aumento da segurança e na extensão da vida útil de produtos cárneos.

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For heat-resistance derterminations, a Neosartorya fischeri ascospore suspension, obtained at three different conditions of temperature (20, 30 and 35ºC) and age (1, 2 and 3 months), was heat treated in pineapple juice with three different ratio conditions. The 1/k (min) values obtained were statistically analysed through the software Statisitca 6.0. At 80ºC, when there was a level change in the ratio factor from a lower (10) to a higher one (38), the thermal resistance (1/k) increased in approximately 41 per cent. The increase of the ascospore production age and temperature caused an increase on the thermal resistance approximately equal to 28 per cent and 14 per cent, respectively. Throufh the results, it could be concluded that N. fischeri presented lower thermal resistance for heating mediums with lower ratio values, or rather, the increase of the soluble solids concentration caused an increase of its thermal resistance. It was also observed that the ascospore production age and temperature increased significantly the thermal resistance of this mould in pineapple juice.

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The objective of this study was to isolate lactic acid bacteria (LAB) from vacuum packaged beef and to investigate their antagonist activity. LAB mean counts of 5.19 log cfu/cm(2) were obtained from five samples of vacuum packaged beef. Two hundred isolates were selected and screened for the inhibitory effect on five ATCC reference Lactobacillus strains. Thirty six isolates showed activity in the agar spot test against at least two of the indicator strains. However, only six cell free supernatants (CFS) from these isolates exhibited activity against the indicator strains using the well-diffusion test and conditions that eliminated the effects of organic acids and hydrogen peroxide. L. acidophilus was the most sensitive indicator tested, whereas L. plantarum and L. fermentum were the most resistant ones. Identification by MIDI system indicated that these LAB isolates were Lactococcus lactis subsp. cremoris, Pediococcus acidilactici, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus casei GC subgroup A. The antagonistic factors produced by most of these LAB against L. acidophilus were resistant to heat treatment (100°C for 10 min) and stable over a wide pH range (4.0 to 9.0). These data suggest that these isolates could be used as promising hurdles aiming increased safety and extended shelf life of meat products.

RESUMO

The influence of the plating medium used for the recovery of survivors, on the D-values for L. monocytogenes, in formulations for beef hamburger was investigated: 10% fat (F1), 20% fat (F2), 10% fat and 1.5% sodium chloride (F3) and 10% fat and 10.5% hydrated texturized soy protein (F4) were prepared and inoculated with a mixture of five strains of L. monocytogenes. Using tryptose phosphate agar containing 1% sodium pyruvate (TPAP) D-values (min) ranged from 36.11 (F1) to 62.76 (F3) at 55.0C, from 2.55 (F2) to 4.32 (F3) at 60.0C and from 0.34 (F1 e F2) to 0.52 (F3) at 65.0C. The D-values, using lithium chloride phenylethanol moxalactam medium (LPM) for enumeration, ranged from 23.05 (F1) to 38.54 (F3) at 55.0C; from 1.81 (F2) to 3.06 (F3) at 60.0C and from 0.25 (F2) to 0.37 (F3) at 65.0C. The z-values (C), using the TPAP recovery data, ranged from 4.81 (F3) to 4.95 (F4) and from 4.95 (F3) to 5.21 (F4) using the LPM recovery data. The authors concluded that the heat resistance of L. monocytogenes in formulations for beef hamburger can be underestimated when LPM is used as the recovery medium.

A influência do meio de cultura na recuperação da população sobrevivente sobre os valores D para L. monocytogenes foi avaliada em formulações de hambúrguer bovino: 10% de gordura (F1), 20% de gordura (F2), 10% de gordura e 1,5% de cloreto de sódio (F3) e 10% de gordura e 10,5% de proteína de soja texturizada hidratada (F4), inoculadas com suspensão mista de 5 cepas de L. monocytogenes. Empregando-se ágar triptose fosfato + 1% de piruvato de sódio (ATFP) os valores D (min) variaram de 36,11 (F1) a 62,76 (F3) a 55,0C, de 2,55 (F2) a 4,32 (F3) a 60,0C e de 0,34 (F1 e F2) a 0,52 (F3) a 65,0C. Os valores D calculados utilizando-se ágar cloreto de lítio feniletanol moxalactam (LFM) variaram de 23,05 (F1) a 38,54 (F3) a 55,0C; 1,81 (F2) a 3,06 (F3) a 60,0C e de 0,25 (F2) a 0,37 (F3) a 65,0C. Os valores z (C) variaram de 4,81 (F3) a 4,95 (F4) com ATFP e 5,08 (F2) a 5,21 (F4) com LFM. Os autores concluíram que a resistência térmica de L. monocytogenes em formulações para hambúrguer bovino foi subestimada até 1,6-1,7 vezes quando o meio LFM foi utilizado.

RESUMO

The objective of this study was to isolate lactic acid bacteria (LAB) from vacuum packaged beef and to investigate their antagonist activity. LAB mean counts of 5.19 log cfu/cm² were obtained from five samples of vacuum packaged beef. Two hundred isolates were selected and screened for the inhibitory effect on five ATCC reference Lactobacillus strains. Thirty six isolates showed activity in the agar spot test against at least two of the indicator strains. However, only six cell free supernatants (CFS) from these isolates exhibited activity against the indicator strains using the well-diffusion test and conditions that eliminated the effects of organic acids and hydrogen peroxide. L. acidophilus was the most sensitive indicator tested, whereas L. plantarum and L. fermentum were the most resistant ones. Identification by MIDI system indicated that these LAB isolates were Lactococcus lactis subsp. cremoris, Pediococcus acidilactici, Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus casei GC subgroup A. The antagonistic factors produced by most of these LAB against L. acidophilus were resistant to heat treatment (100ºC for 10 min) and stable over a wide pH range (4.0 to 9.0). These data suggest that these isolates could be used as promising hurdles aiming increased safety and extended shelf life of meat products.

Este trabalho teve como objetivo isolar bactérias ácido láticas (BAL) de carne bovina embalada a vácuo e investigar sua atividade antimicrobiana. Uma média de 5,19 log ufc/cm² BAL foi obtida de cinco amostras. Duzentos isolados foram selecionados e o efeito inibitório sobre cinco cepas ATCC referência de Lactobacillus foi pesquisado. Trinta e seis isolados apresentaram atividade inibitória pelo teste 'agar spot' de pelo menos duas das cepas indicadoras. Sobrenadantes isentos de células (SIC) destes isolados foram obtidos. Apenas seis SIC apresentaram atividade contra as cepas indicadoras usando o método de difusão em poços e condições que eliminaram a influência do peróxido de hidrogênio e de ácidos orgânicos. L. acidophilus foi a cepa indicadora mais sensível, enquanto L. plantarum e L. fermentum foram as mais resistentes. Identificação pelo sistema MIDI indicaram que as BAL isoladas eram Lactococcus lactis subsp. cremoris, Pediococcus acidilactici, Lactobacillus delbrueckii subsp. bulgaricus e Lactobacillus casei GC subgrupo A. O efeito antagonista ao L. acidophilus dos SIC destas BAL foi resistente ao tratamento térmico (100ºC/10 min) e estável em ampla faixa de pH (4,0 a 9,0). Estes dados sugerem que os isolados obtidos poderiam ser utilizados como barreiras promissoras no aumento da segurança e na extensão da vida útil de produtos cárneos.