Inactivation of Alicyclobacillus acidoterrestris ATCC 49025 spores in apple juice by pulsed light: Influence of initial contamination and required reduction levels / Inactivación de esporas de Alicyclobacillus acidoterrestris ATCC 49025 por luz pulsada en jugo de manzana: Influencia de la contaminación inicial y de las reducciones requeridas

The purpose of this study was to analyze the response of different initial contamination levels of Alicydobadllus acidoterrestris ATCC 49025 spores in apple juice as affected by pulsed light treatment (PL, batch mode, xenon lamp, 3pulses/s, 0-71.6 J/cm²). Biphasic and Weibull frequency distribution models were used to characterize the relationship between inoculum size and treatment time with the reductions achieved after PL exposure. Additionally, a second order polynomial model was computed to relate required PL processing time to inoculum size and requested log reductions. PL treatment caused up to 3.0-3.5 log reductions, depending on the initial inoculum size. Inactivation curves corresponding to PL-treated samples were adequately characterized by both Weibull and biphasic models (R²d j 94-96%), and revealed that lower initial inoculum sizes were associated with higher inactivation rates. According to the polynomial model, the predicted time for PL treatment increased exponentially with inoculum size.

El objetivo del presente trabajo fue evaluar la influencia de la concentración de esporas de Alicyclobacillus acidoterrestris ATCC 49025 en la respuesta de inactivación por acción de la luz pulsada (modo estanco, lámpara de xenón, 3 pulsos/s, 0-71,6 J/cm²) en jugo de manzana comercial. Para caracterizar la relación existente entre la concentración de esporas y el tiempo de tratamiento con las reducciones logarítmicas alcanzadas luego de la exposición a la luz pulsada (LP), se aplicaron 2 modelos: el de Weibull y el bifásico. Adicionalmente, se estimó la relación entre el tiempo de tratamiento con LP y la concentración inicial de inoculo en el jugo con las reducciones logarítmicas logradas mediante regresión múltiple y la metodología de superficie de respuesta (MSR). La inactivación por LP provocó entre 3 y 3,5 reducciones logarítmicas, según la concentración inicial de esporas. Las curvas de inactivación fueron adecuadamente caracterizadas por los modelos matemáticos propuestos (Restado = 94-96%). El análisis por MSR permitió predecir un aumento exponencial del tiempo de tratamiento requerido conforme se incrementa el nivel de contaminación inicial.

Evaluation of different culture media and enrichment in orange juice upon the growth of Alicyclobacillus spp / Avaliação de diferentes meios de cultura e enriquecimento em suco de laranja sobre o crescimento de Alicyclobacillus spp

Bacteria of the genus Alicyiclobacillus spp. form spores and develop in acid media, leading to the spoilage of citrus juices. Brazil is the largest exporter of orange juice concentrate, and yet, it has been extensively studied due to changes in taste and smell. Several investigations have reported different culture media used to detect and enumerate Alicyiclobacillus spp. The objective of the present study was to evaluate the recovery of Alicyiclobacillus spp. spores grown in ALI, BAT, K agar and YSG media using the methodology suggested by ABECitrus. Five inocula were used, two from reference strains and three from pasteurized concentrated orange juice. Cell recovery after the enrichment in reconstituted orange juice was also analyzed. An initial population of 6 log CFU/mL was inoculated. ALI, BAT and YSG media were able to recover the initial population of all different inocula, with no significant differences between the results. When compared to BAT, however, the preparation of ALI and YSG media was simpler and had more advantages. The recovery with K agar medium was lower than the other media for all the tested inocula, with significant differences found for Alicyclobacillus acidocaldarius0298T (3.66 log CFU/mL) and Alicyclobacillus pomorum-like CBMAI 0278 (4.11 log CFU/mL).(AU)

As bactérias do gênero Alicyiclobacillus spp. formam esporos e se desenvolvem em meios ácidos, podendo causar deterioração em sucos cítricos. O Brasil é o maior exportador de suco de laranja concentrado do mundo e, assim, este gênero vem sendo estudado por causar alterações de odor e sabor. Vários estudos relatam diferentes meios de culturas empregados para a detecção e enumeração de Alicyclobacillus spp. Este estudo teve como objetivo avaliar a recuperação de esporos de Alicyclobacillus spp. nos meios ALI, BAT, K ágar e YSG, utilizando a metodologia indicada pela ABECitrus. Cinco inóculos diferentes foram utilizados, sendo dois de linhagens-referência e os outros três isolados de suco concentrado de laranja pasteurizado. Também foi verificada a recuperação das células após o enriquecimento em suco de laranja reconstituído. Foi inoculada uma população inicial de 6 log UFC/mL. Os meios ALI, BAT e YSG conseguiram recuperar esta população nos diferentes inóculos, não existindo diferenças significativas entre os resultados. Contudo, devido à facilidade do preparo, os meios ALI e YSG mostraram-se mais vantajosos quando comparados ao meio BAT. O meio K ágar apresentou recuperação inferior aos outros meios para todos os inóculos, porém, houve diferença significativa apenas para Alicyclobacillus acidocaldarius 0298 T (3,66 log UFC/mL) e Alicyclobacillus pomorum-like CBMAI 0278 (4,11 log UFC/mL).(AU)

Survey of molds, yeast and Alicyclobacillus spp. from a concentrated apple juice productive process

Bacteria and molds may spoil and/or contaminate apple juice either by direct microbial action or indirectly by the uptake of metabolites as off-flavours and toxins. Some of these microorganisms and/or metabolites may remain in the food even after extensive procedures. This study aim to identify the presence of molds (including heat resistant species) and Alicyclobacillus spp., during concentrated apple juice processing. Molds were isolated at different steps and then identified by their macroscopic and microscopic characteristics after cultivation on standard media at 5, 25 and 37ºC, during 7 days. Among the 19 isolated found, 63% were identified as Penicillium with 50% belonging to the P. expansum specie. With regards to heat resistant molds, the species Neosartorya fischeri, Byssochlamys fulva and also the genus Eupenicillium sp., Talaromyces sp. and Eurotium sp. were isolated. The thermoacidophilic spore-forming bacteria were identified as A. acidoterrestris by a further investigation based on 16S rRNA sequence similarity. The large contamination found indicates the need for methods to eliminate or prevent the presence of these microorganisms in the processing plants in order to avoid both spoilage of apple juice and toxin production.

Chemicals and lemon essential oil effect on Alicyclobacillus acidoterrestris viability

Alicyclobacillus acidoterrestris is considered to be one of the important target microorganisms in the quality control of acidic canned foods. There is an urgent need to develop a suitable method for inhibiting or controlling the germination and outgrowth of A.acidoterrestris in acidic drinks. The aim of this work was to evaluate the chemicals used in the lemon industry (sodium benzoate, potassium sorbate), and lemon essential oil as a natural compound, against a strain of A.acidoterrestris in MEB medium and in lemon juice concentrate. The results pointed out that sodium benzoate (500-1000-2000 ppm) and lemon essential oil (0.08- 0.12- 0.16%) completely inhibited the germination of A. acidoterrestris spores in MEB medium and LJC for 11 days. Potassium sorbate (600-1200 ppm) was more effective to inhibit the growth of the microbial target in lemon juice than in MEB medium. The effect of sodium benzoate, potassium sorbate and essential oil was sporostatic in MEB and LJC as they did not affect spore viability.

Propriedade antimicrobiana do eugenol frente às amostras de Alicyclobacillus spp. isoladas de suco de laranja / Antimicrobial properties of eugenol on Alicyclobacillus spp. isolated from orange juice

Foi avaliada a atividade antimicrobiana do eugenol contra Alicyclobacillus hesperidum, Alicyclobacillus cycloheptanicus, Alicyclobacillus acidiphilus, Alicyclobacillus acidoterrestris e de Alicyclobacillus spp., isolados em indústrias de sucos de laranja de várias regiões do Estado de São Paulo. O teste de suscetibilidade para o eugenol foi realizado determinando-se a concentração inibitória mínima (CIM)pela técnica de microdiluição em placas de 96 cavidades. A CIM do eugenol variou de 66,65 a >1066,4μg/mL, evidenciando-se a atividade inibitória forte e moderada frente a todas as amostras testadas e a atividade bactericida. Este estudo demonstrou ser significativa a fonte de isolamento das linhagens de Alicyclobacillus spp. nas indústrias. Conclui-se que o eugenol possui eficaz atividade antimicrobiana contra Alicyclobacillus spp. e sugere-se o uso deste composto para prevenir a deterioração de bebidas acidificadas e para diminuir o custo do controle microbiológico mais especificamente em suco de laranja.

Microbial modeling of thermal resistance of Alicyclobacillus acidoterrestris CRA7152 spores in concentrated orange juice with nisin addition / Modelagem microbiana da resistência térmica de esporos de Alicyclobacillus acidoterrestris CRA7152 em suco de laranja concentrado com adição de nisina

The nisin effect on thermal death of Alicyclobacillus acidoterrestris CRA 7152 spores in concentrated orange juice (64ºBrix) was studied. Concentrations of 0, 50, 75 and 100 IU of nisin/ml juice, at temperatures of 92, 95, 98 and 102ºC were evaluated. The quadratic polynomial model was used to analyze the effects of the factors and their interaction. Verification of surviving spores was carried out through plating in K medium (pH 3.7). The results showed that the D values without nisin addition were 25.5, 12.9, 6.1 and 2.3 min for 92, 95, 98 and 102ºC respectively. With addition of nisin into the juice there was a drop of heat resistance as the concentration was increased at a same temperature. With 30, 50, 75, 100 and 150 IU/ml at 95ºC, the D values were 12.34, 11.38, 10.49, 9.49 and 9.42 min respectively, showing that a decrease in the D value up to 27 percent can be obtained. The second order polynomial model established with r² = 0.995 showed that the microorganism resistance was affected by the action of temperature followed by the nisin concentration. Nisin therefore is an alternative for reducing the rigor of the A. acidoterrestris CRA 7152 thermal treatment.

Estudou-se o efeito da nisina na inativação térmica dos esporos de Alicyclobacillus acidoterrestris CRA 7152 em suco de laranja concentrado (64 ºBrix). Foram avaliadas as concentrações de 0, 50, 75 e 100 UI de nisina/ml de suco nas temperaturas de 92, 95, 98 e 102 ºC. Foi utilizado o modelo polinomial quadrático para analisar os efeitos dos fatores e suas interações. A contagem dos esporos sobreviventes foi feita através de plaqueamento em meio K (pH = 3,7). De acordo com os resultados obtidos encontrouse um valor de D sem adição de nisina de 25,5; 12,9; 6,1 e 2,3 min para as temperaturas de 92, 95, 98 e 102 ºC respectivamente. Quando a nisina foi adicionada ao suco observouse uma queda na resistência térmica em função do aumento da concentração de nisina para os mesmos valores de temperatura. Ao utilizar as concentrações de 30, 50, 75 e 150 IU/ml a 95 ºC, o valor de D obtido foi de 12,34; 11,38; 10,49; 9,49; e 9,42 min respectivamente demonstrando que a adição de nisina provoca um decréscimo de até 27 por cento no valor de D. O modelo polinomial de segunda ordem ajustado com r² = 0,995 mostrou que a resistência do microorganismo foi afetada pela temperatura seguida da concentração de nisina. A adição de nisina é, portanto, uma alternativa para reduzir o rigor do tratamento térmico em A. acidoterrestris CRA 7152.

Survey on and Phytogeny of Alicyclobacillus Species in Hot Springs of Southern China's Guangdong and Yunna Provinces / 微生物学通报

12 isolates of thermoacidophilies were obtained from samples of hot springs of Southern China's Guangdong and Yunna provinces. All isolates are heterotrophic. Cells are rods, Gram positive or variable. The optimal pH and temperature for growth are 3.5-5.5 and 43℃-52℃, respectively. Based on their morphological physiological properties and their 16S rDNA sequences, they were identified as members of Alicyclobacillus.