Viability, Sublethal Injury, and Release of Cellular Components From Alicyclobacillus acidoterrestris Spores and Cells After the Application of Physical Treatments, Natural Extracts, or Their Components

Alicyclobacillus acidoterrestris is a spoiling microorganism regarded as one of the most important causes of spoilage of fruit juices and acidic products. In this paper, four strains of A. acidoterrestris (type strain-DSM 3922; two wild strains isolated from soil-C8 and C24; wild strain isolated from a spoiled pear juice CB1) were treated through natural extracts/active compounds from essential oils (EOs), and physical treatments were used to assess their susceptibility and the presence of sublethal injury. The characterization of damage was also performed. The results suggest that it is possible to control A. acidoterrestris through alternative approaches, although the effect relied upon the age of spores. In addition to the mere antimicrobial effect, some treatments could cause a sublethal injury on spores. Lemon extract was the most effective treatment for both the antimicrobial effect and the sublethal injury, as evidenced by the release of proteins, and calcium dipicolinate [dipicolinic acid (DPA)] by fresh spores and only DPA (with an exception for C8) by old spores. A sublethal injury with protein release was also found for physical treatments [US (ultrasound) or heating]. For the first time, this paper reports on the existence of a sublethal injury for A. acidoterrestris, and this evidence could also be a challenge, because injured microorganisms could restore their metabolism, or an opportunity to design new preserving treatments.

Método alternativo e sustentável para a realização de coloração bacteriana de Gram e Wirtz-Conklin: Relevância ambiental e econômica no ensino prático da microbiologia

As preparações coradas são os exames microscópicos mais comumente empregados em microbiologia, pois proporcionam uma melhor visualização dos microrganismos. Tendo em vista que as técnicas de coloração de Gram e Wirtz-Conklin são comumente utilizadas na rotina de laboratórios de microbiologia e nas aulas de microscopias coradas, este projeto teve como objetivo comparar a técnica padrão de coloração em superfície das lâminas, com a técnica por imersão, que por sua vez pode evitar o desperdício de corantes, impacto ambiental e diminuir custos laboratoriais e no ensino. Dessa forma, foram feitos esfregaços bacterianos (Escherichia coli; Staphylococcus aureus; Alicyclobacillus acidoterrestris e Bacillus subtilis) em lâmina, seguidos de fixação em chama. Após esse procedimento as lâminas foram submetidas à coloração pela técnica de superfície e pela técnica de imersão. Posteriormente as lâminas foram visualizadas em microscópio óptico comum e a resolução das imagens e características tintoriais foram avaliadas e registradas. A técnica de coloração por imersão, tanto para a coloração de Gram quanto para Wirtz-Conklin, evitou desperdício dos corantes utilizados nas técnicas, bem como, a saturação das soluções após muitas passagens de lâminas. Assim, a técnica de coloração por imersão é favorável para o uso nas aulas ou nos laboratórios de microbiologia. A aplicação dessa técnica mostrou resultados significativos para a diminuição do desperdício e, consequentemente, os impactos ao meio ambiente e à economia das instituições de ensino.

Impact of Heating Rates on Alicyclobacillus acidoterrestris Heat Resistance under Non-Isothermal Treatments and Use of Mathematical Modelling to Optimize Orange Juice Processing

Alicyclobacillus acidoterrestris is a spoilage microorganism responsible for relevant product and economic losses in the beverage and juice industry. Spores of this microorganism can survive industrial heat treatments and cause spoilage during posterior storage. Therefore, an effective design of processing treatments requires an accurate understanding of the heat resistance of this microorganism. Considering that industrial treatments are dynamic; this understanding must include how the heat resistance of the microorganism is affected by the heating rate during the heating and cooling phases. The main objective of this study was to establish the effect of heating rates and complex thermal treatments on the inactivation kinetics of A. acidoterrestris. Isothermal experiments between 90 and 105 °C were carried out in a Mastia thermoresistometer, as well as four different dynamic treatments. Although most of the inactivation takes place during the holding phase, our results indicate the relevance of the heating phase for the effectiveness of the treatment. The thermal resistance of A. acidoterrestris is affected by the heating rate during the heating phase. Specifically, higher heating rates resulted in an increased microbial inactivation with respect to the one predicted based on isothermal experiments. These results provide novel information regarding the heat response of this microorganism, which can be valuable for the design of effective heat treatments to improve product safety and stability. Moreover, it highlights the need to incorporate experimental data based on dynamic treatments in process design, as heating rates can have a very significant effect on the thermal resistance of microorganisms.

AVALIAÇÃO DA ATIVIDADE ANTIMICROBIANA DE Stevia rebaudiana FRENTE AO Alicyclobacillus acidoterretris

Alicyclobacillus acidoterrestris é uma bactéria Gram-positiva, termoacidófila, formadora de esporos e deteriorante de bebidas ácidas. Extratos de plantas naturais vêm sendo investigados como alternativa de antimicrobiano, como a Stevia rebaudiana, onde estudos revelam que extratos de suas folhas possuem metabólitos com potencial bioativo. Dessa forma, o objetivo desse trabalho, foi avaliar a atividade antimicrobiana da Stevia rebaudiana frente ao A. acidoterrestris. O extrato etanólico das folhas de Stevia rebaudiana (EES) e fração hexano das folhas de Stevia rebaudiana (FHS) foram testados para verificar concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) frente ao A. acidoterrestris. Fez-se a microscopia eletrônica de varredura (MEV) do sub-CIM e CIM do EES e da FHS, e do controle (células de A. acidoterrestris não tratadas). A atividade inibitória encontrada foi de 31,25 μg/ml para EES e FHS testados contra o A. acidoterrestris. Enquanto que a CBM foi de 1000 μg/ml para ambos. Com a MEV foi possível observar os danos causados nas células vegetativas de A. acidoterrestris, além de verificar que houve redução do crescimento das células vegetativas ao utilizar EES e FHS. Conclui-se que os extratos testados foram efetivos contra A. acidoterrestris, demonstrando potencial biotecnológico como antimicrobiano natural para controle de suas células vegetativas em sucos de frutas cítricas industrializados.

Inactivation Effect of Thymoquinone on Alicyclobacillus acidoterrestris Vegetative Cells, Spores, and Biofilms

Alicyclobacillus acidoterrestris (A. acidoterrestris), a spore-forming bacterium, has become a main challenge and concern for the juices and acid beverage industry across the world due to its thermo-acidophilic characteristic. Thymoquinone (TQ) is one of the active components derived from Nigella sativa seeds. The objective of this study was to investigate antibacterial activity and associated molecular mechanism of TQ against A. acidoterrestris vegetative cells, and to evaluate effects of TQ on A. acidoterrestris spores and biofilms formed on polystyrene and stainless steel surfaces. Minimum inhibitory concentrations of TQ against five tested A. acidoterrestris strains ranged from 32 to 64 μg/mL. TQ could destroy bacterial cell morphology and membrane integrity in a concentration-dependent manner. Field-emission scanning electron microscopy observation showed that TQ caused abnormal morphology of spores and thus exerted a killing effect on spores. Moreover, TQ was effective in inactivating and removing A. acidoterrestris mature biofilms. These findings indicated that TQ is promising as a new alternative to control A. acidoterrestris and thereby reduce associated contamination and deterioration in the juice and acid beverage industry.