Thesis (PhD (Food Science))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Species of Alicyclobacillus are acid-tolerant and heat-resistant bacteria that cause spoilage of heat-treated fruit juices stored at room temperature. During the past decade, Alicyclobacillus spp. have become a major cause of spoilage in pasteurised fruit juices leading to significant economic losses world-wide. Spoilage has been reported in apple, pear, orange, peach, mango and white grape juice, as well as in fruit juice blends, fruit juice containing drinks and tomato products, such as tomato juice and canned tomatoes. Spoilage is characterised by a medicinal smell and guaiacol production. These endospore-formers have been shown to survive pasteurisation conditions of 95 °C for 2 min, grow at temperatures between 25° and 60 °C and a pH range of 2.5 to 6.0. Knowledge of this organism is limited, both locally and internationally and the route of contamination to the final product is not well established. In this study the fruit concentrate processing environment was investigated as a potential source and route of contamination for the final product. Species of Alicyclobacillus were isolated from orchard soil, various stages during processing and from fruit juice and concentrates. The isolates were identified based on morpholological, biochemical and physiological properties. Identification to species level was done by 16S ribosomal RNA gene sequencing and strain differentiation by RAPD-PCR. Results indicate that species of A. acidoterrestris and Alicyclobacillus acidocaldarius were found in orchard soil and throughout the processing environment. This is the first report on the isolation of these species from orchard soil, vinegar flies and the fruit processing environment. The 16 isolates identified as A. acidoterrestris grouped into four clusters based on RAPD-PCR banding patterns, suggesting that they belong to at least four genotypic groups. Isolates from the fruit concentrate, wash water and soil located outside of the fruit processing plant grouped into one cluster. Concluded from these results, A. acidoterrestris found in the wash water and soil outside of the factory could act as a potential reservoir of organisms for the contamination of the final fruit concentrate. Thus good manufacturing practices play an essential role in controlling incidence of spoilage caused by these bacteria. Fruit juices can be treated using ultraviolet (UV-C) light with a wavelength of 254 nm, which has a germicidal effect against micro-organisms. Alicyclobacillus acidoterrestris spores were inoculated into tap water, used wash water from a fruit processing plant and grape juice concentrate. Ultraviolet dosage levels (J L−1) of 0, 61, 122, 183, 244, 305 and 367 were applied using a novel UV-C turbulent flow system. The UV treatment method was shown to reliably achieve in excess of a 4 log10 reduction (99.99%) per 0.5 kJ L-1 of UV-C dosage in all the liquids inoculated with A. acidoterrestris. The applied novel UV technology could serve as an alternative to thermal treatments of fruit juices for the inactivation of Alicyclobacillus spores or in the treatment of contaminated processing wash water. Finally, the thermal inactivation at 95 °C for two strains of A. acidoterrestris isolated from contaminated fruit juice concentrates were investigated in a 0.1% (m/v) peptone buffer solution (pH 7.04) and grape juice (pH 4.02, 15.5 °Brix). The thermal inactivation of A. acidoterrestris spores followed first-order kinetics, suggesting that as the microbial population is exposed to a specific high temperature, the spores inactivated at a constant rate. D-values determined in the buffer solution were calculated to be 1.92 min and 2.29 min, while in grape juice D-values were found to be 2.25 min and 2.58 min for the two strains tested. From this study it is clear that the D-value is dependant on the strain tested, but also on the soluble solids of the solution the cells are suspended in. The results indicated that the spores of A. acidoterrestris isolated from South African fruit juice concentrate may survive after the pasteurisation treatment commonly applied during manufacturing. / AFRIKAANSE OPSOMMING: Spesies van Alicyclobacillus is suur-tolerante en hittebestande bakterieë wat bederf veroorsaak in hitte-behandelde vrugtesappe wat teen kamertemperatuur gestoor word. Gedurende die afgelope dekade het Alicyclobacillus spp. ‘n belangrike oorsaak van bederf in gepasteuriseerde vrugtesappe geword en beduidende ekonomiese verliese wêreldwyd veroorsaak. Bederf is aangeteken in appel-, peer-, lemoen-, perske-, mango- en witdruiwesap, sowel as in vrugtesapversnitte, vrugtesapbevattende drankies en in tamatieprodukte soos tamatiesap en ingemaakte tamaties. Bederf word gekenmerk deur ’n medisinale reuk en guaiacol produksie. Daar is gevind dat hierdie endospoorvormers pasteurisasie teen 95 °C vir 2 min kan oorleef en kan groei by temperature tussen 25° en 60 °C en ‘n pH van 2.5 to 6.0. Plaaslik sowel as internasionaal is kennis van hierdie organisme beperk en die roete van kontaminasie van produkte is nog nie goed vasgestel nie. In hierdie studie is die vrugtekonsentraat-verwerkingsmilieu ondersoek as ‘n moontlike bron en roete van kontaminasie van die finale produk. Spesies van Alicyclobacillus is vanuit vrugteboordgrond, verskeie verwerkingstadia en van vrugtesap en vrugtesapkonsentraat geïsoleer. Die isolate is op grond van morfologiese, biochemiese en fisiologiese eienskappe geïdentifiseer. Identifikasie tot spesiesvlak is deur 16S rDNS sekwensering gedoen en stam differensiasie deur RAPD-PKR. Resultate het aangetoon dat A. acidoterrestris en A. acidocaldarius in vrugteboordgrond sowel as in alle stadia van die verwerkingsmilieu voorkom. Dit is die eerste verslag van die isolering van hierdie spesies uit die Suid-Afrikaanse vrugteverwerkingsmilieu, vrugteboordgrond en asynvlieë. Die 16 isolate, geïdentifiseer as A. acidoterrestris en in vier groepe geplaas op grond van hul RAPD-PKR bandpatrone, dui aan dat hulle aan minstens vier genotipiese groepe behoort. Isolate afkomstig van die vrugtekonsentraat, waswater en die grond buitekant die vrugteverwerkingsaanleg het een groep gevorm. Uit hierdie resultate kan afgelei word dat A. acidoterrestris, wat in die waswater en grond buite die aanleg voorkom, as ‘n moontlike bron van organismes vir die kontaminering van die finale vrugtekonsentraat kan dien. Goeie vervaardigingspraktyke speel dus ‘n noodsaaklike rol in die beheer van bederf veroorsaak deur hierdie bakterieë. Vrugtesappe kan behandel word met ultravioletlig (UV-C) met ‘n golflengte van 254 nm wat ‘n dodende effek op mikro-organismes het. Kraanwater, gebruikte waswater van ‘n vrugtesapvervaardigingsaanleg en druiwesapkonsentraat is met A. acidoterrestris spore geïnokuleer. Ultraviolet toedieningsvlakke (J L−1) van 0, 61, 122, 183, 244, 305 en 367 is aangewend met behulp van ‘n nuwe UV-C drukvloei stelsel. Daar is aangetoon dat die UV-behandelingsmetode ‘n betroubare vermindering (99.99%) van meer as 4 log10 per 0.5 kJ L-1 van ‘n UV-C dosis gee in al die vloeistowwe wat geïnokuleer is met A. acidoterrestris. Die toegepaste nuwe UV-tegnologie kan gebruik word as ‘n alternatief tot die hittebehandeling van vrugtesap vir die deaktivering van Alicyclobacillus spore of in die behandeling van gekontamineerde waswater. Ten slotte is hitte-deaktivering teen 95 °C van twee stamme van A. acidoterrestris, geïsoleer uit gekontamineerde vrugtesapkonsentraat, in ‘n 0.1% (m/v) peptoonbufferoplossing (pH 7.04) en druiwesap (pH 4.02, 15.5 °Brix), ondersoek. Die hitte-deaktivering van A. acidoterrestris spore het eerste-orde kinetika gevolg, wat aandui dat die mikrobe-populasie teen ‘n konstante tempo afsterf, wanneer blootgestel aan ‘n spesifieke hoë temperatuur. Die D-waardes in die bufferoplossing is bereken as 1.92 min en 2.29 min, terwyl daar gevind is dat die D-waardes in druiwesap 2.25 min en 2.58 min is vir die twee betrokke stamme. Vanuit hierdie studie is dit duidelik dat die D-waardes afhang van die betrokke stam, maar ook van die oplosbare vaste stowwe van die oplossing waarin die selle opgelos is. Die resultate dui daarop dat die spore van A. acidoterrestris, wat geïsoleer is uit Suid-Afrikaanse vrugtesapkonsentraat, die pasteurisasiebehandeling wat algemeen tydens vervaardiging toegepas word, kan oorleef. Aangesien die toepassing van strenger hittebehandeling om spore van A. acidoterrestris te deaktiveer onaanvaarbare organoleptiese veranderinge in die produk tot gevolg het, word dit aanbeveel dat die risiko van bederf verminder behoort te word deur die gebruik van goeie vervaardigingspraktyke gedurende vrugteverwerking.

O suco de laranja possui características que dificultariam ou mesmo impediriam o crescimento de microrganismos, mesmo assim já foram isolados fungos filamentosos, leveduras, bem como bactérias lácticas e termorresistentes. Dentre as bactérias termorresistentes e deteriorantes está a Alicyclobacillus acidoterrestris que tem causado prejuízos no setor de suco de laranja concentrado e congelado. Esta bactéria está apta a crescer em temperaturas abaixo de 35°C, constituindo um risco aos sucos de laranja que estejam contaminados. Mesmo os que já passaram por tratamento térmico, se forem estocados em local sem refrigeração, podem sofrer deterioração. Como os métodos tradicionais de descontaminação e conservação não têm se mostrado eficientes na inviabilização dessa bactéria, outras formas de esterilização tornam-se necessárias, dentre elas destacam-se as radiações gama e ultra-sônica. A radiação gama é capaz de esterilizar alimentos e reduzir as cargas microbiológicas, permitindo assim ampliar o período de armazenamento. A aplicação de ultra-som tem sido utilizada atualmente com o objetivo de controle microbiológico, sendo essa técnica eficiente por causar a destruição das células microbianas. Sendo o Brasil o maior produtor e exportador de suco de laranja concentrado congelado, no presente trabalho foram conduzidos dois experimentos com o objetivo de determinar a resistência da bactéria A. acidoterrestris às radiações gama e ultra-sônica, em suco de laranja previamente contaminado. As amostras de suco de laranja foram diluídas a 11,5 °Brix e inoculadas com uma suspensão bacteriana, passando pelos processos de radiação gama com as doses de 0, 2; 4; 6 e 8, kGy e ultra-sônica com as freqüências de 25, 35 e 42 kHz e com os tempos de exposição de 0, 1, 5, 10 e 20 minutos. Após o tratamento, as amostras foram armazenadas em temperatura ambiente (25±2°C) e de refrigeração (4±1°C). Para as análises microbiológicas as amostras foram diluídas em escala decimal, plaqueadas pela técnica de \"pour plate\". As placas de Petri permaneceram em estufa a46±2°C, por 48 horas, após esse período procedeu-se a contagem das UFC mL-1. A qualidade microbiológica do suco foi avaliada em determinados períodos de armazenamento (1, 10, 20, 30 e 40 dias). Através de análise de variância dos experimentos conduzidos com as radiações, observou-se que houve efeito significativo (p<0,05) com relação à dose de radiação gama que apresentou uma redução da carga microbiana com o aumento da dose de radiação e à interação do período com a temperatura de armazenamento demonstrou que houve uma oscilação na contagem total para a temperatura ambiente, mas para a temperatura de refrigeração uma diminuição gradativa com o aumento do período de armazenamento, A freqüência do ultra-som mostrou uma diminuição da contagem microbiana com o aumento da freqüência e o período de armazenamento apresentou a maior contagem no primeiro dia. Os dois tratamentos mostraram-se eficientes na redução bacteriana, mas não na completa eliminação da A. acidoterrestris. / The orange juice has attributes could make that hard or even block the growth. of microorganisms, even so already have been isolated filamentous fungi, yeast, as well lactics and heat resistant bacteria. Alicyclobacillus acidoterrestris is a heat resistant bacteria that has done damage in the orange juice concentrated and frozen business. This bacterium is able the grown in temperatures below 35°C constituting a risk to orange juices that have been contaminated. Even those that already pass trough thermal treatment may deteriorated, if they are storage in place without refrigeration. As the traditional methods of decontamination and conservation have not been effective in inviabilizated this bacterium, others methods of sterilization are need, among them are gamma and ultrasound radiation. The gamma radiation is able to sterilize foods and reduce the microbiology density, allowing in this way to enhance the period of storage. The ultrasound application has been used currently with the aim to microbiology control, been this technique effective in the microbial cell destruction. The Brazil, are the major producer and exporter of concentrated frozen orange juice, due to it, the present work was carried out to two experiments with the aim to determinate the resistance of the bacterium Alicyclobacillus acidoterrestris to gamma and ultrasound irradiation, in the orange juice, priory contaminated. The orange juice samples was diluted to 11,5°Brix and the bacteria suspensions was added, passed by process of radiation gamma with dose of 0, 2, 4, 6 and 8 kGy and ultrasonic with the frequency of 25, 35 and 42 kHz and with the time of exposition of 0, 1, 5, 10 and 20 minutes. After the treatment, the samples were storaged in room temperature (25±2°C) and of refrigeration (4±1°C). For the microbial analyses, the samples were diluted in to decimal scale, plated by \"pour plate\" technique. The Petri plates were storage in warn temperature (46±2°C) by 48 hours, after it the UFC was counted mL. The microbiological quality of the juice was evaluated in determinated period of storaged (1, 10, 20, 30 e 40 days). By statistical of variance of the experiment conducted with the radiations, it was noted that has been significant effect (p>0,05) to gamma radiation doses where occurred microbial density reduction with the raised of radiation doses and the interaction of the period with the storage temperature, where at room temperature occurred a oscillation in the total counting of bacteria, but for refrigeration temperature the total counting of bacteria reduced with the raised of storage period. The ultrasound frequency showed also reduction to the microbial counting with the raised of the ultrasound frequency and the period of storage showed the highest bacteria counting in the first day. Both treatment, were effective to promote bacteria reduction, but not completed elimination of A. acidoterrestris.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Neste trabalho foi desenvolvido um nanocompósito polimérico de óxido de grafeno reduzido e poli(ácido 3 hidroxibenzóico) para a modificação de eletrodos de grafite, visando o desenvolvimento de um genossensor para a detecção do DNA genômico de Alicyclobacillus acidoterrestris. Esta é uma bactéria associada à deterioração de sucos ácidos, como o suco de laranja, do qual o Brasil é o maior produtor mundial. Neste contexto, os biossensores aparecem como dispositivos de detecção rápidos e fáceis de manusear, com grande potencial para serem utilizados em toda a cadeia produtiva do suco. Para a construção do genossensor, óxido de grafeno foi produzido pelo método de Hummers modificado, gotejado sobre a superfície do eletrodo de grafite e reduzido eletroquimicamente. O ácido 3-hidroxibenzóico foi eletropolimerizado sobre esta superfície contendo o nanomaterial. Análises por espectroscopia no infravermelho e voltametria cíclica comprovaram a redução do óxido de grafeno. Além disso, as análises eletroquímicas evidenciaram que o nanocompósito produzido apresenta propriedades eletrônicas superiores às do filme polimérico. Sobre este nanocompósito foi imobilizado um oligonucleotídeo sonda ALIC1, específico para A. acidoterrestris, o qual foi utilizado para detecção de um oligonucleotídeo alvo complementar ALIC2 pela técnica de voltametria de pulso diferencial (VPD), tanto direta quanto indiretamente, esta última utilizando-se o intercalante da dupla fita de DNA Hoechst 33258. Um lisado celular obtido a partir de uma cultura de A. acidoterrestris também foi detectado de maneira indireta pela técnica de VPD, e uma curva de calibração foi construída. O genossensor proposto apresentou um limite de detecção de 174 ng mL-1 e limite de quantificação de 581 ng mL-1, sendo capaz de detectar o DNA genômico em uma amostra real de suco de laranja e de discernir entre amostras de A. acidoterrestris e Escherichia coli. Deste modo, este bioeletrodo apresenta-se como a primeira plataforma de detecção eletroquímica do DNA genômico de A. acidoterrestris na literatura científica. / In this work a polymeric nanocomposite of reduced graphene oxide and poly (3- hydroxybenzoic acid) was developed for the modification of graphite electrodes, aiming the development of a genossensor for the detection of the Alicyclobacillus acidoterrestris genomic DNA. This bacterium is associated with the spoilage of acidic juices, such as orange juice, of which Brazil is the largest producer in the world. In this context, biosensors appear as fast and easy to handle detection devices, with great potential for use throughout the juice production chain. For the construction of the genosensor, graphene oxide was produced by the modified Hummers method, dripped onto the surface of the graphite electrode and reduced electrochemically. The 3-hydroxybenzoic acid was electropolymerized on this surface containing the nanomaterial. Analyzes by infrared spectroscopy and cyclic voltammetry proved the reduction of graphene oxide. In addition, the electrochemical analysis showed that the nanocomposite produced has higher electronic properties than the polymeric film. On this nanocomposite, an oligonucleotide probe ALIC1, specific for A. acidoterrestris, was immobilized, and was used to detect a complementary target oligonucleotide ALIC2, both directly and indirectly, the latter using the Hoechst 33258 double strand DNA intercalator, by the differential pulse voltammetry (DPV) technique. A cell lysate obtained from an A. acidoterrestris culture was also indirectly detected by DPV, and a calibration curve was constructed. The proposed genosensor presented a limit of detection of 174 ng mL-1 and limit of quantification of 581 ng mL-1, being able to detect the genomic DNA in a real sample of orange juice and to distinguish between the samples of A acidoterrestris and Escherichia coli. Thus, this bioelectrode presents as the first platform of electrochemical detection of the genomic DNA of A. acidoterrestris in the scientific literature. / Dissertação (Mestrado)

O suco de laranja possui características que dificultariam ou mesmo impediriam o crescimento de microrganismos, mesmo assim já foram isolados fungos filamentosos, leveduras, bem como bactérias lácticas e termorresistentes. Dentre as bactérias termorresistentes e deteriorantes está a Alicyclobacillus acidoterrestris que tem causado prejuízos no setor de suco de laranja concentrado e congelado. Esta bactéria está apta a crescer em temperaturas abaixo de 35°C, constituindo um risco aos sucos de laranja que estejam contaminados. Mesmo os que já passaram por tratamento térmico, se forem estocados em local sem refrigeração, podem sofrer deterioração. Como os métodos tradicionais de descontaminação e conservação não têm se mostrado eficientes na inviabilização dessa bactéria, outras formas de esterilização tornam-se necessárias, dentre elas destacam-se as radiações gama e ultra-sônica. A radiação gama é capaz de esterilizar alimentos e reduzir as cargas microbiológicas, permitindo assim ampliar o período de armazenamento. A aplicação de ultra-som tem sido utilizada atualmente com o objetivo de controle microbiológico, sendo essa técnica eficiente por causar a destruição das células microbianas. Sendo o Brasil o maior produtor e exportador de suco de laranja concentrado congelado, no presente trabalho foram conduzidos dois experimentos com o objetivo de determinar a resistência da bactéria A. acidoterrestris às radiações gama e ultra-sônica, em suco de laranja previamente contaminado. As amostras de suco de laranja foram diluídas a 11,5 °Brix e inoculadas com uma suspensão bacteriana, passando pelos processos de radiação gama com as doses de 0, 2; 4; 6 e 8, kGy e ultra-sônica com as freqüências de 25, 35 e 42 kHz e com os tempos de exposição de 0, 1, 5, 10 e 20 minutos. Após o tratamento, as amostras foram armazenadas em temperatura ambiente (25±2°C) e de refrigeração (4±1°C). Para as análises microbiológicas as amostras foram diluídas em escala decimal, plaqueadas pela técnica de \"pour plate\". As placas de Petri permaneceram em estufa a46±2°C, por 48 horas, após esse período procedeu-se a contagem das UFC mL-1. A qualidade microbiológica do suco foi avaliada em determinados períodos de armazenamento (1, 10, 20, 30 e 40 dias). Através de análise de variância dos experimentos conduzidos com as radiações, observou-se que houve efeito significativo (p<0,05) com relação à dose de radiação gama que apresentou uma redução da carga microbiana com o aumento da dose de radiação e à interação do período com a temperatura de armazenamento demonstrou que houve uma oscilação na contagem total para a temperatura ambiente, mas para a temperatura de refrigeração uma diminuição gradativa com o aumento do período de armazenamento, A freqüência do ultra-som mostrou uma diminuição da contagem microbiana com o aumento da freqüência e o período de armazenamento apresentou a maior contagem no primeiro dia. Os dois tratamentos mostraram-se eficientes na redução bacteriana, mas não na completa eliminação da A. acidoterrestris. / The orange juice has attributes could make that hard or even block the growth. of microorganisms, even so already have been isolated filamentous fungi, yeast, as well lactics and heat resistant bacteria. Alicyclobacillus acidoterrestris is a heat resistant bacteria that has done damage in the orange juice concentrated and frozen business. This bacterium is able the grown in temperatures below 35°C constituting a risk to orange juices that have been contaminated. Even those that already pass trough thermal treatment may deteriorated, if they are storage in place without refrigeration. As the traditional methods of decontamination and conservation have not been effective in inviabilizated this bacterium, others methods of sterilization are need, among them are gamma and ultrasound radiation. The gamma radiation is able to sterilize foods and reduce the microbiology density, allowing in this way to enhance the period of storage. The ultrasound application has been used currently with the aim to microbiology control, been this technique effective in the microbial cell destruction. The Brazil, are the major producer and exporter of concentrated frozen orange juice, due to it, the present work was carried out to two experiments with the aim to determinate the resistance of the bacterium Alicyclobacillus acidoterrestris to gamma and ultrasound irradiation, in the orange juice, priory contaminated. The orange juice samples was diluted to 11,5°Brix and the bacteria suspensions was added, passed by process of radiation gamma with dose of 0, 2, 4, 6 and 8 kGy and ultrasonic with the frequency of 25, 35 and 42 kHz and with the time of exposition of 0, 1, 5, 10 and 20 minutes. After the treatment, the samples were storaged in room temperature (25±2°C) and of refrigeration (4±1°C). For the microbial analyses, the samples were diluted in to decimal scale, plated by \"pour plate\" technique. The Petri plates were storage in warn temperature (46±2°C) by 48 hours, after it the UFC was counted mL. The microbiological quality of the juice was evaluated in determinated period of storaged (1, 10, 20, 30 e 40 days). By statistical of variance of the experiment conducted with the radiations, it was noted that has been significant effect (p>0,05) to gamma radiation doses where occurred microbial density reduction with the raised of radiation doses and the interaction of the period with the storage temperature, where at room temperature occurred a oscillation in the total counting of bacteria, but for refrigeration temperature the total counting of bacteria reduced with the raised of storage period. The ultrasound frequency showed also reduction to the microbial counting with the raised of the ultrasound frequency and the period of storage showed the highest bacteria counting in the first day. Both treatment, were effective to promote bacteria reduction, but not completed elimination of A. acidoterrestris.

These are the abstracts of the oral presentations of the 6th PhD conference held on 10. June 2022 in Freiberg.