Work was undertaken to examine the antibacterial mechanism of allyl isothiocyanate (AITC), as well as explore the hydrolysis of sinigrin by bacteria and sinigrin metabolism in Escherichia coli O157:H7. Glucosinolates are found in cruciferous plants and react with the plant enzyme myrosinase to form antimicrobial compounds called isothiocyanates (ITCs). The mechanism of bactericidal action of ITCs is not well understood. Inhibition experiments against 12 bacteria including foodborne pathogens and starter cultures indicated that AITC exhibited a minimum inhibitory concentration (MIC) profile most similar to antibiotics ciprofloxacin and polymyxin B. Similarities in mechanisms of action between groups were identified. The sinigrin degradation capacity of a range of bacteria was examined. The effect of glucose in combination with other sugars on the sinigrin degradation capacity of E. coli O157:H7 was assessed. No statistically significant differences in sinigrin utilisation were observed. Results indicated that the metabolism of sinigrin was simultaneous with glucose consumption. / February 2016

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12936号 / 論農博第2816号 / 新制||農||1033(附属図書館) / 学位論文||H27||N4895(農学部図書室) / 32146 / (主査)教授 伏木 亨, 教授 保川 清, 教授 安達 修二 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Since 2006 there have been four Salmonella enterica and one Listeria monocytogenes foodborne outbreaks linked to whole cantaloupe fruit. No post-harvest intervention to reduce potential contamination on cantaloupe currently exists. The complex surface topography of netted cantaloupes aids bacterial attachment. This research evaluates the use of allyl isothiocyanate (AITC; a natural antimicrobial) to reduce populations of S. enterica Michigan and L. monocytogenes on the surface of cantaloupe. Fifty μl of S. Michigan or L. monocytogenes was inoculated onto whole ‗Athena‘ or ‗Hales Best Jumbo‘ (‗HBJ‘) cantaloupe fruit in 22 mm diameter circles and allowed to dry for 90 min. resulting in 6.60 log CFU/g. Cantaloupe received either AITC liquid or vapor, sterile deionized water, 200 ppm sodium hypochlorite per circle, or no treatment. All cantaloupes were stored in separate sealed glass desiccators for 1 or 24 h at 25°C or 35°C. To enumerate the bacteria following treatment, 22 mm sections of the rind were removed, homogenized and plated onto appropriate agar. Headspace analysis using Gas Chromatography-Mass Spectrometry (GC-MS) quantified the concentration of each AITC vapor treatment. The texture quality of the pericarp tissue of whole cantaloupes was evaluated after 24 h treatments, followed by two weeks of storage at 4°C. The concentration of vapor ranged from 3.4 to 19.6 μl AITC/L inside the desiccators. The liquid treatment reduced (P < 0.05) S. Michigan populations on ‗Athena‘ (3 log CFU/g) and L. monocytogenes on ‗HBJ‘ (2.6 log CFU/g). The longer exposure time to the AITC vapor (24 h versus 1 h) resulted in a greater reduction of both S. Michigan and L. monocytogenes on ‗Athena‘ and treatments at 35°C reduced microbial populations up to 4.5 times greater (P < 0.05). The highest vapor concentration reduced (P < 0.05) both pathogens at least 3.0 log CFU/g on ‗Athena‘ at 25°C. Generally, bacterial pathogens from the surface of ‗Athena‘ cantaloupe were reduced more than pathogens inoculated on the surface of ‗HBJ.‘ The application of AITC liquid or vapor is a natural alternative post-harvest treatment to 200 ppm free chlorine to reduce the level of bacterial contamination on cantaloupe surfaces for certified organic production. / Ph. D.

Made available in DSpace on 2015-03-26T13:30:35Z (GMT). No. of bitstreams: 1 texto completo.pdf: 217734 bytes, checksum: 6fd7a4c6b5089dddf92260f4cc930663 (MD5) Previous issue date: 2011-03-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this study was to evaluate the toxicity of the essential oil of mustard (EOM) for adults of five populations of Sitophilus zeamais (Coleoptera: Curculionidae) and the influence of the essential oil on the locomotory behavior of the populations. EOM was used with 90% of allyl isothiocyanate volatile component (ITCA), diluted in soybean oil at 1:9 v/v to obtain a 9% solution of ITCA. EOM was applied on filter paper placed in Petri dishes (5.0 cm diameter). The dishes were covered with organza and placed inside glass jars of 0.8 L, and 50 adult insects were transferred to the jars, which were kept closed for 48 hours. EOM was used at concentrations ranging from 1.2 to 2.8 &#956;L L-1. The S. zeamais populations used were from Abre Campo, Machado, Paracatu, Piracicaba and Tunápolis. The toxicity of EOM was evaluated by determining the lethal concentrations (CL) to 50 (LC50) and 95% (LC95) of adult insects, which were then used to estimate the toxicity ratio. The following locomotory behavioral parameters were studied: walked distance, walking velocity, resting time, and number of stops, as well as the number of takeoffs and flying height of the insects. Additionally, the instantaneous rates of increase (ri), the respiration rate (CO2 production) and body mass were evaluated for all populations. The most susceptible population to EOM was Piracicaba for wich the lowest LC50 was obtained (1.49&#956;L L-1). This concentration was used for all the other assays treated with EOM, except the instantaneous rate of population growth, which hand no EOM treatment. The Paracatu population showed the highest LC50 (2.97 &#956;L L-1) and LC95 (6.82 &#956;L L-1) while Machado showed the lowest LC95 (2.16 &#956;L L-1). The toxicity ratio based on the LC50 ranged from 1.08 to 1.99 times, and from 0.76 and 2.42 times based on the LC95. The slopes of the concentration mortality curves varied among the populations from 4.56 ± 0.72 (Paracatu) to 13.14 ± 0.75 (Tunápolis). The distance walked did not significantly differ between insects treatead with EOM and control or among populations. However, regarless of the population, the resting time of the insects was reduced by treatment with EOM. The other walking parameters (walking velocity and number of stops), flight parameters (flying height and number of takeoffs), respiratory rate, and body mass, the only varied among the populations, but no difference existed for insects exposed or not exposed to EOM. In conclusion, the EOM was toxic to the five populations of S. zeamais and did not promote major changes in the locomotory behavior of the insects. Different locomotory behavioral patterns, respiratory rate, and body mass were observed in the S. zeamais populations, but with uniform response to mustard oil; these differences should be inherent to the genetic make up of the populations. / O objetivo deste trabalho foi avaliar a toxicidade do óleo essencial de mostarda (OEM) para adultos de cinco populações de Sitophilus zeamais (Coleoptera: Curculionidae) e verificar a influência do óleo essencial sobre o comportamento locomotor dos insetos. Foi utilizado OEM com 90% do componente volátil Isotiocianato alilo (ITCA), diluído em óleo de soja, na proporção de 1:9 v/v, para obter solução oleosa de 9% de ITCA. O OEM foi aplicado em papel-filtro colocado em placas de Petri (5,0 cm diâmetro), que foram cobertas com organza e colocadas no interior de frascos de vidro de 0,8 L, aos quais foram acrescentados posteriormente 50 insetos adultos. Os frascos foram mantidos fechados por 48h. Utilizou-se o OEM em concentrações variando de 1,2 a 2,8 &#956;L L-1. As populações de S. zeamais utilizadas foram Abre Campo, Machado, Paracatu, Piracicaba e Tunápolis. Avaliou-se a toxicidade do OEM por meio da estimativa das concentrações letais (CL) para 50 (CL50) e 95% (CL95) dos insetos adultos. As CL50 e CL95 foram usadas para calcular as respectivas razões de toxicidade. Foram avaliadas também as características comportamentais de caminhamento: distância percorrida, velocidade de caminhamento, tempo de repouso e número de paradas, bem como as características de voo como número de decolagens e altura de voo dos insetos. Adicionalmente, foram avaliadas as taxas instantâneas de crescimento populacional (ri), as taxas respiratórias (produção de CO2) e massa corpórea para todas as populações. A população padrão de susceptibilidade foi a da cidade de Piracicaba, pois apresentou menor CL50 (1,49 &#956;L L- 1). Esta concentração foi utilizada para todos os bioensaios tratados com o OEM, exceto para a taxa instantânea de crescimento populacional, em que não se utilizou o tramento com OEM. A população de Paracatu foi a que apresentou maior CL50 (2,97 &#956;L L-1) e CL95 (6,82 &#956;L L-1), enquanto Machado foi a população que apresentou menor CL95 (2,16 &#956;L L-1). A razão de toxicidade com base na CL50 variou entre 1,08 e 1,99 vezes, e entre 0,76 e 2,42 vezes baseada na CL95. As inclinações das curvas de concentração-mortalidade variaram entre as populações, sendo menor (4,56 ± 0,72) para a população de Paracatu e maior (13,14 ± 0,75) para a de Tunápolis. Não houve diferença significativa na distância percorrida entre os tratamentos com OEM e controle, nem entre as populações. No entanto, o tempo de repouso dos insetos foi reduzido pelo tratamento com OEM em todas as populações. Para os outros padrões comportamentais de caminhamento (velocidade de caminhamento e número de paradas), de voo (altura de voo e número de decolagens), taxa respiratória e de massa corpórea, houve diferença apenas entre as populações, não diferindo quando na presença ou ausência do OEM. Com isso, conclui-se que o OEM foi tóxico para as cinco populações de S. zeamais e que este óleo não promoveu grandes alterações no comportamento locomotor dos insetos. Diferentes padrões comportamentais, respiratórios e de massa corpórea foram observados nas populações de S. zeamais, porém com uniformidade de resposta ao óleo de mostarda, sendo que estas diferenças devem ser inerentes à constituição genética das populações.

This study examined the feasibility of using (BETA)-cyclodextrins ((BETA)CDs), which are derived from starch, as agents to stabilize volatile and leachable biocides for the protection of wood and wood composites. The encapsulation of volatile natural antimicrobial compounds, such as allyl isothiocyanate (AITC) and essential oils (EOs), in βCDs was qualitatively confirmed by Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) method and the maximum inclusion yield was quantitatively estimated by ultraviolet-visible spectrophotometry (UV/VIS) while the partial fixation of leachable borates with βCD was confirmed by ATR-FTIR and nuclear magnetic resonance (NMR) techniques. The efficacy of the βCD complexes as wood preservatives of southern yellow pine and randomly Oriented Strand Board (OSB) was examined in soil block tests (AWPA E10-16 and E22-16 standard). The results indicate that (A) (BETA)CDs suppress the premature leaching of otherwise volatile natural compounds and suggest a novel approach to the application of volatile or water-immiscible natural preservatives for wood and wood composites protection; (B) (BETA)CD can be used as an agent for partial fixation of boric acid to form boric acid esters. However, the borate esters are susceptible to leach out after AWPA E11 test and not suitable for protection against Gloeophyllum trabeum and Postia placenta in outdoor conditions.

In this project the factors influencing the stability and antimicrobial activity of allyl isothiocyanate (AITC) against Campylobacter jejuni, Salmonella or Listeria monocytogenes as well as factors that enhance sinigrin (glucosinolate in Oriental mustard) hydrolysis by these pathogens were investigated. The minimum inhibitory concentration (MIC) of AITC against 5 strains of each of Salmonella or L. monocytogenes, ranged from 60-100 ppm at 37 ºC. This was reduced to 10-40 ppm at 21 ºC and a further reduction to 5-10 ppm against strains of L. monocytogenes was observed at 4 ºC. This was attributed to greater stability of AITC as temperature was decreased. C. jejuni strains were more susceptible to AITC with MICs of 0.63-1.25 ppm and 2.5-5 ppm at 37 and 42 ºC, respectively. AITC was more inhibitory at ≤ 21 ºC against Salmonella with acidic pH or against L. monocytogenes with neutral pH. C. jejuni, Salmonella and L. monocytogenes strains and mixtures had the ability to degrade sinigrin to form inhibitory concentrations of AITC, and sinigrin hydrolysis was significantly enhanced by higher incubation temperature (21 ºC > 10 ºC > 4 ºC), the presence of 10 mM ferric or ferrous irons, and the presence of < 0.25% glucose. This project also investigated the antimicrobial activity of AITC or Oriental mustard extract alone or combined with ethylenediamine tetraacetic acid (EDTA), malic acid and acetic acid in edible antimicrobial coatings against C. jejuni and Salmonella on fresh, refrigerated, vacuum-packed chicken breasts or L. monocytogenes on refrigerated, cured roast chicken. Malic acid improved the antimicrobial activity of Oriental mustard extract against L. monocytogenes, while EDTA improved its activity against Salmonella. Incorporation of 25 to 50 µl/g AITC or 100 to 250 mg/g Oriental mustard extract in 0.5%κ-carrageenan/2%chitosan coatings, prepared using 1.5% malic or acetic acid, reduced L. monocytogenes on cooked, cured, vacuum-packed chicken slices 4.2 to > 7.0 log10 CFU/g, compared to uncoated chicken by 70 d at 4 ºC. In addition, 0.2%κ-carrageenan/2%chitosan coatings (prepared using a 1% acetic acid solution) containing 250 mg/g mustard extract or 50 µl/g AITC reduced Salmonella numbers on vacuum-packed chicken breasts 3.0 log10 CFU/g by 21 d at 4 ºC. Further, 0.2%κ-carrageenan/2%chitosan coatings containing 50 or 100 µl/g AITC reduced numbers of C. jejuni on fresh, vacuum-packed chicken breasts > 5.0 log10 CFU/g (C. jejuni cells were not detected) after 5 d storage at 4 ºC, while coatings containing 200 to 300 mg/g Oriental mustard extract or 25 µl/g AITC reduced C. jejuni numbers by 3.6 to 4.6 log10 CFU/g. Numbers of lactic acid and aerobic bacteria on poultry meat products were significantly reduced by the coatings. It is clear that κ-carrageenan/chitosan coatings containing either AITC, mustard extract alone or combined with EDTA, malic or acetic acid significantly reduced C. jejuni and Salmonella on fresh, refrigerated, vacuum-packed chicken breasts and L. monocytogenes on refrigerated, cured roast chicken, and consequently enhanced their safety.