Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O estado de Roraima é dividido em três grandes sistemas ecológicos: floresta, campinas-campinarana e savanas ou cerrados e está localizado no extremo norte da Amazônia brasileira. Apesar da grande variedade de plantas existem poucas informações sobre plantas aromáticas da região. Lippia microphylla Cham., pertence à família Verbenaceae e ao gênero Lippia, e conhecida popularmente em Roraima como salva do campo, sendo encontrada nos lavrados roraimenses. Esta planta pode ser encontrada com facilidade ao longo da BR 174 que liga Boa Vista a Santa Elena de Uairén, na Venezuela. O objetivo desse trabalho foi avaliar a constituição química do óleo essencial de Lippia microphylla Cham. em épocas e horários diferentes e analisar sua atividade antioxidante. As amostras foram coletadas no município de Boa Vista, Roraima, em três horários diferentes (8,12 e 18 horas) nos meses de maio 2009, 2010 e 2011. As folhas extraídas por hidrodestilação com auxílio de um aparelho de clevenger e os óleos foram analisados por cromatografia gasosa com auxílio da espectrometria de massas. Para determinação da atividade antioxidante do óleo essencial foi utilizado o método de capacidade sequestrante do radical livre DPPH. Os maiores rendimentos de óleos foram registrados em maio de 2011, mês que registrou maior precipitação, mas não choveu no dia da coleta. A análise da constituição do óleo revelou que o timol, carvacrol, E-cariofileno, nerolidol e o óxido de cariofileno foram os principais constituintes, sendo o carvacrol majoritário em quase todas as análises. O óleo de melhor capacidade antioxidante foi extraído do material coletado ao meio dia e contem maior concentração de timol (9,22%) e carvacrol (19,80%). Observamos que os óleos coletados nos diferentes anos apresentam diferenças significativas quanto a sua composição química e ao seu rendimento. A chuva do dia da coleta foi mais relevante do que o volume de precipitação no mês. A atividade antioxidante desses óleos pode ser atribuída principalmente à presença dos dois isômeros fenólicos. / The state of Roraima is divided into three major ecological systems: forests, meadow-campinarana and savannas, and are located in the extreme north of the Brazilian Amazon. Despite the wide variety of plants there is little information on herbs of the region. The Lippia microphylla Cham., belongs to the family Verbenaceae and the genus Lippia, popularly known as salva-do-campo being found in the Roraima savannas. This plant is easily found along the margins of the BR 174, the route Boa Vista, Brazil, to the Santa Elena de Uairén, Venezuela. The aim of this study was to evaluate the chemical composition of essential oil of Lippia microphylla Cham. Collected at different times and schedules and analyze their antioxidant activity. The samples were collected in Boa Vista, Roraima, at three different times ( 8, 12 and 18 hours ) in May 2009, 2010 and 2011, for realization of antioxidant activity the samples were collected in the month of March 2011. The leaves were extracted by hydrodistillation with the assistance of a Clevenger apparatus and the oils analyzed by Gas chromatography coupled with mass spectrometry, GC-MS. To determine the antioxidant activity of the essential oil the method of sequestering ability of the free radical DPPH was used. The highest yields of oils were recorded in May 2011, which recorded highest rainfall month, but it did not rain on the day of collection. The analysis of the constitution of the oils revealed that thymol, carvacrol, E-caryophyllene, nerolidol and caryophyllene oxide were the main constituents of which the majority carvacrol was in almost all analyzes. The best antioxidant capacity oil was extracted from material collected at noon containing higher concentration of thymol ( 9.22% ) and carvacrol ( 19.80% ). We observed that the oils collected in different years showed significant differences in their chemical composition and yield. The rain on the day of collection was more important than the volume of rainfall in the month. The antioxidant activity of these oils can be attributed mainly to the presence of two phenolic isomers.

Terpenes are naturally occurring compounds obtained from the plants secondary metabolism. Despite presenting pharmacological effects, structural changes within their skeleton may increasing their pharmacological activity and attenuate the toxicological effects. Carvacrol is a phenolic monoterpene present in essential oils from plants belonging to the Labiatae family. Studies have demonstrated that carvacrol has anti-inflammatory activity. However, sctructural changes may reduce the effective dose of this monoterpene. Thus, in this study, we conducted an extensive systematic review evaluating the antiinflammatory activity of terpenes that suffered an alteration in its structure through synthesis and semi-synthesis, synthesize the carvacrol propionate (CP) from the carvacrol and evaluate its potential antinociceptive, anti-hyperalgesic and anti-inflammatory effects. To build the revision, it was made the search in Scopus, Embase and PubMed databases, using the descriptors anti-inflammatory agents, terpenes and structure activity relationship. In the experimental part, it was used Male Swiss mice (25-35 g) with 2 to 3 months age. The animals were divided in groups and were treated with CP (25, 50 and 100 mg/kg), vehicle (saline solution 0.9% + Tween 80 0.2%) or standard drug, intraperitoneally (i.p.). The antinociceptive effect was evaluated through the formalin (1%) protocol and the hot plate test. The mechanical hyperalgesia was evaluated through the algic agents injection: carragee nan (CG; 300 µg/paw), tumor necrosis factor-a (TNF-a; 100 pg/paw), prostaglandin E2 (PGE2; 100 ng/paw) or dopamine (DA; 30 µg/paw) using a digital analgesimeter (von Frey). To assess the anti-inflammatory effect, it was used the pleurisy and paw edema induced by GC (1 %) in digital plethysmometer. The cytotoxicity of CP was evaluated by the MTT colorimetric method. The experimental protocols were approved by the UFS ethics committee (CEPA/UFS: 35/12). The results are expressed as mean ± SEM and differences between groups were analyzed by one-way or two-way ANOVA test followed by Tukey or Bonferroni tests. Values of p < 0.05 were considered statistically significant. In systematic review, 27 papers were found concerning about terpenes structural modification and the evaluation of their anti-inflammatory activity. In the experimental part, the administration of CP produced a significant decrease (p < 0.01 and 0.001) in the test formalin-induced nociceptive in both phases of the test. In the hot plate test, the reaction time increased significantly at doses 50 and 100 mg/kg (p < 0.05, 0.01 and 0.001). CP inhibited the development of mechanical hyperalgesic induced by all agents tested (p < 0.05, 0.01 and 0.001). In the evaluation of anti-inflammatory activity, the treatment with CP was able to decrease significantly the leukocyte recruitment (p < 0.001), the TNF-a (p < 0.001), the IL-1ß (p < 0.05) and protein leakage (p < 0.01). In addition, the paw edema induced by CG in mice was inhibited significantly by CP (p < 0.05, 0.01 and 0.001). Thus, it is concluded that the CP attenuates nociception, mechanical hyperalgesia and inflammation, through an inhibition of cytokines. Therefore, structural modification terpene can be an interesting alternative for obtaining molecules with pharmacological properties. / Os terpenos sao compostos naturais obtidos do metabolismo secundario das plantas. Apesar de apresentar efeitos farmacologicos, modificacoes estruturais realizadas no seu esqueleto podem levar o aumentando de suas atividades farmacologicas e atenuar os efeitos toxicologicos. Neste contexto, insere-se o carvacrol, um monoterpeno fenolico, presente em oleos essenciais de plantas pertencentes a familia Labiatae. Estudos comprovam a atividade farmacologica deste monoterpeno. No entanto, modificacoes estruturais podem diminuir a dose efetiva deste composto. Desta forma, no presente estudo realizamos uma extensa revisao sistematica que avaliou a atividade anti-inflamatoria de terpenos que sofreram modificacoes em sua estrutura, atraves de sintese. Adicionalmente, sintetizar o propionato de carvacrol (CP), a partir do carvacrol, e avaliar seus possiveis efeitos antinocicepivo, anti-hiperalgesico e anti-inflamatorio. Para construir a revisao, foi realizada a busca nas bases de dados Scopus, PubMed e Embase, utilizando os descritores agentes anti-inflamatorios, terpenos e relacao estrutura atividade. Ja para a parte experimental, foram utilizados camundongos Swiss machos (25-35 g) com 2 a 3 meses de idade. Os animais foram divididos em grupos e foram tratados com CP (25, 50 e 100 mg/kg), veiculo (solucao salina 0,9% + Tween 80 0,2%) ou droga padrao, por via intraperitoneal (i.p.). O efeito antinociceptivo foi avaliado utilizando o protocolo de formalina (1%) e o teste da placa quente. A hiperalgesia mecanica foi avaliada apos a administracao dos agentes algicos carragenina (CG; 300 Êg/pata), fator de necrose tumoral- ¿ (TNF- ¿; 100 pg/pata), prostaglandina E2 (PGE2; 100 .g/pata) ou dopamina (DA; 30 Êg/pata) utilizando o analgesimetro digital Von Frey. Na avaliacao do efeito antiinflamatorio utilizou-se o teste de pleurisia e edema de pata induzido por CG (1%) em pletismometro digital. A citotoxicidade foi avaliada atraves do metodo colorimetrico MTT. Os protocolos experimentais foram aprovados pelo comite de etica da UFS (CEPA/UFS: 35/12). Os resultados foram expressos como media } erro padrao da media e as diferencas entre os grupos foram analisadas por meio do teste de variancia ANOVA, uma via ou duas vias, seguido pelo teste de Tukey ou Bonferroni. Valores de p < 0,05 foram considerados estatisticamente significantes. Na revisao sistematica foram encontrados 27 artigos sobre modificacao estrutural de terpenos e atividade anti-inflamatoria. Na parte experimental, a administracao do CP produziu uma reducao significativa (p < 0,01 ou 0,001) no teste da nocicepcao induzida por formalina, em ambas as fases do teste. No teste da placa quente, o tempo de reacao aumentou significativamente nas doses de 50 e 100 mg/kg (p < 0,05; 0,01 ou 0,001). O CP tambem foi capaz de inibir o desenvolvimento da hiperalgesia mecanica induzida por todos os agentes testados (p < 0,05; 0,01 ou 0,001). Na avaliacao da atividade anti-inflamatoria, o tratamento com CP causou uma diminuicao significativa (p < 0,001) no numero total de leucocitos, diminuindo os niveis de TNF- ¿ (p < 0,001), IL-1 À (p < 0,05) e extravasamento de proteinas (p < 0,01). Alem disso, o edema de pata induzido por CG tambem foi inibido pelo CP (p < 0,05; 0,01 ou 0,001). Desta forma, conclui-se que o CP possui atividade antinociceptiva, anti-hiperalgesica e anti-inflamatoria, provavelmente por inibicao de citocinas. Dessa maneira, a modificacao estrutural em terpeno pode ser uma alternativa interessante para obtencao de moleculas com propriedades farmacologicas.

O concentrado em substituição à parte da forragem na alimentação, principalmente por grãos de cereais ricos em amido, a fim de suprir as necessidades energéticas do animal, pode exceder a capacidade digestiva do intestino delgado e atingir o ceco e cólon, acarretando em mudanças na microbiota e causando distúrbios gastrointestinais. O objetivo desse estudo foi avaliar o efeito da suplementação do óleo essencial carvacrol sobre a digestibilidade aparente dos nutrientes da dieta, resposta glicêmica e insulinêmica pós-prandial, pH fecal, perfil plasmático de triglicerídeos, colesterol total, frações de colesterol ligado à lipoproteína de alta densidade (HDL-C), colesterol ligado à lipoproteína de baixa densidade (LDL-C) e colesterol ligado à lipoproteína de muito baixa densidade (VLDL-C). Foram utilizados oito animais da raça Mini-horse, machos, castrados, com idade de 42±6 meses, peso médio de 135±15 kg, alimentados com dieta contendo alta proporção de concentrado: volumoso, constituída de 60% concentrado comercial peletizado e 40% de feno de gramínea, cuja variação foi a inclusão de um produto contendo 7% de carvacrol, onde os tratamentos foram inclusão de controle, 100, 200 e 300 ppm de óleo essencial contendo 7% de carvacrol. O delineamento experimental utilizado foi em quadrado latino duplo 4x4 com medidas repetidas no tempo e os dados obtidos foram submetidos à análise de variância com nível de significância de 5%. Os resultados dos coeficientes de digestibilidade MS, MO, PB, EE, MM, FDN e FDA foram respectivamente, P= 0,48; P= 0,66; P= 0,58; P= 0,64; P= 0,55; P= 0,58; P= 0,46; valores de pH fecal observados foram adequados (pH = 6,38) para o equilíbrio da microbiota, não sendo observado efeito de tratamento (P=0,84). Na avaliação da área abaixo da curva (AAC), não foi observado efeito de tratamento (P= 0,48) para os valores de glicose. A resposta plasmática de insulina apresentou efeito quadrático para AAC (P=0,05) nos tempos (90, 150 e 210 minutos). Na avaliação de gordura plasmática não foi encontrado efeito de tratamento para triglicérides o valor de (P=0,37), para colesterol total e frações HDL, LDL e VLDL os valores foram respectivamente, P= 0,85; P= 0,57; P= 0,94; P= 0,37. A inclusão de óleo essencial na dieta não promove efeito sobre a digestibilidade, resposta glicêmica, perfil plasmático de gordura plasmática e pH das fezes. A inclusão de óleo essencial promove efeito sobre a resposta insulinêmica. / The concentrate to replace part of the forage in the diet, mainly cereal grains rich in starch, in order to meet the energy needs of the animal, may exceed the digestive capacity of the small intestine and reach the cecum and colon, resulting in changes in the microbiota and causing gastrointestinal disorders. The aim of this study was to evaluate the effect of supplementation of carvacrol as essential oil on the apparent digestibility of nutrients, glucose and insulin responses postprandial fecal pH, plasma levels of triglycerides, total cholesterol and its fractions high density lipoprotein (HDL-C), low density lipoprotein (LDL-C) very low density lipoprotein (VLDL-C). Were used eight animals, Mini-horse breed, geldings, aged 42±6 months, average weight 135±15 kg, fed a diet containing high relation concentrate:roughage, consisting of 60% commercial concentrate and 40 % of grass hay, which variation was the inclusion of essential oil containing 7% carvacrol, with the treatments divided according to the level of inclusion: 0 (control), 100, 200 and 300 ppm. The experimental design was 4x4 latin square with repeated measures over time and the data were subjected to analysis of variance with a significance level of 5%. The results of the digestibility coefficients of dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE), mineral matter (MM), neutral detergent fiber (NDF) and acid deterge nt fiber (ADF) were, respectively, P=0.48; P=0.66; P=0.58; P=0.64; P=0.55; P=0.58 and P=0.46. Faecal pH values were found suitable (pH=6.38) to the balance of microorganisms, and there was no effect of treatment (P=0.84). In evaluation of the area under the curve (AUC), weren´t observed effects of treatment (P=0.48) values for glucose. The response of plasma insulin showed a quadratic effect for AUC (P=0.05) in times 90, 150 and 210 minutes. In the evaluation of plasma lipids wasn´t found effect treatment of the triglyceride value (P=0.37) for total cholesterol, and HDL-C, LDL-C and VLDL-C values were respectively (P=0.85; P=0.57; P=0.94 and P=0.37). The inclusion of essential oil in the diet does not promote effect on digestibility, glycemic response, plasma levels lipids and plasma pH of feces. The inclusion of essential oils promotes effect on the insulin response.

The effect of the natural essential oil carvacrol or the anesthetic propofol on cell viability, cell cycle distribution, reactive oxygen species (ROS), intracellular free Ca2+ concentrations ([Ca2+]i) and caspase-3-associated apoptosis in human normal cells or non-normal cells is unclear. Human gingival fibroblasts (HGF), human oral cancer cell line (OC2) and human glioblastoma cell line (DRTBG-05MG, HGB) were used in this study. Cell viability was measured by detecting reagent water soluble tetrazolium salt-1 (WST-1). Apoptosis was detected by Annexin V/propidium iodide (PI) staining, cell cycle distribution was detected by PI staining, and ROS was detected by membrane-permeable probe dichlorofluorescein diacetate (DCFH-DA) or hydroethidine (HE) staining. Apoptosis, cell cycle distribution and ROS were analyzed by flow cytometry. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Caspase-3 expression was detected by western blotting. Carvacrol at 200-800 £gM decreased the cell viability of OC2 or HGB cells in a concentration-dependent manner and 1,000 £gM carvacrol almost killed all OC2 or HGB cells, but in HGF cells, 200-800 £gM carvacrol did not significantly kill cells and 1,000 £gM carvacrol decreased only about 63% of cell viability. Similarly, propofol at concentrations between 300 and 600 £gM decreased the cell viability of OC2 or HGB cells in a concentration-dependent manner and 700 £gM propofol almost killed all OC2 or HGB cells, but in HGF cells, 300-600 £gM propofol did not significantly kill cells and 700 £gM propofol decreased about 62% of cell viability. In OC2 or HGB cells, carvacrol (200 £gM, 400 £gM or 600 £gM) or propofol (300 £gM, 400 £gM or 500 £gM) induced apoptosis, increased ROS production, evoked cell cycle arrest and activated caspase-3. The caspase-3 inhibitor (DEVD-CHO) partially decreased the apoptotic effect induced by carvacrol or propofol. On the other hand, in OC2 or HGB cells, carvacrol at concentrations between 400 £gM and 1,000 £gM induced a [Ca2+]i rise in a concentration-dependent manner and the signal was reduced by removal of extracellular Ca2+. In HGF cells, carvacrol at 1000 £gM did not induce immediate [Ca2+]i rises in Ca2+-containing or Ca2+-free medium. Similarly, propofol at concentrations between 400 £gM and 1,000 £gM induced a [Ca2+]i rise in a concentration-dependent manner in OC2 or HGB cells, but not in HGF cells. This cytotoxic effect was not reversed in carvacrol-treated groups, but was partially reversed in propofol-treated groups when cytosolic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA-AM) in OC2 or HGB cells. The apoptotic effect of propofol was also partially decreased by BAPTA-AM treatment in OC2 and HGB cells. In OC2 and HGB cells, carvacrol- or propofol-induced Ca2+ signal was not altered by L-type voltage-gated Ca2+ channel blocker nifedipine, store-operated Ca2+ channel blocker econazole or SK&F96365) and protein kinase C (PKC) activator phorbol myristate acetate (PMA), but was inhibited by PKC inhibitor GF109203X. When extracellular Ca2+ was removed, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol- or propofol-induced [Ca2+]i rises. Incubation with carvacrol or propofol also abolished TG or BHQ-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished carvacrol- or propofol-induced [Ca2+]i rises. Together, first, in HGF cells, carvacrol (200-800 £gM) or propofol (300-600 £gM) did not induce [Ca2+]i rises and cell death. Second, in OC2 or HGB cells, carvacrol induced [Ca2+]i rises and cell death that might involve ROS- and caspase-3-associated apoptosis. Third, in OC2 or HGB cells, propofol induced [Ca2+]i rises and cell death that might involve ROS-, Ca2+- and caspase-3-associated apoptosis. Lastly, in OC2 or HGB cells, carvacrol or propofol induced [Ca2+]i rises by inducing PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-sensitive, non store-operated Ca2+ channels.

Tesis para optar al Título Profesional de Ingeniera Agrónoma y al Grado de Magíster en Ciencias Agropecuarias, Mención Producción Agroindustrial / Hoy en día los consumidores tienen mayor conocimiento de la importancia de integrar frutas y hortalizas frescas a la dieta de manera que ésta sea saludable, dado que se conoce el gran aporte nutricional y los beneficios para la salud tales como la prevención de enfermedades cardiovasculares, diabetes, cáncer, etc. El gran problema de las frutas y hortalizas frescas es que tienen un rápido deterioro causado principalmente por enzimas y microorganismos. Las bacterias se consideran como la mayor causa del deterioro de alimentos frescos, seguido por hongos, virus, residuos de pesticidas y micotoxinas (Fallik, 2014).

O concentrado em substituição à parte da forragem na alimentação, principalmente por grãos de cereais ricos em amido, a fim de suprir as necessidades energéticas do animal, pode exceder a capacidade digestiva do intestino delgado e atingir o ceco e cólon, acarretando em mudanças na microbiota e causando distúrbios gastrointestinais. O objetivo desse estudo foi avaliar o efeito da suplementação do óleo essencial carvacrol sobre a digestibilidade aparente dos nutrientes da dieta, resposta glicêmica e insulinêmica pós-prandial, pH fecal, perfil plasmático de triglicerídeos, colesterol total, frações de colesterol ligado à lipoproteína de alta densidade (HDL-C), colesterol ligado à lipoproteína de baixa densidade (LDL-C) e colesterol ligado à lipoproteína de muito baixa densidade (VLDL-C). Foram utilizados oito animais da raça Mini-horse, machos, castrados, com idade de 42±6 meses, peso médio de 135±15 kg, alimentados com dieta contendo alta proporção de concentrado: volumoso, constituída de 60% concentrado comercial peletizado e 40% de feno de gramínea, cuja variação foi a inclusão de um produto contendo 7% de carvacrol, onde os tratamentos foram inclusão de controle, 100, 200 e 300 ppm de óleo essencial contendo 7% de carvacrol. O delineamento experimental utilizado foi em quadrado latino duplo 4x4 com medidas repetidas no tempo e os dados obtidos foram submetidos à análise de variância com nível de significância de 5%. Os resultados dos coeficientes de digestibilidade MS, MO, PB, EE, MM, FDN e FDA foram respectivamente, P= 0,48; P= 0,66; P= 0,58; P= 0,64; P= 0,55; P= 0,58; P= 0,46; valores de pH fecal observados foram adequados (pH = 6,38) para o equilíbrio da microbiota, não sendo observado efeito de tratamento (P=0,84). Na avaliação da área abaixo da curva (AAC), não foi observado efeito de tratamento (P= 0,48) para os valores de glicose. A resposta plasmática de insulina apresentou efeito quadrático para AAC (P=0,05) nos tempos (90, 150 e 210 minutos). Na avaliação de gordura plasmática não foi encontrado efeito de tratamento para triglicérides o valor de (P=0,37), para colesterol total e frações HDL, LDL e VLDL os valores foram respectivamente, P= 0,85; P= 0,57; P= 0,94; P= 0,37. A inclusão de óleo essencial na dieta não promove efeito sobre a digestibilidade, resposta glicêmica, perfil plasmático de gordura plasmática e pH das fezes. A inclusão de óleo essencial promove efeito sobre a resposta insulinêmica. / The concentrate to replace part of the forage in the diet, mainly cereal grains rich in starch, in order to meet the energy needs of the animal, may exceed the digestive capacity of the small intestine and reach the cecum and colon, resulting in changes in the microbiota and causing gastrointestinal disorders. The aim of this study was to evaluate the effect of supplementation of carvacrol as essential oil on the apparent digestibility of nutrients, glucose and insulin responses postprandial fecal pH, plasma levels of triglycerides, total cholesterol and its fractions high density lipoprotein (HDL-C), low density lipoprotein (LDL-C) very low density lipoprotein (VLDL-C). Were used eight animals, Mini-horse breed, geldings, aged 42±6 months, average weight 135±15 kg, fed a diet containing high relation concentrate:roughage, consisting of 60% commercial concentrate and 40 % of grass hay, which variation was the inclusion of essential oil containing 7% carvacrol, with the treatments divided according to the level of inclusion: 0 (control), 100, 200 and 300 ppm. The experimental design was 4x4 latin square with repeated measures over time and the data were subjected to analysis of variance with a significance level of 5%. The results of the digestibility coefficients of dry matter (DM), organic matter (OM), crude protein (CP), ether extract (EE), mineral matter (MM), neutral detergent fiber (NDF) and acid deterge nt fiber (ADF) were, respectively, P=0.48; P=0.66; P=0.58; P=0.64; P=0.55; P=0.58 and P=0.46. Faecal pH values were found suitable (pH=6.38) to the balance of microorganisms, and there was no effect of treatment (P=0.84). In evaluation of the area under the curve (AUC), weren´t observed effects of treatment (P=0.48) values for glucose. The response of plasma insulin showed a quadratic effect for AUC (P=0.05) in times 90, 150 and 210 minutes. In the evaluation of plasma lipids wasn´t found effect treatment of the triglyceride value (P=0.37) for total cholesterol, and HDL-C, LDL-C and VLDL-C values were respectively (P=0.85; P=0.57; P=0.94 and P=0.37). The inclusion of essential oil in the diet does not promote effect on digestibility, glycemic response, plasma levels lipids and plasma pH of feces. The inclusion of essential oils promotes effect on the insulin response.

The use of antibiotics in animal feed is a large cause of concern due to bacterial resistance, which occurs when bacteria change after exposure to antibiotics and become less affected by the drug. Consequently, the desire to find a replacement for these antibiotics has garnered interest in both farmers and consumers. The ideal substance would display antimicrobial activity without promoting bacterial resistance and would still function as a growth promoter in animals. One antimicrobial that fits this criterion is carvacrol, a compound found in oregano extract. Carvacrol exhibits antimicrobial activity in a number of bacteria, including E.coli O157:H7 and S. enteritidis. In this study, the effectiveness of carvacrol nanoemulsions against E.coli and S. enteritidis growth in a micro-broth dilution assay was tested, as well as the use of alamarBlueTM dye reagent as a bacterial viability indicator. The carvacrol nanoemulsion was diluted in TSB to obtain 2000, 1000, 500, 250, 125, and 62.5 ppm, then aliquots of 100 μl of each dilution were added to a 96-well microtiter plate. Test group wells were inoculated with 100 μl of microorganism while control cells remained uninoculated, and the plate was incubated for 24h at 37 °C. After incubation, 10μl of alamarBlueTM was added to each well, and the microtiter plate was incubated for one hour. After one hour, the color changes were analyzed both visually and via fluorescence. Additionally, a traditional plating assay in which samples were plated on tryptic soy agar was done alongside the microtiter plate assay. It was found that the minimum inhibitory concentration of carvacrol needed to inhibit E. coli growth was 500ppm, and to inhibit S. enteritidis growth was 800ppm. These results show that carvacrol may be suitable as a possible replacement for antibiotic in animal feed in the future.

Previous findings showed that Listeria monocytogenes exhibits higher heat tolerance to thermal exposure at 60°C when pre-exposed to sublethal heat stress at 48°C for 30/60 min. The objective of this study was to investigate the morphological changes that occurred in L. monocytogenes serotype 1/2a cells as visualized by transmission electron microscopy (TEM) after exposure to sublethal heat stress at 48°C for 60 min and in combination with lethal concentration of carvacrol for 30 min. The TEM micrographs revealed thickening of cell wall and cell membrane, and clumping of cytoplasm when subjected to sublethal heat stress followed by carvacrol treatment. These studies indicate that L. monocytogenes cells when adapted to heat stress may alter its morphology to protect themselves against carvacrol. Further studies will investigate the effect of increased contact time with carvacrol and with other essential oils on the ultrastructural changes in L. monocytogenes cells adapted to heat stress.

The present work examines the antimicrobial efficacy of the essential oils of thyme, oregano and carvacrol against L. monocytogenes biofilms produced on stainless steel coupons and for control of L. monocytogenes growth on raw catfish fillets stored at 4°C for 10 days. At 0.5%, all three essential oils were highly effective in completely eliminating L. monocytogenes cells from stainless coupons within 24 h as compared to the untreated control yielding ~7 log CFU/cm2 L. monocytogenes. When catfish inoculated with L. monocytogenes were dipped for 30 min at 4°C in essential oil solutions of thyme and oregano at 1%, 2% and 5%, there were no significant reductions in L. monocytogenes counts on the fresh catfish fillets as compared to untreated control. For the same conditions, treatment with 2% carvacrol resulted in a complete reduction of 4 log CFU/g of L. monocytogenes counts from fresh catfish fillets.

The antimicrobial efficacy of carvacrol, trans-cinnamaldehyde, eugenol and thyme oil (0.5, 1, 2 and 5% v/v) was evaluated against Salmonella on turkey breast cutlets as 2 min dip treatments at 4°C. Carvacrol revealed maximum efficacy against Salmonella on turkey cutlets and was selected for 30s and 60s dip treatments at 4°C. Only 5% carvacrol was effective and exhibited 1.0 and 1.8 log CFU/g reduction of Salmonella with 30s and 60s dip treatments, respectively. However, carvacrol showed synergistic activity with carbon dioxide in modified atmosphere packaging (95% CO2/5% O2) against Salmonella and Campylobacter and caused 1.0-2.0 log CFU/g reductions with lower concentrations (0.25, 0.5 and 1.0%). This combination reduced the growth of lactic acid bacteria. In the third experiment, efficacy of lauric arginate (200 and 400ppm) against C. jejuni was tested on chicken breast fillets. Both these concentrations significantly reduced C. jejuni on chicken fillets (1.0-1.5 log CFU/g) at 4°C.