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117 Journal of Oleo Science Copyright ©2015 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess14163 J. Oleo Sci. 64, (1) 117-124 (2015) Essential Oils from Herbs against Foodborne Pathogens in Chicken Sausage Lidiane Nunes Barbosa1*, Isabella Silva Probst1, Bruna Fernanda Murbach Teles Andrade1, Fernanda Cristina Bérgamo Alves1, Mariana Albano1, Vera Lucia Mores Rall2 and Ary Fernandes Júnior1 1 Laboratory of Bacteriology and Natural Products. 2Labor atory of Food Microbiology. Department of Microbiology and Immunology, Biosciences Institute,Universidade Estadual Paulista "Julio de Mesquita Filho", Botucatu,São Paulo, Brazil, CEP 18618-970 1 INTRODUCTION Chicken meat and its products have increased in popu-larity and have become widespread throughout the world, with chicken sausage being one of the most popular cate-gories among these products1. Sausage manufacture in-volves a number of handling steps, which increase the chances of contamination by pathogens or spoilage2. Fresh sausage does not undergo heat treatment and has a highwater activity; giving this food a short shelf life and sub-jecting it directly to the action of the microorganisms pres-ents3. Application of agents with adequate antimicrobial and antioxidant activities has significant potential to extend the shelf life of chicken products and prevent economic losses4. Due to the negative perception of chemical preser-vatives, consumers attention is c hanging to natural alter-*Correspondence to: Lidiane Nunes Barbosa, Department of Microbiology and Immunology, Biosciences Institute, Sao Paulo State University E-mail: lidianebarbosa@ibb.unesp.br Accepted September 2, 2014 (received for review July 25, 2014) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/http://mc.manusriptcentral.com/jjocs natives and particular interest has been focused on the po-tential use of essential oilsEOfrom aromatic plants5. It is well known that most species, especially those be-longing to the Lamiaceae family, have different biological and pharmacological activities, which has meant that for a long time they have been used for improving the taste and organoleptic properties o f different foods6. Ocimum basi-licumbasiland its EO are used as flavoring in tomato-based products and those that are prone to deterioration by acid-tolerant microbiota7, 8 and studies have revealed the potential use of the of Origanum vulgareoreganoEO against several microorganismos9, 10. Foodborne diseases are a growing public health problem worldwide. Salmonella Enteritidis is considered the most important serovar of Salmonella, causing gastrointestinal disease of varying severity in humans11. This pathogen is Abstract: Consumption of chicken meat and its products, especially sausage, have increased in recent years. However, this product is susceptible to microbial contamination during manufacturing, which compromises its shelf life. The flavoring and preservative activities of essential oils (EO) have been recognized and the application of these antimicrobial agents as natural active compounds in food preservation has shown promise. The aim of this study was to evaluate the effect of Ocimum basilicum and Origanum vulgare EO on Listeria monocytogenes and Salmonella Enteritidis strains in artificially inoculated samples of fresh chicken sausage. First, the minimal inhibitory concentration (MIC) of EO in vitro was determined. The sausage was prepared and kept at ± 4°C; then, the inoculation of individual bacteria was carried out. EO were added at 0.3%, 1.0% and 1.5%v/w. After 0, 5, and 24 hours, the most probable number method (MPN) was performed. Transmission electron microscopy (TEM) was used to view the damage caused by these EO on bacterial morphology and/or structure. Only the 1.5% concentration was effective in reducing L. monocytogenes. 0.3% of O. vulgare EO was able to reduce the MPN/g of Salmonella Enteritidis (2 log) after 5 hours trials. O. basilicum EO showed no effect on Salmonella after 5 hours, but decreased by 2 log after 24 hours. O. vulgare EO at 1% gave a greater reduction of S. Enteritidis at 5 hours, increasing or maintaining this effect after 24 hours. The results confirmed the potential benefits of use EO in control of foodborne pathogens. Key words: Ocimum basilicum, Origanum vulgare, Salmonella Enteritidis, Listeria monocytogenes, preservative
L. N. Barbosa, I. S. Probst, B. F. M. T. Andrade et al. J. Oleo Sci. 64, (1) 117-124 (2015) 118 commonly found in chicken, which is the primary vector for transmission of Salmonella to humans12, 13. Listeria monocytogenes, on the other hand, is common in dairy products and red meat, but it can also be found in chicken, adding to the health concerns of Salmonella and Campy-lobacter14. Listeria is an opportunistic pathogen that mainly affects pregnant women, newborns, the elderly and immunocompromised individuals. This pathogen emerged in the late 20th century and has caused many outbreaks with high mortality rates15, 16. Thus the aim was to investigate the antimicrobial activi-ties of O. basilicum and O. vulgare EO against Listeria monocytogenes and Salmonella Enteritidis in artificially inoculated fresh chicken sausage samples after different periods of contact between pathogen and EO. 2 EXPERIMENTAL 2.1 Essential oils Fresh plant samples of O. basilicum and O. vulgare were purchased in the city of Botucatu, São Paulo, Brazil, and used in the preparation of EO by the steam distillation methodology in a Marconi device, Model M480. Dried speci-mens of plants were deposited in the Herbarium Irina Delanova Gemtchujnicov Department of Botany, Institute of Biosciences – IBB/ UNESP, whose numbers were: O. basilicum Botu 26037 and O. vulgare Botu 26287. 2.2 Chemical characterization Chemical analysis of EO was performed by gas chroma-tography-mass spectrometryGC-MSin a Shimadzu device, model QP5050A, using a capillary column, CBP-5, 50 m in length, with an internal diameter of 0.25 mm and 0.25 μm film thickness. The carrier gas was He and the identification of EO compounds was made on the basis of the National Institute of Standards and TechnologyNISTlibrary, analysis of the mass spectra, and also data in the literature17. 2.3 Preparation of fresh chicken sausage samples The formulation comprising 84.55 of boneless chicken breast, 10 lard, 3 water, 1.5 salt, 0.5 polyphos-phate, 0.25 garlic, and 0.2 pepper 18. The mass was in-corporated into in swine casings with a mean diameter of around 30 mm, and the samples produced were divided buds, separated by lots, and stored in a refrigerator at 4. 2.4 Bacterial strains Salmonella EnteritidisATCC-13076and Listeria monocytogenesATCC-15313strains were stored at 80 until their use in microbiological essays. 2.5 Enumeration of L. monocytogenes and S. Enteritidis in chicken sausage assays Susceptibility tests of the EO were performed with the inoculation of bacterial strains on chicken sausage samples 25 gwith suspensions standardized by a 0.5 MacFarland standard, aiming at a bacterial concentration of approxi-mately 105 colony forming unit/garound 5 log CFU/ g. After, volumes of O. vulgare and O. basilicum EO were added separately, to achieve concentrations of 0.3MIC ob-tained in previous microdilution in vitro assays – data not shown, 1.0, and 1.5 in inoculated sausage samples. All phases of assays were performed in sterile Petri plates, all procedures were carried out at laminar flow, and handling of the bacteria and EO homogenization were performed using sterile cutleryknife and forkmade of stainless steel. Following homogenization, sausage samples were kept at 4 refrigerator temperature. After 0, 5, and 24 hours, quantification of the bacteria inoculated in the sausage samples was performed by the most probable number MPNmethod. Despite the inherent characteristics of the MPN techniquee.g., large volume of material required, workload, and the time necessary to complete identifica-tion, this method proved to have high sensitivity and high reproducibility19. Different times were chosen to verify ifthe contact time influences the antibacterial action of es-sential oils. Control tests were also prepared using non-in-oculatednegative controland inoculated sausage samples positive controlboth without EO addition. Assays were performed in triplicate. The detection of Listeria, 25 g were homogenized in stomacher with 225 ml of LEB brothListeria Enrichment Broth - Oxoidand pre incubated a t 30 for 4 hours. The following were added selective agents40 mg/L nalidixic acid 50 mg/L of cycloheximide and 15 mg/L of acriflavinewith reincubation under the same temperature for 48 hours. At 24 and 48 hours, aliquots were plated with the aid of a chromium nickel strap in Palcam agarOxoidincu-bated at 35 for 48 hours. After this period, up to 5 coloniesblack with black halos, due to b reakage Aesculincharacteristics were transferred to a tube with TSA-YE agarTSA plus 0.6 yeast extract, incubated at 35ºC/24 hours. From this stock preliminary evidence of identifica-tion such as Gram stainGram positive rods are, the catalasepositive reactionand plated on agar motility for observation of growth like umbrella were performed. Then, if necessary, suspected colonies are identified with the help of API20. In tests with Salmonella, it is wo
หน้า: 18 117 Journal of Oleo Science Copyright ©2015 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess14163 J. Oleo Sci. 64, (1) 117-124 (2015) Essential Oils from Herbs against Foodborne Pathogens in Chicken Sausage Lidiane Nunes Barbosa1*, Isabella Silva Probst1, Bruna Fernanda Murbach Teles Andrade1, Fernanda Cristina Bérgamo Alves1, Mariana Albano1, Vera Lucia Mores Rall2 and Ary Fernandes Júnior1 1 Laboratory of Bacteriology and Natural Products. 2Labor atory of Food Microbiology. Department of Microbiology and Immunology, Biosciences Institute,Universidade Estadual Paulista "Julio de Mesquita Filho", Botucatu,São Paulo, Brazil, CEP 18618-970 1 INTRODUCTION Chicken meat and its products have increased in popu-larity and have become widespread throughout the world, with chicken sausage being one of the most popular cate-gories among these products1. Sausage manufacture in-volves a number of handling steps, which increase the chances of contamination by pathogens or spoilage2. Fresh sausage does not undergo heat treatment and has a highwater activity; giving this food a short shelf life and sub-jecting it directly to the action of the microorganisms pres-ents3. Application of agents with adequate antimicrobial and antioxidant activities has significant potential to extend the shelf life of chicken products and prevent economic losses4. Due to the negative perception of chemical preser-vatives, consumers attention is c hanging to natural alter-*Correspondence to: Lidiane Nunes Barbosa, Department of Microbiology and Immunology, Biosciences Institute, Sao Paulo State University E-mail: lidianebarbosa@ibb.unesp.br Accepted September 2, 2014 (received for review July 25, 2014) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/http://mc.manusriptcentral.com/jjocs natives and particular interest has been focused on the po-tential use of essential oilsEOfrom aromatic plants5. It is well known that most species, especially those be-longing to the Lamiaceae family, have different biological and pharmacological activities, which has meant that for a long time they have been used for improving the taste and organoleptic properties o f different foods6. Ocimum basi-licumbasiland its EO are used as flavoring in tomato-based products and those that are prone to deterioration by acid-tolerant microbiota7, 8 and studies have revealed the potential use of the of Origanum vulgareoreganoEO against several microorganismos9, 10. Foodborne diseases are a growing public health problem worldwide. Salmonella Enteritidis is considered the most important serovar of Salmonella, causing gastrointestinal disease of varying severity in humans11. This pathogen is Abstract: Consumption of chicken meat and its products, especially sausage, have increased in recent years. However, this product is susceptible to microbial contamination during manufacturing, which compromises its shelf life. The flavoring and preservative activities of essential oils (EO) have been recognized and the application of these antimicrobial agents as natural active compounds in food preservation has shown promise. The aim of this study was to evaluate the effect of Ocimum basilicum and Origanum vulgare EO on Listeria monocytogenes and Salmonella Enteritidis strains in artificially inoculated samples of fresh chicken sausage. First, the minimal inhibitory concentration (MIC) of EO in vitro was determined. The sausage was prepared and kept at ± 4°C; then, the inoculation of individual bacteria was carried out. EO were added at 0.3%, 1.0% and 1.5%v/w. After 0, 5, and 24 hours, the most probable number method (MPN) was performed. Transmission electron microscopy (TEM) was used to view the damage caused by these EO on bacterial morphology and/or structure. Only the 1.5% concentration was effective in reducing L. monocytogenes. 0.3% of O. vulgare EO was able to reduce the MPN/g of Salmonella Enteritidis (2 log) after 5 hours trials. O. basilicum EO showed no effect on Salmonella after 5 hours, but decreased by 2 log after 24 hours. O. vulgare EO at 1% gave a greater reduction of S. Enteritidis at 5 hours, increasing or maintaining this effect after 24 hours. The results confirmed the potential benefits of use EO in control of foodborne pathogens. Key words: Ocimum basilicum, Origanum vulgare, Salmonella Enteritidis, Listeria monocytogenes, preservativeL. N. Barbosa, I. S. Probst, B. F. M. T. Andrade et al. J. Oleo Sci. 64, (1) 117-124 (2015) 118 commonly found in chicken, which is the primary vector for transmission of Salmonella to humans12, 13. Listeria monocytogenes, on the other hand, is common in dairy products and red meat, but it can also be found in chicken, adding to the health concerns of Salmonella and Campy-lobacter14. Listeria is an opportunistic pathogen that mainly affects pregnant women, newborns, the elderly and immunocompromised individuals. This pathogen emerged in the late 20th century and has caused many outbreaks with high mortality rates15, 16. Thus the aim was to investigate the antimicrobial activi-ties of O. basilicum and O. vulgare EO against Listeria monocytogenes and Salmonella Enteritidis in artificially inoculated fresh chicken sausage samples after different periods of contact between pathogen and EO. 2 EXPERIMENTAL 2.1 Essential oils Fresh plant samples of O. basilicum and O. vulgare were purchased in the city of Botucatu, São Paulo, Brazil, and used in the preparation of EO by the steam distillation methodology in a Marconi device, Model M480. Dried speci-mens of plants were deposited in the Herbarium Irina Delanova Gemtchujnicov Department of Botany, Institute of Biosciences – IBB/ UNESP, whose numbers were: O. basilicum Botu 26037 and O. vulgare Botu 26287. 2.2 Chemical characterization Chemical analysis of EO was performed by gas chroma-tography-mass spectrometryGC-MSin a Shimadzu device, model QP5050A, using a capillary column, CBP-5, 50 m in length, with an internal diameter of 0.25 mm and 0.25 μm film thickness. The carrier gas was He and the identification of EO compounds was made on the basis of the National Institute of Standards and TechnologyNISTlibrary, analysis of the mass spectra, and also data in the literature17. 2.3 Preparation of fresh chicken sausage samples The formulation comprising 84.55 of boneless chicken breast, 10 lard, 3 water, 1.5 salt, 0.5 polyphos-phate, 0.25 garlic, and 0.2 pepper 18. The mass was in-corporated into in swine casings with a mean diameter of around 30 mm, and the samples produced were divided buds, separated by lots, and stored in a refrigerator at 4. 2.4 Bacterial strains Salmonella EnteritidisATCC-13076and Listeria monocytogenesATCC-15313strains were stored at 80 until their use in microbiological essays. 2.5 Enumeration of L. monocytogenes and S. Enteritidis in chicken sausage assays Susceptibility tests of the EO were performed with the inoculation of bacterial strains on chicken sausage samples 25 gwith suspensions standardized by a 0.5 MacFarland standard, aiming at a bacterial concentration of approxi-mately 105 colony forming unit/garound 5 log CFU/ g. After, volumes of O. vulgare and O. basilicum EO were added separately, to achieve concentrations of 0.3MIC ob-tained in previous microdilution in vitro assays – data not shown, 1.0, and 1.5 in inoculated sausage samples. All phases of assays were performed in sterile Petri plates, all procedures were carried out at laminar flow, and handling of the bacteria and EO homogenization were performed using sterile cutleryknife and forkmade of stainless steel. Following homogenization, sausage samples were kept at 4 refrigerator temperature. After 0, 5, and 24 hours, quantification of the bacteria inoculated in the sausage samples was performed by the most probable number MPNmethod. Despite the inherent characteristics of the MPN techniquee.g., large volume of material required, workload, and the time necessary to complete identifica-tion, this method proved to have high sensitivity and high reproducibility19. Different times were chosen to verify ifthe contact time influences the antibacterial action of es-sential oils. Control tests were also prepared using non-in-oculatednegative controland inoculated sausage samples positive controlboth without EO addition. Assays were performed in triplicate. The detection of Listeria, 25 g were homogenized in stomacher with 225 ml of LEB brothListeria Enrichment Broth - Oxoidand pre incubated a t 30 for 4 hours. The following were added selective agents40 mg/L nalidixic acid 50 mg/L of cycloheximide and 15 mg/L of acriflavinewith reincubation under the same temperature for 48 hours. At 24 and 48 hours, aliquots were plated with the aid of a chromium nickel strap in Palcam agarOxoidincu-bated at 35 for 48 hours. After this period, up to 5 coloniesblack with black halos, due to b reakage Aesculincharacteristics were transferred to a tube with TSA-YE agarTSA plus 0.6 yeast extract, incubated at 35ºC/24 hours. From this stock preliminary evidence of identifica-tion such as Gram stainGram positive rods are, the catalasepositive reactionand plated on agar motility for observation of growth like umbrella were performed. Then, if necessary, suspected colonies are identified with the help of API20. In tests with Salmonella, it is wo
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