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Registros recuperados: 23
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Adaptation to Cold and Proteomic Responses of the Psychrotrophic Biopreservative Lactococcus piscium Strain CNCM I-4031 ArchiMer
Garnier, Matthieu; Matamoros, Sebastien; Chevret, Didier; Pilet, Marie-france; Leroi, Francoise; Tresse, Odile.
There is considerable interest in the use of psychrotrophic bacteria for food biopreservation and in the understanding of cold adaptation mechanisms. The psychrotrophic biopreservative Lactococcus piscium strain CNCM I-4031 was studied for its growth behavior and proteomic responses after cold shock and during cold acclimation. Growth kinetics highlighted the absence of growth latency after cold shock, suggesting a very high promptness in cold adaptation, a behavior that has never been described before for lactic acid bacteria (LAB). A comparative proteomic analysis was applied with two-dimensional gel electrophoresis (2-DE), and upregulated proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both cold shock and cold...
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Ano: 2010 URL: http://archimer.ifremer.fr/doc/00019/13047/10150.pdf
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Applications of protective cultures, bacteriocins and bacteriophages in fresh seafood and seafood products ArchiMer
Pilet, Marie-france; Leroi, Francoise.
Microbial seafood-borne disease represents 10 to 20% of the total food-borne outbreaks. Most of them are from bacterial origin and involve seafood products that have been contaminated by pathogenic Vibrio, Listeria monocytogenes and histaminogen bacteria. On the other hand, seafood products are very sensitive to the development of spoiling bacteria producing off-odours. Pathogenic and spoiling microflora are not always reduced or limited by the processing steps that are currently used in these foodstuffs, and the interest for alternative techniques such as bioprotection to improve quality and safety of seafood has increased in the last years. Among the microbial flora of lightly preserved seafood products, lactic acid bacteria usually become dominant...
Tipo: Text Palavras-chave: Biopreservation of seafood; Seafood safety and spoilage; Lactic acid bacteria; Sensory quality; Listeria monocytogenes.
Ano: 2010 URL: http://archimer.ifremer.fr/doc/00030/14138/11384.pdf
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Bioprotective Effect of Lactococcus piscium CNCM I-4031 Against Listeria monocytogenes Growth and Virulence ArchiMer
Saraoui, Taous; Leroi, Francoise; Chevalier, Frederique; Cappelier, Jean-michel; Passerini, Delphine; Pilet, Marie-france.
Listeria monocytogenes is a Gram-positive pathogen occurring in many refrigerated ready-to-eat foods. It is responsible for foodborne listeriosis, a rare but severe disease with a high mortality rate (20–30%). Lactococcus piscium CNCM I-4031 has the capacity to prevent the growth of L. monocytogenes in contaminated peeled and cooked shrimp and in a chemically defined medium using a cell-to-cell contact-dependent mechanism. To characterize this inhibition further, the effect of L. piscium was tested on a collection of 42 L. monocytogenes strains. All strains were inhibited but had different sensitivities. The effect of the initial concentration of the protective and the target bacteria revealed that the inhibition always occurred when L. piscium had reached...
Tipo: Text Palavras-chave: Biopreservation; Lactococcus piscium; Listeria monocytogenes; Co-culture; Cell ratio; Scanning electron microscopy; Virulence.
Ano: 2018 URL: https://archimer.ifremer.fr/doc/00449/56065/57588.pdf
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Characterisation of the spoilage microbiota in raw salmon (Salmo salar) steaks stored under vacuum or modified atmosphere packaging combining conventional methods and PCR–TTGE ArchiMer
Mace, Sabrina; Cornet, Josiane; Chevalier, Frederique; Cardinal, Mireille; Pilet, Marie-france; Dousset, Xavier; Joffraud, Jean-jacques.
In order to characterise the spoilage related to microbiota of raw salmon, a combination of culture-dependent and -independent methods, including PCR–TTGE, was used to analyse 3 raw salmon batches stored for 3 days at chilled temperature in modified atmosphere packaging (MAP) (50% CO2/50% N2) or under vacuum. Sensory evaluation, microbiological enumeration and chemical analysis were performed after 3, 7 and 10 days of storage. At the onset of spoilage, 65 bacterial isolates were picked from the plates. Thus, 13 different genera or species were identified by phenotypic and molecular tests: Serratia spp., Photobacterium phosphoreum, Yersinia intermedia, Hafnia alvei, Buttiauxella gaviniae, Pseudomonas sp., Carnobacterium maltaromaticum, Carnobacterium...
Tipo: Text Palavras-chave: Seafood; Culture-independent method (PCR-TTGE); Sensory analysis; Photobacterium phosphoreum; Lactococcus piscium.
Ano: 2012 URL: https://archimer.ifremer.fr/doc/00052/16277/13779.pdf
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Comportement d'une bactérie lactique psychrotrophe à différentes températures ArchiMer
Matamoros, Sebastien; Leroi, Francoise; Prevost, Herve; Pilet, Marie-france.
La souche de Lactococcus sp. EU2241 a été isolée à partir de saumon emballé sous atmosphère modifiée. Elle a montré d'exellentes aptitudes à la biopréservation, augmentant la durée de vie de crevettes décortiquées sous vide, de saumon fumé et permettant d'inhiber de 2 logs la croissance de Listeria monocytogenes et Staphylococcus aureus sur du saumon fumé. L'objectif de ces travaux est de caractériser le comportement à basse température de cette souche, et d'identifier les mécanismes moléculaires intervenant dans sa capacité à s'adapter aux basses températures. Ce travail a été réalisé dans le cadre du projet intégré (IP) SEAFOODplus accordé par la commission européenne sous le numéro FOOD-CT-2004-506359.
Tipo: Text Palavras-chave: Psychrotrophe; Bactérie lactique; Psychrotrophic; Lactic bacteria.
Ano: 2007 URL: http://archimer.ifremer.fr/doc/2007/acte-3986.pdf
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Development of a Rapid Real-Time PCR Method as a Tool To Quantify Viable Photobacterium phosphoreum Bacteria in Salmon (Salmo salar) Steaks ArchiMer
Mace, Sabrina; Mamlouk, Kelthoum; Chipchakova, Stoyka; Prevost, Herve; Joffraud, Jean-jacques; Dalgaard, Paw; Pilet, Marie-france; Dousset, Xavier.
A specific real-time PCR quantification method combined with a propidium monoazide sample treatment step was developed to determine quantitatively the viable population of the Photobacterium phosphoreum species group in raw modified-atmosphere-packed salmon. Primers were designed to amplify a 350-bp fragment of the gyrase subunit B gene (gyrB) of P. phosphoreum. The specificity of the two primers was demonstrated by using purified DNA from 81 strains of 52 different bacterial species. When these primers were used for real-time PCR in pure culture, a good correlation (R-2 of 0.99) was obtained between this method and conventional enumeration on marine agar (MA). Quantification was linear over 5 log units as confirmed by using inoculated salmon samples. On...
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Ano: 2013 URL: https://archimer.ifremer.fr/doc/00135/24623/22772.pdf
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Effect of inoculation of Carnobacterium divergens V41, a biopreservative strain against Listeria monocytogenes risk, on the microbiological, chemical and sensory quality of cold-smoked salmon ArchiMer
Brillet, Anne; Pilet, Marie-france; Prevost, Hervé; Cardinal, Mireille; Leroi, Francoise.
The aim of this study was to develop a biopreservation strategy for cold-smoked salmon (CSS) by the use of lactic acid bacteria previously selected for their capability to inhibit the growth of Listeria monocytogenes in the product. The spoiling potential of three Carnobacterium strains (Carnobacterium divergens V41, Carnobacterium piscicola V1 and SF668) was tested in sterile CSS blocks inoculated by 10(4-5) CFU g(-1) and stored under vacuum for 9 days at 4 degrees C followed by 19 days at 8 degrees C. C. divergens V41 grew a little faster than the other strains and none of the three carnobacteria showed any adverse effect on quality of the product, i.e. no off-odour detected by a trained panel, no total volatile basic nitrogen (TVBN) production, no...
Tipo: Text Palavras-chave: Sensory analysis; Biogenic amine; Spoilage microflora; Carnobacterium; Biopreservation; Cold smoked salmon.
Ano: 2005 URL: http://archimer.ifremer.fr/doc/2005/publication-724.pdf
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Evaluation of the spoilage potential of bacteria isolated from spoiled cooked whole tropical shrimp (Penaeus vannamei) stored under modified atmosphere packaging ArchiMer
Mace, Sabrina; Cardinal, Mireille; Jaffres, Emmanuel; Cornet, Josiane; Lalanne, Valerie; Chevalier, Frederique; Serot, Thierry; Pilet, Marie-france; Dousset, Xavier; Joffraud, Jean-jacques.
The spoilage potential of isolates belonging to five bacterial groups/species (Shewanella baltica, Carnobacterium maltaromaticum, Aeromonas salmonicida, Vibrio sp., “other Gamma-Proteobacteria” [containing one strain of Pseudoalteromonas sp. and one strain of Psychrobacter sp.]) isolated from spoiled cooked and whole tropical shrimp stored under modified atmosphere packaging (MAP) was evaluated by inoculation into ionized cooked and peeled tropical shrimp followed by storage for 32 days at 8°C. Microbial growth and sensory changes were monitored during the storage period. The major spoilage bacterial isolate groups were C. maltaromaticum and S. baltica. In order to characterize their spoilage potential further and to study the effect of their interactions,...
Tipo: Text Palavras-chave: Spoilage potential; Bacterial interaction; Specific spoilage organism; Cooked tropical shrimp; Volatile compounds.
Ano: 2014 URL: https://archimer.ifremer.fr/doc/00168/27926/26200.pdf
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Evaluation of the spoilage potential of bacteria isolated from spoiled raw salmon (Salmo salar) fillets stored under modified atmosphere packaging ArchiMer
Mace, Sabrina; Joffraud, Jean-jacques; Cardinal, Mireille; Malcheva, Mariya; Cornet, Josiane; Lalanne, Valerie; Chevalier, Frederique; Serot, Thierry; Pilet, Marie-france; Dousset, Xavier.
The spoilage potential of eight bacterial groups/species (Serratia spp., Hafnia alvei, Brochothrix thermosphacta, Carnobacterium maltaromaticum, Shewanella baltica, Lactococcus piscium, Photobacterium phosphoreum, "other Enterobacteriaceae" [containing one strain of Moellerella sp., Morganella sp. and Pectobacterium sp.]) isolated from spoiled raw salmon fillets stored under modified atmosphere packaging (MAP) was evaluated by inoculation into sterile raw salmon cubes followed by storage for 12 days at 8 degrees C. Microbial growth and sensory changes were monitored during the storage period. The dominant spoilage bacteria were C maltaromaticum, H. alvei and P. phosphoreum. In order to further characterize their spoilage potential and to study the effect...
Tipo: Text Palavras-chave: Spoilage potential; Bacterial interaction; Specific spoilage organism; Atlantic salmon; Volatile compounds.
Ano: 2013 URL: http://archimer.ifremer.fr/doc/00125/23656/22006.pdf
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Hurdle technology to ensure the safety of seafood products ArchiMer
Leroi, Francoise; Amarita, Felix; Arboleya, Juan Carlos; Bjørkevoll, Ingebright; Cruz, Ziortza; Dousset, Xavier; Izurieta, E; Joffraud, Jean-jacques; Lasagabaster, Amaïa; Lauzon, Hélène-liette; Lorentzen, G; Martínez De Marañón, Iñigo; Matamoros, Sébastien; Miranda, I; Nuin, Maider; Olabarrieta, Idoia; Olsen, R; Pilet, Marie-france; Prevost, Hervé; Skjerdal, Taran.
The microbial safety and stability of most food, are based on an application of preservative factors called hurdles. Each hurdle implies putting microorganisms in a hostile environment, which inhibits their growth or causes their death (Leistner, 2000). Some of those hurdles have been empirically used for years to stabilize meat, fish, milk and vegetables. This sometimes leads to completely different product with its own new taste characteristics. Examples of hurdles in marine products are salt (salted cod, klipfish), smoke (cold or hot smoked salmon, herring), acids (marinated products, pickles), temperature (high or low), fermentative microorganisms (traditional Asian sauces) and more recently redox potential (vacuum-packed products). Those preservative...
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Ano: 2008 URL: http://archimer.ifremer.fr/doc/2006/publication-6492.pdf
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Influence of temperature, pH and NaCl concentration on the maximal growth rate of Brochothrix thermosphacta and a bioprotective bacteria Lactococcus piscium CNCM I-4031 ArchiMer
Leroi, Francoise; Fall, Papa-abdoulaye; Pilet, Marie-france; Chevalier, Frederique; Baron, Regis.
The maximum specific growth rate (μmax) of Brochothrix thermosphacta, a spoilage bacteria of cooked peeled shrimp, and Lactococcus piscium CNCM I-4031, a bioprotective strain, was investigated under different conditions of temperature, NaCl concentrations and pH. The basic modelling approach used was the Gamma concept (γ-concept) and the model developed was then adapted to shrimp. Cardinal growth parameters were quite similar for the two strains, except for NaCl. No NaCl was required for growth and the NaClmax was three-times higher for B. thermosphacta than for L. piscium (62 and 23 g l−1 respectively). However, tolerance to NaCl was higher in seafood than in liquid broth, possibly due to presence of osmoltically active molecules. L. piscium and B....
Tipo: Text Palavras-chave: Modelling; Biopreservation; Shrimp; Lactic acid bacteria; Brochothrix thermosphacta; Spoilage bacteria; Environmental conditions.
Ano: 2012 URL: http://archimer.ifremer.fr/doc/00083/19473/17088.pdf
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Inhibition of Brochothrix thermosphacta and sensory improvement of tropical peeled cooked shrimp by Lactococcus piscium CNCM I-4031 ArchiMer
Fall, Papa-abdoulaye; Leroi, Francoise; Cardinal, Mireille; Chevalier, Frederique; Pilet, Marie-france.
Aims: To investigate the antimicrobial spectrum of Lactococcus piscium CNCM I-4031 and its protective effect in cooked and peeled shrimp against Brochothrix thermosphacta. Methods and Results: Sixteen pathogenic and spoiling bacteria were inhibited in Elliker, but not in shrimp juice agar plates. In shrimp packed under modified atmosphere and stored at 8 degrees C, B. thermosphacta (103 CFU g-1) was inhibited by 4 center dot 1 log CFU g-1 when co-inoculated with L. piscium (106 CFU g-1). Brochothrix thermosphacta spoiled the product after 11 days, with the emission of strong butter/caramel off-odours. In co-culture with L. piscium, sensory shelf-life was extended by at least 10 days. The inhibition was partially explained by a drop in pH from 6 center dot...
Tipo: Text Palavras-chave: Biopreservation; Brochothrix thermosphacta; Inhibition spectrum; Lactococcus piscium; Penaeus vanamei; Shrimp composition.
Ano: 2010 URL: http://archimer.ifremer.fr/doc/00002/11291/7884.pdf
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Lactic acid bacteria selection for biopreservation as a part of hurdle technology approach applied on seafood ArchiMer
Wiernasz, Norman; Cornet, Josiane; Cardinal, Mireille; Pilet, Marie-france; Passerini, Delphine; Leroi, Francoise.
As fragile food commodities, microbial and organoleptic qualities of fishery and seafood can quickly deteriorate. In this context, microbial quality and security improvement during the whole food processing chain (from catch to plate), using hurdle technology, a combination of mild preserving technologies such as biopreservation, modified atmosphere packaging and superchilling, are of great interest. Thirty-five lactic acid bacteria (LAB) belonging to the species Carnobacterium maltaromaticum, Carnobacterium inhibens, Lactococcus piscium, Leuconostoc gelidum, Vagococcus fluvialis, Vagococcus penaei and Aerococcus viridans, known to possess interesting antimicrobial activity, were chosen for their potential application as bioprotective agents as a part of...
Tipo: Text Palavras-chave: Antimicrobial activity; Spoilage; Screening; Chitosan; Fish juice; Safety assessment.
Ano: 2017 URL: https://archimer.ifremer.fr/doc/00382/49342/49732.pdf
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Maîtrise du développement de Listeria monocytogenes dans le saumon fumé : intérêt de la biopréservation par des bactéries lactiques ArchiMer
Richard, Christelle; Leroi, Francoise; Brillet-viel, Anne; Rachman, Cinta; Connil, Nathalie; Drider, Djamel; Pilet, Marie-france; Onno, Bernard; Dousset, Xavier; Prevost, Hervé.
The interest of biopreservation using lactic bacteria to improve the food security of smoked salmon was evaluated. Cold smoked salmon is a non-stable microbiological product whose physicochemical characteristics allow the development of Listeria monocytogenes which constitutes the major microbiological risk depending on the consumption of this product. Biodiversity of the microflora of smoked salmon and its evolution during conservation, the origin of the contamination by Listeria and its consequences, and the parameters of the manufacturing process allowing a better control of microbiological quality are discussed. The innovating concept of biopreservation of smoked salmon is developed. It consists of an inoculation of salmon by a competitive lactic flora...
Tipo: Text Palavras-chave: Saumon fumé; Bactérie lactique; Biopréservation; Listeria; Carnobacterium; Cold smoked salmon; Lactic acid bacteria; Biopreservation; Listeria; Carnobacterium.
Ano: 2004 URL: http://archimer.ifremer.fr/doc/00000/10768/7509.pdf
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Optimization of growth and bacteriocin activity of the food bioprotective Carnobacterium divergens V41 in an animal origin protein free medium ArchiMer
Brillet-viel, Anne; Pilet, Marie-france; Courcoux, Philippe; Prevost, Hervé; Leroi, Francoise.
Optimization of Carnobacterium divergens V41 growth and bacteriocin activity in a culture medium deprived of animal protein, needs for food bioprotection, was performed by using a statistical approach. In a screening experiment, twelve factors (pH, temperature, carbohydrates, NaCl, yeast extract, soy peptone, sodium acetate, ammonium citrate, magnesium sulphate, manganese sulphate, ascorbic acid and thiamine) were tested for their influence on the maximal growth and bacteriocin activity using a two-level incomplete factorial design with 192 experiments performed in microtiter plate wells. Based on results, a basic medium was developed and three variables (pH, temperature and carbohydrates concentration) were selected for a scale-up study in bioreactor. A...
Tipo: Text Palavras-chave: Optimization; Carnobacterium; Bacteriocin; Experimental design; Bioreactor; Growth; Cold-smoked salmon.
Ano: 2016 URL: https://archimer.ifremer.fr/doc/00345/45615/45236.pdf
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Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage ArchiMer
Chaillou, Stephane; Chaulot-talmon, Aurelie; Caekebeke, Helene; Cardinal, Mireille; Christieans, Souad; Denis, Catherine; Desmonts, Marie Helene; Dousset, Xavier; Feurer, Carole; Hamon, Erwann; Joffraud, Jean-jacques; La Carbona, Stephanie; Leroi, Francoise; Leroy, Sabine; Lorre, Sylvie; Mace, Sabrina; Pilet, Marie-france; Prevost, Herve; Rivollier, Marina; Roux, Dephine; Talon, Regine; Zagorec, Monique; Champomier-verges, Marie-christine.
The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota,...
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Ano: 2015 URL: https://archimer.ifremer.fr/doc/00268/37960/37199.pdf
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Protective Effect of a Non-Bacteriocinogenic Lactococcus piscium CNCM I-4031 Strain Against Listeria monocytogenes in Sterilized Tropical Cooked Peeled Shrimp ArchiMer
Fall, Papa-abdoulaye; Leroi, Francoise; Chevalier, Frederique; Guerin, Charline; Pilet, Marie-france.
The protective activity of a non-bacteriocinogenic Lactococcus piscium CNCM I-4031 strain against Listeria monocytogenes was investigated in tropical cooked peeled shrimp stored at 8°C in modified atmosphere packaging (50% N2-50% CO2). When inoculated alone (L. piscium 107 CFU g-1 and L. monocytogenes 104 CFU g-1), protective culture and target strain grew very well on shrimp reaching a maximum cell number of 109 CFU g-1 after 7 and 14 days, respectively. In the presence of L. piscium, growth of L. monocytogenes was totally prevented after 3 days of storage. The count was 3.4 log CFU g-1 lower than in the control after 10 days and until the end of storage (31 days). Using the Seafood Spoilage and Safety Predictor Software (http://sssp.dtuaqua.dk), it was...
Tipo: Text Palavras-chave: Inhibition; Biopreservation; Challenge-test; Lactic acid; Nutritional competition.
Ano: 2010 URL: http://archimer.ifremer.fr/doc/00005/11624/8281.pdf
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Response of Listeria monocytogenes to liquid smoke ArchiMer
Guilbaud, Morgan; Chafsey, Ingrid; Pilet, Marie-france; Leroi, Francoise; Prevost, Herve; Hebraud, Michel; Dousset, Xavier.
Aims: To investigate the effect of liquid smoke on growth, survival, proteomic pattern and haemolytic potential of Listeria monocytogenes. Methods and Results: Growth and survival curves were recorded in brain-heart infusion broth supplemented with three concentrations of liquid smoke. L. monocytogenes growth was inhibited in the presence of 15 mu g ml(-1) phenol while a rapid decrease in cell viability occurred in the presence of 30 mu g ml(-1) phenol. The proteome of L. monocytogenes cytosoluble proteins was slightly modified after 2-h incubation with 30 mu g ml(-1) phenol but no protein already characterized in response to other known stresses was induced, except the protease ClpP. Liquid smoke inhibited the haemolytic potential without affecting hly...
Tipo: Text Palavras-chave: Survival; Stress; Proteome analysis; Listeria monocytogenes; Liquid smoke; Haemolysis.
Ano: 2008 URL: http://archimer.ifremer.fr/doc/2008/publication-4522.pdf
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Salmon Gravlax Biopreservation With Lactic Acid Bacteria: A Polyphasic Approach to Assessing the Impact on Organoleptic Properties, Microbial Ecosystem and Volatilome Composition ArchiMer
Wiernasz, Norman; Leroi, Francoise; Chevalier, Frederique; Cornet, Josiane; Cardinal, Mireille; Rohloff, Jens; Passerini, Delphine; Skırnisdóttir, Sigurlaug; Pilet, Marie-france.
Seafood and fishery products are very perishable commodities with short shelf-lives owing to rapid deterioration of their organoleptic and microbiological quality. Microbial growth and activity are responsible for up to 25% of food losses in the fishery industry. In this context and to meet consumer demand for minimally processed food, developing mild preservation technologies such as biopreservation represents a major challenge. In this work, we studied the use of six lactic acid bacteria (LAB), previously selected for their properties as bioprotective agents, for salmon dill gravlax biopreservation. Naturally contaminated salmon dill gravlax slices, with a commercial shelf-life of 21 days, were purchased from a French industrial company and inoculated by...
Tipo: Text Palavras-chave: Metabarcoding; 16S rRNA gene; Seafood; Antilisterial activity; Volatile organic compounds; Sensory analyses.
Ano: 2020 URL: https://archimer.ifremer.fr/doc/00605/71716/70174.pdf
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Selection and evaluation of seafood-borne psychrotrophic lactic acid bacteria as inhibitors of pathogenic and spoilage bacteria ArchiMer
Matamoros, Sebastien; Pilet, Marie-france; Gigout, Frederique; Prevost, Herve; Leroi, Francoise.
In this study, inhibitory psychrotrophic lactic acid bacteria were isolated and investigated for future use in biopreservation of seafood products. Screening of 5575 colonies isolated from various seafood products resulted in the selection of 132 colonies presenting inhibitory properties. Among them, 52 isolates had characteristics of LAB and showed growth at 15 °C but not at 30 °C. The inhibition spectrum of these 52 isolates against 14 target strains (Gram-positive and -negative) showed inhibition of typical seafood spoiling and pathogenic bacteria and enabled the formation of seven interesting clusters. Sequencing of the 16S rRNA gene of a representative isolate from each cluster identified three Leuconostoc gelidum, two Lactococcus piscium, one...
Tipo: Text Palavras-chave: Carnobacterium alterfunditum; Lactobacillus fuchuensis; Leuconostoc gelidum; Lactococcus piscium; Psychrotrophic; Inhibition; Seafood; Lactic acid bacteria.
Ano: 2009 URL: http://archimer.ifremer.fr/doc/2009/publication-6472.pdf
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