Tesis sobre el tema "Psychrotrophy"

Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d₂, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.

Psychrotrophic bacteria, such as Pseudomonas fluorescens B52, are a major cause of milk spoilage at refrigeration temperature due to the production of lipolytic and proteolytic enzymes. Regulatory mechanisms controlling the production of lipase and protease by the B52 lipA and aprX genes were investigated. Transposon mutagenesis identified the possible involvement of a poly-A polymerase enzyme which destabilises mRNA by 3' polyadenylation. A homologue of the E. coli EnvZ/OmpR two-component sensor/regulator system was identified by transposon mutagenesis and shown to repress lipase and protease production. This system responds to Na+ and K+ concentration in E. coli and these ions were also shown to repress lipase and protease expression in B52, however the EnvZ/OmpR system is not solely responsible for this. Assays of translational lacZ fusions with aprX and lipA were used to speculate on the mechanism by which Na+ and EnvZ/OmpR repress the aprX-lipA operon. A membrane-bound sensor, MspA, which regulates protease production in P. fluorescens LS107d2, was shown to exist in B52 but mutagenesis of the B52 mspA gene had no effect on lipase and protease expression. A homologue of the P. fluorescens CHA0 rsmA gene, encoding an RNA-binding translation repressor, was found in B52. Although aprX and possibly lipA contain consensus sequences for RsmA, mutagenesis of rsmA had no significant effect on lipase and protease expression. Repression of lipase and protease expression by Na+ was increased by expression of the P. fluorescens M114 pbrA sigma-factor gene in B52.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Health Sciences
Full Text

Pseudomonas fluorescens MFO est une souche psychrotrophe isolée du lait cru, qui produit des enzymes exocellulaires (protéase et lipase). Une famille d'enzymes comprenant la protéase et la lipase, mais aussi trois phosphatases endocellulaires, a été caractérisée par une température optimale de production commune (17°C), inferieure à la température optimale de croissance (30°C). Une étude en chémostat des effets respectifs de la température et du taux de croissance nous a permis de montrer que la production de ces enzymes est effectivement régulée par la température. Cependant le mécanisme de régulation de la production des enzymes exocellulaires apparait beaucoup plus complexe que celui des phosphatases cellulaires acides constitutives. Il met en jeu une induction et une répression exercée soit par le substrat énergétique (répression catabolique), soit par le taux (ou la phase) de croissance. C'est à ce dernier niveau qu'intervient la régulation par la température : l'effet de la répression n'est complètement levé à faible taux de croissance qu'a la température intermédiaire de 17°C. D'un point de vue plus général, les effets de la température et du taux de croissance sur la composition macromoléculaire (protéines, ARN) et la taille cellulaire de P. Fluorescens MFO ont été analysés en cultures discontinues et continues, et comparés à ceux observés chez les bactéries mésophiles et psychrophiles. Il apparait que le comportement de P. Fluorescens MFO vis à vis de la température diffère fondamentalement de celui de bactéries mésophiles et psychrophiles. Il est caractérisé par l'existence d'une zone de température critique intermédiaire (17°C-20°C), qui marque la limite entre deux domaines thermiques (domaine inférieur froid et domaine supérieur chaud) différant dans leurs implications cinétiques et physiologiques. Ainsi le comportement de cette souche psychrotrophe apparait comme intermédiaire entre celui de bactéries mésophiles et de bactéries psychrophiles.

$ beta$-Galactosidase (E.C. 3.2.1.23) or lactase was produced by the growth of a selected Bacillus subtilis strain (KL88) which was adapted to grow at 10$ sp circ$C. The growth and enzyme production were maximal at 2% (w/v) lactose supplemented with 0.2% (w/v) yeast extract. A Fast Protein Liquid Chromatography system (FPLC) was used for $ beta$-galactosidase purification. The enzyme was purified to 44-fold over the crude extract with a recovery of $ sim$54%. Native-PAGE and SDS-PAGE using "PhastSystem" showed the presence of two isoenzymes having molecular weights of 88 and 170 kD. The purified enzyme showed high activity at low temperatures (10$ sp circ$C) and recorded an optimum pH of 7.0. The K$ sb{ rm m}$ values were found to be 2.21 mM and 28.08 mM for o-nitrophenyl-$ beta$-D-galactopyranoside (ONPG) and lactose, respectively.
$ beta$-Galactosidase from psychrotrophic Bacillus subtilis was specific to the $ beta$-D-glycosidic linkage normally present in lactose.
To investigate the possibility of producing proteinase-free $ beta$-galactosidase from this psychrotrophic microorganism, FPLC was used for the rapid separation of $ beta$-galactosidase.

Les effets de chocs thermiques sur la croissance et la synthese proteique ont ete etudies chez pseudomonas fragi, bacterie psychotrophe d'alteration des aliments. Les cinetiques de synthese proteique ont ete caracterisees par autoradiographies des proteines intracellulaires marquees a la methionine-#3#5s et separees en electrophorese bidimensionnelle (e-2d). La croissance se poursuivait avec un taux caracteristique de la temperature finale du choc de facon immediate apres les chocs chauds de 5 a 20 ou 30c et de 28 a 34c, et apres 3 et 5 h de latence a la suite d'un transfert de 20 a 5c et de 30 a 5c. Les profils obtenus en e-2d apres les differents chocs ont revele des variations dans le niveau relatif de synthese de 20 a 37 proteines. Ces proteines ont ete repertoriees en fonction de leur poids moleculaire, point isoelectrique et de leur cinetique de variations en reponse a un choc thermique. Les principales proteines impliquees dans la reponse adaptative de p. Fragi a la temperature, ont ainsi ete caracterisees. Les genes de 3 d'entre elles, designees c7. 0, c8. 0, e7. 0, ont ete amplifies pas pcr et sequences. Ces proteines appartiennent a une famille d'activateur de faible masse moleculaire capables de se lier aux acides nucleiques dont le representant type est cspa, la proteine majeure de choc froid d'e. Coli, caracterisee comme un activateur transcriptionnel

Four meat spoilage organisms were grown at 4$ sp circ$C for 7 d, in an aqueous extract of meat (Meat Juice Medium), and the levels of various nutrients in the extracts were measured. At an agitation rate of 50 rev$ cdot$min$ sp{-1},$ the four species reached viable counts of 10$ sp8$ Colony Forming Units (CFU)$ cdot$ml$ sp{-1},$ and the order of nutrient utilization was as follows: (1) glucose, (2) gluconate and urea, (3) glycerol, (4) glucose-6-phosphate. Several substrates were still present in the growth medium at the end of the growth period, namely lactate, glucose-6-phosphate and the two unknowns. At a higher agitation rate (100 rev$ cdot$min$ sp{-1}),$ the non-fluorescent pseudomonad reached final counts of ca. 10$ sp{10}$ CFU$ cdot$ml$ sp{-1},$ 2 logs higher than those of the other three organisms present in the mixed culture. The order of nutrient utilization was: (1) glucose, (2) gluconate, urea and glycerol, (3) lactate and glucose-6-phosphate, (4) unknowns 1 and 2. At day 7, none of the nine substrates studied remained in the growth medium.

Le groupe Bacillus cereus comprend six bactéries phénotypiquement distinctes : B. Anthracis, agent responsable de la maladie du charbon ; B. Cereus, pathogène opportuniste capable de contaminer les produits alimentaires ; B. Thuringiensis, bio-pesticide utilisé pour lutter contre les insectes ; B. Weihenstephanensis, qui possède la capacité de croître à basse température et B. Mycoides et B. Pseudomycoides, qui forment des colonies de forme mycoi͏̈dale. Les membres du groupe B. Cereus influent de manière notable sur les activités humaines. Il est important de caractériser les différences génétiques entre ces bactéries, ainsi que les mécanismes de plasticité génomique qui contribuent à la variabilité des souches du groupe B. Cereus. Au cours de cette étude, la phase finale du séquençage complet du génome de la souche type B. Cereus ATCC 14579 a été achevée. Nous avons étudié deux facteurs potentiels de la plasticité génomique : les opérons ARN ribosomiques et les phages tempérés. Nous avons caractérisé la variabilité du nombre d'opérons ribosomiques. Nos travaux ont mis en évidence l'existence de 2 types d'opérons ribosomiques au sein du groupe B. Cereus. Nous avons identifié et caractérisé deux types de phages tempérés inductibles chez la souche B. Cereus ATCC 14579 et chez des souches proches. Un mécanisme d'interaction entre ces deus types de phages a été proposé. Nos travaux soulignent également le rôle des phages dans la plasticité du génome de B. Cereus. Enfin, l'approche MLST nous a permis de caractériser un large échantillon de souches du groupe B. Cereus, et de mettre en évidence l'existence de 2 complexes clonaux regroupant la plupart des souches psychrotrophes
The Bacillus cereus group includes six phenotypically distinct bacteria : B. Anthracis, the causative agent of anthrax ; B. Cereus, an opportunistic food-borne pathogen ; B. Thuringiensisi, a biopesticide used to struggle against insects ; B. Weihenstephanensis, which is able to grow at low temperature (7ʿC) ; B. Mycoides and B. Pseudomycoides which form rhizoidal colonies. Members of the B. Cereus group have a great impact on human activities. It is important to characterise the genetic differences between these bacteria as well as the effective mechanisms of genome plasticity which contribute to the variability of the strains. The finishing of the complete genome sequencing of the type strain B. Cereus ATCC 14579 was achieved. We characterised two potentially important factors in genome plasticity in the B. Cereus group : ribosomal RNA operons and temperate phages. We identified the number and the structural variability of ribosomal RNA operons of several strains of the B. Cereus group, revealing the existence of two distinct types of ribosomal RNA operons. We identified and characterised two types of inducible prophages in the strain B. Cereus ATCC 14579 and in some related strains. A general mode of interrelation between these temperate phages was proposed. Our work highlighted the role of temperate phages in the genome plasticity of the B. Cereus group bacteria. We characterised a representative collection of strains from diverse origins MLST approach, revealing that most of the psychrotrophic strains of the B. Cereus group are genetically closely related

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第13493号
農博第1670号
新制||農||951(附属図書館)
学位論文||H20||N4318(農学部図書室)
UT51-2007-T869
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 江﨑 信芳, 教授 清水 昌, 教授 阪井 康能
学位規則第4条第1項該当

La croissance a basse temperature a ete etudiee chez la bacterie psychrotrophe arthrobacter globiformis si55. Elle fait intervenir des proteines particulieres appelees caps (cold acclimation proteins) qui persistent pendant toute la duree de la croissance au froid. D'autres proteines appelees csps (cold shock proteins) sont surexprimees transitoirement apres des chocs froids d'amplitude variee. L'analyse en afc (analyse factorielle des correspondances) de leur cinetique d'induction en fonction de la temperature de choc a permis de degager nettement trois groupes de proteines: un groupe de proteines specifiquement induites apres un choc froid de 25 a 4c, un groupe de proteines induites apres des chocs froids d'amplitude moyenne et un troisieme groupe de proteines presentes lors de tous les chocs thermiques etudies. L'implication des proteines de choc froid dans l'adaptation a la croissance aux bases temperatures a ete montree. Un gene appele cspagl codant pour une proteine de 67 acides amines et presentant 64% d'identite avec la proteine cs7,4, a ete clone et sequence. La proteine correspondante a ete identifiee comme etant la csp/cap a9. Elle est surexprimee par a. Globiformis dans l'heure qui suit un choc froid de 25 a 4c et persiste dans les cellules en croissance a 4c. Le blocage de la synthese des csps et caps empeche la reprise de la croissance apres un choc froid. La reponse au choc hypothermique apparait etre une reponse adaptative. De plus, la proteine cspagl est impliquee dans la reprise de la croissance apres un choc froid de 25 a 4c. Parmi les caps mises en evidence a 4c, se trouve une proteine de poids moleculaire similaire a la proteine h-ns et qui presente des epitopes communs avec elle

Top loin steaks were used to determine the influence of packaging procedure on the microbial growth and succession on the top and bottom surfaces of steaks during a 12 day storage period. The following packaging treatments were used: (1) Gas permeable Resinite film overwrap as a control; (2) gas flush with 1% CO, 40% O₂, 59% N₂ for 2 minutes followed by film overwrap; (3) loose packaging in gas impermeable barrier bags with 100 to 150 cc ambient air; (4) 15% CO₂, 40% O₂, 45% N₂ gas atmosphere; (5) 60% CO₂, 40% O₂ gas atmosphere; and (6) 10% CO₂, 5% O₂, 85% N₂ gas atmosphere. Total psychrotrophic counts obtained from the top and bottom surfaces did not differ significantly for most sampling periods. Similar growth patterns were observed on both steak surfaces, increasing (P <.05) primarily between Day 3 and Day 9 of post-treatment storage. The steaks packaged within the gas atmospheres had lower (P<.05) total growth than the control steaks. Comparing atmospheres, the steaks packaged in relatively low CO₂ and O₂ levels (10% and 5%, respectively) had lower (P<.05) microbial growth than steaks packaged in 15 to 60% CO₂ and 40% O₂ when initial contamination was low. Pseudomonas dominated the microflora on the steak surfaces in all packaging treatments during early storage. Other aerobic organisms which survived gas atmosphere treatments include Coryneforms, Micrococcus, and Microbacterium, although they did not show large increases in growth. Coryneforms were also competitive on the bottom surface of the control steaks despite domination by the pseudomonad organisms. Loose packaging in impermeable barrier bags allowed the growth of Serratia liquifaciens after 6 days of storage. This bacterium also tended to dominate the flora within the gas atmosphere packages, although other facultative organisms (Enterobacter aerogenes and Yersenia enterocolitica) were also identified. When initial contamination was low, the gas atmospheres provided an effective means of prolonging the shelf-life of fresh beef steaks.

Les bactéries psychrotrophes représentent une part importante de la microflore des sols des régions tempérées, soumis a des fluctuations de température. De plus si ces souches sont capables de dégrader des composés xénobiotiques, elles présentent un grand intérêt pour la dépollution des sols. Dans le cadre d'une approche physiologique de ce type de souches, une étude de la croissance et de la toxicité du toluène a été réalisée, en fonction de la température. L'étude de l'effet de la température sur la croissance de ces souches psychrotrophes sur différents substrats (citrate, toluène et benzoate) nous a permis de mettre en évidence, grâce à l'utilisation de la représentation d'Arrhénius, l'existence d'une température « Critique »(comprise entre 17°C et 20°C) définissant deux domaines de températures. La variation de l'effet de la température sur la croissance de part et d'autre de cette température critique, semble dépendre non seulement de la souche, mais également du substrat utilisé pour la croissance. L'étude de l'effet de la température sur la toxicité du toluène sur ces souches, a montré que cette toxicité dépend non seulement de la température de croissance, et est plus faible à 28°C qu'à 8°C, et maximale à 17°C. Elle dépend également de la capacité des souches à métaboliser ce composé. La capacité de ces souches à s'adapter au toluène, a également pu être mis en évidence, indépendamment de la capacité à le dégrader. Cette adaptation des souches semble être affectée par la température de croissance et est probablement liée à des modifications au niveau des protéines membranaires, induites lors de l'adaptation.

This study examined the efficacy of potassium lactate and lactic acid to control Campylobacter sp. and psychrotrophic bacteria on chicken. The objectives of the two studies conducted were to determine the optimal combination of potassium lactate and lactic acid to inhibit Campylobacter sp. in a challenge study and to inhibit naturally occurring Campylobacter sp. and psychrotrophic bacteria in a shelf life study.

Boneless, skinless chicken breasts were injected with three levels of potassium lactate (0,1.5,2%), in conjunction with four levels of lactic acid. Lactic acid was injected (0, 0.1, 0.2, 0.3%) as well as applied directly to the surface (0.1% of weight of chicken breast). The chicken breasts were surface inoculated with a mixture of Campylobacter sp. and sampled over a period of 28 days at 11oC. The greatest inhibition was found using 2% potassium lactate in conjunction with any level of lactic acid (injected) or 0.1% lactic acid (surface application). Results of this study indicate that potassium lactate and lactic acid can be used to control the growth and/or survival of Campylobacter sp. on boneless chicken breasts.

The second study eliminated the 1.5% potassium lactate and 0.2% and 0.3% lactic acid treatments and chicken breasts were not inoculated with Campylobacter sp.. This 4oC shelf life study occurred over 32 days, testing for Campylobacter species, psychrotrophic bacteria, as well as testing for sensory perceptions of color and odor changes in the chicken. The most effective treatment was the 2% potassium lactate-0.1% lactic acid surface treatment, demonstrating the most inhibition against both target populations. This treatment also had the greatest impact upon the odor of the chicken breasts. This treatment had the greatest difference from control samples, which was achieved by the inhibition of spoilage organisms on the chicken breasts.
Master of Science

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第16926号
農博第1942号
新制||農||1001(附属図書館)
学位論文||H24||N4687(農学部図書室)
29601
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 栗原 達夫, 教授 喜多 恵子, 教授 阪井 康能
学位規則第4条第1項該当

Le principal objectif etait la recherche et la mise au point d'une methode de determination de l'activite lipasique des laits de collecte permettant de differencier la lipoproteine lipase naturelle et les lipases excretees par les bacteries psychrotrophes, notamment le genre pseudomonas. Ces toutes dernieres annees, plusieurs methodes ont ete proposees dans le but de detecter ces enzymes, le plus souvent presentes en faibles quantites en raison de leur thermoresistance. La complexite du milieu lait, avec la presence de proteines, de matieres grasses et celles d'une autre enzyme lipolytique, rend le probleme difficile. Une methode immunologique pour le dosage des lipases represente une solution particulierement interessante etant donne l'expansion que connaissent actuellement ces methodes

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Este trabalho teve o objetivo de avaliar a eficácia de uma nova ferramenta de gestão da qualidade do leite cru refrigerado, comparativamente à utilização dos testes tradicionais de controle, nas condições da Instrução Normativa 51 (IN 51). Foram utilizadas 374 amostras individuais de leite cru refrigerado (produtor) e 125 amostras de leite cru de conjunto (bocas de tanques de caminhões transportadores, referindo-se a leites desses mesmos produtores). As amostras foram submetidas a duas análises microbiológicas, sendo as individuais submetidas à prova rápida de redução em tubos – o “Tetra-Test”, com o objetivo de estimar a carga microbiana do leite e a microbiota predominante, enquanto as amostras de conjunto foram submetidas à contagem padrão em placas (CPP) como método de referência. Paralelamente, buscou-se verificar os efeitos do processo de centrifugação nas características e/ou propriedades da matériaprima industrial durante a execução do processo por Ultra Alta Temperatura (UAT). Foram utilizadas 56 amostras de leite provenientes da linha de processamento, tomadas imediatamente antes e depois da etapa de centrifugação interposta no início do processo, as quais foram submetidas a ambos os testes microbiológicos (“Tetra- Test” e CPP), além da determinação da variabilidade da composição do leite e determinação do índice proteolítico da k-caseína. O “Tetra-test” se mostrou eficaz na avaliação da qualidade microbiológica do leite cru podendo ser utilizado como uma ferramenta de gestão, uma vez que seus resultados se correlacionaram proporcionalmente aos obtidos pela CPP e possibilitaram informações complementares sobre as características da microbiota dominante, oferecendo vantagens sobre os tradicionais testes de redução. Os resultados mostraram que grande...
This study aimed to evaluate the efficacy of a new tool for quality management of refrigerated raw milk, compared to traditional control tests in accordance to the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA) Normative Instruction No. 51. A total of 374 individual samples of refrigerated raw milk (from producers) and 125 samples of bulk raw milk (from milk transport tankers from these same producers) were studied. Samples were submitted to microbiological analyses, being individual samples submitted to rapid reduction in test tubes - the Tetra-Test, in order to estimate the microbial load of milk and predominant microbiota, while the bulk samples were analyzed by standard plate count as a reference method. At the same time, it was investigated the effects of the centrifugation on the characteristics and/or properties of industrial raw material into the incoming of UHT process. Were analyzed 56 samples of milk from the processing line, obtained immediately before and after centrifugation performed early in the process. Both of them were subjected to microbiological tests (Tetra-Test and PCA). In addition it was determined the variability of milk composition and proteolytic rate of k-casein. The “Tetra-test showed to be effective in assessing the microbiological quality of raw milk and can be used as a management tool, since its results correlated proportionally with those obtained by standard plate count and make possible to obtain more information about predominant... (Complete abstract click electronic access below)

Orientador: Luiz Francisco Prata
Banca: Ana Maria Centola Vidal Martins
Banca: Angela Cleusa de Fátima Banzatto de Carvalho
Resumo: Este trabalho teve o objetivo de avaliar a eficácia de uma nova ferramenta de gestão da qualidade do leite cru refrigerado, comparativamente à utilização dos testes tradicionais de controle, nas condições da Instrução Normativa 51 (IN 51). Foram utilizadas 374 amostras individuais de leite cru refrigerado (produtor) e 125 amostras de leite cru de conjunto (bocas de tanques de caminhões transportadores, referindo-se a leites desses mesmos produtores). As amostras foram submetidas a duas análises microbiológicas, sendo as individuais submetidas à prova rápida de redução em tubos - o "Tetra-Test", com o objetivo de estimar a carga microbiana do leite e a microbiota predominante, enquanto as amostras de conjunto foram submetidas à contagem padrão em placas (CPP) como método de referência. Paralelamente, buscou-se verificar os efeitos do processo de centrifugação nas características e/ou propriedades da matériaprima industrial durante a execução do processo por Ultra Alta Temperatura (UAT). Foram utilizadas 56 amostras de leite provenientes da linha de processamento, tomadas imediatamente antes e depois da etapa de centrifugação interposta no início do processo, as quais foram submetidas a ambos os testes microbiológicos ("Tetra- Test" e CPP), além da determinação da variabilidade da composição do leite e determinação do índice proteolítico da k-caseína. O "Tetra-test" se mostrou eficaz na avaliação da qualidade microbiológica do leite cru podendo ser utilizado como uma ferramenta de gestão, uma vez que seus resultados se correlacionaram proporcionalmente aos obtidos pela CPP e possibilitaram informações complementares sobre as características da microbiota dominante, oferecendo vantagens sobre os tradicionais testes de redução. Os resultados mostraram que grande... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: This study aimed to evaluate the efficacy of a new tool for quality management of refrigerated raw milk, compared to traditional control tests in accordance to the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA) Normative Instruction No. 51. A total of 374 individual samples of refrigerated raw milk (from producers) and 125 samples of bulk raw milk (from milk transport tankers from these same producers) were studied. Samples were submitted to microbiological analyses, being individual samples submitted to rapid reduction in test tubes - the "Tetra-Test", in order to estimate the microbial load of milk and predominant microbiota, while the bulk samples were analyzed by standard plate count as a reference method. At the same time, it was investigated the effects of the centrifugation on the characteristics and/or properties of industrial raw material into the incoming of UHT process. Were analyzed 56 samples of milk from the processing line, obtained immediately before and after centrifugation performed early in the process. Both of them were subjected to microbiological tests ("Tetra-Test" and PCA). In addition it was determined the variability of milk composition and proteolytic rate of k-casein. The "Tetra-test" showed to be effective in assessing the microbiological quality of raw milk and can be used as a management tool, since its results correlated proportionally with those obtained by standard plate count and make possible to obtain more information about predominant... (Complete abstract click electronic access below)
Mestre

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第16927号
農博第1943号
新制||農||1001(附属図書館)
学位論文||H24||N4688(農学部図書室)
29602
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 栗原 達夫, 教授 小川 順, 教授 阪井 康能
学位規則第4条第1項該当

L'extension de la réfrigération a certes amélioré la qualité et la durée de vie microbiologique du lait. Toutefois, ce type de conservation est responsable de la sélection de la flore psychotrophe. Or, ces micro-organismes sont capables de secréter des enzymes extracellulaires thermorésistantes, qui peuvent altérer les principaux composés du lait. Ces enzymes, principalement les protéases et les lipases, entraînent d'une part de graves accidents de conservation et d'autre part réduisent l'aptitude du lait à la transformation. La nécessité de trouver des méthodes alternatives afin de contrôler le développement et les activités enzymatiques de cette flore dans le lait cru stocké à basse température nous a conduit à envisager trois types d'approches. Les deux premières techniques de préservation du lait sont basées sur son traitement d'une part par atmosphères gazeuses à base de dioxyde de carbone et d'azote et d'autre part par ensemencement lactique. Si les deux gaz et particulièrement leur combinaison s'avèrent être d'excellents inhibiteurs, la durée de conservation du lait cru réfrigéré se trouvant nettement prolongée, l'action inhibitrice exercée par les cultures lactiques demeure bien plus faible. La combinaison des deux premières méthodes a finalement conduit à une troisième procédure : l'ensemencement du lait cru par une culture lactique combiné à un traitement sous atmosphère gazeuse CO2/N2 suivi d'une conservation à basse température. Ce procédé a conduit à une inhibition quasi-totale de la croissance des psychrotrophes et de leurs synthèses enzymatiques, entraînant par conséquent une prolongation de la durée de vie du lait cru. Cette technique offre donc de belles perspectives à la conservation du lait sans avoir recours à l'addition de conservateurs chimiques indésirables dans tout produit alimentaire et particulièrement dans le lait.

Orientador: Pilar Rodriguez de Massaguer
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Esta pesquisa teve por objetivos: a. enumerar os Clostridium psicrotróficos na forma vegetativa e esporulada presentes em cortes bovinos embalados a vácuo e resfriados, deteriorados e não deteriorados; b. caracterizar e identificar estes isolados bioquímica e geneticamente, comparando a precisão entre as duas metodologias; c. monitorar as possíveis fontes de Clostridium psicrotróficos junto à linha de produção em frigorífico no interior do estado de São Paulo; d. testar a habilidade dos isolados, identificados como Clostridium psicrotróficos e recuperados tanto a partir dos cortes bovinos quanto a partir de diversas etapas ao longo da linha de produção do frigorífico, em reproduzir o defeito de estufamento quando inoculados em altas populações em cortes bovinos e acondicionados em embalagens a vácuo e termoencolhimento sob condições praticadas pela indústria (6mBar/83°C-3s) ; e. avaliar se diferentes níveis de vácuo e termoencolhimento teriam efeito sobre as características do estufamento dos isolados que melhor reproduziram o defeito; f. analisar as características físicas das embalagens primárias e secundárias bem como possíveis danos após simulação de transporte para distância de 500km que pudessem favorecer a incidência de Clostridium psicrotrófico ou outras espécies contaminantes. Para as enumerações foram utilizadas 8 amostras deterioradas (6 contra-filés, 1 cupim bovino e 1 picanha bovina) e 5 não deterioradas (contra-filé), sendo analisados: exsudato (1mL), superfície (100cm2) e interior do corte cárneo (25g). As subamostras foram submetidas à enumeração de anaeróbios psicrotróficos vegetativos (Reinforced Clostridial Médium - RCA, 5% de sangue de carneiro desfibrilado estéril, 5% de glicose e 1,5% de Agar, RCM modificado), anaeróbios esporulados ativados pelo calor e pelo álcool, em Perfringens Agar Base (PAB, Oxoid, com 5% de solução de gema de ovo estéril, 15000UI polimixina/500mL e 6mg Kanamicina/500mL, PKP; 200mg cicloserina/500mL (PC)). Os 17 isolados caracterizados preliminarmente como Clostridium foram submetidos ao teste de sensibilidade ao metronidazol. Aqueles com halos de inibição = 3,5mm tiveram seu perfil fenotípico analisado mediante kit API 20A (bioMeriéux) e seu perfil genético analisado (sequênciamento do gene 16S rRNA). O isolamento no ambiente do frigorífico foi realizado a partir das fezes, couro e amostras provenientes do corredor de atordoamento e sangria, sala de desossa, esteiras de corte e embalagem, através de coleta por swabs acondicionados em caldo PYGS em anaerobiose por 4 semanas a 4 e 15°C. Após, as amostras foram plaquea das em meio RCA e SFP (Shahidi Ferguson Perfringens) por 1 mês, caracterizadas e isoladas as colônias diferenciadas e ainda submetidas a confirmação, de gênero, para Clostridium. Os 2 isolados, provenientes de cultivo a 4°C confir mados como Clostridium, foram identificados utilizando mesma metodologia descrita anteriormente para isolados provenientes do corte cárneo. O teste de habilidade de reprodução do defeito de estufamento foi realizado com 12 linhagens, isoladas na primeira etapa da pesquisa, sendo 10 provenientes de cortes cárneos (8 identificados como C.gasigenes e 2 como C.algidicarnis) e 2 isoladas da linha de produção em frigorífico (C.gasigenes). Este teste foi realizado sob condições praticadas na indústria (6mBar com termoencolhimento 83°C/3s), inoculando-se nas superfícies dos bifes ( 10x5x2cm) de contra-filé, 1mL de suspensão padronizada em 108UFC/mL, obtendo-se 106UFC/50cm2. A incubação dos cortes bovinos inoculados juntamente, com 2 controles positivos (inoculados com C.estertheticum) e 2 controles negativos (sem inoculo) foi conduzida a 2° e 15°C por 8 semanas. Os 3 isolados (2 provenien tes dos cortes cárneos ¿ C.algidicarnis e C.gasigenes e 1 proveniente do frigorífico ¿ C.gasigenes) que melhor reproduziram as características do estufamento, seguiram para testes de influência de diferentes níveis de vácuo (6 e 9mBar) e temperaturas de termoencolhimento (83, 84 e 87°C) sob o tempo para aparecimento e intensidade do defeito. Também foram avaliados os sistemas de embalagem primário e secundário a fim de detectar falhas em ambos os sistemas que pudessem favorecer a incidência de espécies de Clostridium psicrotróficas. Mediante teste de simulação de transporte (norma ASMD4169-08, para caminhões) simulou-se o efeito de um percurso de 500km sobre as embalagens primárias e secundárias. Nas carnes deterioradas foi notado esverdeamento, aumento nos valores de pH e odores putrefativos e butíricos. As enumerações de psicrotróficos anaeróbios vegetativos, nas amostras deterioradas, atingiram níveis altíssimos (1014UFC/mL de exsudato). Quanto às formas esporuladas ocorreu predominância das ativadas pelo calor (105UFC/mL), sendo também recuperadas nas amostras não deterioradas. Bacillus facultativos psicrotróficos foram a flora dominante nas amostras deterioradas e não deterioradas com 50 e 70%, respectivamente. Das 17 linhagens, provenientes dos cortes cárneos, suspeitas de serem Clostridium apenas 10 foram confirmadas, destas, apenas 4 apresentaram perfis fenotípico e genético coincidentes (C1I13ESUPPKP; C2I2EXRCM; C4I8RCMEX; C2I5EXCHPC, sendo as 3 primeiras C.gasigenes e a última C.algidicarnis). Destes 10, 8 foram identificadas como C.gasigenes e 2 como C.algidicarnis. É importante enfatizar que em apenas 3 das 8 amostras de cortes cárneos deteriorados analisados (com estufamento), foram recuperados Clostridium psicrotrófico, ou seja, nas demais amostras onde estas espécies não foram recuperadas, a microflora causadora do estufamento pode ser um grupo heterogêneo composto por Bacillus psicrotróficos facultativos, que nesta pesquisa corresponderam a 50% da contaminação para as amostras deterioradas e, muito provavelmente, bactérias ácido lácticas e enterobactérias, conforme já detectado, em pesquisa paralela utilizando as mesmas amostras. Nos ambientes do frigorífico, temperaturas de 15°C fo ram medidas em locais que deveriam estar com temperaturas entre 0-6°C ou no máximo 7°C. A partir das superfícies de contato do frigorífico, fezes e chão, foram recuperados 38 isolados. Destes, apenas 2, 1 recuperado do couro e outro do corredor de abate/esteira de corte (mesmo isolado encontrado nos dois ambientes), foram identificados como Clostridium, os demais foram caracterizados como Bacillus facultativos psicrotróficos. Estes 2 foram identificados como C.gasigenes. A ocorrência destes isolados na forma viável junto à esteira de corte representa grande risco a carne pois uma vez arrastado ao produto final será um potencial causador do defeito de estufamento. Quanto aos ensaios de reprodução do defeito de estufamento, observou-se que a temperatura de 15°C acelerou a tempo para aparecimento dos primeiros sinais de estufamento, passando de 30 dias a 2°C pa ra 4 dias a 15°C e o isolado que melhor e mais rapidamente reproduziu as características do estufamento foi o C2I5EXCHPC (C.algidicarnis), com produção de gás, proteólise e alterações de pH (de 5.5 para 7.5) após 4 dias/15°C e 30 dias/2°C. Resultados similares foram obtidos para o controle positivo (C.estertheticum). Os outros 2 isolados que também reproduziram rápida e intensamente o estufamento foram C2I8EXCHPC e LMI13CA/EC ¿ isolado a partir do corredor de abate/esteira de corte junto ao frigorífico, ambos identificados como C.gasigenes, cabendo esclarecer que o defeito, para estes dois isolados, não foi tão intenso quanto para o C2I5EXCHPC. Todos os isolados foram capazes de reproduzir o defeito de estufamento ao final de 8 semanas de incubação a 15°C, enquanto que, a 2°C, somente 5 tiveram esta capacidade. Assim, temperaturas de abuso de refrigeração (15°C) aceleram o tempo para aparecimento dos primeiros sinais de estufamento por Clostridium e a temperatura de 2°C não é capaz de impedir a ocorrência deste defeito. Amostras inoculadas com C2I5EXCHPKP (C.algidicarnis) e submetidas a termoencolhimento a 83°C e 84°C/6mBar apresentaram períodos similares para aparecimento dos primeiros sinais de deterioração (~14 dias), entretanto, aplicando termoencolhimento a 87°C, foi observado um incremento neste tempo para 4 semanas. Assim sendo, considerando-se o nível de vácuo de 6mBar (aplicado pela indústria) a deterioração somente seria retardada, mas não deixaria de existir, aplicando-se um termoencolhimento a 87°C/3s, provav elmente porque nesta condição as células mais termosensíveis são inativadas. Por outro lado, as amostras tratadas com condições similares de termoencolhimento mas com nível de vácuo de 9mBar, apresentaram decréscimo no tempo de deterioração de 14 para 4 dias quando se aplicou 83 e 84°C/3s, d emonstrando que este vácuo maior estimula espécies de Clostridium. Entretanto, nas amostras tratadas com termoencolhimento a 87°C os primeiros sinais de deterioração somente foram observados após 7 semanas a 1°C. Assim, a condição de vácuo/termoencolhimento que mais prolongou a vida de prateleira da carne, quando inoculada, foi 87°C/9mBar, mas esta condição não impediu o desenvo lvimento do C.algidicarnis e o tempo de atraso do defeito de estufamento ainda foi inferior ao prazo de vida útil do produto. As embalagens primárias e secundárias analisadas estavam de acordo com as especificações do fabricante. Quanto à simulação de transporte, após o percurso de 500km apenas 1 embalagem primária demonstrou alterações que pudessem favorecer a atividade de anaeróbios facultativos. Por esta pesquisa, observou-se coincidência entre a flora encontrada nas superfícies do frigorífico e a flora recuperada a partir das amostras deterioradas. Além disso, a condição mais severa de termoencolhimento e vácuo aplicados (87°C/3s/9mBar), somente foi capaz de retardar o tempo para aparecimento do defeito de estufamento não o impedindo. Mesmo este tempo maior ainda foi inferior ao prazo de validade do produto. Sendo assim, pode-se inferir que a prevenção do defeito de estufamento não deve residir apenas na eliminação de células vegetativas de ambientes de planta mas também na eliminação de esporos que, uma vez arrastados para a embalagem, poderão germinar tornandose potenciais causadores do defeito de estufamento, além, é claro, da manutenção de ambientes de produção e estocagem em temperaturas inferiores a 2°C e higiene adequada durante a manipulação das carcaças e retirada do couro. Em adição, ficou comprovado que o abuso de temperatura de refrigeração acelera o estufamento de embalagens no caso de espécies de Clostridium psicrotróficos estarem presentes, principalmente C.algidicarnis, e que, as condições de vácuo/termoencolhimento aplicadas atualmente pela maioria das indústrias (83°C/3s/6mBar) não se apres entam como barreira eficiente à prevenção do defeito de estufamento na carne bovina embalada a vácuo
Abstract: This research aimed at: a. to enumerate psychrotrophic clostridia vegetative and spores from spoiled and unspoiled chilled vacuum packed red meat; b. to characterize and identify the isolates biochemical and genetically, comparing precision between then; c. to determine possible preslaughter and processing sources of psychrotrophic clostridia causing spoilage of vacuum packed red meats; d. assessment of blowing ability of Clostridium isolates at industrial conditions (6mBar/83°C-3s); e. to evaluate different vacuum and heat shrinking treatments on blown characteristics of the best representative isolates; f. to analyze physical characteristics of primary and secondary packs as well as possible damages after transport simulation (500km) that could lead to psychrotrophic Clostridium and other contaminant species incidence. For enumeration 8 spoiled samples (6 strip-loin, 1 hump beef and 1 rump cap beef) and 5 unspoiled (strip-loin): drip (1mL), surface (100cm2) and deep tissue (25g), were analyzed. Sub-samples were analyzed for vegetative psychrotrophic anaerobes (Reinforced Clostridial Medium - RCA, 5% of sterile defibrinated sheep blood, 5% of glucose and 1.5% of Agar, modified RCM); sporeforming anaerobes were counted by alcohol and heat treatment on Perfringens Agar Base (PAB, Oxoid, containing 5% sterile egg-yolk solution, 15000UI polymixin/500mL e 6mg kanamycin/500mL, PKP; 200mg cycloserin/500mL (PC)). For the 17 isolates characteried as clostridia, metronidazole test was applied for genera determination of Clostridium, isolates with inhibition zones = 3.5mm were phenotypical (by API 20A ¿ bioMeriéux) and genetically (16S rRNA sequence) identified. The slaughterhouse survey was conducted from feces, leather, stunning aisle, boning room, meat cutting conveyor belt and packing conveyor belt by sterile swabs inoculated in PYGS medium, incubated anaerobically at 4 and 15°C/4 weeks. Afte r incubation, loopfuls of the enrichments were directly streaked onto the surface of RCA and SFP (Shahidi Ferguson Perfringes), incubated for a month and then the different colonies were characterized and forwarded to genera confirmation. The confirmation of blowing ability was conducted with 12 strains, isolated from first stage of this research: 10 from red meats (8 identified as C.gasigenes and 2 as C.algidicarnis) and 2 from slaughterhouse (C.gasigenes). This test was conducted under industrial practices (6mBar, 83°C/3s), inocul ating sterile strip-loin beefs surfaces (10x5x2cm) with 1mL of vegetative cells suspension (108CFU/mL), obtained 106CFU/50cm2. Inoculated samples with 2 positive controls (inoculated with C.estertheticum) and 2 negative controls (uninoculeted samples) were performed at 2 and 15°C / 8 weeks. The three best representative isolates (2 from meat samples ¿ C.algidicarnis e C.gasigenes and 1 from slaughterhouse ¿ C.gasigenes) were tested for blown characteristics conducted at different vacuum (6 and 9mBar) and heat shrinking (83, 84 and 87°C) treatments to evaluate the time to first sign of blow pack and spoilage magnitude. The primary and secondary pack systems were evaluated for failures that support the psychrotrophic Clostridium incidence. Effects of a 500km route on primary and secondary packs were measured by transport simulation test (ASMD4169-08, for trucks). Spoiled samples showed green discoloration, increase of pH values and putrefactive and butyric odors. For these samples, the psychrotrophic vegetative anaerobic counts reached very high levels (1014CFU/mL of drip). Concerning sporeforming the heat activated ones were predominant (105CFU/mL) and also were recovered for unspoiled samples. Psychrotrophic facultative Bacillus was the dominant flora in analyzed samples (50% for spoiled and 70% for unspoiled). From 17 Clostridium suspect strains only 10 were confirmed and these 4 showed phenothypical and genetical coincident profiles (C1I13ESUPPKP, C2I2EXRCM and C4I8RCMEX ¿ C.gasigenes; C2I5EXCHPC ¿ C.algidicarnis). Concerning these 10 Clostridium isolates, 8 were identified as C.gasigenes and 2 as C.algidicarnis. It is important to emphasize that only 3 spoiled samples provided psychrotrophic Clostridium isolation, others spoiled samples flora were heterogeneous group, composed by facultative psychrotrophic Bacillus, that represented 50% of isolates from spoiled samples, and, probably, lactic acid bacteria and enterobacteria, according to Chaves (2010), in parallel research using same samples. Inside the slaughterhouse, ambient temperatures of 15°C were observed for places that should be at 0-6°C or 7°C (maximum). From slaugther house ambient and contact surfaces 38 isolates were recovered but only 2, 1 from leather and 1 for stunning aisle/meat cutting conveyor belt (same isolate from both points), were identified as Clostridium, other were characterized as psychrotrophic facultative Bacillus. These 2 isolates were identified as C.gasigenes. The occurrence of viable Clostridium on meat cutting conveyor belt is a risk for meat as a focus of blown pack spoilage clostridia. For confirmation blowing ability tests, abuse temperature decrease the time to first sign of blown pack from 30 days at 2°C to 4 days at 15°C and the isolate that better reproduce (more quickly) the spoilage was C2I5EXCHPC (C.algidicarnis) showing gas production, proteolysis and pH alteration (5.5 to 7.5) after 4 days/15°C and after 30 days/2°C. Similar results we re obtained for C.estertheticum (positive control). In addition, others two isolates that also quickly reproduced the spoilage were C2I8EXCHPC and LMI13CA/EC ¿ isolated from stunning aisle/meat cutting conveyor belt slaughterhouse, both identified as C.gasigenes, however the defect was not intense than for samples inoculated with C2I5EXCHPC (C.algidicarnis). All isolates were capable to reproduce the spoilage at the end of 8 weeks at 15°C while at 2°C, only 5 iso lates reproduce the defect. So, abuse temperature (15°C) accelerate the onset of Clostridium mediate blown pack spoilage and 2°C does not control it. Samples inocu lated with C2I5EXCHPKP (C.algidicarnis) treated with heat shrink of 83 e 84°C/6mBar showed similar perio ds for initial spoilage (~14 days), but applying heat treatment at 87°C, it was observed an increase in t his period to 4 weeks. So, using vacuum at 6mBar (industrial practice) the deterioration is retarded, but does not cease to exist, applying heat shrink of 87°C/3s, probably because this condition inactivated some heat sensitive spores. On other side, samples treated with same conditions of heat shrink but applying vacuum at 9mBar showed a decrease of time to spoilage from 14 to 4 days at 83 and 84°C/3s as lower vacuum may stimulate clostridia germination. However, for samples heat shrinked at 87°C the initial spoilage was only observed after 7 weeks at 1°C. So the condition th at more extended meat shelf-life, when inoculated, was 87°C /9mBar, but this condition not prevented C.algidicarnis development and delay of blown pack was still smaller than product shelf-life. Primary and secondary packs were according to manufacture¿s specifications. Concerning transport simulation, after 500km only one primary pack showed alterations that could support facultative anaerobes activity. In this research, it was observed coincidence between slaughterhouses and spoiled samples flora. Besides, the more rigorous condition applied (87°C/3s/9mBar) was only capable to retard the blown pack not inhibit and the time to deterioration first sign was smaller than product shelf-life. In regard to this fact, the defect prevention of blown pack spoilage is associated with the elimination of vegetative and sporeformers from slaughterhouses that after germination process could induce spoilage, besides, temperature control of production lines and storage environment at = 2°C, must be apply. In addition, abuse temperature accelerate the onset of Clostridium mediate blown pack spoilage, if psychrotrophic clostridia are present, in special, C.algidicarnis, and the industry process condition (vacuum and heat shrink - 6mBar/83°C/3s) are not capable to prevent the spoilage, being necessary the application of more efficient hurdles as efficient sanitization of process line as well as reduce temperature of the refrigerate environment
Doutorado
Doutor em Ciência de Alimentos

Le stockage du lait au froid permet le développement spécifique des microorganismes psychrotrophes, principalement du genre Pseudomonas, qui sécrètent des protéases extracellulaires thermorésistantes, responsables d'une protéolyse des laits lors de leur conservation. La souche psychrotrophe protéolytique isolée de lait cru industriel, Pseudomonas sp. LBSA1 produit une seule protéase extracellulaire caséinolytique de masse moléculaire apparente de 49 kDa en présence de lait. Divers facteurs, température de croissance, agitation, présence de certains composés, influent sur sa production. L'étude taxonomique de la souche LBSA1, révèle qu'elle est phylogénétiquement proche de l'espèce de P. Poae, mais elle possède un sidérotype original et pourrait donc appartenir à une nouvelle espèce de Pseudomonas. La protéase de la souche LBSA1 a été purifiée et son gène séquencé ; elle est identifiée comme étant une métalloendopeptidase à zinc de la famille des serralysines (E. C. 3. 4. 24. 40). Elle est active sur une large gamme de pH et sa température optimale d'activité est voisine de 40°C. Une inactivation thermique de la protéase est observée pour des températures voisines de 45°C, mais elle est plus stable à 60-80°C. L'enzyme hydrolyse rapidement les caséines bovines sans présenter de réelle spécificité d'hydrolyse mais les protéines du lactosérum sont résistantes à son action
Milk storage at refrigeration temperature permits the growth of psychrotrophic microorganisms, mainly belonging to the genus Pseudomonas that can generally produce heat-resistant extracellular proteases, causing milk proteolysis during storage. The proteolytic psychrotrophic strain isolated from raw milk, Pseudomonas sp. LBSA1 produces a unique caseinolytic extracellular protease with an apparent molecular mass of 49 kDa with milk addition. Different factors, such as temperature, agitation, and addition of many components can affect its production. A polyphasic taxonomic study of the LBSA1 strain reveals that it is phylogenetically related to the species P. Poae, but, it has an original siderovar and then it could belong to a new Pseudomonas species. The protease of the strain LBSA1 was purified, the corresponding gene was sequenced; the enzyme is identified as a zinc metalloprotease of the serralysin family (E. C. 3. 4. 24. 40). It is active under broad pH values and that optimal temperature for activity is nearly 40°C. The protease presented a low temperature inactivation around 45°C but it showed a greater stability around 60-80°C. The bovine caseins are rapidly hydrolysed by the enzyme without a real specificity but whey proteins are resistant to the action of the protease

Pectobacterium atrosepticum, pathogène majeur de la pomme de terre, cause des symptômes de pourriture molle et de jambe noire. Il occasionne d’importantes pertes économiques pour la filière pomme de terre, notamment en France où le Comité Nord occupe une place prépondérante. Les lacunes en matière de lutte et le potentiel d’expansion de la maladie justifient l’urgence de trouver des solutions de protection efficaces. Les priorités de la politique agricole nous ont mené à affiner nos connaissances sur ce pathogène et sur la flore associée aux racines de pommes de terre, ceci afin de proposer une méthode de lutte originale soucieuse de l’environnement. Comme beaucoup d’autres bactéries phytopathogènes, la virulence de cette bactérie fait intervenir un système de quorum sensing basé sur les N-Acyl homosérine Lactone (N-Acyl-HSL). Chez la bactérie psychrotrophe P. Atrosepticum 6276 la N-3oxo-octanoyl-L-homosérine lactone est la principale N-Acyl-HSL. La seule inactivation du gène responsable de sa synthèse rend la souche non virulente. Située à l’interface entre les 2 évènements de la pathogénie (multiplication cellulaire et macération), la production des N-Acyl-HSL intervient au moins de 8 à 28°C et est optimale à 24°C, comme la croissance bactérienne, suggérant l’absence de thermorégulation particulière. Face au rôle clé de ce signal, nous nous proposons de développer une méthode de lutte biologique employant des bactéries dégradant les N-Acyl-HSL. La microflore associée aux racines de pomme de terre et 2 souches modèles de Pseudomonas et de Rhodococcus ont servi de source de protecteurs
Pectobacterium atrosepticum is a major pathogen of Solanum tuberosum that causes tuber soft rot and potato plant blackleg that are responsible for important economic losses. As methods of control are limited to avoidance of contamination and pathogen expansion ability is high, potato industry – notably Comité Nord – promotes researches that aim at finding efficient control strategies. Concerns about environment preservation in European agricultural policies has incited us to detail our knowledge of this telluric pathogen and of the microflora associated to potato roots in order to develop a new control strategy environmentally respectful. As many other phytopathogens, P. Atrosepticum virulence is controlled by a quorum sensing regulation system based on N-Acyl homoserine Lactone (N-Acyl-HSL) signalling molecules. The psychrotrophic bacteria P. Atrosepticum 6276 uses N-3-oxo-L-octanoyl-homoserine lactone as major N-Acyl-HSL. Mutation of the unique NAcyl- HSL synthase gene leads to an avirulent phenotype. The N-Acyl-HSL production occurs between 2 key events of pathogenicity which are cellular multiplication and maceration. As bacterial growth, N-Acyl-HSL production is observed at least from 8 to 28°C and is optimal production of N-Acyl-HSL is observed at 24°C. Considering the importance of N-Acyl-HSL for pathogenicity, we develop a biocontrol method using NAcyl- HSL degrading bacteria. Potato root associated microflora and 2 model strains belonging to Pseudomonas and Rhodococcus genera were used as a source of biocontrol agents

The use of microfiltration as an alternative to pasteurisation to reduce the microbial load of raw skimmed milk is a well established technology. However, its application in reducing bacteria from highly viscous dairy based solutions has not due to issues of low flux and high fouling tendency. This study involves the application of microfiltration to remove spores from high solids content Milk Protein Isolate (MPI) solutions. MPI feeds were inoculated with Bacillus mycoides spores a safer alternative to Bacillus cereus, a psychrotrophic spore forming bacteria found in dairy feeds. Suitable protocols for MPI resolubilisation, Bacillus mycoides cell and spore preparations were established and the membranes, MPI and spores were fully characterised by scanning electron microscopy (SEM), particle size distribution, rheology and pure water flux (PWF) measurements. Feed and permeate samples collected during experiments were analysed for solids content by oven drying, protein content using the Bradford assay and spore content using PetrifilmTM Aerobic count plates. To try and determine an optimum protocol for MPI filtration, a variety of filtration rig set-ups, modules and membranes were tested. Experiments were carried out at different MPI concentrations (4 – 16 wt%), cross flow velocities (CFV’s) (0.7 – 2.0 m s-1) and transmembrane pressures (TMP’s ) (1 and 2 bar). The filtration of 15 wt% MPI proved challenging. The best set of results were obtained using the 12.0 μm membrane at 1.4 m s-1, producing a 27 LMH flux, 96.5% protein transmission and a 2.1 log spore reduction. These results indicate that large pore ceramic microfiltration may be a suitable technology to replace or augment pasteurisation for high solids content dairy feeds. The effect of backwashing using different durations and frequencies was investigated. Backwashing parameters of 10 seconds every 5 minutes at 1 bar were found to be the most effective. The optimum cleaning regime found for MPI fouled ceramic membranes involved a long rinsing backflush at 1 bar, acid and alkali steps without backwashing, which produced a 99.6% flux recovery.

Thesis (BTech (Food Technology))--Cape Technikon, 1999.
The Dairy industry, one of the larger food industries in South Africa processes probably the most perishable and possibly the most regulated foodstuff, namely mille The unique combination of vitamins, proteins, carbohydrates, lipids, moisture and near neutral pH, offers a suitable environment for the proliferation of microbes. Milk is therefore highly susceptible to microbiological activity resulting in the irreversible spoilage of this food (Frazier & Westhoff, 1988). The coliform group of organisms comprises all aerobic and anaerobic, gram-negative, non-spore-forming rods that are able to ferment lactose with the production of acid and gas at 32°C within 48 hours (Richardson, 1985). The primary purpose of the coliform detection test is to measure the quality of the practices used to minimise bacterial contamination of processed dairy products (Richardson, 1985). IDF Standard 132A: (1991) defines psychrotrophic organisms as organisms forming countable colonies when incubated aerobically at 6.5°C for 10 days under the conditions specified in IDF standard 101A. Shelf-life tests conducted in the fresh milk laboratory of a processing plant, revealed significant growth of coliforms in samples stored at 5°C. Luch, (1985) reported that other contaminating psychrotrophs together with the coliforms reduce the shelf-life of the milk when the storage temperature thereof is above 10°C.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第21379号
農博第2303号
新制||農||1071(附属図書館)
学位論文||H30||N5152(農学部図書室)
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 栗原 達夫, 教授 植田 充美, 教授 小川 順
学位規則第4条第1項該当

Des souches de bactéries lactiques ont été isolées à partir de produits de la mer et regroupées en fonction de leur pouvoir inhibiteur contre 14 bactéries appartenant à la flore d'altération ou pathogène de ces produits, dans le but de développer des applications en biopréservation. Elles ont été identifiées majoritairement comme leuconostoc gelidum et Lactococcus piscium. L'identification approfondie d'une souche de Lc. Piscium montre qu'il s'agit d'une nouvelle espèce de Lactococcus. Des analyses sensorielles ont permis de montrer que 2 souches appartenant à l'espèce Leuconostoc gelidum permettait de prolonger la durée de vie de crevettes cuites emballées sous vide et qu'une souche de Lactococcus sp. Avait le même effet sur du saumon fumé. Un test d'inhibition de bactéries pathogènes (Listeria monocytogenes, Staphylococcus aureus et Vibrio cholerae) a été effectué sur du saumon fumé. La souche de Lc. Sp. EU2241 (non productrice de bactériocine) s'est révélée plus efficace que la souche de Ln. Gelidum EU 2247 (productrice de bactériocine) pour inhiber ces pathogènes, avec une inhibition atteignant 2 log par rapport au temoin non biopréservé. Les caractéristiques de croissance de la souche Lc sp. EU2241 à différentes températures ont montré un optimum de croissance à 26°C et aucune croissance à 29°C ou au dessus. L'analyse du protéome a révélé la surproduction d'une protéine de choc froid lors de la croissance à 5°C, et le gène correspondant a pu être identifié et séquencé. Il s'agit de la première mise en évidence de la surproduction d'une protéine de choc froid lors de la croissance à température suboptimale pour une bactérie lactique
Lactic acid bacteria strains were isolated from seafood products and clustered according to their inhibiting capacities against 14 food borne spoiling and pathogenic strains in order to develop applications in biopreservation. Most of the strains were identified as Leuconostoc gelidum and Lactococcus piscium. Detailed identification of one strain of the Lactococcus piscium strains suggests that it is a new specie. According to sensory analysis results, 2 strains belonging to the Leuconostoc gelidum specie were the most efficient for the enhancement of the shelf life of vacuum packed peeled and cooked shrimps. Similar results were obtained in cold smoked salmon with one strains of Lactococcus sp. A challenge test against three pathogenic strains (Listeria monocytogenes, Staphylococcus aureus and Vibrio cholerae) was performed in cold smoked salmon. The Lc. Sp. EU2241 strain (non bacteriocinogenic) enabled a more efficient inhibition of the pathogens than the Ln. Gelidum EU2247 strain (bacteriocinogenic). The maximum recorded inhibition was 2 logs compared to the uninoculated samples. Growth characteristics of strain Lc. Sp. EU2241 were determined at different temperatures showed an optimal growth at 26°C and no growth at 29°C or above. Proteomic analysis showed a cold shock protein overproduction during growth at 5°C. The gene coding for this protein was identified and sequenced. Overproduction of a cold shock protein during growth at suboptimal temperature has never been described in a lactic acid bacterium before

Sept souches de Pseudomonas ont été isolées à partir d'un sol pollué par du toluène pour leur capacité à dégrader ce composé à 10\c. Parmi celles-ci, la souche Pseudomonas putida 01g3 qui degrade le toluène à 4\c a été sélectionnée. La première partie de l'étude concerne la recherche de la voie de dégradation par des analyses physiologiques. Celle-ci est initiée par une dioxygénase périphérique proche de la toluène dioxygénase de p. Putida f1 (souche mésophile) et l'ouverture du cycle aromatique est réalisée par une catéchol 2,3-dioxygénase. La seconde partie a concerné la caractérisation génétique de la voie catabolique du toluène. Des expériences de mutagenèse avec le transposon mini-tn5lacz1 ont permis d'isoler 15 mutants tol. Les différents gènes ebd ont été clonés en deux fragments et séquences afin d'établir l'organisation génétique. Le premier renferme le gène ebdr dont le produit est une protéine de régulation positive impliquée dans la dégradation des alkylbenzènes. La seconde séquence renferme 6 orf dont les protéines putatives sont responsables de la dégradation de l'isopropylbenzène. Les gènes définis ebdaabcdbc codent respectivement pour une dioxygénase périphérique, une cis-dihydrodiol deshydrogénase et une catéchol 2,3-dioxygénase toutes similaires à celles présentées chez les souches Pseudomonas re204, jr1 et ip01. Le meilleur inducteur de la voie ebd est l'ethylbenzène. Dans la dernière partie, l'influence de la température sur les capacités dégradatives a été étudiée. Les cinétiques de croissance ont montré que la souche 01g3 ainsi que les souches dégradant l'isopropylbenzène minéralisent les alkylbenzènes de 4 à 30\c. Ces quatre souches sont psychrotrophes, alors que la majorité des souches connues jusqu'alors.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
Extended storage of refrigerated milk can lead to reduced quality of raw and processed milk, which is a consequence of the growth and metabolic activities of psychrotrophic bacteria, able to grow under 7oC or lower temperatures. Although most of these microorganisms are destroyed by heat treatment, some have the potential to produce termoresistant proteolytic and lipolytic enzymes that can survive even UHT processing and reduce the processed products quality. Recently, the IN 51 determineds that milk should be refrigerated and stored at the farm what increased the importance of this group of microorganisms. In this work, psychrotrophic bacteria were isolated from 20 communitarian bulk tanks and 23 individual bulk tanks from dairy farms located at Zona da Mata region of Minas Gerais State and from southeastern Rio de Janeiro. Selected milk dilutions were plated on standard agar and after incubation for 10 days at 7oC, five colonies were isolated, firstly using nutrient agar and after using McConkey agar for 24 hours at 21oC. The isolates were identified by morphology, Gram stain method, catalase production, fermentative/oxidative metabolism and by API 20E, API 20NE, API Staph, API Coryne or API 50 CH (BioMerieux). In order to ensure reproductibility, API was repeated for 50% of the isolates. Species identification was considered when APILAB indexes reached 75% or higher. 309 strains were isolated, 250 Gram negative and 59 Gram positive. 250 Gram negative isolates were identified as: Acinetobacter spp. (39), Aeromonas spp. (07), A. Hydrophila (16), A. sobria (1), A. caviae (1), Alcaligenes feacalis (1), Burkholderia cepacia (12), Chryseomonas luteola (3), Enterobacter sp. (1), Ewingella americana(6), Hafnia alvei (7), Klebsiella sp. (1), Klebsiella oxytoca (10), Yersinia spp. (2), Methylobacterium mesophilicum (1), Moraxella spp. (4), Pantoea spp. (16), Pasteurella sp. (1), Pseudomonas spp. (10), P. fluorescens (94), P. putida (3), Serratia spp. (3), Sphigomonas paucomobilis (1). Five isolates kept unidentified. Pseudomonas was the predominant bacteria found (43%) and P. fluorescens the predominant species (37.6%), in accordance with previous reports. Qualitative analysis of proteolytic and lipolytic activity was based on halo formation using caseinate agar and tributirina agar during 72 hours at 21oC and during 10 days at 4?C, 10oC and 7?C. Among 250 Gram negative bacteria found, 104 were identified as Pseudomonas spp. and 60,57% of this group showed proteolytic and lipolytic acitivities over all four studied temperatures. 20% of Acinetobacter, Aeromonas, Alcaligenes, Burkholderia, Chryseomonas, Methylobacterium, Moraxella presented only lipolytic activity. Some isolates presented enzymatic activity in one or more studied temperatures. Among Gram positive bacteria, 30.51% were proteolytic and lipolytic at 10oC, 8.47% were proteolytic at 7oC, 10oC, and 21oC, 8.47% were proteolytic at all studied temperatures (4oC, 7oC, 10oC and 21oC) and 3.38% were proteolytic only at 21oC. At 4oC, only one isolate showed proteolytic activity and six isolates were lipolytic. In relation to Gram negative microorganisms, 4% were proteolytic and lipolytic at 7oC, 10oC and 21oC, 10% were proteolytic at 10oC and 4.4% were lipolytic at 4oC, 7oC, 10oC and 21oC, while 6.4% of all isolates were proteolytic and lipolytic at 10oC and 21oC as well as lipolytic at 4oC and 7oC. These findings are in accordance with previous researches that pointed out Pseudomonas as the predominant psycrotrophic flora in stored refrigerated raw milk
O processo de conserva??o do leite cru em temperaturas de refrigera??o por per?odos prolongados pode acarretar perda de qualidade do leite e, conseq?entemente, de seus produtos derivados, fato associado ao crescimento e ? atividade enzim?tica de bact?rias psicrotr?ficas, as quais podem se desenvolver em temperaturas abaixo de 7?C. Estes microrganismos geralmente s?o eliminados durante o tratamento t?rmico do leite, por?m suas enzimas proteol?ticas e lipol?ticas s?o termoest?veis, podendo resistir at? mesmo ao tratamento UAT e permanecerem ativas causando perda de qualidade dos produtos processados. Recentemente a INSTRU??O NORMATIVA 51, estabelece que o leite deve ser refrigerado e armazenado na propriedade rural, o que aumentou a import?ncia desse grupo de bact?rias. Neste trabalho, foram isoladas e identificadas bact?rias contaminantes de leite cru refrigerado proveniente de 20 tanques comunit?rios e 23 tanques individuais de propriedades leiteiras da regi?o da Zona da Mata do Estado de Minas Gerais e sudeste do Rio de Janeiro. Dilui??es selecionadas foram plaqueadas em agar padr?o e ap?s incuba??o a 7?C/10 dias, 5 col?nias foram isoladas em placas contendo agar nutriente, com incuba??o a 21?C/24 h. Os isolados foram identificados de acordo com a morfologia, colora??o de Gram, produ??o de catalase, oxidase, metabolismo fermentativo/oxidativo, crescimento em meio Mac Conkey, rea??o de KOH 3%, hidr?lise da arginina, motilidade, produ??o de H2S e indol em meio SIM e presen?a de esporos, e pelo Sistema API 20E, API 20NE, API Staph, API Coryne ou API 50 CH (BioM?rieux). O sistema API foi repetido para 50% dos isolados para garantir a reprodutibilidade dos resultados. A identifica??o em n?vel de esp?cie foi considerada quando o programa APILAB indicou identidade ≥ 75%. Foram obtidos 309 isolados, sendo 250 Gram negativos e 59 Gram positivos. Os 250 isolados Gram negativos obtidos foram identificados como: Acinetobacter spp. (39), Aeromonas spp. (07), A. Hydrophila (16), A. sobria (1), A. caviae (1), Alcaligenes feacalis (1), Burkholderia cepacia (12), Chryseomonas luteola (3), Enterobacter sp. (1), Ewingella americana(6), Hafnia alvei (7), Klebsiella sp. (1), Klebsiella oxytoca (10), Yersinia spp. (2), Methylobacterium mesophilicum (1), Moraxella spp. (4), Pantoea spp. (16), Pasteurella sp. (1), Pseudomonas spp. (10), P. fluorescens (94), P. putida (3), Serratia spp. (3), Sphigomonas paucomobilis (1). Cinco isolados n?o puderam ser identificados por meio dos testes utilizados. Pseudomonas foi o g?nero mais isolado (43%) sendo Pseudomonas fluorescens a esp?cie predominante (37,6%), o que corrobora dados da literatura. A produ??o de enzimas proteol?ticas e lipol?ticas foi avaliada qualitativamente pela presen?a de halo em torno da col?nia em agar caseinato e agar tributirina durante 72 horas a 21?C e 10 dias a 4?C, 7?C e 10oC. Do total de 250 Gram negativos, 104 foram identificados como Pseudomonas spp., das quais 60,57% apresentaram atividade proteol?tica e lipol?tica nas quatro temperaturas estudadas. 20% das esp?cies pertencentes aos g?neros: Acinetobacter, Aeromonas, Alcaligenes, Burkholderia, Chryseomonas, Methylobacterium, Moraxella, demonstraram apenas atividade lipol?tica nas quatro condi??es estudadas. Dos 59 isolados Gram-positivas obtidos, foram identificados os seguintes g?neros: Kurthia spp (7), Levedura (1), B. stearothermophilus (1), Bacillus coagulans (1), Bacillus lentus (1), Brevibacterium spp (1), Cellum/Microbacterium (6), Staphylococcus spp (3). Trinta e sete isolados n?o puderam ser identificados por meio dos testes utilizados. Alguns isolados apresentaram atividade enzim?tica em uma ou mais das quatro temperaturas estudadas: Gram-positivos- 30,51% foram proteol?ticas a 7?, 10 e 21?C e lipol?tica a 10?C; 8,47% foram proteol?ticos a 7?, 10? e 21?C; 8,47% foram lipol?tica nas quatro temperaturas estudadas; 3,38% proteol?ticos s? a 21?C e somente um isolado tiveram atividade proteol?tica a 4?C e seis atividade lipol?tica a esta mesma temperatura. Gram negativos - 4% foram proteol?tico e lipol?tico a 7?, 10? e 21?C; 10% proteol?ticas a 10?C; 4,4% lipolitico a 4?, 7? e 21?C; 6,4% foram proteol?tico e lipol?tico a 10? e 21?C e tamb?m lipol?tico a 4? e 7?C. Estes resultados s?o consistentes com os de outras pesquisas, em que tamb?m foram constatados que esp?cies do g?nero Pseudomonas representam a microbiota psicrotr?fica deterioradora mais freq?ente do leite refrigerado

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The milk is rich in proteins, lipids, water and carbohydrates, making it an excellent environment for bacterial contamination from different origins and for its proliferation. Bacterial multiplication depends on its capacity of adaptation to the environment and time available. In dairy farms, milk can be storage under cold in bulk tanks models of two and four milking capacity, with a chilling capacity of 50% and 25% respectively of its total volume, in each milking process. The use of cold storage in milk production reduced the occurrence of acid milk, however, increased the proliferation of psychrotrophic microorganisms. These microorganisms presents proteolytic capacity over casein without modifing the milk acidity. This proteolytic capacity could be responsible for unstable non-acid milk (UNAM). The purpose of this study was to evaluate the effect of the cold storage length of time and temperature of raw milk in bulk tanks of two and four milking models on psychrotrophic bacterial count (PBC), casein stability in alcohol test and occurrence of UNAM. The study was conducted in 19 dairy farms in two rehearsal and each farm had a direct expansion bulk milk tank of two or four milking models. Dairy farms with an interval of four milking between milk transportation to the processing industry were selected, thus the milk had a 36 hours of cold storage in the dairy farm. Samples were collected before and after each milking for bacterial total count (BTC), physical-chemical analysis and PBC. Samples of 12 from a total of 19 dairy farms were collected for casein quantification. The length of time of cold storage did not affect the BTC and PBC (P > 0,05). The bulk tank model only affected the PBC (P < 0,05), presenting a lower mean tank s model of two milking when compared with models of four milking (3,61±0,104 and 4,00±0,120 CFU/mL(log10)). The PBC did not affected the casein fractions, however the length of time of cold storage affected the қ and β casein fraction concentrations. The reductions in these casein fractions did not affect the casein stability in the alcohol test. The length of time of cold storage affected the casein stability (P < 0,05) in the alcohol test without affecting the UNAM occurrence. Bulk tanks, when used properly in concern of raw milk volume to be chilled, allow the BTC and PBC rates to remain stable. The length of time of cold storage does not affect BTC and PBC but affects the casein concentration and casein stability in the alcohol test without affecting the UNAM occurrence
O leite é um meio nutritivo facilmente colonizado por bactérias de diferentes origens, com taxa de proliferação condicionada à capacidade do micro-organismo adaptar-se ao ambiente e do tempo disponível. Na propriedade rural o leite pode ser armazenado por até 48 horas em tanques de expansão direta modelo de duas ou quatro ordenhas, os quais diferem na capacidade de resfriamento a cada ordenha, respectivamente, esses equipamentos são capazes de resfriarem 50% e 25% de seu volume a cada ordenha. A conservação do leite sob resfriamento pode não ser suficiente para o controle de micro-organismos psicrotróficos, os quais apresentam atividade proteolítica sobre a caseína sem alterar a acidez do leite, podendo ser um fator desencadeador do leite instável não ácido (LINA). O objetivo do presente estudo foi avaliar o efeito do tempo e temperatura de estocagem do leite cru em tanques de expansão direta modelos de duas e quatro ordenhas sobre a contagem bacteriana total (CBT) e contagem de bactérias psicrotróficas (CBP), assim como, determinar o efeito do tempo e da CBP sobre a estabilidade da caseína ao teste do álcool e a ocorrência de LINA. O experimento foi conduzido em 19 propriedades leiteiras localizadas nas regiões Serrana e Meio-oeste Catarinense, as quais possuíam equipamentos de refrigeração do leite por expansão direta modelo duas ou quatro ordenhas com taxa de ocupação de no mínimo 60%. Nas propriedades rurais foram avaliadas informações referentes ao intervalo de 36 horas entre a captação de leite a granel para a indústria de laticínios, as quais incluíam monitoramento temperatura leite, temperatura ambiente e umidade relativa do ar, aplicação de questionários aos responsáveis pela ordenha e coletas de amostras de leite antes e após a cada ordenha para análise de CBT, CBP, composição e teste físico-químicos (pH, acidez titulável e concentração alcoólica), sendo que em 12 das propriedades as frações de caseína foram quantificadas em duplicata. O tempo de estocagem do leite cru não afetou a CBT e CBP (P > 0,05). O modelo de tanque de expansão afetou somente a CBP (P < 0,05), com média inferior para amostras de leite de tanques de duas ordenhas em relação a quatro ordenhas (3,61±0,104 e 4,00±0,120 UFC/mL(log10)). A CBP não afetou as frações das caseínas e caseína total, entretanto, o tempo de estocagem do leite cru reduziu (P < 0,05) a concentração das frações қ e β da caseína e caseína total. A redução das frações de caseína não demonstrou efeito sobre a estabilidade do leite ao teste do álcool. O tempo de estocagem reduziu a (P < 0,05) a estabilidade do leite ao teste do álcool, entretanto não cursou com o aumentou a ocorrência de LINA. Conclui-se que tanques de resfriamento por expansão, utilizados dentro de suas especificações quanto ao volume de leite a ser resfriado, permitem a manutenção da CBT e CBP . O tempo de estocagem não afeta a CBT e CBP, entretanto reduz a concentração de caseína e a estabilidade do leite ao teste do álcool, sem alterar a ocorrência de LINA

Este estudo objetivou analisar o potencial de deterioração da microbiota psicrotrófica presente em presunto cozido fatiado comercializado entre Maio e Junho de 2015 e Fevereiro e Março de 2016 no mercado público da cidade de Porto Alegre/RS e avaliar a influência de fatores ambientais na qualidade microbiológica do mesmo. Os presuntos foram coletados em 4 bancas desse local e foram realizadas contagens de bactérias psicrotróficas de 8 amostras e pesquisa de Listeria monocytogenes. Selecionaram-se 134 colônias de psicrotróficos isolados de presunto fatiado, 71 deles apresentaram atividade proteolítica, 58 atividade lipolítica e 12 apresentaram produção de exopolissacarídeo. Selecionaram-se 2 bactérias com a presença dessas atividades para identificação molecular, as quais foram identificadas como Kluyvera sp. e Carnobacterium sp. Além delas, mais 2 Listeria monocytogenes isoladas nesse trabalho foram submetidas ao teste de produção de biofilme, resultando como fracas formadoras e também ao teste de aderência em aço inoxidável, todas apresentando capacidade de adesão. A pesquisa de Listeria monocytogenes nos presuntos fatiados mostrou 100% de presença, sendo que 50% foram identificadas como L. monocytogenes, as quais pertenceram aos sorotipos 1/2a (1), 1/2b (2), 1/2c (2). Realizou-se análise de presunto cozido inteiro, em sua embalagem original, sendo que não foram encontrados micro-organismos Tratou-se o presunto fatiado com extrato de hibisco a 40% e pediocina a 0,5% e 1,0% e realizou-se contagem de mesófilos, psicrotróficos, Listeria spp., S. aureus e E.coli. O extrato de hibisco reduziu a carga desses micro-organismos. Pediocina 0,5% e 1% apresentaram pouca ação frente ao controle de mesófilos, psicrotróficos e E. coli, mas mantiveram a carga de S. aureus controlada e foram eficazes contra Listeria spp. Foram realizadas também contagens para Listeria monocytogenes, E. coli, S. aureus, mesófilos e psicrotróficos em suabes oriundos de fatiador de alimentos, superfície de contato e utensílio utilizados nas bancas do mercado público. Em conclusão grande parte dos psicrotróficos apresentou atividade proteolítica e lipolítica, as quais alteram organolepticamente o alimento. Alguns apresentaram produção de biofilme e capacidade de aderência, fato indesejado, pois sua remoção é mais difícil no ambiente industrial, com isso nota-se que a legislação brasileira apresenta carência na contagem de psicrotróficos em produtos cárneos.
This study aimed to evaluate the microbiota present in sliced cooked ham sold in the public market in Porto Alegre/RS and evaluate the ability of compounds with antimicrobial activity of hibiscus extract and pediocin to control the microbiota found. Ham collected was stored refrigerated until to arrive the laboratory for analysis. Psychrotrophic bacteria counts were performed. Were selected 134 colonies of psychrotrophic microorganisms isolated for sliced ham and 71 of them showed proteolytic activity, 58 lipase activity, 12 showed production of exopolysaccharide. Two of these bacteria were selected for molecular identification which were identified as Kluyvera sp. and Carnobacterium sp. These two bacteria plus two Listeria monocytogenes isolated for sliced ham were subjected to testing of biofilm production (resulting as weak forming of biofilm) and were tested for adhesion in stainless steel and all showed this property. The research of Listeria spp. in sliced cooked ham showed 100% of presence, which 50% were identified as L. monocytogenes to serotypes 1/2a (1) 1/2b (2), 1/2c (2). Analysis was carried out of a whole piece of cooked ham in its original packaging and none microorganisms were found. The sliced ham was treated with hibiscus extract of 40% and pediocin of 0.5% and 1.0% and has been mesophilic, psychrotrophic, Listeria spp., S. aureus and E. coli counts The hibiscus extract reduced the quantity of these microorganisms. Pediocin 0.5% and 1,0% had little action against the control of mesophilic, psychrotrophic and E. coli, but in S. aureus counts were controlled bacteria charge and were effective against Listeria spp.. Also counts of Listeria monocytogenes, E. coli, S. aureus, mesophilic and psychrotrophic bacteria were performed from swabs of slicer food, contact surface and food tool (knife or spatula) used in public market stalls. In conclusion, most of the psychrotrophs presented proteolytic and lipolytic activity, which alter organoleptically the food. Some of them have presented biofilm production and adhesion capacity, undesirable fact because when the biofilm is formed is more difficult to remove it in the industrial environment. With this it is showed that the brazilian legislation presents a lack in the research of psychrotrophs in meat products kept refrigerated.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Among the psychrotrophic micro-organisms that are able to grow during refrigeration, some of them produce thermostable proteases which can cause many technological problems in the dairy industry. The food industries need to high throughput methods for the quality control raw of milk, thus, fast and sensitive methods for psychrotrophic detection, specially Pseudomonas fluorescens, predominant specie, are economically interesting to the dairy industry. In order to establish a protocol for the detection of P. fluorescens by using the polymerase chain reaction (PCR), five DNA extraction methods for milk samples, two target genes for the PCR amplification and the detection limit in raw milk and sterilized milk inoculated with P. fluorescens were evaluated. A commercial DNA extraction kit and a modified filtration method were the most appropriate for successfully bacterial DNA extraction from milk samples. These methods were selected and used for the detection of the target genes. Nevertheless, the modified filtration method was more efficient showing a lower detection limit in raw milk and sterilized milk inoculated with P. fluorescens. By means of PCR amplification of the 16S rRNA target gene, an 850 bp amplicon was observed and it was possible to detect as few as 102 UFC/ml of P. fluorescens from inoculated milk. In raw milk samples, Pseudomonas and psychrotrophic bacteria were detected at 106 UFC/mL and 107 to 109 UFC/mL, respectively. The relationship between the psychrotrophic population and the degradation of milk proteins was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed the proteolysis degree in raw milk doesn t depend just on the psychrotrophic bacteria count.
Entre os micro-organismos psicrotróficos que são capazes de crescer durante a refrigeração, alguns produzem proteases termorresistentes relacionadas com problemas tecnológicos nos derivados lácteos. Visto que a demanda por métodos que permitem alta taxa de processamento exigida em indústrias de alimentos para controle da qualidade de leite cru é crescente, a avaliação de métodos mais sensíveis e mais rápidos para detecção de psicrotróficos, especialmente Pseudomonas fluorescens, espécie predominante, é de grande importância. Com a finalidade de estabelecer um protocolo para detecção de P. fluorescens pela reação em cadeia da polimerase (PCR), foram avaliados cinco métodos de extração de DNA total do leite, dois genes alvos na PCR e o limite de detecção dos métodos para análise de leite cru e leite esterilizado e inoculado com P. fluorescens. O kit comercial e o método de filtração modificado foram os métodos mais adequados para extração de DNA total de amostras de leite, por isso, foram utilizados nas análises seguintes para detecção dos genes alvos. No entanto, a utilização do método de filtração modificado para análise de leite inoculado com P. fluorescens e leite cru resultou em um menor limite de detecção quando comparado com o kit comercial. Por meio da amplificação do gene alvo rDNA 16S, foi possível observar um amplificado de 850 pb quando a população do leite inoculado com P. fluorescens era da ordem de 102 UFC/ml. Em amostras de leite cru, foi possível detectar o produto quando a contagem de Pseudomonas era da ordem de 106 UFC/ml e a população de psicrotróficos estava entre 107 e 109 UFC/ml. A análise do grau de proteólise das amostras de leite cru por eletroforese em gel de poliacrilamida demonstrou que a relação entre a população de psicrotróficos e a degradação das proteínas do leite depende de outros fatores além da contagem de psicrotróficos.

La bactérie psychrotrophe P. Fluorescens MF0 est plus sensible aux antibiotiques de type β-lactamines à une température de croissance de 8°C qu'à une température de 28°C. Cette différence est parfaitement corrélée avec les valeurs de conductance obtenues pour les porines majoritaires de la membrane externe OprF à 8°C et 28°C puisqu'elles sont respectivement de 80 pS et 250 pS dans du NaCl 1M. Pour expliquer ce phénomène, les OprFs 8°C et 28°C ont été purifiées et les études de spectrométrie de masse et de dichroïsme circulaire ne montrent pas de différence dans leur structure primaire. Des mesures de cinétiques de digestion à la pronase ont conduit à une différence des valeurs de constantes de vitesse de dégradation impliquant ainsi une modification de la conformation protéique en fonction de la température de croissance. Les fragments protéolytiques de 18 kDa, résistant à l'action de la pronase, ont été purifiés et identifiés comme la partie N-terminale de la protéine native (177 acides aminés). Les deux fragments forment des canaux ioniques avec des valeurs de conductances similaires (65 et 75 pS dans NaCl 1M) impliquant que la région C-terminale est seule responsable de la modulation des canaux formes par l'OprF en fonction de la température de culture. La caractérisation du LPS associé aux OprFs met en évidence une différence de phosphorylation des LPS obtenus à partir de culture à 28°C et 8°C. Ainsi, l'association LPS-porine serait différente selon les températures de culture. Nous avons ensuite voulu rechercher une éventuelle généralisation de ce phénomène et nous avons choisi la bactérie psychrotrophe Chryseomonas luteola MCF10. La protéine majoritaire de la membrane externe (31 kDa) a été purifiée à 8°C et 28°C et son étude fonctionnelle montre des valeurs de conductances unitaires similaires quelle que soit la température de croissance. Cependant, la détermination de la séquence N-terminale ainsi que la séquence déduite du gène la caractérise comme une OmpA. L'utilisation des anticorps montre que C. Luteola est plus proche des entérobactéries que des pseudomonas. Ce résultat montre que le mécanisme de résistance décrit chez P. Fluorescens n'est pas systématiquement applicable à toute bactérie psychrotrophe.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Milk production is an important income for family farmers, but to act competitively in the market the dairy sector needs to raise the quality of its raw material. The study aimed to evaluate the factors that influence water quality and milk quality and unstable non-acid milk (UNAM) occurrence in family farms of Planalto Norte of Santa Catarina, Brazil. The study also aimed to assess the influence of water quality used in farm on the milk quality, and to establish relation between bacterial contamination and concentration of CMP in milk. The farms were characterized by a semi-structured questionnaire guide. Samples were collected for analysis of milk composition, somatic cell count (SCC), total bacterial count (TBC) and psychrotrophic bacterial count (PBC). It carried out the alcohol test, titratable acidity and pH, and CMP index was determined by high-performance liquid chromatography. Analyzes performed in water were CBP, Escherichia coli, coliforms at 35ºC and mesophilic aerobic. The food offered to animals also were analyzed. Data were subject to factor and cluster analysis, and broken-line model analysis using SAS statistical package. The most appropriate strategies of animal feed, with consequent improvement of animal productivity and increase of lactose content in milk were related to a lower occurrence of UNAM, which had little relation with farm structure and with season. The farm structure also had little influence on milk microbiological quality. Already the milking management influenced the bacterial contamination, and these were related to CMP concentration in milk. The water quality used in dairy farms was not related to milk quality. Rainfall and water source affected the microbiological contamination in water
A produção de leite é uma importante fonte de renda para agricultura familiar, mas para atuar de modo competitivo no mercado o setor lácteo precisa elevar a qualidade da sua matéria-prima. O estudo objetivou avaliar os fatores que influenciam na qualidade da água e do leite e na ocorrência de leite instável não ácido (LINA) em propriedades de agricultura familiar do Planalto Norte de Santa Catarina. O trabalho também objetivou verificar a influência da qualidade da água utilizada na propriedade sobre a qualidade do leite produzido, além de estabelecer a relação entre a contaminação bacteriana e a concentração de caseinomacropeptídeo (CMP) no leite. As propriedades foram caracterizadas por meio de um questionário guia semi-estruturado. Foram coletadas amostras de leite para analisar sua composição, contagem de células somáticas (CCS), contagem bacteriana total (CBT), e contagem de bactérias psicrotróficas (CBP). Realizou-se o teste do álcool, análises de acidez titulável e pH, e determinou-se o índice CMP por cromatografia líquida de alta eficiência. Na água, foram realizadas análises de CBP, Escherichia coli, coliformes a 35ºC e aeróbios mesófilos. Também foram analisados os alimentos oferecidos aos animais. Os dados foram submetidos à análise fatorial e de agrupamento, e regressão segmentada (broken-line) utilizando o pacote estatístico SAS. As estratégias mais adequadas de alimentação animal, com consequente melhoria da produtividade e aumento do teor de lactose no leite, tiveram influência na menor ocorrência de LINA, que por sua vez apresentou pouca relação com a infra-estrutura das propriedades e com as estações do ano. A infra-estrutura das propriedades também teve pouca relação com a qualidade microbiológica do leite. Já o manejo de ordenha mostrou influência na contaminação bacteriana, que relacionou-se com a concentração de CMP no leite. A qualidade da água utilizada nas propriedades leiteiras não apresentou relação com a qualidade do leite. A precipitação pluviométrica e a origem da água afetaram a sua contaminação microbiológica

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Milk is nutritionally complete, but a highly perishable food and its characteristics easily altered by the action of microorganisms and by the manipulation to which it is submitted. The quality and shelf life of the product that reaches the consumer is directly related to the raw material received for industrialization, and consequently to the conditions and practices carried out in obtaining, storing and transporting. The objective of this study was to evaluate the influence of transport of raw milk on the count of psychrotrophic microrganisms, total bacterial count, physical-chemical and compositional analyses of the refrigerated raw milk received in a dairy industry of Vale do Itajaí – SC, Brazil. Other objective of this study was to evaluate the use of the preservative azidiol ®, under controlled conditions and under field conditions, in the count of psychrotrophic microorganisms and in the total bacterial count of refrigerated raw milk. Two experiments were carried out. In the first one, samples were collected, with and without the use of the preservative azidiol®, in bulk tanks of 3 dairy farms, which were stored under laboratory refrigeration (7ºC) and analyzed at pre-defined times (0, 6, 12 and 24 hours). In the second experiment, samples were collected with and without the use of the azidiol® preservative in the raw milk capture at routes of a dairy (in the storage tanks of the farms, in the isothermal compartments of the trucks and in the storage silo of the industry), doing analysis at varying times. Data were analyzed by ANOVA, regression analysis, means comparison and multivariate factorial analysis. Samples collected without preservatives showed increasing psychrotrophic microrganisms counts and total bacterial count as the time between collection and analysis increased. In the samples conserved by azidiol ®, psychrotrophic microrganisms counts and total bacterial count remained constant during the time between collection and analysis. The transport of raw milk in bulk worsened the microbiological quality of the milk, but did not show influence on the compositional and physical-chemical results of the milk received in the industry. Poor hygiene conditions in storage rooms and in expansion tanks influenced the increase of psychrotrophic microrganisms counts and total bacterial count. This increase was also related to farms with lower production, but with less representative influence. It was observed a relation of milk transport in bulk in cleaner trucks with lower counts of psychrotrophic microorganisms. The transport route time and temperature increase during the transport increased counts of total bacteria and psychrotrophic bacteria, with more pronounced effect for total bacterial count
O leite é um alimento nutricionalmente completo, porém altamente perecível, tendo suas características facilmente alteradas pela ação de microrganismos e pela manipulação a que é submetido. A qualidade e tempo de prateleira do produto que chega ao consumidor estão diretamente relacionados à matéria-prima recebida para industrialização e, consequentemente, às condições e práticas realizadas na obtenção, armazenamento e seu transporte. Este estudo teve como objetivo a avaliação da influência do transporte do leite cru a granel sobre a contagem de microrganismos psicrotróficos, contagem bacteriana total, análises físico-químicas e composição do leite cru refrigerado recebido em uma indústria de laticínios da Região do Vale do Itajaí – SC. Também objetivou avaliar o uso do conservante azidiol®, em condições controladas e em condições a campo, na contagem de microrganismos psicrotróficos e na contagem bacteriana total de leite cru refrigerado. Foram realizados dois experimentos. No primeiro, foram coletadas amostras, com e sem o uso do conservante azidiol®, em tanques de expansão de três propriedades rurais, as quais foram armazenadas em laboratório e sob refrigeração (7ºC) e analisadas em tempos pré-definidos (0, 6, 12 e 24 horas). No segundo experimento foram coletadas amostras com e sem a adição do agente bacteriostático azidiol® em rotas de captação de matéria-prima de um laticínio (nos tanques de armazenamento das propriedades rurais, nos compartimentos isotérmicos dos caminhões e no silo de armazenamento da indústria), analisando-as em tempos variáveis. Os dados foram avaliados por análise de variância e de regressão, comparação de médias e pela técnica de análise multivariada fatorial. Amostras coletadas sem conservante demonstraram contagem de microrganismos psicrotróficos e CBT crescentes à medida que o tempo entre a coleta e a análise aumentava. Já nas amostras adicionadas do conservante azidiol® a contagem de microrganismos psicrotróficos e a CBT se mantiveram constantes durante o tempo entre a coleta e a análise. O transporte do leite cru a granel piorou a qualidade microbiológica do leite; porém, não demonstrou influência nos resultados das análises composicionais e físico-químicas do leite recebido na indústria. Condições ruins de higiene nas salas de armazenamento e nos tanques expansão influenciaram o aumento da contagem de microrganismos psicrotróficos e a CBT. Este aumento também foi relacionado com propriedades com menor produção de leite, porém, com influência menos representativa. Observou-se relação do transporte do leite a granel em caminhões mais limpos com menores contagens de microrganismos psicrotróficos. Maior tempo da rota de transporte e o aumento da temperatura do leite durante o transporte aumentaram as contagens de bactérias totais e de microrganismos psicrotróficos, sendo este efeito mais pronunciado para a CBT

Des bactéries adaptées au froid ont été sélectionnées pour leur capacité à dégrader des polysaccharides naturels. L'influence de la température de croissance, sur la régulation de la production des enzymes exocellulaires, a été étudiée. Nous avons mis en évidence l'existence de cinq profils de régulation qui ne sont corrélés ni a l'origine taxonomique des souches, ni au type d'activité produite. D'autre part, chez une même souche, le profil de production d'une activité donnée peut varier suivant la composition du milieu de culture, notamment en substrat carboné, ainsi qu'en fonction de la présence d'inducteur spécifique. La dépendance des profils de production à l'égard des conditions nutritionnelles, démontre également qu'aucune loi générale ne peut être établie quant à l'influence de la température sur la production des enzymes exocellulaires, chez les bactéries adaptées au froid. Cependant des comportements différents peuvent être observés suivant le groupe thermique des souches. En utilisant la représentation selon Arrhénius, du taux de croissance en fonction de la température, nous avons démontré l'existence d'une température critique de rupture de pente chez toutes les souches psychrotrophes, celle-ci étant absente chez les psychrophiles. Sur la base de ce critère distinctif nous avons établi que, chez les psychrotrophes, la production des enzymes exocellulaires varie de façon discontinue sur l'ensemble de la gamme de températures suboptimales de croissance, ce qui se traduit par l'existence de deux domaines thermiques de production. En revanche chez les psychrophiles la production varie de façon monotone sur la gamme de températures inférieures à l'optimum de croissance. Ces profils pourraient résulter d'adaptation en réponse aux différentes amplitudes de température caractérisant les environnements d'origine des représentants de chacun de ces deux groupes thermiques.

Este trabalho avaliou os efeitos de diferentes períodos de armazenamento do leite de cabra in natura sobre a qualidade do produto em pó. Foram avaliadas as alterações microbiológicas, físico-químicas e bioquímicas do leite cru e a influência nas características microbiológicas, físicas, bioquímicas e sensoriais do leite em pó durante o armazenamento por 0, 60, 120 e 180 dias. Foram realizados 3 ensaios idênticos nos quais cerca de 105 L de leite de cabra recém-ordenhado foram igualmente divididos em 3 partes e armazenados a temperatura controlada de 4 ºC por até 5 dias. Nos dias 1, 3 e 5 após a coleta do leite in natura, uma alíquota de 500 mL foi coletada para a realização das análises. O restante da fração (aproximadamente 35L) foi submetido à pasteurização (65 ºC por 30 min), concentração sob vácuo (40% de sólidos totais) e secagem por atomização. Os lotes de leite de cabra em pó obtidos foram avaliados através de análises de composição (umidade, teores de proteína, gordura, lactose e cinzas), dispersibilidade, cor, atividade de água, índice de peróxidos, atividades proteolítica e lipolítica e análise sensorial por uma equipe de provadores treinados. Foram observados efeitos (P < 0,05) do período de armazenamento do leite in natura e/ou do leite em pó, ou mesmo interação destes efeitos sobre determinadas características durante o armazenamento do leite em pó, como: aumento linear das populações de micro-organismos mesófilos, psicrotróficos lipolíticos e psicrotróficos proteolíticos do leite in natura, aumento da intensidade da cor branca (L*) do leite em pó, da atividade lipolítica e da oxidação do leite em pó. Também foram observados efeitos (P < 0,05) em características sensoriais como: redução da coloração amarela do pó de do leite reconstituído, aumento do odor cáprico e dos sabores rançoso e amargo do leite reconstituído. Considerando-se a avaliação global das variáveis estudadas, recomenda-se que o período de armazenamento a 4 oC do leite de cabra in natura não ultrapasse 3 dias, para que ocorra a preservação da qualidade do leite de cabra em pó por até 180 dias.
This study evaluated the effects of different storage periods of raw goat milk on the quality of the powder product. Alterations in microbiological and physical-chemical properties of raw milk and their influence on the microbiological, physical, biochemical and sensory characteristics of milk powder during storage for 0, 60, 120 and 180 days were evaluated. There were 3 identical tests in which about 105 L of recently milked goat milk were divided into 3 parts and stored at controlled temperature of 4 ºC for up 5 days. On days 1, 3 and 5 after storage, an aliquot (500 mL) of raw milk was collected to perform microbiological, physico-chemical and biochemical analysis. The remaining fraction (about 35 L) was subjected to pasteurization (65 ºC for 30 min), vacuum concentration (40% of total solids) and spray drying. The powders produced were evaluated through analysis of composition (moisture, protein, fat, lactose and ash), dispersibility, color, water activity, granulometry, peroxide value, proteolytic and lipolytic activities and sensory analysis by a selected team of panelists. Effects of storage of raw milk or/and powdered milk or their interaction were observed (P <0.05) on certain characteristics during storage of milk powder, as the increasing of mesophilic, lipolytic psychrotrophic and proteolytic psychrotrophic microorganisms populations in raw milk, increasing of the white color (L*), the lipolytic activity and the peroxide value of milk powder. There were also observed effects (P < 0.05) on sensory characteristics such as decreasing of yellow color of milk powder and reconstituted milk, increasing of capric smell, rancid and bitter flavour of reconstituted milk. Considering the overall evaluation of studied variables, it\'s recommended that the raw goat milk storage at 4 oC does not exceed 3 days to preserve the quality of goat milk powder until 180 days.

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The raw milk storage under cooling temperatures selects the psychrotropic bacteria growth including species able to produce heat resistant proteases and lipases that are usually associated with milk quality deterioration. The decrease in the cheese production yield is an important problem associated with the psychrotrophic bacteria growth in the refrigerated raw milk. This work aimed to evaluate the relation between the psychrotrophic bacteria population present in the cooled raw milk and the yield of Minas Frescal cheese production. The raw milk was stored at 10 °C, for up to four days, it was pasteurized, and daily used for the Minas Frescal cheese production. Concomitantly, the number of psychrotrophic, proteolytic and lipolytic psychrotrophic bacteria and protease and lipase activity was evaluated in the raw milk. The raw milk maintenance at 10 °C made possible the psychrotrophic bacteria growth, with an increase of two logarithmic cycles in the proteolytic psychrotrophic bacteria population and of, approximately, three logarithmic cycles in the lipolytic psychrotrophic bacteria population, after four days of storage. The protease and lipase activities increased during the refrigerated raw milk storage, presenting superior values to the ones of the recently milked milk. A decrease of 6.78% in liters of milk per kg of cheese and 6.38 % in grams of total solids of cheese per liter of milk when the product was done with cooled milk stored for four days, was observed. The psychrotrophic bacteria number present in that milk varied from 108 CFU mL-1 to 109 CFU mL-1, and the proteolytic and lipolytic psychrotrophic bacteria populations were higher than 107 CFU mL-1. For the same storage period, an increase in the total nitrogen, fat and total solids concentrations in the serum from the Minas Frescal cheese production, was verified. It was estimated that, for an industry able to process 50.000 liters of milk per day, designating 33% of this amount to produce Minas Frescal cheese, the economic losses associated with the decrease in the cheese production are estimated in US$ 15,480.00 per month.
A estocagem do leite cru sob refrigeração favorece a seleção de bactérias psicrotróficas, incluindo espécies produtoras de proteases e lipases termorresistentes que são associadas à perdas de qualidade do leite e seus derivados. Para as indústrias de queijos, um dos principais problemas causados em decorrência da contaminação e do crescimento de bactérias psicrotróficas no leite cru refrigerado é a redução do rendimento durante a fabricação. O objetivo deste trabalho foi relacionar o rendimento do queijo Minas Frescal com a população de bactérias psicrotróficas no leite cru refrigerado granelizado. O leite cru foi estocado a 10 °C, por até quatro dias, posteriormente pasteurizado e, diariamente, utilizado para a fabricação de queijo Minas Frescal. Paralelamente, foram quantificadas as microbiotas psicrotrófica, psicrotrófica proteolítica e lipolítica e a atividade proteolítica e lipolítica do leite cru. A manutenção do leite cru a 10 °C possibilitou um crescimento da microbiota psicrotrófica, com um aumento de dois ciclos logarítmicos na população de bactérias psicrotróficas proteolíticas e de, aproximadamente, três ciclos logarítmicos na população de bactérias psicrotróficas lipolíticas, após quatro dias de estocagem. As atividades proteolítica e lipolítica aumentaram durante a estocagem do leite cru refrigerado, apresentando valores superiores aos do leite recém-ordenhado. Foi constatada uma redução de 6,78 % no rendimento em termos de litros de leite por quilograma de queijo e de 6,38 % em gramas de sólidos totais no queijo por litro de leite quando o queijo Minas Frescal foi fabricado com leite refrigerado armazenado por quatro dias. O leite cru que resultou nestas perdas de rendimento apresentava uma população de 108 UFC mL-1 a 109 UFC mL-1 de bactérias psicrotróficas e a população de bactérias psicrotróficas proteolíticas e lipolíticas era superior a 107 UFC mL-1. Verificou-se ainda, para o mesmo período de estocagem, um aumento das concentrações de nitrogênio total, gordura e sólidos totais no soro proveniente da fabricação do queijo Minas Frescal. As perdas econômicas decorrentes da redução do rendimento da fabricação de queijo Minas Frescal foram estimadas em US$ 15,480.00 por mês, para uma indústria de laticínios processadora de 50.000 litros de leite por dia e que destine 33% do leite captado a fabricação de queijo Minas Frescal.

Au cours de ce travail, les principaux micro-organismes psychrotrophes phytopathogènes de la flore microbienne se trouvant à la surface de céleri-raves conservés en chambre froide ont été isolés et identifiés. Parmi ceux-ci, une souche d'Erwinia carotovora (MFCL0) a servi de modèle pour l'étude de la physiologie bactérienne en fonction des variations physico-chimiques de l'environnement. Elle a été identifiée par sidérotypage, séquençage de l'ADNr 16S, hybridation ADN/ADN et détermination de ces caractéristiques physiologiques et phénotypiques. Il s'agit d'une souche, capable de se développer sur une large gamme de température (0°C-37°C) et de produire de nombreuses enzymes impliquées dans la dégradation des tissus végétaux. Elle sécrète au moins une cellulase, une pectine lyase, une pectine méthylestérase, une polygalacturonase (Peh I) et 3 pectate lyases (Pel I, Pel II et Pel III). La température optimale pour la production de PehI, Pel I et Pel III se situe aux alentours de 20-24°C en bouillon nutritif ordinaire, alors qu'elle est de 14°C en PGA. L'étude de courbes obtenues par extrapolation de la relation d'Arrhénius à la croissance bactérienne nous montre que cette bactérie présente une température critique qui varie avec la source de carbone et d'énergie utilisée. Le résultat le plus intrigant est la coïncidence entre les températures critiques et les températures optimales de production enzymatique dans chacun des milieux testés. Un tel phénomène pourrait être la conséquence d'un nouveau type de thermorégulation dont les mécanismes restent à élucider. Deux gènes de pectate lyase ont d'ores et déjà été clonés et séquencés. L'étude de différents mutants affectés dans la production des différents enzymes est en cours afin de déterminer leur implication dans la phytopathogénicité ainsi que la manière dont ils sont régulés par les conditions de l'environnement.