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The use of page and gas chromatography of cellular fatty for the rapid identification of Fusobacteria and CapnocytophagaLitz, Julie S. January 1987 (has links)
The use of PAGE of soluble cellular proteins and gas chromatography of cellular fatty acids was studied to determine the possible use of both methods for rapid identification of anaerobic bacteria. Species of <u>Fusobacterium</u> and <u>Capnocytophaga</u> were analyzed to determine the identification accuracy of each system.
The electrophoretic patterns of soluble cellular proteins and the cellular fatty acid patterns determined by gas chromatography were found to remain relatively constant within the species examined. This similarity allowed for the development of automated systems using computer programs to analyze the patterns, and compare them to the patterns of known species. Procedures for gas chromatography of cellular fatty acids were developed by Myron Sasser, University of Delaware, and Microbial Identification Systems (Suite 115 Barksdale Professional Center, Newark, Delaware 19711), in cooperation with Hewlett Packard.
From this study it was determined that the accuracy of identification of the PAGE analyses was not high, and therefore this method would have limited use in a clinical laboratory. Gas chromatography of cellular fatty acids had a relatively high accuracy, which is still being improved. / Master of Science
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Identification of lactic acid bacteria isolated from vinegar flies and Merlot grapesGroenewald, W. H. 10 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Thirty lactic acid bacteria were isolated from the intestinal tract of Drosophila simulans
Stuvervant and nine lactic acid bacteria from Merlot grapes collected from the same
winery in the Stellenbosch region, South Africa.
The isolates were grouped according to morphological, biochemical and physiological
characteristics. Isolates selected from each group were identified to species level by PCR
with species-specific primers, PCR-based DGGE and 16S rDNA sequencing. The
majority of isolates from the intestinal tract of Drosophila simulans Stuvervant belonged
to the species Lactobacillus plantarum, but Lactobacillus paracasei, Lactobacillus
sanfranciscensis, Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis
subsp. lactis, Enterococcus faecalis and Pediococcus pentosaceus were also identified.
As far as we could determine, this is the first report on the isolation of L. paracasei, L.
sanfranciscensis, L. mesenteroides subsp. mesenteroides, L. lactis subsp. lactis, E.
faecalis and P. pentosaceus from vinegar flies. Lactobacillus plantarum has previously
been isolated from Merlot grapes.
The genotypic relatedness among isolates of L. plantarum isolated from the intestinal
tract of vinegar flies and from Merlot grapes were determined by RAPD-PCR. The
isolates were grouped into four genotypically well-separated clusters. Thirteen isolates
from grape must and five from flies yielded identical RAPD-PCR banding patterns and
grouped into one cluster, suggesting that they are descendants from the same strain. This
suggests that L. plantarum has the ability to use vinegar flies as a vector. / AFRIKAANSE OPSOMMING: Dertig melksuurbakterieë is vanuit die dermkanaal van Drosophila simulans Stuvervant
geïsoleer en nege melksuurbakterieë vanuit Merlot-druiwe. Die druiwe is afkomstig van
dieselfde wynkelder in die Stellenbosch-area van Suid-Afrika.
Die isolate is volgens morfologiese, biochemiese en fisiologiese eienskappe gegroepeer.
Verteenwoordigende isolate vanuit die fenotipiese groepe is tot spesievlak met behulp
van lukraak ge-amplifiseerde polimorfe-DNA (RAPD) polimerase ketting-reaksie (PKR),
PKR met spesie-spesifieke inleiers, PKR-gebaseerde denaturerende gradient-jel
elektroforese (DGGE) en 16S rDNA sekwensering geïdentifiseer.
Die meerderheid isolate uit die ingewande van Drosophila simulans Stuvervant is as
Lactobacillus plantarum geklassifiseer. Stamme van Lactobacillus paracasei,
Lactobacillus sanfranciscensis, Leuconostoc mesenteroides subsp. mesenteroides,
Lactococcus lactis subsp. lactis, Enterococcus faecalis en Pediococcus pentosaceus is
ook geïdentifiseer. Sover bekend, is dit die eerste keer dat L. paracasei, L.
sanfranciscensis, L. mesenteroides subsp. mesenteroides, L. lactis subsp. lactis, E.
faecalis en P. pentosaceus uit asynvlieë geïsoleer is. Lactobacillus plantarum is
voorheen uit Merlot-druiwe geïsoleer.
Die genotipiese ooreenkoms tussen die stamme van L. plantarum wat uit die asynvlieë en
Merlot-druiwe geïsoleer is, is deur middel van RAPD-PKR bepaal. Hiervolgens is die
stamme in vier genotipies goed-gedefinieerde groepe geplaas. Dertien isolate vanuit
druiwemos en vyf vanuit asynvlieë het identiese RAPD-PKR bandpatrone vertoon en het
in een groep gesorteer. Hierdie resultate dui daarop dat die stamme heel moontlik uit een
voorouer ontstaan het en dat asynvlieë heel moontlik as vektor vir L. plantarum dien.
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Detection of NheA from Bacillus spp. in food and soil isolates using real-time and rep-PCR / Detection of non-hemolytic enterotoxin A from Bacillus spp. in food and soil isolates using real-time and rep-polymerase chain reactionBeer, Matthew R. 06 August 2011 (has links)
Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Rep-PCR was used to compare the banding patterns of each sample against B. cereus ATCC14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe, and were capable of causing gastroenteritis in consumers. / Department of Biology
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Feasibility study on the application of Fourier transform infrared spectroscopy for the rapid identification of bacteria of public health significanceTao, Jin, 1948- January 1994 (has links)
No description available.
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Schnelle Identifizierung von oralen Actinomyces-Arten des subgingivalen Biofilms mittels MALDI-TOF-MSBorgmann, Toralf Harald 25 November 2015 (has links) (PDF)
Aktinomyzeten sind ein Teil der residenten Flora des menschlichen Verdauungstraktes, des Urogenitalsystems und der Haut. Die zeitraubende Isolation und Identifikation der Aktinomyzeten durch konventionelle Methoden stellt sich häufig als sehr schwierig dar. In den letzten Jahren hat sich jedoch die Matrix-unterstützte Laser-Desorption/Ionisation-Flugzeit-Massenspektrometrie (MALDI-TOF-MS) als Alternative zu etablierten Verfahren entwickelt und stellt heutzutage eine schnelle und simple Methode zur Bakterienidentifikation dar. Unsere Studie untersucht den Nutzen dieser Methode für eine schnelle und zuverlässige Identifizierung von oralen Aktinomyzeten, die aus dem subgingivalen Biofilm parodontal erkrankter Patienten isoliert wurden. In dieser Studie wurden elf verschiedene Referenzstämme aus den Stammsammlungen ATCC und DSMZ und 674 klinische Stämme untersucht. Alle Stämme wurden durch biochemische Methoden vorab identifiziert und anschließend ausgehend von den erhobenen MALDI-TOF-MS-Daten durch Ähnlichkeitsanalysen und Klassifikationsmethoden identifiziert und klassifiziert. Der Genotyp der Referenzstämme und von 232 klinischen Stämmen wurde durch Sequenzierung der 16S rDNA bestimmt. Die Sequenzierung bestätigte die Identifizierung der Referenzstämme. Diese und die zweifelsfrei durch 16S rDNA Sequenzierung identifizierten Aktinomyzeten wurden verwendet, um eine MALDI-TOF-MS-Datenbank zu erstellen. Methoden der Klassifikation wurden angewandt, um eine Differenzierung und Identifikation zu ermöglichen. Unsere Ergebnisse zeigen, dass eine Kombination aus Datenerhebung mittels MALDI-TOF-MS und deren Verarbeitung mittels SVM-Algorithmen eine gute Möglichkeit für die Identifikation und Differenzierung von oralen Aktinomyzeten darstellt.
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Selected applications of Fourier transform infrared spectroscopy to the study of cells and cellular componentsDubois, Janie. January 1999 (has links)
The potential applicability of Fourier transform infrared (FM) spectroscopy for the study of biomolecules, has been investigated through the investigation of three specific applications. Two of the applications selected involve the classification of whole cells, whereas the third concerns the study of peptides and proteins isolated from tissues as well as synthetic peptides. In the microbiological application, for the differentiation of bacteria on the basis of their FTIR spectra, it has been found that classification is most efficiently achieved through the utilisation of artificial neural networks, and that a wide, variety of bacteria can be correctly identified with minimal sample preparation after being grown on a single growth medium. The suitability of low-cost, disposable materials as supports for the deposition of bacteria samples has also been demonstrated. In contrast to the successful classification of bacteria, it has been found that the examination of cytological smears by FTIR spectroscopy does not allow the classification of cervical cell populations into the recognised diagnostic categories. In a microscopic investigation of the same cell populations utilising infrared imaging, localised areas of the smear were found to exhibit distinct characteristics not observed in the spectra of the entire population. This indicates that infrared imaging system may be required for this type of application because the infrared spectrum recorded from a whole cervical smear does not allow the detection of the small spectral features arising from the molecular changes associated with a localised pathological state. Finally, the investigation of factors affecting the stability of peptides related to amyloidosis has shown that temperatures above 70°C, hydrostatic pressure greater than 6 kbar, and alkaline pH promote the disaggregation of the typical intermolecular beta-sheet structure of amyloid peptides. Peptides utilised as model systems for amyloidosis showed
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Feasibility study on the application of Fourier transform infrared spectroscopy for the rapid identification of bacteria of public health significanceTao, Jin, 1948- January 1994 (has links)
The infrared spectra of 14 bacteria were recorded by Fourier Transform Infrared (FTIR) spectroscopy. The effects of changes in growth conditions, such as temperature, growth medium, and incubation time, sampling conditions on the reproducibility of the spectra were investigated. The spectra of bacteria suspended in saline solution, in D$ sb2$O-saline and on plate films were obtained and stored in separate spectral libraries. The application of library search routines for differentiation and identification of the 14 bacteria was investigated. The spectral regions used for the library search include 1750-1560 cm$ sp{-1}$ and 1480-960 cm$ sp{-1}$, which contain bands mainly associated with proteins (secondary structure), lipids, and polysaccharides. An index produced by the library search, which indicates how much an "unknown spectrum" matches a library spectrum, is suggested as a criterion for the differentiation and identification of bacteria. A correct identification of five unknown samples shows the feasibility of using spectral library searching routines for identification of bacteria down to the species level by FTIR spectroscopy. This technique is rapid, easier, and more efficient than conventional microbiological and biochemical methods. Quantitative analysis of a mixture of Escherichia coli and Staphylococcus aureus by the partial-lest-square (PLS) technique was also evaluated. The analytical error was about 10%.
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Optimization of pre-processing variables for hyperspectral analysis of focal plane array Fourier transform infrared imagesPinchuk, Tommy. January 2006 (has links)
A genetic algorithm was employed to select the optimal combination of preprocessing variables, including data pretreatment, data manipulation and feature extraction procedures, for eventual clustering of a data set consisting of hyperspectral images acquired by a focal plane array Fourier transform infrared (FPA-FTIR) spectrometer. The data set consisted of infrared images of bacterial films, and the classification task investigated was the discrimination between Gram-positive and Gram-negative bacteria. The genetic algorithm evaluated combinations of variables pertaining to bacterial film thickness tolerances, baseline correction, pixel co-addition, outlier removal, smoothing, mean centering, normalization, derivatization, integration and principal component selection. Following numerous iterations of unsupervised processing, the genetic algorithm arrived at a sub-optimal solution yielding a clustering accuracy of 97.8% and a data utilization of 28.6%. The results provided insight into the co-dependencies of the pre-processing variables and their consequential effect on the selected data. The robustness of the classification model was evaluated and reinforced by the successful classification of two distinct validation sets. The overall success of the genetic algorithm suggests that it is an effective time saving resource for the optimization of pre-processing variables that does not require operator intervention.
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Identification of bacteria isolated from malt, with the emphasis on lactic acid bacteria and their influence on brewer's yeastBooysen, Clifford 12 1900 (has links)
Thesis (MScAgric.)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Changes in the bacterial population throughout the malting process of two barley cultivars, i.e.
Clipper (local cultivar) and Prisma (imported cultivar), malted at Southern Associated Maltsters
(SAM), Caledon, South Africa, were studied. Samples were taken from four individual runs of each
cultivar at ten different stages, i.e. dry barley before steep, water from the first steep water-stand,
barley after draining the first steep, water from the second steep water-stand, barley from the second
steep water-stand, barley after draining of the second steep, barley from the first, second and third
days of germination in the germination vessels (GV), and malt after kilning. Emphasis was placed
on the taxonomy and composition of the lactic acid bacteria (LAB) isolated from the ten different
phases.
The LAB were identified to species level by using numerical analysis of total soluble cell protein
patterns, RAPD-PCR banding patterns and 16S rRNA sequencing. The Gram-negative bacteria were
identified to genus level by using the API 20E system and included Citrobacter spp., Enterobacter
spp., Pantoea spp., Proteus spp., Seratia spp., Kluyvera spp., Klebsiella spp., Vibrio spp. and
Escherichia coli. The number of viable bacteria throughout the malting process of the two cultivars
did not differ significantly, although the LAB counts in the barley before steep and on the kilned
malt were higher in Prisma than in Clipper. Leuconostoc argentinum, Leuconostoc laetis and
Weissella confusa were the most predominant in both cultivars. A few strains of Weissella
paramesenteroides, Lactobacillus casei, Lactococcus laetis and Lactobacillus rhamnosus were also
isolated. Lb. casei and Lb. rhamnosus were not isolated from the Prisma cultivar, whilst W
paramesenteroides and Le. laetis were absent in the Clipper cultivar. Kilned malt of the Clipper
cultivar contained predominantly Le. argentinum, whereas the Prisma cultivar contained mainly Le.
lactis.
The effect of these bacteria on the fermenting ability of the brewer's yeast Saccharomyces
cerevisiae SAB 05, was also studied. Fermentations were conducted in wort prepared from Clipper
and Prisma malt. Yeast in combination with the different bacteria were used in the fermentation
studies. Wort with only yeast was used as control. Emphasis was placed on the effect the bacteria
has on the gravity, pH, yeast- and bacterial- counts and the different volatile aroma compounds produced throughout the fermentations.
The presence of LAB and Gram-negative bacteria had no effect on the yeast to reduce the gravity of
the fermenting wort, whilst the LAB caused a decrease in the pH of the fermentations in both
Clipper and Prisma wort. The cell numbers of the Gram-negative bacteria decreased throughout the
fermentations, whilst the LAB cell numbers remained constant. Comparisons could be drawn
between the volatile aroma compounds produced in the control fermentation and fermentations with
yeast and Gram-negative bacteria, yeast and Lactobacillus spp. and yeast and Weissella spp.
Leuconostoc spp. had a much greater influence on the aromatic composition of fermented malt, with
much more clear variations between Prisma and Clipper. No major differences were recorded in the
aroma profiles of Prisma and Clipper malt fermented in the presence and absence of Lactococcus
spp. The Gram-negative bacteria had no significant effect on the volatile aroma compounds
produced by the yeast, whilst the LAB had a definite effect on aroma composition in both cultivars.
The levels of four of the five principle aroma compounds, present in beer, were in the acceptable
concentration range on the fmal day of fermentation. The compounds with the highest
concentrations were iso-amyl alcohol, acetic acid and acetoin, with acetic acid being present in the
highest concentration in all the fermentations. / AFRIKAANSE OPSOMMING: Veranderinge in die bakteriese populasie van die gars kultivars, Clipper (plaaslik) en Prisma
(ingevoer), vermout by Southern Associated Maltsters (SAM), Caledon, Suid Afrika, is ondersoek.
Monsters is van vier individuele lopies van elke kultivar en tydens tien verskillende fases van die
vermoutingsproses geneem. Die tien verskillende stadia het die volgende ingesluit: Droë gars voor
benatting, water van die eerste benattingsfase, gars nadat water van die eerste benattingsfase
gedreineer is, water van die tweede benattingsfase, gars van die tweede benattingsfase, gars na die
dreinering van water in die tweede benattings fase, gars na die eerste, tweede en derde dag van
ontkieming binne die ontkiemingstenke, en mout na droging. Klem is geplaas op die taksonomie en
samestelling van melksuurbakterieë (MSB) wat tydens die tien verskillende fases geïsoleer is.
Die MSB is tot spesievlak geïdentifiseer deur gebruik te maak van numeriese analise van totale
oplosbare selproteïen bandpatrone, RAPD-PKR bandpatrone en 16S rRNA volgorde-bepaling.
Gram-negatiewe bakterieë is tot op genusvlak geïdentifiseer deur gebruik te maak van die API 20E
toetssisteem. Spesies van die genera Citrobacter, Enterobacter, Pantoea, Proteus, Seratia,
Kluyvera, Klebsiella, Vibrio asook Escherichia coli is geïdentifiseer. Tydens die vermoutingsproses
van die twee kultivars is geen beduidende verskille in die lewensvatbare bakterietellings gevind nie,
alhoewel die MSB-tellings in die gars voor benatting en mout na droging in Prisma hoër was as in
Clipper. Leuconostoc argentinum, Leuconostoc laetis en Weissella confusa het die meeste
voorgekom in beide kultivars. Kleiner hoeveelhede van Weissella paramesenteroides, Lactobacillus
casei, Lactococcus laetis en Lactobacillus rhamnosus is ook geïsoleer. Lb. casei en Lb. rhamnosus
het nie in die Prisma-kultivar voorgekom nie, terwyl W paramesenteroides en Le. laetis nie in die
Clipper-kultivar teenwoordig was nie. Le. argentinum het meestal in die gedroogde mout van die
Clipper-kultivar voorgekom, terwyl Le. laetis meestal in die Prisma-kultivar waargeneem is.
Die effek van hierdie bakterieë op die fermentasievermoë van die brouersgis Saccharomyces
cerevisiae SAB 05 is ook bestudeer. Die fermentasies is in Clipper- en Prisma- wort gedoen. Vir die
fermentasiestudies is gis in kombinasie met verskillende bakterieë gebruik. Wort met slegs gis het as
kontrole gedien. Klem is geplaas op die effek van die bakterieë op die digtheid, pH, gis- en bakterietellings
en die verskillende vlugtige komponente wat tydens die fermentasies geproduseer is. Die
teenwoordigheid van MSB en Gram-negatiewe bakterieë het geen effek gehad op die vermoë van die gis om die digtheid van die gefermenteerde wort te verlaag nie. Die MSB het wel 'n verlaging
van die pH in beide Clipper- en Prisma- wort teweeggebring. Tydens die fermentasie het die Gramnegatiewe
bakterietellings verminder, terwyl die MSB-tellings konstant gebly het. 'n Verband is
gevind tussen vlugtige komponente geproduseer in die kontrole-fermentasie en fermentasies met gis
en Gram-negatiewe bakterieë, gis en Lactobacillus spp. en gis en Weissella spp. Leuconostoc spp.
het groter veskille in die samestelling van die gefermenteerde wort teweeg gebring met duidelike
verskille tussen Clipper en Prisma. Die teenwoordigheid van Lactococcus spp. het nie groot
verskille in die samestelling van die gefermenteerde wort getoon nie. Op die laaste dag van die
fermentasies was die vlakke van vier uit die vyfbelangrikste vlugtige aroma komponente wat in bier
voorkom in die kontrole fermentasies in aanvaarbare konsentrasies teenwoordig. Die Gramnegatiewe
bakterieë het geen beduidende invloed gehad op die vlugtige aroma komponente wat deur
die gis geproduseer is nie, terwyl die MSB 'n besliste effek in die aroma-samestelling van beide die
kultivars gehad het. Die komponente met die hoogste konsentrasies was, isoamiel-alkohol, asynsuur
en asetoin. Asynsuur was in al die fermentasies in die hoogste konsentrasie teenwoordig.
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Identificação de bactérias láticas deteriorantes e avaliação do efeito inibitório de nisina e pediocina em presunto cozido fatiado embalado a vácuoKalschne, Daneysa Lahis 02 April 2013 (has links)
Dissertação composta por 2 artigos. / INTRODUÇÃO E OBJETIVOS ─ Nos produtos cárneos industrializados cozidos a maior parte da microbiota é inativada pela aplicação de calor em processo que simula uma pasteurização. Esses produtos também recebem aditivos em sua composição que geram efeito inibidor no crescimento de determinados grupos de microrganismos. Com a microbiota patogênica reduzida a níveis aceitáveis, em amostras embaladas a vácuo ou em atmosfera modificada, criam-se condições para o crescimento de bactérias ácido láticas (BAL), descritas como microrganismos Gram positivos, não formadores de esporos, catalase negativa, desprovidos de citocromos, anaeróbios facultativos, fastidiosos, mesofílicos, ácidos tolerante, com metabolismo estritamente fermentativo, que produzem ácido láctico como o principal produto final da fermentação de carboidratos. O desenvolvimento desses microrganismos gera aroma, sabor e aspecto indesejável nos produtos cárneos. Tendo em vista estas considerações, o objetivo geral desse trabalho foi avaliar a possibilidade da inibição do crescimento de BAL em amostras de presunto fatiado embalado a vácuo, pela aplicação de bacteriocinas. Os objetivos específicos incluíram: a quantificação e identificação das espécies de BAL predominantes nas amostras de presunto cozido fatiado embalado a vácuo; o estudo dos ingredientes que compõem a formulação do presunto cozido combinados com nisina e pediocina, realizados em caldo Man Rogosa e Sharp (MRS) pela aplicação da estratégia de planejamento experimental aliada à microbiologia preditiva na avaliação do crescimento do Lactobacillus sakei; a validação dos resultados obtidos nos caldos MRS na matriz alimentar a partir da elaboração de três formulações envolvendo a utilização da nisina e uma formulação controle, submetidas a análises físico-químicas e microbiológicas durante a vida de prateleira do produto. MÉTODOS ─ Inicialmente foi realizada a quantificação das BAL totais presentes, para 4 lotes semanais de presunto cozido fatiado embalado a vácuo, nos tempos de 1 e 45 dias de armazenamento a 4 C e 8 C. Paralelamente foi realizada a identificação fenotípica dos gêneros presentes; adicionalmente, no tempo de 45 dias foram realizadas análises moleculares do gene 16S rRNA para a identificação das espécies predominantes, por meio da qual selecionou-se a espécie Lactobacillus sakei. Posteriormente, utilizou-se uma estratégia sequencial de planejamento experimental, que objetivou estudar a inibição do crescimento celular da espécie Lactobacillus sakei em caldo MRS, aplicando-se um Planejamento Fatorial Fracionário 26-2 com as variáveis cloreto de sódio, tripolifosfato de sódio, lactato de sódio, eritorbato de sódio e as bacteriocinas nisina e pediocina. Adicionalmente foi realizado um Delineamento Composto Central Rotacional (DCCR) 22 objetivando otimizar a aplicação de nisina e pediocina por meio da redução das concentrações aplicadas que apresentassem eficiência semelhante, considerando os altos custos desses ingredientes. As respostas estudadas em ambos delineamentos experimentais foram os parâmetros preditos pelos ajuste das curvas de crescimento ao Modelo de Gompertz Modificado, a citar a população máxima atingida (A), velocidade específica máxima de crescimento () e a duração da fase lag (). Para a validação dos resultados obtidos na etapa de estudo em caldo MRS, foram elaboradas três formulações teste (com diferentes concentrações de nisina) e uma controle (sem adição de nisina) para o acompanhamento da vida de prateleira do produto por meio de análises físico-químicas e microbiológicas. PRINCIPAIS RESULTADOS ─ Foram observadas contagens médias de 1,98 log UFC.g-1 no tempo 1 dia; 7,59 log UFC.g-1 a 4 C e 8,25 log UFC.g-1 a 8 C no tempo 45 dias para a contagem total de BAL. As espécies predominantemente identificadas no tempo de 45 dias foram o Lactobacillus curvatus e o Lactobacillus sakei. Na etapa de estudo em caldo MRS, os resultados do Planejamento Fatorial Fracionário evidenciaram que o tripolifosfato de sódio, a nisina e a pediocina, foram as variáveis com efeito estatisticamente significativo (p ≤ 0,05) na inibição do crescimento do Lactobacillus sakei. No Delineamento Composto Central Rotacional, a nisina foi a variável que apresentou efeito significativo na inibição do crescimento do Lactobacillus sakei, indicando a possibilidade de redução na concentração aplicada para níveis inferiores ao indicado pela legislação brasileira (12,5 mg/Kg), com efeito semelhante na inibição. A validação dos estudos em caldo MRS pela aplicação das quantidades reduzidas de nisina (indicadas pelo DCCR) na matriz alimentar, mostrou que a quantidade mínima aplicada (0,001%) apresentou um efeito semelhante na inibição das BAL em relação à quantidade máxima aplicada (0,013%); sendo em ambos os casos eficiente quando comparada à amostra controle. DISCUSSÃO E CONCLUSÃO ─ De acordo com os resultados obtidos, verificou-se que os principais gêneros de BAL envolvidos na deterioração das amostras de presunto cozido fatiado embalado a vácuo, armazenadas durante 45 dias a 4 C e 8 C, foram o Lactobacillus curvatus e Lactobacillus sakei. A contagem de BAL totais após 45 dias mostrou-se significativamente influenciada pela temperatura de armazenamento, pois as contagens foram maiores para as amostras armazenadas a 8 C quando comparadas às armazenadas a 4 C. Dentre os ingredientes comumente adicionados na formulação do presunto cozido, o tripolifosfato de sódio mostrou efeito significativo na inibição do Lactobacillus sakei nos estudos em caldo MRS. De forma semelhante, a nisina e a pediocina mostraram efeito significativo na redução das contagens do microrganismo estudado quando avaliados pela aplicação de um Planejamento Fatorial Fracionário. No Delineamento Composto Central Rotacional envolvendo as variáveis nisina e pediocina com faixas de estudos redefinidas, a nisina apresentou efeito significativo na inibição do Lactobacillus sakei, quando aplicada em concentrações inferiores ao estipulado pela legislação brasileira. Os resultados obtidos no Delineamento Composto Central Rotacional foram validados pela aplicação das quantidades otimizadas de nisina na matriz alimentar, que mesmo na concentração mínima (0,001%) mostrou um efeito similar ao obtido pela aplicação da concentração máxima (0,013%). A contagem de BAL e bactérias aeróbias mesófilas (BAM) totais mostrou-se inferior para as amostras contendo nisina quando comparada com a amostra controle, em torno de 2 a 3 ciclos logarítmicos para as BAL, e 3 ou 4 ciclos logarítmicos para as BAM; em relação ao pH, o mesmo manteve-se praticamente inalterado até o final da vida de prateleira (60 dias), enquanto o pH da amostra controle diminuiu de forma significativa. De um modo geral constatou-se que o presunto cozido fatiado embalado a vácuo é um produto com condições favoráveis ao desenvolvimento de BAL deteriorantes, a citar as espécies Lactobacillus curvatus e Lactobacillus sakei, e que dentre as bacteriocinas estudadas, a nisina apresentou efeito na inibição do Lactobacillus sakei nos estudos em caldo MRS e também na inibição das BAL deteriorantes totais no presunto cozido fatiado embalado a vácuo, mesmo em concentrações inferiores ao indicado pela legislação brasileira. / INTRODUCTION AND AIMS ─ In processed cooked meat products most of the microbiota is inactivated by heat application process which simulate a pasteurization. These products also receive additives in composition that generate inhibitory effect on the certain microorganisms groups growth. With the reduce of pathogenic microorganisms on vacuum packed or modified atmosphere samples, conditions are created for the lactic acid bacteria (LAB) growth, as described Gram positive, non-spore forming, catalase negative, devoid of cytochromes, facultative anaerobic, fastidious, mesophilic, acid tolerant, strictly fermentative metabolism that produce lactic acid as the major end product of fermentation of carbohydrates. The development of these microorganisms causes aroma, flavour and appearance undesirable in meat products. In view of these considerations, the aim of this work was to evaluate the LAB inhibition growth in samples of sliced vacuum-packed cooked ham, by the application of bacteriocins. Specific aims included: the quantification and identification of species of predominate LAB in the samples of sliced vacuum-packed cooked ham; the study of the ingredients of the formulation of cooked ham combined with nisin and pediocin conducted in Man Rogosa and Sharp (MRS) broth using a strategy of experimental design combined with predictive microbiology on evaluation of Lactobacillus sakei growth; and a validation of results obtained in MRS broths on food matrix by the elaboration of three formulations involving the use of nisin and a control formulation, which were submitted for microbiological and physico-chemical analysis during the shelf life of the product. METHODS ─ Initially, the quantification of total LAB present was performed for 4 weekly batches of sliced vacuum-packed cooked ham at time 1 and 45 days of shelf life storage at 4 C and 8 C. Simultaneously was performed phenotypic identification of the genera present; additionally, at the time of 45 days of storage, was carried out a molecular analyze of 16S rRNA gene for identification of the predominant species, whereby selected the specie Lactobacillus sakei. Subsequently, a strategy of sequential experimental design was performed to evaluate the Lactobacillus sakei cell growth inhibition in MRS broth, applying a Fractional Factorial Design 26-2 with the variables sodium chloride, sodium tripolyphosphate, sodium lactate, sodium erythorbate and the bacteriocins nisin and pediocin. The statistically significant variables in relation to Lactobacillus sakei growth inhibition were selected and a Central Composite Rotatable Design (CCRD) 22 was performed to optimize the application of nisin and pediocin by reducing the concentration applied to present similar efficiency, considering the high cost of these ingredients. The responses studied in both experimental design were the parameters logarithmic increase of population (A), maximum specific growth rate (µ) and lag phase duration (), obtained by the adjustment of Modified Gompertz predictive model. For validation of the results obtained in MRS broth, three test formulations were prepared (with different concentrations of nisin) and a control (without addition of nisin) for monitoring the shelf life of the product by physico-chemical and microbiological analyzes. MAIN RESULTS ─ Mean counts around log 1.98 CFU.g-1 at time 1 day, log 7.59 CFU.g-1 at 4C, and log 8.25 CFU.g-1 at 8C on time 45 days, was detected for the total LAB. The species predominantly identified on time 45 days were Lactobacillus curvatus and Lactobacillus sakei. In step of study in MRS broth, the results of the Fractional Factorial Design showed the sodium tripolyphosphate, nisin and pediocin like variables with statistically significant effects in Lactobacillus sakei growth inhibition. In the Central Composite Rotatable Design, nisin was the variable that had a significant effect in the Lactobacillus sakei growth inhibition, indicating that it may be reduced to smaller amounts than indicated by Brazilian law (12.5 mg/Kg) with similar effect on inhibition. The validation of MRS broth studies for applying the reduced amounts of nisin (indicated by the CCRD) in the food matrix, showed that the minimum amount applied (0.001%) presented a similar effect on LAB growth inhibition related to the maximum quantity applied (0.013%); being efficient in both cases when compared to the control sample. DISCUSSION AND CONCLUSION ─ From the results obtained, was possible to conclude that the LAB comprise the microbiota of sliced vacuum-packed cooked ham and the Lactobacillus curvatus and Lactobacillus sakei were the major species that cause deterioration on the samples stored at 4 C and 8 C for 45 days. Total LAB counts after 45 days was significantly affected by storage temperature, because the counts were higher for samples stored at 8 C compared to the samples stored at 4 C. Among the ingredients added in the formulation of cooked ham, sodium tripolyphosphate showed significant effect in Lactobacillus sakei inhibition in MRS broth. Similarly, nisin and pediocin showed significant effects on microorganism reducing, evaluated by applying a Fractional Factorial Design. The Central Composite Rotatable Design involving the variables nisin and pediocin with redefined ranges of concentrations showed that nisin presented significant effect on the Lactobacillus sakei growth inhibition, when applied in smaller amounts in comparison to the stipulated by Brazilian law. The results obtained in Central Composite Rotatable Design was validated by applying the optimized quantities of nisin in food matrix, that even when applying minimal concentration (0.001%) showed a similar effect to that obtained by applying the maximum concentration (0.013%). The count of LAB and mesophilic aerobic bacteria (MAB) total were lower for the samples containing nisin than control sample, approximately 2 to 3 log cycles for LAB, and 3 or 4 log cycles for MAB, while the pH was maintained until the end of shelf life (60 days); at the same time the pH of the control sample had a significant decrease. Altogether the sliced vacuum-packed cooked ham is a favorable product to the LAB spoilage development, principally to the species Lactobacillus curvatus and Lactobacillus sakei, and that among the studied bacteriocins against the inhibition of LAB, nisin showed effect on the Lactobacillus sakei growth inhibition by studies in MRS broth, and also on the LAB spoilage growth inhibition in sliced vacuum-packed cooked ham, even in smaller quantities than indicated by Brazilian law. / 5000
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