Дисертації з теми "Spoilage microorganisms"

Thesis (MScAgric (Viticulture and Oenology))--Stellenbosch University, 2008.
The production of good quality wine is essential to ensure competitiveness on an international level. Wine quality is usually evaluated for the visual, olfactory and taste characteristics of that specific wine. The winemaking process starts with the grapes in the vineyard followed by oenological practises in the winery until the final wine is bottled. Factors that could influence wine quality include the grape quality from which the wine is made and different techniques used during wine production. Other factors include the presence as well as the interaction between microorganisms found in the grape juice and wine, and the biochemical effect these microorganisms have on certain chemical compounds in the wine. The different microorganisms found in grape juice and wine can either have a negative or positive contribution to the final quality of the wine. During certain stages of the winemaking process the growth and metabolic activity of certain microorganisms is a necessity to produce good wine. During other stages the presence of certain microorganisms can lead to the development of compounds that is regarded as off-flavours and therefore lead to unpalatable wines of low quality. Yeast strains that naturally present on the grapes and in the winery can also contribute to the final quality of the wine. Brettanomyces yeasts are part of the natural flora of winemaking and can drastically influence the aroma characters of a wine through the production of volatile phenols. The general aroma descriptions of volatile phenols include "smoky", "spicy", "barnyard", "animal" and "medicinal". Although some wine drinkers believe that these characters can add to the complexity of a wine, high levels of volatile phenols is mostly regarded as off-flavours and mask the natural fruity flavours of a wine. With this study we wanted to generate a better understanding of the effect of different winemaking practises on the production of volatile phenols by B. bruxellensis. We evaluated the difference in volatile phenol production when B. bruxellensis was introduced before or after alcoholic fermentation. We have shown that B. bruxellensis could grow and produce volatile phenols during alcoholic fermentation. Results obtained also showed that commercial wine yeast strains could produce the vinyl derivatives that serve as precursors for Brettanomyces yeast to produce the ethyl derivatives. The commercial yeast strains differed in their ability to produce vinyl derivatives. Different malolactic fermentation scenarios were evaluated, namely spontaneous versus inoculated, and with or without yeast lees. Results showed that spontaneous malolactic fermentation had higher volatile phenol levels in the wine than inoculated malolactic fermentation. The treatment with lees reduced the level of volatile phenols, probably due to absorption by yeast cells. The presence of the phenyl acrylic decarboxylase (PAD1) gene and the production of volatile phenols by S. cerevisiae commercial yeast strains were evaluated in Shiraz grape juice and in synthetic grape juice. The results indicated that the yeast strains differ in their ability to produce 4-vinylphenol and 4-vinylguaiacol. All the yeast strains tested had the PAD1 gene. We also evaluated the presence of the phenolic acid decarboxylase (padA) gene and the ability of different lactic acid bacteria strains to produce volatile phenols in synthetic wine media. Although some of these strains tested positive for the phenolic acid decarboxylase gene most of them only produced very low levels of volatile phenols. This study made a valuable contribution on the knowledge about the effect of Brettanomyces yeast on the volatile phenol content of red wines during different stages of the winemaking process and when applying different winemaking practices. It also showed the effect between Brettanomyces yeast and other wine microorganisms and the possible influence it could have on the final quality of wine. Research such as this can therefore aid the winemaker in making certain decisions when trying to manage Brettanomyces yeast spoilage of wines.

La present tesi doctoral es centra en l'aplicació dels bacteris de l'àcid lactic (BAL) com a agents bioprotectors davant microorganismes patògens i deteriorants.Es van aïllar i seleccionar BAL de fruites i hortalisses fresques i es van assajar in vitro davant 5 microorganismes fitopatògens i 5 patògens humans.Es van realitzar assajos d'eficàcia en pomes Golden Delicious amb tots els aïllats enfront les infeccions causades pel fong Penicillium expansum. La soca més eficaç era Weissella cibaria TM128, que reduïa el diàmetre de les infeccions en un 50%.Les soques seleccionades es van assajar enfront els patògens Salmonella typhimurium, Escherichia coli i Listeria monocytogenes en enciams Iceberg i pomes Golden Delicious.Els BAL interferien eficientment amb el creixemet de S. typhimurium, and L. monocytogenes, però van mostrar poc efecte enfront E. coli.Finalment, es van realitzar assajos dosi-resposta amb les soques Leuconostoc mesenteroides CM135, CM160 and PM249 enfront L. monocytogenes. De totes les soques assajades, la soca CM160 va ser la més efectiva.
The present thesis focuses on the use of lactic acid bacteria as bioprotective cultures to inhibit pathogenic and spoilage microorganisms.
Lactic acid bacteria were isolated and selected from fresh fruit and vegetables and tested in vitro against five plant pathogens and five human pathogen test bacteria.Efficacy trials with all the isolates were performed in Golden Delicious apples against the blue mould rot infections, caused by Penicillium expansum. The highest effectivity found in this assay was of about 50%, with strain Weissella cibaria TM128.Selected lactic acid bacteria were tested against Salmonella typhimurium, Escherichia coli and Listeria monocytogenes in Iceberg lettuce and Golden Delicious apples. Lactic acid bacteria interfered efficiently with the growth of S. typhimurium, and L. monocytogenes, but showed little effectivity over E. coli.Finally, dose-response assays were done with Leuconostoc mesenteroides strains CM135, CM160 and PM249 against L. monocytogenes.Among the three strains tested, strain CM160 showed the highest effectivity.

The control of microbial contamination in the food industry is critical, as contamination of food produce, surfaces and equipment can lead to acquisition of foodborne infections. Microbial contamination can also result in food spoilage, which can cause both product and financial loss. Consequently novel decontamination technologies are being sought to help reduce contamination. Initial investigations examined the efficacy of 405 nm light for inactivation of a range of common foodborne microorganisms, both in suspension and on agar surfaces. All exposed populations were significantly reduced following 405 nm light exposure. The hypothesised inactivation mechanism involves photoexcitation of endogenous porphyrin molecules within the microorganisms, resulting in production of reactive oxygen species, oxidative cell damage and microbial inactivation. This theory was investigated by exposing fungi to 405 nm light under both aerobic and anaerobic conditions. Results displayed significant reduction in inactivation rates under oxygen depleted conditions, highlighting the critical role of oxygen during 405 nm light inactivation. This study also demonstrated inactivation of bacterial contamination and biofilms on a range of surfaces, demonstrating potential environmental decontamination applications. Further work highlighted the enhanced bacterial inactivation efficacy of 405 nm light when bacteria were exposed under sub-lethal environmental conditions, typical of those present in the food processing industry. Further studies also demonstrated the synergistic effect of TiO2 with 405 nm light, thereby enabling significantly enhanced bacterial inactivation rates. Studies also investigated potential applications for food decontamination and preservation, with preliminary results highlighting successful prevention of spoilage on a range of food products and significant decontamination of E. coli on fresh fruit. This study has confirmed the microbicidal efficacy of 405 nm light, whilst demonstrating a range of potential applications for use within the food industry for improved environmental decontamination. In conclusion 405 nm light has potential to be used safely and effectively as an additional decontamination technology in the food industry.

Thesis (MTech (Food Technology))--Cape Peninsula University of Technology, 2015.
Rooibos iced tea (RIT), as one of the products of Rooibos is fast becoming very popular as a beverage in society due to the benefits of the phenolic compounds that are associated with this herbal tea. Some of the commercially available products have been found to contain, if any, lower contents of the major phenolic compounds, namely aspalathin and its oxidation products, iso-orientin and orientin. Their presence is considered as indicators of a good quality product. The purpose of this study was to investigate the effect of ultraviolet-C (UV-C) light as an alternative treatment to heat treatment on the shelf life, pH, phenolic composition, colour and microorganisms associated with Rooibos. Two formulations of RIT were used in order to determine the efficacy of the UV-C on the shelf life whilst three formulations were used for the physicochemical analysis. Only one formulation was used for inoculation with three spoilage bacteria, yeast and mould spoilage microorganisms namely; Escherichia coli K12, Staphylococcus aureus, Salmonella sp., Saccharomyces cerevisiae and Cladosporium sp. The UV-C dosages of 0, 918, 1 836, 2 754 and 3 672 J.l -1 were used to treat the RIT using a pilot-scale UV-C system with a turbulent flow at a constant flow rate of 4000 l.hr-1 . A log count of 4 log10 was considered the limit for the spoilage growth since it is the average log10 afternormal pasteurisation. The use of UV-C treatment was found to have significantly (p1) effect on the overall colour difference of the RIT in formulations A, B, and C. All the spoilage microorganisms were significantly reduced by UV-C dosage to less than 4 log10 except the Cladosporium sp. The S. cerevisiae was the most sensitive microorganism whilst Cladosporium sp. was the most resistant. The effect of UV-C on the spoilage microorganism followed the sequence: S. cerevisiae>Salmonella sp.>S. aureus>E. coli K12>Cladosporium sp. This study indicated that microbiological reduction was achieved as a function of increasing UV-C dosage. In order to achieve the highest log10 reduction, the highest UV-C dosage of 3 672 J.l-1 may be used. However, the dosage may need to be increased in order to achieve the desired results in the treatment of Cladosporium sp. It can thus be concluded from the above investigations that UV-C dosage treatment of 3 672 J.l-1 is optimum in the non-thermal treatment of RIT
South African Association for Food Science & Technology Cape Peninsula University of Technology Bursary

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2008.
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Durante a produção, armazenamento, transporte e embalagem de produtos alimentícios, a presença de microrganismos é inevitável. A carne de frango, especialmente, é um alimento altamente perecível e, juntamente com outros tipos de carnes provenientes de ave, apresenta grande variedade de bactérias patogênicas e deteriorantes quando comparada com outros alimentos. Assim, a avaliação do crescimento microbiano e o controle da temperatura de armazenamento são importantes para garantir a segurança e vida útil dos alimentos. O trabalho objetivou caracterizar microbianamente três peitos de frango (cru, temperado com NaCl e cozido), produzidos no Brasil e exportados para a Europa, bem como, estimar a vida útil de tais produtos após o descongelamento, quando armazenados em diferentes condições de temperatura. Foi simulada a cadeia de abastecimento dos peitos de frango congelados, durante 20 dias a -18 ± 0,5 C para simulação do transporte destes produtos por navio até a Europa, desde sua expedição no Brasil e, após descongelamento, durante 21 dias a 4 ± 0,5 C para simulação da vida útil em gôndola de supermercado. Os produtos foram analisados quanto a Pseudomonas spp., Salmonella spp., Listeria monocytogenes, Staphylococcus spp. e microrganismos viáveis totais (mesófilos e psicrotróficos). Em termos de contagens de número de colônias viáveis totais, durante os primeiros 20 dias (a -18°C), a presença de microrganismos se manteve estável em baixos níveis de detecção. Após o descongelamento foram observadas as curvas de crescimento dos microrganismos, onde as fases lag ocorreram durante os primeiros 6 dias. A máxima concentração microbiana foi atingida após 15-18 dias, dependendo do produto. Em nenhuma das amostras foi detectada a presença de Salmonella spp. e Listeria monocytogenes. Após este estudo inicial foi avaliado o tempo de vida útil dos três produtos em diferentes condições de temperatura de armazenamento (2, 4, 7, 10, 15 e 20ºC). O estudo foi baseado na determinação de microrganismos aeróbios psicrotróficos, Pseudomonas spp., aeróbios mesófilos e Staphylococcus spp. O aumento da temperatura fez reduzir a vida útil dos três produtos estudados, em relação a todos os microrganismos. De modo geral, os produtos apresentaram faixas de vida útil de 10 até mais de 26 dias, a 2ºC; de 9 até mais de 21 dias, a 4ºC; de 6 até 12 dias, a 7ºC; de 4 até 8 dias, a 10ºC; de 2 até 4 dias, a 15ºC; e de 1 a 2 dias, a 20ºC. Quando armazenadas em temperaturas de refrigeração (2, 4 e 7°C), as amostras apresentaram pouca variação no tempo de vida útil, especialmente a 2 e 4°C. Já quando armazenadas à temperatura ambiente (temperaturas iguais ou superiores a 10ºC), a cada 5ºC de elevação na temperatura de armazenamento, a vida útil reduziu-se à metade do tempo.
During production, storage, transport and packaging of food products, the presence of microorganisms is unavoidable. The chicken meat, especially, is a highly perishable food and, together with other types from poultry meat presents great variety of pathogenic bacteria and spoilage when compared with other foods. Like this, the evaluation of the microbial growth and the control of the storage temperature are important to guarantee the safety and shelf life of the foods. The work aimed to characterize microbially, three chicken breast (raw, tempered with NaCl and cooked), produced in Brazil and exported to Europe, as well as, to estimate the shelf life of such products after thawing, when stored in different temperature conditions. The supply chain of frozen chicken breast was simulated for 20 days at -18±0,5oC for simulation of the transport of these products by ship to Europe, from its expedition in Brazil and, after thawed, during 21 days at 4 ± 0,5oC for simulation of the supermarket shelf life. Pseudomonas spp., Salmonella spp., Listeria monocytogenes, Staphylococcus spp. and total viable microorganisms (mesophilic and psicrotrophic) were analyzed in the products. In terms of accountings of total viable colonies number, during the first 20 days (-18°C), the presence of microorganisms was stable at low levels of detection. After thawing the microorganism growth curves showed that the lag phases happened during the first 6 days. The highest microbial concentration was reached after 15-18 days, according to the product. In none of the samples the presence of Salmonella spp. and Listeria monocytogenes were detected. After this initial study it was evaluated the shelf life time of the three products in different conditions of storage temperature (2, 4, 7, 10, 15 and 20ºC). The study was based on the determination of aerobic psicrotrophic, Pseudomonas spp., aerobics mesophilic and Staphylococcus spp. The increase of temperature reduce the shelf life of the three studied products, in relation to all of the microorganisms. In general, the products presented shelf life ranges from 10 to more than 26 days, at 2ºC; from 9 to more than 21 days, at 4ºC; from 6 to 12 days, at 7ºC; from 4 to 8 days, at 10ºC; of 2 to 4 days, at 15ºC; and, from 1 to 2 days at 20ºC. When stored in refrigerating temperature (2, 4 and 7°C), the samples presented a few variation in the shelf life time, especially at 2 and 4°C. But when stored to room temperature (at 10ºC or higher temperature), to each 5ºC of elevation in the storage temperature, the shelf life was reduced to half time.

Seafood permits the transmission of many bacterial pathogens. In order to reconcile consumer demands with important safety standards, traditional means of regulating microbial spoilage and safety hazards in foods are combined with novel technologies. These include biological antimicrobial systems, such as the use of lactic acid bacteria (LAB) and/or their bacteriocins, such as Carnobacterium maltaromaticum CS526 and its bacteriocin piscicocin CS526. The aims of this study were to investigate the presence of Listeria monocytogenes in temperate seafood, namely fresh and smoked salmon, fresh and smoked haddock, and fresh mussels and oysters. Additionally, there was an aim to recover, characterise and use bacteriocin-like-substance to control Listeria monocytogenes in cold smoked haddock. Vibrio spp., Enterobacteriaceae representatives, total aerobic heterotrophic counts and Listeria monocytogenes were isolated from commercially prepared smoked and fresh Atlantic salmon, smoked and fresh haddock, live mussels and oysters using selective media and tryptone soya agar (TSA). Vibrio spp. occurred in high densities (>106 CFU gˉ1) in mussels and Enterobacteriaceae representatives were recorded at >106 CFU gˉ1 in fresh salmon. Total aerobic heterotrophic counts in fresh salmon, live mussels and oysters reached 107, > 107, and > 106 CFU gˉ1, respectively. Listeria monocytogenes was recorded at 5.0 x 104 CFU gˉ1 in mussels. In total sixty one bacterial isolates were recovered from the seafood examined. The results revealed 19 genera of bacteria, i.e. Acinetobacter, Aerococcus, Aeromonas, Bacillus, Brochothrix, Carnobacterium, Citrobacter, Corynebacterium, Enterobacter, Escherichia coli, Moraxella, Micrococcus, Pseudomonas, Psychrobacter, Serratia, Shewanella, Staphylococcus, Vibrio and Listeria. The prominent characteristics of fish spoilage isolates were demonstrated by the ability of the isolates to reduce trimethylamine oxide (TMAO) to trimethylamine, and to produce H₂S. Sh. baltica OS185, Aeromonas spp. HB-6, Sh. baltica, Sh. putrefaciens, A. hydrophila HX201006-3, A. salmonicida subsp. achromogenes, A. hydrophila, C. freundii, Enterobacter cloacae were strong producers of TMA and H₂S. The spoilage microorganisms were tested for potential pathogenicity. The result revealed that 6/15 of the spoilage microorganisms produced proteolytic, lecithinase, blood (β and α haemolysin) and elastinase activity, respectively, whereas 7/15 of the spoilage microorganisms showed lipolytic activity. Cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances of Carnobacterium maltaromaticum MMF-32 and KOPRI 25789 producing strains isolated from commercially prepared smoked salmon were investigated for their potential antimicrobial activity against potentially pathogenic and food spoilage microorganisms. Generally, a broad spectrum of activity was revealed against potentially pathogenic and food spoilage microorganisms in vitro. Cold-smoked haddock treated with bacteriocin producing C. maltaromaticum MMF-32, C. piscicola A9b bacˉ phenotype nonbacteriocin producing strain a mutant of C. piscicola A9b bac+, cell free supernatants, ammonium sulphate precipitated supernatants and semi-purified bacteriocin-like substances was challenged with L. monocytogenes ATCC 19114 up to 103 CFU gˉ1, respectively. Samples were stored at 4 °C for 10 days. L. monocytogenes and total bacterial counts were determined along with changes in total volatile base nitrogen (TVBN) and biogenic amines production as well as texture, colour and odour. Although the study on anti-listerial effects of C. maltaromaticum MMF-32 was not successful, this organism did have a positive effect on retention of firmness and sensory perception in cold smoked haddock.

A utilização de embalagens biodegradáveis, tais como os filmes e coberturas comestíveis, apresenta-se como alternativa ao uso de recursos não-renováveis como material de embalagem. A incorporação de substâncias antimicrobianas em embalagens tem como objetivo minimizar o problema da contaminação microbiana em alimentos e, entre elas, os óleos essenciais (OE) têm recebido atenção especial por serem substâncias naturais e atenderem à preferência dos consumidores. Porém, a utilização de OE como um agente antimicrobiano natural é limitada por critérios organolépticos, sendo necessário determinar a concentração mínima necessária para inibir o desenvolvimento de microrganismos sem afetar sensorialmente as características do alimento. Assim, os objetivos desta pesquisa foram: desenvolver um filme comestível à base de alginato com incorporação de agentes antimicrobianos naturais e avaliar a adição de diferentes concentrações de cloreto de cálcio (CaCl2) como agente crosslinking na formulação do filme e na etapa complementar de formação do filme; caracterizar o filme frente às propriedades mecânicas e propriedades de barreira; determinar a concentração mínima inibitória (CIM) de óleos essenciais para Pseudomonas spp., Salmonella spp. e Listeria monocytogenes presentes em carne de frango, e verificar a aceitação pelo consumidor, através da análise sensorial (aroma), de pedaços de peito de frango in natura embalado com o filme antimicrobiano O OE de cravo foi o que se apresentou mais eficiente para os microrganismos testados com CIM de 0,2% sendo este o limite mínimo estudado no planejamento experimental para o desenvolvimento do filme antimicrobiano. As variáveis independentes neste planejamento foram: CaCl2 na faixa de concentração de 0,02 a 0,1% e OE cravo na faixa de 0,2 a 1,0%. Valores acima de 0,0316% de CaCl2, independente da concentração de OE estudada, diminuiram a zona de inibição do crescimento microbiano em testes realizados in vitro, possivelmente devido a formação de um gel muito forte que pode ter dificultado a incorporação da emulsão de OE na matriz polimérica dos filmes. Os resultados de permeabilidade ao vapor de água mostraram que a adição de CaCl2 à formulação dos filmes diminuiu a permeabilidade enquanto a adição de OE cravo foi responsável pelo aumento dessa propriedade. Com relação às propriedades mecânicas, tanto a adição de CaCl2 como a de OE cravo à formulação dos filmes aumentou a resistência máxima à tração. Porém, com relação ao alongamento máximo na ruptura, valores menores foram obtidos com a adição de CaCl2, enquanto maiores valores foram encontrados com a adição de OE cravo à formulação dos filmes. A avaliação da atividade antimicrobiana dos filmes em carne de frango foi realizada somente com a formulação que apresentou os maiores valores de zona de inibição in vitro (CaCl2=0,0316% e OE cravo=0,884%). Após 5 dias de armazenagem a 7º C, observou-se que a utilização do filme adicionado de OE de cravo como embalagem primária em amostras de carne de peito de frango promoveu o controle da multiplicação de L. monocytogenes o mesmo não ocorrendo para as populações de Salmonella spp. e Pseudomonas spp. A análise sensorial relacionada ao aroma da carne de peito de frango mostrou que o uso do filme à base de alginato incorporado de OE cravo é viável. Porém, este filme poderá sofrer interferência da matriz alimentar caso esta matriz apresente exsudação.
The use of biodegradable packaging such as edible films and coatings are an alternative to the use of non-recyclable packaging. The incorporation of antimicrobial substances in packaging aims at reducing food microbial contamination among which, essential oils (EO) have received special attention being natural and attending consumer demand. However, the use of EO as a natural antimicrobial agent is limited by organoleptic criteria making it necessary to determine the minimum concentration to inhibit the multiplication of microorganisms without affecting the sensory characteristics of the food. Therefore, the aims of this research were: to develop an alginate based edible film with natural antimicrobial agents, evaluating the addition of different concentrations of calcium chloride as a crosslinking agent in the formulation of the film and in the complementary stage; to characterize the mechanical properties and barrier properties; to determine the minimum inhibitory concentration (MIC) of EO for Pseudomonas spp, Salmonella spp and Listeria monocytogenes found in chicken meat and to verify consumer acceptance of the product through sensorial analysis (aroma). Among the studied EO, the concentration of 0.2% of clove oil was effective in inhibiting the microorganisms tested, this concentration being the minimum limit used in the experimental design for film development. The independent variables studied in this design were calcium chloride in the range of 0.02 to 0.01% and clove EO in the range of 0.2 to 1.0%. Concentrations of CaCl2 above 0.0316%, independent of the EO concentration, reduced the inhibition zone of microbial growth in in vitro tests, possibly due to the formation of a very strong gel which could have made the incorporation of the EO emulsion in the polymeric matrix of the film very difficult. The results of water vapor permeability tests showed that the addition of CaCl2 to the formulation of the films reduced the permeability while the addition of clove EO increased this property. Regarding to mechanical properties, the addition of CaCl2 as well as clove EO to the film formulation increased the values of tensile strength. On the other hand, relating to elongation at the break, smaller values were obtained with the addition of the salt while the addition of EO provided higher values. The evaluation of antimicrobial activity of the films in chicken meat was performed only with the formulation that showed the highest inhibition values presented in vitro (CaCl2=0.0316% and clove EO=0.0884%). After five days of storage at 7° C, it was observed that the use of the film added by clove EO as primary packaging provided the control of L. monocytogenes growth in samples of chicken meat but not of Salmonella spp and Pseudomonas spp. The sensorial analysis - aroma - showed that the use of alginate based film incorporated with clove EO is viable in food. However, when the food matrix presents exudation, it can interfere in this film.

Mestrado em Biotecnologia - Biotecnologia Alimentar
Hyperbaric storage (HS) is a preservation methodology of food products in which pressure is used as a determining factor in spoilage inhibition. With this new preservation methodology significant energy saving might be achieved, namely when the storage occurs at room temperature (RT). As such, the objective of this study focused on the evaluation of HS as an alternative to refrigeration for sliced cooked ham and minced pork meat preservation by using different combinations of pressures (0.1-150 MPa), temperatures (4-37 ºC) and storage times (4-24 h). In general, it was observed an increase of the microbial counts of at least 1 Log CFU/g for both sliced cooked ham and minced pork meat stored at RT and 0.1 MPa whereas under refrigeration the counts remained equal or slightly higher than before storage. On the other hand, the samples stored under HS conditions presented equal or lower counts than the initial samples, regardless of the storage temperature employed. Nevertheless, a storage pressure of at least 50 MPa is required in order to inhibit microbial growth similarly to refrigeration. In the case of sliced cooked ham, no significant differences were observed between the different storage conditions and the initial samples concerning physicochemical parameters analysed (pH, water holding capacity, lipid oxidation and colour) whereas for minced pork meat HS inhibited lipid oxidation when compared to the storage at 0.1 MPa at the same temperature. Therefore, HS shows to be effective in preventing meat products ham spoilage, by microbial growth inhibition, as or more efficiently than refrigeration, depending on the storage pressure used. As such, these results points towards the use of HS as an efficient alternative to refrigeration in meat products preservation.
O armazenamento hiperbárico (AH) é uma metodologia de conservação de alimentos na qual a pressão é usada como fator determinante no retardamento da deterioração. A esta nova metodologia de conservação poderão estar associadas poupanças energéticas significativas, nomeadamente quando o armazenamento ocorre à temperatura ambiente (TA). Desta forma, o objetivo deste estudo focou-se na avaliação do AH como alternativa à refrigeração na conservação de fiambre fatiado e de carne picada de porco utilizando diferentes combinações de pressão (0.1-150 MPa), temperatura (4-37 ºC) e tempo (4-24 h). No geral observou-se um aumento da carga microbiológica em pelo menos 1 Log CFU/g para o fiambre bem como a carne picada armazenados à TA e 0.1 MPa enquanto que sob refrigeração a carga microbiológica manteve-se igual ou ligeiramente superior à inicial. Por outro lado, as amostras sujeitas a AH apresentaram cargas iguais ou menores do que as amostras iniciais, independentemente da temperatura de armazenamento empregue. Contudo verificou-se que pressões mínimas de 50 MPa são necessárias de forma a inibir o crescimento microbiológico similarmente à refrigeração. No caso do fiambre, não foram verificadas diferenças significativas nos parâmetros físico-químicos analisados (pH, capacidade de retenção de água, oxidação lipídica e cor) entre as diferentes condições de armazenamento e as amostras iniciais. Por outro lado, na carne picada o AH inibiu a oxidação lipídica quando comparado ao armazenamento a 0.1 MPa à mesma temperatura. Assim, o AH demonstra-se eficaz na prevenção da deterioração de produtos cárneos, por inibição do crescimento microbiológico, com igual ou maior eficiência do que a refrigeração, dependendo da pressão de armazenamento usada. Como tal, estes resultados apontam o uso do AH como uma alternativa eficiente à refrigeração, na conservação de produtos cárneos.

M. Tech. (Biotechnology, Department of Biosciences, Faculty of Applied and Computer Sciences), Vaal University of Technology.
The Organization for Economic Co-operation and Development (OECD) and the Food and Agriculture Organization (FAO) of the United Nations predict that milk production and the dairy sector will remain one of the fastest-growing agricultural subsectors over the coming decade. The global milk production is projected to expand over the 2011-2020 period at an annual rate of 2%. In South Africa alone, approximately 14 – 15 million litres of milk are wasted annually due to microbial spoilage. Therefore, the identification of the spoilage microorganisms in the milk products is necessary. This will contribute towards the design of appropriate measures to prevent wastage due to spoilage and in turn contribute towards sustainability of the sector. Accordingly, one hundred samples of spoiled full cream UHT milk were collected from two plants of each of the two largest milk processors. These samples were examined visually, and the pH was measured. A presumptive identification up to genus level was conducted by examining morphological features and conducting Gram-stain, catalase and oxidase tests. Species-specific identification was done by using the Analytical Profile Index and Biolog system. Molecular profiling was done by sequencing the rDNA genes. The main spoilage organisms identified in the samples were Pseudomonas, Micrococcus, Bacillus, Enterococcus and Lactobacillus. All organisms belonging to the five genera were psychrotrophs, which are commonly found in biofilms in UHT milk processing equipment. Therefore, according to the study, the spoilage bacteria apparently entered into the milk due to inadequate cleaning-in-place (CIP) processes. More importantly, further studies should be conducted in order to identify the spoilage microbes and how CIP processes can be improved.

Les eaux florales sont des matières premières aromatiques issues de la distillation, contenant généralement moins de 1 g/L de composés volatils leur conférant leurs propriétés organoleptiques. Elles sont utilisées principalement en industrie agroalimentaire et cosmétique. Elles sont sujettes à des problèmes d’instabilité microbienne incompatibles avec leurs applications. Ces microorganismes, leurs dynamiques, ainsi que les nutriments disponibles nécessaires à leur croissance restent mal connus. Les eaux florales sont actuellement stabilisées par ajout de conservateurs dont certains sont controversés et visent à être retirés du marché. De plus, leur efficacité dans les eaux florales n’a pas été évaluée.L’objectif de cette thèse est d’apporter une meilleure connaissance de la composition des eaux florales et de ses contaminants afin de proposer une méthode de stabilisation adaptée.La composition en huile essentielle et le microbiote de 22 échantillons d’eau de fleur d’oranger (Citrus aurantium L. ssp. amara L.) et de rose (Rosa damascena Miller et Rosa centifolia L.), provenant de différents producteurs autour du bassin méditerranéen, ont été analysés afin de déterminer les facteurs responsables de leur altération. Bien que les composés volatils soient connus pour leurs propriétés antimicrobiennes, leurs concentrations dans les hydrolats ne sont pas suffisantes pour assurer la stabilité microbiologique. En plus des composés volatils, les hydrolats contiennent des composés non-volatils tels que des sucres, entraînés vers le distillat par effet de primage ou de moussage pendant la distillation, et pouvant être utilisés comme substrat de croissance par les microorganismes. La population microbienne peut atteindre 106 à 107 UFC/mL en quelques jours à température ambiante et jusqu’à 3 mois à 5°C. Des bactéries environnementales, oligotrophes, et acido-tolérantes, appartenant principalement aux genres Pseudomonas sp. et Burkholderia sp. ont été isolées et identifiées. Parmi ces bactéries, B. vietnamiensis et Novosphingobium capsulatum ont été capables de métaboliser des composés volatiles tels que le géraniol ou l’acétate de 2-phényléthyle pour produire la 6-méthyl-5-heptèn-2-one ou le 2-phényléthanol, et modifier ainsi les propriétés organoleptiques des hydrolats. Enfin, la capacité de croissance de bactéries pathogènes et d’altération dans les hydrolats a été évaluée, et différents conservateurs ont été testés sur les souches capables de se multiplier dans les hydrolats.Une distillation aseptique et un conditionnement stérile permettrait d’assurer la stabilité des hydrolats sans ajout de conservateurs. En l’absence de conditions aseptiques, l’ajout de conservateurs est nécessaire pour assurer la stabilité des hydrolats
Hydrosols are hydrodistillation products mainly used as food flavoring agents or ingredient in cosmetics. They contain less than 1 g/L of dispersed essential oils giving the organoleptic properties. These are subjected to microbial proliferation that can prevent use due to non-compliance to professional microbiological standards. The microorganisms, their growth dynamics, and the available nutrients in hydrosols remain unknown. Hydrosols can contain few preservatives, but there is no data about their efficiency in hydrosols.The aim of this study was to have a better knowledge on hydrosols composition, their microbiota, and spoilage conditions, in order to propose an adapted stabilization method.The composition in volatile compounds and the microbiota of 22 hydrosol samples of Citrus aurantium L. ssp. amara L. (orange blossom), Rosa damascena Miller (rose D.), and Rosa centifolia L. (rose C.) flowers were analyzed to determine factors responsible for decay. Some non-volatile compounds were likely carried over during distillation by a priming and foaming effect, and could be used as nutrients by microorganisms. Concentrations of volatile compounds in hydrosols are not high enough to prevent microbial proliferation, and bacteria concentrations can reach up to 106 CFU/mL in both hydrosols. The isolated microbial population was composed of oligotrophic Gram-negative bacteria, arranged in four major genera: Pseudomonas sp., Burkholderia cepacia complex, and presumably two new genera belonging to Acetobacteraceae and Rhodospirillaceae. Among those bacteria, Burkholderia vietnamiensis and Novosphingobium capsulatum were able to metabolize volatile compounds, such as geraniol to produce 6-methyl-5-hepten-2-one or geranic acid, or 2-phenylethyl acetate to produce 2-phenylethanol. Finally, the growth potential of a range of bacteria isolated from hydrosols and of pathogenic micro-organisms was evaluated, then the anti-microbial activity in nutrient broth and/or in hydrosols of a range of chemical preservatives authorized for food and cosmetic applications was tested.Additional hurdles such as chemical preservatives or aseptic packaging will be necessary to insure microbial stability

Microbial cross-contamination remains an on-going challenge in the food sector despite implemented sanitation programs. Antimicrobial coatings with inherent self-sanitizing properties have been explored to enhance current cleaning practice and support food safety. Prior work has demonstrated successful incorporation of dual antimicrobial characters, cationic polymers and N-halamines, into one coating system. In addition to the rechargeable nature of N-halamines, the coating was reported to exhibit biocidal effects due to the inherently antimicrobial cationic moieties and the chlorinated N-halamines. However, while these polymer coatings were able to retain antimicrobial activity after repeated chlorination, signs of hydrolysis was observed for the N-halamine bonds, indicating potential issues for long-term usage. Herein, we introduced varied molecular weight cross-linkers in an adaption of the established fabrication method to evaluate cross-linker molecular weight (styrene maleic anhydride (SMA) of 6, 8, 120, 250 kDa) influence on surface properties of the coating. All antimicrobial polymer coatings exhibited similar FTIR spectra, with a prominent absorption band at ~1650 cm-1 suggesting successful cross-link of the polyethyleneimine and SMA. Surface concentration of primary amines ranged from 350-900 nmol/cm2, and N-halamines from 90-130 nmol/cm2. Surface energy decreased with increasing molecular weight of SMA, but were not statistically different from one another. In the end, optimal cross-linker molecular weight was determined based on antimicrobial performance, where the coated PPs with 6 kDa SMAs demonstrated enhanced biocidal effects against E. coli O157:H7 in its chlorinated form. Further, the antimicrobial coating demonstrated efficacy of ~3 to >5 log reductions of microbial load in its unchlorinated and chlorinated form against E. coli O157:H7, L. monocytogenes, and P. fluorescens. Storage studies support the stability of the chlorinated halamines, with full retention of chlorinated N-halamines over a 24 h study (representative of time between sanitation cycles). These results support the potential application of this antimicrobial polymer coating in food processing and handling operations, in support of reducing cross-contamination of spoilage and pathogenic microorganisms.

Microgard, a commercially-available pasteurized fermented milk, was found inhibitory to gram negative bacteria such as Pseudomonas putida, some well-known foodborne pathogens (e.g. Salmonella, Yersinia and Aeromonas) and some fungi as revealed by an agar incorporation assay method. Gram positive bacteria, however, were not inhibited. Rather, some bacteria in this group were stimulated by Microgard. The inhibitory activity of Microgard was optimum at pH 5.3 and below. Thermal stability and protease sensitivity were characteristic of Microgard. Addition of albumin or some emulsifying substances antagonized Microgard whereas treatment with proteases at an initial pH of 11.0 enhanced its inhibitory ability. Purified eluates of Microgard from a Sephadex Column (gel filtration) exhibited thermal stability and maximum UV absorbance. Furthermore, they gave positive results with the Biuret test, indicating the presence of protein type substance(s). Diacetyl, known to be inhibitory for psychotrophic bacteria, was present in Microgard as well as several organic acids. Filter-sterilized fermentation growth metabolites of Pediococcus spp., Propionibacterium spp., Bifidobacterium longum, Lactobacillus plantarum and a black-pigmented yeast were tested for antimicrobial activity as Microgard had been tested. Results revealed that some of these metabolites were inhibitory to Pseudomonas putida and Listeria monocytogenes, which represented food spoilage and pathogenic bacteria, respectively.
Graduation date: 1989

The success in using a food product as a delivery vehicle for probiotics depends on its ability to maintain required level of viable cells (at least 107 cfu g-1) and to suppress the growth of spoilage and pathogenic organism. Cheese-based dips could deliver probiotic bacteria owing to its stable pH, buffering capacity of ingredients and the presence of prebiotics. The anti-microbial properties of probiotics can also be employed for controlling the spoilage organisms such as yeast and mould. The work described in this thesis focused on the survival of probiotics and their anti-microbial effects in dips. Effective selective enumeration methods were first identified for specific probiotic cultures to enumerate their numbers, the ideal conditions in which the organisms survive better were evaluated and the mechanism by which the probiotic organisms antagonise pathogenic and spoilage organisms were then elucidated. The cultures of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Bifidobacterium spp. and propionibacteria were tested in selected bacteriological media to evaluate their suitability as selective media. Nineteen bacteriological media were evaluated at different incubation conditions, including Streptococcus thermophilus (ST) agar, pH-modified MRS agar, MRS - vancomycine agar (MRS-V agar), MRS-bile agar, MRS-NaCl agar, MRS-lithium chloride agar, MRS - NNLP agar, RCA agar, sugar-based (such as maltose, galactose, sorbitol, manitol, esculin) agar media, sodium lactate agar (NaLa), arabinose agar, raffinose agar, xylose agar and LC agar. Aerobic and anaerobic incubations were carried out at temperatures of 27°C, 30°C, 37°C, 43°C and 45°C for the duration of 24h, 72h and 7-9 days. ST agar and aerobic incubation at 37°C for 24h were suitable for S. thermophilus. L. delbrueckii ssp bulgaricus can be enumerated in MRS agar (pH 4.58 or pH 5.20) and anaerobic incubation at 45°C for 72h. MRS-V agar and anaerobic incubation at 43°C for 72h was suitable to enumerate L. rhamnosus. Anaerobic incubation in MRS-V agar at 37°C for 72h was selective to enumerate L. casei. It is recommended that subtraction method should be implemented when L. rhamnosus is present in the product. To do this, the count of L. rhamnosus recorded on MRS-V agar at 43°C for 72h under anaerobic incubation should be subtracted from the total count of L. casei. and L. rhamnosus recorded on MRS-V agar 37°C for 72h under anaerobic incubation.

Several lactic acid bacteria (LAB) of the Lactococcus, Lactobacillus, Leuconostoc and Pediococcus genera were screened for inhibition of food-borne pathogens and spoilage microorganisms in raw milk and dairy products. Listeria monocytogenes was killed by Lactococcus lactis subsp. lactis and Pediococcus pentosaceus due to their production of bacteriocin-type inhibitors. Staphylococcus aureus was not able to grow in raw milk at temperatures below 5°C even without LAB being present. Gram negative Salmonella enteritidis. Salmonella typhimurium and Escherichia coli, along with spoilage bacteria of the genus Pseudomonas were dramatically inhibited by a Lactobacillus species, designated AS-1, in raw and pasteurized milk as well as in cottage cheese. However, other LAB were not able to inhibit these organisms. Lactobacillus AS-1, did not produce hydrogen peroxide but carbon dioxide was produced. The AS-1 strain was a gram positive coccobacillus, catalase and oxidase negative and produced DL-lactic acid. It deaminated arginine and grew over a temperature range of 5°C to 45°C. It was also able to ferment glucose, galactose, fructose and lactose in addition to 17 other carbohydrates. High numbers (107 CFU/ml) of AS-1 were required to obtain complete inhibition of gram negative bacteria. A selective medium (ASLM) for Listeria monocytogenes was developed to follow the fate of this particular pathogen in association with LAB in raw milk; other selective media were not able to inhibit the growth of background flora of raw milk. ASLM was superior to four other media in allowing only the growth of the target pathogen. For the Lactococcus genus, a selective and differential agar medium (Alsan) was formulated to selectively allow growth of Lactococcus spp. and to differentiate between Lactococcus lactis subsp. lactis and the biovariety diacetylactis, based on citrate utilization.
Graduation date: 1993

碩士
國立中興大學
食品科學系
85
The objectives of this study were to use immobilized antimicrobial agent（ nisin, acetic acid or combination of both ）against pre- or post- contaminated pathogens such as Listeria monocytogenes Scott A and Yersinia enterocolitica CCRC10807 in cooked pork tenderloin stored at 4℃. In addition, effectivenss of combination system of immobilized antimicrobial agents with modified atmosphere packaging（ MAP ）against the pathogens（ L. monocytogenes and Y. enterocolitica ）and the spoilage microorganisms on raw pork tenderloin stored at 4℃ was also investigated. In the pre-contaminated cooked pork model, the use of immobilized technique can extend the effectiveness time of antimicrobial agents. Nisin did not inhibit the growth of Y. enterocolitica, however, it had bactericidal effect against L. monocytogenes. On the other hand, the growth of Y. enterocolitica was inhibited by acetic acid. When immobilized nisin and acetic acid were used, the growth of both pathogens was inhibited effectively. The results of post-contaminaed cooked pork tenderloin showed that the effectiveness of antimicrobial agents can be extended by immobilization. The immobilized gel can increase the time for the bacteria to diffuse and absorbe on meat surface and it can lower the attachment ability of the bacteria. This cause the bacteria to remain in the water film, where it is much more sensitive to antimicrobial agent. Overall, the immobilized gel can prevent post-contamination of pathogen. In raw pork system, the growth of spoilage microorganism, Pseudomonas spp., was inhibited by MAP；however, since lactic acid bacteria were able to grow under anaerobic condition, they soon become predominant. The growth of lactic acid bacteria and there meetabolic products were detrimental to the growth of L. monocytogenes. But large population of lactic acid bacteria will cause undesirable flavor in pork, treatment with immobilized antimicrobial agent will decrease the lactic acid bacteria number. On the other hand, Y. enterocolitica showed slow growth in MAP. From the extention of shelf-life and the safety point of view of meat products, the immobilized antimicrobial agents can lower the bacterial count in both the pathogen and the spoilage microorganisms on meat. In combination with MAP, the immobilized system was the most effective in terms of lowering bacteria count.

The inherent volatility and/or heat sensitivity of many natural antimicrobial (AM) additives can be detrimental to their widespread use in commodity polymer packaging film formulations. In this study, beta-cyclodextrin (β-CD) inclusion complexes with naturally-derived AM agents: thymol, carvacrol, and linalool were prepared using a co-precipitation technique. The complexes were optimised and then characterised by techniques including thermal analysis. They were then incorporated into low-density polyethylene (LDPE) films with AM agents added directly for comparison. The subsequent release of the AM agents into food simulants was studied followed by an investigation of the efficacy of the films in vitro against selected bacteria. The films were later tested on real foods to assess their potential for controlling microbial growth and lipid oxidation. In the initial experiments, conditions for synthesising the β-CD/AM agent complex including solvent composition, temperature, reaction time, and total solvent volume were investigated to optimise the inclusion efficiency (IE) and yield. Electrospray ionization mass spectrometry and gas chromatography were used to confirm the formation and quantify the amount AM agents that were encapsulated, absorbed onto the surface, or remaining in the filtered solvent. The systematic optimisation of the conditions improved both the yield of the complex and the IE of the AM agents. Using a 1:1 mole ratio of the AM agent to β-CD, the optimised parameters resulted in maximum yields of 87, 84 and 86% (w/w) for thymol, carvacrol and linalool respectively with IE’s close to 100% (w/w) for each agent. The kinetics of the thermal decomposition of the optimised β-CD and complexes of the three AM agents were then investigated using thermogravimetric analysis. Under a linear temperature ramp and in the degree of conversion, α, domain: 0.1 ≤ α ≤ 0.8, the major decomposition steps of the β-CD, and complexes with carvacrol, linalool and thymol occurred at ca. 300°C and followed Avrami-Erofeev kinetics with apparent activation energies, Ea, of: 156 ± 6, 107 ± 7, 96 ± 3 and 110 ± 3 kJ mol-1 respectively. Below ca. 300°C there were staged mass losses from each of the complexes that were not observed for the neat β-CD. These were attributed to lower energy binding interactions and accounted for a little over half of the available guest species in the complex in each case. Lower temperature mass losses for β-CD complexes with carvacrol (ca. 140 to 230°C) and linalool (ca. 95 to 150°C) were analysed and found to be adequately fitted by second-order kinetics with apparent Ea values of: 37 ± 1 and 69 ± 6 kJ mol-1 respectively. The results suggest the optimized complexes are generally thermally stable and would potentially be suitable for high-temperature extrusion processes with acceptably low losses. The next experiments involved the incorporation of the AM agents into LDPE films either directly or encapsulated in β-CD. Quantification of the AM agents was performed immediately following thermal processing, then six and thirty days after the film samples were stored in an open atmosphere. After six days, no AM agent was detected in the films where the agent was added directly to the film whereas the films containing encapsulated agents showed only small decreases in the concentrations of the agents up to 30 days. The migration of AM agents from LDPE films into 95% (v/v) ethanol/water mixtures food simulants at 4℃ was adequately described using first-order kinetics and Fick’s second law of diffusion. For the AM agents added directly to the film, the initial release rates were between four and eight times greater than those of the encapsulated agents. Similarly, the diffusion coefficients of the free agents were ca. four to five times greater than the encapsulated agents. The free and encapsulated natural AM agents incorporated into LDPE film were tested against Escherichia coli (ATCC 25922) in order to assess the potential of the AM inclusion complexes for use in food packaging films. The direct incorporation of the complexes in the film formulations resulted in little inhibition of the target bacterium as assessed by the agar diffusion method even with AM levels as high as 5% (w/w). In comparison, levels of 2% (w/w) of free thymol and carvacrol added directly to the film demonstrated inhibition. The addition of glycerol to the film formulations was investigated as a means of facilitating the AM agent from the complex. A concentration of 1% (w/w) of glycerol in the film formulation was found to result in microbial inhibition which increased with additional glycerol. The use of 2% (w/w) glycerol resulted in a more pronounced inhibition of targeted microorganism. Upon the addition of glycerol, all of the films showed AM activity against the target bacterium with the exception of those containing linalool in either the free or encapsulated forms. Upon the addition of 2% (w/w) of glycerol to the film formulation, encapsulated thymol at a concentration of 2% (w/w) was more effective than encapsulated carvacrol at a concentration of 3% (w/w) against E. coli with zones of inhibition of 30.70 ± 0.72 and 29.61 ± 0.86 mm respectively. In the final experiments, the LDPE films containing encapsulated thymol were tested on real food systems. The level of thymol was 1 to 3% (w/w) relative to the LDPE and glycerol was added in order to obtain the optimum controlled release. In the case of packaged minced beef inoculated with E. coli, no inhibition was observed when the concentration of encapsulated thymol was 3% (w/w) with 2% (w/w) glycerol, however, the same film reduced E. coli growth by 0.7 log10 CFU g-1 on chicken breast fillets compared with the control during storage at 4℃ for 12 days. The growth of E. coli was found to be affected by the temperature at 4℃ whereby the bacterial counts remained relatively low with slow growth over the test period under refrigeration. However, when the temperature increased to 10℃ it was also found that the presence of coliforms interfered with growth of E. coli and, in general, the films containing encapsulated thymol effectively reduced coliform growth. Analysis of the antioxidant (AO) activity of films using the diphenyl-picrylhydrazyl (DPPH) radical assay showed a 71% reduction in DPPH concentration for the LDPE/thymol/β-CD films containing 3% (w/w) thymol. Furthermore, a film comprised of 1% (w/w) thymol with glycerol stored at room temperature for 20 months showed a reduction by 23% in DPPH concentration confirming that the films are suitable for extended storage. Analysis of the formation of thiobarbituric acid reactive substances on packaged minced beef showed decreases in lipid oxidation of 60 and 75% for films containing 1% and 3% (w/w) thymol in the film respectively. The films therefore show promise for the dual purpose of AO and AM activity in order to prolong the shelf-life of selected food products.