Chan, Wing Hin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 126-143). / Abstracts in English and Chinese. / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.vi / Table of Content --- p.viii / List of Figures --- p.xiv / List of Tables --- p.xvi / Abbreviations --- p.xviii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Food and herb authentication --- p.1 / Chapter 1.1.1 --- Background and definition --- p.1 / Chapter 1.1.2 --- Importance of species identification in food and herb authentication --- p.2 / Chapter 1.1.2.1 --- Primary health care --- p.2 / Chapter 1.1.2.2 --- Food and herb safety --- p.3 / Chapter 1.1.2.3 --- Conservation --- p.4 / Chapter 1.1.3 --- Methods for species identification in food and herb authentication --- p.4 / Chapter 1.1.3.1 --- Morphological identification --- p.5 / Chapter 1.1.3.2 --- Chemical analysis --- p.6 / Chapter 1.1.3.3 --- Molecular analysis --- p.9 / Chapter 1.1.4 --- Legislation --- p.11 / Chapter 1.1.4.1 --- Labeling ´ب --- p.11 / Chapter 1.1.4.2 --- Chinese medicine : --- p.12 / Chapter 1.1.4.3 --- Conservation --- p.12 / Chapter 1.2 --- Dongchongxiacao --- p.13 / Chapter 1.2.1 --- Background information of Dongchongxiacao --- p.13 / Chapter 1.2.2 --- Classification of fungal part of Dongchongxiacao --- p.14 / Chapter 1.2.3 --- Dongchongxiacao as a Traditional Chinese Medicine. --- p.15 / Chapter 1.2.4 --- The Dongchongxiacao market --- p.16 / Chapter 1.2.5 --- Adulteration and contamination of Dongchongxiacao --- p.18 / Chapter 1.2.6 --- Authentication of Dongchongxiacao --- p.19 / Chapter 1.2.6.1 --- Morphological identification --- p.19 / Chapter 1.2.6.2 --- Chemical analysis --- p.20 / Chapter 1.2.6.3 --- Molecular analysis --- p.22 / Chapter 1.2.6.3.1 --- "FINS analysis with genomic ITS, nrLSU, EF-lα and rpbl regions for fungal analyses" --- p.22 / Chapter 1.2.6.3.2 --- FINS analysis with mitochondrial CytB and COI regions for caterpillar analyses --- p.24 / Chapter 1.3 --- Abalone --- p.26 / Chapter 1.3.1 --- Background information of abalone --- p.26 / Chapter 1.3.2 --- Abalone as food --- p.27 / Chapter 1.3.3 --- The abalone market --- p.28 / Chapter 1.3.4 --- Adulteration of abalone --- p.31 / Chapter 1.3.5 --- Authentication of abalone --- p.32 / Chapter 1.3.5.1 --- Morphological identification --- p.32 / Chapter 1.3.5.2 --- Chemical analysis --- p.32 / Chapter 1.3.5.3 --- Molecular analysis --- p.33 / Chapter 1.3.5.3.1 --- FINS analysis with mitochondrial COI and 16S rDNA --- p.33 / Chapter 1.3.5.3.2 --- Haliotis-specific detection --- p.34 / Chapter 1.4 --- Aim and Objectives --- p.35 / Chapter Chapter 2 --- Materials and Methods --- p.36 / Chapter 2.1 --- Materials used in this sutdy --- p.36 / Chapter 2.1.1 --- Dongchongxiacao and Cordyceps samples --- p.36 / Chapter 2.1.2 --- Downloaded sequences from NCBI database included in Dongchongxiacao study. --- p.45 / Chapter 2.1.3 --- Abalone and gastropod samples --- p.48 / Chapter 2.1.4 --- Downloaded sequences from NCBI database included in abalone study --- p.54 / Chapter 2.2 --- Reagents and equipments : --- p.56 / Chapter 2.2.1 --- Chemical test on the presence of potassium alum in Dongchongxiacao --- p.56 / Chapter 2.2.2 --- Sample preparation and DNA extraction --- p.57 / Chapter 2.2.3 --- Polymerase Chain Reaction --- p.57 / Chapter 2.2.4 --- Agarose gel electrophoresis and Gene Clean --- p.57 / Chapter 2.2.5 --- Cloning --- p.58 / Chapter 2.2.6 --- Cycle sequencing --- p.58 / Chapter 2.3 --- Experimental procedures --- p.58 / Chapter 2.3.1 --- Morphological observation of Dongchongxiacao and abalone --- p.59 / Chapter 2.3.2 --- Chemical test of potassium in Dongchongxiacao --- p.59 / Chapter 2.3.3 --- Sample preparation and DNA extraction --- p.60 / Chapter 2.3.4 --- Polymerase Chain Reaction --- p.61 / Chapter 2.3.5 --- Agarose gel electrophoresis and Gene Clean --- p.64 / Chapter 2.3.6 --- Cloning --- p.65 / Chapter 2.3.7 --- Cycle sequencing --- p.67 / Chapter 2.3.8 --- Sequence analyses --- p.67 / Chapter 2.3.9 --- Haliotis-specific primer design and PCR test --- p.68 / Chapter Chapter 3 --- Results --- p.71 / Chapter 3.1 --- Dongchongxiacao --- p.71 / Chapter 3.1.1 --- Morphological observations --- p.71 / Chapter 3.1.2 --- Chemical test of potassium alum --- p.77 / Chapter 3.1.3 --- Sequence analyses --- p.79 / Chapter 3.1.4 --- The dendrograms --- p.81 / Chapter 3.2 --- Abalone --- p.91 / Chapter 3.2.1 --- Morphological observations --- p.91 / Chapter 3.2.2 --- Sequence analyses --- p.92 / Chapter 3.2.3 --- The dendrograms --- p.94 / Chapter 3.2.4 --- Haliotis-specific PCR --- p.96 / Chapter Chapter 4 --- Discussion --- p.98 / Chapter 4.1 --- Dongchongxiacao --- p.98 / Chapter 4.1.1 --- Species identification of Dongchongxiacao and related Cordyceps species --- p.98 / Chapter 4.1.1.1 --- Ophiocordyceps sinensis --- p.98 / Chapter 4.1.1.2 --- Cordyceps gunnii --- p.100 / Chapter 4.1.1.3 --- Metacordyceps taii --- p.102 / Chapter 4.1.1.4 --- Cordyceps militaris --- p.103 / Chapter 4.1.2 --- Adulteration of Dongchongxiacao and labeling --- p.104 / Chapter 4.1.3 --- Hosts of Dongchongxiacao fungi and relationship between them --- p.107 / Chapter 4.2 --- Abalone --- p.109 / Chapter 4.2.1 --- Species identification of abalones and other gastropod species by FINS analysis --- p.109 / Chapter 4.2.1.1 --- Haliotis species --- p.109 / Chapter 4.2.1.1.1 --- Haliotis diversicolor --- p.110 / Chapter 4.2.1.1.2 --- Haliotis discus --- p.110 / Chapter 4.2.1.1.3 --- Haliotis asinina --- p.111 / Chapter 4.2.1.1.4 --- Haliotis rufescens --- p.111 / Chapter 4.2.1.1.5 --- Haliotis midae --- p.111 / Chapter 4.2.1.1.6 --- Haliotis madaka --- p.112 / Chapter 4.2.1.1.7 --- Haliotis rubra --- p.113 / Chapter 4.2.1.1.8 --- Haliotis iris --- p.113 / Chapter 4.2.1.1.9 --- Haliotis corrugata --- p.114 / Chapter 4.2.1.2 --- Concholepas concholepas --- p.114 / Chapter 4.2.1.3 --- Hemifusus species --- p.115 / Chapter 4.2.1.4 --- """Dried abalone slice"" samples (D1 to D3) and canned top-shell (E5)" --- p.115 / Chapter 4.2.2 --- Haliotis-speciflc PCR --- p.115 / Chapter 4.2.3 --- Adulteration of abalone and labeling --- p.116 / Chapter 4.3 --- Significance and limitation of molecular approaches in authentication of food and herbs --- p.117 / Chapter 4.3.1 --- FINS analysis --- p.117 / Chapter 4.3.1.1 --- High interspecific variability but low intraspecific variations --- p.118 / Chapter 4.3.1.2 --- Amplification with universal primers --- p.118 / Chapter 4.3.1.3 --- Insufficient DNA sequence available in database --- p.119 / Chapter 4.3.1.4 --- Contamination by foreign DNA and amplification of undesirable DNA in sample mixture --- p.120 / Chapter 4.3.1.5 --- Amplification of degraded DNA --- p.121 / Chapter 4.3.1.6 --- Suggested regions for authentication of Dongchongxiacao and abalone based on FINS analysis results --- p.121 / Chapter 4.3.2 --- PCR with specific primers for targeted amplicons --- p.122 / Chapter 4.3.3 --- Other limitations of molecular approaches in authentication of food and herbs --- p.123 / Chapter 4.4 --- Further investigation --- p.124 / Chapter 4.5 --- Conclusion --- p.124 / References : --- p.126 / Chapter Appendix 1 --- Sequence alignment of 16S rDNA gene sequences of abalone for Haliotis-specific primer design --- p.144 / Chapter Appendix 2 --- Accession numbers of sequences of Dongchongxiacao and Cordyceps samples in this study --- p.149 / Chapter Appendix 3 --- Search results of CytB sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.150 / Chapter Appendix 4 --- Search results of COI sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.151 / Chapter Appendix 5 --- Search results of COI sequences of caterpillar host of Cordyceps samples based on BLAST search results from GenBank --- p.152 / Chapter Appendix 6 --- Sequence alignment of ITS sequences of Cordyceps samples and related sequences --- p.153 / Chapter Appendix 7 --- Sequence alignment of nrLSU sequences of Cordyceps samples and related sequences --- p.161 / Chapter Appendix 8 --- Sequence alignment of EF-lα sequences of Cordyceps samples and related sequences --- p.168 / Chapter Appendix 9 --- Sequence alignment of rpbl sequences of Cordyceps samples and related sequences --- p.173 / Chapter Appendix 10 --- "Sequence alignment of combined dataset of three regions (nrLSU, EF-lα and rpbl) of Cordyceps samples and related sequences" --- p.179 / Chapter Appendix 11 --- Sequences alignment of CytB sequences of caterpillar host of Cordyceps samples and related sequences --- p.188 / Chapter Appendix 12 --- Sequence alignment of COI sequences of caterpillar host of Cordyceps samples and related sequences --- p.191 / Chapter Appendix 13 --- Sequence alignment of COI sequences of Cordyceps samples D12-2 and D14 and related sequences --- p.195 / Chapter Appendix 14 --- Sequence distance matrix of ITS sequences of Cordyceps samples and related samples based on K2P algorithm --- p.196 / Chapter Appendix 15 --- Sequence distance matrix of nrLSU sequences of Cordyceps samples and related samples based on K2P algorithm --- p.203 / Chapter Appendix 16 --- Sequence distance matrix of EF-lα sequences of Cordyceps samples and related samples based on K2P algorithm --- p.208 / Chapter Appendix 17 --- Sequence distance matrix of rpbl sequences of Cordyceps samples and related samples based on K2P algorithm --- p.213 / Chapter Appendix 18 --- "Sequence distance matrix of combined dataset of three regions (nrLSU, EF-lα and rpbl) sequences of Cordyceps samples and related samples based on K2P algorithm" --- p.217 / Chapter Appendix 19 --- Sequence distance matrix of CytB sequences of caterpillar host of Cordyceps samples and related samples based on K2P algorithm --- p.219 / Chapter Appendix 20 --- Sequence distance matrix of COI sequences of caterpillar host of Cordyceps samples and related samples based on K2P algorithm --- p.223 / Chapter Appendix 21 --- Sequence alignment of chloroplast trnH-psbA sequences of Cordyceps sample D12-2 and related sequences --- p.226 / Chapter Appendix 22 --- Accession numbers of sequences of abalone and gastropod samples in this study --- p.227 / Chapter Appendix 23 --- Search results of 16S rDNA sequences of the abalone and gastropod samples based on BLAST search results from GenBank --- p.228 / Chapter Appendix 24 --- Search results of COI sequences of the abalone and gastropod samples based on BLAST search results from GenBank --- p.229 / Chapter Appendix 25 --- Search results of COI sequences of the abalone and gastropod samples based on BOLD-IDS --- p.230 / Chapter Appendix 26 --- Sequence alignment of 16S sequences of abalone samples and related sequences --- p.231 / Chapter Appendix 27 --- Sequence alignment of COI sequences of abalone samples and related sequences --- p.234 / Chapter Appendix 28 --- Sequence alignment of COI sequences of abalone product sample D2 and related sequences --- p.238 / Chapter Appendix 29 --- Sequence distance matrix of 16S sequences of abalone samples and related samples based on K2P algorithm --- p.239 / Chapter Appendix 30 --- Sequence distance matrix of COI sequences of abalone samples and related samples based on K2P algorithm --- p.243

INTRODUÇÃO Produtos intermediários da reação de Maillard e da peroxidação, como os compostos dicarbonílicos, reagem facilmente com grupamentos aminas de proteínas e ácidos nucléicos levando a modificações biológicas que podem resultar em patologias observadas no diabetes, aterosclerose e doenças neurodegenerativas. O consumo de Produtos da Reação de Maillard (PRM) aumentou nas últimas décadas e há evidências de que estas substâncias são absorvidas e podem tomar parte em processos patológicos, embora ainda não haja consenso sobre os possíveis efeitos deletério à saúde a partir do aumento de sua ingestão. Ressalta-se a necessidade de estimar o consumo destes PRMs a partir de dados sobre os conteúdos e a ingestão habitual do alimento em questão como cereais matinais e café. Objetivos: a) validar metodologia para quantificar indicadores da reação de Maillard: hidroximetilfurfural (HMF), furosina (FUR), carboximetilisina (CML) e Compostos Intermediários Fluorescentes (CIF) em cereais matinais (flocos e granola) e café; b) avaliar se há diferenças nos teores desses compostos nas diferentes marcas destes produtos comercializados em São Paulo; METODOLOGIA: Foram analisados dois lotes de três marcas de cereais do tipo flocos, três marcas de cereais do tipo granola e cinco marcas de café presentes em 100 por cento dos hipermercados visitados no município de São Paulo. A validação da metodologia para quantificação, empregando HPLC, consistiu no cálculo da exatidão (recuperação), repetibilidade e sensibilidade para os compostos: HMF e FUR. Foram determinados os teores de CIF por espectrofotometria de fluorescência e os teores de CML por teste imunoenzimático. RESULTADOS: Os métodos de determinação de FUR e HMF foram validados conforme o Instituto Nacional de Metrologia, Normalização e 9 Qualidade Industrial (INMETRO). O teor médio de CIF livre e total foram maiores para as amostras de café, com média de 232CIF/mg e 765CIF/mg respectivamente. Não houve diferença (p>0,05) no teor de CIF livre e total entre os flocos F1 e F2. O mesmo foi observado para a granola marcas. G1, G2 e G3 A granola foi o produto com maior teor médio de HMF (67,5mg/Kg) e furosina (301mg/100g de proteína). A FUR não foi detectado nas amostras de café. Todas as marcas dos alimentos estudados para os indicadores HMF e FUR apresentaram diferença estatisticamente significativa (p<0,05). O café apresentou maior teor médio de CML (1823,5ng/mg de proteína), sem diferença entre as marcas (p>0,05) CONCLUSÕES: Os cereais do tipo flocos contribuem para maior ingestão de PRMs da fase inicial da reação de Maillard (RM), a granola contribui para maior ingestão de PRMs da fase intermediária da RM e o café contribui de forma significativa para maior ingestão de PRMs da fase avançada da RM. O café, por ser submetido a tratamento térmico mais severo apresenta maior concentração de PRMs da fase avançada da reação / INTRODUCTION Maillard reaction products and lipid peroxidation, such as dicarbonyl compounds easily react with amino groups of proteins and nucleic acids leading to biological changes that can result in complications in diseases such as diabetes, atherosclerosis and neurodegenerative. The consumption of Maillard Reaction Products (MRP) has increased in recent decades and there is evidence that these substances are absorbed and can participate in pathological processes, although there is no consensus about the possible harmful health effects from their intake. We highlight the need to identify the consumption of MRP, mainly in vulnerable populations like children and diabetics, in order to establish acceptable daily intakes and guidelines for the food industry. OBJECTIVES: a) validate the methodology to measure indicators of the Maillard reaction: hydroxymethylfurfural (HMF), Furosine (RUF) carboxymethylysine (CML) and fluorescent intermediate compounds (FIC) in breakfast cereals (corn flakes and granola) and coffee, b) to evaluate if there are differences in the levels of these compounds contents among brands commercialized in São Paulo METHODS: two lots of 3 brands of flakes cereal, 3 brands of granola and 5 coffee brands present in 100 per cent of supermarkets visited in the city Sao Paulo were analyzed. HPLC methodology validation was assessed by determining accuracy (recovery), repeatability, and sensibility (linearity, limits of detection and quantitation) for the compounds: HMF and FUR. The contents of the Fluorescent Intermediary compounds (FIC) was measured by spectrophotometric method and the levels of CML by ELISA. RESULTS: Calibration curves determination coefficient (r 2) were higher than 0,99 for all compounds. Recovery ranged from 84 to 110 per cent and repeatability average was 3,5 per cent. The average content of free and total FIC was higher for coffee 232CIF/mg and 765CIF/mg respectively. The brands of granola and flakes was similar but just brands F1 and F2 was similar between brands (p<0,05). For HMF the higher values were for granola 67,5mg/kg. The presence of dried fruit in these grains may 11 have contributed significantly to the higher rate of this indicator. For indicator FUR average was higher in granola samples (301mg/100g of protein) and it was not possible to quantify the levels of FUR for coffee. All brands analyzed for HMF and FUR was similar (p<0,05). CML average was higher for coffee (1823,5ng/mg of protein). The brands analyzed was similar for all samples (p <0.05). CONCLUSIONS: Flakes contribute to higher intake of MRPs from early stage of the Maillard reaction (MR), the granola contributes to higher intake of MRPs from intermediate phase of MR and coffee contributes significantly to higher intake of final MRPs. Data suggest that coffee has more severe thermal treatment causing a higher concentration of MRPs from the final phase of the MR

<p>Water is the most consumed foodstuff around the world. Therefore it is very important to analyze possible faecal contamination of water, and Enterococci are an indicator for that. They are also used as an indicator for possible faecal contamination in food. Normally you find Enterococci in the intestines of humans and animals, but Enterococci can also give infections like urinary tract infection.</p><p>In this study, varying number of colonies and colours of Enterococci on different media were evaluated. The purpose was to investigate if three different methods would give the same numbers of colonies. Another interesting perspective was to investigate if one medium could be used for two methods. Membrane filter techniques and surface spreading techniques were used to detect Enterococci. These methods were compared with a most probable number method.</p><p>None of the strains showed an optimal result on all media, however one medium, ChromoCult, showed good results for all investigated strains.</p>

Water is the most consumed foodstuff around the world. Therefore it is very important to analyze possible faecal contamination of water, and Enterococci are an indicator for that. They are also used as an indicator for possible faecal contamination in food. Normally you find Enterococci in the intestines of humans and animals, but Enterococci can also give infections like urinary tract infection. In this study, varying number of colonies and colours of Enterococci on different media were evaluated. The purpose was to investigate if three different methods would give the same numbers of colonies. Another interesting perspective was to investigate if one medium could be used for two methods. Membrane filter techniques and surface spreading techniques were used to detect Enterococci. These methods were compared with a most probable number method. None of the strains showed an optimal result on all media, however one medium, ChromoCult, showed good results for all investigated strains.

A cold fiber solid phase microextraction device was designed and constructed based on the use of a thermoelectric cooler (TEC). A three-stage thermoelectric cooler was used for cooling a copper rod coated with a polydimethylsiloxane (PDMS) hollow fiber, which served as the SPME fiber. The copper rod was mounted on a commercial SPME plunger and exposed to the cold surface of the TEC, which was enclosed in a small aluminum box. A heat sink and a fan dissipated the generated heat at the hot side of the TEC. By applying an appropriate DC voltage to the TEC, the upper part of the copper rod, which was in contact to the cold side of the TEC, was cooled and the hollow fiber reached a lower temperature through heat transfer. A thermocouple was embedded in the cold side of the TEC for indirect measurement of the fiber temperature. A portable cold fiber SPME device was made by using a car battery as the power supply. The cold fiber SPME device with thermoelectric cooling was applied in quantitative analysis of off-flavors in rice. Hexanal, nonanal, and undecanal were chosen as three test analytes in rice. These analytes were identified according to their retention times and analyzed with a GC/FID instrument. Headspace extraction conditions (i.e. extraction temperature and extraction time) were optimized. Standard addition calibration graphs were obtained at the optimized conditions and the concentrations of the three analytes were calculated. The developed method was compared to a conventional solvent extraction method. The applicability of the portable cold fiber SPME with TEC for field sampling was tested. The effect of cooling on extraction recovery and the reproducibility of extraction were examined for extractions from an n-alkane flow through system. It was found that the extraction recoveries were significantly higher when the fiber was cooled. To further investigate the effect of cooling on the sensitivity of SPME in field sampling, the portable cold fiber SPME was used for extraction of volatile components from living wisteria flowers. Both the number of identified compounds and the related peak areas increased for extractions with cold PDMS fiber relative to without cooling and commercial PDMS and PA fibers. The portable cold fiber SPME device was also used for field sampling of volatile components of living lily-of-the-valley flowers and the extracted compounds were analyzed with GC/MS. The desorption kinetics of hydrophobic organic compounds (HOCs) from environmental solid matrices was investigated using cold fiber SPME with CO2 cooling. Polycyclic aromatic hydrocarbons (PAHs) and selected volatile organic compounds (i.e. toluene, ethylbenzene, o-xylene) were used as test analytes. Sand, silica gel, and clay were used as laboratory model solid matrices and were contaminated by the test analytes. Certified sediments were used as naturally contaminated samples. In this approach, the organic compounds, released from contaminated solid samples at different elevated temperatures, were exhaustively extracted with cold fiber SPME over different extraction times. The extraction data were used to obtain desorption and Arrhenius plots. The rate constants of desorption and activation energies of desorption were measured for each contaminant using these plots. The results were comparable to those reported in the literature.

A cold fiber solid phase microextraction device was designed and constructed based on the use of a thermoelectric cooler (TEC). A three-stage thermoelectric cooler was used for cooling a copper rod coated with a polydimethylsiloxane (PDMS) hollow fiber, which served as the SPME fiber. The copper rod was mounted on a commercial SPME plunger and exposed to the cold surface of the TEC, which was enclosed in a small aluminum box. A heat sink and a fan dissipated the generated heat at the hot side of the TEC. By applying an appropriate DC voltage to the TEC, the upper part of the copper rod, which was in contact to the cold side of the TEC, was cooled and the hollow fiber reached a lower temperature through heat transfer. A thermocouple was embedded in the cold side of the TEC for indirect measurement of the fiber temperature. A portable cold fiber SPME device was made by using a car battery as the power supply. The cold fiber SPME device with thermoelectric cooling was applied in quantitative analysis of off-flavors in rice. Hexanal, nonanal, and undecanal were chosen as three test analytes in rice. These analytes were identified according to their retention times and analyzed with a GC/FID instrument. Headspace extraction conditions (i.e. extraction temperature and extraction time) were optimized. Standard addition calibration graphs were obtained at the optimized conditions and the concentrations of the three analytes were calculated. The developed method was compared to a conventional solvent extraction method. The applicability of the portable cold fiber SPME with TEC for field sampling was tested. The effect of cooling on extraction recovery and the reproducibility of extraction were examined for extractions from an n-alkane flow through system. It was found that the extraction recoveries were significantly higher when the fiber was cooled. To further investigate the effect of cooling on the sensitivity of SPME in field sampling, the portable cold fiber SPME was used for extraction of volatile components from living wisteria flowers. Both the number of identified compounds and the related peak areas increased for extractions with cold PDMS fiber relative to without cooling and commercial PDMS and PA fibers. The portable cold fiber SPME device was also used for field sampling of volatile components of living lily-of-the-valley flowers and the extracted compounds were analyzed with GC/MS. The desorption kinetics of hydrophobic organic compounds (HOCs) from environmental solid matrices was investigated using cold fiber SPME with CO2 cooling. Polycyclic aromatic hydrocarbons (PAHs) and selected volatile organic compounds (i.e. toluene, ethylbenzene, o-xylene) were used as test analytes. Sand, silica gel, and clay were used as laboratory model solid matrices and were contaminated by the test analytes. Certified sediments were used as naturally contaminated samples. In this approach, the organic compounds, released from contaminated solid samples at different elevated temperatures, were exhaustively extracted with cold fiber SPME over different extraction times. The extraction data were used to obtain desorption and Arrhenius plots. The rate constants of desorption and activation energies of desorption were measured for each contaminant using these plots. The results were comparable to those reported in the literature.

published_or_final_version / Environmental Management / Master / Master of Science in Environmental Management

Thesis (MScFoodSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The preservation of various fresh fish products is achieved by either smoking, salting, canning, freezing or fermenting a highly perishable raw product. Since many of these facilities are not readily available, the use of fermentation as a means of preserving the product has been extensively practiced. However, the fermentation of fish is a time consuming practise and only by accelerating the process would it be possible to ensure the production of a more cost effective and readily available safe end-product. The quality of the fermented fish product is partially determined by the fermentation conditions and the metabolic activity of the microbes present. The rapid identification of the microbes present during the fermentation would enable the selection of possible starters to ensure an accelerated production of high quality fermented fish products. This study was thus undertaken to develop identification fingerprints for bacteria isolated from fermented fish products. A 1300 bp fragment of the 16S rRNA genes of each of the bacteria previously isolated was successfully amplified using the PCR technique. The isolates included strains of the genera Bacillus, Staphylococcus, Sphingomonas, Kocuria, Brevibacillus, Cryseomonas, Vibrio, Stenotrophomonas and Agrobacterium. The data obtained can, therefore, be used in the identification of these microbes isolated from other similar fermented fish products. The fingerprints could also be used to assist in determining the dominant microbial populations responsible for the characteristic qualitative changes occurring in the fish product during fermentation. The microbial composition of a fermenting fish product partially determines the quality of the end-product, therefore, the use of selected bacterial starters could result in the accelerated production of a microbial safe fermented fish product. A further objective of this study was to accelerate the production of a fermented fish product by inoculating macerated trout with either selected lactic acid bacteria (LAB) or with selected bacteria with high proteolytic activity over a 30 day fermentation period. The LAB included a combination of Lactobacillus plantarum, Lactococcus diacetylactis and Pediococcus cerevisiae strains, whereas the bacteria with high proteolytic activity included strains of Kocuria varians, Bacillus subtilis, two strains of B. amyloliquefaciens and a combination of these bacterial species. The quality of the fermented product was determined using changes in product pH, titratable acidity (%TA) and free amino nitrogen (FAN) formation as efficiency parameters. The data obtained during the fermentation of the macerated trout showed that the selected starters did not have a significant effect on the pH decrease in the product over a 30 day fermentation period. The LAB strains did not have a significant effect on the %TA of the fermenting fish product, yet the presence of these bacteria appeared to limit the FAN production in the product. The bacteria with high proteolytic activity resulted in slightly enhanced %TA values and a higher FAN content in the fermented product. It was also determined that the LAB and Kocuria varians, in contrast to the Bacillus spp. inoculums, did not survive the fermentation conditions well, possibly due to the low pH environment. The presence of the starter bacteria in the fermenting fish mixture at the end of the fermentation was also successfully determined with the use of the PCR-RFLP technique. The fermented fish product, obtained at the end of the fermentation period, had a good aroma and compared favourably to similar commercially available fermented fish products. The use of different microbial starters could in future enable the production of a diverse range of high quality products, which could be produced and marketed locally. / AFRIKAANSE OPSOMMING: Die preservering van ‘n verskeidenheid vars vis produkte word bereik deur die hoogs bederfbare produk te rook, te sout, te blik, te vries of te fermenteer. Aangesien baie van hierdie fasiliteite nie geredelik beskikbaar is nie, is die gebruik van fermentasie as ‘n preserverings metode al ekstensief beoefen. Die fermentasie van vis is egter 'n tydsame proses en slegs deur die versnelling van die proses sal dit moontlik wees om die produksie van ‘n meer koste effektiewe en geredelike beskikbare veilige eindproduk te verseker. Die kwaliteit van die gefermenteerde vis produk word gedeeltelik bepaal deur die fermentasie kondisies en die metaboliese aktiwiteit van die mikrobes teenwoordig. Die vinnige identifikasie van die mikrobes teenwoordig gedurende die fermentasie sal die seleksie van moontlike suursels om die versnelde produksie van hoë kwaliteit gefermenteerde vis produkte moontlik maak. Hierdie studie is dus onderneem om identifikasie vingerafdrukke vir bakteriee wat gei'soleer is van gefermenteerde vis produkte moontlik te maak. ‘n 1300 bp fragment van die 16S rRNA gene van elkeen van die bakteriee wat voorheen gei'soleer is, is suksesvol geamplifiseer deur die PCR tegniek. Die isolate sluit in stamme van die genera Bacillus, Staphylococcus, Sphingomonas, Kocuria, Brevibacillus, Cryseomonas, Vibrio, Stenotrophomonas en Agrobacterium. Die data kan dus gebruik word in die identifikasie van hierdie mikrobes as dit gei'soleer word van ander gefermenteerde vis produkte. Die vingerafdrukke kan ook gebruik word om die dominante mikrobiese populasies wat verantwoordelik is vir die kenmerklike kwalitatiewe veranderinge wat plaasvind in die vis produk gedurende die fermentasie, te identifiseer. Die mikrobiese samestelling van ‘n fermenterende vis produk bepaal gedeeltelik die kwaliteit van die eindproduk, daarom kan die gebruik van geselekteerde bakteriese suursels die versnelde produksie van ‘n mikrobies veilige gefermenteerde vis produk teweeg bring. ‘n Verdere doel van hierdie studie was om die produksie van ‘n gefermenteerde vis produk te versnel deur fyngemaakte forel met of geselekteerde melksuurbakteriee of met geselekteerde bakteriee met hoë proteolitiese aktiwiteit te inokuleer oor ‘n 30 dag fermentasie periode. Die melksuurbakteriee het ingesluit ‘n kombinasie van Lactobacillus plantarum, Lactococcus diacetylactis en Pediococcus cerevisiae, terwyl die bakterieë met hoë proteolitiese aktiwiteit stamme van Kocuria varians, Bacillus subtilis, twee stamme van Bacillus amyloliquefaciens en ‘n kombinasie van hierdie bakteriese stamme ingesluit het. Die kwaliteit van die gefermenteerde produk is bepaal deur die veranderinge in die pH, titreerbare suur (%TS) en vrye amino stikstof (VAS) vorming van die produk as effektiwiteits parameters te gebruik. Die data wat verkry is gedurende die fermentasie van die fyngemaakte forel het gedui daarop dat die geselekteerde suursels nie ‘n merkwaardige effek op die afname in pH in die produk oor ‘n 30 dag fermentasie periode het nie. Die melksuurbakteriee het nie ‘n merkwaardige effek op die %TS van die gefermenteerde vis produk gehad nie, terwyl dit geblyk het dat die teenwoordigheid van hierdie bakterieë die produksie van VAS in die produk belemmer het. Die bakteriee met hoe proteolitiese aktiwiteit het ‘n effense verhoogde %TS en ‘n hoër VAS inhoud in die gefermenteerde produk veroorsaak. Dit is ook bepaal dat die melksuurbakteriee en Kocuria varians, in teenstelling met die Bacillus spp. inokulums, nie die fermentasie kondisies goed oorleef het nie, moontlik as gevolg van die lae pH omgewing. Die teenwoordigheid van die suursel bakteriee in die fermenterende vis mengsel aan die einde van die fermentasie is ook suksesvol bepaal met die PKR-RFLP tegniek. Die gefermenteerde vis produk, verkry aan die einde van die fermentasie periode, het ‘n goeie aroma gehad en het goed vergelyk met soortgelyke kommersieel beskikbare gefermenteerde vis produkte. Die gebruik van verskillende mikrobiese suursels kan in die toekoms die produksie van ‘n diverse reeks hoë kwaliteit produkte wat plaaslik geproduseer en bemark kan word moontlik maak.

O objetivo do presente estudo foi avaliar diferentes agentes ligantes para elaboração de barras de cereais salgadas e caracterizá-las quanto aos parâmetros físicos, físico-químicos e sensoriais. Foram desenvolvidas quatro formulações de barras de cereais utilizando diferentes agentes ligantes. Os agentes ligantes avaliados foram colágeno, goma guar, goma xantana e psyllium. As barras desenvolvidas foram avaliadas quanto as características físicas (cor e textura), físico-químicas (pH, umidade, cinzas, proteínas, lipídeos, Aw, fibra bruta) além do valor calórico, composição em ácidos graxos e concentração dos principais minerais. Dentre os quatro agentes ligantes avaliados o psyllium se destacou quanto as características físico-químicas, apresentando uma barra de cereal com alto teor de proteínas e fibras; com baixo teor de carboidratos e atividade de água. Os agente ligantes gomas guar e xantana apresentaram características próximas ao psyllium, principalmente quanto ao teor de fibras. O colágeno como agente ligante conferiu ao produto final alto teor de proteínas e lipídeos. As análises de cor e de textura revelaram que as barras de cereais salgadas apresentaram coloração e crocância características para este tipo de produto. Com relação a composição em ácidos graxos as barras desenvolvidas oferecem um bom aporte de ácidos graxos essenciais e teor de minerais. Sensorialmente, as barras de cereais salgadas elaboradas com chia apresentaram boa aceitabilidade, com destaque para as barras elaboradas com os agentes ligantes psyllium e goma xantana. Os diferentes agentes ligantes demonstraram eficiência tecnológica na elaboração de barras de cereais salgadas. O agente ligante psyllium em detrimento dos demais, apresentou melhores características físico-químicas e sensoriais. No entanto, o produto de modo geral apresentou características nutritivas e saudáveis podendo ser indicado para uma dieta proteica, com alto teor de fibras e livre de açúcares. / The objective of this study was to evaluate different binders when preparing salt cereal bars and to characterize them as physical, physico-chemical and sensory parameters. Four formulations of different cereal bars using binders have been developed. The evaluated binders were collagen, guar gum, xanthan gum and psyllium. The developed cereal bars were evaluated according to their physical characteristics (color and texture), physicochemical (pH, moisture, ash, protein, lipids, Aw, crude fiber) beyond their calorie, fatty acid composition and concentration of the main minerals. Among the four binding agents evaluated, psyllium stood out due to its physicochemical characteristics. A cereal bar high in protein and fiber; low in carbohydrates and water activity. The binding agent guar gum and xanthan showed characteristics similar to psyllium, especially regarding to fiber content. Collagen as binder gave the final product a high level in protein and lipid. The color and texture analyzes showed that the salt cereal bars had the color and crispness characteristics for this type of product. Regarding to the composition in the fatty acid, the developed bars offer a good supply of essential fatty acids to the human body. The same was observed regarding to mineral contents. Sensory, salt cereal bars made with chia showed good acceptability, highlighting the elaborate bar with psyllium binder. Different binders demonstrated technological efficiency in the preparation of salt cereal bars. The binder psyllium agent over others showed better physical-chemical and sensory characteristics. However, in general the product has healthy and nutritional characteristics it may be indicated for a protein diet with high fiber content and free sugars.

Dissertação composta por 5 artigos. / CAPES / A quitosana é um aminopolissacarídeo natural, biodegradável, hidrofílico, atóxico e biocompatível que pode ser encontrada na parede de micro-organismos, especialmente nas espécies Mucor, mas principalmente nas cascas de crustáceos oriundos dos resíduos da indústria pesqueira. Por possuir diversas características funcionais a quitosana tem se destacado como um excelente agente encapsulante, seja através da formação de microcápsulas ou de microesferas. Diversos estudos têm sido realizados a fim de demonstrar a eficiência das microesferas de quitosana em adsorver, proteger e liberar compostos ativos e resíduos, beneficiando assim, diversos segmentos como: farmacêutico, alimentar, agroindustrial, químico, biomédico e de cosméticos. / Chitosan is a natural amino polysaccharide, biodegradable, hydrophilic, biocompatible and low toxicity and can be found in the wall of microorganisms, especially in Mucor species, but especially in crustacean shells waste from the fishing industry. By owning several functional characteristics chitosan has emerged as an excellent encapsulating agent, either through the formation of microcapsules or microspheres. Several studies have been conducted to demonstrate the efficiency of chitosan microspheres in adsorption, protection and release of active compounds, thus benefiting, several segments, including pharmaceutical, food, agribusiness, chemical, biomedical and cosmetics. / 5000