Orientador: Marta Cristina Teixeira Duarte / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-26T00:33:23Z (GMT). No. of bitstreams: 1 Gasperini_AlessandraMarcon_M.pdf: 1292764 bytes, checksum: fcbdfc6ab370ac6744e8002a57b4a69e (MD5) Previous issue date: 2014 / Resumo: O fungo Aspergillus flavus é responsável pela degradação de alimentos, além de produzir aflatoxinas que são reconhecidas como potentes carcinógenos. Atualmente, frente ao aumento acelerado de cepas resistentes aos antifúngicos sintéticos e à pressão dos consumidores pela substituição destes por produtos naturais, a busca de novos antimicrobianos a partir de óleos essenciais (OE) tem se tornado uma opção promissora. Neste contexto, o objetivo deste trabalho foi avaliar a capacidade de 13 OE, provenientes de plantas medicinais e aromáticas no controle do crescimento de A. flavus CCT 0614 e produção das aflatoxinas AFB1 e AFB2. Ensaios in vitro realizados pelo teste da microdiluição mostraram que os OE de Cinamommum burmanii (MIC 0,2 mg.mL-1) e Cymbopogon citratus, C. martinii, C. winterianus, Eugenia caryophyllata e T. vulgaris (MIC 0,5 mg.mL-1) apresentaram elevada ação anti ¿ A. flavus. Em relação à produção de biomassa seca pelo fungo na presença dos OE, nas concentrações correspondentes a de mínima concentração inibitória (MIC) e 2xMIC, houve diminuição significativa em relação ao controle (p > 0,05). A avaliação do crescimento radial de A. flavus quando submetido à ação dos compostos voláteis (AV) do OE de C. burmanii a 0,2 mg.mL-1 mostrou inibição total até 336 h. Por outro lado, os OE de E. caryophyllata, T. vulgaris e C. citratus reduziram o crescimento radial do fungo quando incorporados ao meio de cultura e quando submetidos a ação dos voláteis. Os OE mais ativos foram analisados através de CG-EM, que permitiu identificar nos óleos compostos conhecidos pela ação antimicrobiana. Em seguida, o OE de C. citratus foi selecionado para estudo dos efeitos sobre a produção de aflatoxinas por A. flavus, quantificada por CLAE-FL. Os resultados mostraram que o OE de C. citratus foi capaz de reduzir a produção de AFB1 em 95% quando incorporado ao meio a 0,5 mg.mL-1, enquanto houve um aumento de cerca de 53% na produção da micotoxina a 0,25 mg.mL-1, após o mesmo período. Não foi detectada a presença da AFB2. Os resultados deste trabalho indicam que os OE estudados representam uma alternativa promissora no controle do crescimento A. flavus e o OE de C. citratus à produção de AFB1. A possibilidade de aplicação dos OE por AV é uma alternativa atrativa, pelo fato de não afetar as propriedades físicas e sensoriais do alimento / Abstract: The fungus Aspergillus flavus is responsible for damage in foods, and by aflatoxins production that are recognized as potent carcinogens. Currently, due to rapid increase of resistant strains to synthetics antifungal, and consumer pressure to replacing these synthetics compounds by natural products, the search for new antimicrobial from essential oils (EO) has become a promising option. This study aimed evaluate the capacity of 13 EO from medicinal and aromatic plants in the control of growth and aflatoxins B1 and B2 production by A. flavus CCT 0614. In vitro assays performed by microdilution method demonstrated that EO of Cinnamomum burmanii (MIC 0.2 mg.mL-1) and Cymbopogon citratus, C. martinii, C. winterianus, Eugenia caryophyllata and T. vulgaris (MIC 0.5 mg.mL-1) showed strong action anti - A. flavus. In relation to fungal biomass dry weight in the presence of OE in the concentrations corresponding to minimum inhibitory concentration (MIC) and 2xMIC, occurred a significant decrease compared to control (p< 0.05). The radial growth of A. flavus was inhibited totally until 336 h when submitted to the action of the volatile compounds (AV) of C. burmanii at 0.2 mg.mL-1. In contrast, the OE of E. caryophyllata, T. vulgaris and C. citratus reduced the radial growth of the mould when were incorporated to medium (IP) and when used in AV. EO that showed higher action were analysed by CG-MS this allowed identify the compounds known by antimicrobial action. The EO of C. citratus was selected to evaluation of the effects on the aflatoxins production by A. flavus, which was quantified by HPLC-FL. The results showed that C. citratus EO (0.5 mg.mL-1) was able to reduce 95% of AFB1 production, while the production increase about 53% with 0.25 mg.mL-1 after the same period. The presence of AFB2 was not detected. The most active EO were identified by GC-MS, which show the presence of compounds with known antimicrobial activity. The results of this study indicate that the EO studied represent a promising alternative to control the growth and AFB1 production by A. flavus. The possibility of application of EO by AV is an attractive alternative, in view of the fact that it does not affect the physical and sensory properties of food / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

Orientador: Marta Cristina Teixeira Duarte / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-26T00:07:11Z (GMT). No. of bitstreams: 1 Possari_CamilaKopezky_M.pdf: 2524622 bytes, checksum: e715e6d4f298fb93b3325792c5427282 (MD5) Previous issue date: 2014 / Resumo: A castanha do Brasil (Bertholletia excelsa H.B.K.) é a segunda mais importante fonte extrativista da floresta amazônica. Desta forma, não são utilizados defensivos químicos e a produção da castanha do Brasil é considerada orgânica tendo sua extração ambientalmente correta, porém este baixo nível tecnológico favorece o crescimento de fungos potencialmente produtores de micotoxinas como os do grupo Aspergillus section flavi. Visando reduzir esta contaminação, uma das possibilidades é a utilização de óleos essenciais, que apresentam propriedades antimicrobianas. Assim, o objetivo do presente trabalho foi avaliar a atividade antifúngica de 11 óleos essenciais de espécies medicinais e aromáticas sobre cepas aflatoxigênicas de A. flavus, A. nomius e A. arachidicola isoladas de castanha do Brasil. O ensaio de microdiluição mostrou que os óleos que apresentaram melhor atividade antifúngica contra estes os isolados foram Cinnamomum burmannii e Eugenia caryophyllata, com MIC de 250 'mu'g/mL e 500 'mu'g/mL, respectivamente. A inibição do crescimento micelial dos fungos foi melhor quando estes foram submetidos à ação por contato com os óleos essenciais, do que por ação de seus compostos voláteis, sendo as concentrações referentes às de MIC capazes de inibir totalmente o crescimento dos fungos. A produção de aflatoxina por A. flavus foi inibida somente pelo óleo essencial de E. caryophyllata, na concentração de 500 'mu'g/mL. Ensaios in vivo conduzidos com a casca da castanha do Brasil mostraram que o óleo essencial de C. burmannii inibiu o fungo a 500 'mu'g/mL e propiciou maior redução na porcentagem de infecção pelos isolados de A. flavus. Quanto aos isolados de A. nomius e A. arachidicola, a menor porcentagem de infecção foi obtida após tratamento com o óleo essencial de E. caryophyllata na concentração de 1000 'mu'g/mL. Os óleos de E. caryophyllata e C. burmanni mostraram ser alternativas naturais para controle do crescimento de A. flavus, A. nomius e A. arachidicola e da produção de aflatoxinas por A. flavus, através da ação na redução da contaminação fúngica das castanhas do Brasi / Abstract: The Brazil nut (Bertholletia excelsa H.B.K.) is the second most important forest source of the Amazon rainforest. Thus, no chemical pesticides are used and the production of Brazil nuts is considered organic and an environmentally friendly extraction. However, this low technological level favors the growth of potentialy mycotoxin producing fungi such as Aspergillus section flavi. In order to reduce this contamination, one possibility is the use of essential oils that exhibit antimicrobial properties. The objective of this study was to evaluate the antifungal activity of 11 essential oils of medicinal and aromatic species on aflatoxigenics strains of Aspergillus flavus, A. nomius and A. arachidicola isolated from Brazil nuts. The microdilution assay showed that the oils from Cinnamomum burmannii and Eugenia caryophyllata presented the best antifungal activity against all isolates, with MIC of 250 'mu'g/mL and 500 'mu'g/mL, respectively. The inhibition of mycelial growth of the fungi was better when submitted to action by contact than by action of oils volatile compounds, with concentrations of MIC able to completely inhibit the growth of fungi. The aflatoxin production was inhibited only by E. caryophyllata essential oil at a concentration of 500 'mu'g/mL . In vivo assays conducted with shells of Brazil nut showed that the essential oil of C. burmannii at 500 'mu'g/mL provided greater reduction in the percentage of infection by A. flavus isolates. As the isolates of A. nomius and A. arachidicola, the infection rate was lower after the treatment with the essential oil of E. caryophyllata in the concentration of 1000 'mu'g/mL. The essential oils of E. caryophyllata and C. burmannii showed to be natural alternatives for controlling the growth of A. flavus, A. nomius and A. arachidicola and aflatoxin production by A. flavus, through the reduction of the fungal contamination of Brazil nuts / Mestrado / Ciência de Alimentos / Mestra em Ciência de Alimentos

M.Tech. (Biomedical Technology) / Aflatoxins (AFs) are naturally occurring secondary metabolites produced principally by Aspergillus flavus and Aspergillus parasiticus in food and feed commodities worldwide. Contaminations of compound feeds by AFs do not only affect animal health, but the economy as well. It is for this purpose that a study was carried out to establish the quality of South African feeds with respect to AF-producing fungi, establish a correlation between levels of AFs and determinant gene (nor-1) responsible for producing these toxins. To this end, compound feeds (n=92) from various feed manufacturers in South Africa were sampled and analysed for aflatoxigenic fungi (Aspergillus flavus and Aspergillus parasiticus) and nor~1 genes using the conventional identification and real time- polymerize reaction (RT-PCR) methods, respectively. Data obtained revealed that 66.5 and 53.1% of samples were positive for A. flavus and A. parasiticus, respectively. Aflatoxins levels in similar samples were estimated by high performance liquid chromatography (HPLC) following an immune-affinity clean-up and multi mycotoxin extraction procedures. Accordingly, levels established ranged from 0.06 – 77.97 ppb (mean: 16.8 ppb) with feeds for poultry being the main contaminating substrate and no correlation (overall R2=0.093) was established between the concentrations of AFs and those of nor~1. The cytotoxic effect of some selected AF extracts from these feeds on human lymphocyte cells was performed in comparison to that of AFB1 standard. Data obtained from the cytotoxic assay revealed that cell viability was affected significantly (P<0.001) by both the dose and duration of exposure, which was much more noticeable when cells were exposed to AFB1 standard than for individual extracts. In conclusion, even though none of the feeds analysed contained levels of AFs above regulatory limits established in South Africa, such feeds when consumed on a continuous basis may pose some serious health problems especially when AFs is found in co-contamination with such significant mycotoxins as ochratoxins (OTs) and fumonisins (FBs). Thus, the continuous need to limit AFs levels in feed commodities from South Africa is imperative.

This thesis presents the development of chlorophyll, cholic acid, aflatoxin B1 molecularly imprinted polymer (MIP) particles and cholic acid MIP nanofibers for application as selective solid phase extraction (SPE) sorbents. The particles were prepared by bulk polymerization and the nanofibers by a novel approach combining molecular imprinting and electrospinning technology. The AFB1 MIP particles were compared with an aflatoxin specific immunoextraction sorbent in cleaning-up and pre-concentrating aflatoxins from nut extracts. They both recorded high extraction efficiencies (EEs) of > 97 % in selectively extracting the aflatoxins (AFB1, AFB2, AFG1 and AFG2). High reproducibility marked by the low %RSDs of < 1% and low LODs of ≤ 0.02 ng/g were calculated in all cases. The LODs were within the monitoring requirements of the European Commission. The results were validated with a peanut butter certified reference material. The chlorophyll MIP on the other hand selectively removed chlorophyll that would otherwise interfere during pesticide residue analysis (PRA) from > 0.6 to <0.09 Au in green plants extracts. The extracted chlorophyll was removed to far below the level of ≥ 0.399 Au that is usually associated with interference during PRA. Furthermore, the MIP demonstrated better selectivity by removing only chlorophyll (> 99%) in the presence of planar pesticides than the currently employed graphitized carbon black (GCB) that removed both the chlorophyll (> 88%) and planar pesticides (> 89%). For the interfering cholic acid during drug residue analysis, cholic acid MIP electrospun nanofibers demonstrated to be more sensitive and possessing higher loading capacity than the MIP particles. 100% cholic acid was removed by the nanofibers from standard solutions relative to 80% by the particles. This showed that the nanofibers have better performance than the micro particles and as such have potential to replace the particle based SPE sorbents that are currently in use. All the templates were optimally removed from the prepared MIPs by employing a novel pressurized hot water extraction template removal method that was used for the first time in this thesis. The method employed only water, an environmentally friendly solvent to remove templates to ≥ 99.6% with template residual bleeding of ≤ 0.02%.

<p>Aflatoxins (AFs) are highly toxic second metabolites produced by <i>Aspergillus flavus </i>and<i> A. parasiticus</i>. They are widely detected in cereals, spices, and drinks worldwide. Aflatoxin contamination of foods and crops poses a high health risk for humans and livestock. It is well known that environmental conditions and substrates could influence fungal growth and aflatoxin production. This study tested the effect of water activity (0.82, 0.86, 0.90, 0.94, and 0.98 a_w) and incubation temperatures (20°, 27°, and 35°C) on the growth and aflatoxin production of <i>A. flavus </i>and <i>A. parasiticus</i> on ground flax seeds and ground niger seeds. The effect of oil contents of ground niger seeds on fungal growth and aflatoxin production was also investigated in this study.</p><p> These two fungal species could not grow on any of the tested substrates with 0.82 a_w at 20°, 27°, or 35°C. <i>Aspergillus flavus</i> grew most rapidly on flax seeds with 0.90 a_w at 27°C and also 0.94 a_w at 27° or 35°C. However, on niger seeds, <i>A. flavus </i>grew best at 0.90 or 0.94 a_w incubated at 35°C as well as at 0.94 or 0.98 a_w incubated at 27°C. <i>Aspergillus parasiticus</i> showed the optimum growth on flax seeds with 0.90 a_w at 35°C, whereas on niger seeds, the optimum occurred on seeds with 0.90 a_w at 35°C and also on seeds with 0.94 a_w at 27° or 35°C. The optimum conditions for <i>A. flavus </i>to produce high levels of aflatoxins (270-299 μg/kg) on flax seeds were 0.90 a_w at 35°C; whereas, the optimum conditions for <i>A. flavus </i>to produce aflatoxin (203-278 μg/kg) on niger seeds were 0.90 or 0.98 a_w at 27°C and also 0.90 a_w at 35°C. <i>Aspergillus parasiticus</i> produced high levels of aflatoxins (284-365 μg/kg) on flax seeds under the following three conditions, 0.86 or 0.98 a_w at 35°C and 0.94 a_w at 27°C; <i>A. parasiticus</i> produced 200-384 μg/kg of aflatoxins on niger seeds under nine out of 12 tested incubation conditions.</p><p> Reducing mean oil contents from 35.2 to 10.5% of ground niger seeds had very little effect on the growth of the two fungi but significantly decreased their aflatoxin production under certain incubation conditions. On de-oiled niger seeds inoculated with <i>A. flavus</i>, only 13μg/kg of AFB1 was found on seeds with 0.94 a_w at 27°C; whereas, on de-oiled niger seeds inoculated with <i>A. parasiticus</i>, high levels of aflatoxins (245-345 μg/kg) were only detected under the three following incubation conditions, 0.90 or 0.94 a_w at 27°C, and 0.86 a_w at 35°C.</p> This study showed that the optimum growth and aflatoxin production by <i>A. flavus </i>and<i> A. parasiticus </i>were not identical and influenced by incubation conditions, including temperature, water activity, and growth substrates. The results of this study could help establish guidelines for post-harvest and storage conditions for oil seeds to prevent fungal growth and aflatoxin formation.

Supplemental feeding of deer is a common management action. However, concentrating animals, as feeding does, is known to promote the transmission of disease. We examined how feeding alters three sources of disease: aflatoxins, gastrointestinal parasites, and ticks. To do this, we paired 79 feeder sites throughout Mississippi with ecologically-equivalent sites without feeders. Wildlife visitation increased at feeders compared to sites without feeders. For aflatoxins, we sampled during the summer and hunting season and found low prevalence and levels in feeders and bagged/bulk feed. The greater concern was environmental exposure to aflatoxins. All corn piles exposed to environmental contamination in July contained toxic levels of aflatoxins after eight days. The environmental load of gastrointestinal parasites was elevated for coccidia (4x) and strongylids (3x). Finally, feeding reduced the number of ticks at feeder sites, but did not alter the prevalence of tick-borne diseases within captured ticks compared to sites without feeders.

Lipid oxidation is one of the leading causes of food quality degradation. Manufacturers typically add antioxidants or purge a product's package of oxygen to inhibit oxidation and the resulting off-flavors. Synthetic antioxidants (e.g. BHT, BHA) and some natural antioxidants (e.g. α-tocopherol) have found widespread use in this application. Unfortunately, the public views synthetic additives in a negative light and the current natural antioxidants have been unable to match the protection afforded by the synthetic antioxidants. The search for underutilized and natural antioxidants has led scientists to investigate many different plant-based extracts for use in food and in the treatment and prevention of disease. The objectives of this research were (1) to use ORAChromatography to identify peanut root extract fractions with high antioxidant capacity, (2) identification of compounds in peanut root extracts using HPLC and mass spectrometry, (3) test for the presence of aflatoxins in the extracts, (4) test peanut root extract in food model system for oxidation reduction capabilities, and (5) Testing peanut root extract's ability to decrease protein oxidation in cell culture. Crude peanut root extracts have high antioxidant activities that do not vary by cultivar. The ORAC activities of the peanut root fractions separated by HPLC with a C18 column varied (600.3 – 6564.4 μM TE/g dry extract), as did the total phenolic contents (23.1 – 79.6 mg GAE/g dry extract). Peanut root fractions had aflatoxins contamination well above the 20 ppb limit. Peanut root extracts and the known antioxidants tested were found to have no significant effect in inhibiting oxidation of peanut paste or HBMEC. Peanut root extracts were not shown to have any positive effects, but further research is necessary to eliminate peanut root extracts as a possible food ingredient and health supplement. / Ph. D.

This study assessed the mycological and aflatoxin contamination of peanuts collected from Kinshasa, DRC and Pretoria, South Africa. Forty peanut samples were collected randomly at informal markets in the two cities and analysed for mycoflora and aflatoxins (B1, B2, G1 and G2) using standard methods. The results indicated that 95% and 100% of peanut samples collected from Kinshasa and Pretoria, respectively were contaminated with aflatoxigenic fungi with Kinshasa’s samples being the most contaminated (up to 49, 000 CFU/g). Seventy percent (70 %) of Kinshasa-samples and 35% of Pretoria-samples exceeded the maximum allowable limit of aflatoxin B1 set by JECFA (5 ppb). Statistical evidence showed a significant positive correlation between mycoflora and aflatoxin level for Kinshasa-samples (r = 0.4743, p < 0.005) while Pretoria-samples showed no correlation. The study reveals that high level of contamination in Kinshasa-samples could be due to the tropical nature of the climate and poor storage conditions as compared to Pretoria which is sub-tropical and sanitary regulations are enforced. / Life & Consumer Sciences / M. Sc. (Life Sciences)

This study assessed the mycological and aflatoxin contamination of peanuts collected from Kinshasa, DRC and Pretoria, South Africa. Forty peanut samples were collected randomly at informal markets in the two cities and analysed for mycoflora and aflatoxins (B1, B2, G1 and G2) using standard methods. The results indicated that 95% and 100% of peanut samples collected from Kinshasa and Pretoria, respectively were contaminated with aflatoxigenic fungi with Kinshasa’s samples being the most contaminated (up to 49, 000 CFU/g). Seventy percent (70 %) of Kinshasa-samples and 35% of Pretoria-samples exceeded the maximum allowable limit of aflatoxin B1 set by JECFA (5 ppb). Statistical evidence showed a significant positive correlation between mycoflora and aflatoxin level for Kinshasa-samples (r = 0.4743, p < 0.005) while Pretoria-samples showed no correlation. The study reveals that high level of contamination in Kinshasa-samples could be due to the tropical nature of the climate and poor storage conditions as compared to Pretoria which is sub-tropical and sanitary regulations are enforced. / Life and Consumer Sciences / M. Sc. (Life Sciences)

Thesis (M.Tech.: Biotechnology)- Dept. of Biotechnology & Food Technology, Durban University of Technology, 2007 xv, 102 leaves / For decades the genus Aspergillus (of fungi) has been classified based on morphological and growth criteria. Members of the Aspergillus section Flavi are economically valuable and methods of differentiating them are thus very important. Several molecular methods have been developed to distinguish these strains. Also, a number of biochemical and genetic studies have been used in order to provide a better means of classification (Lee et al., 2004). Aflatoxins, the most frequently studied mycotoxins, are produced by certain Aspergillus species/strains/isolates of fungi. The aflatoxin biosynthetic pathway studies have led to a number of discoveries. Several structural and regulatory genes (and their enzymes) involved in the biosynthesis of aflatoxins have been discovered and purified (Trail et al., 1995). Aflatoxin production and contamination of agricultural crops are major causes of economic losses in agriculture. Thus, better methods of characterization/differentiation are required for both aflatoxigenic and non-aflatoxigenic isolates. Molecular biology is one of the current tools used to differentiate between these isolates. Polymerase Chain Reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) analysis has been used successfully in the analysis of DNA relatedness of species of fungi, bacteria, plants and animals. Dendograms which evaluate/assess the likeness between different isolates has also been used (Martinez et al., 2001). Restriction fragment length polymorphism (RFLP) analysis has been applied to a number of studies to detect differences between fungi and to establish relationships between them. Therefore, the scope of this study was to investigate RAPD analysis (with dendograms) and detection of RFLPs by hybridization as molecular methods that can distinctly differentiate or characterize the aflatoxigenic and non-aflatoxigenic Aspergillus isolates.