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Item Control of citrinin and pigments produced by Monascus purpureus during the fermentation of red fermented rice : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatu Campus, New Zealand(Massey University, 2024) Abdul Halim, FarawahidaMonascus spp. is a fungal starter used to produce red fermented rice (RFR). This product has some health benefits and contains pigments that can act as natural colour and flavouring agents. However, Monascus spp. can also produce the mycotoxin, citrinin (CIT) which is believed to have adverse effects on human health. CIT in RFR has been reported worldwide by using different methods of detection. In this current research, Coconut Cream Agar (CCA) was developed as a simple and rapid semiquantitative method to screen CIT–producing Monascus spp. isolates from RFR. Two Monascus purpureus isolates, MF1 and MS1, were isolated. These isolates were identified based on the macroscopic and microscopic observations, and deoxyribonucleic acid (DNA) sequencing [internal transcribed spacer (ITS) and beta–tubulin (β–tubulin) genes]. Further analysis showed that these isolates contained polyketide synthase (pksCT) and ctnA genes, which are CIT biosynthesis genes. These isolates exhibited fluorescence on CCA and were confirmed as CIT producers by Ultra–high performance liquid chromatography with a fluorescence detector (UHPLC–FLD). A toxicity test was conducted using Artemia salina to determine the toxicity of CIT. The results showed that LC50 was 66 µg/mL. The fungal growth, CIT, pigments production, and pH of M. purpureus isolates were characterized on CCA for 30 days. Decreasing CIT levels were observed after incubation of MF1 and MS1 on CCA for 8 and 7 days, respectively. The pigments increased during this incubation time. There were similar trends for CIT and pigments observed during the production of RFR. CIT increased to a maximum level after 5 days of incubation and pigments increased from 5–9 days. There appears to be a relationship between pigments production and CIT levels during the growth of M. purpureus. Mixing CIT and pigments extracted from MF1 and MS1 resulted in a reduction of CIT by 26–68% and 16–45%, respectively. The results on specific pigments and their effect on CIT showed that ankaflavin, monascin, monascorubrin, rubropunctatin, and monascorubramine significantly reduced CIT, with the highest reduction produced by ankaflavin. The findings of this study suggest pigments production could be optimized to control CIT levels in Monascus–fermented products.Item Monascus spp. and citrinin: Identification, selection of Monascus spp. isolates, occurrence, detection and reduction of citrinin during the fermentation of red fermented rice(Elsevier BV, 2022-10-16) Farawahida AH; Palmer J; Flint SRed fermented rice (RFR) is rice fermented using Monascus spp. This product contains monacolin K, providing health benefits including mitigation of diarrhoea and improving blood circulation. RFR can produce pigments that can act as natural colour and flavouring agents. However, Monascus spp. (a fungal starter to ferment RFR) can also produce the mycotoxin, citrinin (CIT) which is believed to have adverse effects on human health. CIT in RFR has been reported worldwide by using different methods of detection. This review focuses on the production of RFR by solid-state fermentation (SSF) and submerged fermentation (SmF), the occurrence of CIT in RFR, CIT quantification, the factors affecting the growth of Monascus spp., pigments and CIT production in RFR, and possible methods to reduce CIT in RFR. This review will help the food industries, researchers, and consumers understand the risk of consuming RFR, and the possibility of controlling CIT in RFR.Item Synthesis of an aryl phosphonate via the anionic phospho-fries rearrangement : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at Massey University, Palmerston North, New Zealand(Massey University, 2002) Watson, Amy JaneZearalenone (1) is a mycotoxin, which reduces fertility in sheep and leads to substantial production losses in New Zealand. Catalytic antibodies are proposed as a potential approach to reducing the problem. This thesis describes progress toward the synthesis of the aromatic fragment of a transition-state analogue for the hydrolysis of the zearalenone lactone. The key step in the synthesis is an anionic phospho-Fries rearrangement; this relatively novel transformation is reviewed. α-Resorcylic acid (36) was converted to three different substrates, each with potential to undergo the O→C transfer of the dimethylphosphoryl moiety. Methyl 3-dimethylphosphato-5-[[(1,1- dimethylethyl)dimethylsilyl]oxy]benzoate (117) was prepared in three steps and 40% overall yield, but the tert-butyldimethylsilyl (TBDMS) protecting group was found to be unstable to phenolate anions. (2-Bromo-3,5-dibenzyloxyphenyl)-1,3-dioxolane (154) was prepared but further elaboration required hydrogenolytic cleavage of the benzyl ethers which was incompatible with the Ar-Br linkage. Finally, Ethyl 3- dimethylphosphate-5-(p-methoxybenzoxy)benzoate (167) was prepared in three steps and 25% overall yield. Treatment with LDA at -78 °C led to the formation of 166, rather than the desired regioisomer 165, as a result of lithium preferentially coordinating to the phosphoryl and OPMB groups.Item Identification of putative dothistromin biosynthetic genes : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biology at Massey University, Palmerston North, New Zealand(Massey University, 1998) Monahan, Brendon JosephDothistromin is a polyketide-derived mycotoxin produced by the Pinus pathogen Dothistroma pini, and is thought to be important in the development of the necrotic disease Dothistroma needle blight. Targeted disruption of dothistromin biosynthetic genes will allow the direct assessment of the role of the toxin in D. pint pathogenicity. Dothistromin displays structural and biochemical similarities to the aflatoxins (AF) and sterigmatocystin (ST) which are produced by various Aspergillus species. In our laboratory, knowledge from the well characterised ST/AF pathway is being used to isolate and characterise genes likely to be involved in dothistromin production. The D. pini lambda clone, λCGV1. was isolated from a D. pini genomic library by heterologous hybridisation with a fragment of the Aspergillus parasiticus ver 1 gene (Gillman, 1996). In this study, the complete nucleotide sequence of XCGV1 was determined. Analysis revealed that five genes are located within the 13.3 kb genomic region sequenced Three of these genes (dkr1, dox1 and dte1) display strong similarities to genes contained within the ST/AF biosynthetic gene clusters. The dtp1 gene, located between dox1 and dtp 1, shows similarities to transmembrane efflux pumps and is proposed to be a dothistromin toxin pump. The ddh1 gene, located upstream of dkr1, shows similarities to bacterial dehydrogenases. However, the ddh1 coding sequence contains a premature stop codon (encoding a product of 63 amino acids), indicating that the product may be non-functional. Expression analysis of each gene identified in this study confirmed that dkr1, dox1, dte1 and dtp1 are expressed. However, no obvious expression was detected for the ddh1 gene. Southern blot analysis confirmed the genomic clustering of the genes and indicated that a single copy of each gene was present in the D. pini genome. Due to the biogenetic relationship between dothistromin and ST/AF biosynthesis, and because genes identified in this study show similarities to genes involved in ST/AF production, it is thought that these genes are likely to be involved in dothistromin biosynthesis and constitute part of a dothistromin biosynthetic gene cluster.Item Paxilline negative mutants of Penicillium paxilli generated by heterologous and homologous plasmid integration : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand(Massey University, 1998) Young, Carolyn AnneUsing a monoclonal antibody-based ELISA, 600 pAN7-1 plasmid-tagged mutants of Penicillium paxilli were screened for paxilline accumulation and one paxilline negative mutant, YI-20, was identified (Itoh, unpublished data). A molecular analysis of this mutant showed that pAN7-1 was inserted at a single site but was present as 4-6 copies arranged in a head-to tail tandem repeat. Rescue of flanking sequences and analysis of the corresponding genomic region revealed that YI-20 has an extensive deletion at the site of pAN7-1 integration. Probing of a CHEF gel with the same sequences showed that associated with the deletion is a rearrangement of chromosome Va. Targeted gene disruption of wild-type sequences adjacent to the site where pAN7-1 inserted, resulted in the generation of two additional paxilline-negative mutants; both were single crossovers with deletions extending outside the region mapped. Neither of these new mutants had a rearrangement of chromosome Va, suggesting that deletion of genes on this chromosome is responsible for the paxilline-negative phenotype.Item Further characterisation of the Dothistromin gene cluster of Dothistroma pini : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand(Massey University, 2004) Teddy, Olivia RachelThe polyketide dothistromin is a toxin produced by the filamentous fungus Dothistroma pini that is thought to play a role in causing Dothistroma needle blight in Pinus radiata. Dothistromin is structurally similar to aflatoxin B1 (AF), a highly carcinogenic toxin with no known function that is produced by the fungus Aspergillus parasiticus and also to versicolorin, an intermediate of the well characterised biosynthetic pathways of AF and sterigmatocystin (ST). The structural similarities between AF/ST and dothistromin suggest that genes homologous to AF biosynthetic genes will be involved in dothistromin biosynthesis. AF/ST biosynthetic genes of A. parasiticus and A. nidulans are clustered and hence it is likely that the dothistromin biosynthetic genes are also clustered in a similar manner. Two λ clones, λKSA and λCGV1 containing portions of the putative dothistromin cluster have been isolated in previous studies. Another λ clone λCGV2 was also identified using an aflatoxin gene probe but it is unknown whether it is part of the dothistromin biosynthetic cluster. The λKSA clone contains part of a putative polyketide synthase pks dot (64% identical to A. parasiticus AF biosynthetic gene pksA). Two crucial domains required for functioning are contained within λKSA, the β-keto acyl synthase (KS) and acyl transferase (AT) domains. The putative pks dot is thought be involved in the beginning of the dothistromin biosynthetic pathway, working in a complex with a fatty acid synthase (FAS) to produce the intermediate noranthrone. A gene replacement construct was made using Multisite Gateway TM Recombination, replacing the AT and KS domains with an hph cassette. Disruption of the pks dot gene will confirm it's involvement in dothistromin biosynthesis and could also confirm the role of dothistromin in pathogenicity as if the putative polyketide synthase (pks dot ) is involved in the first step of the dothistromin pathway thus a knockout would form a mutant devoid of any intermediates. Confirming the involvement of pks dot would also provide evidence that like λCGV1, λKSA contains a portion of the dothistromin biosynthetic gene cluster. As the positioning of the three lambda clones λKSA, λCGV1 and λCGV2 relative to one another in the D. pini genome was unknown Southern blot analysis was implemented to identify any relationship between the three lambda clones. No evidence was found to suggest the close linkage of the three lambda clones however this does not discount any linkage at all. Southern blot analysis did provide evidence that ver-2 (77% identity to melanin biosynthetic gene phn1 of Cochliobolus heterostrophus) of λCGV2 is within dose proximity to a putative aflR gene (regulatory gene for activating gene transcription in AF/ST biosynthesis) suggesting a regulatory role of this putative aflR gene in melanin biosynthesis and not dothistromin biosynthesis. Further nucleotide sequencing of the λKSA clone revealed three putative dothistromin genes. Mox dot and ord dot have high amino acid identity to genes involved in the AF/ST pathways (70% identity to moxY and 51% identity to avfA of A. parasiticus respectively), suggesting similar roles in dothistromin biosynthesis. Epox dot showed high amino acid identity to an epoxide hydrolase of A. niger (hyll) suggesting it has a unique role in dothistromin biosynthesis as no homologs are seen in the AF/ST clusters. Southern blotting was also used to confirm the arrangements of genes from the λKSA clone within the D. pini genome. Further characterisation of genes involved in dothistromin biosynthesis will firstly enable understanding of the role of dothistromin in needle blight and secondly will enable further comparative studies between AF/ST and dothistromin.Item Identification of dothistromin biosynthetic pathway genes : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand(Massey University, 1996) Gillman, Carmel JaneDothistromin is a polyketide-derived toxic secondary metabolite produced by the filamentous fungus Dothistroma pini which causes the disease Dothistroma needle blight in Pinus radiata. Dothistromin is considered to be an important component in the disease process, although its exact function is yet to be identified. By isolating and identifying genes involved in dothistromin biosynthesis, and subsequently obtaining mutants blocked or altered in the synthesis of dothistromin, the role of this toxin in pathogenicity will be able to be assessed. Dothistromin is structurally related to the mycotoxins, aflatoxin (AF) from Aspergillus parasiticus and A. flavus, and sterigmatocystin (ST) from A. nidulans. Three intermediates in the ST and AF biosynthetic pathways (averantin, averufin, and versicolorin B) are thought to also be intermediates dothistromin biosynthesis. Due to these similarities, cloned AF pathway genes were used as heterologous probes in Southern hybridisation analysis to provide a direct method for identifying dothistromin biosynthetic genes. A fragment of the A. parasiticus nor-1 gene, encoding a reductase involved in the conversion of norsolorinic acid (NA) to averantin (AVN) in the AF biosynthetic pathway, was used as a probe to detect a region of sequence similarity to D. pini genomic DNA. A D. pini genomic library was then constructed and screened, resulting in clone λCGN2. However, Southern hybridisation analysis suggested that this clone did not contain a homologue of the nor-1 gene from A. parasiticus. A fragment of the Aspergillus parasiticus ver-1 gene, encoding a reductase involved in the conversion of versicolorin A (VA) to ST in the AF biosynthetic pathway, was also used as a probe to detect a region of sequence similarity to D. pini genomic DNA. The D. pini genomic library was then screened. Two clones, λCGV1 and λCGV2, were isolated and Southern hybridisation analysis confirmed that these clones contained sequences hybridising to the A. parasiticus ver-1 gene fragment. Fragments of these clones which hybridised were then sequenced and compared to the GenBank database. The amino acid coding sequence of a 0.8 kb SalI region from clone λCGV1 exhibited a high degree of similarity with the A. nidulans verA and A. parasiticus ver-1 genes, involved in the ST and AF biosynthetic pathways, and the Magnaporthe grisea ThnR, and Colletotrichum lagenarium Thr1 genes, involved in melanin biosynthesis. This data suggested a ver-1 homologue is present in the D. pini genome. Limited sequence analysis of a 2.1 kb region from clone λCGV2 suggested that a second independent copy of a ver-1-like gene may also be present in the genome.Item Characterisation of a putative dothistromin biosynthetic cluster : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Biology at Massey University, Palmerston North, New Zealand(Massey University, 1999) Laarakkers, SethThe fungus Dothistroma pini is a key pathogen in New Zealand (and international) softwood plantations, most notably P. radiata. The mycotoxin dothistromin produced by this saprophytic fungus is believed to play a major role in its pathogenesis. Dothistromin shares functional groups and pathway intermediates with those of sterigmatocystin and aflatoxin, secondary metabolites of Aspergillus sp. As the sterigmatocystin and aflatoxin biosynthetic pathways are characterised this provided us with a model pathway and potential probes for the isolation of dothistromin genes. The verl gene is critical to the completion of aflatoxin biosynthesis in Aspergillus sp. as its disruption prevented the synthesis of aflatoxin. Assuming similar enzymes act in the dothistromin biosynthetic pathway a probe for ver1 was obtained and used to probe a D. pini genomic library. This led to the isolation of two lambda clones named λCGV1 and λCGV2 (Gillman 1996). A second library screen was completed using an aflatoxin polyketide synthase (PKS) probe and led to the isolation of the lambda clone λBMKSA (Morgan 1997). The λCGV1 clone has been studied in detail and shown to contain a gene similar to aflatoxin ver1 (named dkr1) and other potential dothistromin biosynthetic genes (Monahan 1998). This study looks in greater detail at the lambda clones λCGV2 and λBMKSA and determines whether they contain putative dothistromin biosynthetic genes and are part of the anticipated gene cluster. In this project the lambda clone λCGV2 was partially characterised which revealed that the other potential ver gene showed a greater similarity to the melanin biosynthetic gene phn than to the aflatoxin gene ver-1. This implied that the clone was unlikely to contain dothistromin biosynthetic genes so no further sequence was generated. However, a partial restriction map was constructed. The other lambda clone, λBMKSA was then further characterised. Double stranded sequence of the putative pks gene region was completed. The remainder of the lambda clone was subcloned and exploratory sequence revealed a gene with high similarity to stcW. The next stage was to determine how the three lambda clones were related. This was approached by probing genomic Southern blots with the ends of the lambda clones to determine the presence of commonly hybridised fragments. The presence of common fragments suggests that the three clones are very close together in the genome, although the evidence which links λCGV2 and λBMKSA is stronger than the evidence that links λCGV2 and λCGV1. This is the first evidence that the three lambda clones isolated using aflatoxin probes are close together in the genome of D. pini. The genes present on these lambda clones show a high degree of similarity to their aflatoxin counterparts and could potentially contain a dothistriomin biosynthetic cluster.Item Zearalenone in pasture and its effects on reproduction in ewes : a thesis presented in partial fulfilment for the degree of Master of Applied Science in Animal Science at Massey University(Massey University, 1997) Kramer, RichardZearalenone is an oestrogenic mycotoxin which has the potential to cause reproductive disorders in sheep. Zearalenone-producing Fusarium species are present in New Zealand pasture and it is likely that the amount of zearalenone present during the mating period may be sufficient to cause reproductive dysfunction in the grazing sheep. This study consisted of three trials which aimed to measure zearalenone levels in the pasture and sheep, and determine the subsequent effects on reproductive performance. The first trial investigated the levels of zearalenone during April in various components of the ryegrass plant at various pasture sites, which included urine-patch, dung-patch and inter-excreta sites. Zearalenone taken up by the ryegrass plant was also determined. The second trial comprised of 6 groups of ewes (n=10), and compared levels of zearalenone and related metabolites in the blood and urine of ewes grazed on pasture or chicory and either orally (5 mg/ewe)or intravenously dosed (2 or 0.5 mg/ewe) daily with zearalenone. The subsequent effects on ovulation rate, conception rate, and number of lambs carried was also determined. The third trial comprised of 4 groups (n=110) of ewes, of which two groups were grazed on grass-dominant pasture and the remaining 2 groups were grazed on chicory for two weeks prior to mating at which time one of the groups on each grazing treatment was interchanged and the ram introduced. The levels of free and conjugated zearalenone in the blood and urine were determined and the subsequent effects on ovulation rate, conception rate and the number of lambs carried were measured. In the first trial it was shown that zearalenone concentration within sites was highly variable at that time of the year, however, urine-patch and dung patch sites yielded significantly higher quantities of zearalenone. Zearalenone appeared to be readily taken up by the ryegrass plant through the roots and translocated into the young growing tissue of the plant. The distribution of zearalenone in the pasture and the plant are discussed with regards to zearalenone intake by the animal. The zearalenone dosing trial showed that significant levels of zearalenone, α-and α-zearalenol, zeranol and taleranol were present in the blood and urine of dosed ewes and that levels of all compounds analysed were higher in ewes grazed on pasture. Ewes grazing pasture had a significantly lower (P<0.05) ovulation rate than ewes grazed on chicory. The third trial showed that chicory was effective in reducing the levels of free zearalenone present in the ewe around the time of mating with levels in ewes grazed on chicory being significantly lower (P<0.05) in both the urine and blood, than in ewes grazed on grass pasture. There were no significant differences in reproductive performance. Zearalenone levels in the pasture were generally lower in 1995 than in previous years and might have reduced possible differences in reproductive performance between ewes on the different feed types. The implications of higher zearalenone concentrations in the pasture are discussed with regards to reproductive performance and the use of chicory as a feed prior to mating. Further research is required to identify and clarify links with zearalenone and metabolites produced in pasture and reproductive dysfunction in ewes.Item Toxigenic fungi and mycotoxin production in Maldive fish (smoked dried tuna fish) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology, Massey University, Palmerston North, New Zealand(Massey University, 2013) Mohamed, ShazlaThis is the first study on the mycological safety of “Maldive fish”, a smoked dried tuna product that is both economically and nutritionally important to the Maldives. The most obvious concern with this product is the effect of fungal contamination. The initial aim of the current study was therefore to determine if Maldive fish supports the growth of toxigenic fungi and production of mycotoxins. The uncontrolled mycoflora on the product were characterised and related to the physiological parameters of the Maldive fish. Ninety six percent of the samples (n=25) were contaminated with one or more mycotoxigenic fungi with Aspergillus flavus (92%), A. tamarii (96%), A. niger (40%), A. ochraceus (12%) and Penicillium citrinum (60%) identified as the significant species. Subsequently, the potentially toxigenic isolates were screened for their corresponding mycotoxins aflatoxins, ochratoxin A (OTA), cyclopiazonic acid (CPA) and citrinin. A high proportion (72%) of isolates was able to produce toxic metabolites in vitro indicating possible contamination of the product with mycotoxins. Almost half (46%) of the A. flavus isolates were able to produce the potent carcinogen, aflatoxin B. All species on the surface were also found invading the product. The huge variability in aw levels (0.951 to 0.720) of the samples would support growth of a wide range of species. Furthermore, the slightly acidic pH (5.65 to 6.68) and low salt content (1.48 to 4.29%) together with the high ambient temperatures of the Maldives were eminently suitable for fungal growth and mycotoxin production. Quantification of aflatoxins from the product revealed two of the 25 samples to be contaminated above the legal limits and confirms potential exposure to significant levels of this toxin from Maldive fish infected with fungi. These results led to a new question: can fungal growth and mycotoxin production in Maldive fish be eliminated or reduced to safe levels? The most practical approach would be to reduce the aw to sufficiently low levels that inhibit fungal growth and mycotoxin production. The limiting aw levels for the most important species were therefore evaluated. The limiting aw for growth of A. tamarii was between 0.82 and 0.85 on NaCl media and between 0.79 and 0.75, on media containing sugars at ambient storage temperatures (25 to 35°C). The aw of Maldive fish should be maintained below 0.75 to prevent the growth of A. tamarii. The physiology of A. flavus has been extensively studied previously but the limiting values are dependent on the food matrix. A smoked fish agar was used to simulate Maldive fish for fungal growth (A. flavus) and mycotoxin production (aflatoxin and CPA) under varying conditions. No growth occurred at an aw of 0.75 while the toxin production was limited at an aw 0.80 under all incubation conditions (25°C to 40°C). Hence, control of A. flavus can be achieved by rapid drying of Maldive fish to an aw of 0.75 or below. This study has provided scientific evidence that the mycoflora on Maldive fish produce aflatoxins and other mycotoxins that are a food safety risk. Hence, control of toxigenic fungi is imperative and can be achieved through adequate drying. This information is crucial for the Maldives as well as other developing countries that consume hot smoked dried fish while it potentially has a broader application for other food products.