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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 Biofilm formation of Vibrio parahaemolyticus : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Microbiology at Massey University, Campus Manawatū, New Zealand(Massey University, 2024-05-20) Wang, DanVibrio parahaemolyticus in seafood can cause food poisoning. There is increasing concern with the increase in reports of illness globally believed to be due to climate change affecting sea temperatures. Biofilm formation of V. parahaemolyticus is an additional concern as biofilms are more resistant to cleaning and sanitation than planktonic cells. However, little is known about the biofilm formation of V. parahaemolyticus. Strain variation and the factors determining biofilm formation were investigated in this study with the aim to provide information that can be used to design more effective control strategies. This study identified two robust biofilm forming strains (PFR30J09 and PFR34B02) from nine V. parahaemolyticus seafood isolates. Comparative genome analysis unveiled 136 unique accessory genes in robust biofilm formers. Protein-protein-interaction analysis showed interactions between UDP-glucose metabolism (Gene ontology (GO): 0006011), cellulose biosynthesis (GO: 0030244), rhamnose metabolism (GO: 0019299) and O antigen biosynthesis (GO: 0009243). Cellulose contributed to robust biofilm formation. Cellulose biosynthesis was identified as being acquired from within the order Vibrionales. The cellulose synthase operons consisting of genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC were present in 15.94% (22/138) of V. parahaemolyticus. Strong biofilm-forming V. parahaemolyticus showed greater resistance to sanitizers of biofilm cells than the weaker biofilm forming cells. The effective concentrations of sodium hypochlorite for inactivating most V. parahaemolyticus biofilm cells were higher than the recommended concentration. Available chlorine of 1176 mg/L inactivated 1.74-2.28 log10 CFU/cm2 of biofilm on stainless steel surfaces and 4704 mg/L inactivated > 7.00 log10 CFU/cm2 of biofilm (to undetectable levels, < 10 CFU/cm2), except for biofilms formed by the strong biofilm formers. Peracetic acid (PAA) at 200 ppm (89.56 mg/L PAA, 471.64 mg/L hydrogen peroxide) inactivated > 5.00 log10 CFU/cm2 of biofilm from stainless steel surfaces (except for those the strong biofilm formers, see Figure 4.4). RNA sequencing (RNA-seq) identified 74 differentially expressed genes when comparing planktonic and biofilm cells of V. parahaemolyticus. These represented the rearrangement of nucleotide and energy metabolism in biofilm cells. Biosynthesis of secondary metabolites, purine and pyrimidine metabolism, propanoate metabolism, and valine, leucine and isoleucine degradation were deemed essential in the young V. parahaemolyticus biofilms. Genes of purH, purF, pdhA are potential genetic targets for biofilm prevention and control of V. parahaemolyticus. Understanding V. parahaemolyticus biofilm formation will help to design strategies to overcome the limitations of chemical sanitizers, improving product safety and quality in the seafood industry.Item Consumer perception and behavior toward food safety risk in Vietnam : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Economics at Massey University, Manawatu Campus New Zealand(Massey University, 2020) Ha, Thi Thanh MaiPerception of food safety risk is heightened in Vietnam. The main objective of this thesis is to gain an understanding of consumer perception of food safety risk and the relationship between risk perception and behaviour toward food safety risk in Vietnam. The thesis used the primary data that comes from our survey of 498 consumers and group discussions. Data were collected during 2017 in Hanoi, Vietnam. Results from Structural Equation Modelling (SEM) analysis confirmed that extensive media coverage of food safety scandals decreased trust in institutions and heightened risk perception of common food and risk perception of hazards directly. Negative food safety information indirectly amplified perception of food safety risk in general. Using the mixed method, we found that risk perception was shaped by the fear of hazards, risk perceived from common foods, and food risk information. This finding was supported by those generated from SEM. Region was the most important determinant of risk perception, where urban consumers perceived a higher food safety risk than their rural counterparts. Applying Principle Component Analysis and ordered logit regression, we found differences and similarities in the determinants of vegetable risk perception between the rural and urban regions. The Kruskal-Wallis test shows that higher risk perception was associated with a larger decline in vegetable consumption. To reduce the perceived risk, consumers avoided eating vegetables that were believed to be unsafe and switched to safer ones. We used the contingent valuation method to predict the willingness to pay (WTP) for organic vegetables. Results show that the WTP of urban consumers was higher than that of rural respondents. Perceived values of organic food, trust in organic labels, and income increased the WTP across the regions. Growing own vegetables reduced the WTP in the rural region only. Our findings suggest that regional differences need to be considered when designing risk communication and food safety policy. Urban farming should be encouraged as a mean to reduce food safety concerns in cities.Item Persistent contamination of Salmonella, Campylobacter, Escherichia coli and Staphylococcus aureus at a broiler farm in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Albany, New Zealand(Massey University, 2017) Castaneda, Kristine MejiaThe public demand for poultry products has increased over the years due to their health benefits and relatively low cost. Intensive production of poultry in broiler farms gives an opportunity for contamination of the birds, thus creating potential foodborne hazards to consumers. Foodborne cases are therefore extensively monitored to implement mitigating strategies to control the outbreaks. Therefore, the main aim of this project was to determine the prevalence and microbial loads of contaminating Campylobacter spp., Salmonella spp., S. aureus and E. coli, in different locations of four broiler sheds at a selected poultry farm in Auckland New Zealand. Standard microbiological methods and multiplex quantitative polymerase chain reaction (qPCR) were used in the analyses. Swab samples were collected in three cycles from March 2016 to June 2016. During each cycle of the cleaning and disinfection regime, 248 swab samples were collected from feeders, feed loaders, drinkers, fans, vents, annex floor, and wall crevices to determine the extent of contamination before cleaning and after disinfection. The collected samples (n = 744) were analysed for the presence of Salmonella spp. and Campylobacter spp. using standard microbiological methods. Suspected isolates of Salmonella spp. were confirmed by latex agglutination test, whilst Campylobacter spp. was confirmed by both latex agglutination and oxidase tests. The swab samples were also analysed for viable S. aureus and E. coli cell counts using Petrifilm™ plates. Multiplex qPCR was developed and validated to enumerate Salmonella spp. and Campylobacter spp. positive samples. Results of this study showed that all collected samples were contaminated with Salmonella spp., Campylobacter spp., S. aureus and E. coli before performing cleaning. After disinfection, different areas of the shed were still contaminated, posing real danger for infection of the new flock. Crevices and drinkers were the most contaminated areas after disinfection. Organic matter that accumulates in crevices and drinkers during rearing are likely to protect pathogens against disinfectants, which may then contribute to residual contamination and biofilm formation. The ventilation system of the farm was also heavily contaminated. After disinfection, dusts were trapped between the wires of the ventilation screen, making air vents a potential source of contamination in poultry sheds. Feed loaders had higher contamination rates than feeders, even though it was elevated, away from direct contact to birds. When the ventilation system was open, contaminated dusts settle into various areas of the shed, thereby increasing contamination levels before cleaning, thus affecting the efficacy of the disinfectant used. Meanwhile, fans and the annex were less contaminated, indicating that the cleaning regime could effectively disinfect these areas. However, results showed that microbial concentration in the annex was higher after disinfection. This was probably caused by the introduction of pathogens from the outside environment, highlighting the importance of erecting hygiene barriers before entering the main shed. Multiplex qPCR is an important quantification tool due to its ability to detect, identify and quantify multiple pathogens in one assay. The standard curves generated from inoculated samples determined the detection limit to be 3.24 - 8.24 Log10 CFU/mL for Salmonella spp., and 2.97 - 7.97 Log10 CFU/mL for Campylobacter spp. respectively. The agreement of results using the standard and qPCR methods was investigated by comparing S. aureus counts obtained from100 environmental samples through Bland-Altman analysis. The two methods showed agreement, but the qPCR was limited to the detection of S. aureus from 3.5 to 6 Log10 CFU/mL. The concentration of Salmonella spp. and Campylobacter spp. enumerated by multiplex qPCR, had no significant difference between the mean counts of each location before cleaning and after disinfection. Concentration of Salmonella spp. and Campylobacter spp. in the samples subjected to analysis by qPCR post-disinfection, were below the detection limit of the method. However, the qPCR method may be suitable for analysis of samples collected before cleaning. Pre-enrichment of samples analysed post-disinfection is recommended to improve the detection and enumeration of Salmonella spp. and Campylobacter spp. by qPCR analysis.Item Germination of psychrotolerant clostridia responsible for red meat spoilage : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand(Massey University, 2012) Adam, Katharine HelenPsychrotolerant clostridia are responsible for spoilage of fresh chilled vacuum-packed red meat (beef, lamb and venison). Red meat is one of New Zealand’s primary exports, and spoilage results in financial loss. Spoilage by psychrotolerant clostridia is difficult to control due to the ability of these bacteria to grow at cold temperatures, down to -1.5 °C. They can also form spores that have increased resistance to heat, chemicals, oxygen and desiccation compared to vegetative bacterial cells. As clostridia are strict anaerobes, it is considered highly likely that initial contamination of meat is primarily with spores. The main objective of this work was to determine the triggers of germination of spores, of those psychrophilic and psychrotrophic clostridia, associated with spoilage of New Zealand red meat. Germination of psychrotolerant clostridia was studied using a range of techniques including molecular, in vitro, and on meat methods. In this study in vitro germinant systems were identified for Clostridium frigidicarnis, and a New Zealand species designated LA1, consisting of lactate in combination with an amino acid. Some of the amino acids identified, including valine and cysteine, are naturally present on the surface of red meat. Failure to chill to, or maintain meat at, the recommended temperature, of -1.5 °C and a pH of above 5.5 were identified as being important factors leading to spoilage by Cl. frigidicarnis. Germination in Clostridium estertheticum was extremely poor in media, compared with meat slurry or fresh meat, preventing the identification of a specific germinant system(s), and indicating a non-nutrient factor may be involved. Two distinct nonchemical interventions, hot water wash (HWW) and cold water wash (CWW), were found to reduce spoilage of vacuum-packed chilled lamb inoculated with spores of Cl. estertheticum. Vegetative cells of psychrotolerant clostridia survived exposure to air longer than expected, upwards of seven days in the case of Cl. estertheticum subsp. estertheticum, suggesting that they play a greater role in initial contamination of meat than originally thought. From an industry point of view the results highlight the importance of preventing initial contamination and proper chilling, as well as the need for further investigation of HWW and CWW interventions.Item A novel model developed for quantitative microbial risk assessment in the pork food chain : a dissertation presented in partial fullfilment [sic] of the requirements for the degree of Doctor of Philosophy at Massey University, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, New Zealand(Massey University, 2007) Titus, Simone MeganFood-borne diseases contribute substantially to morbidity and mortality rates worldwide. The deleterious impact of these diseases on human health, concurrent with the associated socioeconomic cost has led to an increased demand for the production of safe food globally. Consequently, agencies such as the World Health Organization (WHO) and the Food and Agriculture Organization (FAO) have resolved to address this issue. In this vein, scientific, risk-based approaches which facilitate estimation of the probability of disease occurrence, the magnitude of the disease and efficacious control measures have been recommended for use internationally. Many pathogens have been implicated as aetiological agents of food-borne disease. The WHO has identified non-typhoidal Salmonella, Escherichia coli and thermophilic Campylobacter as zoonotic food-borne pathogens of greatest importance. These pathogens can be transmitted to humans through pork consumption. This thesis therefore proposes a suite of novel, mechanistic, semi-stochastic, quantitative, modular process risk models describing the propagation of these three pathogens from the live pig at the abattoir, to pork chops sold at retail. The model is developed for use in risk-based, quantitative microbial exposure assessments in New Zealand and can be employed to explore different intervention strategies targeted at mitigating contamination levels of these pathogens on pork chops. The models comprise multiple, coupled, differential and difference equations. These equations explicitly describe bacterial growth, inactivation, removal, cross-contamination and food partitioning occurring in continuous and discrete time in abattoirs and at retail. Distributions of pathogen numbers on the surface of carcasses, and prevalence levels are output by the models at different stages of abattoir processing and pork chop production. Both dressed pork carcases exiting abattoirs in New Zealand and pork chops at retail are predicted to contain low surface contamination levels of the pathogens under consideration, while a small percentage is estimated to be highly contaminated. Median contamination levels on dressed pork exiting the abattoir are predicted to be less than one cfu/cm2. Generally, there are large reductions in surface bacterial numbers for all three organisms from the time the live pig enters the abattoir, to sale of the pork chop at retail. The introduction of a second singeing procedure immediately postevisceration in the abattoir is predicted by our models, to be an effective mitigation strativ egy, with estimated reductions in median pathogen levels of 100%. This control measure is considered to be more effective than coverage of the anal region of the pig during evisceration. This latter mitigation strategy was predicted to result in 10% – 44% reduction of median pathogen contamination levels. At retail, pork chops are also estimated to contain low numbers of these pathogens. Therefore handling of the raw pork chop soon after purchase from retail outlets may be associated with a low risk of contracting salmonellosis, colibacillosis and campylobacteriosis. This risk can be further reduced by placing pork chops in a blast chiller for 12 hours prior to display. When this mitigation strategy was modelled the outputs indicated a 15% – 61% reduction in the maximum pathogen levels on pork chops, 44 – 100% reduction in the 10th – 90th range and 14% – 50% reduction in pathogen prevalence levels. Detailed investigation revealed the limitations of a specific modelling approach. We determined that the population-based modelling approach is not an appropriate alternative to the individual-based modelling approach when there is a large disparity in contamination levels between processed carcasses. Therefore the former technique should not be used in the presence of large heterogeneity with respect to the number of bacteria on the food unit of interest, or when bacterial populations input into the model are described with large variances. This thesis demonstrates the application of a suite of novel risk models in the pork food chain. We propose use in quantitative microbial exposure assessments. The applicability of these models is not only limited to the pork chain or to the above mentioned pathogens, but by modification of parameters, the entire model, or portions thereof can be extrapolated to other animal species undergoing similar abattoir procedures with pathogens of analogous epidemiological patterns. Finally the information provided by the models can be instrumental in assisting risk managers in their decision-making and policy development undertakings and provide guidance to effectively and strategically funnel limited resources.Item Thermophilic Campylobacter in animals and man : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Pathology and Public Health at Massey University(Massey University, 1984) Kakoyiannis, Charalambos KleanthousDuring the course of the work reported in this thesis, more than 2,000 samples and isolates from animals and from humans, were examined for the presence of intestinal thermophilic Campylobacter. This figure does not include samples and isolates examined during the course of experimental studies. Culture of either rectal swabs from pigs or cloacal swabs from poultry usually provides an accurate assessment of the prevalence of distal gastro-intestinal (G.I.) tract infection by C. coli and C. jejuni, provided that the swabs are kept at 4°C and cultured within 6 hours. Cultures of isolates of C. jejuni can be preserved for at least 21 months if stored in FBP broth or PBS at -70°C. The major site of colonisation by C. coli in pigs, is the distal part of the G.I. tract and the proximal G.I tract in the case of C. jejuni. In poultry, the major sites of colonisation by both C. jejuni and C. coli are the distal ileum and the large intestine, especially the caeca. Surveys of pigs showed that 60 - 100% of post weaning pigs are infected with C. coli. In neonates the rates of infection increase with age, and infected sows are the major source of infection for piglets. C. jejuni is rarely isolated from pigs. Twenty eight percent (28%) of all poultry flocks examined were infected with intestinal thermophilic Campylobacter. In infected broiler flocks the rate of infection is almost 100%, while in older birds lower rates were found. Of all the isolates examined, 86% were C. jejuni and 14% C. coli. All poultry carcases and edible viscera, derived from infected broiler flocks, are contaminated with Campylobacter. The levels of contamination of these products by both C. jejuni and C. coli are approximately 106 and 104 cfu per whole carcase and packet of edible viscera respectively. Poultry chilled products for sale in supermarkets were also heavily contaminated by these two species Campylobacter which remained viable for the 'shelf life' of the products (ten days). The prevalence of infection by intestinal thermophilic Campylobacter in gulls was 59%, in ducks 29%, and in rats 60%. Infected sparrows were not detected. With the exception of an isolate from one pig, C. laridis is a species apparently host specific for sea birds, and isolates from gulls of what, on conventional taxonomic criteria, would be classified as C. coli are shown on more detailed examination to be C. laridis. The level of excretion in faeces of Campylobacter in most infected pigs is between 102 - 105 cfu/g, in poultry approximately 108 cfu/g, in rats 105 cfu/g, and in gulls 103 cfu/g. The infectivity of C. jejuni isolated from poultry for experimental chickens is in the region of 5 x 102 cfu and that of C. coli approximately 5 x 105. Both species of Campylobacter were a 100 to 1000-fold less infective for rats. Infection in both chickens and rats was self-limiting and attempts to reinfect chickens with the same species or organism, were usually unsuccessful. C. laridis was not infective for either chickens or rats at dose rates of up to 109 organisms. Campylobacter infections are the most common notified human enteric infection in New Zealand with an annual incidence rate of 31/100,000. Approximately 94% of the human cases of campylobacteriosis are due to C. jejuni and 6% to C. coli. People between 1 to 4, and 15 to 35 years of age are the most commonly affected, and patients who no longer have clinical signs of infection may shed up to 108 cfu of C. jejuni/g of faeces. By use of a bacterial restriction endonuclease DNA technique (BRENDA), up to 80 different types C. jejuni and C. coli were identified from 338 isolates from humans. One hundred and ninety four (61%) of the 316 isolates of C. jejuni from humans had similar BRENDA patterns to isolates of C. jejuni from animals. Poultry appear to be the major source of C. jejuni, and possibly C. coli, for humans, while pigs apparently are an insignificant source of C. coli for humans. Rats can be infected with strains of C. jejuni which can infect poultry, humans and other animals. BRENDA types recovered from gulls and ducks were not similar to any of the isolates from humans or other animals examined. Some strains of C. jejuni and C. coli developed an in-vitro resistance to nalidixic acid. This is an important finding in relation to conventional taxonomic criteria for differentiating C. laridis from other intestinal thermophilic Campylobacter. Isolates of 'C. coli' from gulls are phenotypic variants of C. laridis which may be either resistant, or non resistant, to nalidixic acid. Only by determining whether or not, anaerobic growth in the presence of TMAO occurs, can C. coli be differentiated from C. laridis.