Molecular and aflatoxigenicity analyses of Aspergillus flavus isolates indigenous to grain corn in Malaysia; potentials for biological control.

AIMS: The present work aimed to distinguish the indigenous Aspergillus flavus isolates obtained from the first (pioneer) grain corn farms in Terengganu, Malaysia, into aflatoxigenic and non-aflatoxigenic by molecular and aflatoxigenicity analyses, and determine the antagonistic capability of the non-aflatoxigenic isolates against aflatoxigenic counterparts and their aflatoxin production in vitro. METHODS AND RESULTS: Seven A. flavus isolates previously obtained from the farms were characterized molecularly and chemically. All isolates were examined for the presence of seven aflatoxin biosynthesis genes, and their aflatoxigenicity was confirmed using high performance liquid chromatography with fluorescence detector. Phylogenetic relationships of all isolates were tested using ITS and ß-tubulin genes. Of the seven isolates, two were non-aflatoxigenic, while the remaining were aflatoxigenic based on the presence of all aflatoxin biosynthesis genes tested and the productions of aflatoxins B1 and B2. All isolates were also confirmed as A. flavus following phylogenetic analysis. The indigenous non-aflatoxigenic isolates were further examined for their antagonistic potential against aflatoxigenic isolates on 3% grain corn agar. Both non-aflatoxigenic isolates significantly reduced AFB1 production of the aflatoxigenic isolates. CONCLUSION: The indigenous non-aflatoxigenic A. flavus strains identified in the present work were effective in controlling the aflatoxin production by the aflatoxigenic A. flavus isolates in vitro and can be utilized for in situ testing.

Risk Assessment of Polycyclic Aromatic Hydrocarbons and Heterocyclic Aromatic Amines in Processed Meat, Cooked Meat and Fish-Based Products Using the Margin of Exposure Approach.

Background: The objective of this study is to assess the risk of exposure of polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines (HCAs) in meat and fish-based products marketed in Malaysia using the margin of exposure (MOE) approach. Methods: Benchmark Dose (BMD) software was used to model the BMD at a lower end of a one-sided 95% confidence interval with a 10% incremental risk (BMDL10) of PAHs and HCAs from different target organ toxicities. The MOEs of PAHs and HCAs in meat and fish-based products were determined by utilising the calculated BMDL10 values and estimated daily intake of meat and fish-based products from published data. Results: The calculated BMDL10 values of PAHs (i.e. benzo[a]pyrene [BaP] and fluoranthene [FA]) and HCAs (i.e. 2-amino-3,8,dimethylimidazo[4,5-f]quinoxaline [MeIQx] and 2-amino-1-methyl-6-phenylimidazo[4,5,6]pyridine [PhIP]) ranged from 19 mg/kg bw/day to 71,801 mg/kg bw/day. The MOE of BaP ranged from 41,895 to 71,801 and that of FA ranged from 19 to 1412. As for MeIQx and PhIP, their MOEs ranged from 6,322 to 7,652 and from 2,362 to 14,390, respectively. Conclusion: The MOEs of FA, MeIQx and PhIP were lower than 10,000, indicating a high concern for human health and therefore demanding effective risk management actions.

Dietary exposure to acrylamide among the Malaysian adult population.

This study aimed to estimate the Malaysian adult population's current dietary exposure and margin of exposure (MOE) to the carcinogenic processing contaminant, acrylamide. A total of 448 samples from 11 types of processed foods were collected randomly throughout Malaysia in the year 2015 and 2016. Acrylamide was analysed in samples using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) with a limit of detection (LOD) of 10 µg/kg and a limit of quantification (LOQ) of 25 µg/kg. The highest average level of acrylamide (772 ± 752 µg/kg) was found in potato crisps, followed by French fries (415 ± 914 µg/kg) and biscuits (245 ± 195 µg/kg). The total acrylamide exposure for the adult Malaysian was 0.229 and 1.77 µg/kg body weight per day for average and high consumers, respectively. The MOE were 741 and 1875 for the average consumer based on cancer and non-cancer effects of acrylamide, respectively. Meanwhile, for high consumers, the MOE is 96 for cancer and 243 for non-cancer effects. These findings indicate potential carcinogenic risks from acrylamide exposure among Malaysian adults, especially in Malay and other Bumiputra groups compared to Chinese, Indian, and other ethnic groups, while non-cancer effects appeared less concerning.

Mycotoxigenic fungal growth inhibition and multi-mycotoxin reduction of potential biological control agents indigenous to grain maize.

The present work investigated the potential of fungal species from grain maize farms in Malaysia as antagonists against the indigenous mycotoxigenic fungal species and their subsequent mycotoxin production. Dual-culture assay was conducted on grain maize agar (GMA) with 12 strains of potential fungal antagonists namely Bjerkandra adusta, Penicillium janthinellum, Schizophyllum commune, Trametes cubensis, Trichoderma asperelloides, Trichoderma asperellum, Trichoderma harzianum, and Trichoderma yunnanense against seven mycotoxigenic strains namely Aspergillus flavus, Aspergillus niger, Fusarium verticillioides, and Fusarium proliferatum producing aflatoxins, ochratoxin A, and fumonisins, respectively. Based on fungal growth inhibition, Trichoderma spp. showed the highest inhibitory activity (73-100% PIRG, Percentage Inhibition of Radial Growth; 28/0 ID, Index of Dominance) against the tested mycotoxigenic strains. Besides, B. adusta and Tra. cubensis showed inhibitory activity against some of the tested mycotoxigenic strains. All fungal antagonists showed varying degrees of mycotoxin reduction. Aflatoxin B1 produced by A. flavus was mainly reduced by P. janthinellum, Tra. cubensis, and B. adusta to 0 ng/g. Ochratoxin A produced by A. niger was mainly reduced by Tri. harzianum and Tri. asperellum to 0 ng/g. Fumonisin B1 and FB2 produced by F. verticillioides was mainly reduced by Tri. harzianum, Tri. asperelloides, and Tri. asperellum to 59.4 and 0 µg/g, respectively. Fumonisin B1 and FB2 produced by F. proliferatum were mainly reduced by Tri. asperelloides and Tri. harzianum to 244.2 and 0 µg/g, respectively. This is the first study that reports on the efficacy of Tri. asperelloides against FB1, FB2, and OTA, P. janthinellum against AFB1, and Tra. cubensis against AFB1.

Assessment of multi-mycotoxin contamination throughout the supply chain of maize-based poultry feed from selected regions of Malaysia by LC-MS/MS.

The objective of this study was to examine the occurrence of multi-mycotoxin contamination throughout the supply chain of maize-based poultry feed. Different sampling points throughout the feed supply chain were selected from two companies that manufactured the poultry feed. A total of 51 samples, consisting of grain maize and maize-based poultry feeds, were collected. The samples were analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the occurrence of multi-mycotoxin. The results revealed that 100% of samples throughout the maize-based poultry feed supply chain were spoiled with more than one mycotoxin. Fumonisin B1 (8.02-1,220 µg/kg) and fumonisin B2 (11.1-1,109 µg/kg) were the main mycotoxins detected at all sampling points throughout the feed supply chain. Zearalenone (ZEA) (6.63-7.50 µg/kg) was also detected in 11.7% (n = 6) (out of a total of 51) samples. As the supply chain progresses, a reduction in mycotoxin contamination was observed. Aflatoxins, ochratoxin A (OTA), deoxynivalenol (DON), HT-2, and T-2 toxin were not detected. The levels of mycotoxins detected throughout the supply chain were below the international regulatory limits, thus indicating the low risk of exposure to mycotoxins in maize-based poultry feed in Malaysia. Nevertheless, due to the presence of multiple ingredients in most food and feed, efforts to understand and address challenges associated with mycotoxins throughout the entire supply chain need to be more holistic to protect public health.

Vitamin A and E Homologues Impacting the Fate of Acrylamide in Equimolar Asparagine-Glucose Model System.

This study aims to evaluate the influence of Vitamin A and E homologues toward acrylamide in equimolar asparagine-glucose model system. Vitamin A homologue as ß-carotene (BC) and five Vitamin E homologues, i.e., α-tocopherol (AT), δ-tocopherol (DT), α-tocotrienol (ATT), γ-tocotrienol (GTT), and δ-tocotrienol (DTT), were tested at different concentrations (1 and 10 µmol) and subjected to heating at 160 °C for 20 min before acrylamide quantification. At lower concentrations (1 µmol; 431, 403, 411 ppm, respectively), AT, DT, and GTT significantly increase acrylamide. Except for DT, enhancing concentration to 10 µmol (5370, 4310, 4250, 3970, and 4110 ppm, respectively) caused significant acrylamide formation. From linear regression model, acrylamide concentration demonstrated significant depreciation over concentration increase in AT (Beta = -83.0, R2 = 0.652, p ≤ 0.05) and DT (Beta = -71.6, R2 = 0.930, p ≤ 0.05). This study indicates that different Vitamin A and E homologue concentrations could determine their functionality either as antioxidants or pro-oxidants.

Assessing the Performance of Food Safety Management System Using Food Safety Management System Diagnostic Tools and Microbial Assessment Scheme: A Case of Powdered Beverage Manufacturers.

BACKGROUND: The objective of the study is to assess the performance of the Food Safety Management System (FSMS) among powdered beverage manufacturers using Food Safety Management System Diagnostic Tools (FSMS-DI) and Microbial Assessment Scheme (MAS). METHODS: FSMS-DI was used to evaluate the context factors, core control and core assurance activities of five powdered beverage manufacturers with different types of FSMS certification. Manufacturer A is not certified with any FSMS, while manufacturers B, C, D and E are complied with MeSTI, GMP, HACCP and ISO 22000, respectively. For MAS, samples were collected from the selected critical sampling locations of two manufacturers who complied FSMS with the least (manufacturer B) and the most stringent (manufacturer E) requirements. The samples consisted of two different types of powdered beverage products were analysed for total plate count (TPC), Salmonella, Escherichia coli, Staphylococcus aureus, yeast and mould count (YMC). Results: The food safety (FS) output of powdered beverages for manufacturer E was better (overall score of 3) than manufacturer B (overall score of 2-3). Manufacturer E was able to achieve their FS objectives. The FSMS activities of manufacturer C, D and E were better (overall score of 2-3) than manufacturer A and B (overall score of 1-2). CONCLUSION: The study demonstrated that FSMS-DI and MAS can be used to differentiate the FSMS performance of powdered beverage manufacturers with different types of FSMS certification. Higher scores of FSMS activities obtained by the manufacturer who complied with stringent FSMS certifications contributed to better microbiological safety performance of powdered beverages.

Morphological, Structural, Thermal, Permeability, and Antimicrobial Activity of PBS and PBS/TPS Films Incorporated with Biomaster-Silver for Food Packaging Application.

The development of antimicrobial film for food packaging application had become the focus for researchers and scientists. This research aims to study the characteristics and antimicrobial activity of novel biofilms made of poly (butylene succinate) (PBS) and tapioca starch (TPS) added with 1.5% or 3% of Biomaster-silver (BM) particle. In morphological examination, the incorporation of 3% BM particle was considerably good in forming well-structured PBS film. Meanwhile, the functional groups analysis revealed the 3% BM particle was effectively interacted with PBS molecular chains. The flame retard behavior of BM metal particle also helped in enhancing the thermal stability for pure PBS and PBS/TPS films. The nucleating effect of BM particles had improved the films crystallinity. Small pore size features with high barrier property for gas permeability was obtained for BM filled PBS/TPS films. From antimicrobial analysis, the BM particles possessed antimicrobial activity against three bacteria Staphylococcus aureus, Escherichia coli, and Salmonella Typhimurium in which PBS/TPS 3% BM film exhibited strong antimicrobial activity against all tested bacteria, however, PBS/TPS 1.5% BM film exhibited strong antimicrobial activity against E. coli only. Hence, the incorporation of BM into PBS/TPS film could be a sustainable way for developing packaging films to preserve food products.

Selection of Vegetable Oils and Frying Cycles Influencing Acrylamide Formation in the Intermittently Fried Beef Nuggets.

This study aims to investigate the effect of different vegetable oils and frying cycles on acrylamide formation during the intermittent frying of beef nuggets. Different vegetable oils, palm olein (PO), red palm olein (RPO), sunflower oil (SFO), and soybean oil (SBO), were used for a total of 80 frying cycles. Oil was collected at every 16th frying cycle and analyzed for peroxide value (PV), p-anisidine value (p-AV), free fatty acid (FFA), total polar compound (TPC), polar compound fractions, and fatty acid composition (FAC). Total oxidation (TOTOX) value was calculated, and acrylamide content was quantified in the nuggets. Regardless of the oil type, PV, p-AV, and TOTOX initially increased but gradually decreased. However, FFA and TPC continued to develop across the 80 frying cycles. The C18:2/C16:0 remained almost unchanged in PO and RPO but dropped progressively in SFO and SBO. The lowest acrylamide content in fried products was observed in the PO, while the highest content was observed in RPO. Bivariate correlation analysis showed no significant (p ≤ 0.05) correlation between oil quality attributes and acrylamide concentration. The oil type but not the frying cycle significantly affected the acrylamide concentration in beef nuggets.

Prevalence of mycotoxigenic fungi and assessment of aflatoxin contamination: a multiple case study along the integrated corn-based poultry feed supply chain in Malaysia.

BACKGROUND: Corn, a main feed ingredient in the livestock industry, is one of the most susceptible crops to fungal infection and aflatoxin contamination. Livestock feeding on aflatoxin (AF)-contaminated feed have been shown to experience feed refusal, and decreased growth rate, milk production, and feed efficiency. In poultry, AF poisoning causes weight loss, poor feed efficiency, and reduced egg production and egg weight. The present work therefore aimed to determine the prevalence of mycotoxigenic fungi and the occurrence of AF contamination along the integrated corn-based poultry feed supply chain in Malaysia. A total of 51 samples were collected from different points along the feed supply chain from integrated poultry feed companies. The samples were subjected to mycological analyses (fungal isolation, enumeration, identification), and AFs were quantified by high-performance liquid chromatography equipped with a fluorescence detector (HPLC-FLD). RESULTS: Samples collected from sampling point 1 (company A) and sampling point 9 (company B) yielded the highest total fungal load (>log 4 CFU g-1 ). The prevalent fungal genera isolated were Aspergillus, Fusarium, and Penicillium spp. Aflatoxin B1 was detected in 8.3% of corn samples, and 7.4% of corn-based poultry feed samples along the feed supply chain, whereas AFs B2 , G1 , and G2 were not detected. CONCLUSION: The incidence of mycotoxigenic fungi along the integrated poultry feed supply chain warrant continuous monitoring of mycotoxin contamination to reduce the exposure risk of mycotoxin intake in poultry. © 2020 Society of Chemical Industry.

The Inhibitory Effects of Heterotrigona Itama Honey Marinades on the Formation of Carcinogenic Heterocyclic Amines in Grilled Beef Satay.

Heterocyclic amines (HCAs) are carcinogenic food toxicants formed in cooked meats, which may increase the risk of cancer development in humans. Therefore, in this study, the effect of stingless bee honey from different botanical origins on the formation of HCAs in grilled beef satay was investigated. HCAs concentration in grilled beef satay was determined by using high performance liquid chromatography (HPLC). In total, six of the most toxigenic HCAs representing aminoimidazo-azaarenes (AIAs) (MeIQx, 4,8-DiMeIQx, and PhIP) and amino carbolines (norharman, harman, and AαC) groups were identified in all the beef samples investigated. A significant reduction in HCAs was observed in grilled beef marinated in honey as compared to beef samples marinated in table sugar (control), in which the reduction of 95.14%, 88.45%, 85.65%, and 57.22% was observed in gelam, starfruit, acacia, and Apis honey marinades, respectively. According to the partial least squares regression (PLS) model, the inhibition of HCAs in grilled beef was shown to be significantly correlated to the antioxidant activity (IC50) of the honey samples. Therefore, the results of this study revealed that the addition of stingless bee honey could play an important role in reducing HCAs in grilled beef.

A Comparative Characterization of Physicochemical and Antioxidants Properties of Processed Heterotrigona itama Honey from Different Origins and Classification by Chemometrics Analysis.

Stingless bee honey produced by Heterotrigona itama from different botanical origins was characterised and discriminated. Three types of stingless bee honey collected from acacia, gelam, and starfruit nectars were analyzed and compared with Apis mellifera honey. The results showed that stingless bee honey samples from the three different botanical origins were significantly different in terms of their moisture content, pH, free acidity, total soluble solids, colour characteristics, sugar content, amino acid content and antioxidant properties. Stingless bee honey was significantly different from Apis mellifera honey in terms of physicochemical and antioxidant properties. The amino acid content was further used in the chemometrics analysis to evaluate the role of amino acid in discriminating honey according to botanical origin. Partial least squares-discriminant analysis (PLS-DA) revealed that the stingless bee honey was completely distinguishable from Apis mellifera honey. Notably, a clear distinction between the stingless bee honey types was also observed. The specific amino acids involved in the distinction of honey were cysteine for acacia and gelam, phenylalanine and 3-hydroxyproline for starfruit, and proline for Apis mellifera honey. The results showed that all honey samples were successfully classified based on amino acid content.

Verification and evaluation of monochloropropanediol (MCPD) esters and glycidyl esters in palm oil products of different regions in Malaysia.

This study was conducted to investigate on the effect of different sampling regions of palm-refined oils and fats on the 2- and 3-monochloropropanediol fatty acid esters (MCPDE) and glycidol fatty acid esters (GE) levels. The American Oil Chemists' Society (AOCS) Official Method Cd 29a-13 on the determination of MCPDE and GE in edible oils and fats by acid transesterification was successfully verified and optimised, with slight modification using 7890A Agilent GC system equipped with 5975C quadrupole detector. The determined limits of detection (LOD) for MCPDE were 0.02 mg kg-1 and 0.05 mg kg-1 for GE. The method performance has showed good recovery between 80% and 120% for all pertinent compounds with seven replicates assayed in three separate days. Round robin test with two European laboratories, i.e. Eurofins and SGS, has shown compliance results with those of the present study. Among the sampling regions, only one refinery located in the central region of Malaysia showed a significant increment of the MCPDE and GE levels after refining process. The GE level averaging at 2.5 mg kg-1 was slightly higher than that of 3-MCPDE averaging at 1.3 mg kg-1. Both esters were preferentially partitioned into the liquid phase rather than the solid phase after fractionation. However, the overall results exhibited no direct correlation between the esters content and the different sampling locations of the palm oil products in Malaysia. Analysis of total chlorine content also displayed significant variations between sampling locations which clearly show its effect on the chlorine content in the CPO samples.

Effects of different types of soy sauce on the formation of heterocyclic amines in roasted chicken.

The objective of the study was to determine the effect of different types of soy sauce and marinating time on the formation of heterocyclic amines (HCAs) in roasted chicken. Chicken breast samples were marinated with sweet, salty, light and dark soy sauce at 0, 3, 6 and 12 h (control treatment was the chicken without marinade). The concentrations of free amino acids, sugars and creatinine were determined before roasting while HCA concentrations were determined after roasting. All types of soy sauce significantly increased (p ≤ 0.05) the concentration of HCAs in roasted chicken with increasing marinating time. The highest increment of total concentration of HCAs was found in samples marinated with light soy sauce (887%) followed by dark (375%), salty (193%) and sweet (169%) at 12 h. PhIP (2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine) showed a substantial reduction in samples only momentarily marinated with sweet, salty and dark soy sauce (0 h). Free amino acids were found to be more strongly correlated with the formation of HCAs than reducing sugars or creatinine.

Simultaneous formation of polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines (HCAs) in gas-grilled beef satay at different temperatures.

This study investigated the simultaneous formation of polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines (HCAs) in gas-grilled beef satay at different temperatures (150, 200, 250, 300, and 350°C). Solid-phase extraction (SPE) was used for sample clean-up. Fifteen PAHs were determined using high performance liquid chromatography with fluorescence detection (HPLC-FLD) and nine HCAs were quantified using liquid chromatography tandem-mass spectrometry (LC-MS/MS) with a gradient programme. The lowest significantly concentrations of PAHs and HCAs were generated at 150°C; the formation of PAHs and HCAs simultaneously increased with temperatures. Benzo[a]pyrene was detected in all samples and increased markedly at 300 and 350°C. The sums of 4 PAHs (PAH4) in marinated beef satay at 300 and 350°C exceeded the maximum level in Commission Regulation (EU) 2015/1125. Significant reductions of polar and non-polar HCAs (except PhIP) were found in marinated beef satay across all temperatures. Overall, PAHs and HCAs showed opposite trends of formation in beef satay with marination.

Effect of Fat-Soluble Anti-oxidants in Vegetable Oils on Acrylamide Concentrations During Deep-Fat Frying of French Fries.

BACKGROUND: This study attempted to evaluate the effect of fat-soluble anti-oxidants in vegetable oils on acrylamide during the deep-fat frying of French fries. METHODS: Three vegetable oils with different fat-soluble anti-oxidant contents were selected and par-fried potato strips were fried in these oils. Acrylamide in the French fries at different frying times (at 180 °C) and over 10 consecutive frying sessions were measured. The anti-oxidant contents and quality degradation of oils were monitored before and after the 5th and 10th consecutive frying sessions. RESULTS: The effect of the fat-soluble anti-oxidants in red palm oil on the acrylamide was more apparent when a prolonged frying time was used for consecutive frying sessions than when different frying conditions were used. Using red palm oil, acrylamide concentration in French fries significantly dropped to the lowest level, at 524 ng g-1, after the 10th frying session. The ß-carotene content after the 10th frying session was the highest in red palm oil. CONCLUSION: The use of red palm oil for deep-fat frying French fries can be a mitigation strategy to reduce acrylamide formation, but further studies are necessary to investigate the influence of different types of fat-soluble anti-oxidants on the inhibition of acrylamide formation.