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Fulltext Application of nanomaterials in the development of biosensors for food safety and quality control

Farah, A.A., Sukor, R., Fatimah, A.B., Jinap, S.

International Food Research Journal, 2016;23(5):1849-1856.
MyJurnal

Nanotechnology contribute to significant impacts in every way in our daily life. Recently,
the application of nanotechnology in biosensors has been a trend in developing a highly
sensitive, selective, quick response, inexpensive, high volume production, great reliability
and miniaturized sensors. High demands on the production of rapid sensors for food safety
and quality control purposes are increasingly become the interest for researchers all over the
world. This is because, in food sector, the quality of a certain product is based on their periodic
chemical and microbilogical analysis. The uses of nanomaterials in biosensors are very
promising because they mediate current flow. Surface modification of the electrode based on
various nanomaterials including nanoparticle, nanofiber, nanowire and nanotube significantly
increase the performance of the biosensor. Ultimately, this implementation will enhance the
sensor’s sensitivity and stability. This review explores the previous research and development
work on nanomaterials-based sensors for food applications.
Fulltext Antifungal activity of Boesenbergia Rotunda (temukunci) extract against filamentous spoilage fungi from vegetables

Zakuan, Z., Mustapa, S.A., Sukor, R., Rukayadi, Y.

International Food Research Journal, 2018;25(1):433-438.
MyJurnal

The filamentous spoilage fungi in vegetables can lead to significant impact in food and economic loss. In order to overcome this problem, chemical fungicide has been implemented in vegetable farming and processing but it causes problems towards environment and food safety. Thus, the utilization of natural products such as plants extracts, which exhibit antimicrobial and antifungal activity, is more acceptable to solve this problem. The aim of this study is to investigate the antifungal activity of Boesenbergia rotunda extract against ten filamentous spoilage fungi isolated from five vegetables. The extract was used to treat fungal isolates from vegetables; CRb 002 (Penicillium sp.), CHa 009 (Aspergillus sp.), TMa 001 (Geotrichum sp.), TMa 002 (Aspergillus sp), ONb 001 (Aspergillus sp.), WBb 003 and WBb 004 (Fusarium sp.) WBb 007 (unidentified), WBb 008 (Aureobasidium sp.) and WBb 010 (Penicillium sp.). The results showed that the yield of the extract of B. rotunda using ethanol (95%) was 11.42% (w/v). The 10% of B. rotunda extract exhibited antifungal activities against ten filamentous fungi after 5 days treatment with growth reduction of 41.56%, 30.68%, 86.20%, 50.62%, 26.67%, 47.44%, 50.74%, 36.39%, 42.86%, and 39.39% for WBb 008, WBb 004, WBb 007, WBb 003, CRb 002, WBb 010, CHa 009, TMa 001, ONb 001, and TMa 002, respectively. B. rotunda extract showed highest antifungal activity against fungi isolated from winged bean (WBb 007) with percentage reduction in growth was 86.20%, while the lowest activity was against fungi isolated from the carrot (CRb 002) with 26.67% reduction in growth. Generally, the TPC of fungi in the vegetable samples were reduced after treatment with 5% of B. rotunda extract at 5 min and 10 min of exposure time. The results suggested that B. rotunda extract has high potential to become natural food preservative which can reduce the fungi spoilage of vegetables.
Fulltext The extraction of crude enzyme of lipase from Penicillium candidum PCA 1/TT031 by way of solid state fermentation

Alhelli, A. M., Abd Manap, M. Y., Mohammed, A. S., Mirhosseini, S. H., Sukor, R., Khalil, E. S., et al.

International Food Research Journal, 2018;25(101):1-11.
MyJurnal

Solid-state fermentation (SSF), which is an excellent alternative for industrial enzyme production, entails the exploitation of cheap agro-industrial residues (low priced culture media). This paper delves into the cultivation of mould (GRAS Penicillium candium PCA 1/TT031) using wheat bran (WB) as the culture media. The parameters for crude enzymatic extraction of lipase production were optimized to achieve the highest possible lipase activity. An incubation period of 7 days (3.06 U/g WB), 2% of tributyrin (5.43 U/g WB), meat peptone equal to 2% (5.2 U/g WB), moisture content of 50% (v:w) (6.6 U/g WB), initial pH of 9.0( 8.6 U/g WB), inoculum size of 5×106 spore/g of WB (11.3 U/g WB), an incubation temperature of 20°C (13.6 U/g WB), and an extractant consisting of phosphate buffer pH 7.0 (14.7 U/g WB) were the conditions applied for this study.
Fulltext Electrochemical Immunosensor for the Detection of Aflatoxin B₁ in Palm Kernel Cake and Feed Samples

Azri FA, Selamat J, Sukor R

Sensors (Basel), 2017 Nov 30;17(12).
PMID: 29189760 DOI: 10.3390/s17122776

Palm kernel cake (PKC) is the solid residue following oil extraction of palm kernels and useful to fatten animals either as a single feed with only minerals and vitamins supplementation, or mixed with other feedstuffs such as corn kernels or soy beans. The occurrence of mycotoxins (aflatoxins, ochratoxins, zearalenone, and fumonisins) in feed samples affects the animal's health and also serves as a secondary contamination to humans via consumption of eggs, milk and meats. Of these, aflatoxin B₁ (AFB₁) is the most toxically potent and a confirmed carcinogen to both humans and animals. Methods such as High Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) are common in the determination of mycotoxins. However, these methods usually require sample pre-treatment, extensive cleanup and skilled operator. Therefore, in the present work, a rapid method of electrochemical immunosensor for the detection of AFB₁ was developed based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). Multi-walled carbon nanotubes (MWCNT) and chitosan (CS) were used as the electrode modifier for signal enhancement.N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (EDC) andN-hydroxysuccinimide (NHS) activated the carboxyl groups at the surface of nanocomposite for the attachment of AFB₁-BSA antigen by covalent bonding. An indirect competitive reaction occurred between AFB₁-BSA and free AFB₁ for the binding site of a fixed amount of anti-AFB₁ antibody. A catalytic signal based on horseradish peroxidase (HRP) in the presence of hydrogen peroxide (H₂O₂) and 3,3',5,5'-tetramethylbenzidine (TMB) mediator was observed as a result of attachment of the secondary antibody to the immunoassay system. As a result, the reduction peak of TMB(Ox)was measured by using differential pulse voltammetry (DPV) analysis. Based on the results, the electrochemical surface area was increased from 0.396 cm² to 1.298 cm² due to the electrode modification with MWCNT/CS. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.0001 to 10 ng/mL with limit of detection of 0.1 pg/mL. Good recoveries were obtained for the detection of spiked feed samples (PKC, corn kernels, soy beans). The developed method could be used for the screening of AFB₁ in real samples.
Fulltext Dispersive Liquid-Liquid Microextraction (DLLME) and LC-MS/MS Analysis for Multi-Mycotoxin in Rice Bran: Method Development, Optimization and Validation

Salim SA, Sukor R, Ismail MN, Selamat J

Toxins (Basel), 2021 04 15;13(4).
PMID: 33920815 DOI: 10.3390/toxins13040280

Rice bran, a by-product of the rice milling process, has emerged as a functional food and being used in formulation of healthy food and drinks. However, rice bran is often contaminated with numerous mycotoxins. In this study, a method to simultaneous detection of aflatoxins (AFB1, AFB2, AFG1, and AFG2), ochratoxin A (OTA), deoxynivalenol (DON), fumonisins (FB1 and FB2), sterigmatocystin (STG), T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS) and zearalenone (ZEA) in rice bran was developed, optimized and validated using dispersive liquid-liquid microextraction (DLLME) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In DLLME, using a solvent mixture of methanol/water (80:20, v/v) as the dispersive solvent and chloroform as the extraction solvent with the addition of 5% salt improved the extraction recoveries (63-120%). The developed method was further optimized using the response surface methodology (RSM) combined with Box-Behnken Design (BBD). Under the optimized experimental conditions, good linearity was obtained with a correlation coefficient (r2) ≥ 0.990 and a limit of detection (LOD) between 0.5 to 50 ng g-1. The recoveries ranged from 70.2% to 99.4% with an RSD below 1.28%. The proposed method was successfully applied to analyze multi-mycotoxin in 24 rice bran samples.
Fulltext Effects of simultaneous UV-C radiation and ultrasonic energy postharvest treatment on bioactive compounds and antioxidant activity of tomatoes during storage

Esua OJ, Chin NL, Yusof YA, Sukor R

Food Chem, 2019 Jan 01;270:113-122.
PMID: 30174024 DOI: 10.1016/j.foodchem.2018.07.031

The effects of a novel technology utilizing a simultaneous combination of Ultraviolet-C radiation and ultrasound energy postharvest treatment on tomato bioactive compounds during 28 days' storage period was investigated by varying Ultraviolet-C radiation intensities of 639.37 or 897.16 µW/cm2 at a constant ultrasound intensity of 13.87 W/L from a 40 kHz-1 kW transducer. A minimal treatment time of 240 s at Ultraviolet-C dosage of 2.15 kJ/m2 was observed to provoke a considerable increase in bioactive compounds content, proportionated to treatment time. Although treatment led to temperature increase in the system reaching 39.33 °C due to heat generation by ultrasonic cavitation, the extractability and biosynthesis of phytochemicals were enhanced resulting in 90%, 30%, 60%, 20%, and 36% increases in lycopene, total phenols, vitamin C, hydrophilic and lipophilic antioxidant activities respectively. Results present the potential use of the combined non-thermal technologies as post-harvest treatment to improve bioactive compounds and antioxidant activity during storage.
Fulltext Effect of Fat-Soluble Anti-oxidants in Vegetable Oils on Acrylamide Concentrations During Deep-Fat Frying of French Fries

Kamarudin SA, Jinap S, Sukor R, Foo SP, Sanny M

Malays J Med Sci, 2018 Sep;25(5):128-139.
PMID: 30914869 MyJurnal DOI: 10.21315/mjms2018.25.5.12

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

Shamsudin S, Selamat J, Sanny M, Jambari NN, Sukor R, Praveena SM, et al.

Molecules, 2020 Aug 26;25(17).
PMID: 32858787 DOI: 10.3390/molecules25173874

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.
Determination of minimal sequence for zearalenone aptamer by computational docking and application on an indirect competitive electrochemical aptasensor

Azri FA, Selamat J, Sukor R, Yusof NA, Raston NHA, Eissa S, et al.

Anal Bioanal Chem, 2021 Jun;413(15):3861-3872.
PMID: 34021369 DOI: 10.1007/s00216-021-03336-1

Aptamers are short single-stranded oligonucleotides (either DNA or RNA) that can fold into well-defined three-dimensional (3D) spatial structures which enable them to capture their specific target by complementary shape interactions. Aptamers are selected from large random libraries through the SELEX process and only a small fraction of the sequence is involved in direct docking with the target. In this paper, we describe the possible truncation variants of zearalenone (ZEA) aptamer which might be an effective binding region for the target. The originally selected zearalenone (ZEA) aptamer was 80-mer in length and shown to bind the target with a high affinity (Kd = 41 ± 5 nM). Herein, computational docking simulation was performed with 15 truncated variants to determine the predicted binding energy and responsible binding site of the aptamer-analyte complex. The results revealed that 5 truncated variants had binding energy lower than - 7.0 kcal/mol. Circular dichroism analysis was performed on the shortlisted aptamer and the conformational change of aptamers was observed with the presence of an analyte. Aptamer Z3IN (29-mer) was chosen as the most enhanced affinity for its target with a dissociation constant of 11.77 ± 1.44 nM. The aptamer was further applied in the electrochemical aptasensor of ZEA based on an indirect competitive format. The results demonstrated that the truncated aptamer leads to an enhancement of the sensitivity of the biosensor.
Fulltext In Vivo Toxicity Evaluation of Sugar Adulterated Heterotrigona itama Honey Using Zebrafish Model

Fakhlaei R, Selamat J, Razis AFA, Sukor R, Ahmad S, Amani Babadi A, et al.

Molecules, 2021 Oct 15;26(20).
PMID: 34684803 DOI: 10.3390/molecules26206222

Honey is prone to be adulterated through mixing with sugars, cheap and low-quality honey, and other adulterants. Consumption of adulterated honey may cause several health issues such as weight gain, diabetes, and liver and kidney dysfunction. Therefore, studying the impact of consumption of adulterated honey on consumers is critical since there is a lack of study in this field. Hence, the aims of this paper were: (1) to determine the lethal concentration (LC50) of adulterated honey using zebrafish embryo, (2) to elucidate toxicology of selected adulterated honey based on lethal dose (LD50) using adult zebrafish, (3) to determine the effects of adulterated honey on histological changes of zebrafish, and (4) to screen the metabolites profile of adulterated honey by using zebrafish blood serum. The LC50 of Heterotrigona itama honey (acacia honey) and its sugar adulterants (light corn sugar, cane sugar, inverted sugar, and palm sugar in the proportion of 1-3% (w/w) from the total volume) was determined by the toxicological assessment of honey samples on zebrafish embryos (different exposure concentrations in 24, 48, 72, and 96 h postfertilization (hpf)). Pure H. itama honey represents the LC50 of 34.40 ± 1.84 (mg/mL) at 96 hpf, while the inverted sugar represents the lowest LC50 (5.03 ± 0.92 mg/mL) among sugar adulterants. The highest concentration (3%) of sugar adulterants were used to study the toxicology of adulterated honey using adult zebrafish in terms of acute, prolong-acute, and sub-acute tests. The results of the LD50 from the sub-acute toxicity test of pure H. itama honey was 2.33 ± 0.24 (mg/mL). The histological studies of internal organs showed a lesion in the liver, kidney, and spleen of adulterated treated-honey groups compared to the control group. Furthermore, the LC-MS/MS results revealed three endogenous metabolites in both the pure and adulterated honey treated groups, as follows: (1) S-Cysteinosuccinic acid, (2) 2,3-Diphosphoglyceric acid, and (3) Cysteinyl-Tyrosine. The results of this study demonstrated that adulterated honey caused mortality, which contributes to higher toxicity, and also suggested that the zebrafish toxicity test could be a standard method for assessing the potential toxicity of other hazardous food additives. The information gained from this research will permit an evaluation of the potential risk associated with the consumption of adulterated compared to pure honey.
Fulltext Effect of Different Amino Acids and Heating Conditions on the Formation of 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and Its Kinetics Formation Using Chemical Model System

Ishak AA, Selamat J, Sulaiman R, Sukor R, Abdulmalek E, Jambari NN

Molecules, 2019 Oct 24;24(21).
PMID: 31652883 DOI: 10.3390/molecules24213828

The formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was investigated using a kinetic study approach as described by first-order, Arrhenius, and Eyring equations. Chemical model systems with different amino acid precursors (proline, phenylalanine, and glycine) were examined at different times (4, 8, 12, and 16 min) and temperatures (150, 180, 210, 240, and 270 °C). PhIP was detected using high-performance liquid chromatography equipped with fluorescence detector (HPLC-FLD). The good fit in first-order suggested that PhIP formation was influenced by the types of amino acids and PhIP concentration significantly increased with time and temperature (up to 240 °C). PhIP was detected in proline and phenylalanine model systems but not in the glycine model system. The phenylalanine model system demonstrated low activation energy (Ea) of 95.36 kJ/mol that resulted in a high rate of PhIP formation (great amount of PhIP formed). Based on the ∆S‡ values both proline and phenylalanine demonstrated bimolecular rate-limiting steps for PhIP formation. Altogether these kinetic results could provide valuable information in predicting the PhIP formation pathway.
Fulltext Etlingera elatior-Mediated Synthesis of Gold Nanoparticles and Their Application as Electrochemical Current Enhancer

Azri FA, Selamat J, Sukor R, Yusof NA, Ahmad Raston NH, Nordin N, et al.

Molecules, 2019 Aug 29;24(17).
PMID: 31470528 DOI: 10.3390/molecules24173141

This work presents a simple green synthesis of gold nanoparticles (AuNPs) by using an aqueous extract of Etlingera elatior (torch ginger). The metabolites present in E. elatior, including sugars, proteins, polyphenols, and flavonoids, were known to play important roles in reducing metal ions and supporting the subsequent stability of nanoparticles. The present work aimed to investigate the ability of the E. elatior extract to synthesise AuNPs via the reduction of gold (III) chloride hydrate and characterise the properties of the nanoparticles produced. The antioxidant properties of the E. elatior extract were evaluated by analysing the total phenolic and total flavonoid contents. To ascertain the formation of AuNPs, the synthesised particles were characterised using the ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray (EDX) microscopy, and dynamic light scattering (DLS) measurement. The properties of the green synthesised AuNPs were shown to be comparable to the AuNPs produced using a conventional reducing agent, sodium citrate. The UV-Vis measured the surface plasmon resonance of the AuNPs, and a band centered at 529 nm was obtained. The FTIR results proved that the extract contained the O-H functional group that is responsible for capping the nanoparticles. The HRTEM images showed that the green synthesized AuNPs were of various shapes and the average of the nanoparticles' hydrodynamic diameter was 31.5 ± 0.5 nm. Meanwhile, the zeta potential of -32.0 ± 0.4 mV indicates the high stability and negative charge of the AuNPs. We further successfully demonstrated that using the green synthesised AuNPs as the nanocomposite to modify the working surface of screen-printed carbon electrode (SPCE/Cs/AuNPs) enhanced the rate of electron transfer and provided a sensitive platform for the detection of Cu(II) ions.
Fulltext Generation of High Affinity Anti-Peptide Polyclonal Antibodies Recognizing Goat αs1-Casein

Mohsin AZ, Sukor R, Selamat J, Meor Hussin AS, Ismail IH, Jambari NN, et al.

Molecules, 2020 Jun 05;25(11).
PMID: 32516919 DOI: 10.3390/molecules25112622

The chemical, technological and allergy properties of goat's milk are significantly affected by the level of αs1-casein. Detection and quantification of αs1-casein requires high-specificity methods to overcome high-sequence similarity between this protein and others in the casein family. Unavailability of antibodies with high affinity and specificity towards goat αs1-casein hinders the development of immuno-based analytical methods such as enzyme-linked immunosorbent assay (ELISA) and biosensors. Here, we report the generation of polyclonal antibodies (or immunoglobulins, IgGs) raised towards goat αs1-casein N- (Nter) and C-terminal (Cter) peptide sequences. The Nter and Cter peptides of goat αs1-casein were immunized in rabbits for the generation of antisera, which were purified using protein G affinity chromatography. The binding affinity of the antisera and purified IgGs were tested and compared using indirect ELISA, where peptide-BSA conjugates and goat αs1-casein were used as the coating antigens. The Nter antiserum displayed higher titer than Cter antiserum, at 1/64,000 and 1/32,000 dilutions, respectively. The purification step further yielded 0.5 mg/mL of purified IgGs from 3 mL of antisera. The purified Nter IgG showed a significantly (p < 0.05) higher binding affinity towards peptide-BSA and goat αs1-casein, with lower Kd value at 5.063 × 10-3 μM compared to 9.046 × 10-3 μM for the Cter IgG. A cross-reactivity test showed that there was no binding in neither Nter nor Cter IgGs towards protein extracts from the milk of cow, buffalo, horse and camel. High-quality antibodies generated will allow further development of immuno-based analytical methods and future in vitro studies to be conducted on goat αs1-casein.
A highly selective two-way purification method using liquid chromatography for isolating αS2-casein from goat milk of five different breeds

Mohsin AZ, Sukor R, Selamat J, Meor Hussin AS, Ismail IH, Jambari NN, et al.

J Chromatogr B Analyt Technol Biomed Life Sci, 2020 Dec 01;1160:122380.
PMID: 32971369 DOI: 10.1016/j.jchromb.2020.122380

The main challenges in the purification of αS2-casein are due to the low quantity in milk and high homology with other casein subunits, i.e., αS1-casein, β-casein, and κ-casein. To overcome these challenges, the aim of this study was to develop a two-step purification to isolate native αS2-casein in goat milk from five different breeds; British Alpine, Jamnapari, Saanen, Shami, and Toggenburg. The first step of the purification was executed by anion-exchange chromatography under optimal elution conditions followed by size exclusion chromatography. Tryptic peptides from in-gel digestion of purified αS2-casein were sequenced and analyzed by LC-ESI-MS/MS. From 1.05 g of whole casein, the highest yield of αS2-casein (6.7 mg/mL) was obtained from Jamnapari and the lowest yield (2.2 mg/mL) was from Saanen. A single band of pure αS2-casein was observed on SDS-PAGE for all breeds. The αS2-casein showed coverage percentage of amino acid sequence from 76.68 to 92.83%. The two-step purification process developed herein was successfully applied for isolating native αS2-casein from goat milk with high purity, which will allow for future in vitro studies to be conducted on this protein.
Assessment of Fumonisin B1 Concentrations in Wheat and Barley Products in the Punjab Region of Pakistan

Iqbal SZ, Rehman B, Selamat J, Akram N, Ahmad MN, Sanny M, et al.

J Food Prot, 2020 Aug 01;83(8):1284-1288.
PMID: 32678886 DOI: 10.4315/0362-028X.JFP-19-361

ABSTRACT: A total of 133 samples of whole wheat and barley grains and wheat and barley flour collected from retail markets in the main cities of Punjab, Pakistan, were analyzed for the mycotoxin fumonisin B1 (FB1) using reverse phase high-performance liquid chromatography with fluorescence detection. Of these samples, 120 (90%) were positive for FB1, and 75 (63%) of the 120 positive samples had FB1 concentrations higher than the European Union maximum (200 μg/kg). The limit of detection was 4 μg/kg. The highest mean (±SD) concentration of FB1 was found in whole wheat samples, 980.5 ± 211.4 μg/kg. The calculated dietary intakes of FB1 from wheat and barley flours were 4,456 and 503.7 ng/g of body weight per day, respectively.
Fulltext Development of Methyl Ester Antibody-Based Competitive Indirect ELISA for Quantitative Detection of Mitragynine in Human Urine

Mustafa RR, Sukor R, Mohd Nor SM, Saari N, Mohsin AZ

ACS Omega, 2023 Dec 19;8(50):47412-47426.
PMID: 38144118 DOI: 10.1021/acsomega.3c02734

Mitragynine is the main psychoactive compound of Mitragyna speciosa Korth. (kratom). This alkaloid could render psychotropic effects and is often misused as a substitute for commercial drugs. Nowadays, the increasing popularity of kratom has led to the development of a rapid and effective detection method. The detection of mitragynine in a biological sample such as urine requires a highly sensitive and specific method due to the complex nature of mitragynine in urine. Enzyme-linked immunosorbent assay (ELISA) is well known as a rapid screening method for biological samples. In this study, a competitive indirect ELISA was successfully developed using MG-22-OCH3 IgG as a detection antibody for mitragynine in human urine. The mitragynine immunoassay showed a limit of detection and a limit of quantification of 0.412 and 1.25 μg/mL, respectively. The measurement range was between 0.01 and 100.0 μg/mL, with a minimal inhibition (IC50) value of 0.152 μg/mL. The developed ELISA was validated using a gold method such as high-performance liquid chromatography-mass spectrometry (HPLC-MS). The percentage of recovery and the coefficient of variation (CV) for the ELISA and LCMS/MS analyses were 84.0-95.70%, 99.20-112.0%, 7.69-9.78%, and 2.86-6.62%, respectively. This indicates that the developed ELISA is a reliable method that can be used as a rapid approach for quantifying mitragynine content in biological samples.
Fulltext Electrochemical Immunosensor for Detection of Aflatoxin B₁ Based on Indirect Competitive ELISA

Azri FA, Sukor R, Selamat J, Abu Bakar F, Yusof NA, Hajian R

Toxins (Basel), 2018 May 11;10(5).
PMID: 29751668 DOI: 10.3390/toxins10050196

Mycotoxins are the secondary toxic metabolites produced naturally by fungi. Analysis of mycotoxins is essential to minimize the consumption of contaminated food and feed. In this present work, an ultrasensitive electrochemical immunosensor for the detection of aflatoxin B₁ (AFB₁) was successfully developed based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). Various parameters of ELISA, including antigen⁻antibody concentration, blocking agents, incubation time, temperature and pH of reagents, were first optimized in a 96-well microtiter plate to study the antigen⁻antibody interaction and optimize the optimum parameters of the assay. The optimized assay was transferred onto the multi-walled carbon nanotubes/chitosan/screen-printed carbon electrode (MWCNTs/CS/SPCE) by covalent attachment with the aid of 1-Ethyl-3-(3-dimetylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Competition occurred between aflatoxin B₁-bovine serum albumin (AFB₁⁻BSA) and free AFB₁ (in peanut sample and standard) for the binding site of a fixed amount of anti-AFB₁ antibody. Differential pulse voltammetry (DPV) analysis was used for the detection based on the reduction peak of TMB(ox). The developed immunosensor showed a linear range of 0.0001 to 10 ng/mL with detection limit of 0.3 pg/mL. AFB₁ analysis in spiked peanut samples resulted in recoveries between 80% and 127%. The precision of the developed immunosensor was evaluated by RSD values (n = 5) as 4.78% and 2.71% for reproducibility and repeatability, respectively.
Development of a zebrafish model for toxicity evaluation of adulterated Apis mellifera honey

Fakhlaei R, Selamat J, Abdull Razis AF, Sukor R, Ahmad S, Khatib A, et al.

Chemosphere, 2024 May;356:141736.
PMID: 38554873 DOI: 10.1016/j.chemosphere.2024.141736

Since ancient times, honey has been used for medical purposes and the treatment of various disorders. As a high-quality food product, the honey industry is prone to fraud and adulteration. Moreover, limited experimental studies have investigated the impact of adulterated honey consumption using zebrafish as the animal model. The aims of this study were: (1) to calculate the lethal concentration (LC50) of acid-adulterated Apis mellifera honey on embryos, (2) to investigate the effect of pure and acid-adulterated A. mellifera honey on hatching rate (%) and heart rate of zebrafish (embryos and larvae), (3) to elucidate toxicology of selected adulterated honey based on lethal dose (LD50) using adult zebrafish and (4) to screen the metabolites profile of adulterated honey from blood serum of adult zebrafish. The result indicated the LC50 of 31.10 ± 1.63 (mg/ml) for pure A. mellifera honey, while acetic acid demonstrates the lowest LC50 (4.98 ± 0.06 mg/ml) among acid adulterants with the highest mortality rate at 96 hpf. The treatment of zebrafish embryos with adulterated A. mellifera honey significantly (p ≤ 0.05) increased the hatching rate (%) and decreased the heartbeat rate. Acute, prolong-acute, and sub-acute toxicology tests on adult zebrafish were conducted at a concentration of 7% w/w of acid adulterants. Furthermore, the blood serum metabolite profile of adulterated-honey-treated zebrafish was screened by LC-MS/MS analysis and three endogenous metabolites have been revealed: (1) Xanthotoxol or 8-Hydroxypsoralen, (2) 16-Oxoandrostenediol, and (3) 3,5-Dicaffeoyl-4-succinoylquinic acid. These results prove that employed honey adulterants cause mortality that contributes to higher toxicity. Moreover, this study introduces the zebrafish toxicity test as a new promising standard technique for the potential toxicity assessment of acid-adulterated honey in this study and hazardous food adulterants for future studies.
Fulltext Muscle and Serum Metabolomics for Different Chicken Breeds under Commercial Conditions by GC-MS

Tan C, Selamat J, Jambari NN, Sukor R, Murugesu S, Khatib A

Foods, 2021 Sep 14;10(9).
PMID: 34574284 DOI: 10.3390/foods10092174

Globally, village chicken is popular and is known as a premium meat with a higher price. Food fraud can occur by selling other chicken breeds at a premium price in local markets. This study aimed to distinguish local village chicken from other chicken breeds available in the market, namely, colored broiler (Hubbard), broiler (Cobb), and spent laying hen (Dekalb) in pectoralis major and serum under commercial conditions using an untargeted metabolomics approach. Both pectoralis major and serum were analyzed using gas chromatography-mass spectrometry (GC-MS). The principal component analysis (PCA) results distinguished four different chicken breeds into three main groups for pectoralis major and serum. A total of 30 and 40 characteristic metabolites were identified for pectoralis major and serum, respectively. The four chicken breeds were characterized by the abundance of metabolites such as amino acids (L-glutamic acid, L-threonine, L-serine, L-leucine), organic acids (L-lactic acid, succinic acid, 3-hydroxybutyric acid), sugars (D-allose, D-glucose), sugar alcohols (myo-inositol), and fatty acids (linoleic acid). Our results suggest that an untargeted metabolomics approach using GC-MS and PCA could discriminate chicken breeds for pectoralis major and serum under commercial conditions. In this study, village chicken could only be distinguished from colored broiler (Hubbard) by serum samples.
Electrochemical determination of zearalenone using a label-free competitive aptasensor

Azri FA, Eissa S, Zourob M, Chinnappan R, Sukor R, Yusof NA, et al.

Mikrochim Acta, 2020 04 12;187(5):266.
PMID: 32279134 DOI: 10.1007/s00604-020-4218-7

An electrochemical aptasensor is described for determination of the phytohormone of zearalenone (ZEA). The gold electrode was modified with ZEA via covalent attachment using cysteamine-hydrochloride and 1,4-phenylene diisocyanate linker. A truncated ZEA aptamer with a dissociation constant of 13.4 ± 2.1 nM was used in an aptasensor. The electrochemical property was investigated using square wave voltammetry for monitoring the change in the electron transfer using the ferro/ferricyanide system as redox probe. Under optimal experimental conditions, the response was best measured at a potential of 0.20 V (vs. Ag/AgCl). The signals depended on the competitive mechanism between the immobilised ZEA and free ZEA for the aptamer binding site. The aptasensor works in the range 0.01 to 1000 ng·mL-1 ZEA concentration, with a detection limit of 0.017 ng·mL-1. High degree of cross-reactivity with the other analogues of ZEA was observed, whereas none towards other mycotoxins. The aptasensor was further applied for the determination of ZEA in the extract of maize grain and showed good recovery percentages between 87 and 110%. Graphical abstract Schematic representation of the electrochemical determination of zearalenone based on indirect competitive assay. Step a Immobilisation of ZEA on the surface of gold electrode via covalent attachment, b competition for the ZEA aptamer binding site between immobilised and free ZEA, and c current signal of the binding event based on SWV technique.

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