Red-emitted AIE fluorescent microspheres based lateral flow immunoassay for the visual and quantitative detection of aflatoxin M1 in milk.

Background. Aflatoxins are found in various types of food and animal feed. Food contamination with aflatoxin toxin is of particular importance today. Various studies have reported different prevalence of aflatoxin M1 in animal milk. Therefore, due to the importance of this toxin, its role in health, and lack of general statistics about it worldwide, the present study aimed to determine the prevalence of aflatoxin M1 in milk worldwide with a systematic review and meta-analysis study. Methods. In this review study, national and international databases were extracted from SID, MagIran, IranMedex, IranDoc, Embase, ScienceDirect, Scopus, PubMed, and Web of Science (ISI) between January 1988 and February 2020. A random effects model was used for analysis, and heterogeneity of studies with an I2 index was investigated. Data were analyzed using Comprehensive Meta-Analysis (version 2). Results. The prevalence of aflatoxin M1 in milk worldwide from January 1988 to February 2020 in 122 articles with a sample size of 18921 was 79.1% (95% CI: 75.5–82.3%). Regarding the heterogeneity based on metaregression, there was a significant difference between the effect of the year of study (p≤0.001) and sample size (p≤0.001) with the prevalence of aflatoxin M1 in animal milk. Conclusion. The results of this study show that the prevalence of aflatoxin M1 in milk is high worldwide. Therefore, considering the importance of the milk group and its products, special measures should be taken to protect the ration from aflatoxin molds and milk quality.

The objective of this network meta-analysis was to determine the efficacy of different mycotoxin binders (MTB) to reduce aflatoxin M1 (AFM1) in milk. A literature search was conducted to identify in vivo research papers from different databases. Inclusion criteria were in vivo, dairy cows, description of the MTB used, doses of MTB, aflatoxin inclusion in the diet, and concentration of AFM1 in milk. Twenty-eight papers with 131 data points were selected. Binders used in the studies were hydrated sodium calcium aluminosilicate (HSCAS), yeast cell wall (YCW), bentonite, and mixes of several MTB (MX). The response variables were AFM1 concentration, AFM1 reduction in milk, total AFM1 excreted in milk, and transfer of aflatoxin from feed to AFM1 in milk. Data were analyzed with CINeMA and GLIMMIX procedures with the WEIGHT statement of SAS (SAS Inst. Inc.). The AFM1 concentration in milk decreased for bentonite (0.3 µg/L ± 0.05; mean ± SE) and HSCAS (0.4 µg/L ± 0.12), and tended to decrease for MX (0.6 µg/L ± 0.13) but was similar for YCW (0.6 µg/L ± 0.12), compared with control (0.7 µg/L ± 0.12). The percentage reduction of AFM1 in milk was similar for all MTB and different from control with a range of reduction from 25% for YCW to 40% for bentonite. The excretion of AFM1 in milk was lower in YCW (5.3 µg/L ± 2.37), HSCAS (13.8 µg/L ± 3.31), and MX (17.1 µg/L ± 5.64), and not affected by bentonite (16.8 µg/L ± 3.33) compared with control (22.1 µg/L ± 5.33). The transfer of aflatoxin B1 from feed into AFM1 in milk was lowest in bentonite (0.6% ± 0.12), MX (1.04% ± 0.27), and HSCAS (1.04% ± 0.21), and not affected in YCW (1.4% ± 0.10), compared with control (1.7% ± 0.35). The meta-analysis results indicate that all MTB reduced the AFM1 transfer into milk, where bentonite had the highest capacity and YCW the lowest.

The prevalence of aflatoxin M1 in milk of Middle East region: A systematic review, meta-analysis and probabilistic health risk assessment

Aflatoxins, produced by multiple fungal species, are present in several kinds of food items and animal feed. Several studies conducted in Pakistan have reported the presence of aflatoxin M1 (AFM1) in milk. Hence, owing to the public health concern and absence of general statistics regarding the prevalence of AFM1 contamination, current study was aimed to investigate the prevalence of AFM1 in milk in Pakistan. For this study, various databases were searched from 2007 to 2020. A random effect model was applied for analytical purpose and heterogeneity of selected studies was investigated with an I2 index. Comprehensive meta-analysis (version 3) was used for analysis of data. According to the results, prevalence of AFM1 in milk was 84.4% (95% CI 75.0-90.7%). Regarding the heterogeneity based on meta-regression, it has been observed that there was a significant difference between the effect of year of study and sample size with prevalence of AFM1 in animal milk. These results suggest that AFM1 contamination in animal milk is high in Pakistan. Hence, continuous monitoring of AFM1 in animal milk requires utmost attention from the respective food and drug regulatory authorities of Pakistan so that the strict actions and preventive measures should be taken to prevent the prevalence of exposure of AFM1 in animal milk.

Aflatoxin M1 (AFM1) in milk poses a significant threat to human health. This study examined the capacity of Bacillus licheniformis CotA laccase to oxidize AFM1. The optimal conditions for the CotA laccase-catalyzed AFM1 oxidation were observed at pH 8.0 and 70 °C, achieving an AFM1 oxidation rate of 86% in 30 min. The Km and Vmax values for CotA laccase with respect to AFM1 were 18.91 μg mL-1 and 9.968 μg min-1 mg-1, respectively. Computational analysis suggested that AFM1 interacted with CotA laccase via hydrogen bonding and van der Waals interactions. Moreover, the oxidation products of AFM1 mediated by CotA laccase were identified as the C3-hydroxy derivatives of AFM1 by HPLC-FLD and UPLC-TOF/MS. Toxicological assessment revealed that the hepatotoxicity of AFM1 was substantially reduced following oxidation by CotA laccase. The efficacy of CotA laccase in removing AFM1 in milk was further tested, and the result showed that the enzyme agent achieved an AFM1 removal rate of 83.5% in skim milk and 65.1% in whole milk. These findings suggested that CotA laccase was a novel AFM1 oxidase capable of eliminating AFM1 in milk. More effort is still needed to improve the AFM1 oxidase activity of CotA laccase in order to shorten the processing time when applying the enzyme in the milk industry.

Aflatoxin M1 in milk and dairy products, occurrence and recent challenges: A review

Immobilization of Bacillus megaterium spores on Eppendorf tubes through physical adsorption has been used in the detection of aflatoxin M1 (AFM1) in milk within real time of 45 ± 5 min using visual observation of changes in a chromogenic substrate. The appearance of a sky-blue colour indicates the absence of AFM1 in milk, whereas no colour change indicates the presence of AFM1 in milk at a 0.5 ppb Codex maximum residue limit. The working performance of the immobilized spores was shown to persist for up to 6 months. Further, spores immobilized on 96-well black microtitre plates by physical adsorption and by entrapment on sensor disk showed a reduction in detection sensitivity to 0.25 ppb within a time period of 20 ± 5 min by measuring fluorescence using a microbiological plate reader through the addition of milk and fluorogenic substrate. A high fluorescence ratio indicated more substrate hydrolysis due to spore-germination-mediated release of marker enzymes of spores in the absence of AFM1 in milk; however, low fluorescence ratios indicated the presence of AFM1 at 0.25 ppb. Immobilized spores on 96-well microtitre plates and sensor disks have shown better reproducibility after storage at 4 °C for 6 months. Chromogenic assay showed 1.38% false-negative and 2.77% false-positive results while fluorogenic assay showed 4.16% false-positive and 2.77% false-negative results when analysed for AFM1 using 72 milk samples containing raw, pasteurized, and dried milk. Immobilization of spores makes these chromogenic and fluorogenic assays portable, selective, cost-effective for real-time detection of AFM1 in milk at the dairy farm, reception dock, and manufacturing units of the dairy industry.

A quantitative fluorescence-labeled immunosorbent assay and qualitative on-site column tests were developed for the determination of aflatoxin M1 in milk products. The use of liposomes loaded with quantum dots as a label significantly increased the assay sensitivity by encapsulating multiple quantum dots in a single liposome and, therefore, amplifying the analytical signal. Two different techniques were compared to obtain aflatoxin-protein conjugates, used for further coupling with the liposomes. The influence of nonspecific interactions of the liposome-labeled conjugates obtained with the surface of microtiter plates and column cartridges was evaluated and discussed. The limit of detection for fluorescence-labeled immunosorbent assay was 0.014 μg kg(-1). For qualitative on-site tests, the cutoff was set at 0.05μg kg(-1), taking into account the EU maximum level for aflatoxin M1 in raw milk, heat-treated milk, and milk for the manufacture of milk-based products. The direct addition of labeled conjugate to the milk samples resulted in an additional decrease of analysis time. An intralaboratory validation was performed with sterilized milk and cream samples artificially spiked with aflatoxin M1 at concentrations less than, equal to and greater than the cutoff level. It is shown that milk products can be analyzed without any sample preparation, just diluted with the buffer. The rates for false-positive and false-negative results were below 5% (2.6% and 3.3%, respectively).

Excretion of Aflatoxin M1 in Milk of Dairy Ewes Treated with Different Doses of Aflatoxin B1

A spore germination-based concept and its transformation into a field level prototype for monitoring aflatoxin M1 (AFM1) in milk was developed. Initially, 15 strains of Bacillus spp. procured from different culture collection were screened for AFM1 sensitivity using spot assay and marker strain showing inhibition at 0.5 ppb was selected based upon maximum zone of inhibition. The selected strain B. megaterium 2949 was further screened for different enzymes activities and subsequently its spores were produced to an extent of 73.13% ± 3.197% in newly developed sporulation medium containing beef extract (0.0075% ± 0.0004%), yeast extract (0.015% ± 0.001%), peptone (0.0375% ± 0.0016%), and sodium chloride (0.0375% ± 0.0018%). A spore germination-based concept/ assay was optimized by immobilizing spores in eppendorf with pretreated milk (80°C/15 min) containing germinant and chromogenic substrate followed by incubation at 37°C. The appearance of sky blue color within real time of 45 min indicated spores germination and release of specific marker enzyme such as acetyl esterase and its specific action on chromogenic substrate which demonstrates absence of AFM1 in milk. However, if there was no color change, presence of AFM1 at 0.5 ppb MRL was denoted by Codex. The developed concept on AFM1 detection was validated and a correlation of 0.97 was established with AOAC approved Charm 6602 and ELISA at Codex MRL with minimal false positive and negative results. The cost effective test has potential application in dairy farms, manufacturing, and R&D units for routine monitoring of AFM1 in milk.

A label-free silver wire based impedimetric immunosensor for detection of aflatoxin M1 in milk

Control of aflatoxin M1 in milk by novel methods: A review

Aflatoxin M1 (AFM1) in milk is a problem of great concern. Current methods of detection require large instruments and need specific test sites. Therefore, it is necessary to establish a fast, convenient, and accurate detection method for AFM1. We established a system based on fluorescent microspheres containing a Eu3+ chelate named AFM1-POCT. These components comprised the AFM1-POCT kits. After refrigeration at 4°C for 12 months, the intra and inter assay coefficients of variability (CVs) for the kits were 4% and 5%, respectively. AFM1-POCT compared well with ultra-high-performance liquid chromatography (UHPLC) determination in the range 0.0121–2 μg/kg (paired samples test, P > 0.05). Aflatoxin B1 and G1 do not react with aflatoxin M1. By using the AFM1-POCT method, the detection time is shortened to 5 min, the accuracy is comparable to that of UHPLC, and convenience and range of application are improved.

Detection of aflatoxin M1 in milk products from China by ELISA using monoclonal antibodies

An experiment was carried out to study the excretion of aflatoxin M1(AFM1) in milk of three goats fed a single dose (0.8mg/head) of pure aflatoxin B1 (AFB1). The values of AFM1 concentration excreted in milk was highly variable among goats, even if the pattern of excretion over time was very similar among the three animals. AFM1 was first detected at the milking performed 1h after the AFB1 administration. The highest values of AFM1 concentration in milk were reached 3 and 6h after the AFB1 intake. The trend of clearance of AFM1 in milk over time was expressed by a decreasing exponential equation. AFM1 concentration was below the EU maximum allowed level (50 ng/L) in milk collected 36 h after the AFB1 administration.