Aflatoxin in feedstuffs and raw milk in Republic of North Macedonia, occurrence and assessment

The main objective of this research is to estimate the situation of occurance of aflatoxins in feedstuffs and aflatoxin M1 raw cow`s milk in Republic of North Macedonia. Detected average concentrations of total aflatoxins in the examined period 2014-2017 were below legal prescribed level. Within the official monitoring programme in the country, 1718 samples of raw cow's milk have been analyzed during 2017 and 2018. Comparison of the data showed that the number of examined samples with values for aflatoxin M1 above the allowed limits, according to the regulation is in 2018 (10 samples) and 2017 (5 samples). Additionally, the calculation of exposure assessment of the human population in North Macedonia demonstrated that the exposure to AFM1 of the adult population in N. Macedonia for 2017 is 0.0563 ng/kg bw/daily, and for 2018 is 0.0824 ng/kg bw/daily. The calculated hazard index (HI) In both years, was lower than one, which means that there are no significant health risks for the consumers. It is important to have continuously low levels of aflatoxins in animal feed and nutrition, especially that the climate changes create more favorable conditions for the development of moulds of Aspergillus.

The Protective Effects of Lactoferrin on Aflatoxin M1-Induced Compromised Intestinal Integrity

Aflatoxin M1 (AFM1), the only toxin with maximum residue levels in milk, has adverse effects on the intestinal barrier, resulting in intestinal inflammatory disease. Lactoferrin (LF), one of the important bioactive proteins in milk, performs multiple biological functions, but knowledge of the protective effects of LF on the compromised intestinal barrier induced by AFM1 has not been investigated. In the present study, results using Balb/C mice and differentiated Caco-2 cells showed that LF intervention decreased AFM1-induced increased intestinal permeability, improved the protein expression of claudin-3, occludin and ZO-1, and repaired the injured intestinal barrier. The transcriptome and proteome were used to clarify the underlying mechanisms. It was found that LF reduced the intestinal barrier dysfunction caused by AFM1 and was associated with intestinal cell survival related pathways, such as cell cycle, apoptosis and MAPK signaling pathway and intestinal integrity related pathways including endocytosis, tight junction, adherens junction and gap junction. The cross-omics analysis suggested that insulin receptor (INSR), cytoplasmic FMR1 interacting protein 2 (CYFIP2), dedicator of cytokinesis 1 (DOCK1) and ribonucleotide reductase regulatory subunit M2 (RRM2) were the potential key regulators as LF repaired the compromised intestinal barrier. These findings indicated that LF may be an alternative treatment for the compromised intestinal barrier induced by AFM1.

Lactoferrin Alleviated AFM1-Induced Apoptosis in Intestinal NCM 460 Cells through the Autophagy Pathway

Aflatoxin M1 (AFM1) is the only mycotoxin with maximum residue limit in milk, which may result in serious human diseases. On the contrary, lactoferrin (Lf) is an active protein with multiple functions. Studies have confirmed that Lf has a powerful potential to protect the intestines, but the influence of Lf on mycotoxins is not clear. This study aims to explore whether Lf can protect the cytotoxicity induced by AFM1, and determine the underlying mechanisms in human normal colonic epithelial NCM460 cells. The results indicated that AFM1 decreased the cell viability, and increased the levels of apoptosis and autophagy of NCM460 cells. Lf can alleviate the cytotoxicity induced by AFM1 through enhancing cell viability, significantly down-regulated the expression of apoptotic genes and proteins (BAX, caspase3, caspase9, caspase3, and caspase9), and regulated the gene and protein expression of autophagy factors (Atg5, Atg7, Atg12, Beclin1, ULK1, ULK2, LC3, and p62). Furthermore, interference of the key gene Atg5 of autophagy can reduce AFM1-induced apoptosis, which is consistent with the role of Lf, implying that Lf may protect AFM1-induced intestinal injury by inhibiting excessive autophagy-mediated apoptosis. Taken together, our data indicated that Lf has a mitigating effect on apoptosis induced by AFM1 through the autophagy pathway.

The use of the two different mycotoxin deactivators in the nutrition of dairy cows

The aim of this study was to investigate the effect of two different commercial products for mycotoxin deactivation in the dairy cows’ nutrition. The research was done on the 18 dairy cows in the different phase of lactation, with the average production of 14 L of milk per day. The diet contained in average 8 kg of concentrate feed, 10 kg of mixed peas and wheat forage, 4 kg hay of alfalfa and cereal straw ad libitum. Before starting the trials all components of the cows’ ration were analysed for the presence of aflatoxin B1, while cow’s milk was analysed for the presence of aflatoxin M1. In the first phase of the trial cows were fed diet containing aflatoxin B1 at level of 5±0.9 µg/kg, which resulted in the average aflatoxin M1concentration of 181±3.5 ng/kg in the milk. In the second phase of the trail 0.5 % of the commercial product 1 (Neozel®) was added into the feed ration of cows and after the period of adaptation, the aflatoxin M1 concentration in milk was measured. In the third phase of the trail 0.3 % of the commercial product 2 (miko-Stop 0.3®) was added into the feed ration of cows and also, after the period of adaptation, the concentration of aflatoxin M1 in milk was measured. The last phase was the control phase in which cows were fed diets with aflatoxin B1 (5 µg/kg) without added mycotoxin deactivators. The use of both commercial products for ten days resulted in the reduction of aflatoxin M1 levels for 35.9 % and 53.6 %, respectively.

Research of microstructure and microbiological indicators of pasta products from non-traditional raw materials

Modern trends in the development of pasta production are based on the use of starch-containing raw materials for the production of medicinal and preventive pasta products. In this connection, an important observation is the state of protein and starch in pasta flour, which can be achieved by electron microscopy of the microstructure of macaroni products. Therefore, the study of microstructures and microbiological indicators of pasta from non-traditional raw materials will be relevant. The practical value of the research is characterized by improved consumer properties of pasta made from non-traditional raw materials. The novelty of the research is the development of scientific and technological bases for the production of pasta from non-traditional polycereal raw materials. The results obtained: the analysis of the obtained electronic micro-drawings showed that the microstructure of pasta from non-traditional raw materials corresponds to the standards in comparison with the control sample. The following types of mycotoxins were found in pasta from non-traditional raw materials in permitted quantities: aflatoxin B1, aflatoxin M1, zearalenone, deoxynivalenol, ochratoxin A, fumonisin, patulin, T-2. For example, the amount of zearalenone (mg/kg): in pasta according to recipe No. 1 – 0.0637, according to recipe No. 2 – 0.0251 and according to recipe No. 3 – 0.0758; in flour raw materials – according to recipe No. 1 – 0.1485, according to recipe No. 2 – 0.0261 and according to recipe No. 3 – 0.2.

Innovative application of postbiotics, parabiotics and encapsulated Lactobacillus plantarum RM1 and Lactobacillus paracasei KC39 for detoxification of aflatoxin M1 in milk powder

This study aimed to evaluate aflatoxin M1 (AFM1) level in milk powder and infant milk formulae, in addition to applying innovative methods for AFM1 & AFB1 detoxification. Fifty random samples of milk powder and infant formulae (25 of each) were collected from the Egyptian markets for assessing AFM1 level using ELISA technique. Bioactive components comprising cell free supernatants (postbiotic), acid-dead cells (parabiotic) and the encapsulated-cells of Lactobacillus plantarum RM1 and Lactobacillus paracasei KC39 were evaluated for their antifungal activity against toxigenic mold strains and their impact on AFB1 and AFM1 reduction in reconstituted milk powder. AFM1 concentration in unpacked milk powder was higher than that of packed samples and infant formulae, although these differences were not significant (P > 0.05). About 96.0, 29.4 and 25.0% of the tested infant formulae, unpacked, and packed milk powder were unacceptable in terms of the AFM1 limit defined by Egyptian and European standards, while all samples were in accordance with the USA/FDA standard. All tested mycotoxigenic strains were sensitive to the different treatments of the probiotics with the highest sensitivity regarding Fusarium strain with L. paracasei KC39 compared to other genera. The degradation ratios of AFM1 using the bioactives of the L. paracasei KC39 were higher than that of L. plantarum RM1 bioactives. Additionally, KC39 parabiotic manifested the best AFB1 reduction (60.56%). In conclusion, the positive and highly significant relationship (P < 0.05) between these effective biocompounds mirrors their major detoxification role which gives a safe solution for AFs contamination issues in milk and milk products.

The contamination rate of Aflatoxin M1 in milk samples of Iran: A systematic review and meta-analysis protocol

Aflatoxin M1 (AFM1) as a 4-hydroxylated metabolite of carcinogenic mycotoxin Aflatoxin B1 (AFB1) is considered as an acute toxic compound found in milk and its derived products (1). Regarding the critical public health issue induced by AFM1 and as milk is essential in human food chain, its maximum limit has been set by regulatory agencies considering the economic status and development of the countries (4). The maximum permissible limit for AFM1 is 50 ng/kg (different countries including European Union (EU) members) to 100 ng/kg (different countries including USA) (5). In Iran, it has been regulated as 100 ng/kg (6). To the best of our knowledge, there is several similar systematic review and meta-analysis about the contamination of campylobacter in milk, globally and specifically in Iran (4, 5, 7-10). However, we will try to examine the mean and prevalence of AFM1 in different type of milk (cow, sheep, goat, raw, processed) in different region of Iran with long time interval (1974-2021). Moreover, more general and specific databases and grey literature with excellent eligibility criteria will be applied to have a more comprehensive review leading to an interesting finding by techniques such as risk of bias assessment (for assessing quality of included studies), publication bias assessment, finding the source of heterogeneity by sub-group analysis in addition to meta-analysis process.

Development of Gold Nanoparticles Decorated Molecularly Imprinted–Based Plasmonic Sensor for the Detection of Aflatoxin M1 in Milk Samples

Aflatoxins are a group of extremely toxic and carcinogenic substances generated by the mold of the genus Aspergillus that contaminate agricultural products. When dairy cows ingest aflatoxin B1 (AFB1)−contaminated feeds, it is metabolized and transformed in the liver into a carcinogenic major form of aflatoxin M1 (AFM1), which is eliminated through the milk. The detection of AFM1 in milk is very important to be able to guarantee food safety and quality. In recent years, sensors have emerged as a quick, low–cost, and reliable platform for the detection of aflatoxins. Plasmonic sensors with molecularly imprinted polymers (MIPs) can be interesting alternatives for the determination of AFM1. In this work, we designed a molecularly–imprinted–based plasmonic sensor to directly detect lower amounts of AFM1 in raw milk samples. For this purpose, we prepared gold–nanoparticle–(AuNP)−integrated polymer nanofilm on a gold plasmonic sensor chip coated with allyl mercaptan. N−methacryloyl−l−phenylalanine (MAPA) was chosen as a functional monomer. The MIP nanofilm was prepared using the light–initiated polymerization of MAPA and ethylene glycol dimethacrylate in the presence of AFM1 as a template molecule. The developed method enabled the detection of AFM1 with a detection limit of 0.4 pg/mL and demonstrated good linearity (0.0003 ng/mL–20.0 ng/mL) under optimized experimental conditions. The AFM1 determination was performed in random dairy farmer milk samples. Using the analogous mycotoxins, it was also demonstrated that the plasmonic sensor platforms were specific to the detection of AFM1.