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Article

The Influence of Binding of Selected Mycotoxin Deactivators and Aflatoxin M1 on the Content of Selected Micronutrients in Milk

1
The Dr Andrija Stampar Teaching Institute of Public Health, Mirogojska Cesta 16, 10000 Zagreb, Croatia
2
Department of Epidemiology and Public Health, University of Applied Health Sciences in Zagreb, Mlinarska Cesta 38, 10000 Zagreb, Croatia
3
Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia
*
Author to whom correspondence should be addressed.
Processes 2022, 10(11), 2431; https://doi.org/10.3390/pr10112431
Received: 17 September 2022 / Revised: 13 November 2022 / Accepted: 15 November 2022 / Published: 17 November 2022

Abstract

:
Milk containing aflatoxin M1 (ATM1) in quantities above 0.05 µg/kg is considered unsuitable for consumption. It is possible to use mycotoxin deactivators that bind aflatoxin M1 and allow the further use of milk. The study aimed to examine the impact of selected mycotoxin deactivators (beta-glucan from yeast and oats, and live and dead lactic acid bacteria) on the nutritional composition of milk after binding to aflatoxin M1 intentionally added to milk. The study used consumption milk with 2.8% milk fat intentionally contaminated with aflatoxin M1. Furthermore, 0.05% and 0.1% solutions of beta-glucan from yeast and beta-glucan from oats were added to the contaminated milk, as well as live and dead lactic acid. Concentrations of Na, K, Mg, and Ca were monitored at the zero hour of binding of mycotoxin deactivators and ATM1, after 2 h of binding, and after 4 and 24 h of binding. The largest deviations were found in Na, K, and Mg, while the minimum changes were observed in Ca. Live lactic acid bacteria were found to have the least impact on micronutrients, except in Na (difference = 40, p = 0.029, GES = 0.083), where the 0.1% solution from oats had the least impact on micronutrient content. The results of this study suggest that it is best to use live lactic acid bacteria where the different duration of action regarding nutrients, with the possible exception of Na, is not relevant, which indicates that, when using this mycotoxin deactivator, milk contaminated with ATM1 can be further used.

1. Introduction

Milk is a product of mammals’ mammary glands, obtained by one or more milking sessions, to which nothing has been added or subtracted. It is a white liquid, with a complex chemical composition, naturally rich in certain micro and macronutrients such as calcium, magnesium, phosphorus, proteins, fats, carbohydrates, and vitamins [1]. Micronutrients such as Ca, Mg, Na, K, P, and Cl; Fe; Cu; Zn; and Se are necessary for several vital functions in the body. The microelements are differently distributed in the aqueous and micellar phases of milk. Ions K, Na, and Cl are in the aqueous phase, while Ca, P, and Mg are partially bound to casein micelles. Approximately a third of Ca, half of P, and two-thirds of Mg are in the aqueous phase of milk. Studies have shown that micronutrients in the human body make up only 4% of total body weight but are part of every tissue, body fluid, cell, and organ [2]. They act as catalysts in many biological reactions in the body, such as muscle contraction and nerve impulse transmission, and help to utilize nutrients from food [3,4]. The mineral composition of milk can be affected by the heat treatment of milk, so mineral concentrations in raw milk are higher compared to heat-treated milk [5,6]. The exception is iron, which has been found to be present in greater quantities in heat-treated milk than in raw milk [7]. In milk and dairy products, mineral elements can be present as inorganic ions and salts, or as part of organic molecules such as proteins, fats, carbohydrates, and nucleic acids. The chemical form in which the mineral elements are present in milk is extremely important because it affects their absorption in the stomach and thus their biological utilization. The mineral composition of milk is not constant, as it varies depending on the stage of lactation and nutritional status of the animal, as well as environmental conditions and genetic factors [7]. The highest amount of calcium in cow’s milk [99%] can be found in skimmed milk. Two-thirds of total calcium is most often in the form of calcium phosphate in the colloidal phase (casein micelles) or as calcium ions bound to phosphoserine. The remaining calcium is present in the soluble phase of milk. About 10% of total calcium is ionic calcium present in the soluble phase of milk, and the remaining soluble calcium is present in the form of calcium citrate [8,9]. Of the total phosphorus found in cow’s milk, 20% is present as organic phosphate bound to casein and 80% as inorganic phosphate. Of the total inorganic phosphate content, 44% is bound to calcium phosphate casein micelles, while 56% is in soluble form as free phosphate ions. Magnesium in cow’s milk is present in the skimmed fraction of milk (98–100%), of which 65% is in the soluble phase (40% as magnesium citrate, 7% as magnesium phosphate, and 16% as free ions). The remainder in the colloidal phase is bound to casein micelles (50% to colloidal calcium phosphate and 50% to phosphoserine in casein) [10]. Sodium is a major cation in extracellular fluids and is an important regulator of pressure, acid-base balance, and membrane potential, and plays an important role in the active transport of substances across the cell membrane. The contribution of cow’s milk to the daily intake of sodium in the human diet is low, but some dairy products that contain additional amounts of salt are a significant source of sodium. Potassium is one of the most important intracellular cations, and extracellular potassium is important for transmitting nerve impulses, muscle contractions, and maintaining blood pressure. Potassium intake has been found to positively affect the quality of human bones [11]. The content of sodium, potassium, and chloride in milk has physiological significance in the nutrition of infants. Conversely, excessive intake of these three mineral elements can cause issues related to renal capacity in infants [6]. Milk and dairy products are rich sources of calcium [12], while their absorption in the body depends on the amount of vitamin D and the person’s age [13]. About 20–30% of the calcium ingested in the body is absorbed [14], with the rate of absorption depending on the form in which calcium is present in food, its quantity, solubility, and interaction with other food ingredients. Considering all the above factors, calcium from milk has a high degree of bioavailability [15]. Phosphorus is an element that is associated with a number of important biological functions in the human body. It is integral to many biological components such as fats, proteins, carbohydrates, and nucleic acids. It is involved in building bones and regulating many enzymes and is an integral part of ATP (adenosine triphosphate) [16,17].
Milk as a food, as well as other types of food [18,19], can be contaminated with different types of contaminants, among which mycotoxins are prominent, especially aflatoxin M1 (ATM1). It is a metabolic product of aflatoxin B1 (AFB1), or dihydroderivative ATB1 produced by the toxicogenic mold Aspergillus flavus and belongs to the group of carcinogenic compounds [20]. In view of the above, such contaminated milk poses a danger to human health, especially young children, where milk is one of the basic foodstuffs, as was confirmed by the risk assessment of the European Food Safety Authority (EFSA) [21]. Animal feed is very often contaminated with various types of mycotoxins, most often AFB1, which research has proven to be a very toxic compound for animals [22,23,24,25,26], and which the International Agency for Research on Cancer has classified in the category of potentially carcinogenic compounds [27]. By consuming contaminated food, said mycotoxin enters the animal’s digestive system, where it is transformed in the mammary glands into the hydroxylated form of ATM1 [28]. Milk containing ATM1 in quantities above 0.05 µg/kg is considered unhealthy and unsuitable for human and animal consumption [29].
To prevent major economic harm and protect human and animal health, various types of mycotoxin deactivators are used to remove certain mycotoxins from food and thus make them suitable for consumption. Studies have shown that aluminosilicates [30,31], clay [32,33], and beta-glucan [34,35,36] can be used as effective mycotoxin deactivators, as well as some types of lactic acid bacteria (BMK) [37,38,39] which have a certain affinity to remove mycotoxins from food. Thus, it was found that food contaminated with ochratoxin A intended for the broiler diet causes various changes in internal organs (spleen, liver, kidneys), while in broilers fed with feed containing the yeast Trichosporon mycotoxinivorans, as a mycotoxin deactivator, damage to internal organs was significantly lower, which confirms the fact that it is an effective mycotoxin deactivator, and it binds mycotoxins well [38]. Similar studies have been conducted on other types of feed using different types of mycotoxin deactivators for the purpose of binding various types of mycotoxins [39,40,41]. In addition to these mycotoxin deactivators, studies also mention nanotechnology that can be used to effectively remove mycotoxins from food and feed [42]. Since milk is a food that is contaminated with a specific form of mycotoxins—ATM1, which in milk binds to proteins, only some of the mycotoxin deactivators are suitable for its removal. Previous research has indicated that beta-glucans and LABs have a high affinity for ATM1 binding, but very little research has been associated with their effect on milk’s nutritional composition [43,44,45]. Contamination of milk with aflatoxin M1 because of feeding animals with feed contaminated with aflatoxin B1 is relatively common. To prevent major economic harm and destruction of contaminated milk, it is possible to use mycotoxin deactivators that bind aflatoxin M1 and allow further use of milk.

2. Materials and Methods

2.1. Aim and Hypothesis

The study used consumption milk with 2.8% milk fat from a Croatian producer, which was intentionally contaminated with aflatoxin M1 with a purity of 99.1 ± 1% (manufactured by Romer Labs Diagnostic GmbH, Tulln an der Donau, Austria) in acetonitrile (manufactured by PanReac AppliChem, Castellar del Vallès, Spain) to obtain a concentration of 50 ng/mL.
The milk samples were selected considering the size of the market coverage by the largest producer in the country of the research, which holds almost 50 percent of the milk market.
The study aimed to examine the impact of selected mycotoxin deactivators (beta-glucan from yeast and oats, and live and dead lactic acid bacteria) on the nutritional composition of milk after binding to intentionally added aflatoxin M1 to milk.
The hypothesis of this research assumes that the selected mycofixants bind ATM1 well, but do not significantly affect the content of micronutrients, and milk can be used as food for humans and animals after removing ATM1.

2.2. Laboratory Methodology

For this purpose, the changes related to the number of selected micronutrients such as Na, K, Mg, and Ca were monitored. Their concentrations were monitored at zero hours of binding, and after two, four, and 24 h of binding and the values obtained were compared with the values of micronutrients in milk analyzed before ATM1 contamination. Additionally, this study aimed to determine whether there are statistically significant differences between individual micronutrients and type of mycotoxin deactivator, as well as differences in the length of binding of mycotoxin deactivators and ATM1, and the amount of ATM1 contaminating the milk.
Furthermore, 0.05% and 0.1% solution of beta-glucan from yeast (Solgar, New Jersey, NJ, USA, SXENB7023502B) and beta-glucan from oats (Darvitalis d.o.o., Zagreb, Croatia) were added to the contaminated milk, as well as live and dead lactic acid bacteria in the amount of 106 for live cells and 1 mg biomass/mL for dead cells. Beta-glucan suppliers were selected in accordance with the availability on the Croatian market and annual commercial contracts with suppliers at the institution level were made through mandatory public binding. Concentrations of Na, K, Mg, and Ca were monitored at the zero hour of binding of mycotoxin deactivators and ATM1, after 2 h of binding, and after 4 and 24 h of binding. For comparison, the amounts of micronutrients investigated were also analyzed before ATM1 milk contamination. A total of fifteen milk samples were analyzed for each mycotoxin deactivator used. The samples were prepared at room temperature by weighing 5 mL of the sample into a Teflon cuvette of a microwave device after homogenization and adding 5 mL of concentrated nitric acid (min. 65% w/w, Scharlab, S.L., Barcelona, Spain) and 1 mL of 30% hydrogen peroxide (Fisher, Loughborough, UK). The cuvette was closed and placed in a drum for microwave digestion; after cooling, the clear solution was quantitatively transferred through a glass funnel into a volumetric flask, while rinsing with deionized water, and filled up to the mark with deionized water. Inductively coupled plasma with a mass detector (ICP-MS 7900), manufactured by Agilent, Santa Clara, CA, USA, was used for the analysis of the elements. An overview of the conditions of recording on ICP-MS is presented in Table 1.
All micronutrients were analyzed and quantified in milk in the same manner and under the same conditions before the addition of mycotoxin deactivators. Before adding aflatoxin AF M1 to milk for human consumption with 2.8% milk fat, the amounts of micronutrients were determined and the following values were obtained: Na 302.97 mg/L; Mg 75.16 mg/L; K 1188.86 mg/L; and Ca 864.7 mg/L, which is within the acceptable and expected values for milk on the Croatian market. The stated values are mean values based on 5 measurements of micronutrients’ composition using the same initial sample. All the analyses in the further experiments were carried out in relation to the same initial milk sample, which is the reason for the basic values of micronutrients being considered the same in all the conducted experiments. The design of the experiment and the analyses and the subsequent statistical processing of the data was carried out so as to monitor and compare the changes between the micronutrients’ composition at 4 time points, and not in relation to the initial sample.
Individual standards were used to prepare the calibration curves. Linearity of ≥0.999 was achieved for the calibration curve of each element, while used for the operation of the instrument were argon gases with a purity of 99.9995% (manufactured by Messer, Gumpoldskirchen, Austria), helium with a purity of 6.0 (manufactured by Messer, Gumpoldskirchen, Austria), internal standard (Bi, Sc, Y, Ge) 100 µg L−1, and certified reference materials: On (purity: 1001.3 ± 3.9, manufactured by Cpa Chem Ltd., Bogomilovo, Bulgaria), K (purity: 1002.2 ± 4.5, manufactured by Cpa Chem Ltd., Bogomilovo, Bulgaria), Ca (purity: 1001.5 ± 2.9, manufactured by Cpa Chem Ltd., Bogomilovo, Bulgaria), and Mg (purity: 1003.4 ± 2.2, manufactured by Cpa Chem Ltd., Bogomilovo, Bulgaria).

2.3. Statistical Analysis

A repeated measures design was used in the study, testing six different mycotoxin deactivators across four time points. The aim was to assess the lowest variation in mineral content due to mycotoxin deactivators. Data are presented descriptively as mineral values by mycotoxin deactivator and time point. The analysis was conducted on difference scores with respect to the baseline to find mycotoxin deactivators that had the least influence on mineral content at 2 h, 4 h, and 24 h time points. Repeated measures ANOVA was used to analyze the data, Mauchly’s test of sphericity was used to assess the assumption of sphericity, and Greenhouse–Geisser correction was applied to the degrees of freedom as needed. The effect size was calculated as generalized eta squared (GES). The probability of type I error was set to 5%. Analysis was conducted using R statistical software, version 4.2.1. Code and data are available in the Supplementary Materials.

3. Results

The results of the research are shown in Table 2 and Table 3. Table 2 shows the means and standard deviations of all mycotoxin deactivators across four time points. Table 3 shows the results of the effect of aflatoxin M1 binding in milk on the quantity of sodium, magnesium, calcium, and potassium, and LAB to live and dead cells depending on the time of binding.
According to the analysis in Table 3, we cannot reject the null hypothesis for the sample group that had one percent of oats added as a mycotoxin deactivator, indicating that there is less change in mineral content over time than in other sample groups. For all other minerals, the null hypothesis held true for the live bacteria sample. Of all the minerals, calcium mineral content was least affected by mycotoxin deactivators, with live bacteria having the smallest impact indicated by the generalized eta squared value.
Figure 1 shows mean differences in relation to the baseline. The live bacteria sample group is represented by the green line. The general trend indicates a decrease in mineral content across time points.
It is also important to present the results of aflatoxin reduction based on the bacteria and beta-glucans in Table 4 because that result is essential to access the efficiency of deactivators.
The initial concentration of aflatoxin M1 (ATM1) contaminated with milk was 0.5 µg/kg.
The best removal of AFM1 was demonstrated using 0.01% β-glucan from oats and 0.005% β-glucan isolated from yeast which is 0.169 µg/kg and 0.178 µg/kg, respectively. The results indicate the fact that aflatoxin binding is a very fast process and reaches its maximum in the first hours of binding. This is most likely due to the different molecular structure of β-glucan from oats compared to β-glucan isolated from yeast.
The results of ATM1 binding to lactic acid bacteria (LAB) indicate that dead LAB cells have a higher ability to bind ATM1 in intentionally contaminated milk than live LAB. This ability to bind is most pronounced in the second hour of binding, while no difference in their binding was observed during the fourth hour. In the period from 4 to 24 h, dead LAB cells showed better binding power than living LAB.
The use of mycotoxin deactivators to reduce mycotoxins in food intended for animal consumption is increasingly used, and the number of mycotoxin deactivators and their mixtures is increasing. Inorganic mycotoxin deactivators based on aluminosilicate or clay are most used, while organic mycotoxin deactivators are used to a lesser extent due to their relatively high market price [46,47,48]. Mycotoxin deactivators can be added to foods also intended for human consumption, especially those of organic origin [49]. Since milk is a common and widely used food, primarily by children and the elderly, due to the specific physiology of the digestive system of dairy animals, especially cows, can be contaminated with aflatoxin M1, especially where insufficient attention is paid to animal feed security [50], the issue of aflatoxin M1 removal is of paramount public health importance. This is more significant since it is a carcinogenic substance, making the removal of aflatoxin M1 from milk a challenging process aimed at removing AFM1 from milk without changing the nutritional composition of milk to a greater extent or having milk retain its nutritional properties so that it can continue to be used for human or animal consumption.

4. Discussion

This study used live and dead LAB cells and beta-glucan derived from yeast and beta-glucan from oats as mycotoxin deactivators. Previous studies have shown that LABs are good mycotoxin deactivators, but there are scarce studies on their effect on the composition and quantity of micronutrients after their binding to mycotoxins [51]. Similarly, studies have been conducted that indicate the effectiveness of using beta-glucan as a mycotoxin deactivator in various types of mycotoxin-contaminated foods. Just like LABs, they belong to the group of organic mycotoxin deactivators that are increasingly preferred in everyday practice due to their binding efficiency to mycotoxins, as well as due to their chemical structure. In addition to binding mycotoxins well, LABs and beta-glucans also bind well to some other toxic substances from food, such as metals. LABs have shown a high percentage of binding of aluminum from food, especially in the case of dead cells, and high binding efficiency was present as early as zero hours, 0 o’clock, showing it is a fast-binding process or passive binding mechanism [52]. Equally, from the literature data of previous research, it is clear that LABs to a greater or lesser extent bind other metals such as mercury, cadmium, lead, copper, and zinc to a greater or lesser extent. Binding efficiency varies from metal to metal, as does the type of bacteria. In mercury, the binding efficiency to LABs was determined after a longer incubation period, while dead cells have a higher affinity for mercury. Bacteria are considered as having a higher affinity for components other than mercury, resulting in poorer binding of mercury [53]. For lead binding, it is significant that a large part of ions can bind to LABs within an hour, which indicates the important role of bio-absorption in the binding mechanism; the process of bioaccumulation steps in after most metal-binding sites on the cell surface is taken [54]. The results of this study, which looked at the effect of live and dead LABs that bind AFM1 as mycotoxin deactivators, on naturally present micronutrients in milk, showed that LABs have very little effect on changing the number of micronutrients in milk. It was found experimentally that when using dead LABs, there was no negative deviation for Ca during the zero hours of binding, while for other micronutrients, deviations of 21% for Na, 15% for K, and 5% for Mg were observed. When using live LABs, negative deviations for Ca and Mg in the same period were also not found, while for K and Na they are 6%. Equally, no negative deviations for Ca were observed in live bacteria during a longer binding time, or after 2, 4, and 24 h binding, while in Mg the quantity decreased by 3% after 2 h of binding, by 5% after 4 h, and by 6% at 24 h. The amount of K during 2 h of binding remained unchanged compared to the zero hours, and after 4 and 24 h, it decreased by 2%. The only statistically significant change in micronutrients by live bacteria was for sodium (p = 0.029, GES = 0.083) which increased at 4 h by 13%. The obtained results suggest that dead LABs have a weak effect on the change in the composition of the micronutrients naturally present in milk. This can be explained by the fact proven in previous studies that LABs bind AFM1 best in the first hour of action as well as contaminants in milk, especially lead, cadmium, aluminum, copper, and zinc, which leads to saturation on the cell surface, and it can be assumed that there occurs an inability to bind to other substances, such as milk micronutrients [52,53,54]. The binding affinity of metals and micronutrients can be affected by temperature, as confirmed by the results of studies related to copper binding. It was found that at higher temperatures, there is an increase in biomass, which increases the number of sites available for binding of metal ions, but if temperatures are too high, the cell wall becomes damaged, thus reducing the number of binding sites [55,56,57]. In zinc binding, binding was found to be affected by changes in pH, biomass concentration, and temperatures [58]. Studies related to the binding of iron to LABs have indicated the possibility of creating a negative charge on the surface of dead cells, thus creating greater affinity for metal cations, resulting in better binding to dead cells [59], which may further explain differences in binding of individual micronutrients during this research.
Beta-glucans have also been recognized as good natural biofixes of mycotoxins and metals. [60,61], while in vitro studies have shown that the cell wall of yeast Saccharomyces cerevisiae can bind a wide range of toxins and is considered one of the most efficient model organisms for toxin binding [62]. The interaction with metals arises most likely due to the high carbon content and the available metal binding sites through specific hydroxyl groups when it comes to polysaccharides and porous surfaces [63]. Studies have shown that beta-D-glucans are in the form of a random coil or in the form of an ordered structure, which depends on the available binding sites, and with the spatial organization of beta-D-glucans changing, there is higher or lower adsorption of toxic molecules. Carboxylic, amino, hydroxyl, and amide groups of proteins and carbohydrates found in the cell wall of beta-glucans are involved in the binding of metals to yeasts [64,65].
This study also monitored the effect of binding microelements naturally present in milk with beta-glucans added to milk to primarily remove AFT M1. The number of micronutrients was monitored during the first hour of binding with 0.005 and 0.01% solutions of yeast-derived beta-glucan and oats beta-glucan with AFT M1, after two, four, and twenty-four hours. It was found that in the studied micronutrients, sodium, magnesium, potassium, and calcium, during the zero hours of binding of beta-glucan from the yeast with ATM1, there was a change in the quantity of Na, which decreased by 15% compared to the initial quantity (beta-glucan concentration 0.005%), while there were no significant deviations in other micronutrients. At the concentration of 0.01%, a larger deviation in the quantity of Na was recorded; there was a decrease of 27.1% and a decrease in Mg by 15.4%. In other micronutrients, there was no decrease in quantities. In beta-glucan from oats, at a concentration of 0.005%, the quantity of Na decreased by 24.8% and of K by 12%, while in other micronutrients there was no decrease. This can be explained by the fact that ATM1 has a higher affinity for beta-glucans than for naturally present micronutrients on the one hand, and the fact that the natural bonds of the studied micronutrients within milk are much stronger compared to added contaminants. It was found experimentally that during the first hour of binding between the analyzed mycotoxin deactivators and ATM1, there were no significant changes in the nutritional composition of milk, especially when LABs were used as mycotoxin deactivators. Changes occur after the prolonged action of mycotoxin deactivators where the quantity of investigated micronutrients deviates from the quantities measured in untreated milk, except when using live lactic acid bacteria, and the largest deviations were found for Na, K, and Mg, while the smallest changes were observed in Ca.

5. Conclusions

This study found that the analyzed mycotoxin deactivators—beta-glucan from yeast, beta-glucan from oats, and lactic acid bacteria (live and dead) —had a varying effect on the nutritional composition of milk, after binding to intentionally added aflatoxin M1 in milk. The smallest effect on the nutritional value came from live lactic acid bacteria except in the sodium micronutrient, where 1% concentration of beta-glucans from oats appeared the have a lesser impact. The largest deviations are found in Na, K, and Mg, while the minimum changes are observed in Ca. Interestingly, both 1% and 5% concentrations of beta-glucans from oats had a statistically significant impact on Ca micronutrient.
In the upcoming studies, it would be necessary to extend the impact of mycotoxin deactivators to also include other micronutrients important for milk quality, such as phosphorus (P) and iron (Fe). Emphasis should also be put on the importance of further research into the impact of mycotoxin deactivator binding with ATM1 in milk on changes in fatty acid composition as important parameters in assessing milk quality.

6. Limitations of the Study

A limitation of the study can be the fact that all micronutrients were analyzed and quantified in milk in the same manner and under the same conditions before adding the mycotoxin deactivators. The following amounts of micronutrients and values were obtained before adding aflatoxin AF M1 to milk for human consumption with 2.8% milk fat: At 302.97 mg/L, Mg 75.16 mg/L, K 1188.86 mg/L, and Ca 864.7 mg/L, which are within the acceptable and expected values for milk on the Croatian market. The stated values are mean values based on five measurements of micronutrients’ composition using the same initial sample. All the analyses in the further experiments were carried out in relation to the same initial milk sample, which is the reason for the basic values of micronutrients being considered as the same in all the conducted experiments. The design of the experiment and the analyses and the subsequent statistical processing of the data were carried out so as to monitor and compare the changes between the micronutrients’ composition at four time points, and not in relation to the initial sample.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pr10112431/s1

Author Contributions

Conceptualization, Z.P., J.B., Z.J.; methodology, D.L., K.M., Z.J.; software, validation, J.B., Z.P., J.F.; formal analysis, I.J., Z.K., K.M., Z.P.; investigation, resources, Z.P., J.B., A.R.; data curation, I.J.; writing—original draft preparation, Z.P., J.B., Z.K., A.R., D.L.; writing—review and editing, Z.J., J.F., D.L.; visualization, I.J.; supervision, Z.J., J.F., D.L., J.B., A.R.; project administration, J.B., K.M., D.L.; funding acquisition J.B., D.L. All authors have read and agreed to the published version of the manuscript.

Funding

This study was carried out within the “Food Safety and Quality Centre” (K.K.01.1.1.02.0004) project funded by the European Regional Development Fund.

Data Availability Statement

Code and data are available on a reasonable request through the corresponding author.

Acknowledgments

This study was carried out within the “Food Safety and Quality Centre” (K.K.01.1.1.02.0004) project funded by the European Regional Development Fund.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

ATM1aflatoxin M1
AFB1aflatoxin B1
EFSAEuropean Food Safety Authority

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Figure 1. Mean differences in minerals per time point from baseline values.
Figure 1. Mean differences in minerals per time point from baseline values.
Processes 10 02431 g001
Table 1. Conditions of recording on ICP-MS.
Table 1. Conditions of recording on ICP-MS.
ON ICP-MSThe Conditions of Recording
AtomizerMicroMist
Injectorquartz
Conesnickel
RF power1180 W
Plasma gas flow15.0 L/min
Atomizer gas flow1.07 L/min
Auxiliary gas flow0.90 L/min
Integration time1000 ms
Peak points100
Number of replicas5
Delay time30 s
Rinse time70 s
Table 2. Descriptive parameters of minerals by time points.
Table 2. Descriptive parameters of minerals by time points.
Time 1Time 2Time 3Time 4
Sodium
Yeast 5258 (22.8)263 (23.2)251 (19.1)215 (31.8)
Yeast 1222 (20.3)239 (24.4)249 (26.5)226 (24.7)
Oats 5228 (24.8)235 (23.2)222 (25.6)230 (24.8)
Oats 1205 (38.6)195 (38.0)206 (34.2)202 (26.2)
Live bac288 (36.8)295 (40.6)328 (66.2)297 (47.5)
Dead bac240 (11.1)253 (15.7)264 (26.6)230 (17.4)
Magnesium
Yeast 578.0 (3.35)73.6 (3.74)67.8 (4.32)64.6 (4.84)
Yeast 178.3 (7.13)70.3 (5.40)65.2 (4.08)62.6 (4.23)
Oats 584.6 (3.98)76.9 (5.97)81.3 (4.19)66.9 (3.51)
Oats 179.0 (4.84)72.2 (3.87)80.9 (4.10)110 (10.6)
Live bac75.6 (12.3)73.3 (12.4)72.2 (8.85)71.2 (6.59)
Dead bac71.9 (1.27)77.3 (1.88)72.1 (2.36)66.9 (3.60)
Potassium
Yeast 51460 (171)1220 (163)1010 (91.4)1090 (146)
Yeast 1931 (73.1)1100 (108)952 (108)1050 (112)
Oats 51240 (57.3)1080 (63.9)1260 (39.8)1080 (52.6)
Oats 11090 (82.8)913 (70.3)1130 (88.3)901 (67.9)
Live bac1190 (228)1190 (193)1210 (120)1100 (97.4)
Dead bac1180 (46.3)1100 (61.7)1280 (75.2)1120 (109)
Calcium
Yeast 5811 (37.8)744 (51.0)783 (56.8)761 (53.6)
Yeast 1766 (42.9)728 (46.4)760 (59.3)739 (79.6)
Oats 5846 (35.5)887 (34.4)790 (59.6)818 (51.1)
Oats 1807 (73.9)839 (76.7)715 (79.8)747 (66.6)
Live bac288 (36.8)295 (40.6)328 (66.2)297 (47.5)
Dead bac240 (11.1)253 (15.7)264 (26.6)230 (17.4)
Table 3. Repeated measures ANOVA test.
Table 3. Repeated measures ANOVA test.
FdfN/dfDp-ValueGES
Sodium
Dead bac18.912/28<0.001 *0.325
Live bac4.0162/280.029 *0.083
Yeast 115.7382/28<0.001 *0.152
Yeast 515.4831.37/19.14<0.001 *0.393
Oats 10.7752/280.4710.032
Oats 510.6141.06/14.880.005 *0.152
Magnesium
Dead bac66.6241.42/19.85<0.001 *0.672
Live bac0.2232/280.8020.005
Yeast 146.7041.12/15.67<0.001 *0.188
Yeast 548.4991.38/19.25<0.001 *0.51
Oats 1165.8031.15/16.04<0.001 *0.85
Oats 5131.1762/28<0.001 *0.755
Potassium
Dead bac36.5451.4/19.67<0.001 *0.526
Live bac2.2281.32/18.440.1480.035
Yeast 116.5761.29/18.07<0.001 *0.192
Yeast 517.9162/28<0.001 *0.204
Oats 155.9172/28<0.001 *0.688
Oats 5105.5572/28<0.001 *0.783
Calcium
Dead bac2.0871.02/14.230.170.072
Live bac0.7242/280.4940.024
Yeast 12.3451.23/17.290.140.046
Yeast 53.9711.18/16.530.0580.091
Oats 141.922/28<0.001 *0.471
Oats 562.6471.16/16.28<0.001 *0.537
Legend: *—statistically significant; GES—generalized eta squared.
Table 4. Amount of ATM1 (µg/kg) in milk after binding to selected mycotoxin deactivators.
Table 4. Amount of ATM1 (µg/kg) in milk after binding to selected mycotoxin deactivators.
Type of Mycotoxin Deactivatorsβ-Glucan from Oatsβ-Glucan from YeastDead BacteriaLive Bacteria
The concentration of mycotoxin deactivators0.01%0.005%0.01%0.005%106 BMK1 mg biomass/mL
Time of binding (hours)0.1690.3010.4730.1780.1450.119
0
20.2230.3420.3620.3150.0920.101
40.2870.2970.4940.4370.0120.100
240.3000.2260.2550.2690.0220.087
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Pavlek, Z.; Bosnir, J.; Kuharic, Z.; Racz, A.; Jurak, I.; Lasic, D.; Markov, K.; Jakopovic, Z.; Frece, J. The Influence of Binding of Selected Mycotoxin Deactivators and Aflatoxin M1 on the Content of Selected Micronutrients in Milk. Processes 2022, 10, 2431. https://doi.org/10.3390/pr10112431

AMA Style

Pavlek Z, Bosnir J, Kuharic Z, Racz A, Jurak I, Lasic D, Markov K, Jakopovic Z, Frece J. The Influence of Binding of Selected Mycotoxin Deactivators and Aflatoxin M1 on the Content of Selected Micronutrients in Milk. Processes. 2022; 10(11):2431. https://doi.org/10.3390/pr10112431

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Pavlek, Zeljka, Jasna Bosnir, Zeljka Kuharic, Aleksandar Racz, Ivan Jurak, Dario Lasic, Ksenija Markov, Zeljko Jakopovic, and Jadranka Frece. 2022. "The Influence of Binding of Selected Mycotoxin Deactivators and Aflatoxin M1 on the Content of Selected Micronutrients in Milk" Processes 10, no. 11: 2431. https://doi.org/10.3390/pr10112431

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