IN VITRO STUDY OF THE EFFICACY OF MYCOTOXINS DEGRADATION BY FEED ENZYMES

Providing healthy and safe food in terms of mycotoxicological safety is an imperative for not only good and sustainable livestock production, but also for the population that consumes food of both plant and animal origin. Climate change in the temperate regions of southern Europe has led to frequent occurrence of afl atoxins, deoxynivalenol and zearalenone in cereals. In order to reduce harmful eff ects of these toxins on animal health as well as to avoid large economic losses, various feed additives are increasingly being used. All of them must fi rst of all be safe, and then have certain effi ciency in the fi ght against mycotoxins. Although in vivo experiments are mandatory to assess the effi cacy, in vitro test off ers the advantage of rapid screening of the effi cacy of a large number of food additives. In this paper, the effi ciency of two commercial products belonging to the enzyme group for animal nutrition was investigated for degradation of afl atoxin B1, zearalenone and deoxynivalenol using in vitro experiments. For this purpose, two diff erent methodologies were used according to the recommendation of the enzyme manufacturer. Th e percentage of mycotoxin degradation was recorded by high pressure liquid chromatography and ELISA methods. One of the tested enzymes showed a very high effi ciency in zearalenone degradation being as much as 96%. Both tested enzyme samples showed similar percentage of afl atoxin B1 degradation (about 35%). Deoxynivalenol was not signifi cantly degradable under the applied test conditions.


INTRODUCTION
Enzymes are considered biological catalysts; they are proteins capable of accelerating the speed of chemical reactions, which are essential for the proper cellular functioning of all living beings. Th eir use has shown benefi ts in the food industry, while in animal feed could improve the consistency and nutritional value of feed, increase digestibility, animal performance and reduce the eff ect of antinutrients. (Velázquez-De Lucio et al., 2021). Th e purpose of adding enzymes in animal feed is to improve food effi ciency, production, and consequently to reduce the cost of feeding (Bedford 2018). Enzymes can be obtained from animals, plants or microorganisms. Th e development of recombinant DNA technology has allowed for the isolation and expression of genes of some microorganisms, and production of enzymes for animal feed (Sarder et al., 2005). Enzymes used in the production of animal feed are considered zootechnical additives in the Republic of Serbia, their use is allowed by the Rulebook on the quality of animal feed (Offi cial Gazette of the Republic of Serbia, 2010).
Mycotoxins are a numerous group of secondary metabolic products of fungi or molds that pose a serious risk to human and animal health. Mycotoxin contamination is widespread in animal feed, especially in cereals (Wu et al., 2015). Currently, more than 300 mycotoxins have been identifi ed, and scientifi c attention has been focused mainly on mycotoxins that have been shown to be carcinogenic and/or toxic to human and animal health. Afl atoxins (AF), zearalenone (ZEA) and deoxynivalenol (DON) are of great public health concern due to their high prevalence, teratogenic, carcinogenic, mutagenic and immunosuppressive eff ects (Oueslati et al., 2012). Increased amounts of mycotoxins in animal feed can result in huge economic losses on an annual basis, including declining livestock production as well as increasing human and animal mortality (Zain, 2011). An eight-year study based on the determination of mycotoxins (AF, ZEA, DON, fumonisin and ochratoxin A) in animal feed and feed materials worldwide showed that 72% of the samples were positive for at least one mycotoxin, and 38% is simultaneously contaminated with multiple mycotoxins (Streit et al., 2013).
Prevention of mycotoxin contamination begins in the fi eld. However, when speaking of the protection of cereals from mold and mycotoxins during storage, further measures are necessary. Diff erent treatments (physical, chemical and biological) are used for this purpose (Stoev, 2013;Jevtić et al., 2021). Mycotoxin adsorbents are oft en used as feed additives (Nešić et al., 2020). Although mycotoxins are stable compounds, research on their degradation is very topical today. Eff ective degradation of mycotoxins must provide irreversible degradation of mycotoxins to less toxic or non-toxic products. One of the frequently studied techniques is the biological detoxifi cation of mycotoxins, using microorganisms and/or enzymes to degrade mycotoxins into non-toxic or less toxic compounds (Taylor and Draughon, 2001). Th e advantage of mycotoxin degradation using enzymes is the simplicity of the process, without the potential risk of contamination and operator safety compared to the use of live microorganisms (Loi et al., 2017). Th e main conversion paths are hydroxylation, hydrogenation, hydrolysis, oxidation, esterifi cation, glucuronidation and glucosidation, de-epoxidation, methylation, sulfation, demethylation and deamination (Nešić et al., 2021). Th e following enzymes are used for this purpose: oxidase, peroxidase, laccase, reductase (AF), carboxylesterase and aminotransferase (fumonisins), glucosyltransferase (trichotecenes), laccase, lactonohydrolase (ZEA), lypase, protease (ochratoxin) (Loi et al., 2017). Europen Union (EU, 2014; 2017; 2018) and European Food Safety Authority (EFSA, 2020) established the regulations regarding fumonisin esterase (the enzyme that degrade fumonisins) as a feed additive for animal species in accordance with rules for additives for use in animal nutrition (EU, 2003).
Climate change in the temperate climate of southern Europe has led to frequent occurrences of AF, DON and ZEA in cereals and in Serbia. In order to reduce the harmful eff ects of these toxins on animal health, as well as to avoid large economic losses, various feed additives are increasingly being used. All of them must fi rst of all be safe, and then have a certain effi ciency in the fi ght against mycotoxins. Although in vivo experiments are mandatory to assessing the effi cacy, the advantage of the in vitro test is its capacity to rapidly screen the effi cacy of a large number of food additives. In this way, the reduction of mycotoxin toxicity is indirectly confi rmed. In this work, the degradation efficiency of AFB1, ZEA and DON was investigated in vitro using two commercial products belonging to the enzyme feed additives.

Chemicals and enzymes
Two samples of diff erent feed enzymes were provided by INBERG ltd (Belgrade, Republic of Serbia). Th e tested enzymes are intended for use as feed additives in order to reduce the harmful eff ects of mycotoxins.
Standard substances were used for degradation tests: AFB1 Cat No A6636, ZEA Cat No Z2125, and DON Cat No D0156. All standards were purchased from "Sigma Aldrich", Saint Louis, USA.

In vitro experiments
For the purpose of in vitro testing of the possibility of mycotoxin degradation by enzymes, two diff erent methodologies were used. Th e effi ciency of sample No. 1 was tested according to the manufacturer's recommendation applying the methodology No. 1. Its effi ciency for the degradation of AFB1, ZEA and DON was examined. Th e effi ciency of sample No. 2 for the degradation of the same mycotoxins was tested according to the manufacturer's recommendation and using the methodology No. 2.
Th e fi rst methodology involved incubation of mycotoxin standards and enzyme in phosphate buff er solution (0.1M PBS, pH 6.5). Th e test solution for ZEA degradation assay consisted of 1980 μL (1900 μL) of buff er (with appropriate enzyme weighing in suspension), in which 20 μL (100 μL) of ZEA standard solution (100 μg/mL) was added. For testing the degradation of DON 100 μL of standard solution (100 μg/mL in a mixture of ethyl acetate and methanol) was evaporated and reconstituted in 2000 μL buff er (with appropriate weighing of enzymes in suspension). Th e test solution for AFB1 degradation assay consisted of 1980 μL of buff er (with appropriate enzyme weighing in suspension), in which 20 μL of standard solution of AFB1 (10 μg/mL) was added. All tested solutions were incubated with shaking for 4 h at 37 °C and then centrifuged, fi ltered, and mycotoxins were determined by liquid chromatography.
In both cases, the experiment consisted of a toxin and enzyme assay and a control assay with toxins only. Th e degradation effi ciency expressed with standard deviation (STD) is the result of measurement in three replications.

Mycotoxins analysis
Aft er in vitro tests, the effi ciency of the tested enzymes for mycotoxins degradation was evaluated. Th e percentage of degradation was recorded by quantitative measurement of residual mycotoxin in the supernatant by using optimized and validated methods. In the case of ZEA and AFB1, there high pressure liquid chromatography with fl uorescence detection (HPLC-FLD) was used, while HPLC-DAD and ELISA were applied for DON.
HPLC Dionex UltiMate 3000 Series system with FLD 3100 and DAD detector (Th ermo Scientifi c, Germany) was used for quantitative measurement of mycotoxins in the solution before and aft er adsorption. Th e system was controlled with Chromeleon ® 7 soft ware (Th ermo Scientifi c, Germany).
For the determination of AFB1 Supelcosil column, 250 x 4.6 mm, 5μm, was used for separation with mobile phase 50% ACN and fl ow rate 1.2 mL/ min. Fluorescence detection was done on λex 365 nm, and λem 435 nm. For ZEA determination Hypersil Gold aQ column, 150 x 3 mm, 3 μm, (Th ermo Scientifi c, Germany), with mobile phase 60% ACN, and fl ow rate 1 mL/min was used, while for detection the wavelengths λex 275 nm and λem 455 nm were set. Th e method for the determination of DON used the same column as for ZEA, mobile phase 10% ACN, fl ow rate 1 mL/min, and detection was done at λ 220 nm.

Determination of the percentage of toxin degradation
Aft er HPLC determination, the peak areas of the determined mycotoxins in the test samples were compared with the corresponding areas of control samples without added enzyme. Th e percentage of adsorption was calculated by using the equation: % degradation = (1 -PI / P0) x 100% Where: PI = peak area of toxins aft er incubation with enzyme; P0 = surface area of toxin peaks in control solution without enzyme addition.

RESULTS
Th e tested enzymes are commercially available as feed additives. Th eir use is based on the eff ect of reducing the harmful eff ects of mycotoxins, that is, they degrade mycotoxins in the conditions of the digestive tract of animals. Th ere were no available data on their activity and effi ciency, the exact composition and origin. Th e only available information was about the conditions under which those enzymes work, i.e., the pH values, in which medium, and at what mycotoxins concentrations. Th ese data were used for in vitro testing thereof.
Because of diff erent testing conditions as well as diff erent concentrations of mycotoxins and enzymes, the results for each enzyme were presented separately.

Effi ciency of mycotoxin degradation by enzyme No. 1
Th e results of the study of the infl uence of enzyme number 1 on the degradation of ZEA are shown in Table 1. It can be seen that increasing the mass of the enzyme increases the degradation effi ciency to a signifi cant 93%. Given such a high effi ciency with a high amount of enzyme, and in order to optimize the ratio of enzyme and ZEA, the possibility of degradation of a larger amount of ZEA was tested ( Table 2). As can be seen, a 96% degradation of ZEA was achieved at a toxin: enzyme ratio of 1 μg: 1.8 mg. Th e effi cacy of the same enzyme for AFB1 degradation was examined with three diff erent enzyme amounts, and the results showed maximum effi ciency of 34.8% (Table 3).

Effi ciency of mycotoxin degradation by enzyme No. 2.
Testing the effi cacy of enzyme sample No. 2 was examined by incubating the enzyme and toxin in a nutritionally rich medium Lysogeny Broth (LB). Th e degradation effi ciency of ZEA alone and ZEA and AFB1 in the mixture was examined. Th e results are shown in Figure 1. Th ere was no diff erence in the effi ciency of ZEA degradation in case where ZEA alone was present in the reaction mixture (13 ± 6%) as compared to the mixture with AFB1 (11%).

DISCUSSION
A modern and economically justifi ed approach to the fi ght against mycotoxins also involves the use of additives that enzymatically lead to their degradation. Before using these supplements, it is necessary to confi rm their activity or effi ciency for the decomposition of mycotoxins. Although regulations in the EU on enzymes as additives against mycotoxins are available, national regulations in Serbia and other countries do not include requirements pertaining to the quality and degradation effi ciency of enzymes used against mycotoxins in animal feed. Also, there are no unique methodologies for analyzing enzyme effi ciency. In in vitro tests, it is important to defi ne the conditions under which these experiments are performed. Experimental conditions should mimic the biotransformation of toxins in the animal's body. Th e levels of toxin and enzyme to be tested in in vitro reaction system are also important. A range of various experiments to check the activity of enzymes for the degradation of mycotoxins is described in the literature. Two diff erent methodologies, with diff erent concentrations of toxins and enzymes, were used in this study. Th erefore, it is diffi cult to compare the obtained results with each other. Th e conclusions of experiments on the effi cacy of enzymes should be stated with reference to the Arhiv veterinarske medicine, Vol. 15, No. 1, 5 -17, 2022 Jakšić, S… et al.: In vitro study of… conditions under which they were obtained. According to the available literature, eff ective degradation of AFB1 and ZEA (86% and 100% respectively) is achieved by laccase, using redox mediators, while under the same conditions the degradation of DON was not possible (Loi et al., 2018). A high percentage of AFB1 degradation (90 -100%) using the enzymes peroxidase and oxidase, while ZEA reduction was achieved by using lactono hydrolase (100%) has also been reported in the literature (Loi et al., 2017).
Here give analyses of the obtained results comparing to the results and opinions of other authors, pointing the importance of this research, without giving a conclusion. Th e Discussion section is not used to summarize current knowledge. Th e Discussion should clearly identify the main Conclusions of the study. Authors are to provide a clear explanation of the importance and relevance of these Conclusions. Any new information should be distinguished from the previous fi ndings, and relevant hypotheses can be generated.

CONCLUSION
Th e result of this research is the confi rmation of a quality of the enzyme that has been proven to be highly effi cient for the decomposition of ZEA, a toxin oft en present in cereals and responsible for reproductive disorders in livestock production. Based on the results obtained in in vitro studies, the optimal ratio of enzyme and ZEA was obtained, which gives maximum effi ciency in application.
Control of enzymes, similar to adsorbents used as feed additives, before mixing into complete mixtures is necessary, and conscientious producers are aware of the cost-eff ectiveness of such approach and conscious production strategies. Th e signifi cance of ensuring the health of animals and humans by using proven components of animal feed to prevent the occurrence of mycotoxicosis and mycotoxin-contaminated food is an imperative in the production of safe food.

АCKNOWLEDGEMENT
Th is paper is published as part of the project fi nanced by the Inovation Fund of the Republic of Serbia. Th e authors cordially thank INBERG d.o.o., Belgrade, Serbia for providing tested adsorbents.