1. Are mycotoxins only highly toxic in high rainfall years, or is it also due to stress in drought years?
Fungal infection of crop plants is generally favoured by wet conditions but spread of infection and mycotoxin formation does not require rain. A well-documented example is the high aflatoxin contamination in maize from Serbia in the 2012 drought year. Drought was also reported to cause, for example, higher fumonisin concentrations to accumulate in maize (Vaughan et al. (2016) PLoS ONE 11(7): 1-24).
In the EU, there is a regulation for maximum tolerable aflatoxin contamination in animal feedingstuff: Directive 2003/100/EC. For the Fusarium mycotoxins, there are “guidance values”: 2006/576/EU. There is a separate “Recommendation” for T2 andHT-2 toxins: 2013/165/EU. EC No 1881/2006 regulates the maximum aflatoxin M1 concentration in milk. EU Directive 2002/32 defines that in the EU, chemical decontamination processes are not legal to use, and mixing of batches to reduce mycotoxin concentrations under the limit concentrations is also not allowed.
Some authors have reported binding of vitamins and trace elements to bentonite, but other authors reported that no reduction of vitamin concentrations or trace mineral concentrations in blood was measurable.
4. What are the main biomarkers that we can measure in the commercial life to determine which toxin control solution is more suitable?
If biomarkers are to be used for comparison of anti-mycotoxin feed additives, a properly designed experiment is essential. Absolute values of concentrations of biomarker-metabolites have little meaning, and comparison of treatment and control groups is necessary. Biomarker analysis further requires thorough consideration and execution of correct sampling, because concentrations of Fusarium mycotoxins including deoxynivalenol and zearalenone in blood and urine vary greatly with time after feeding. Biomarkers that have been used in scientific studies include aflatoxin M1 concentration in urine or milk, aflatoxin-lysine conjugate concentration derived from blood serum albumin, concentrations of deoxynivalenol and deoxynivalenol-derived metabolites including deoxynivalenol-glucuronide in blood or urine, concentrations of zearalenone and zearalenone-derived metabolites in blood or urine, and sphingolipid concentrations for fumonisins. A future scenario under current development is to measure biomarkers of mycotoxin exposure from blood spots dried on filter paper.
5. If the fumonisin esterase you mentioned is sensitive to being inactivated, how viable is this technology for processed feeds?
The fumonisin esterase has been engineered for increased thermostability so that it tolerates feed pelleting. It can be stored without loss of activity, and only requires to be stored dry. It can be used in animal feed just like any other feed enzyme, such as phytase. However, also just like phytase, the fumonisin esterase will not tolerate extrusion at very high temperature as it is used for manufacturing of fish feed. A preparation of the enzyme for liquid post-pellet spray application is also available.
6. At what temperature ranges have the enzymes been evaluated and what are critical temperatures that will reduce the enzyme actions?
Temperature – activity correlations showed that the enzymes had good activity at body temperature, but also at low temperatures relevant for cold water fish. Pelleting experiments showed that, with the right formulation, pelleting at 85°C caused little loss of activity.
The kcat of fumonisin esterase is over 200 s-1, which means that at saturating fumonisin concentration, one molecule of fumonisin esterase catalyses 200 hydrolytic fumonisin-cleavage reaction per second. The enzyme concentration typically used in animal feed, 30 units per kilogram, takes less than one minute to completely hydrolyse 5 ppm fumonisins in a buffer solution. In an animal, the reaction speed would be very difficult to measure. That is why biomarkers, and in particular the sphinganine to sphingosine ratio in blood, were used to determine the required fumonisin esterase concentration for complete hydrolysis of fumonisins in feed.
The enzyme activity requires water. Enzyme and substrate have to be dissolved so that they can meet by diffusion, and the enzymes also need water for activity. If feed is mixed and processed dry, activation with water will only occur after ingestion. However, activation with water is instant.
9. Can the activation of the enzymes be influenced by high rumen flow through diet? And does rumen PH effect the activation of these Enzymes?
The enzymes will be instantly active in rumen, and variation of the pH that is typical for rumen will have little effect on enzyme activity, even under SARA conditions. However, the enzyme will not remain indefinitely active in rumen, but will also be digested and degraded over time.