AGRO-MOS YEAST. Beer & Spirits:

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1 MYCOTOXIN DETECTION 37+ applied in Denmark By Ole Jensen Welcome Established in 1980 by Dr. Pearse Lyons 1

2 The Alltech Advantage Top 5 animal health company Only company that is: -Privately-held -Non-pharmaceutical Growing consistently over 36 years Moving towards a $4bn company Our OPTIONAL Core HEADER is FERMENTATION Yeast: BIO-MOS, INTEGRAL A +, NUPRO YEA-SACC, MYCOSORB A + LIQUI-PLEX AGRO-MOS Bacteria: ALL-LAC, LACTO-SACC SOIL-SET Enzymes: ALLZYME SSF ALLZYME VEGPRO YEAST Algae: ALL G-RICH Beer & Spirits: KENTUCKY ALE Range including KENTUCKY BOURBON BARREL ALE LYONS SPIRITS including TOWN BRANCH BOURBON, PEARSE LYONS RESERVE 2

3 Beverage 13. Division juni 2016 First Kentucky Ale brewed in 2000 Kentucky Bourbon Barrel Ale available across the U.S. and in Ireland and China Spirits include PearseLyons Reserve malt whiskey, Bluegrass Sundown and Town Branch Bourbon Alltech: Growth through acquisition 3

4 HVORFOR TØRRING? Hvis ikke afgrøden nedtørres kan følgende bl.a. forekomme: Høj temperatur: Beskadigelse af spireevne, ca. 40 C Marksvampe: Fusarium svampe fra våd høst over 18-20% Fusarium toxiner (kan ikke fjernes ved nedtørring) Lagersvampe: Skadedyr: Penicillum gruppen Ochratoxin svampe m.fl. Mykotoksiner: Giftstoffer dannet af svampe. Kornsnudebiller Kornmider 4

5 Skadedyr Mider i rapsfrø Kornsnudebiller Bemærk de gnavede huller Ochratoxin 5

6 Zearalenone Аbortions and stillborn piglets 6

7 Rectal and vaginal prolapses Tjekliste Reduceret foderudnyttelse Reduceret libido Opkast Aborter Dårlig immunitet Uensartet huld Pludselige dødsfald Reduceret tilvækst Uforklarlig ændring i fertilitet Færre levendefødte Ringere malkning Øget sygdomsfrekvens Rektal eller vaginal prolaps Hvis du kan svare JA til en af ovennævnte påstande kan mykotoxiner være forklaringen. 7

8 Analyses is key what do we test for? Aflatoxins Vomitoxin, & T-2 toxin Zearalenone and fumonisin B1 Using ELISA or TLC methodologies Needfor a more accurate way to identify and quantify mycotoxins in feed, grains and forages INCREASE ANALYTICAL PRECISION/ MULTI-DETECTION Qualitative test Kit Semi-Quantitative test Kit FIA RIA ELISA Nano-Biosensors GC-UV HPLC-UV HPLC-PDA HPLC-ECD GC-MS LC-MS 1.00e6 9.00e5 LC/LC-MS/MS >300 pesticides 8.00e5 7.00e5 Intensity, cps 6.00e5 5.00e5 4.00e5 3.00e5 2.00e5 1.00e5 Yes No Time, min 8

9 IT ENDS-UP BEING A BIASED DECISION Aflatoxins Aflatoxicol Aflatoxin B1,B2,B2α Aflatoxin G1,G2,G2α Aflatoxin M1 Austocystin A Averufin 5-Methoxysterigmatocystin Dihydroxysterigmatocystin Methoxysterigmatocystin Sterigmatocystin Norsolorinic acid Parasiticol Trichothecenes Nivalenol Fusarenone X Deoxynivalenol 3-Acetyldeoxynivalenol 15-O-Acetyl-4-DON Scirpentriol 15-Acetoxyscirpenol Diacetoxyscirpenol 3α -DAS Neosolaniol T-2 Triol HT-2 Toxin T-2 Toxin Iso-T-2 toxin Acetyl-T-2 toxin Trichodermin Trichodermol 7-a-Hydroxytrichodermol Verrucarol 4,15-Diacetylverrucarol Trichothecin Trichothecolone Macrocyclic trichothecens Isosatratoxin F Roridin A,E,H,L-2 Satratoxin G,H Trichoverrin A,B Trichoverrol A,B Verrucarin A,J Ochratoxins Ochratoxin A,B,α Ochratoxin A,B,α-ethyl ester Ochratoxin A,B,α-methyl ester Sphinganinmyotoxins AAL toxin TA1,TA2 AAL toxin TB1,TB2 Fumonisin B1,B2,B3,B29 Cytochalasins Chaetoglobosin A,C Cytochalasin A,B,C,D,E,H,J Dihydrocytochalasin B Dihydrocytohalasin B γ lactone Zeralenons α,β-zearalanol Zearalanone α,β-zearalenol Zearalenone Roquefortines, ergot amines and alkaloids Agroclavine-I Auranthine Aurantiamine (alkaloid 302) Aurantioclavine Chanoclavine-I Costaclavine Cyclopenin Cyclopenol Cyclopeptin Dihydroergotamin Elymoclavine Epoxyagroclavine-I Ergocristine Ergometrin Ergotamin Fumigaclavine B,C Marcfortine A,B Meleagrin Oxalin Pyroclavine Roquefortine A,B,C,D Rugulosuvine Rugulovasine A,B Secoclavine α-ergocryptin C -7,12-diene-one Tremorgens Fumitremorgen B Lolitrem B Lolitriol Territrem A,B,C Penitrem A Penitremone A 19-Acetoxypaxillin Deoxypaxillin Paxillin a-paxitriol b-paxitriol C Janthitrem B Peptides Alamethicin α-amanitin Antimycin A1,A2,A3,A4,A5 Apicidin Cyclo(Phe-Ser) Cyclosporin Distamycin A Enniatin A1,A2,B,B1 Malformin A1,A2,B1,B2,C Phalloidin Valinomycin Atranone& precursors Atranone A,B,E,F,H,J 6-Hydroxydolaballa-3,7,12- -trien-14-one 3,4-Epoxy-6-hydroxy- dolabella- Others 2-(4-Hydroxyazobenzene)- benzoic acid 2,4,6-Trihydroxytoluene 2,5-Dihydroxybenzoic acid 3,5-Dihydrotoluene 3,7-Dimethyl-8-hydroxy-6- methoxy-isochroman 3-Methylsalicylic acid 5-Methylbenzene-1,2,3 triol 6-Methylcitreoisocumarin Abscisic acid 2-Acetyl-4(3H)-quinazolone α-acetyl-g-methyltetronic acid Aflatrem Alizarin Altenuene Alternariol Alternariol-methyl ether Altersolanol A Altertoxin I Amphotericin B Anacine Anhydrofusarubin p-anisaldehyde Antibiotic Y Arabenoic acid (Verrucolone) Aurofusarine Ascochitine Aspergillic acid Aspergillic acid-polymer Asperthecin Asteltoxin Asterric acid Austamide Austdiol Baccatin III Barnol Beauvericin 1,3-Benzendiol Benzoic acid Bikaverin Bis-dethio-bis(methylthio)- gliotoxin Bostrycin Bostrycoidin Brassicasterol Brefeldin A Brevianamide A Butenolide Byssochlamic acid Caffeic acid Caffeine Calphostin C Canadensolide Candidusin C Canescin Carlosic acid Carolic acid Catenarin Chaetocin Chetomin Chromanol 1, 2, 3 Chrysazin Chrysogine Chrysophanol Cinnamic acid Circumdatin A, B, C Citreoisocumarin Citreomontanin Citreoviridin A, X Citrinin Citromycetin Cladofulvin Cladosporin Clerocidin Communesin A, B Compactin Culmorin Curvularin Cyclochlorotine Cyclopaldic acid Cyclopiazonic acid Cynodontin Daldinin D Bis-dechlorogeodin Dechlorogriseofulvin Dehydrocarolic acid Dehydrocurvularin Deoxybostrycin Dermoglaucin Des-acetylpebrolide Desertorin A,B, C Des-methoxyviridiol Desmosterol Dethiosecoemestrin Diaporthin Diaportin acid Diaportinol Dichlorodiaportin cis-dihydrofusarubin trans-dihydrofusarubin Dihydrojasmonic acid 29,39-Dihydrosorbicillin Dihydroxyaflavinine 3,5-Dimethyl-6- hydroxyphthalide Dimethylphthalate Dipicolinic acid Dithiosilvatin Duclauxin Dustanin Echinulin Emestrin Emestrin B Emindole DA,DB Emodin Epicorazine A Epi-dechlorogriseofulvin Epoxysuccinic acid Equisetin Ergosterol Erythroglaucin Ethisolide Expansolide Ferulic acid Flavoglaucin Formyl-xanthocillin X Frequentin Fructigenine Fucosterol Fulvic acid Fumagillin Fumagillol Fumiguinazoline F Fusaproliferin Fusaric acid Fusarin C Fusarochromanone C Fusarubin Fusicoccin Fusidic acid Fusoxysporone Gallic acid Gentisylalcohol Gibberellic acid Gibepyrone A,F Gliotoxin Gliovirin Griseofulvin Griseophenone C Helminthosporin Helvolic acid Histamin 59-Hydroxyasperentin p-hydroxybenzoic acid p-hydroxycinnamic acid 5-Hydroxymaltol 4-Hydroxymellein ω-hydroxypachybasin p-hydroxyphenylacetic acid Indolacetic acid Insulicolide A,B Insulicolide B Iodinine Islandicin Isoemodin Isomarticin Italicic acid Italicic acid-methyl ester Italinic acid Jasmonic acid Jasmonic acid-methyl ester Javanicin Kojic acid Kotanin Lactone 1,2 Lambertellin Lanosterol Ascorbic acid Leucyltryptophanyldiketopi perazine Lichexanthone L-Phenylalanin L-tyrosin Luteoskyrin Macrosporin Maculosin Marticin 3-Methoxyviridicatin 6-Methylsalicylic acid Mevinolin Mitomycin C Mitorubrin Mitorubrinic acid Mitorubrinol Mitorubrinol acetate C Mizoribine Mollisin Moniliformin Monorden Mycophenolic acid Myriocin Nectriafurone Nidulin Nigragillin 3-Nitropropionic acid Nominine Norjavanicin Norlichexanthone Nortryptoquivaline Oleic acid Oosporein Ophiobolin G Orsellinic acid Oxalic acid Pachybasic acid Pachybasin Palitantin Paracelsin Paspaline Paspalinin Penicillic acid Penicillin G Phaseic acid Phoenicin Phomarin Physcion PI-3 PR-1635 Preechinulin PR-imine Propyl-3,4,5- trihydroxybenzoate PR Toxin Puberuline Puberulonic acid Purpurin Purpurogenone Pyrogallol 2- Pyruvoylaminobenzamide Questin Questinol Radicinin Ravenelin Riboflavin Roseopurpurin Rubratoxin A,B Rugulosin Salicin Salicyclic acid Sclerotigenin Scytalidine Scytalone Secalonic acid D Shikimic acid Skyrin SMTP-6,-7,-8 Solaniol Soranjidiol Sorbicillin Spinulosin Staplabin Steckiin Stemphone Stipitatic acid Sulochrin Taxol Tentoxin Tenuazonic acid Terrein Terrestric acid Terretonin Thiamin, hydrochloride Toluhydroquinone Tomatine Torreyol 3,4,5-Trihydroxy-7- methoxy-2- methylanthraquinone Trisdechloronornidulin Trypacidin Tryptophan Tryprostatin B Tyramine Usnic acid Vermiculin Verrucine A,B Verrucofortine Verrucosidin Verrucosine Verruculogen Verruculotoxin 10-Epi-verruculotoxin Vertinolide Violaceic acid Viomellein Vioxanthin Viridamine Viridicatic acid Viridicatin Viridicatumtoxin Viridiol Viriditoxin Visoltricin Walleminol Walleminone Wortmannin Xanthocillin X Xanthomegnin EXTENDED LC-ESI-MS/MS CAPABILITIES PROGRAM Research setting: Dual Acquity UPLC H-class coupled to: Xevo TQD MS/MS System FLD/UV Systems Commercial setting: Binary Acquity UPLC coupled to: TQD MS/MS System 9

10 What is ISO/IEC 17025: International Standard designed to recognize the competence of Testing Laboratories. This accreditation is accepted worldwide by Governments, businesses and laboratories. 2. Objective third-party assessment of the managerial and technical requirements of the lab ensuring the accuracy and impartiality of analytical results. 3. Technical competence of personnel and availability of technical resources. This ensures personnel are qualified for their positions and are provided with proper training, instrumentation and environment to perform their duties. 4. Quality system that continually improves ability to consistently produce valid test results. Includes review and updating of work instructions and forms. Includes an established process for corrective and preventive actions within the lab. 5. Ensures confidentiality and impartiality of analytical results. Parent company has no influence on results. PROGRAM: MYCOTOXIN SELECTION 1 Aflatoxin B1 2 Aflatoxin B2 3 Aflatoxin G1 4 Aflatoxin G2 5 Sterigmatocystin 6 Gliotoxin 7 Ochratoxin A 8 Ochratoxin B 9 Deoxynivalenol 10 3-AcDon AcDon 12 DON-3-Glucoside 13 Nivalenol 14 Fusarenon X 15 T2 toxin 16 HT2 toxin 17 Diacetoxyscirpenol 18 Neosolaniol Coming soon 39 Fusaric acid 19 Fumonisin B1 20 Fumonisin B2 21 Fumonisin B3 22 Zearalenone 23 α-zearalanol 24 β-zearalanol 25 Zearalanone 26 Patulin 27 Mycophenolic Acid 28 Roquefortine C 29 Penicillic Acid 30 Wortmannin 31 Alternariol 32 Verruculogen 33 2-Br-α-ergocryptine 34 Ergocornine 35 Ergometrine/Ergonovine 36 Ergotamine 37 Lysergol 38 Methylergonovine 10

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12 RISK ANALYSIS Aflatoxin (B1) Aflatoxins (B1+B2+G1+G2) Ochratoxins (A+B) Type B Trichothecenes* Type A Trichothecenes** Fumonisins (B1+B2+B3) Zearalenone Group Fusaric Acid Other Penicillium Mycotoxins*** Other Aspergillus Mycotoxins**** Ergot Toxins REQ (Risk Equivalent Quantity) INTERPRETATION: RISK EVALUATION TEF/TEQ WHAT ARETEF ANDTEQ? Toxic Equivalency compare to a single reference: Aflatoxin B1 TEF: TOXIC EQUIVALENT FACTORS Poultry Swine Dairy Mycotoxins Groups High risk (ppb) TEFs High risk (ppb) TEFs High risk (ppb) TEFs Aflatoxins B Total Aflatoxins Ochratoxins Other Aspergillus Toxins Trichothecenes B Trichothecenes A Fumonisins Zearalenones Penicillium Toxins Ergot Alkaloids TEQ: TOXIC EQUIVALENT QUANTITIES Evaluate the total toxicity given mycotoxin mixture, based on amount and TEF: TEQ total = C tox1 TEF tox1 + C tox2 TEF tox2 + + C toxn TEF toxn 12

13 Pratical Limits Sows/Gilts/Boars Feed, ppb (µg/kg feed) Mycotoxin Low Moder ate High Aflatoxin B FDA = 100ppb for Corn in breeding pigs, for feed it will 50 be 50% of 100ppb Aflatoxins Aflatoxin B1 and G1 are additive (Bonomi et al., 1996) Ochratoxins EU, 50ppb Type B Trichothecenes EU, 900ppb Type A Trichothecenes Canada/Russia, 1000/50ppb EU, 5000ppb; LOAEL is 5000ppm (EFSA, 2005), based on Fumonisins Sa/So ratio Zearalenone Group EU,100ppb Other Penicillium mycotoxins two times ochratoxins Other Aspergillus mycotoxins two times total aflatoxins Ergot alkaloids Canada, 6000ppb Interpretation: Risk Evaluation Feed: Corn Target species: Swine Safe Apply Remediation Strategy Apply Rules & CCP Levels Proportion Risk Assessment Mycotoxins (ppb) (%) very low low high very high Aflatoxins <5 20 >20 Ochratoxins <500 1,000 1,500 Trichothecenes B <500 1,000 >1,000 Trichothecenes A <50 75 >100 Fumonisins 32, <1,500 2,500 10,000 >10,000 age >4 months <4 months Zearalenone < >500 Ergot alkaloids 3, <500 1,000 >2,000 Others < >200 Available regulatory limits or guidance or practical limits according to scientific literature 13

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15 European grains 2014:Risk to sows Low Medium High Main moulds and mycotoxins Field Origin Storage Origin Fusarium Deoxynivalenol Zearalenone T-2 Toxin Fumonisin Moniliformin Nivalenol Fusaric Acid Aspergillus Aflatoxin Ochratoxin Sterigmatocystin Fumitremorgens Fumigaclavines Fumitoxins Gliotoxin Penicillium Ochratoxin PR Toxin Patulin Penicillic Acid Citrinin Penitrem Cyclopiazonic acid 15

16 Maksimal sikker lagringstid (døgn) m.h.t. tilvækst af lagersvampe Temperat ur ºC Antal døgn ved afgrødens vandindhold % ,5 1,7 1, , ,5 tørring Luften (evt. opvarmet) anvendes for at transportere vandet bort Vandindhold % Mængde vand (ton) at tørre bort ved forskellige mængder korn (ton): ,7 18,6 37,2 55, ,9 55,8 83,7 16

17 Ligevægtstabel mellem relativ luftfugtighed og vandindhold i korn og frøafgrøder Relativ luftfugtighed i % Byg 10,5 11,1 11,8 12,6 13,4 14,3 15,2 16,3 17, ,9 Hvede 10,6 11,3 12,3 13,1 13,5 14,3 15,2 16,3 17,4 19,1 20,5 Havre 9,7 10,3 11,3 11,9 12,5 13,3 14,2 15,4 16,7 18,5 21 Rug 10,3 10,9 11,6 12,4 13,2 13,9 14, , ,5 Raps 5,7 6,1 6,6 7 7, ,3 12,7 14,3 Ærter 10, ,9 12,7 13,5 14, , Rajgræs 8,6 9,3 10, , ,2 15,6 17,4 19,8 23,3 Opvarmning af luften med 1 C = 4% reduktion af relativ luftfugtighed Plantørring: max. 6ºC opvarmning af udeluften inkl. blæserens egenopvarmning (ca. 1ºC) Betydning af varmetilsætning 750 ton hvede, 6,5 meter lagringshøjde i silo med omrører Nedtørring % giver 35 ton vand at tørre bort 22 kw blæser og Jumbo 200 olieovn 18 C og 70% luftfugtighed Betyder følgende: Tørretid uden varme, ca. 30 døgn Tørretid med varme, ca. 6 døgn Samme omkostning til energi, men meget kortere tørretid! 17

18 Kondens på indersiden af siloen dårligt/muggent korn Skidt (støv) under silogulv? Støv kan lægge sig under silogulvene i mindre omfang. Billeder fra 5 år gammel silo. Støvet vil ved en tæt silo være meget tørt, da al anvendt tørreluft er passeret forbi. 18

19 Rengøring under silogulv? Mulig løsning: Udtagning af silogulv Rengøring Fugning af silo/fundament Montering af gulv igen I nye siloer fuges der i dag på indvendig side mellem silovæg og fundament. Fugning af silo / fundament 19

20 Storage Contamination Score 1 clean silo with no accumulated crusts on its walls. Score 2 Silo with crusts accumulated on its walls. Score 3 Many old crusts On the walls of the silo. dålig opbevaring 20

21 GENERELT TØRRING HUSK ALTID: 1. TILSTRÆKKELIG LUFTMÆNGDE 2. (OMRØRING) 3. TØRRING STRAKS AFGRØDEN ER HØSTET 4. VARME (HVIS NØDVENDIGT) 5. KOLD LUFT EFTER TØRRING = NEDKØLING Jo bedre en råvare, der kommer ind, des bedre en færdigvare får man ud i den anden ende. Evt. rensning inden lagring gælder for både planlager og siloer. Afgrøderne er plejet og passet optimalt sæsonen igennem så sørg for at gøre arbejdet færdigt. DAN-CORN A/ Hvad gør man hvis man har problemer? Destruktion af råvaren Fortynde den kontaminerede råvare Brug af mykotoxinbinder 21

22 Stabilitet ved ændringer af ph % Absorberet EffektafpH påmycosorbabsorbtionafaflatoxinb ph 4.5 ph Devegowda et al., 1994 Mycosorb inclusion, (mg) Mycosorb binder IKKE Mineraler og Vitaminer Vitamin levels Vitamin A (IU/g) Vitamin E (mg/g) Negative Control Control + Mycosorb Contaminated feed Contaminated feed + Mycosorb Treatments Meissonnier et al.,

23 MYCOTOXIN FORSKER ÅR Aflatoxins Stanley and Co-workers 2004 Aflatoxins Diaz and Whitlow 2004 Zearalenone Yiannikouris and Co-workers 2004 T-2 toxin Manoj and Devegowda 1999 T-2 toxin Dvorska and Surai 2001 DAS Pavicic and Spring 2001 Ergot alkaloids Merrill and Co-workers 2007 KOMBINATIONER AF MYKOTOXINER Aflatoxin, ochratoxin Raju and Devegowda 2000 & T-2 toxin DON and Zearalenone Swamy and Smith 2002 DON and Zearalenone Chowdhury and Smith 2005 DON and Zearalenone Raymond, Smith and Swamy 2003 DON and Zearalenone Diaz and Smith 2006 DON and Zearalenone Korosteleva and Smith, DON and Zearalenone Girish and Smith, Evne til at binde en bred vifte af mykotoxiner Evne til at binde 30 minutter Efter fodring. Forskellige absorbenters evne til hurtigt at binde Zearalenone % Absorberet min 24 h Time intervals Mycosorb Clay A Clay B Clay C Völkl and Karlovsky,

24 Effektivt ved lav iblanding % of aflatoxin adsorbed ppb Aflatoxin ppb Aflatoxin ppb Aflatoxin % 0.20% 0.40% % 0.025% 0.05% Clay A Mycosorb Mahesh & Devegowda, 1996 Peer-reviewed in vivo data 24

25 Krav til absorbent Stabilitet Ikke binde mineralet/vitaminer Dokumenteret effekt Hurtig binding In vivo data 25

26 Funktionelle ingredienser fra gær Saccharomyces cerevisiae 1026 Indvendig (polymer) Indvendig og udvendig cellevæg Hel gær Ekstrakt udvendig (manno-proteins) THEYEAST CELL WALL AND THEALGAE CONTAIN VARIOUS MONOSACCHARIDES COMPOSING THEIR CARBOHYDRATE NETWORK Yeast Cell Wall Extract Glucose Mannose Glucosamine + Mannose + Glucose Arabinose + Glucosamine Rhamnose Galactose Xylose Heterotrophic Algae 26

27 Molecular details of the mode of action Detoxification et al, Biomacromolecules 2004; 2006 Interaction energy (kcal/mol) Mycotoxins ZEA DON AFB1 Van der Waals Electrostatic Total Critical similarity in geometry favors the interaction Aluminosilicates? Bentonite? Zeolite? Bentonite mine Only effective against Aflato High levels of inclusion for efficacy (10X) No specific to mycotoxins (minerals and vitamins such a Mn are adsorbed Antibiotics? Contaminants (dioxins, PCBs, Pb) Zeolite mine 27

28 Aluminosilicates? Bentonite? Zeolite? Bentonite mine Only effective against Aflatoxin High levels of inclusion for efficacy (10X) No specific to mycotoxins (minerals and vitamins such as Mn are adsorbed Antibiotic Interactions EU 29 October 2013 Contaminants (dioxins, PCBs, Pb) Zeolite mine Krav til absorbent Stabilitet Ikke binde mineralet/vitaminer Dokumenteret effekt Hurtig binding In vivo data 28

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