Applied Microbiology and Biotechnology

, Volume 98, Issue 17, pp 7583–7596 | Cite as

Development of an in vitro method for the prediction of mycotoxin binding on yeast-based products: case of aflatoxin B1, zearalenone and ochratoxin A

  • Virginie Faucet-Marquis
  • Claire Joannis-Cassan
  • Kheira Hadjeba-Medjdoub
  • Nathalie Ballet
  • Annie Pfohl-Leszkowicz
Methods and protocols


To date, no official method is available to accurately define the binding capacity of binders. The goal is to define general in vitro parameters (equilibrium time, pH, mycotoxin/binder ratio) for the determination of binding efficacy, which can be used to calculate the relevant equilibrium adsorption constants. For this purpose, aflatoxin B1 (AFB1), zearalenone (ZEA) or ochratoxin A (OTA) were incubated with one yeast cell wall in pH 3, pH 5 or pH 7 buffers. The percentage of adsorption was recorded by quantitation of remaining mycotoxins in the supernatant and amount of mycotoxin adsorbed on the residue. The incubation of yeast cell wall in the presence of mycotoxins solved in buffer, lead to unexpected high adsorption percentage when the analysis was based only on remaining mycotoxins in the supernatant. The decrease of mycotoxins in the supernatant was not correlated to the amount of mycotoxins found in the residue. For this reason we modified the conditions of incubation. Yeast cell wall (5 mg) was pre-incubated in buffer (990 μl) at 37 °C during 5 min and then 10 μl of an alcoholic solution of mycotoxin (concentration 100 times higher than the final concentration required in the test tube) were added. After incubation, the solution was centrifuged, and the amount of mycotoxins were analysed both in the supernatant and in the residue. A plateau of binding was reached after 15 min of incubation whatever the mycotoxins and the concentrations tested. The adsorption of ZEA was better at pH 5 (75 %), versus 60 % at pH 3 and 7. OTA was only significantly adsorbed at pH 3 (50 %). Depending on the pH, the adsorptions of OTA or ZEA were increased or decreased when they were together, indicative of a cooperative effect.


Binder Aflatoxin Ochratoxin Zearalenone Yeast In vitro screening method 



This study was supported by Program county “Midi-Pyrénées” (Food Safety, 2008–2012). ARC (association de la recherchecontre le cancer, France) is acknowledged for KHM’s doctoral grant. The authors thank GM for English proofreading.

Supplementary material

253_2014_5917_MOESM1_ESM.pdf (489 kb)
ESM 1 (PDF 488 kb)


  1. Armando MR, Pizzolitto RM, Escobar F, Dogi RP, Peirano MS, Salvano MA, Sabini II, Combina M, Dalcero AM, Cavaglieri LR (2011) Saccharomyces cerevisiae strains from animal environment with in vitro aflatoxin B1 binding ability and antipathogenic bacterial influence. World Mycotoxin J 4:59–68CrossRefGoogle Scholar
  2. Armando MR, Pizzolitto RM, Dogi RP, Cristofolini A, Merkis C, Polini V, Dalcero AM, Cavaglieri LR (2012) Adsorption of ochratoxin A and zearalenone by potential probiotic Saccharomyces cerevisiae strains and its relation with cell wall thickness. J Appl Microbiol 113:256–264PubMedCrossRefGoogle Scholar
  3. Avantaggiato G, Solfrizzo M, Visconti A (2005) Recent advances of the use of adsorbent materials for detoxification of Fusarium mycotoxins. Food Addit Contam 22:379–388PubMedCrossRefGoogle Scholar
  4. Bazin I, Marquis-Faucet V, ElKhoury M, Marty JL, Pfohl-Leszkowicz A (2013) Impact of pH on the stability and cross reactivity of ochratoxin A and Citrinin. Toxins 5:2325–2340CrossRefGoogle Scholar
  5. Binder EM (2007) Managing the risk of mycotoxins in modern feed production. Anim Feed Sci Technol 133:149–166CrossRefGoogle Scholar
  6. Bueno D, Casale C, Pizzolitto R, Salvano M, Oliver G (2007) Physical adsorption of aflatoxin B1 by lactic acid bacteria and Saccharomyces cerevisiae: a theoretical model. J Food Prot 70(9):2148–2154PubMedGoogle Scholar
  7. Carvet S, Laurent N, Videmann B, Mazallon M, Lecœur S (2010) Assessment of deoxynivalenol (DON) adsorbents and characterisation of their efficacy using complementary in vitro tests. Food Addit Contam 27(1):43–53CrossRefGoogle Scholar
  8. Dakovic A, Tomasevic-Canovic M, Dondur V, Rottinghaus GE, Medakovic V, Zaric S (2005) Adsorption of mycotoxins by organozeolites. Colloids Surf B: Biointerfaces 46(1):20–25PubMedCrossRefGoogle Scholar
  9. Di Natale F, Gallo M, Nigro R (2009) Adsorbents selection for aflatoxins removal in bovine milks. J Food Eng 95:186–191CrossRefGoogle Scholar
  10. EFSA (2004a) Opinion of the scientific panel on contaminants in the food chain on a request from the Commission related to Zearalenone as undesirable substance in animal feed. EFSA J 89:1–35Google Scholar
  11. EFSA (2004b) Opinion of the scientific panel on contaminants in the food chain on a request from the Commission related to ochratoxin A (OTA) as undesirable substance in animal feed. EFSA J 101:1–36Google Scholar
  12. EFSA (2009). Review of mycotoxin-detoxifying agents used as feed additives: mode of action, efficacy and feed/food safety. Scientific report submitted to EFSA. (
  13. Fink-Gremmels J, Malekinejad H (2007) Clinical effects and biochemical mechanisms associated with exposure to the mycoestrogen zearalenone. Anim Feed Sci Technol 137:326–341CrossRefGoogle Scholar
  14. Firmin S, Gandia P, Morgavi DP, Houin G, Jouany JP, Bertin G, Boudra H (2010) Modification of aflatoxin B1 and ochratoxin A toxicokinetics in rats administered a yeast cell wall preparation. Food Addit Contam 27:153–1160CrossRefGoogle Scholar
  15. Firmin S, Morgavi DP, Yiannikouris A, Boudra H (2011) Effectiveness of modified yeast cell wall extracts to reduce aflatoxin B1 absorption in dairy ewes. J Dairy Sci 94:5611–5619PubMedCrossRefGoogle Scholar
  16. Fruhauf S, Schwartz H, Ottner F, Krska R, Vekiru E (2012) Yeast cell based feed additives: studies on aflatoxin B1 and zeralenone. Food Addit Contam 29(2):217–231CrossRefGoogle Scholar
  17. Guo C, Yue T, Hatab S, Yuan Y (2012) Ability of inactivated yeast powder to adsorb patulin from apple juice. J Food Protect 75(3):585–590CrossRefGoogle Scholar
  18. Huwig A, Freimund S, Käppeli O, Dutler H (2001) Mycotoxin detoxication of animal feed by different adsorbents. Toxicol Lett 122:179–188PubMedCrossRefGoogle Scholar
  19. IARC, International Agency for Research on Cancer, Geneva. 1993, 56, p. 489.Google Scholar
  20. Joannis-Cassan C, Tozlovanu M, Hadejba-Medjdoub K, Ballet N, Pfohl-Leszkowicz A (2011) Binding of Zearalenone, Aflatoxin B1 and Ochratoxin A by yeast based-products: a rapid method for quantification of adsorption performance. J Food Prot 74(7):1175–1185PubMedCrossRefGoogle Scholar
  21. Jouany JP (2007) Methods for preventing, decontaminating and minimizing the toxicity of mycotoxins in feeds. Anim Feed Sci Technol 137:342–362CrossRefGoogle Scholar
  22. Kabak B, Dobson ADW, Var I (2006) Strategies to prevent mycotoxin contamination of food and animal feed: a review. Crit Rev Food Sci Nutr 46:593–619PubMedCrossRefGoogle Scholar
  23. Kabak B, Dobson ADW (2009) Biological strategies to counteract the effects of mycotoxins. J Food Prot 72:2006–2016PubMedGoogle Scholar
  24. Karaman M, Basmocioglu H, Ortatali M, Oguz H (2005) Evaluation of the detoxifying effect of yeast glucomannan on aflatoxicosis in broilers as assessed by gross examination and histopathology. Br Poult Sci 46:394–400PubMedCrossRefGoogle Scholar
  25. Keppel G (1973) Design and analysis: researcher’s handbook. Prentice-Hall, Englewood Cliffs, NJ, pp 658–663Google Scholar
  26. Kollar R, Reinhold BB, Petrakova E, Yeh HJC, Ashwell G, Drgonova J, Kapteyn JC, Klis FM, Cabib E (1997) Architecture of the yeast cell wall. β(1,6)-d-Glucan interconnects mannoprotein, β-(1,3)-d-glucan, and chitin. J Biol Chem 272:17762–17775PubMedCrossRefGoogle Scholar
  27. Kolosova A, Stroka J (2011) Substances for reduction of the contamination of feed by mycotoxins: a review. World Mycotoxin J 4(3):225–256CrossRefGoogle Scholar
  28. Korosteleva SN, Smith TK, Boermans HJ (2007) Effects of feed borne Fusarium mycotoxins on the performance, metabolism, and immunity of dairy cows. J Dairy Sci 90:3867–3873PubMedCrossRefGoogle Scholar
  29. Kurtbay H, Bekçi Z, Merdivan M, Yurdkoç K (2008) Reduction of ochratoxin A levels in red wine by bentonite, modified bentonite and chitosan. J Agric Food Chem 56(7):2541–2545PubMedCrossRefGoogle Scholar
  30. Manafi M, Narayanaswamy HD, Pirany N (2009) In vitro binding ability of mycotoxin binder in commercial broiler feed. Afr J Agric Res 4(2):141–143Google Scholar
  31. Manners DJ, Masson AJ, Patterson JC (1973) The structure of a β(1,3)-d-glucan from yeast cell walls. Biochemistry J135:19–30Google Scholar
  32. Matur E, Ergul E, Akyazi I, Eraslan E, Cirakli ZT (2010) The effects of Saccharomyces cerevisiae extract on the weight of some organs, liver, and pancreatic digestive enzyme activity in breeder hens fed diets contaminated with aflatoxins. Poult Sci 89:2213–2220PubMedCrossRefGoogle Scholar
  33. Mirocha CJ, Christensen CM, Nelson GH (1971) Algal and fungal toxins. In: Kadis S, Ciegler A, Ajl SS (eds) Microbial toxins, Vol. VII. New York, Academic Press, pp 107–117Google Scholar
  34. Ostry V, Malir F, Ruprich J (2013) Producers and important dietary sources of ochratoxin A and citrinin. Toxins 5:1574–1586PubMedCentralPubMedCrossRefGoogle Scholar
  35. Pereyra CM, Cavaglieri LR, Chiacchieria SM, Dalcero A (2012) The corn influence on the adsorption levels of aflatoxin B1 and zearalenone by yeast cell wall. J Appl Microbiol 114:655–662Google Scholar
  36. Pfohl-Leszkowicz A, Manderville RA (2007) Review on ochratoxin A: an overview on toxicity and carcinogenicity in animals and humans. Mol Nutr Food Res 51:61–99PubMedCrossRefGoogle Scholar
  37. Pfohl-Leszkowicz A, Manderville RA (2012) An update on direct genotoxicity as molecular mechanism of ochratoxin A carcinogenicity. Chem Res Toxicol 25:252–262PubMedCrossRefGoogle Scholar
  38. Pizzolitto RM, Bueno DJ, Armando MR, Cavaglieri L, Dalcero AM, Salvano MA (2011). Binding of aflatoxin B1 to lactic acid bacteria and Saccharomyces cerevisiae in vitro: a useful model to determine the most efficient microorganism, aflatoxins — biochemistry and molecular biology. Ramon G. Guevara-Gonzalez (Ed.), ISBN: 978-953-307-395-8, InTech,Google Scholar
  39. Pizzolitto RM, Armando MR, Combina M, Cavaglieri LR, Dalcero AM, Salvano MA (2012) Evaluation of Saccharomyces cerevisiae strains as probiotic agent with aflatoxin B1 adsorption ability for use in poultry feedstuffs. J Environ Sci Health Part B 47:933–941CrossRefGoogle Scholar
  40. Regulation (EC) No 386/2009 of 12 May 2009 amending Regulation (EC) No 1831/2003 of the European Parliament and of the Council as regards the establishment of a new functional group of feed additives. Official Journal of the European Union. L118/66.Google Scholar
  41. Ringot D, Lerzy B, Bonhoure JP, Auclair E, Oriol E, Larondelle Y (2005) Effect of temperature on in vitro ochratoxin biosorption onto yeast cell wall derivatives. Process Biochem 40:3008–3016Google Scholar
  42. Ringot D, Lerzy B, Chaplain K, Bonhoure JP, Auclair E, Larondelle Y (2007) In vitro biosorption of ochratoxin A on the yeast industry by-products: comparison of isotherm models. Bioresour Technol 98:1812–1821Google Scholar
  43. Sabater-Vilar M, Malekinejad H, Selman MH, van der Doelen MA, Fink-Gremmels J (2007) In vitro assessment of adsorbents aiming to prevent deoxynivalenol and zearalenone mycotoxicoses. Mycopathologia 163:81–90PubMedCentralPubMedCrossRefGoogle Scholar
  44. Santos RR, Vermeulen S, Haritova A, Fink-Gremmels J (2011) Isotherm modeling of organic activated bentonite and humic acid polymer used as mycotoxin adsorbents. Food Addit Contam 28(11):1578–1589CrossRefGoogle Scholar
  45. Shetty PH, Jespersen L (2006) Saccharomyces cerevisiae and lactic bacteria as potential mycotoxin decontamination agents. Trends Food Sci Technol 17:48–55CrossRefGoogle Scholar
  46. Shetty PH, Hald B, Jespersen L (2007) Surface binding of aflatoxin B1 by Saccharomyces cerevisiae strains with potential decontaminating abilities in indigenous fermented food. Int J Food Microbiol 113:41–46PubMedCrossRefGoogle Scholar
  47. Varga J, Rigo K, Teren J, Mesterhazy A (2001) Recent advances in ochratoxin research: II. Biosynthesis, mode of action and control of ochratoxins. Cereal Res Commun 29:93–100Google Scholar
  48. Varga J, Rigo K, Toth B, Teren J, Kozakiewicz Z (2003) Evolutionary relationships among Aspergillus species producing economically important mycotoxins. Food Technol Biotechnol 41:29–36Google Scholar
  49. Ye S-Q, Lv X-Z, Zhou A-G (2009) In vitro evaluation of the efficacy of sodium humate as an aflatoxin B1 adsorbent. Australian journal of basic and applied sciences 3(2):1296--1300Google Scholar
  50. Yiannikouris A, Jouany JP (2002) Mycotoxins in feeds and their fate in animals: a review. Anim Res 51:81–99CrossRefGoogle Scholar
  51. Yiannikouris A, Poughon L, Cameleyre X, Dussap CG, François J, Bertin G, Jouany JP (2003) A novel technique to evaluate interactions between Saccharomyces cerevisiae cell wall and mycotoxins: application to zearalenone. Biotechnol Lett 25:783–789PubMedCrossRefGoogle Scholar
  52. Yiannikouris A, André G, Buléon A, Jeminet G, Canet I, François J, Bertin G, Jouany JP (2004) Comprehensive conformational study of key interactions involved in zearalenone complexation with beta-d-glucans. Biomacromolecules 5:2176–2185PubMedCrossRefGoogle Scholar
  53. Yiannikouris A, André G, Poughon L, Francois J, Dussap CG, Jeminet G, Bertin G, Jouany JP (2006) Chemical and conformational study of the interactions involved in mycotoxin complexation with beta-d-glucans. Biomacromolecules 7:1147–1155PubMedCrossRefGoogle Scholar
  54. Yiannikouris A, Kettunen J, Apajalahti E, Pennala A, Moran CA (2013) Comparison of the sequestering properties of yeast cell wall extract and hydrated sodium calcium aluminosilicate in three in vitro models accounting for the animal physiological bioavailability of zearalenone. Food Addit Contam 30(9):1641–1650Google Scholar
  55. Zekovic DB, Kwiatkowski S, Vrvic MM, Jakovljevic D, Moran CA (2005) Natural and modified (1–3)-beta-d-glucans in health promotion and disease alleviation. Crit Rev Biotechnol 25:205–230PubMedCrossRefGoogle Scholar
  56. Zinedine A, Soriano JM, Moltó JC, Mañes J (2007) Review on the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone: an oestrogenic mycotoxin. Food Chem Toxicol 45:1–18PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Virginie Faucet-Marquis
    • 1
    • 2
    • 3
  • Claire Joannis-Cassan
    • 1
    • 4
  • Kheira Hadjeba-Medjdoub
    • 1
    • 2
  • Nathalie Ballet
    • 5
  • Annie Pfohl-Leszkowicz
    • 1
    • 2
  1. 1.Laboratory Chemical EngineeringUniversity of Toulouse, UMR-CNRS/INPT/UPS 5503Auzeville-TolosaneFrance
  2. 2.Department Bioprocess and Microbial systemENSATAuzeville-TolosaneFrance
  3. 3.AnabiotoxRamonville-St AgneFrance
  4. 4.Department Bioprocess and Microbial systemENSIACETToulouseFrance
  5. 5.Lesaffre InternationalMarcq-en-BaroeulFrance

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