Food Analytical Methods

, Volume 11, Issue 5, pp 1267–1280 | Cite as

TLC-Digital Image-Based Fluorometric Analysis of Ergosterol and Chitin Content in Food Grains Artificially Infested with Aspergillus flavus and Fusarium verticillioides

  • Tanuja Kosuri
  • Sujatha Nayak
  • Mir Zahoor Gul
  • Karuna Rupula
  • Sashidhar Rao Beedu
Article
First Online:
Received:
Accepted:
  • 75 Downloads

Abstract

Novel thin-layer chromatography-digital image-based analytical methods were developed for the quantitation of ergosterol and chitin content in six food matrices (rice, wheat, maize, sorghum, groundnut, and sunflower), artificially infested with Aspergillus flavus (MTCC 6513)/Fusarium verticillioides (MRC 826). For ergosterol, single-step method, based on liquid/liquid extraction, was followed by thin-layer chromatography (TLC). Chitin was solubilized using lithium chloride (5%) in dimethyl acetamide and converted to chitosan using 5 N NaOH and subsequently complexed with calcofluor white dye. The absorption and emission maxima of chitosan-calcofluor complex were recorded at λ 350/230 and 430 nm, respectively. The sensitivity based on the limit of detection (LOD) was found to be 100 ng both for ergosterol and chitin analysis. Based on ergosterol and chitin analysis, groundnut and maize were found to be suitable substrates for A. flavus (p < 0.013 and p < 0.01), while sorghum followed by groundnut and sunflower were found to be ideal for F. verticillioides (p < 0.01 and p < 0.0001) and rice was established as poor substrate as there was no growth on it up to 12 days of incubation. A strong correlation was found between ergosterol and chitin contents with regression (r 2) values of 0.974 and 0.997 in food grains inoculated with A. flavus and F. verticillioides, respectively, during the period of infection. The authenticity of the two methods developed was further confirmed by applying them to commercial food grains and flours. Thus, ergosterol in combination with chitin analysis could be successfully used as an index of fungal contamination employing TLC-digital-based analytical methods.

Keywords

Food grains Ergosterol Chitin Calcofluor white Fungal contamination TLC 
This is a preview of subscription content, log in to check access

Notes

Acknowledgements

The authors are thankful to CSIR, New Delhi, for funding the research project, vide sanction No. [38 (1155)/07/EMR-II] and for infrastructural facility provided under DST - PURSE programme at Osmania University.

Compliance with Ethical Standards

Conflict of Interest

Tanuja Kosuri declares that she has no conflict of interest. Sujatha Nayak declares that she has no conflict of interest. Mir Zahoor Gul declares that he has no conflict of interest. Karuna Rupula declares that she has no conflict of interest. Sashidhar Rao Beedu declares that he has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed Consent

Not applicable.

References

  1. Adeyeye SAO, Yildiz F (2016) Fungal mycotoxins in foods: a review. Cogent Food Agric 2(1):1213127Google Scholar
  2. Anonymous (2003) Mycotoxins: risks in plant, animal and human systems. CAST - Council for Agricultural Science and TechnologyGoogle Scholar
  3. Austin PR (1977) Chitin solution, Vol. 4. U.S. Patent, pp 059457Google Scholar
  4. Bankole S, Schollenberger M, Drochner W (2006) Mycotoxins in food systems in sub Saharan Africa: a review. Mycotoxin Research 22(3):163–169.  https://doi.org/10.1007/BF02959270 CrossRefGoogle Scholar
  5. Booth C (1971a) Fungal culture media. In: Booth C (ed) Methods in microbiology, Academic Press, vol 4. Cambridge, Massachusetts, pp 49–94Google Scholar
  6. Booth C (1971b) Introduction to general methods. In: Booth C (ed) Methods in microbiology, Academic press, vol 4. Cambridge, Massachusetts, pp 1–47Google Scholar
  7. Börjesson T, Stenberg B, Schnürer J (2007) Near-infrared spectroscopy for estimation of ergosterol content in barley: a comparison between reflectance and transmittance techniques. Cereal Chem 84(3):231–236.  https://doi.org/10.1094/CCHEM-84-3-0231 CrossRefGoogle Scholar
  8. Braid GH, Line MA (1981) A sensitive chitin assay for the estimation of fungal biomass in hardwoods. Holzforschung 35(10–15):10–15.  https://doi.org/10.1515/hfsg.1981.35.1.10 CrossRefGoogle Scholar
  9. Bulawa CE (1993) Genetics and molecular biology of chitin synthesis in fungi. Annu Rev Microbiol 47(1):505–534.  https://doi.org/10.1146/annurev.mi.47.100193.002445 CrossRefGoogle Scholar
  10. Castro MFPM, Bragagnolo N, Valentini SRT (2002) The relationship between fungi growth and aflatoxin production with ergosterol content of corn grains. Braz J Microbiol 33(1):22–26.  https://doi.org/10.1590/S1517-83822002000100004 CrossRefGoogle Scholar
  11. Cegielska-Radziejewska R, Stuper-Szablewska K, Szablewski T (2013) Microflora and mycotoxin contamination in poultry feed mixtures from western Poland. Ann Agric Env Med 20(1):30–35Google Scholar
  12. Christensen CM, Kaufmann HH (1969) Grain storage: the role of fungi in quality loss. University of Minnesota PressGoogle Scholar
  13. Coppock R, Jacobsen BJ (2009) Mycotoxins in animal and human patients. Toxicol Ind Health 25(9–10):637–655.  https://doi.org/10.1177/0748233709348263 CrossRefGoogle Scholar
  14. Costa-de-Oliveira S, Silva AP, Miranda IM, Salvador A, Azevedo MM, Munro CA, Rodrigues AG, Pina-Vaz C (2013) Determination of chitin content in fungal cell wall: an alternative flow cytometric method. Cytometry A 83(3):324–328.  https://doi.org/10.1002/cyto.a.22250 CrossRefGoogle Scholar
  15. Cousin M (1995) Chitin as a measure of mold contamination of agricultural commodities and foods. J Food Prot 59(1):73–81CrossRefGoogle Scholar
  16. Dastjerdi R, Karlovsky P (2015) Systemic infection of maize, sorghum, rice and beet seedlings with fumonisin-producing and nonproducing Fusarium verticillioides strains. Plant Pathol J 31(4):334–342.  https://doi.org/10.5423/PPJ.OA.05.2015.0088 CrossRefGoogle Scholar
  17. de Nobel H, van Den Ende H, Klis FM (2000) Cell wall maintenance in fungi. Trends Microbiol 8(8):344–345.  https://doi.org/10.1016/S0966-842X(00)01805-9 CrossRefGoogle Scholar
  18. De Ruiter GA, Notermans SW, Rombouts FM (1993) New methods in food mycology. Trends Food Sci Technol 4(4):91–97.  https://doi.org/10.1016/0924-2244(93)90089-S CrossRefGoogle Scholar
  19. Donald WW, Mirocha CJ (1977) Chitin as a measure of fungal growth in stored corn and soybean seed. Cereal Chem 54:466–474Google Scholar
  20. Dong Y, Steffenson BJ, Mirocha CJ (2006) Analysis of ergosterol in single kernel and ground grain by gas chromatography-mass spectrometry. J Agric Food Chem 54(12):4121–4125.  https://doi.org/10.1021/jf060149f CrossRefGoogle Scholar
  21. Ekblad ALF, Wallander H, NÄSholm T (1998) Chitin and ergosterol combined to measure total and living fungal biomass in ectomycorrhizas. New Phytol 138(1):143–149.  https://doi.org/10.1046/j.1469-8137.1998.00891.x CrossRefGoogle Scholar
  22. Fakruddin M, Chowdhury A, Hossain MN, Ahmed MM (2015) Characterization of aflatoxin producing Aspergillus flavus from food and feed samples. SpringerPlus 4(1):159.  https://doi.org/10.1186/s40064-015-0947-1 CrossRefGoogle Scholar
  23. Gagnaire D, Saint-Germain J, Vincendon M (1982) NMR studies of chitin and chitin derivatives. Die Makromol Chem 183(3):593–601.  https://doi.org/10.1002/macp.1982.021830309 CrossRefGoogle Scholar
  24. Golubchuk M, Cuendet LS, Geddes WF (1960) Grain storage studies: chitin content of wheat as an index of mold contamination and wheat deterioration. Cereal Chem 37:405–411Google Scholar
  25. Gourama H, Bullerman LB (1995) Relationship between aflatoxin production and mold growth as measured by ergosterol and plate count. LWT - Food Sci Technol 28(2):185–189.  https://doi.org/10.1016/S0023-6438(95)91372-6 CrossRefGoogle Scholar
  26. Hackman RH (1960) Studies on chitin. Aust J Biol Sci 13(4):568–577.  https://doi.org/10.1071/BI9600568 CrossRefGoogle Scholar
  27. Holton WC (2000) Fresh ideas for food safety. Environ Health Perspect 108(11):A516–A519Google Scholar
  28. Hubbard JO, Seitz LM, Mohr HE (1979) Determination of hexosamines in chitin by ion-exchange chromatography. J Food Sci 44(5):1552–1553.  https://doi.org/10.1111/j.1365-2621.1979.tb06487.x CrossRefGoogle Scholar
  29. IARC (2002) IARC monographs on the evaluation of carcinogenic risks to humans. World Health Organization, International Agency for Research on Cancer press, Lyon, FranceGoogle Scholar
  30. Jambunathan R, Kherdekar MS, Vaidya P (1991) Ergosterol concentration in mold-susceptible and mold-resistant sorghum at different stages of grain development and its relationship to flavan-4-ols. J Agric Food Chem 39(10):1866–1870.  https://doi.org/10.1021/jf00010a037 CrossRefGoogle Scholar
  31. Jarvis B (1977) A chemical method for the estimation of mould in tomato products. Int J Food Sci Technol 12(6):581–591CrossRefGoogle Scholar
  32. Jay JM, Loessner MJ, Golden DA (2005) Culture, microscopic, and sampling methods. Modern Food Microbiology. Springer US, Boston, pp 217–240Google Scholar
  33. Jedlickova L, Gadas D, Havlova P, Havel J (2008) Determination of ergosterol levels in barley and malt varieties in the Czech Republic via HPLC. J Agric Food Chem 56(11):4092–4095.  https://doi.org/10.1021/jf703675q CrossRefGoogle Scholar
  34. Matcham SE, Jordan BR, Wood DA (1985) Estimation of fungal biomass in a solid substrate by three independent methods. Appl Microbiol Biotechnol 21(1):108–112Google Scholar
  35. Milani JM (2013) Ecological conditions affecting mycotoxin production in cereals: a review. Veterinarni Medicina 58(8):405–411CrossRefGoogle Scholar
  36. Mille-Lindblom C, von Wachenfeldt E, Tranvik LJ (2004) Ergosterol as a measure of living fungal biomass: persistence in environmental samples after fungal death. J Microbiol Methods 59(2):253–262.  https://doi.org/10.1016/j.mimet.2004.07.010 CrossRefGoogle Scholar
  37. Muller HM, Schwadorf K (1990) Ergosterol as a measure for fungal growth in feed. 2. Ergosterol content of mixed feed components and mixed feed. Arch Tierernahr 40(4):385–395CrossRefGoogle Scholar
  38. Musa HM, Aidoo KE, Hunter CA (2013) Ergosterol and chitin levels as potential markers for mold contamination of building substrate. Architecture and Environment 1:14–20CrossRefGoogle Scholar
  39. Muzzarelli RAA, Peter MG (1997) Chitin handbook. AtecGoogle Scholar
  40. Nandi B (1978) Glucosamine analysis of fungus-infected wheat as a method to determine the effect of anti-fungal compounds in grain preservation. Cereal Chem 55:121–126Google Scholar
  41. Newell SY (1992) Estimating fungal biomass and productivity in decomposing litter. In: Carroll GC, Wicklow DT (eds) The fungal community, 2nd edn. Marcel Dekker, Inc, New York, pp 521–561Google Scholar
  42. Newell SY (1994) Total and free ergosterol in mycelia of saltmarsh ascomycetes with access to whole leaves or aqueous extracts of leaves. Appl Environ Microbiol 60(9):3479–3482Google Scholar
  43. Ng HE, Raj SS, Wong SH, Tey D, Tan HM (2008) Estimation of fungal growth using the ergosterol assay: a rapid tool in assessing the microbiological status of grains and feeds. Lett Appl Microbiol 46(1):113–118.  https://doi.org/10.1111/j.1472-765X.2007.02279.x CrossRefGoogle Scholar
  44. Pallavi RMV, Ramana D, Sashidhar RB (1997) Synthesis of the antigen bovine serum albumin-ergosterol and its immunocharacterization. Food Agric Immunol 9(2):85–95.  https://doi.org/10.1080/09540109709354939 CrossRefGoogle Scholar
  45. Pasikatan MC, Dowell FE (2001) Sorting systems based on optical methods for detecting and removing seeds infested internally by insects or fungi: a review. Appl Spectrosc Rev 36(4):399–416.  https://doi.org/10.1081/ASR-100107719 CrossRefGoogle Scholar
  46. Perkowski J, Buśko M, Stuper K, Kostecki M, Matysiak A, Szwajkowska-Michałek L (2008) Concentration of ergosterol in small-grained naturally contaminated and inoculated cereals. Biologia 63(4):542–547CrossRefGoogle Scholar
  47. Pitt JI, Hocking AD (2009) Fungi and food spoilage. Springer US, DOI:  https://doi.org/10.1007/978-0-387-92207-2
  48. Rao BS, Rao VS, Ramakrishna Y, Bhat RV (1989) Rapid and specific method for screening ergosterol as an index of fungal contamination in cereal grains. Food Chem 31(1):51–56.  https://doi.org/10.1016/0308-8146(89)90150-7 CrossRefGoogle Scholar
  49. Rao MS, Stevens WF (2005) Chitin production by Lactobacillus fermentation of shrimp biowaste in a drum reactor and its chemical conversion to chitosan. J Chem Technol Biotechnol 80(9):1080–1087.  https://doi.org/10.1002/jctb.1286 CrossRefGoogle Scholar
  50. Ratnavathi CV, Sashidhar RB (2007) Inhibitory effect of phenolics extracted from sorghum genotypes on Aspergillus parasiticus (NRRL 2999) growth and aflatoxin production. J Sci Food Agric 87(6):1140–1148.  https://doi.org/10.1002/jsfa.2827 CrossRefGoogle Scholar
  51. Ravelet C, Grosset C, Alary J (2001) Quantitation of ergosterol in river sediment by liquid chromatography. J Chromatogr Sci 39(6):239–242.  https://doi.org/10.1093/chromsci/39.6.239 CrossRefGoogle Scholar
  52. Reid LM, Nicol RW, Ouellet T, Savard M, Miller JD, Young JC, Stewart DW, Schaafsma AW (1999) Interaction of Fusarium graminearum and F. moniliforme in maize ears: disease progress, fungal biomass, and mycotoxin accumulation. Phytopathology 89(11):1028–1037.  https://doi.org/10.1094/PHYTO.1999.89.11.1028 CrossRefGoogle Scholar
  53. Rudall KM, Kenchington W (1973) The chitin system. Biol Rev 48(4):597–633.  https://doi.org/10.1111/j.1469-185X.1973.tb01570.x CrossRefGoogle Scholar
  54. Sashidhar RB, Sudershan RV, Ramakrishna Y, Nahdi S, Bhat RV (1988) Enhanced fluorescence of ergosterol by iodination and determination of ergosterol by fluorodensitometry. Analyst 113(5):809–812.  https://doi.org/10.1039/an9881300809 CrossRefGoogle Scholar
  55. Saxena J, Munimbazi C, Bullerman LB (2001) Relationship of mould count, ergosterol and ochratoxin A production. Int J Food Microbiol 71(1):29–34.  https://doi.org/10.1016/S0168-1605(01)00584-0 CrossRefGoogle Scholar
  56. Schwadorf K, Muller HM (1989) Determination of ergosterol in cereals, mixed feed components, and mixed feeds by liquid chromatography. J Assoc Off Anal Chem 72(3):457–462Google Scholar
  57. Seitz LM, Mohr HE, Burroughs R, Sauer DB (1977) Ergosterol as an indicator of fungal invasion in grains. Cereal Chem 54:1207–1217Google Scholar
  58. Seitz LM, Sauer DB, Burroughs R, Mohr HE, Hubbard JD (1979) Ergosterol as a measure of fungal growth. Phytopathology 69:202–1203CrossRefGoogle Scholar
  59. Sharma PD, Fisher PJ, Webster J (1977) Critique of the chitin assay technique for estimation of fungal biomass. Trans Br Mycol Soc 69(3):479–483.  https://doi.org/10.1016/S0007-1536(77)80087-9 CrossRefGoogle Scholar
  60. Sinha R, Anima D, Dubey A (1991) Aflatoxin production in different varieties of paddy and wheat by Aspergillus parasiticus NRRL-3240. J Mycol Plant Pathol 21:259–262Google Scholar
  61. Srzednicki G, Craske J, Nimmuntavin C, Mantais LG, Wattananon S (2004) Determination of ergosterol in paddy rice using solid phase extraction. J Sci Food Agric 84(15):2041–2046.  https://doi.org/10.1002/jsfa.1909 CrossRefGoogle Scholar
  62. Tardieu D, Bailly G, Guerre P (2007) Comparison of two extraction methods for ergosterol determination in vegetable feeds. Revue de Medecine Veterinaire 158(8–9)Google Scholar
  63. Weete J, Gandhi S (1996) Biochemistry and molecular biology of fungal sterols. In: Brambl R, Marzluf G (eds) The Mycota III biochemistry and molecular biology. Springer, Berlin, pp 421–438.  https://doi.org/10.1007/978-3-662-10367-8_20 CrossRefGoogle Scholar
  64. Whipps JM, Lewis DH (1980) Methodology of a chitin assay. Trans Br Mycol Soc 74(2):416–418.  https://doi.org/10.1016/S0007-1536(80)80174-4 CrossRefGoogle Scholar
  65. Zorzete P, Castro RS, Pozzi CR, Israel ALM, Fonseca H, Yanaguibashi G, Corrêa B (2008) Relative populations and toxin production by Aspergillus flavus and Fusarium verticillioides in artificially inoculated corn at various stages of development under field conditions. J Sci Food Agric 88(1):48–55.  https://doi.org/10.1002/jsfa.2930 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of BiochemistryUniversity College of Sciences, Osmania UniversityHyderabadIndia

Personalised recommendations