Journal of Food Science and Technology

, Volume 56, Issue 4, pp 2195–2204 | Cite as

Evaluation of different hurdles on Penicillium crustosum growth in sponge cakes by means of a specific real time PCR

  • Mariana GondaEmail author
  • Caterina Rufo
  • Gianna Cecchetto
  • Silvana Vero
Original Article


Limited shelf life of bakery products, caused by microbial deterioration, is a concern for industries due to economic losses. Fungal spoilage of sponge cakes industrially produced in Montevideo was caused mainly by Penicillium species, in particular by Penicillium crustosum. The combination of different hurdles was studied to inhibit P. crustosum growth in sponge cakes. A full factorial design was performed to study the effect of the concentration of potassium sorbate, pH, packaging atmosphere and storage time. The results showed that packaging atmosphere and storage time were the significant factors in the ranges tested. No growth was detected in cakes stored in modified atmosphere packaging (MAP) (N2:CO2 50:50) at room temperature (25 °C) for 15 days. The effect of MAP on P. crustosum growth in cakes at room temperature was compared with the effect of air-packaging and storage at low temperature (4 °C) for 30 days. P. crustosum growth was not detected in cakes packaged in MAP, whereas it was detected after 20 days in cakes packaged in air and stored at 4 °C. This growth was quantified by a specific real time PCR developed in this work. Specific primers were designed using the sequence of β-tubulin gene of P. crustosum as a target and PCR conditions were adjusted to ensure specificity. PCR efficiency was 107%, with a detection limit of 0.0014 ng of DNA. The qPCR method presented here, resulted specific and sensitive enough to detect the growth of P. crustosum even before biodeterioration signs were visible.


Bakery products Penicillium crustosum Hurdle technology Real time PCR 



Potassium sorbate


Modified atmosphere packaging


Full factorial design



We thank the Comisión Sectorial de Investigación Científica (CSIC, Uruguay) and the Agencia Nacional de Investigación e Innovación (ANII, Uruguay) for financial aid. We also thank OLASO (Uruguay) for providing cakes samples.


  1. Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefGoogle Scholar
  2. Anderson MJ, Whitcomb PJ (2016) DOE simplified: practical tools for effective experimentation. CRC Press, LondonCrossRefGoogle Scholar
  3. Arquiza JMRA, Hunter J (2014) The use of real-time PCR to study Penicillium chrysogenum growth kinetics on solid food at different water activities. Int J Food Microbiol 187:50–56CrossRefGoogle Scholar
  4. Branquinho MR, Ferreira RTB, Cardarelli-Leite P (2012) Use of real-time PCR to evaluate two DNA extraction methods from food. Food Sci Technol 32:112–118CrossRefGoogle Scholar
  5. Carter AT, Peck MW (2015) Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II. Res Microbiol 166:303–317CrossRefGoogle Scholar
  6. CRL-GMFF (2008) Report on the validation of a DNA extraction method for maize seeds and grains. Community Reference Laboratory for GM Food and Feed. European Union Reference Laboratory for GM Food and Feed Publishing. Accessed 10 Oct 2018
  7. Decreto Nº315/994(1994) Reglamento Bromatológico Nacional, 3rd edn. IMPO Publisher. Accessed 10 Oct 2018
  8. Dodds KL (1989) Combined effect of water activity and pH on inhibition of toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes. Appl Environ Microbiol 55:656–660Google Scholar
  9. Dorak MT (2006) Real-time PCR. Taylor & Francis Group, UKGoogle Scholar
  10. Forsythe SJ, Hayes PR (2000) Food spoilage. In: Forsythe SJ, Hayes PR (eds) Food hygiene, microbiology and HACCP, 3rd edn. Springer, New York, pp 86–149CrossRefGoogle Scholar
  11. Gonda M (2015) New technologies, alternative to preservatives, to inhibit biodeterioration of bakery products during packaging and storage at room temperature (Tecnologías alternativas para inhibir el biodeterioro en productos panificados envasados y conservados a temperatura ambiente. Master in Biotechnology. Facultad de Ciencias, Universidad de la República, Uruguay. Colibrí UdelaR publishing. Accessed 10 October 2018
  12. Guynot ME, Ramos AJ, Sala D et al (2002) Combined effects of weak acid preservatives, pH and water activity on growth of Eurotium species on a sponge cake. Int J Food Microbiol 76:39–46CrossRefGoogle Scholar
  13. Guynot ME, Marín S, Sanchis V, Ramos AJ (2003) Modified atmosphere packaging for prevention of mold spoilage of bakery products with different pH and water activity levels. J Food Prot 66:1864–1872CrossRefGoogle Scholar
  14. Guynot ME, Marin S, Sanchis V, Ramos AJ (2004) An attempt to minimize potassium sorbate concentration in sponge cakes by modified atmosphere packaging combination to prevent fungal spoilage. Food Microbiol 21:449–457CrossRefGoogle Scholar
  15. Guynot ME, Marín S, Sanchis V, Ramos AJ (2005) An attempt to optimize potassium sorbate use to preserve low pH (4.5–5.5) intermediate moisture bakery products by modelling Eurotium spp., Aspergillus spp. and Penicillium corylophilum growth. Int J Food Microbiol 101:169–177CrossRefGoogle Scholar
  16. Hasan S, Abdolgader R (2012) Study of weak acid preservatives and modified atmosphere packaging (MAP) on mold growth in modal agar system. Food Nutr Sci 3:802–809Google Scholar
  17. Huang Y, Wilson M, Chapman B, Hocking AD (2010) Evaluation of the efficacy of four weak acids as antifungal preservatives in low-acid intermediate moisture model food systems. Food Microbiol 27:33–36CrossRefGoogle Scholar
  18. Janjarasskul T, Tananuwong K, Kongpensook V et al (2016) Shelf life extension of sponge cake by active packaging as an alternative to direct addition of chemical preservatives. LWT Food Sci Technol 72:166–174CrossRefGoogle Scholar
  19. Marín S, Guynot ME, Neira P et al (2002) Risk assessment of the use of sub-optimal levels of weak-acid preservatives in the control of mould growth on bakery products. Int J Food Microbiol 79:203–211CrossRefGoogle Scholar
  20. Mensah-Attipoe J, Reponen T, Veijalainen A-M et al (2016) Comparison of methods for assessing temporal variation of growth of fungi on building materials. Microbiology 162:1895–1903CrossRefGoogle Scholar
  21. Mohammadzadeh-Aghdash H, Sohrabi Y, Mohammadi A et al (2018) Safety assessment of sodium acetate, sodium diacetate and potassium sorbate food additives. Food Chem 257:211–215CrossRefGoogle Scholar
  22. Pasukamonset P, Pumalee T, Sanguansuk N et al (2018) Physicochemical, antioxidant and sensory characteristics of sponge cakes fortified with Clitoria ternatea extract. J Food Sci Technol 55:2881–2889CrossRefGoogle Scholar
  23. Pitt JI, Hocking AD (2009) Spoilage of stored, processed and preserved foods. In: Pitt JI, Hocking AD (eds) Fungi and food spoilage, 3rd edn. Springer, New York, pp 401–421CrossRefGoogle Scholar
  24. Qiu L, Zhang M, Tang J et al (2019) Innovative technologies for producing and preserving intermediate moisture foods: a review. Food Res Int 116:90–102CrossRefGoogle Scholar
  25. Saranraj P, Geetha M (2012) Microbial spoilage of bakery products and its control by preservatives. Int J Pharm Biol Arch 3:38–48Google Scholar
  26. Smith JP, Daifas DP, El-Khoury W et al (2004) Shelf life and safety concerns of bakery products—a review. Crit Rev Food Sci Nutr 44:19–55CrossRefGoogle Scholar
  27. Stratford M, Plumridge A, Nebe-von-Caron G, Archer DB (2009) Inhibition of spoilage mould conidia by acetic acid and sorbic acid involves different modes of action, requiring modification of the classical weak-acid theory. Int J Food Microbiol 136:37–43CrossRefGoogle Scholar
  28. Suhr KI, Nielsen PV (2004) Effect of weak acid preservatives on growth of bakery product spoilage fungi at different water activities and pH values. Int J Food Microbiol 95:67–78CrossRefGoogle Scholar
  29. Suppakul P, Miltz J, Sonneveld K (2003) Bigger SW (2003) Active packaging technologies with an emphasis on antimicrobial packaging and its applications. J Food Sci 68:408–420CrossRefGoogle Scholar
  30. Tancinová D, Barboráková Z, Masková Z et al (2012) The occurrence of micromycetes in the bread samples and their potential ability produce mycotoxins. J Microbiol Biotechnol Food Sci 1:813–818Google Scholar
  31. Taniwaki MH, Pitt JI, Hocking AD, Fleet GH (2006) Comparison of hyphal length, ergosterol, mycelium dry weight, and colony diameter for quantifying growth of fungi from foods. In: Hocking AD, Pitt JI, Samson RA, Thrane U (eds) Advances in Food Mycology. Springer, New York, pp 49–67CrossRefGoogle Scholar
  32. Tannous J, Atoui A, El Khoury A et al (2015) Development of a real-time PCR assay for Penicillium expansum quantification and patulin estimation in apples. Food Microbiol 50:28–37CrossRefGoogle Scholar
  33. Valle Garcia M, Sonnenstrahl Bregão A, Parussolo G et al (2019) Incidence of spoilage fungi in the air of bakeries with different hygienic status. Int J Food Microbiol 290:254–261CrossRefGoogle Scholar
  34. van Beilen JWA, Teixeira de Mattos MJ, Hellingwerf KJ, Brul S (2014) Distinct effects of sorbic acid and acetic acid on the electrophysiology and metabolism of Bacillus subtilis. Appl Environ Microbiol 80:5918–5926CrossRefGoogle Scholar
  35. Ye J, Coulouris G, Zaretskaya I et al (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134–144CrossRefGoogle Scholar

Copyright information

© Association of Food Scientists & Technologists (India) 2019

Authors and Affiliations

  • Mariana Gonda
    • 1
    Email author
  • Caterina Rufo
    • 2
  • Gianna Cecchetto
    • 1
    • 3
  • Silvana Vero
    • 1
  1. 1.Área Microbiología, Departamento de Biociencias, Facultad de QuímicaUniversidad de la RepúblicaMontevideoUruguay
  2. 2.Alimentos y Nutrición, Instituto Polo Tecnológico, Facultad de QuímicaUniversidad de la RepúblicaPandoUruguay
  3. 3.Microbiología, Facultad de CienciasUniversidad de la RepúblicaMontevideoUruguay

Personalised recommendations