The Effect of Yeast Antagonist Isolated from the Fermentation of Cocoa Beans (Theobroma cacao) from Lampung, Indonesia, on the Growth of Aspergillus flavus UNJCC F-55

Abstract

Mold is one of the microorganisms that causes damage to cocoa fruit because it produces mycotoxins as secondary metabolites that can decrease the crop yield. One type of pathogenic mold that destroys cocoa fruit is Aspergillus flavus. A. flavus produces aflatoxin B1 which is the most potent hepatotoxin and carcinogen to humans. Amongst the most effective ways to avoid alpha-toxin contamination in cocoa beans is to inhibit the growth of A. flavus in the beans. However, the use of pesticides and fungicides can increase the development of resistant strains of fungi and have a negative impact on the environment and human health. Therefore, we need other alternatives such as bio-control agents using antagonistic microorganisms, including yeasts. Thus, this study aims to characterize yeasts isolated from the fermented cocoa beans (Theobroma cacao) from Lampung, Indonesia, and evaluate their ability to inhibit the growth of Aspergillus flavus UNJCCF-55. The methods used were yeast screening, dual culture technique for antagonistic test, and morphological characterization. The 37ºC-growth screening gave 89 out of 98 yeast isolates. The subsequent antagonist test resulted in 13 isolates with the highest inhibition zone against A. flavus UNJCCF-55. These yeast isolates were macroscopically of smooth surface, butyrous texture, milky white color, irregular edges, and convex elevation. Microscopic observation showed that the isolates have oval cell shape, asexual reproduction of budding, and non-hyphae structure.


Keywords: yeast antagonist, cocoa fermentation, aspergillus flavus

References
[1] D.G. of E. Indonesia, “Cocoa Production by Province in Indonesia 2017 - 2021,” (2021).

[2] Ardhana MM, Fleet GH. The microbial ecology of cocoa bean fermentations in Indonesia. Int J Food Microbiol. 2003 Sep;86(1-2):87–99.

[3] Nigam PS, Singh A. “Cocoa and Coffee Fermentations.,” In: Encyclopedia of Food Microbiology. pp. 485–492. Elsevier (2014). https://doi.org/10.1016/B978-0-12- 384730-0.00074-4.

[4] Sukmawati D, Andrianto MH, Arman Z, Ratnaningtyas NI, Al Husna SN, El-Enshasy HA, et al. Antagonistic activity of phylloplane yeasts from Moringa oleifera Lam. leaves against Aspergillus flavus UNJCC F-30 from chicken feed. Indian Phytopathol. 2020;73(1):79–88.

[5] Bailey BA, Meinhardt LW. Cacao Diseases. Cham: Springer International Publishing; 2016. https://doi.org/10.1007/978-3-319-24789-2.

[6] Ky OK, Sunjaya SD, Retnowati IN, Ambarwati S. Stored cocoa beans quality affected by fermentation and Ephestia cautella Walker (Lepidoptera: Phycitidae) infestation. Biotropia (Bogor). 2000;0(15):58–75.

[7] Persons K, Raines JM, Rodriguez JM. Antagonistic effects of Saccharomyces cerevisiae on the growth of Aspergillus flavus and Aspergillus parasiticus at varying temperatures. Mycology. 2013;4(1):38–43.

[8] Reddy KR, Reddy CS, Muralidharan K. Potential of botanicals and biocontrol agents on growth and aflatoxin production by Aspergillus flavus infecting rice grains. Food Control. 2009;20(2):173–8.

[9] Adhikari BN, Bandyopadhyay R, Cotty PJ. Degeneration of aflatoxin gene clusters in Aspergillus flavus from Africa and North America. AMB Express. 2016 Dec;6(1):62.

[10] Droby S, Wisniewski M, Macarisin D, Wilson C. Twenty years of postharvest biocontrol research: is it time for a new paradigm? Postharvest Biol Technol. 2009;52(2):137–45.

[11] Sukmawati D, Setyaningsih A. T. Handayani K, et al., “Isolation and characterization of aflatoxigenic Aspergillus spp. from maize of livestock feed from Bogor.,” IOP Conference Series: Materials Science and Engineering. vol. 434, no. 1, p. 012105, 2018.

[12] Amorim JC, Piccoli RH, Duarte WF. “Probiotic potential of yeasts isolated from pineapple and their use in the elaboration of potentially functional fermented beverages.,” Food Research International. vol. 107, no. 2017, pp. 518–527, 2018. https://doi.org/10.1016/j.foodres.2018.02.054.

[13] Sukmawati D, Arman Z, Sondana GA, Fikriyah NN, Hasanah R, Afifah ZN, et al. Potential amylase-producing yeast isolated from indigenous fermented beverages originating from Bali, Indonesia. J Phys Conf Ser. 2019;1402(5):055021.

[14] Shetty PH, Hald B, Jespersen L. Surface binding of aflatoxin B1 by Saccharomyces cerevisiae strains with potential decontaminating abilities in indigenous fermented foods. Int J Food Microbiol. 2007 Jan;113(1):41–6.

[15] Abdel-Kareem MM, Rasmey AM, Zohri AA. The action mechanism and biocontrol potentiality of novel isolates of Saccharomyces cerevisiae against the aflatoxigenic Aspergillus flavus. Lett Appl Microbiol. 2019 Feb;68(2):104–11.

[16] Sukmawati D. Antagonism mechanism of fungal contamination animal feed using Phylloplane yeasts isolated from the Bintaro Plant (Cerbera manghas) Bekasi in Java, Indonesia. Int J Curr Microbiol Appl Sci. 2016;5(5):63–74.

[17] Kurtzman CP, Fell JW. Yeast Systematics and Phylogeny — Implications of molecular identification methods for studies in ecology. Biodiversity and Ecophysiology of Yeasts. Berlin, Heidelberg: Springer-Verlag; 2006. pp. 11–30.

[18] Agarbati A, Canonico L, Marini E, Zannini E, Ciani M, Comitini F. Potential probiotic yeasts sourced from natural environmental and spontaneous processed foods. Foods. 2020 Mar;9(3):287.

[19] Joannis-Cassan C, Tozlovanu M, Hadjeba-Medjdoub K, Ballet N, Pfohl-Leszkowicz A. Binding of zearalenone, aflatoxin B1, and ochratoxin A by yeast-based products: a method for quantification of adsorption performance. J Food Prot. 2011 Jul;74(7):1175– 85.

[20] Somai BM, Belewa V. Aqueous extracts of Tulbaghia violacea inhibit germination of Aspergillus flavus and Aspergillus parasiticus conidia. J Food Prot. 2011 Jun;74(6):1007–11.

[21] Lima JR, Gondim DM, Oliveira JT, Oliveira FS, Gonçalves LR, Viana FM. Use of killer yeast in the management of postharvest Papaya anthracnose. Postharvest Biol Technol. 2013;83:58–64.

[22] Mostafa AA, Al-Rahmah AN, Abdel-Megeed A, Sayed SR, Hatamleh AA. Antagonistic activities of some fungal strains against the toxigenic Aspergillus flavus isolate and its aflatoxins productivity. J Pure Appl Microbiol. 2013;7:169–78.

[23] Rosa-Magri MM, Tauk-Tornisielo SM, Ceccato-Antonini SR. Bioprospection of yeasts as biocontrol agents against Phytopathogenic molds. Braz Arch Biol Technol. 2011;54(1):1–5.