Souvik Roy¹*, Dyutishmita Bhattacharjee¹, Heeya Gupta¹, Lopamudra Choudhury² and Subham Sarkar¹*

¹Post-Graduate Department of Biotechnology, St. Xavier’s College (Autonomous),
30, Mother Teresa Sarani, Kolkata-700016, West Bengal, India
²Department of Microbiology, Sarsuna College (under University of Calcutta), Ho Chi Minh Sarani, Sarsuna Upanagari, Kolkata - 700061, West Bengal, India

*Corresponding Author: Souvik Roy, Subham Sarkar (Post-Graduate Department of Biotechnology, St. Xavier’s College (Autonomous), 30, Mother Teresa Sarani, Kolkata-700016, West Bengal, India).

Received: March 09, 2026; Published: April 16, 2026

Abstract

A study questioned the assumption that cocoa and low water activity limit contamination in Indian chocolate bars (C1, C2, C3). Each chocolate bar was tested as intact refrigerated (C1 Intact ref, C2 Intact ref, C3 Intact ref), half-eaten refrigerated (C1Href, C2Href, C3Href), or half-eaten at room temperature (C1HRT, C2HRT, C3HRT). After five days, isolation and characterization of notorious food-borne bacterial and fungal pathogens were carried out on them. Tests showed the presence of colonies from C3HRT on Eosin Methylene Blue (EMB) agar plates, typical of Enterobacter species, a common food contaminant. Preliminary identification of species of Staphylococcus was made from greyish black to completely black colonies with prominent halos on Baird Parker Agar (BPA) plates; Pseudomonas was made from green colonies on Hektoen Enteric Agar (HEA) plates; and Citrobacter from bright-orange (salmon) colonies on HEA plates. Growth in glucose-free nutrient broth (GFNB) tubes, followed by staining to reveal endospores, indicated the presence of Bacillus species. Potentially harmful Enterobacter, Pseudomonas, Alcaligenes, Bacillus, and Staphylococcus (like S. aureus and S. epidermidis) were finally confirmed by characteristic biochemical tests. Besides, highly menacing food-borne fungal pathogens, including species of Rhizoctonia, Aspergillus and Alternaria, were also isolated on Yeast Extract Peptone Dextrose Agar (YPDA) plates and presumptively identified microscopically and confirmed using ITS based molecular method sequencing. Antibiotic susceptibility tests (ASTs) of the confirmed bacterial pathogens recorded the highest (80%) resistance against amoxicillin (AMX). Thus, our key findings of high microbial activity and the presence of some of the deadliest food-borne pathogens, including some drug-resistant ones, challenge the long-nurtured notion stated earlier and establish the fact that the overall microbiological quality of the chocolates depends much on their mode of storage.

Keywords: Antibiotic Susceptibility Test; Chocolates; Food-Borne Bacterial And Fungal Pathogens; MPN Index; Total Heterotrophic Count

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Citation

Citation: Souvik Roy., et al. “Microbial Characterization of Chocolates of Three Popular Commercial Brands: A Complete Profiling of Notorious Bacterial and Fungal Contaminants". Acta Scientific Microbiology 9.5 (2026): 03-19.

Copyright

Copyright: © 2026 Souvik Roy., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.