Microbial and Mycotoxin Contamination in Packaged and Unpackaged Smokeless Tobacco and Non-tobacco Chewing Products in India

A bstract Objectives: Smokeless tobacco (SLT) and non-tobacco chewable products such as Gutka, Khaini, and Pan Masalas are widely consumed in India but are rarely assessed for microbial safety, despite the presence of toxin-producing microorganisms that may increase carcinogenic risk. The study was aimed to investigate bacterial and fungal contamination and quantify aflatoxin B 1 (AFB 1 ) levels in commercially available chewable products. Methods: Cross-sectional laboratory-based study analyzing SLT and non-tobacco products collected from North, South, West, and East India. Fourteen samples, including Gutka, Khaini, Pan Masalas, Loose Tobacco, and Areca Nut mixes, were assessed. Bacillus spp. were identified using HiCrome™ Bacillus Agar and API 50CHB/E profiling. Fungal contaminants were characterized by colony morphology and lactophenol cotton blue microscopy. AFB 1 levels were measured in duplicate using competitive ELISA. Median and interquartile ranges (IQR) for AFB 1 ; Kruskal–Wallis H test with Dunn’s post hoc test and Bonferroni correction ( P < 0.05). Results: Bacillus licheniformis was the most frequently isolated bacterium, followed by Bacillus subtilis/amyloliquefaciens and Bacillus pumilus . Nitrate-reducing activity suggested potential nitrosamine formation. Fungal contaminants included Aspergillus flavus , A. niger , Penicillium spp ., Mucor spp ., and Rhizopus spp ., with colony counts ranging from 1.2 × 10 4 to 5.5 × 10 4 colony-forming units (CFU)/g. AFB 1 was detected in 13 of 14 samples (0.085–0.459 ng/mL), with pan masala with tobacco (0.456 ng/mL, IQR 0.455–0.458) and gutka (0.400 ng/mL, IQR 0.341–0.450) showing the highest levels, whereas Khaini exhibited the lowest contamination (0.088 ng/mL, IQR 0.087–0.090). Non-tobacco pan masalas also showed notable contamination. Conclusion: Both SLT and non-tobacco chewable products harbor microbial and aflatoxin hazards, challenging assumptions about the safety of non-tobacco products. These findings highlight the need for product-specific microbial and mycotoxin regulations under FSSAI regulations, along with market surveillance and public awareness initiatives.