Isolation, Morphological, Biochemical, and Molecular Characterisation of Food Spoilage Bacteria from Selected Categories of Spoiled Indian Food Samples

Microbial food spoilage causes enormous economic losses and public health burdens worldwide, with foodborne illnesses affecting an estimated 600 million people annually. India is particularly vulnerable to food spoilage due to warm climatic conditions, inadequate cold chain infrastructure, and dependence on fresh, minimally processed foods. The identification of food spoilage bacteria from representative Indian food categories is an essential first step toward developing effective natural bio- preservative strategies. Ten categories of spoiled Indian food samples were collected aseptically from retail markets in Chh. Sambhajinagar, Maharashtra. Bacterial isolation was performed by enrichment in Nutrient Broth followed by plating on Nutrient Agar, MacConkey Agar, and Mannitol Salt Agar. Isolates were characterised by colony morphology, Gram staining, cell morphology, IMViC biochemical tests (Indole, Methyl Red, Voges- Proskauer, Citrate utilisation), and Catalase test. Definitive species identification was performed by 16S rRNA gene sequencing using universal primers 27F and 1492R, followed by BLASTn analysis against the NCBI 16S rRNA database and neighbour- joining phylogenetic analysis in MEGA v11.0. Ten morphologically distinct bacterial isolates were recovered from ten spoiled food categories. Colony morphology revealed six Gram-positive and four Gram-negative isolates. IMViC biochemical characterisation differentiated enteric Gram-negative organisms and confirmed the coccal Gram-positive isolates as Staphylococcus-like. Molecular identification by 16S rRNA gene sequencing achieved species-level assignment (≥99% identity) for eight isolates and genus-level assignment for two. The identified organisms included Bacillus cereus (spoiled Peas), Enterobacter cloacae (Carrot), Staphylococcus epidermidis (Spinach), Bacillus subtilis (Papaya), Pseudomonas fluorescens (Bottle Gourd), Staphylococcus aureus (Mayonnaise), Klebsiella pneumoniae (Schezwan Sauce), Salmonella enterica (Chilli), Enterococcus faecalis (Ginger-Garlic Paste), and Serratia marcescens (Tomato). The isolation of major food safety pathogens including Bacillus cereus, Staphylococcus aureus, Klebsiella pneumoniae, and Salmonella enterica from commonly consumed Indian food products underscores the critical need for effective, naturally derived bio-preservatives. The characterised isolates were subsequently used as target organisms for evaluating the antimicrobial activity of food-grade plant extracts, establishing an ecologically valid experimental system for bio-preservative research.

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