Use of Chitosan Nanoparticles as Bio-Preservatives in the Inhibition of Spoilage Microorganisms in Egusi and Okra Soups

Strain Bootstrap Value (%) Phylogenetic Relationship EG1 Bacillus cereus Bacillus cereus ATCC 14579 86 Clustered with reference Bacillus cereus strains EG2 Staphylococcus aureus Staphylococcus aureus NCTC 8325 82 Grouped with reference S. aureus isolates EG3 Escherichia coli Escherichia coli K-12 MG1655 88 Clustered with E. coli reference strains EG4 Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1 84 Grouped with Pseudomonas reference isolates EG5 Salmonella enterica Salmonella enterica LT2 81 Clustered with Salmonella reference strains EGF1 Aspergillus niger Aspergillus niger CBS 513.88 85 Grouped with reference A. niger isolates EGF2 Penicillium chrysogenum Penicillium chrysogenum NRRL 1951 80 Clustered with Penicillium reference strains EGF3 Candida tropicalis Candida tropicalis MYA-3404 79 Grouped with Candida reference isolates P-ISSN 2695-222X OK1 Bacillus subtilis Bacillus subtilis 168 87 Clustered with reference B. subtilis strains OK2 Pseudomonas fluorescens Pseudomonas fluorescens Pf-5 83 Grouped with Pseudomonas fluorescens isolates OK3 Klebsiella pneumoniae Klebsiella pneumoniae MGH 78578 81 Clustered with Klebsiella reference strains OKF1 Rhizopus stolonifer Rhizopus stolonifer NRRL 1477 84 Grouped with Rhizopus reference isolates OKF2 Mucor indicus Mucor indicus CBS 226.29 82 Clustered with Mucor reference strains The molecular characterization and phylogenetic analysis results confirmed the accurate identification of the spoilage microorganisms isolated from egusi and okra soups. The DNA quality values ranging from 1.78–1.91 indicated good-quality genomic DNA suitable for molecular analysis and sequencing. The successful PCR amplification of the bacterial 16S rRNA genes and fungal ITS regions with expected band sizes of approximately 1500 bp and 600–800 bp respectively demonstrated effective amplification of the target genetic regions. BLAST analysis showing 97–99% similarity confirmed the identities of the isolates as Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Aspergillus niger, Penicillium chrysogenum, Candida tropicalis, Bacillus subtilis, Pseudomonas fluorescens, Klebsiella pneumoniae, Rhizopus stolonifer, and Mucor indicus. The phylogenetic relationships with bootstrap values greater than 70% further established strong evolutionary relationships between the isolates and known reference strains in public databases. Table 4.2: Characterization of Synthesized Chitosan Nanoparticles Characterization Parameter Observation / Result Interpretation Appearance of Nanoparticles Milky white colloidal suspension Successful nanoparticle formation Particle Size (nm) 85.4 ± 2.1 nm Nanoparticles were within nano-size range Polydispersity Index 0.312 ± 0.01 Moderate uniformity and stability of particles Zeta Potential (mV) +32.6 ± 1.4 mV Good nanoparticle stability and positive surface charge FTIR Peak (O–H Stretching) 3432 cm−1 Presence of hydroxyl functional group FTIR Peak (N–H Bending) 1635 cm−1 Presence of amino groups characteristic of chitosan FTIR Peak (C–O Stretching) 1082 cm−1 Confirmation of polysaccharide backbone SEM Morphology Spherical and smooth particles Uniform nanoparticle morphology P-ISSN 2695-222X Aggregation Status Minimal aggregation observed Stable nanoparticle dispersion The characterization results confirmed the successful synthesis and stability of the chitosan nanoparticles produced through ionic gelation method. The milky white colloidal appearance observed indicated successful nanoparticle formation resulting from ionic cross-linking between chitosan and sodium tripolyphosphate. The particle size of 85.4 ± 2.1 nm showed that the synthesized particles were within the nano-size range, which is important for enhanced antimicrobial activity, increased surface area, and improved interaction with microbial cells. The polydispersity index value of 0.312 ± 0.01 indicated moderate uniformity and acceptable particle distribution, suggesting stable nanoparticle formation. The positive zeta potential value of +32.6 ± 1.4 mV implied strong surface charge and good nanoparticle stability, which enhances electrostatic interaction between the nanoparticles and negatively charged microbial cell membranes. The FTIR peaks observed at 3432 cm−1, 1635 cm−1, and 1082 cm−1 confirmed the presence of hydroxyl, amino, and polysaccharide functional groups characteristic of chitosan structure. SEM analysis further revealed spherical and smooth nanoparticle morphology with minimal aggregation, indicating stable nanoparticle dispersion and uniform particle formation. Table 4.3: Inhibitory Effects of Chitosan Nanoparticles on Spoilage Microorganisms Isolated from Egusi and Okra Soups Microorganisms Isolated Control (Distilled Water) (mm) 0.5% Chitosan NP (mm) 1.0% Chitosan NP (mm) 1.5% Chitosan NP (mm) 2.0% Chitosan NP (mm) Positive Control (Antibiotic) (mm) Bacillus cereus 0.0 ± 0.0 8.2 ± 0.2 12.4 ± 0.3 17.6 ± 0.4 22.1 ± 0.5 25.4 ± 0.5 Staphylococcus aureus 0.0 ± 0.0 7.5 ± 0.1 11.8 ± 0.2 16.9 ± 0.3 21.4 ± 0.4 24.8 ± 0.4 Escherichia coli 0.0 ± 0.0 6.8 ± 0.2 10.5 ± 0.3 15.3 ± 0.2 19.8 ± 0.3 23.6 ± 0.4 Pseudomonas aeruginosa 0.0 ± 0.0 5.9 ± 0.1 9.6 ± 0.2 13.8 ± 0.3 18.2 ± 0.4 22.5 ± 0.3 Salmonella enterica 0.0 ± 0.0 6.4 ± 0.2 10.9 ± 0.3 15.7 ± 0.4 20.0 ± 0.5 23.9 ± 0.4 Klebsiella pneumoniae 0.0 ± 0.0 6.1 ± 0.1 10.2 ± 0.2 14.9 ± 0.3 19.1 ± 0.4 22.8 ± 0.4 Aspergillus niger 0.0 ± 0.0 7.1 ± 0.1 11.5 ± 0.2 16.1 ± 0.3 20.7 ± 0.4 24.1 ± 0.5 Penicillium chrysogenum 0.0 ± 0.0 6.9 ± 0.2 11.1 ± 0.2 15.8 ± 0.3 20.2 ± 0.5 23.7 ± 0.4 Candida tropicalis 0.0 ± 0.0 6.7 ± 0.2 10.8 ± 0.2 15.5 ± 0.3 19.5 ± 0.4 23.3 ± 0.4 Rhizopus stolonifer 0.0 ± 0.0 5.8 ± 0.1 9.9 ± 0.2 14.3 ± 0.3 18.6 ± 0.4 22.4 ± 0.3 Mucor indicus 0.0 ± 0.0 5.6 ± 0.1 9.5 ± 0.2 13.9 ± 0.3 18.1 ± 0.4 22.0 ± 0.4 The results demonstrated that chitosan nanoparticles possessed strong concentration-dependent antimicrobial activity against the spoilage microorganisms isolated from egusi and okra soups. The absence of inhibition zones in the negative control confirmed that sterile distilled water had no antimicrobial effect, while the positive control produced the highest inhibition zones against all P-ISSN 2695-222X tested organisms. The inhibitory activity of the chitosan nanoparticles increased progressively from 0.5% to 2.0% concentration, indicating that higher nanoparticle concentrations enhanced antimicrobial effectiveness. The results further showed that Bacillus cereus and Staphylococcus aureus were the most susceptible bacterial isolates, while Pseudomonas aeruginosa and Mucor indicus exhibited relatively lower susceptibility to the nanoparticles. The fungal isolates including Aspergillus niger, Penicillium chrysogenum, Candida tropicalis, Rhizopus stolonifer, and Mucor indicus were also effectively inhibited by the nanoparticles, demonstrating broad-spectrum antifungal activity. The stronger inhibitory effects observed at 2.0% concentration suggested improved interaction between the positively charged chitosan nanoparticles and negatively charged microbial cell membranes, resulting in membrane disruption and inhibition of microbial growth. Table 4.5: Effects of Chitosan Nanoparticles on Shelf Stability, Physicochemical Properties, and Sensory Quality of Egusi and Okra Soups during Storage Treatment Storage Period Total Viable Count (cfu/ml) Fungal Count (cfu/ml) pH Titrata ble Acidity (%) Viscosity (cP) Sensory Acceptabi lity Score Shelf Stability Control 21 8.6 × 106 ± 0.18 3.2 × 103 ± 0.04 5.92 ± 0.03 0.28 ± 0.01 1420 ± 12 5.1 ± 0.2 5 days 0.5% Chitosan NP 21 6.4 × 106 ± 0.15 2.1 × 103 ± 0.03 5.74 ± 0.02 0.34 ± 0.01 1565 ± 14 6.4 ± 0.1 9 days 1.0% Chitosan NP 21 4.8 × 106 ± 0.12 1.4 × 103 ± 0.02 5.61 ± 0.02 0.41 ± 0.01 1710 ± 15 7.2 ± 0.2 13 days 1.5% Chitosan NP 21 2.9 × 106 ± 0.10 8.0 × 102 ± 0.01 5.48 ± 0.01 0.47 ± 0.02 1885 ± 16 8.1 ± 0.1 17 days 2.0% Chitosan NP 21 1.5 × 106 ± 0.08 3.0 × 102 ± 0.01 5.36 ± 0.01 0.53 ± 0.02 2050 ± 18 8.9 ± 0.2 21 days The results showed that chitosan nanoparticles significantly improved the shelf stability, physicochemical properties, and sensory quality of egusi and okra soups during refrigerated storage. The untreated control samples recorded the highest total viable count and fungal count, indicating rapid microbial spoilage and reduced storage stability. In contrast, soups treated with chitosan nanoparticles showed progressive reductions in microbial load as nanoparticle concentration increased. The 2.0% chitosan nanoparticle treatment recorded the lowest total viable count and fungal count, demonstrating strong antimicrobial activity against spoilage microorganisms during storage. The physicochemical properties of the soups were also positively influenced by chitosan nanoparticle treatment. The gradual decrease in pH and corresponding increase in titratable acidity observed in treated samples suggested controlled microbial and biochemical activities during storage. Increased viscosity values recorded at higher nanoparticle concentrations indicated improved texture stability and consistency of the soups. Furthermore, P-ISSN 2695-222X sensory evaluation results showed that soups treated with chitosan nanoparticles maintained better appearance, aroma, taste, texture, and overall acceptability compared to the untreated control samples throughout storage. The highest sensory acceptability score was recorded in soups treated with 2.0% chitosan nanoparticles. The shelf stability results further demonstrated that increasing concentrations of chitosan nanoparticles significantly prolonged the storage life of the soups from 5 days in the control sample to 21 days at 2.0% nanoparticle concentration. Discussion The results obtained in this study demonstrated that egusi and okra soups contained several bacterial and fungal spoilage microorganisms capable of reducing food quality and posing serious public health risks. The isolation of organisms such as Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella sp., Klebsiella pneumoniae, Aspergillus niger, Penicillium chrysogenum, Candida tropicalis, Rhizopus stolonifer, and Mucor indicus indicated contamination associated with poor handling practices, environmental exposure, and improper storage conditions. Similar observations were reported by Ray and Bhunia [11], who stated that traditional soups and ready-to-eat foods commonly harbour spoilage and pathogenic microorganisms due to their high nutrient and moisture contents. Adams and Moss [12] also reported that Pseudomonas, Bacillus, and fungal species are among the major spoilage organisms associated with perishable foods. Jay et al. [13] further stated that foods with high moisture content provide favourable environments for rapid microbial growth and spoilage. The occurrence of Escherichia coli, Salmonella sp., and Staphylococcus aureus further suggested possible faecal contamination and poor hygienic handling during preparation and storage of the soups. Frazier and Westhoff [14] reported that contamination by enteric bacteria in foods is commonly associated with inadequate sanitation and poor food handling practices. The fungal isolates identified in this study further suggested susceptibility of the soups to fungal spoilage and possible mycotoxin contamination during storage. Pitt and Hocking [15] similarly reported that Aspergillus and Penicillium species are major fungal contaminants responsible for spoilage and toxin production in food products. The molecular characterization and phylogenetic analysis carried out in this study confirmed the accurate identification of the spoilage microorganisms associated with the soups. The good DNA quality values and successful PCR amplification of the bacterial 16S rRNA genes and fungal ITS regions demonstrated efficient molecular analysis of the isolates. Similar findings were reported by Madigan et al. [16], who stated that molecular techniques such as PCR amplification and sequencing provide accurate identification of foodborne microorganisms. Janda and Abbott [17] also reported that phylogenetic analysis improves microbial identification and understanding of evolutionary relationships among food spoilage organisms. The high BLAST similarity values and strong phylogenetic relationships obtained in this study confirmed that the isolates were closely related to known reference strains in public databases. Woese et al. [18] further stated that 16S rRNA sequencing remains one of the most reliable molecular tools for bacterial identification and evolutionary analysis. The molecular identification of fungal species such as Aspergillus niger, Penicillium chrysogenum, and Candida tropicalis further suggested possible fungal spoilage and mycotoxin risks associated with prolonged soup storage. Samson et al. [19] reported that molecular characterization of fungi is essential for accurate identification of toxin-producing food contaminants. The results obtained in this study demonstrated that egusi and okra soups contained several bacterial and fungal spoilage microorganisms capable of reducing food quality and posing serious public health risks. The isolation of organisms such as Bacillus cereus, Staphylococcus aureus, P-ISSN 2695-222X Escherichia coli, Pseudomonas aeruginosa, Salmonella sp., Klebsiella pneumoniae, Aspergillus niger, Penicillium chrysogenum, Candida tropicalis, Rhizopus stolonifer, and Mucor indicus indicated contamination associated with poor handling practices, environmental exposure, and improper storage conditions. Similar observations were reported by Ray and Bhunia [11], who stated that traditional soups and ready-to-eat foods commonly harbour spoilage and pathogenic microorganisms due to their high nutrient and moisture contents. Adams and Moss [12] also reported that Pseudomonas, Bacillus, and fungal species are among the major spoilage organisms associated with perishable foods. Jay et al. [13] further stated that foods with high moisture content provide favourable environments for rapid microbial growth and spoilage. The occurrence of Escherichia coli, Salmonella sp., and Staphylococcus aureus further suggested possible faecal contamination and poor hygienic handling during preparation and storage of the soups. Frazier and Westhoff [14] reported that contamination by enteric bacteria in foods is commonly associated with inadequate sanitation and poor food handling practices. The fungal isolates identified in this study further suggested susceptibility of the soups to fungal spoilage and possible mycotoxin contamination during storage. Pitt and Hocking [15] similarly reported that Aspergillus and Penicillium species are major fungal contaminants responsible for spoilage and toxin production in food products. The molecular characterization and phylogenetic analysis carried out in this study confirmed the accurate identification of the spoilage microorganisms associated with the soups. The good DNA quality values and successful PCR amplification of the bacterial 16S rRNA genes and fungal ITS regions demonstrated efficient molecular analysis of the isolates. Similar findings were reported by Madigan et al. [16], who stated that molecular techniques such as PCR amplification and sequencing provide accurate identification of foodborne microorganisms. Janda and Abbott [17] also reported that phylogenetic analysis improves microbial identification and understanding of evolutionary relationships among food spoilage organisms. The high BLAST similarity values and strong phylogenetic relationships obtained in this study confirmed that the isolates were closely related to known reference strains in public databases. Woese et al. [18] further stated that 16S rRNA sequencing remains one of the most reliable molecular tools for bacterial identification and evolutionary analysis. The molecular identification of fungal species such as Aspergillus niger, Penicillium chrysogenum, and Candida tropicalis further suggested possible fungal spoilage and mycotoxin risks associated with prolonged soup storage. Samson et al. [19] reported that molecular characterization of fungi is essential for accurate identification of toxin-producing food contaminants. The results further demonstrated that chitosan nanoparticles significantly improved the shelf stability, physicochemical properties, and sensory quality of egusi and okra soups during refrigerated storage. The progressive reduction in total viable count and fungal count observed with increasing nanoparticle concentration indicated effective inhibition of spoilage microorganisms during storage. Similar observations were reported by Elsabee and Abdou [20], who stated that chitosan nanoparticles prolong shelf life of foods by reducing microbial proliferation and delaying spoilage. Dutta et al. [21] also reported that nano-chitosan coatings improve microbial safety and reduce deterioration in perishable food products. The gradual reduction in pH and increase in titratable acidity observed in treated soups suggested controlled biochemical activities during storage. The increased viscosity values obtained at higher nanoparticle concentrations further indicated improved texture stability and consistency of the soups. No et al. [22] reported that chitosan-based preservatives improve water-holding capacity and maintain textural quality of food products during storage. Sensory evaluation results showed that soups treated with higher concentrations of chitosan nanoparticles maintained better appearance, aroma, taste, texture, and overall acceptability throughout storage compared to P-ISSN 2695-222X untreated samples. Ojagh et al. [23] similarly reported that chitosan nanoparticles preserve sensory quality and consumer acceptability of stored foods. The extended shelf stability from 5 days in the control sample to 21 days at 2.0% nanoparticle concentration confirmed the effectiveness of chitosan nanoparticles as natural bio-preservatives capable of improving microbial safety, maintaining food quality, reducing spoilage, and prolonging storage life of traditional soups. Conclusion The findings of this study demonstrated that egusi and okra soups are highly susceptible to contamination by diverse bacterial and fungal spoilage microorganisms including Bacillus cereus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella pneumoniae, Aspergillus niger, Penicillium chrysogenum, Candida tropicalis, Rhizopus stolonifer, and Mucor indicus. Molecular characterization and phylogenetic analysis confirmed the accurate identification and evolutionary relationships of the isolates using PCR amplification, sequencing, and bioinformatics tools. The study further established that chitosan nanoparticles synthesized through ionic gelation method possessed desirable physicochemical characteristics including nano-sized particles, positive surface charge, structural stability, and uniform morphology suitable for antimicrobial applications. The inhibitory studies revealed that chitosan nanoparticles exhibited strong concentration-dependent antibacterial and antifungal activities against the spoilage microorganisms isolated from the soups. Higher concentrations of the nanoparticles produced larger zones of inhibition and greater microbial suppression. Furthermore, application of chitosan nanoparticles significantly improved the shelf stability, physicochemical properties, and sensory quality of egusi and okra soups during refrigerated storage. The 2.0% chitosan nanoparticle treatment produced the best preservative effect by reducing microbial load, maintaining desirable pH and viscosity, improving sensory acceptability, and extending shelf life from 5 days in untreated samples to 21 days. The study therefore demonstrated that chitosan nanoparticles are effective natural bio-preservatives capable of improving microbial safety, reducing spoilage, maintaining quality, and prolonging the storage life of traditional soups. Recommendations Based on the findings, the following were recommended: i. Chitosan nanoparticles should be adopted as natural bio-preservatives in the preservation of egusi and okra soups due to their strong antimicrobial activity and ability to extend shelf stability. ii. Food processors and local soup vendors should maintain proper hygienic practices during preparation, handling, and storage of soups in order to minimize contamination by spoilage and pathogenic microorganisms. iii. Further studies should be carried out on the toxicity, safety evaluation, and large-scale industrial application of chitosan nanoparticles in different traditional food products. iv. Refrigerated storage combined with appropriate concentrations of chitosan nanoparticles should be encouraged for preservation of perishable soups and other ready-to-eat foods to reduce spoilage and food wastage. P-ISSN 2695-222X References [1] M. Z. Elsabee and E. S. Abdou, “Chitosan based edible films and coatings: a review,” Mater. Sci. Eng. C Mater. Biol. Appl., vol. 33, no. 4, pp. 1819–1841, 2013. [2] J. Ray and A. K. Bhunia, Fundamentals of Food Microbiology, 5th ed. Boca Raton, FL, USA: CRC Press, 2018. [3] R. K. Adams and M. O. Moss, Food Microbiology, 5th ed. London, UK: Royal Society of Chemistry, 2016. [4] J. R. Jay, M. J. Loessner, and D. A. Golden, Modern Food Microbiology, 7th ed. New York, NY, USA: Springer, 2015. [5] D. C. Frazier and D. C. Westhoff, Food Microbiology, 5th ed. 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