Author : Nursanty Risa

Summary : Foodborne disease caused by ingested foodborne pathogens is the most serious public health problem in many countries. Food decontamination has been used directly or indirectly during production or processing to help increase the self-life of the food. However, many studies have found that chemical decontamination harms human health. Natural preservatives have recently gained popularity, particularly those derived from edible medicinal plants. One of the plants is guava (Psidium guajava L.). This study aimed to determine the antimicrobial activity, stability, and effects of ethanolic P. guajava L. leaf extract, identify its phytochemicals, and evaluate the toxicity and effect of the extract on the microbial population in raw food. The samples were extracted using ethanol as a solvent with the maceration method. The antimicrobial activities of the extract were evaluated against nine foodborne pathogens and eight food spoilage fungi using standard methods of the Clinical and Laboratory Standard Institute (CLSI). The stability of the antimicrobial activity of the extract was evaluated at different pHs and temperatures. The toxicity of the extract was determined by using a brine shrimp lethality assay. The results showed that the yield of extracts was 12.90 ± 0.02%. The extract exhibited antimicrobial activity with an inhibition zone range of 7.00-12.75 mm for bacteria strains and 7.00-8.50 mm for fungi strains. The extract can inhibit the growth of bacteria strains with MICs ranging from 0.15-1.25 mg/mL and 0.31-2.50 mg/mL for fungi, respectively. The extract can kill the bacteria with MBCs ranging from 0.31-> 5.00 mg/mL and MFCs ranged 5.00 - >5.00 mg/mL for fungi strains. Time-kill curve study showed that Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, and Proteus mirabilis were killed by the extract at 2x MIC for 4 h, 4x MIC for 2 h, 4x MIC for 0.5 h, and 4x MIC for 4 h, respectively. However, the microbial population of Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, and Salmonella Typhimurium showed 3 log 10 reductions after being treated at 4x MIC for 4 h. While the population of Candida strains reduced by about 3 log10 reductions after being treated at 4x MIC for 4 h. Based on the inhibition conidial germination assay, the germination of filamentous fungi in the quantitative assay shows that the extract can completely inhibit Aspergillus fumigatus, Rhizopus oryzae, and Rh. oligosporus at 8x MIC. In the qualitative assay, the Rhizopus species showed no growth after being treated with extract started at 8x MIC for 14 days. Furthermore, the antimicrobial activities of the extract were not affected by different pHs and temperatures. The effect of the extract on cells of tested microorganisms showed changes in cell wall structure. The main volatile compounds identified using GC-MS were pyrogallol, α-copaene, caryophyllene, aromadendrene, a-humulene, alloaromadendrene, γ-muurolene, β-selinene, α-selinene, α-muurolene, β-bisabolene, (-) globulol, caryophyllene oxide, α -muurolol, and epiglobulol. LC-MS analysis has identified eight non-volatile compounds namely quinic acid, catechin, apigenin-7-O- β -D­glucuronopyranoside, quercetin, luteolin-7-O-glucuronide, epigallocatechin (4β, β)­gallocatechin, naringenin4'-O-glucopyranoside, and myrcetin. Moreover, the toxicity assay showed that the extract had an LC50 of 15.417 mg/mL, which is recognized as non­toxic to the eukaryote cells. Meanwhile, it was discovered that the effect of 1% extract on the microbial population in food samples exhibited a better effect in controlling microbial survival throughout the storage time. In conclusion, 1% ethanolic P. guajava L. leaf extract has antimicrobial activity against tested foodborne pathogens and food spoilage fungi with no significant effect from different pHs and temperatures and no toxicity effect, implying that it can be developed as a natural sanitizer for washing raw food materials.