Bioactivities and phytochemical and toxicity analyses of ethanolic Kayu Manis Hutan [Cinnamomum iners (Reinw. ex Nees and T. Nees) Blume] leaf extract and its application in food

In the food industry, the foodborne bacteria and spoilage microorganisms have capabilities to cause foodborne diseases and spoilage. This has been prevented with utilization of synthetic antimicrobial agents in food products. However, its application has long term affected human health. This concern needs to be controlled which involves the utilization of safe natural plant such as Kayu Manis Hutan [Cinnamomum iners (Reinw. ex Nees and T. Nees) Blume] which reported to have antimicrobial and antioxidant activities. The study aims to determine the antimicrobial activity of ethanolic young and old C. iners leaves extracts against selected pathogenic microorganisms namely Bacillus cereus, B. megaterium, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Aspergillus flavus, A. niger, Rhizopus oligosporus and R. oryzae. The result of antibacterial activity has exhibited inhibition zone at range between 9.00 to 10.00 mm against all tested bacteria strains. The tested bacteria growths were inhibited at MICs ranged from 0.16 to 2.50 mg/mL, and were killed at MBCs ranged from 1.25 to 5.00 mg/mL. Time kills curve study showed that all tested bacteria can be killed at 4× MIC within 4 h. The antifungal activity of the extracts has exhibited zones of inhibition at range of 6.50 to 7.25 mm against all tested food spoilage microorganisms. The MICs of the extracts ranged from 0.63 to 5.00 mg/mL, and the MFCs at ranges of 1.25 to 5.00 mg/mL. In qualitative analysis, A. niger and R. oryzae showed no growth after being treated with young leaves extracts at 4× MIC while R. oligosporus and A. flavus at 2× MIC for 14 days. Meanwhile, no growth was shown on all tested fungi after being treated with old leaves extracts at 2× MIC for 14 days. In quantitative analysis, using 4× MIC for 24 h, the conidia germination was fully inhibited for all tested fungi. The antimicrobial activities of both extracts were shown not significantly different after being treated at different pHs and temperatures. The SEM analysis showed morphological features in selected treated microorganisms namely, B. cereus, K. pneumoniae and A. flavus were changed in the cell wall shape, ruptured, and the cytoplasm leaked. Both leaves extracts have exhibited high total phenolic content and antioxidant activities. The toxicity study demonstrated that both extracts was not toxic to Artemia salina with LC50 at 19.62 mg/mL and 13.20 mg/mL, respectively. GC-MS analysis has identified the presence of active volatile compounds in both extracts included phytol, hexadecanoic acid, dihydro(-)-neoclovene-(II), 14-β-pregnane and 2-hexadecenal. LC-MS have detected coniferyl alcohol, isorhamnetin-3-O-rutinoside in young leaf extract while α-terpineol, and 4-methylcatechol in old leaf extract. The decrease of 3 Log10 CFU/g of total place count, total viable mold count, B. cereus, E. coli, and K. pneumoniae count on raw chicken meat sample was observed when exposed to 5.00% (v/v) of C. iners extracts and maintain its microbiological quality with longer exposure within 21 days in different storage conditions. In conclusion, C. iners extracts and its compounds have shown potential of antimicrobial and antioxidant activities against foodborne pathogens and spoilage microorganism and thus can be developed as natural antimicrobial agent for food preservatives.

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