Antibacterial, antispore and phytochemical analyses of ethanolic Jambu Batu (Psidium Guajava L.) leaf extract and applications in tofu

Spore-forming bacteria can survive various stages during food processing; thus, eliminating the vegetative bacterial cells and spores become the primary concern to the food industry. Bacillus species (spp.) spores are resistant to heat, radiation and disinfectants, which necessitate potent antibacterial agents. The crude extracts of jambu batu leaves exhibited profound antimicrobial activities against various foodborne pathogens. Thus, the current research aimed to determine the antibacterial and antispore activities of jambu batu leaves extracts on the vegetative cells and spores of B. cereus ATCC® 33019™, B. megaterium ATCC® 14581™, B. pumilus ATCC® 14884™ and B. subtilis ATCC® 6633™. The phytochemical constituents of the extracts were also analysed and the effect of the extracts on bacterial population in tofu were evaluated. The jambu batu leaves were collected from the Taman Pertanian, Universiti Putra Malaysia. The dried powder of jambu batu leaves was extracted with the ultrasonicassisted maceration method using ethanol as solvent. The extracts were tested for the antibacterial activity against Bacillus spp. vegetative cells in terms of well diffusion assay (inhibition zone), minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC), as recommended by the Clinical and Laboratory Standard Institute (CLSI). The Time-kill curve analyses were conducted at the concentration of the extract with ½, 1, 2 and 4×MICs at 0, ½, 1, 2 and 4 h of exposure time. The antispore activity of the extract was determined at different concentrations and time exposures. The stability of the extract on the antibacterial activity was conducted by exposing the extracts to various pHs (pH3, 5, 6.7, 7 and 11) and different temperatures (10ºC, 28ºC, 30ºC, 50ºC and 80ºC). The phytochemicals constituents in the extracts were identified using GC-MS and LC-MS/MS analysis. The effect of jambu batu leaves extract on bacterial population in tofu samples was evaluated at different extract concentrations (tap water, 0.00%, 0.05%, 0.50% and 5.00%) at different exposure times (1, 2 and 4 h). The jambu batu leaves extract generated the minimum and maximum inhibition zones of 13.75 ± 0.95 mm to 16.25 ± 0.95 mm, respectively. It showed that all the tested Bacillus spp. were susceptible to the jambu batu leaves extract. The growth inhibition pattern was detected in all tested Bacillus spp. with MIC ranging from 0.195 mg/mL to 0.781 mg/mL, while killed by an MBC value of 0.781 mg/mL. The bactericidal activities of jambu batu leaves extract were determined by the time-kill curve analysis for B. cereus, B. megaterium, B. pumilus and B. subtilis at 4.0×MIC for 4 h, 4×MIC for 2 h, 1×MIC for 4 h and 4×MIC for 2 h, respectively. The spores of B. cereus were inactivated more than 3-log10 (99.9%) at a concentration of 1.0% extract. Subsequently, spores were killed at a concentration of >2.0% upon 4 h incubation. Jambu batu leaves extract caused a stable antibacterial activity at various pHs and temperatures against Bacillus spp. The GC-MS analysis identified 30 compounds in the extract; mainly six bioactive compounds were caryophyllene, aromadendrene, alpha-humulene, betaselinene, alpha-selinene and globulol. However, LCMS/MS analysis detected 26 compounds and the antimicrobial bioactive compounds detected were lasalocid, cannabichromene, asiatic acid, curcumin, 18-β-Glycyrrhetinic acid, acetoxyurs, apigenin and aloesin. Tofu sample resulted in a reduction of 3-log10 of Bacillus spp. bacteria when exposed to 0.05% (v/v) jambu batu leaves extract for 2 h, and an incubation period of 4 h inhibited all the bacteria. In conclusions, jambu batu leaves extract yielded good antibacterial and antispore activities, suggesting that jambu batu leaves extract can be utilised or explored as a potential anti-Bacillus agent in food.

Text 122556.pdf Download (1MB) Official URL or Download Paper: http://ethesis.upm.edu.my/id/eprint/18609

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