Antibacterial and antispore activities of crude extract and bioactive compounds of Piper cubeba L. berries against Bacillus sp. and application of the extract for food preservation

In the food industry, the Bacillus species, in particular, is
known as organisms that cause foodborne diseases and food
spoilage. Bacillus species are produce of spores and many of the
spores of the Bacillus species have been shown to be resistant to
heat, radiation, and disinfectants. A previous study has shown
that crude extracts of tailed pepper (Piper cubeba L.) have potential
antimicrobial activities against some of Bacillus species.
The present study aims to analyze the antibacterial and antispore
activities of Piper cubeba L. berries extracts on the vegetative
cells and spores of Bacillus cereus ATCC33019, B. subtilis
ATCC6633, B. pumilus ATCC14884, and B. megaterium
ATCC14581. Results showed that exposing of Bacillus sp. to P.
cubeba L. extract and its compounds resulted in an inhibition
zone with a large diameter which ranged between 9.50 to 11.40
mm for the extract and 7.21 to 9.61 mm for the compounds.
The MIC of the extract ranged between 0.156 – 0.313 mg/mL
and the MBC at 2.5 mg/mL. Moreover, for the compounds
the MIC range was between 63.0 to 125.0 µg/mL and MBC at
250.0 to 500.0 µg/mL against Bacillus sp. The time-kill curve
plots showed that exposing Bacillus sp. to a concentration of
8× MIC for a period of four hours resulted in the death of
all cells. The values of MIC and MBC showed a fluctuating
trend when the bacteria were exposed to P. cubeba L. extract
treated with different temperature in comparison to untreated
extract. Generally, the pH altered extracts caused a variation in
the MIC and MBC values of the Bacillus sp. The effect of using
varying concentrations of extracts and compounds against the
Bacillus sp. spores for varying periods of incubation were determined.
Glutaraldehyde, which is a chemical sporicidal agent,
was used as a positive control. P. cubeba L. extract at a concentration
of 1.0% inactivated more than 3- Log10 (90.99%) of
the Bacillus sp. spores after an incubation period of four hours,
and all the spores were killed at a concentration of 2.5%. The
image of scanning electron microscope showed that the structure
of spores were destroyed after treatment with 1% P. cubeba
L. extract for one hour. The major volatile compounds, as determined
using GC-MS, are β-cubebene, cubebol, α-copaene, α-
cubebene, caryophyllene, 9,12-octadecadienoic acid, β-asarone,
and germacrene-D. The non- volatile compounds identified
through LC-MS are gallic acid, quinic acid, asaronaldhyde,
epicatechin, clusin, cubebininolide, hemiarensin, β-asarone,
hinokinin, ellagic acid, myricetin, and β-cubebene. The identified
phytochemical compounds are similar with those in the
literature and MS/MS databases. β-Asarone, asaronaldehyde,
cubebin mixture and linoleic acid were successfully isolated and
identified from the methanol extract of P. cubeba L. In general,
a decrease of 3 Log10 of Bacillus sp., total plate count, E. coli
and coliform bacteria on the tofu sample was observed when
these bacteria were exposed to 0.50% (v/v) P.cubeba L. extract.
In conclusion, P.cubeba L. extracts and its compounds show
a promising potential of antibacterial and sporicidal activities
against the Bacillus sp. and thus can be developed as an anti-
Bacillus agent.

Author(s): Fatimah Alqadeeri , Y. Rukayadi , F. Abbas , K. Shaari

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