| Summary: | Pediocin PaF-11 produced by Pediococcus acidilactici F-11 has potency as a
food preservative because of its ability in controlling the growth of food spoilage and
pathogene bacteria, but its use is still limited. To support the application development
of pediocin PaF-11, research that aims to (1) increase the effectiveness of pediocin
PaF-11 purification through pH adsorption and desorption treatment and the addition
of biomass of heat killed cells of P. acidilactici F-11 during adsoprtion (2) know the
characters of pediocin PaF-11 include the stability of the pH, temperature and storage
conditions (3) know the molecular character of pediocin PaF-11 includes a nucleotide
sequence and the location of pediocin PaF-11 encoding gene, as well as the sequence
of amino acids and the molecular weight and (4) find out the initially mechanism of
pediocin PaF-11 inhibitation as an antibacterial, were conducted.
P. acidilactici F-11 and Lactobacillus pentosus LB42 were used as pediocin
PaF-11 producer and indicator bacteria respectivelly, both of which were obtained
from the Food and Nutrition Culture Collection (FNCC), Center for Food and
Nutrition Studies of Gadjah Mada University in Yogyakarta. P. acidilactici F-11
produced pediocin PaF-11 in the TGE broth (trypticase [1%], glucose [1%], yeast
extract [1%], tween 80 [0.2%], Mn2+ [0.033 mM], Mg2+ [0.02 mM] of pH 6.5
within 18 hours incubation at 37 °C. Partial purification of the pediocin PaF-11
was performed by the adsoprtion desorption methods and its activity was determined
by well diffusion agar methods.
This research was conducted in four experimens: (1) Partial purification of
pediocin PaF-11 with treatment variation in pH adsorption desorption and
concentarion of heat killed cells (2) Characterization of pediocin PaF-11
parameters that lead to its application as a food preservative, include stability in
temperature, pH and storage (3) Molecular characterization, including
determination of the nucleotide sequence and the location of pediocin PaF-11
encoding gene, the amino acid sequence and molecular weight of pediocin PaF-11.
(4) Study the initially mechanism of pediosin PaF-11 inhibition as an antibacterial,
by analysis the influence of gadolinium toward activity of pediocin PaF-11 and the
content of Gd3+ on the cell wall as well as its influence on the morphology of the
target cell.
Pediocin PaF-11 activity produced by P. acidilactici F-11 and purified at pH
adsorption 6,5 and desorption 2,0 was 1500AU/ml. Pediocin PaF-11 activity
obtained from purification without addition of heat killed cells was 1500AU/ml,
while by addition of heat killed cells of 3, 6 and 11 times of the original
concentrations were 3000AU/ml. Therefore it was suggested that addition of heat
killed cells of P. acidilactici F-11during adsorption with 3 times of original
concentration was able to increase the pediocin PaF-11 obtained. Total activity of
the pediocin PaF-11 found in the culture was 6000 AU/ml. This means that the
effectiveness of the pediocin PaF-11 purification with the both methods can still be
optimized.
Pediocin PaF-11 with activity of 1500 AU/ml, was stable after heated at
100°C for 30 minutes and 121°C for 15 minutes, and during 11 and 13 weeks
storage at room temperatur (30
o
C) and refrigerator (4
o
C). Pediocin PaF-11 was
found to be stable over a wide pH range between 3 and 8. These characteristics
make pediocin PaF-11 suitable as a food preservative, especially products that
involve processing on pH such as in the process of fermentation and processing at
high temperatures as well as storage.
Pediocin PaF-11 from the cured cell of P. acidilactici F-11 loss the activity,
suggested that the pediocin PaF-11 gene was carried in the plasmid. Agarose gel
electrophoresis of P. acidilactici F-11 plasmid DNA with marker λDNA/HindIII
showed that pediocin PaF-11 gene was carried in 12 kb plasmid. Amplification
pediocin PaF-11 gene from P. acidilactici F-11 showed that uncured P. acidilactici
F-11 culture contain plasmid DNA, indicated by amplification of the papA gene
(256 bp). Cured P. acidilactici F-11 culture, plasmid eliminated, indicted by no
aplicon DNA detected. This result also suggested that pediocin PaF-11 gene in P.
acidilactici F-11 was carried in plasmid.
Nucleotide of pediocin PaF-11 encoding gene was sequenced : atgaaaaaaa
ttgaaaaatt aactgaaaaa gaaatggcca atatcattgg tggtaaatac tacggtaatg gggttacttg
tggcaaacat tcctgctctg ttgactgggg taaggctacc acttgcataa tcaataatgg agctatggca
tgggctactg gtggacatca aggtaatcat aaatgctag. The alignment of that sequence with
the nucleotide sequence of pediocin PA.1 encoding gene (pedA) in P. acidilactici
PAC1.0
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