Antioxidant Activity of Fermented Buffalo Milk Using Selected Lactic Acid Bacteria
Lactic acid bacteria (LAB) in milk fermentation can generate bioactive peptides including antioxidative peptides. However limited studies about production of bioactive peptides from buffalo milk fermentation generated by selected LAB. Thus, this study was to screen the proteolytic activity and pr...
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| Summary: | Lactic acid bacteria (LAB) in milk fermentation can generate bioactive peptides
including antioxidative peptides. However limited studies about production of bioactive
peptides from buffalo milk fermentation generated by selected LAB. Thus, this study
was to screen the proteolytic activity and probiotic potentials of isolated LAB and
antioxidative activity of skimmed milk whey using LAB isolates as well as to evaluate
the effect of culturing methods and fermentation time on antioxidant activity of whey
generated by buffalo milk fermented with selected LAB. Proteolytic activity of LAB
was screened using skimmed milk agar (SMA) method and the antioxidant activities of
whey skimmed and buffalo milk were measured using scavenging of 1,1-diphenyl-2-
picrylhydrazyl (DPPH) and ferrous chelating activity (FICA) assays. Molecular weight
(Mw) of antioxidative peptides from whey buffalo milk produced by LAB was estimated
using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) method.
Twenty-five LAB isolates from different sources (homemade yogurt, banana, grape,
dates, soil, fermented shrimp, and fermented salted fish) were screened for their ability
to hydrolyse protein in skim milk. Preliminary screening indicated that eight (Yg, Bn2,
WG2, BG, Dt, Pk2, S1, and Bd2) of LAB isolates showed protein hydrolysis on SMA
with diameter of clear zone between 4.0 mm and 14.5 mm. These LAB were fermented
in skimmed milk produced whey had antioxidant activity analysed with DPPH and
FICA assays after 24 h incubation at 37 °C. The WG2, Pk2, S1, and Bd2 isolates
generated antioxidant activity in whey skimmed milk with DPPH values ranged
between 12.59 and 19.04 % and FICA values ranged between 78.61 and 84.81 %
compared to other isolates were further evaluated for their probiotic potentials to
reassure LAB viability and functionality remained. All four LAB isolates tolerated in
acid and bile environments with at least slight reduction (maximally <1.05 log cycle) in
viable cell counts for both acid and bile challenge assays. Isolate WG2 showed more
ability to survive in acidic stress than other isolates with viable count loss less than 0.10
log reduction in the acidic environment at pH 2.0 while isolate Bd2 showed an
increment in viable count with the log cycle 0.37 cfu/ml after exposure in bile condition
at pH 6.5. These LAB isolates able to act as antimicrobial activity with wide range of
inhibition zone by both dual culture overlay and agar well diffusion methods against
Bacillus cereus (B. cereus), Bacillus subtilis (B. subtilis), Staphilucoccos aureus (S.
aureus), Staphilucoccus typhimurium (S. typhimurium) and Escherichia coli (E. coli),
respectively. All four LAB isolates were greatly inhibited S. typhimurium and E. coli
with the largest diameter of clear zone of 85.00 mm by dual culture overlay method.
The largest diameter of clear zone by agar well diffusion method was observed for LAB
isolates of Pk2, S1, and Bd2 against E. coli which was 13.83 mm. Culturing approach
of LAB isolates significantly (p<0.05) affected antioxidative value of whey buffalo milk
by scavenging DPPH radical activity and FICA assays. Whey produced by direct
cultured LAB in fermented buffalo milk resulted in high DPPH value compared to whey
produced by precultured LAB. In contrast, whey produced by direct cultured LAB in
fermented buffalo milk resulted in low FICA value compared to whey produced by
precultured LAB. The DPPH value of whey buffalo milk generated by direct cultured
LAB ranged between 22.61 and 33.62 % while the FICA value of whey buffalo milk
generated by precultured LAB ranged between 59.41 and 78.22 % for WG2, Pk2, S1,
and Bd2 isolates. Identification of isolates WG2, Pk2, S1, and Bd2 were identified as
Lactobacillus plantarum (L. plantarum WG2), Lactobacillus paracasei (L. paracasei
Pk2), Lactobacillus plantarum (L. plantarum Sl) and Enterococcus faecium (E. faecium
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Bd2) using 16S rDNA sequencing method with 99 % similarity. These LAB were
further studied in fermentation of buffalo milk to generate whey with antioxidant
activity by different culture approach and different fermentation time. Addition of LAB
to buffalo milk either direct culturing or preculturing methods affected the antioxidant
activity of whey buffalo milk. Direct culturing of LAB into buffalo milk produced whey
with high DPPH values ranged between 21.87 and 55.03 % and FICA values ranged
between 50.13 and 65.52 %. However, precultured LAB to buffalo milk produced whey
with lower DPPH values ranged between 3.43 and 12.28 % compared to FICA values
ranged between 56.58 and 84.45 % for L. plantarum WG2, L. paracasei Pk2, L.
plantarum S1, and E. faecium Bd2. Buffalo milk fermented with precultured L.
plantarum WG2 and E. faecium Bd2 produced whey with high FICA values and were
influenced by fermentation time. After 24 h fermentation process by precultured E.
faecium Bd2 produced whey buffalo milk with FICA value 78.22 % but after 48 h
fermentation process, precultured L. plantarum WG2 produced whey buffalo milk with
the highest FICA value in this finding (84.45 %). The whey buffalo milk generated by
both precultured L. plantarum WG2 and E. faecium Bd2 were determined the
antioxidant activity by half maximal inhibitory concentration (IC50) in FICA assay
resulted with IC50 values ranged between 0.37 and 0.41 mg/ml which was lower than
IC50 value for standard EDTA (0.29 mg/ml). Using SDS PAGE analysis, the Mw of
peptides from whey buffalo milk either by precultured L. plantarum WG2 or by
precultured E. faecium Bd2 was estimated between 150 and 50 kDa, 20, and 10 kDa for
both LAB, respectively. This study concluded that L. plantarum WG2 and E. faecium
Bd2 can be applied to generate peptides from whey buffalo milk with relatively good
antioxidant activity. |
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