57NISR Research GRANT
2009 Young Investigator Research Grant

Identification of Antioxidant(s) from Lactic Acid Bacteria andScreening of High Antioxidative Strain(s)Yuji YAMAMOTO
Department of Animal Science, School of Veterinary Medicine, Kitasato University

Research aims

antixoidative activities of the selected strains.

Oxygen is a powerful oxidant and therefore a good electron acceptor for respiration metabolism. However oxygen
can also be toxic to all living organisms. Incomplete reduction of oxygen generates reactive oxygen species (ROS)
such as superoxide, hydrogen peroxide, and hydroxyl radicals, which can have deleterious effects on biomolecules including DNA, RNA, membrane lipid, and protein. Although higher organisms including human have developed
their own defense systems against ROS, many clinical andepidemiological studies have suggested that intake of dietary antioxidants, including polyphenolic compounds, Vitamins E and C, and carotenoids, have an important role in
the prevention of the oxidative stress related diseases suchas inflammation, cardiovascular disease, cancer, and agingrelated disorders.1)
Lactic acid bacteria (LAB) are a group of bacteria whichconvert more than fifty percents of carbohydrate to lactic
acid through fermentation metabolism, and have been studied for their possible beneficial physiological effects onhuman and animals. To date, various health promoting effects of LAB such as intestinal regulator activity, immunepotentiating activity, anti-tumor activity, and resistance topathogens, have been reported. Recent several studies haveshown that ingestion of LAB alleviates pathological condition of oxidative stress related model diseases, implyingthat LAB can serve as antioxidant to animals.2,3) To date,several antioxidant enzymes, such as manganese superoxide
dismutase and NADH peroxidase, have been reported inLAB. We previously found a ferritin-like iron-binding protein (designed as Dpr) as a new LAB antioxidative protein,and demonstrated that Dpr confers oxygen tolerance toLAB by sequestering intracellular free iron ions which otherwise promotes generation of hydroxyl radicals throughfenton reaction.4) During the course of study on Dpr, we
also detected activity, which inhibits fenton reaction, in low
molecular weight fraction of cell-free extract. In case ofbacteria including LAB, few low molecular antioxidants except glutathione have been reported, though many proteinous antioxidants have been extensively studied. Since
low molecular weight antioxidants derived from LAB is expected to be highly useful for food industry and humanhealth, it is important to verify the chemical nature of theantioxidant(s). To identify low molecular weight antioxidants from LAB, we screened high antioxidative LAB
strains from our laboratory stock and analyzed properties ofMethods
LAB cell-free extracts were prepared as follows. LAB
strains were grown in M17 or MRS broth at 30 or 37 degrees. An overnight culture of each LAB strain was diluted
100-fold by fresh medium, and then incubated for 24 hrs.
LAB cells were collected by centrifugation at 8,000 g for 10
min and washed with 50 mM potassium phosphate buffer
pH 7.0 (for ORAC assay) or 50 mM MOPS-NaOH buffer
pH 7.0 (for TBARS assay) two times. The LAB cell pellets
were then suspended in either 50 mM potassium phosphatebuffer pH 7.0 (for ORAC assay) or 50 mM MOPS-NaOHbuffer pH 7.0 (for TBARS assay), and the LAB cells wasdisrupted by the BeadBeater at 4,200 rpm for 2 min. The
cell debris and Zirconium beads were removed by centrifugation at 13,000 g for 15 min. The supernatant was used ascell-free extract sample for TBARS and ORAC assays andthe protein concentration of each extract was determined by
BCA assay.
TBARS assay was performed as follows. The basal reaction mixture contained 10 mM MOPS-NaOH buffer (pH7.0), 63 mM NaCl, and 4 mM 2-deoxyribose in a total volume of 0.3 ml. To the basal reaction mixture was added cell
free extract of LAB to give a final concentration of 10m g/ml. The reaction was started by adding hydrogen peroxide and ferrous ammonium sulfate to the reaction mixture
at final concentrations of 100 and 10 m M, respectively.
After incubation at 37°C for 15 min, 0.25 ml of 1% (wt/vol)thiobarbituric acid and 2.8% (wt/vol) trichloroacetic acid
were added to the reaction mixture. After the mixture wasboiled for 10 min and rapidly cooled, the amount of chromogen formed in the sample was measured by its fluorescence emission at 553 nm (ex 532 nm).
ORAC assay was performed by the method described by
Gillespie et al.3) ORAC value of cell free extract of LAB
was calculated by using trolox, a standard antioxidative
compound, as a control, and is indicated as m mol trolox
equivalent (TE) per gram total protein.
Results
Antioxidative activity of cell-free extract of LAB wasmeasured by TBARS and ORAC assays as described inmethods. TBARS assay was used to detect both hydroxyl
radical scavenging activity and inhibitory effect on iron-de-58Table 1. Antioxidative activity of intracellular cell-free extract of lactic
acid bacteria screened by TBARS assay
StrainLactobacillus sakei PP6-S
Lactobacillus casei JCM8129
Leuconostoc mesenteroides PP9-1
Pediococcus pentosaceus JCM5890
Lactobacillus paraplantarum PP7-4
Leuconostoc mesenteroides HS86-1
Lactobacillus sakei PP2-2
Lactobacillus casei JCM1134
Lactobacillus plantarum PP7-3
Leuconostoc lactis HS32-1

Table 2. Antioxidative activity of intracellular cell-free extract of lactic
acid bacteria screened by ORAC assay

Inhibition (%)

Strain69.564.7
61.3
60.7
60.2
59.1
56.3
55.2
53.6
51.4

Lactobacillus casei JCM20024
Lactobacillus sakei PP4-S
Lactobacillus plantarum HS47-1
Lactobacillus plantarum JCM1149
Lactobacillus rhamnosus ATCC53103
Lactobacillus plantarum HS66-1
Lactobacillus paracasei JCM2769
Leuconostoc mesenteroides PP9-1
Lactobacillus casei JCM1134
Lactobacillus sakei PP4-1

m mol TE/g total protein
572
566
469
466
457
422
412
394
394
392

A list of 10 high antioxidative activity strains, out of 100 strains tested,
is shown.

A list of 10 high antioxidative activity strains, out of 72 strains tested, isshown.

pendent hydroxyl radical formation (Fenton reaction).
ORAC assay is a method of measuring antioxidative capacity of biological sample, and is used to measure antioxidative capacity of foods in the United States Department ofAgriculture (USDA).5,6) Laboratory stock of LAB strains
including 41 strains of Lactobacillus, 9 strains of Streptococcus, 20 strains of Enterococcus, 14 strains of Lactococcus, 13 strains of Leuconostoc, 3 strains of Pediococcus,
were tested for their antioxidative activities. All 100 strainstested by TBARS assay demonstrated antioxidative activity
with inhibition rate in the range from 9.5 to 69.5%. The average inhibition rate was 32.812.9%. Among 100 strains,
10 high antioxidative strains were shown in Table 1. Lactobacillus sakei PP6-S which isolated from a fermented fish
food product showed highest activity out of 100 strains.
ORAC assay was performed to measure total antioxidant
capacity of cell-free extract of LAB. All 72 strains tested byORAC assay demonstrated detectable ORAC values in therange from 82.5 to 572.2 m mol TE/g total protein. The average ORAC value of 72 strains was 258.4107.6 TE/g totalprotein. Table 2 shows a list of 10 strains which demonstrated high ORAC values. Lactobacillus casei JCM20024
showed highest ORAC value out of 72 strains tested.
Among the 10 strains listed in Table 2, all the strains wereLactobacillus except Leuconostoc mesenteroides PP9-1.
We next analyzed properties of antixodative activities ofcell free extract of these high antioxidative strains. As theresult of fractionation of cell-free extract by ultrafiltration,
both antioxidative activities measured by TBARS andORAC assays were recovered in low molecular weight fraction (less than 10 KDa) with the range of 32–77%. Antioxidative activity detected by TBARS was resistant to heat
treatment (boiling for 10 min) and Protenase K treatment.
ORAC value of cell-free extract was also relatively heat stable. Interestingly, Protenase K treatment increased ORAC
values of the cell-free extract by about 2.8-fold compared tothat of the non-treated sample, suggesting that peptide de-

rived from LAB protein might have antioxidative activity.
Conclusion
In this study we successfully detected anti-oxidative activity from cell-free extract of 100 strains of LAB by using
TBARS and ORAC assays. The highest antioxidative activity strain screened by TBARS assay was Lactobacillus
sakei PP6-S. The strain which demonstrated highest ORAC
value was Lactobacillus casei JCM20024. Several preliminary analyses indicated that antioxidative activities detected
by TBARS and ORAC assays were low molecular weightcompound(s) and relatively heat stable. We are currently
trying to identify the chemical nature of the compound(s)of antioxidative activity from the strains screened by thisstudy.
References
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