Food Sci. Biotechnol.

25(1): 199-204 (2016)

DOI 10.1007/s10068-016-0030-x

Production of Sesaminol and Antioxidative Activity of

Fermented Sesame with Lactobacillus plantarum P8,
Lactobacillus acidophilus ATCC 4356,
Streptococcus thermophilus S10
Jin-Ju Bae, Su-Jung Yeon, Woo-Joon Park, Go-Eun Hong, and Chi-Ho Lee*
Department of Food Science and Biotechnology of Animal Resources, Konkuk University, Seoul 05029, Korea

Received July 16, 2015

Abstract This study was carried out to select the most competent bacterial cultures that could
Revised October 19, 2015
Accepted October 20, 2015 convert sesaminol glycosides to aglycone by β-glucosidase produced by lactic acid bacteria such as
Published online February 29, 2016 Lactobacillus acidophilus, Lactobacillus plantarum (LP), and Streptococcus thermophilus in sesame
fermented at 37oC for 24 h. The pH of fermented sesame was decreased compared to non-fermented
*Corresponding Author
Tel: +82-2-450-3681 controls. The pH of LP was lower than that of the other two during fermentation. Fermented sesame
Fax: +82-2-453-1948 had higher antioxidant activity compared to non-fermented controls during the entire fermentation
E-mail: [email protected] time. Total phenol content, DPPH free radical scavenging assay, reducing power assay of sesame
pISSN 1226-7708
fermented by LP was the highest compared to the others. In addition, sesame fermented by LP had
eISSN 2092-6456 more bioconversion of sesaminol glycoside to aglycone compared to the others. Therefore, LP was the
best bacterial culture of the three strains studied for producing functional fermented sesame for good
© KoSFoST and Springer 2016 health.

Keywords: sesame, fermentation, lactic acid bacteria, sesaminol, aglycone

Introduction compounds with antioxidant activity in vivo (11,12). Experiments

have proved that STG could be metabolized by human and rat
Sesame is an important health food in Korea, China, and other Asian intestinal microbiota and form phenolics that possess anti-inflammatory,
countries. Korea has the highest consumption of sesame in the world antioxidant, and estrogenic activities. They can be hydrolyzed by
with approximately 6-7 g/day per capita (1). Sesame seed contains intestinal β-glucosidase to sesaminol, an antioxidant. According to
50% lipid, 20% protein, and 14% carbohydrate. It is one significant oil the Jan et al. (13), the transformation of STG in gut involved the
seed crop in the world. Sesame seed provides nutritious meal and demethylation of a methoxy group, hydrolysis of glucoside and
protein as well as highly stable oil. (2). Sesame exhibits many reductive cleavage of furfuran ring. And then STG incorporated
beneficial physiological effects due to its bioactive lignan compounds. through lymphatic absorption into the cardiovascular system (14-18).
Sesame seed contains abundant lignan glycosides such as sesamin, Many scientific studies have proved that sesaminol in sesame is
sesamolin, and sesaminol glucosides as significant functional mostly in the glycoside form (3). β-Glucosidic bond of glycoside can
components (1,3,4). Sesamin is a major lignan in the sesame oil. Its be hydrolyzed by β-glucosidase produced by microorganism to form
beneficial biological effects have been studied extensively. Many aglycone after fermentation. In other words, higher β-glucosidase
studies have reported that sesamin has anti-hypertensive effect (5) activity will release more aglycone contents (19). The formed aglycone
and anti-hypercholesterolemic activity through its inhibitory activity will lead to more active biological activity. Since the intestinal
on cholesterol absorption and synthesis (6). Sesamolin and sesaminol absorption of aglycone is faster than glycoside, aglycone is more
glucoside have antioxidant activities related to their high stability effective in preventing diseases. Aglycones have peroxyl radical
against oxidation during long period storage of sesame oil (7). The scavenging activities by β-glucosidase after being liberated in the
major lignan glycoside exists mainly in sesame seed, hydrophilic gastrointestinal tract (8,20). Lactobacillus plantarum, Lactobacillus
antioxidants are sesaminol triglucosides (STG) that have less acidophilus, and Streptococcus thermophilus have been reported to
bioactivity in vitro (8-10). Although sesaminol glucosides have no be able to produce β-glucosidase. Therefore, they are typically used
antioxidant activity in vitro, they can be converted to phenolic for fermentation (21). For example, S. thermophilus and Lactobacillus
200 Bae et al.

have been used to produce sesame yogurt from sesame milk by presented as percent lactic acid (24).
Afaneh et al. (22). Several studies have demonstrated antioxidant
activity of sesame, however, there is no sufficient study on fermented Proximate analysis The proximate analysis of non-fermented and
sesame with lactic acid bacteria. Unlike previous studies, the objective fermented sesame was analyzed by using AOAC method (24). Total
of this study was to investigate whether sesaminol triglucoside was solid contents were estimated by subtracting the moisture percent
converted to sesaminol by β-glucosidase produced by lactic acid from 100. Crude ash contents were measured by burning at 450.
bacteria such as Lactobacillus acidophilus, Lactobacillus plantarum, Protein and crude fat contents were measured by Kjeldal method
and Streptococcus thermophilus in sesame fermented at 37oC for and Soxhlet method, respectively. Carbohydrate content was
24 h. This study is carried out to select the most competent cultures calculated by deducting the sum of crude ash, crude protein, and
among Lactobacillus acidophilus, Lactobacillus plantarum, and crude fat contents from the total solid contents.
Streptococcus thermophilus that could convert sesaminol glycosides
to aglycone in fermented sesame. HPLC analysis of lignans The amount of lignan was measured by
HPLC using procedures described by Zhu et al. (25). Dried sample was
extracted with 80% methanol (1:20, w/v) using a Sonicator (Branson
Materials and Methods Ultrasonics Co., Danbury, CT, USA) for 60 min followed by centrifugation
at 3,000×g for 20 min. The supernatant was filtered through PVDF
Materials and reagents Sesame was purchased at a local market in syringe filter (0.45 µm, Pall Corporation, Port washington, NY, USA).
Gwangjin-gu, Seoul, Korea. Sesaminol triglucoside and sesaminol at Filtered samples were analyzed with an Agilent 1100 series HPLC
HPLC standard grade were obtained from Nacalai USA Company system equipped with a Binary pump, Autosampler, multiple
(Kyoto, Japan). Mobile phase reagents of HPLC analysis such as wavelength detector, and a ChemStation software. The analyses were
methanol was purchased from J. T. Barker Company (Phillipsburg, NJ, carried out on a Higgins 100 C18 column (4.6×250 mm, 5 µm). The
USA). All other chemicals used were of analytical grade. oven temperature was set at 35. The mobile phase of HPLC was
methanol/water (60:40, v/v) and the flow rate was 0.6 mL/min. The
Microorganisms Streptococcus thermophilus, Lactobacillus plantarum detector was set at 290 nm. Injection volume was 10 µL.
P8 (LP), and Streptococcus thermophilus S10 (ST) were isolated from
the Laboratory of Diary Biotechnology and Bioengineering, Inner Determination of total phenol contents The amount of total
Mongolia Agricultural University. Pure cultures of Lactobacillus phenol contents for antioxidative activity was determined with
acidophilus ATCC 4356 (LA) was obtained from Korean Collection for procedures described by Folin-Ciocalteu (26) with slight modifications.
Type Culture (Daejeon, Korea). Three strains were activated in de Same as the extraction method in HPLC, sample extraction was
Mann Rogosa Sharps (MRS) broth (Oxoid Ltd., Basingstoke, England) processed by the methanol method. Briefly, a 0.5 mL of sample
at 37oC for 24 h aerobically. Each microorganism concentration was extract was added to 2.5 mL of 0.2 N Folin-Ciocalteu’s phenol reagent
adjusted to 108 CFU/mL. and mixed. After standing for 5 min at room temperature, 2 mL of
75 g/L sodium carbonate was added to the mixture and incubated at
Sample preparation Sesame seeds were extracted with n-hexane room temperature for 2 h. The samples were measured with a
(1:10, w/v) by stirring at room temperature for 24 h to obtain spectrophotometer at 760 nm. The results were expressed as mg of
defatted sesame (23). Sesame was soaked in distilled water at 1:10 gallic acid equivalents (GAEs) per 10 g using gallic acid as the
(w/v) ratio, boiled at 100oC for 40 min, and ground with a blender standard.
(KNJ-991100R; NUC Co., Ltd., Daegu, Korea). To extract sesame juice,
the resultant slurry was filtered with cotton cloth. The sesame juice DPPH free radical scavenging assay This experiment was to
was sterilized at 121oC for 15 min, chilled, and refrigerated at 4oC. identify antioxidative activity. The 1,1-diphenyl-2-picrylhydrazyl
The sesame juice was then inoculated (1%, v/v) with LP, ST and LA (DPPH, Sigma-Aldrich Co., St. Louis, MO, USA) radical has an odd
and incubated at 37oC for 24 h. Samples were collected at intervals of electron that has a deep violet color. When the radical accepts a
4 and 6 h and stored at −80oC after freeze-drying. hydrogen radical or an electron, its absorbance value is changed. In
this study, the DPPH radical scavenging activities of the extract of
Determination of total count of lactic acid bacteria, pH, and fermented sesame were measured with the method of Blois (27)
titratable acidity Cell viability of lactic acid bacteria (LAB) was with slight modifications. The sample was extracted by the methanol
determined by using a spread plate method with MRS agar (Oxoid method and analyzed by HPLC A 0.5 mL aliquot of sample extract
Ltd.) after incubation at 37 for 24 h. The number of total viable cells and a 2.5 mL of 75 µM DPPH solution were reacted in the dark at
was expressed in log values. The pH of sesame sample was analyzed room temperature for 30 min. The absorbance value was analyzed
by using a pH meter (pH 900; Precisa Co, London, UK). Titratable with a spectrophotometer (Otizen 2120UV; Mecasys Co., Ltd., Daejeon,
acidity (TA) was measured by titration with 0.1N NaOH. Result was Korea) at 517 nm. Free radical scavenging activity was determined

Food Sci. Biotechnol.

 Fermentation of Sesame 201

using the following: DPPH free radical scavenging effect (%)=(1−

absorbance of sample)/absorbance control×100.

Reducing power assay The reducing power of sample was

determined according to the method of Oyaizu (28). Briefly, a 250 µL
of sample extract was mixed with a 0.2 M sodium phosphate buffer
(250 µL, pH 6.6) and a 250 µL of potassium ferricyanide (1%, w/v,
Sigma-Aldrich). After the mixture was reacted at 50oC for 30 min,
10% trichloroacetic acid (250 µL, Sigma-Aldrich) was added to the
mixture. The mixture was then centrifuged at 1,500×g for 10 min. A
500 µL of distilled water and a 100 µL of 0.1% ferricchloride (Junsei
Chemical Co., Ltd., Tokyo, Japan) were mixed with 500 µL of the
supernatant. The absorbance was measured at 700 nm using a UV-
visible spectrophotometer.

Statistical analysis Statistical analyses were performed using SPSS Fig. 2. Changes in cell viability of LAB in fermented sesame during
different fermentation time. Con, control, non-fermented sesame; LP,
statistics 18.0 (SPSS Inc., Chicago, IL, USA). All data were analyzed by
fermented sesame with Lactobacillus plantarum P8; ST, fermented
one-way analysis of variance (ANOVA) and expressed as mean± sesame with Streptococcus thermophilus S10; LA, fermented sesame
standard deviation (SD). A Turkey test was used to determine with Lactobacillus acidophilus ATCC4356.
whether there is a significant difference between samples. Significant
difference was considered when the p value was less than 0.05.
the end of fermentation. The initial titratable acidity (TA) was 0.26-
0.28. It was increased to 0.61-0.83, among which TA of the sesame
Results and Discussion fermented with LP was the highest after the fermentation. As the
time of fermentation was increased, TA of fermented sesame was
Acid production and microorganism growth during fermentation significantly (p<0.05) increased. It is possible to identify the quality of
Results of pH and titratable acidity of fermented and non-fermented fermented sesame through the production of acid. Wu et al. (29)
sesame are shown in Fig. 1. The pH of fermented sesame was have mentioned that the pH value of yogurt fermented by lactic acid
significantly (p<0.05) decreased during fermentation at 37oC for 24 h bacteria such as Streptococcus and Lactobacillus have reached 4.0 to
due to the production of lactic acid by three bacteria. The initial pH 4.7. Although ST and LA exceeded the usual pH range of yogurt,
ranged was 6.44 to 6.52. It was decreased to 4.69 and 5.34 after 24 h fermented sesame with LP showed an appropriate pH during
fermentation. The pH of sesame fermented with LP was the lowest fermentation. Results of lactic acid bacteria count of fermented
compared to that fermented with the other two strains of bacteria at sesame are shown in Fig. 2. The count of LAB was significantly

Fig. 1. Changes in acid production in fermented sesame during different fermentation time. Con, control, non-fermented sesame; LP, fermented
sesame with Lactobacillus plantarum P8; ST, fermented sesame with Streptococcus thermophilus S10; LA, fermented sesame with Lactobacillus
acidophilus ATCC4356.

February 2016 | Vol. 25 | No. 1

202 Bae et al.

(p<0.05) increased during the fermentation at 37oC for 24 h. The sesaminol triglucoside and sesaminol contents of fermented sesame
initial LAB count ranged from 7.1 to 7.3 log CFU/mL. It reached up to or non-fermented sesame are summarized in Table 2. While the
8.1 to 8.6 log CFU/mL during the fermentation. The minimum concentration of sesaminol triglucoside was significantly (p<0.05)
accepted microflora counts ranged at 106-109 CFU/mL in yogurt and decreased, the concentration of sesaminol was significantly (p<0.05)
thus sesame is a proper growth medium for LAB. Afaneh et al. (22) increased. No significant difference was found in sesame fermented
have reported similar LAB results. with LP or ST after 18 h of fermentation or after 12 h of fermentation
with LA. This indicated that more aglycone was produced in sesame
Proximate analysis Proximate analyses for moisture, crude fat, fermented with LP compared to that of the other two strains of
crude protein, ash, and carbohydrate of fermented and non-fermented bacteria. Zhu et al. (25) have reported that STG should be considered
sesame are summarized in Table 1. There was no significant as suitable substrate for intestinal microbiota. Sesaminol aglycone
difference in crude fat or carbohydrate of sesame fermented with was also produced during the in vitro fermentation of STG. The
three strains of bacteria as compared with non-fermented sesame. maximal production of sesaminol was observed at 12 h of fermentation.
However, crude protein contents were significantly (p<0.05) increased These results suggested that the bioconversion by lactic acid bacteria
in sesame fermented with LP compared to the control or sesames fermentation could be through hydrolysis of sesaminol glucoside.
fermented with the other two strains. On the contrary, ash contents Hence, fermented sesame that produces sesaminol might have the
decreased significantly (p<0.05) in sesame fermented with LP potential as functional food ingredients. L. plantarum could be used
compared to control or that with the other two strains of LAB. No as a starter culture for fermented sesame to produce aglycones.
significant difference was found in sesame fermented with ST or LA.
Lee et al. (30) showed similar results that fermentation with Total phenol contents and antioxidant activities Sesame is good
Lactobacillus plantarum decreased in ash and increase in crude source of carbohydrate, fat, protein, and lignan glycoside. However, it
protein after fermentation at 37oC for 24 h. is difficult to extract and isolate sesaminol glycosides from sesame
due to the existence of carbohydrate, fat, and protein. To extract
Changes in lignan contents HPLC analysis results of lignan such as sesaminol triglucoside from sesame effectively, ethanol or methanol

Table 1. Proximate component of fermented and non-fermented sesame (unit: g/100 g)

Group1) Total solid Crude fat Crude protein Ash Carbohydrate
AB2) A 0 AB B
Con 000.23±0.00 71.93±2.52 9.95±0.15 2.73±0.23 15.17±2.76A
LP 0.17±0.05B 72.50±3.70A 10.63±0.38A0 3.59±0.21A 13.12±3.41A
ST 0.31±0.06A 75.15±2.12A 7.37±0.55C 2.56±0.20B 14.61±2.26A
LA 0.15±0.05B 69.12±2.73A 9.11±0.14B 2.48±0.51B 19.15±2.33A
1)
Con, control; non-fermented sesame; LP, fermented sesame with Lactobacillus plantarum P8; ST, fermented sesame with Streptococcus thermophilus
S10; LA, fermented sesame with Lactobacillus acidophilus ATCC4356
2)
Values are mean±SD (n=3); Values in the same column with different superscripts are significantly (p<0.05) different.

Table 2. Changes in lignan contents of sesame with and without fermentation with LAB (unit: mg/10 g)
Strains
Lignan Time (h) 1)
Con LP ST LA
ABns2) A A
0 104.9±0.22 104.8±0.28 104.5±0.16 104.5±0.30A
4 105.1±0.02ABa 088.3±0.10Bc 092.9±0.09Bb 088.3±0.71Bc
Sesaminol 8 104.6±0.41Ba 068.7±0.06Cc 074.5±0.08Cb 074.2±0.10Cb
triglucosides 12 104.9±0.45ABa 058.6±0.07Dd 064.1±0.14Dc 065.6±0.15Db
18 105.3±0.23ABa 051.8±0.12Ed 057.4±0.19Ec 065.6±0.11Db
24 105.6±0.50Aa 051.7±0.14Ed 057.8±0.45Ec 065.6±0.12Db
0 0 NDNSns NDE NDE NDD
4 NDd 19.5±0.04Da 17.3±0.04Db 15.2±0.05Cc
8 NDd 25.1±0.07Ca 22.9±0.07Cb 22.0±0.03Bc
Sesaminol
12 NDd 29.1±0.14Bb 28.9±0.05Bc 30.5±0.16Aa
18 NDd 35.1±0.04Aa 32.9±0.20Ab 30.6±0.52Ac
24 NDd 34.1±0.05Aa 32.6±0.14Ab 30.6±0.09Ac
1)
Con, control; non-fermented sesame; LP, fermented sesame with Lactobacillus plantarum P8; ST, fermented sesame with Streptococcus thermophilus
S10; LA, fermented sesame with Lactobacillus acidophilus ATCC4356
2)
Values are mean±SD (n=3); ND, not detected; Values with different superscripts capital letters are significantly different during fermentation time
(p<0.05). Values with different superscripts small letters are significantly different among strains (p<0.05); NS, not significant

Food Sci. Biotechnol.

 Fermentation of Sesame 203

phenolic compounds that possess antioxidant activity after oral

administration. As DPPH free radical scavenging assay has been
widely used to determine the antioxidant activity, DPPH was used to
verify the proton radical scavenging action of fermented and non-
fermented sesame in this study. DPPH radical scavenging activities of
sesame fermented with LAB are shown in Fig. 4. The DPPH free
radical scavenging activity of fermented sesame was significantly
(p<0.05) higher than that of non-fermented sesame. DPPH free
radical scavenging activities of fermented sesame were increased
significantly (p<0.05) during 24 h fermentation. The DPPH radical
scavenging assay revealed the highest activity (80.56±0.52) in
sesame fermented with LP at 24 h. Miyake et al. (11) have reported
that the medium that contains sesaminol triglucoside exhibits high
DPPH radical scavenging activity by culturing with Aspergillus.
Reducing power is closely related to antioxidant activity. In this
Fig. 3. Change in total phenol contents in fermented sesame during
different fermentation time. Con, control, non-fermented sesame; LP, method, depending on the reducing power of the compound, the
fermented sesame with Lactobacillus plantarum P8; ST, fermented yellow color of the test solution will change to green and blue.
sesame with Streptococcus thermophilus S10; LA, fermented sesame Reducing activity presents the capacity of giving electrons to active
with Lactobacillus acidophilus ATCC4356.
oxygen species. Compound containing Fe3+ and free radicals can yield
hydrogen to stabilize the free radical, thus producing Fe2+ (32,34).
has been used to remove proteins, sugars, sesaminol glycosides, and The changes of reducing power in fermented sesame during the 24 h
sesaminol. Suja et al. (31) have reported that 80% methanol is the of fermentation are shown in Fig 4. The reducing power of
most effective solvent to extract lignans from sesame. The total fermented sesame was significantly (p<0.05) increased during the
phenol contents appeared as GAE of fermented sesame with LAB are 24 h fermentation at 37oC. In addition, reducing power of fermented
shown in Fig. 3. It is known that the antioxidant activity of plant sesame was significantly (p<0.05) higher than that of non-fermented
corresponds to the content of their phenolic compounds (32). Total sesame. Reducing power of fermented sesame LP (2.16±0.01) was
phenol contents of fermented sesame were higher than those of the highest at 24 h of fermentation. Sesame fermented with LP
non-fermented sesame. Total phenol contents of fermented sesame contained the highest amounts of total phenolic compounds
were increased significantly (p<0.05) during the fermentation time. (14.79±0.01), that showed the highest reducing power among all
The total phenolic compounds had the highest amount in sesame samples. Additionally, the effects of anticancer, antithrombotic and
fermented with LP (14.79±0.01 mg/10 g GAE) at 24 h. Nakai et al. ACE inhibitory activity should be investigated for fermented sesame.
(33) have reported that sesaminol glucosides could be changed to To summarize, STG was converted into aglycone (sesaminol) due to

Fig. 4. Changes in antioxidant activities of fermented sesame during different fermentation time. Con, control, non-fermented sesame; LP,
fermented sesame with Lactobacillus plantarum P8; ST, fermented sesame with Streptococcus thermophilus S10; LA, fermented sesame with
Lactobacillus acidophilus ATCC4356.

February 2016 | Vol. 25 | No. 1

204 Bae et al.

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