The Comparative Evaluation of Fermented and Non-fermented Soybean Extract on Antioxidation and Whitening.

The present study was performed to compare the antioxidative and whitening activities of fermented soybean extract (FSB) and non-fermented soybean extract (SB). Antioxidative and whitening activities of FSB and SB were evaluated by the determination of DPPH, superoxide radical and hydroxyl radical scavenging activities, linoleic acid inhibition activity, and tyrosinase inhibition activity. FSB showed the higher effect than SB in the antioxidative activities. Also FSB showed the better effect than SB in whitening activity. These results demonstrated that the fermentation played a more excellent role than the non-fermentation in antioxidation and whitening. Therefore, this study suggested that FSB could be a useful cosmetic ingredient for antioxidation and skin whitening.

INTRODUCTION

Nowadays, the skin aging is attracting growing interest because the preservation of the youthful appearance of skin is the prerequisite for beauty (Rittié and Fisher, 2002; Zouboulis and Makrantonaki, 2011).

Skin aging was caused by various environmental, physical, chemical and mechanical insults (Makrantonaki and Zouboulis, 2008). It was divided into intrinsic aging and photoaging. Intrinsic aging of the skin is a naturally occurring process relating to chronological age, and photo aging results from cumulative sun exposure. It is commonly associated with increased dryness, roughness, laxity, dyschromia, telangiectasia, wrinkles, and irregular pigmentation (Trookman ; Zhong .

The causes and mechanisms of intrinsic aging are far less understood than photoaging (Oender . Photoaging of the skin is mediated by the sensitization of reactive oxygen species (ROS) and lipid peroxides (Miyachi, 1995; Peres . Among several reactive oxygen species, superoxide and hydroxyl radicals play important roles in photoaging (Kim .

Recently, many studies have focused on the development of the ROS- eliminating natural antioxidants which are not harmful to human body and have strong antioxidative effects (Kim . Soybean (Glycinemax (L.) Merrill) is one of the major crops containing antioxidant components such as phenolic acids, tocopherols, phytic acids, trypsin inhibitor, amino acid and isoflavones containing daidsein, genistein, daidzin and genistin (Hayes ; Lee . It has been reported that these nutritional components in soybean were associated with human health benefits such as decreased risks of various cancers, heart disease, cardiovascular disease, and increased antioxidative effects (Choi ; Wang .

Fermentation has been reported to cause a general improvement in the natural nutritive values of soybean products, increasing total soluble solids, vitamins, free fatty acids, soluble nitrogen, free amino acids and vitamins E and C (Frias ; Wang . For example, the antioxidative activities of fermented soybean products such as miso, tempeh and natto, inoculated with Aspergillus oryzae, Rhizopus oligosporum and Bacillus natto were significantly higher than those of non-fermented steamed soybean (Lin .

The present study was performed to make the comparative evaluation on the antioxidation and whitening activities of fermented soybean extract (FSB) and non-fermented soybean extract (SB). Antioxidation and whitening activities of FSB and SB were investigated by the determination of DPPH, superoxide radical and hydroxyl radical scavenging activities, linoleic acid inhibition activity and tyrosinase inhibition activity.

MATERIALS AND METHODS

Bacillus subtilis 168 (ATCC 33234, KCTC 2217) was obtained from Korean Culture Center of Micro oranisms (KCCM). B.subtilis 168 were maintained in Luria-Bertani broth (LB Difco Laboratories, Detroit, MI) at 35℃.

20 g of soybean (SB) was sterilized in the autoclave for 15 min and Bacillus subtilis 168 was proliferated for 3 hr. The sterilized SB was inoculated with 1% of Bacillus subtilis168 at absorbance of 1.0 at 600 nm, and the inoculated SB was fermented at 40℃ for 36 hr. The fermented SB was extracted by reflux-condensing in 80% ethanol for 4 hr. The extract of SB was concentrated with an evaporator and freezing-dried. The nonfermented SB was extracted by reflux-condensing in 80% ethanol for 4 hr. Then it was concentrated with an evaporator and freezing-dried.

The free radical scavenging activity was evaluated by method of Blosis (Zhu . One ml of sample and 0.5 ml of 1,1-diphenyl-2-picrylhydrazyl (DPPH) alcoholic solution (0.2 mM) were mixed and incubated in a test plate at 37℃ for 10 min. The absorbance at 517 nm was measured on automated microplate reader Synergy HT (Bio-Tek instruments Inc, Vermonts, USA).

This test was measured by method of superoxide radical scavenging (Kang . 10 μl of FSB and SB, 130 μl of 50 mM potassium phosphate buffer, and 10 μl of 20 mM menadion (Sigma-Aldrich, St. Louis, MO), were mixed and to which was added 50 μl of DCFH (Sigma-Aldrich, St. Louis, MO) solution. Fluorescence of DCF was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm for 30 min on microplate fluorescence spectrophotometer Synergy HT (Bio-Tek instruments Inc, Vermonts, USA).

This test was measured by method of hydroxyl radical scavenging (Kang . 10 μl of FSB and SB, and 190 μl of reagent A (540 μl of 10 mM FeSO4 and 20 ml of 1.35 mM H2O2 were mixed) were mixed and to which was added 50 μl of DCFH solution Fluorescence of DCF was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm for 40 min on microplate fluorescence spectrophotometer Synergy HT (Bio-Tek instruments Inc, Vermonts, USA).

This test was measured by method of autoxidation (Cho . The sample were settled in 1:250 volumes of D.D.W. 0.75 ml of pre-treated sample was added 3 ml of 10 mM linoleic acid (Fluka, Buchs, Switzerland) and the solution was left at 4℃ for 24 hours. 0.1 ml of its were added to 4.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate (Sigma-Aldrich, St. Louis, MO), and 0.1 ml of 20 mM iron chloride (I) (Sigma-Aldrich, St. Louis, MO). And then, it was kept at room temperature for 3 min. The absorbance at 500 nm was measured on automated microplate reader Synergy HT (Bio-Tek instruments Inc, Vermonts, USA).

15 μg of sample was add to 150 μl of 0.1 M PBS (pH 6.86), 2.5 μl of 1.5 mM Ltyrosinase (Sigma-Aldrich, St. Louis, MO) and 7 μl of mushroom tyrosinase (2,380 unit/ml), (Sigma-Aldrich, St. Louis, MO) at Elisa’ well. The absorbance at 490 nm was measured on automated microplate reader Synergy HT (Bio-Tek instruments Inc, Vermonts, USA). And then, it was kept at 30℃ for 3 min and absorbance at 490 nm was measured on automated microplate reader Synergy HT (Bio-Tek instruments Inc, Vermonts, USA).

Inhibition activity (%) = (A control - A sample)/A control × 100)

All data were expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Duncan’s post hoc test (SPSS Inc., Chicago, IL, USA).

RESULTS AND DISCUSSION

The optimal condition of fermentation was established by DPPH radical scavenging activity. DPPH free radical scavenging activities of fermented SB (FSB) at temperature and time are shown in Table 1. FSB at 40℃ showed much higher antioxidative activity than FSB at 30℃. The highest free radical scavenging effect was 83.5% at 36 hr. So the optimal condition of fermentation was 40℃ and 36 hr. Cell growth rates at different fermentation temperature in SB fermented by Bacillus subtilis168 are shown in Fig. 1. The rates of cell growth at 40℃ are higher than those of cell growth at 35℃.

Table 1.

DPPH radical scavenging effect of fermented soybean extract

Time	DPPH radical scavenging effect (%)
	FSB at 40℃	FSB at 30℃
6	52.17 ± 1.27a	35.58 ± 1.41b
12	59.90 ± 0.84b	35.12 ± 1.41b
24	64.50 ± 0.00c	58.75 ± 0.98f
30	81.76 ± 1.55g	69.25 ± 0.42g
34	80.07 ± 0.84f	74.08 ± 1.27h
36	83.50 ± 0.56h	60.12 ± 0.56f
48	79.46 ± 1.27f	43.16 ± 0.70e
54	72.50 ± 1.12e	31.62 ± 0.65a
60	74.00 ± 0.28e	38.25 ± 0.28cd
66	69.50 ± 1.41d	39.16 ± 0.14d
72	60.00 ± 0.00b	37.15 ± 0.02c

a, b, c, d, e, f, g, h are different group by one-way ANOVA followed by Duncan’s post hoc test.

Fig. 1.

Bacillus subtilis168 growths of fermented soybean extract. O.D : optical density, variation of O.D at 40℃ (■), variation of O.D at 30℃ (◆).

Free radicals are known to be a major factor in biological damages and DPPH has been used to evaluate the free radical scavenging activity of natural antioxidants (Yokozawa . DPPH radical scavenging activities of FSB, SB, and vitamin C were presented in Table 2. They showed the increasing scavenging activity in a dose-dependent manner from 1 to 100 μl/ml of FSB, SB, and vitamin C. DPPH scavenging activity of FSB was 20% higher than SB. In comparison with vitamin C (1 μl/ml), FSB (1 μl/ml) was much higher. It was reported that fermented Codonopsis lanceolata increased DPPH radical scavenging activity, compared to non-fermented Codonopsis lanceolata (Park, 2009). Antioxidative effect was showed in proportion to cell growth rate (Choi This study demonstrated the similar pattern of the result.

Table 2.

DPPH scavenging activity of fermented soybean extract, non-fermented soybean extract and vitamin C

Concentration (μl/ml)	DPPH scavenging activity (%)
	FSB	SB	Vitamin C
100	78.60 ± 0.38d	57.16 ± 0.35a	83.39 ± 1.02e
10	74.33 ± 3.06c	55.82 ± 0.79a	83.01 ± 1.39e
1	72.60±3.71c	55.44 ± 0.26a	66.79 ± 1.02b

FSB: fermented soybean extract, SB: non-fermented soybean extract, VitaminC : positive control, a, b, c, d, e are different group by one-way ANOVA followed by Duncan’s post hoc test.

Superoxide radical generates metabolic processes of many biological cells (Masaki . Superoxide anion radical is a precursor to active free radicals that have potential of reacting with biological macromolecules and thereby inducing tissue damage (Gülçin, 2006). Xanthine oxidase response with xanthine conversing uric acid, resulting superoxide radical (Kuppusamy and Zweier, 1989). The superoxide radical scavenging activities of FSB, SB, and vitamin C are shown in Table 3. FSB showed the higher scavenging activity of superoxide radical than SB and vitamin C.

Table 3.

O2- scavenging activity of fermented soybean extract, non-fermented soybean extract and vitamin C

Concentration (μl/ml)	O2- scavenging activity (%)
	FSB	SB	Vitamin C
100	89.73 ± 1.13g	45.72 ± 0.00c	76.41 ± 0.36f
10	80.14 ± 0.00f	30.26 ± 4.93b	69.39 ± 1.32e
1	48.79±5.70cd	18.58 ± 1.13a	53.36 ± 2.97d

FSB: fermented soybean extract, SB: non-fermented soybean extract, Vitamin C: positive control, a, b, c, d, e, f, g are different group by one-way ANOVA followed by Duncan’s post hoc test.

Hydroxyl radical was one of the highest reactive free radical, which was known to play important role in oxidation of various tissue and cell membrane in vivo (Heo and Wang, 2008). Hydroxyl radical scavenging activities are shown in Table 4. Scavenging activities of FSB in all kinds of concentrations were higher than SB and vitamin C. It was reported that the increase of radical scavenging activity resulted from physiological active substance such as peptide, maillard browing material and mucilages during the fermentation with multiple mechanisms (Shon .

Table 4.

OH- scavenging activity of fermented soybean extract, non-fermented soybean extract and vitamin C

Concentration (μl/ml)	OH- scavenging activity (%)
	FSB	SB	Vitamin C
100	79.19 ± 0.39f	44.76 ± 1.52c	65.66 ± 1.26e
10	63.85 ± 4.17f	36.54 ± 3.60b	32.35 ± 0.23b
1	51.93 ± 2.89d	30.22 ± 5.13ab	24.17 ± 1.57a

FSB: fermented soybean extract, SB: non-fermented soybean extract, Vitamin C: positive control, a, b, c, d, e, f are different group by one-way ANOVA followed by Duncan’s post hoc test.

Lipid peroxidation can be catalyzed by enzymes (lipoxygenases, cyclooxygenases) or initiated by sources of reactive oxygen species (Goupy . Linoleic acid also influences skin pigmentation by stimulating epidermal turnover and increased desquamation of melanin pigment from the epidermis (Ebanks . Table 5 shows the inhibition effect of linoleic acid of FSB, SB, and vitamin C. FSB showed the higher inhibition activity of linoleic acid than SB, but the lower than vitamin C.

Table 5.

Inhibition of autoxidation by using linoleic acid of fermented soybean extract, non-fermented soybean extract and vitamin C

Concentration (μl/ml)	Inhibition of autoxidation by using linoleic acid (%)
	FSB	SB	Vitamin C
100	79.60 ± 0.38d	57.16 ± 0.35a	83.78 ± 1.09e
10	74.33 ± 3.06c	55.85 ± 0.79a	82.43 ± 1.36e
1	72.60 ± 3.71c	55.44 ± 0.26a	66.79 ± 1.02b

FSB: fermented soybean extract, SB: non-fermented soybean extract, Vitamin C: positive control, a, b, c, d, e are different group by one-way ANOVA followed by Duncan’s post hoc test.

Tyrosinase (monophenol, 3,4-dihydroxyphenylalanine: oxidants, E.C.1.14.18.1), which catalyses the synthesis of the melanin precursor dihydroxyphenylalanine (DOPA), has been reported to be specific for melanocytes (Brichard . The inhibitory effects of FSB, SB, and vitamin C are shown in Table 6. Tyrosinase catalyzed biosynthesis of melanin which the hydroxylation of monophenols to o-phenols (monophenolase activity), and the oxidation of the o-phenols to o-quinones (diphenolase activity), both using molecular oxygen (Nerya . Antioxidants was considered preventing skin damage by theses oxidation progress. The inhibitory effects of FSB were higher than those of SB in the dose-dependent manner, which suggested that FSB could play a role in the skin whitening by the inhibition of melanin production and supported previous reports that antioxidants are good inhibitors of tyrosinase.

Table 6.

Tyrosinase inhibition of fermented soybean extract, non-fermented soybean extract and vitamin C

Concentration (μl/ml)	Tyrosinase inhibition (%)
	FSB	SB	Vitamin C
100	75.55 ± 0.38d	53.21 ± 4.36b	79.09 ± 0.58d
10	72.22 ± 3.06d	52.14 ± 4.56b	73.24 ± 0.44d
1	32.22 ± 3.71a	50.71 ± 6.06b	60.86 ± 2.52c

FSB: fermented soybean extract, SB: non-fermented soybean extract, Vitamin C: positive control, a, b, c, d are different group by one-way ANOVA followed by Duncan’s post hoc test.

Collectively, on the basis that the fermented soybean extract had the better effects than the non-fermented soybean extract in DPPH scavenging activity, superoxide radical, hydroxyl radical, inhibition of autoxidation and inhibitory tyrosinase, it could be concluded that the fermentation played the important role in the improvement of antioxidation and skin whitening.