Physicochemical Characteristics and Antioxidant Capacity in Yogurt Fortified with Red Ginseng Extract

Market milk and skim milk powder were purchased from Seoul Milk Co. (Korea), and red ginseng extract was purchased from Fine Korea Co. (Korea). Lactobacillus acidophilus and Streptococcus thermophilus was used as commercial yogurt starter culture (Culture Systems, Inc., USA). Pectin from citrus was purchased from Daejung Chemicals & Metals Co., Ltd. (Korea). Ascorbic acid and 3,5-di-tert-4-butylhydroxytoluene (BHT) were purchased from Sigma-Aldrich Corp. (USA).

Yogurts were produced with different concentrations of red ginseng extract (0.5, 1, 1.5, and 2%). Yogurt base was made of market milk and skim milk powder and 2% skim milk powder was fortified in milk. Yogurt base was mixed with red ginseng extract, and 0.1% pectin and then pasteurized at 90℃ for 10 min. Pasteurized milk was cooled and inoculated with 0.02% commercial yogurt starter culture containing L. acidophilus and S. thermophilus. Inoculated milk was incubated at 40℃ until a pH of 4.4 to 4.5 and then stored in a refrigerator (4℃) overnight.

Yogurt samples were incubated at 40℃ for 12 h and analyzed at 0, 2, 4, 6, 8, 10, and 12 h. Samples were analyzed pH, titratable acidity, and viable cell count. The pH value and titratable acidity were determined by using pH meter and the method of Collins et al. (1991), respectively. The viable cells of yogurt were determined by using selected medium. L. acidophilus was colonized on MRS medium containing 1% maltose and S. thermophilus was colonized on M17 medium containing 10% lactose (Ashraf and Shah, 2011). Also, yogurt samples were storaged for 31 d at 4℃ and collected at 1, 6, 15, 22, and 31 d. Samples were determined pH, titratable acidity, and viable cell counts of L. acidophilus and S. thermophilus.

The composition of market milk was determined by MilkoScan^TM minor (Type 78110; FOSS Analytical A/S, Denmark). The yogurt components, namely, lactose, protein, fat, moisture, total solids, and ash were measured by the Official Methods of Analysis of the Association of Official Analytical Chemists (AOAC, 2000). Color was measured using a colorimeter (Chroma Meter CR-400 series; Konica Minolta Sensing Inc., Japan).

The yogurt samples were centrifuged at 20,760 g at 4℃ for 30 min and the supernatants were then filtered through a 0.45 μm filter (cellulose acetate, Toyo Roshi Kaisha Ltd., Japan) for analysis of antioxidant activity. The supernatants were stored in freezer at −20℃ before determination of antioxidant activity. Ascorbic acid and BHT were used as a positive control at a concentration of 0.1 mg/mL.

A modified method of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was used to assay (Ye et al., 2013). The sample (200 μL) was mixed with 1 mL of 100 μM DPPH solution. The mixture was shaken and incubated for 15 min in the dark condition. The absorbance of the mixture was measured at 517 nm.

$\text{DPPH radical scavenging activity}\left(\%\right)=\left(1-{\displaystyle \frac{\text{Absorbance of sample}}{\text{Absorbance of control}}}\right)\times 100$

The antioxidant activity of the yogurt was evaluated by the modified β-carotene bleaching assay (Shon et al., 2003). A solution of β-carotene was prepared by dissolving 2 mg of β-carotene, 44 μL of refined linoleic acid, and 200 μL of Tween 80 in 10 mL of chloroform. Then, 5 mL of this solution was transferred to a round bottom flask and chloroform was removed using a rotary evaporator at 40℃. The obtained mixture was diluted with 100 mL of distilled water. Next, 4.5 mL aliquots were transferred to test tubes containing 0.5 mL of sample. Each tube was placed in a water bath at 50℃. The absorbance of the reaction mixture was measured at 470 nm until it was discolored.

$\text{Antioxidant activity}\left(\%\right)={\displaystyle \frac{\text{β-Carotene content after }6\text{ h}}{\text{Initial β-carotene content}}}\times 100$

The antioxidant activity of the yogurt samples was also determined by the ferric thiocyanate (FTC) assay with a modification of the procedure reported previously (Lee et al., 2004). Yogurt samples (100 μL), 200 μL of linoleic acid solution in ethanol (25 mg/mL), 400 μL of phosphate buffer (40 mM, pH 7.0), and 200 μL of distilled water were prepared as the reaction mixture. All mixtures were incubated at 37℃. At 0, 24, 48, and 72 h, 0.1 mL aliquots were taken from the mixtures and diluted with 4 mL of 70% ethanol, followed by addition of 0.1 mL of 30% ammonium thiocyanate and 0.1 mL of 20 mM FeCl₂ in 3.5% HCl. The absorbance was measured at 500 nm.

Sensory evaluation of samples was carried out at 1 d and samples were yogurt fortified with red ginseng extract (0.5, 1, 1.5, and 2%). A trained panel of 27 members are composed of adult male (13, age ranged from 20 to 30) and female (14, age ranged from 20 to 30). The sensory evaluations were carried out using quantitative descriptive analysis (Lawless and Heymann, 1999). The quantitative descriptive analysis was investigated sweetness, bitterness, sour taste, texture, flavor and after taste of yogurt and red ginseng extract having graphic line scale.

The obtained data were analyzed statistically using the SPSS statistical software program version 18 (SPSS Inc., USA). Analysis of variance (ANOVA) and Duncan’s multiple range tests were used to determine differences among results.

The physicochemical properties such as pH and titratable acidity for the studied yogurts are included in Fig. 1. The pH and titratable acidity were affected significantly (p<0.05). The pH value was decreased wherease titratable acidity was increased during fermentation. The pH of all samples was similar value in initial and final fermentation, but it was difference between yogurt and yogurt added with red ginseng extract (0.5, 1, 1.5, and 2%) during fermentation (Fig. 1A). The titratable aicidity was difference in initial value (Fig. 1B), however this value became similar after 8 h because of the reduction of pH value. Previous study reported that the pH value declined wherease titratable acidity rose because of the production of acid and this results were similar to our results (Bae and Nam, 2006).

Fig. 1. The changes of (A) pH and (B) titratable acidity in yogurt fortified with red ginseng extract during fermentation. ○ , control without red ginseng extract; ● , yogurt fortified with 0.5% red ginseng extract; □ , yogurt fortified with 1% red ginseng extract; ■ , yogurt fortified with 1.5% red ginseng extract; △, yogurt fortified with 2% red ginseg extract.

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The viable cells of lactic acid bacteria were determined as microbial property. Fig. 2 presents L. acidophilus and S. thermophilus in yogurt samples during fermentation. The viable cell counts of L. acidophilus and S. thermophilus ranged from 6.93 to 8.29 Log CFU/mL and 6.97 to 9.23 Log CFU/mL, respectively. The viable cells of L. acidophilus were similar level, however, the viable cell counts of S. thermophilus had a little increase in proportion to concentration of red ginseng extract (0.5, 1, 1.5, and 2%). Many researchers investigated about fermentation of ginseng and yogurt added with ginseng or red ginseng and their results had the similar pattern (Bae and Nam, 2006; Lee and Paek, 2003; Park et al., 2006). The growth of lactic acid bacteria including L. acidophilus KCTC3150, L. salivarius subsp. salivarius CNU27, Lactobacillus sp., Bifidobacterium longum MG723, and mixed strains (L. bulgaricus KCTC3188 and S. thermophilus KCTC3658) was influenced by concentration of ginseng and red ginseng. A small amount of red ginseng extract promoted the growth of lactic acid bacteria, however, a large amount inhibited the growth of them (Bae et al., 2005).

Fig. 2. The viable cells number of (A) Lactobacillus acidophilus and (B) Streptococcus thermophilus in yogurt fortified with red ginseng extract during fermentation. ○, control without red ginseng extract; ●, yogurt fortified with 0.5% red ginseng extract; □ , yogurt fortified with 1% red ginseng extract; ■ , yogurt fortified with 1.5% red ginseng extract; △, yogurt fortified with 2% red ginseg extract.

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The composition of yogurt base was 6.39% lactose, 3.78% protein, 3.72% fat, 14.43% total solids, pH 6.6, and 0.13% titratable acidity. The red ginseng extract consisted of 46.3% carbohydrate, 11.3% protein, and 0.8% fat. The composition of the yogurt supplemented with red ginseng extract was analyzed on 1 d. The lactose, protein, fat, moisture, total solids, and ash content of yogurt samples ranged from 1.51 to 2.24%, 3.93 to 4.73%, 2.60 to 3.17%, 84.43 to 85.55%, 14.45 to 15.57%, and 0.87 to 0.95%, respectively (Table 1). The component content of yogurt supplemented with red ginseng extract (0.5, 1, 1.5, and 2%) was significantly higher than that of control yogurt (p<0.05). When the concentration of red ginseng extract increased, the content of lactose, protein, total solids, and ash in yogurt increased, however, the content of fat and moisture decreased. Composition of yogurt added sour cherry changed that the increase of sour cherry concentration had increase of total solids content, but fat content decreased as our data (Şengül et al., 2012). Previous studies reported that fat content was decreased during fermentation because fat was changed to flavor compounds (Tuorila et al., 1993; Ye et al., 2013).

The color of yogurt supplemented with red ginseng extract (0.5, 1, 1.5, and 2%) was analyzed on 1 d (Table 2). The L, a, and b values indicate changes from darkness to lightness, greenness to redness, and blueness to yellowness, respectively. Compared to the yogurts, the L values decreased from 93.99 to 77.86, whereas a and b values increased from −3.04 to 2.46 and 6.13 to 22.42, respectively, when the concentration of the red ginseng extract increased from 0 to 2%. In correlation with the amount of red ginseng extract, lightness decreased, whereas redness and yellowness increased. Thus, the color values of yogurt were affected by the addition of red ginseng extract. Kim et al. (2008) found that color was influenced by the concentration of red ginseng extract.

The pH of the yogurt samples on 1 d ranged from 4.43 to 4.46 (Table 3). Significant differences were noted between the samples over the storage period, the pH of the yogurt decreased during storage (p<0.05) ranging from 4.15 to 4.29 on 31 d. The titratable acidity of control and yogurt supplemented with red ginseng extract increased significantly during the storage period (Table 3). There were no significant differences in titratable acidity between control and samples supplemented with red ginseng extract. The viable cell counts of L. acidophilus and S. thermophilus in yogurts supplemented with red ginseng extract were determined for 31 d. The changes of lactic acid bacteria of yogurt samples are represented in Table 3. At 1 d of storage, the microbial counts of L. acidophilus and S. thermophilus were not significantly different between control and yogurt supplemented with red ginseng extract; they ranged from 9.04 to 9.49 Log CFU/mL and 9.00 to 9.30 Log CFU/mL, respectively. The viable cell counts of L. acidophilus and S. thermophilus were decreased slightly during storage time, and they were ranged from 8.84 to 9.04 Log CFU/mL and 8.76 to 9.04 Log CFU/mL on 31 d.

The antioxidant activity of yogurts supplemented with red ginseng extract was determined by the DPPH radical scavenging activity, β-carotene bleaching activity, and FTC test.

The DPPH radical scavenging activity is easy and potentially accurate with regard to measuring the free radical scavenging activity of beverages. This methodology is based on the reduction of the stable free radical DPPH that strongly absorbs at 517 nm, to the corresponding hydrazine by the free radical scavenging activity of the antiradicals (Sendra et al., 2006). The results for the DPPH radical scavenging activity of the yogurt are presented in Table 4. The antioxidant activity of yogurt supplemented with red ginseng extract (89.44 to 94.80%) was significantly higher than that of control sample (63.63%). The DPPH radical scavenging activity of the yogurt increased in proportion to increasing red ginseng extract concentration. Significant differences in the DPPH radical scavenging activity were found among the yogurt (p<0.05). The antioxidant activity of red ginseng extract (0.5, 1, 1.5, and 2%) was 67.14, 91.41, 92.67, and 91.21%, respectively. The DPPH radical scavenging activity of 0.1 mg/mL of ascorbic acid was 96.29±0.18%. The skim milk fermented with Lactobacillus plantarum Gr1 and Gr2 showed DPPH radical scavenging activity of 50% (Abubakr et al., 2012). However, our data showed that milk with skim milk powder fermented with L. acidophilus and S. thermophilus showed strong DPPH radical scavenging activity over 94%. Red ginseng extract had DPPH radical scavenging activity and the value was different according to extraction method. Also, DPPH radical scavenging activity of red ginseng extract depended on concentration like our results (Kim et al., 2008). The antioxidant activity remained over 85% in yogurt supplemented with red ginseng extract during storage. Amirdivani and Baba (2011) and Şengül et al. (2012) found that herbal yogurt and yogurts supplemented with sour cherry pulp had higher antioxidant activity than plain yogurt and that the antioxidant activity increased during 7 d of storage.

The β-carotene bleaching assay is widely used to measure antioxidant activity. It is based on the principle that lipid radicals as auto-oxidation products of linoleic acid attack double bonds of β-carotene, leading to its oxidation and discoloration; however, in the presence of antioxidative substances and depending on their antioxidant ability, β-carotene retains its yellowish-orange color (Kato et al., 2009). The total antioxidant activity of 5 yogurt samples is presented in Table 4. The high absorbance values indicated that the yogurts possessed antioxidant activity. Yogurt samples supplemented with red ginseng extract had higher antioxidant activity than that of control yogurt; and the results were significantly different (p<0.05). The antioxidant activity of red ginseng extract (0.5, 1, 1.5, and 2%) was 16.29, 19.97, 25.09, and 26.38%, respectively. Antioxidant activity of BHT (0.1 mg/mL) was 80.91±2.42% by β-carotene bleaching assay.

The FTC test was based on determination of the amount of peroxide produced at the initial stage of lipid peroxidation, a lower absorbance indicates a higher level of antioxidant activity (Lee et al., 2004). Fig. 3 shows the changes in the absorbance for each fraction during incubation at 37℃. The absorbance of the control and test samples increased in proportion to the incubation time. The antioxidant activity of red ginseng extract-supplemented yogurt was always higher than that of control yogurt. Comparison of the different yogurt revealed that the antioxidant activity increased depending on the concentration of red ginseng extract, ranging from 38.41 to 92.80% on 1 d. However, a decrease in the antioxidant activity during storage was observed. On 6 d, yogurt did not show antioxidant activity, but yogurt supplemented with red ginseng extract showed antioxidant activity, ranging from 23.64 to 75.06%. The yogurts added with red ginseng extract had antioxidant effect until 22 d. BHT (0.1 mg/mL) had 76.89±0.69% antioxidant activity in FTC test. The previous study reported that whey of skim milk fermented with L. plantarum Gr1 and Gr4 was measured by ferrous chelating activity (FCA) and the FCA value was 94% (Abubakr et al., 2012).

Fig. 3. Antioxidant activity of yogurt fortified with red ginseng extract during storage at 4℃ determined by ferric thiocyanate assay. (A), 1 d; (B), 6 d; (C), 15 d; (D), 22 d; (E), 31 d. ○, control without red ginseng extract; ●, yogurt fortified with 0.5% red ginseng extract; □ , yogurt fortified with 1% red ginseng extract; ■ , yogurt fortified with 1.5% red ginseng extract; △, yogurt fortified with 2% red ginseg extract.

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The yogurt samples were mixed with syrup that decreases bitter taste of red ginseng extract. Results from sensory evaluation did not show significant differences (p>0.05) in yogurt fortified with red ginseng extract (data not shown). An increase in red ginseng extract concentration resulted in increased bitterness, flavor of red ginseng extract, and after taste of red ginseng extract. However, the sweetness, flavor of yogurt, and after taste of yogurt were felt weakly.