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Quality Characteristics and Antioxidant Activity of Yogurt Supplemented with Aronia (Aronia melanocarpa) Juice

We investigated the quality characteristics and antioxidant activities of yogurt supplemented with 1%, 2%, and 3% aronia juice and fermented for 24 h at 37°C. The total acidity increased with increasing levels of aronia juice and incubation time. Lightness and yellowness of the yogurt decreased, but redness increased, with increasing aronia juice content and incubation time. The number of lactic acid bacteria (LAB) increased with increased incubation time, and yogurt containing 2% and 3% aronia juice showed higher LAB counts than 1% aroinia juice-supplemented yogurt. The total polyphenol and flavonoid contents increased proportionally with increasing levels of aronia juice. Antioxidant activity of aronia-containing yogurt was significantly higher than that of the control and increased proportionally with aronia juice concentration. Yogurt with 2% aronia juice had the best taste (P<0.05). Aronia juice may be a useful additive for improving the taste and antioxidant potential of yogurt.


INTRODUCTION
Yogurt is a coagulated milk product obtained from fermentation by lactic acid bacteria (LAB), Streptococcus thermophilus and Lactobacillus delbrueckii spp. bulgaricus (1). Yogurt is traditionally consumed as a healthy food due to its high nutritional value and health benefits as well as its sensory properties (2,3). Yogurt is a rich source of bioactive peptides with antioxidant activity that are produced during fermentation (4). Compared with milk, yogurt is more nutritious and is an excellent source of protein, calcium, phosphorus, riboflavin, thiamin, vitamin B 12 , folate, niacin, magnesium, and zinc (3). Probiotic bacteria can tolerate acid and bile and can survive in the intestinal tract. Thus, they have a beneficial effect on intestinal function and offer health benefits such as decreasing cholesterol absorption, reducing blood pressure, and ameliorating intestinal disorders and chronic diseases (5). Since lactose in milk is converted to lactic acid during fermentation and yogurt contains lactose-fermenting bacteria, lactose intolerant people can consume yogurt without adverse effects (6). Moreover, consumption of fermented milk products causes a slight reduction in stomach pH, which reduces the risk of pathogen transit and ameliorates the effects of low gastric juice secretion (7). Aronia (Aronia melanocarpa) berries, also known as black chokeberries, contain many bioactive compounds, including anthocyanins, carotenoids, fatty acids, flavonoids, phenolic compounds, and vitamins (8,9). Originating from the eastern parts of North America, aronia berries have been cultivated and medicinally used in the former Soviet Union since the middle of the 20th century. The health-promoting potential of this berry is widely recognized, and increasing numbers of preclinical and clinical studies support the health benefits of aronia (10,11). Aronia berries have received significant attention in recent years due to their antioxidant, anti-inflammatory, and anti-cancer activities, which are associated with a reduced risk of high blood pressure, cardiovascular diseases, and cancer (12,13).
Usually, yogurt is flavored by adding natural ingredients or synthetic flavor compounds (14). Yogurt is mainly pigmented and flavored by adding fruit juices or pulp from berry fruits such as strawberries, blueberries, and raspberries, which provide natural color and flavor as well as bioactive compounds. Fruit juices, powders, and extracts have potential as functional ingredients in the food industry, including the dairy sector (15). The effects of different types of plant materials added as functional ingredients on the quality and antioxidant properties of yogurt have been studied (16,17), but aronia has not been studied in this context. The objective this study was to determine the quality and sensory characteristics, bioactive compound contents, and antioxidant activities of yogurt supplemented with aronia juice.
Preparation of aronia juice Aronia (Aronia melanocarpa cv Nero) berries were harvested at optimal ripeness from a local farm in Yeongcheon, Korea in August of 2015. The berries were washed with tap water, ground with a food grinder (HMF-3260S, Hanil, Seoul, Korea), and filtered with cheese cloth 2 times to obtain juice.

Preparation of yogurt
To prepare aronia yogurt, a mixture of market milk (450 mL) and skim milk (50 mL) was heated until boiling, then was cooled until it reached 30ºC. To the mixture, aronia juice and oligosaccharides were added at 0%, 1%, 2%, and 3%. The mixture was well-mixed and inoculated with 0.4% Kefir starter powder which consisted of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus lactis, and yeast as starters, and the mixture was incubated at 37 o C for 24 h. Samples were collected after 0, 3, 6, 12, and 24 h for analysis of several parameters. For the sensory evaluation, yogurts stored at 4 o C after 24 h incubation were used. The mixing ratios for yogurt fortified with different amounts of aronia juice are presented in Table 1.
Changes in pH, titratable acidity, and sugar contents of yogurt during incubation The pH values of yogurt were measured during fermentation using a calibrated glass electrode pH meter (420 Benchtop, Orion Research Inc., Beverly, MA, USA). For determining titratable acidity, 10 mL of yogurt was titrated with 0.1 M sodium hydroxide solution. The titratable acidity was expressed as gram of lactic acid/100 g of yogurt and was calculated using the following equation:

Total acidity=
V×NaOH factor×A×D ×100 volume of sample where V is volume of NaOH added (mL), A is conversion factor (0.009 for lactic acid), D is dilution factor, and F is factor of 0.1 N NaOH. Sugar content was measured using a refractometer (PR-201α, ATAGO Co., Ltd., Tokyo, Japan) calibrated with distilled water. Sugar content was expressed as o Brix.

Color determination
Changes in yogurt color during fermentation were measured using a colorimeter (Chrome meter CR-400, Minolta, Osaka, Japan) calibrated with a standard calibration slide. L, a, and b values for standard tile were 97.10, +0.24, and +1.75, respectively. Results were expressed as L * (lightness/darkness), a * (redness/greenness), and b * (yellowness/blueness).
Determination of LAB count MRS agar was used to quantify the viable LAB in the yogurt. Yogurt (10 g) was diluted 10 times with 0.85% saline solution and homogenized with a Stomacher (Stomacher R400, INTERSCIENCE, Saint-Nom-la-Bretèche, France) for 2 min at speed 4. After further serial dilution, 1 mL of sample was added to MRS agar in petri dishes and then incubated for 48 h at 37 o C. Colonies appearing on the plates were counted, multiplied by the dilution factor, and expressed as the log colony forming units per milliliter (log CFU/mL).
Determination of total phenolic contents Total polyphenol content was measured with Folin-Ciocalteu's phenol reagent using the method described by Zhou et al. (18) with slight modifications. Briefly, 200 μL of the appropriately diluted sample was mixed with 400 μL of 2% 2 N Folin-Ciocalteu's phenol reagent. After incubation for 3 min at room temperature, 800 μL of 10% Na2CO3 was added to the mixture. The mixture was covered with aluminum foil and kept in the dark at room temperature for 1 h. The mixture was vortexed, and the absorbance was measured at 750 nm with a microplate reader (Infinite M200 Pro, Tecan Systems Inc., San Jose, CA, USA). A standard curve was created using gallic acid, and results were expressed as gallic acid equivalents (GAE) per gram of dry weight (mg/g GAE).
Determination of total flavonoid content The total flavonoid contents were measured using the method described by Woisky and Salatino (19) with slight modifications. Briefly, 500 μL of sample was mixed with 30 μL of 5% NaNO 2 , and the solution was incubated for 6 min at room temperature. Then, 60 μL of 10% AlCl3･6H2O was added, and the mixture was again incubated at room temperature for 6 min. NaOH (1.0 M, 200 μL) was added to the mixture. Finally, 110 μL of distilled water was added, and the solution was mixed. The absorbance of the colored flavonoid-aluminum complex was measured immediately at 510 nm with a microplate reader. A standard curve was generated using catechin, and results were expressed as catechin equivalents (CE) per gram of dry weight (mg CE/g).

DPPH assay
The DPPH radical scavenging activities of yogurt samples were determined using the method of Cheung et al. (20) with slight modifications. First, 192 μL of 50 μM DPPH was mixed with 48 μL of diluted sample. The mixture was covered with aluminum foil and kept in the dark at room temperature for 30 min. The control consisted of 48 μL distilled water in 192 μL of 50 μM DPPH for the ascorbic acid standard or 48 μL of 94% ethanol in 192 μL of 50 μM DPPH for the test samples. Decolorization of DPPH was measured at 517 nm with a microplate reader. The DPPH radical scavenging activity was calculated according to the following equation: where Acontrol is the absorbance of the control reaction (containing all reagents except the test compound) and A sample is the absorbance of the test compound.

ABTS assay
The ABTS radical scavenging activities of yogurt samples were determined using the method of Re et al. (21) with slight modifications. First, ABTS was dissolved in distilled water to a concentration of 7 mM. ABTS radical cations were produced by reacting the ABTS stock solution with 2.45 mM K 2 S 2 O 8 (in a 2:1 ratio) in the dark, covered with aluminum foil, for 24 h before use. The ABTS reagent was diluted with 94% ethanol to the ap-propriate absorbance (0.17±0.03), which was measured at 734 nm. The ABTS reagent (950 μL) was mixed with 50 μL of the indicated concentrations of test samples. The mixture was covered with aluminum foil and kept in the dark at room temperature for 10 min. The absorbance at 734 nm was recorded with a microplate reader. Each sample was measured in triplicate, and percent inhibition was calculated using the following equation: where Acontrol is the absorbance of the control reaction (containing all reagents except the test compound) and A sample is the absorbance of the test compound.

Reducing power assay
The Fe 3+ reducing power of yogurt samples was determined using the method of Oyaizu (22) with slight modifications. Various concentrations of samples (0.25 mL) were mixed with 0.25 mL of 0.2 M phosphate buffer (pH 6.6) and 0.25 mL of 1% (w/v) K3Fe(CN)6. The mixture was incubated in a 50 o C water bath for 20 min, and the reaction was stopped by adding 0.25 mL of 10% (w/v) trichloroacetic acid solution. The solution was then centrifuged at 3,000 rpm for 10 min. The supernatant (0.5 mL) was mixed with 0.5 mL of distilled water and 0.1 mL of 0.1% (w/v) FeCl3 for 10 min. The reducing power was determined by measuring the absorbance at 700 nm with a microplate reader. A standard curve of ascorbic acid was generated with concentrations ranging from 0 to 200 μg/mL.

Sensory evaluation
Yogurt samples were subjected to sensory evaluation by 20 panelists after 24 h of fermentation and storage at 4ºC. Each panelist was asked to rank the 4 samples for color, flavor, taste, mouth feel, thickness, and overall acceptance using a 9-point hedonic scale, ranging from 1 (dislike extremely) to 9 (like extremely). All samples were placed into 50 mL white plastic cups with random 3 digit codes and were served to the panelists in a random order. Water was provided for panelists to rinse their mouth between samples.

Statistical analysis
The results are presented as mean±standard deviation. Statistical analysis was performed with the statistical analysis system (version 22.0, SPSS Statistics software package, IBM, Armonk, NY, USA). Data were compared using one-way analysis of variance; P<0.05 was considered significant.

RESULTS AND DISCUSSION
Changes in pH, titratable acidity, and sugar contents of yogurt during incubation The pH values of yogurt during incubation for 24 h at 37ºC are presented in Table 2. An overall decline in yogurt pH occurred during the 24 h period. The initial pH values for all samples were 6.33∼6.46, with control showing higher pH values than samples with aronia juice added. The pH began to drop significantly after 6 h of fermentation. The pH gradually decreased during fermentation and reached 4.25∼4.29 in yogurt containing 1∼3% aronia juice after 24 h incubation. In the yogurt samples containing aronia juice, pH values were lower than in the plain yogurt with no aronia juice. Reductions in pH were essentially proportional to the aronia juice concentration. Aronia-containing yogurt had faster rates of pH reduction than control yogurt. Aronia juice contains several acidic compounds such as acetic, malic, succinic, citric, tartaric, and phosphoric acids (23,24), and these acids may contribute to the lower pH values in yogurts containing aronia juice. The increase in total acidity, which represents the amount of lactic acid present in the yogurt during fermentation, is shown in Table 2. In general, the total acidity increased during incubation, and this upward trend was almost linear compared to the pH change. From 12 h of incubation onward, the difference between control and aronia yogurt became more pronounced. The yogurt fortified with 2% and 3% aronia juice showed the great-er increases in total acidity from 6∼24 h of fermentation. Total acidity of the control sample was 1.01%, while yogurt containing 2% and 3% aronia juice had 1.13% and 1.16% total acidity after 24 h incubation, respectively.
The sugar content of yogurt samples during incubation at 37ºC is presented in Table 2. The initial sugar content in yogurt samples was 23.45∼23.50 o Brix and there were no significant differences between control and aronia juice yogurt samples. Sugar content was reduced during fermentation in each yogurt sample. Until 6 h of fermentation, sugar contents were not changed much in any treatment, but after 12 h of fermentation, sugar content dropped significantly. The sugar content in control yogurt was 15 (26). This is the reason why yogurt containing 2∼3% aronia juice had greater sugar content than control yogurt or yogurt containing 1% aronia juice.

Color evaluation
Color properties of the yogurt samples are shown in Ta   The b * value in yogurt with 3% aronia juice also increased from 0.88 to 2.00 after 24 h of fermentation. Overall, the results showed that the yogurts supplemented with aronia juice were redder, darker, and less yellow than control yogurt. Aronia is a dark purplish fruit (8) and its color undoubtedly contributed to the color of yogurt by decreasing the L * and b * values and increasing the a * value. Table 4 shows the LAB counts in yogurts fortified with various amounts of aronia juice. The LAB counts were 6.45∼6.46 CFU/mL at 0∼3 h in yogurt samples, and the initial number of LAB was not different among samples. After 3 h of incubation, the number of LAB increased rapidly in both control and aronia juice containing samples. The LAB count after 3 h of incubation was 9.00∼ 9.59 log CFU/mL, and yogurt containing 2% and 3% ar-   (17). Aronia juice does not inhibit the growth of LAB and actually improves the growth of LAB during the incubation from 0∼24 h, possibly by providing some sugars for LAB growth. Previous studies showed that adding flowering cherry into yogurt improves LAB growth (16), but adding acanthopanax powder to yogurt prevents LAB growth (17). Some plant materials contain antimicrobial compounds that may affect LAB growth (17). Table 5 shows the total polyphenol and flavonoid contents of yogurt fortified with different amounts of aronia juice after 24 h incubation. The highest total polyphenol content (54.05 mg GAE/g dry weight) was found in the yogurt fortified with 3% aronia juice, and the lowest polyphenol content (16.34 mg GAE/g dry weight) was found in the control yogurt. The total polyphenol content increased 2.5-and 3.3-fold in yogurt containing 2% and 3% aronia juice, respectively. The total flavonoid content in the control yogurt was 117.71 mg CE/g, while yogurt containing 1∼3% aronia juice had higher flavonoid contents than the control, with values ranging from 122.40 to 152.10 mg CE/g. Compared to the control, the total flavonoid content increased 1.2-and 1.3-fold in yogurt with 2% and 3% aronia juice, respectively.

Total phenolic and flavonoid contents
Incorporation of aronia juice into yogurt resulted in higher polyphenol and flavonoid contents. Aronia contains a relatively high amount of polyphenols, particularly tannins (27,28), and a previous study reported the phenolic content of aronia to be 690.2 mg/100 g fresh weight (29).

Antioxidant activity
The antioxidant activities of yogurt samples containing various amounts of aronia juice are shown in Table 6. Aronia-containing yogurt samples had higher antioxidant activity than plain yogurt. The DPPH radical scavenging activity of plain yogurt was 59.47%, and DPPH radical scavenging activity of yogurt increased in an aronia juice concentration-dependent manner. The average inhibition of DPPH radical formation in yogurt containing 2% and 3% aronia juice was 72.26% and 77.87%, respectively. The average inhibition of ABTS radical formation in plain yogurt was 45.96%, which increased to 59.98% in yogurt with 1% aronia juice added. The average ABTS radical scavenging activities of yogurt containing 2% and 3% aronia juice were 69.79% and 70.90%, respectively. The reducing power activity in control yogurt was 27.62%, which increased up to 29.55∼29.86% in yogurt with aronia juice added, but there was no statistically significant difference between yogurt with aronia juice and control yogurt. The higher antioxidant activity in aronia-containing yogurt might result from the phyto-chemical content of the juice and microbial metabolic activity. Table 7 shows the sensory scores of the yogurt samples fortified with various amount of aronia juice. Fortifying yogurt with aronia juice was associated with a statistically significant effect on sensory parameters such as color, taste, and overall acceptance. The color score of control showed the lowest value of 3.00 while scores increased with increasing amounts of aronia juice. The color scores were 4.45, 6.35, and 7.35 in yogurt containing 1%, 2%, and 3% aronia juice, respectively. Aronia contains a purple color from anthocyanins, which positively contributes to yogurt color. The taste score of yogurt with 2% aronia juice showed the highest score of 6.00, while taste scores for yogurt with 1% and 3% aronia juice were 5.50 and 5.74, respectively. However, there were no statistically differences in flavor, mouth feel, thickness, or overall acceptance between control yogurt and aronia-containing yogurt. Aronia itself usually has bitter or astringent taste, but even 3% aronia juice in the yogurt did not have adverse effects on flavor, mouthfeel, thickness, or overall acceptance of the yogurt. Moreover, aronia contained several bioactive compounds that may contribute to the antioxidative effects. The addition of 1∼3% aronia juice into yogurt is recommended for health benefits.