Prospecting Antioxidant Capacities and Health-Enhancing Phytonutrient Contents of Southern Highbush Blueberry Wine Compared to Grape Wines and Fruit Liquors

Blueberry wines may have a multitude of health benefits, but few studies have quantified the health-enhancing antioxidants, total phenols, anthocyanins and flavonoids in blueberry wines, especially the Southern highbush blueberry wine, in comparison to grape wines and fruit liquors. This study was initiated to fill such a gap by measuring the antioxidant capacity and key phytonutrients of Southern highbush blueberry wine as compared to red, Rose and white wines and fruit liquors. The Oxygen Radical Absorbance Capacity (ORAC) of the Southern highbush blueberry wine tested in this study ranged from 18.54 to 25.48 mmol TE/L, with an average of 22.57 ± 2.92 mmol TE/L. This was higher than the ORAC values of over 80% of the red wines and 100% of the Rose and white wines reported in literature. A majority of the red wines were higher, but all the Rose and white wines and most fruit liquors were lower in total phenolic content than the Southern highbush blueberry wine. Anthocyanin contents of the blueberry wines were generally comparable to those of the red wines. Results show that blueberry wines could be more potent than most red, Rose and white wines in health enhancement and disease prevention from the antioxidant perspective.


Introduction
Blueberry (Vaccinium spp.) is a popular natural food product consumed worldwide.For centuries, native American tribes have used the leaves, roots, and fruits from the blueberry plant for medicinal purposes (Sanchez-Moreno et al., 2003), and blueberries continue to be used in many types of dietary health products as pharmaceutical or food supplements in modern society (Kalt & Dufour, 1997).In the U.S., blueberries rank only second to strawberries in terms of berry consumption, while in Canada, blueberry is the largest fruit crop grown nationally, accounting for over ½ of entire Canadian fruit acreage (Scrivener, 2008).
In North America, the most commonly cultivated species is the (Northern) highbush blueberry, but with adaption to the Southern U.S. climate, its hybrid like the Southern highbush blueberry, which was developed by crossing northern highbush varieties from Michigan and New Jersey with wild blueberries native in Florida and other southeastern states (Williamson & Lyrene, 2004), has been created and quickly spread to Florida, Georgia, California, and even the Mediterranean regions of Europe, Southern Hemisphere countries and China.According to the U.S. blueberry (cultivated and wild) production and utilization data (USDA, 2009), of the total utilized production of 204 million tons in 2009, 101 million tons were for fresh use, while 103 million tons were processed, including blueberry wine production.
Since blueberries are on the top of most everyday fruits grown or marketed in North America in terms of antioxidant capacities (USDA, 2010), blueberry wines also contain significant amounts of phenols and has significantly higher antioxidant activity compared to many other wine products (Saucier et al., 1999).Antioxidants are critical to health protection by their ability to combat the free radicals that can cause damages to cellular structures or DNA.Literature shows a multitude of health benefits of blueberries and blueberry wine.
Due to the presence of anthocyanin, blueberries can considerably improve cardiovascular health.The anthocyanin and resveratrol contents in blueberry, which have natural cardiovascular protective qualities, are comparable or higher than in red wine (Sanchez-morendo et al., 2003).Also, blueberry wine has marginally higher antioxidant ability than red wine and much higher antioxidant capacity than white wine (Sanchez-morendo et al., 2003), so blueberry wine may be more potent than red wine in preventing heart diseases.
Literature shows that blueberries have improved the learning capacity and motor skills in aging animals, thus reducing the chances of dementia or Alzheimer's (Robert et al., 1977;Joseph et al., 1999;Shukitt-Hale et al., 1999;Greenwell, 2000).Blueberries have also been found to offer protection from urinary tract infections caused by bacteria adhering to the mucosal lining of the bladder or urinary tract (Ofek et al., 1991;Jepson & Craig, 2007).Highly beneficial to the digestive and excretory system, antioxidants in blueberries help combat free radicals that can cause inflammation on the digestive pathways, and thus prevents occurrence of peptic ulcers (Watson, 2001).Eating blueberries, drinking blueberry juice, or taking blueberry wine in an appropriate quantity may help heal the existing ulcer or hemorrhoid condition (Seeram et al., 2008).
In addition, blueberries can improve day or nighttime vision, reduce the restoration time after exposure to glare, and prevent weakness of eyes (macular degeneration) arising from aging.The various phytonutrients present in blueberries, such as anthocyanin, flavonoids and phenolics, part of which are carried forward to wine during fermentation, are known for their benefits to the eyes and canhelp protect against occurrences of "Cataracts" (Robert et al., 1977;Greenwell, 2000).Studies also show that some compounds, such as polyphenols (flavonoids, proanthocyanidins, ellagitannins, etc.), stilbenoids,lignans and triterpenoids, present in blueberries are capable of inhibiting cell proliferation in the human breast, colon and ovarian cancers (Damianaki et al., 2000;Seeram et al., 2008).
Blueberry wine is usually produced by fermentation through nontraditional methods.During blueberry wine processing, an initial press of the berries provides the juice used for fermentation along with its skin and seeds.Sulfur dioxide and pectin enzymes are usually added.After the primary fermentation, usually 2-3 weeks, wine is separated from the dregs on a free run basis and the residuals are lightly pressed to extract the remaining wine.
During fermentation, phenolic compounds including catechins and other components, flavonoids and anthocyanins are transferred from the juice, skins or seeds into the blueberry wine.Su and Chien (2007) reported that the blueberry wine making process did not really lower the anthocyanin content.There are only a few studies that have quantified the antioxidant capacity, anthocyanins and total phenols in blueberry wines, e.g., Sanchez-Moreno et al. (2003), Su and Chien (2007) and Johnson et al. (2011).However, no analytical quantification of the foregoing antioxidant activity and phytonutrients was found in literature for the Southern highbush blueberry wine.Therefore, the objective of this study was to prospect the antioxidant capacity and key phytonutrients of Southern highbush blueberry wine and systematically compare with grape wines and fruit liquors.

Southern Highbush Blueberry Wine Samples
Southern highbush blueberry wine was obtained from a local blueberry winery for two batches of experiments conducted at the University of Florida.The blueberries used to produce the wine were a mixture of several cultivars including Star, Windsor, Emerald, Primadonna and Jewel.In the first batch, three 750 mL bottles were obtained for each of the different processing conditions: low sulfite, unfiltered; sulfite, unfiltered; and sulfite, filtered.Low sulfite consisted of 30 ppm total sulfites, while normal levels of sulfite were 150 ppm total sulfites.Filtering was performed first with a crude filter (>1 μm) with subsequent filtration by a fine filter (>0.45 μm).Two 10 mL portions of wine from each bottle were sampled for analysis, making 6 total observations for each wine.In a second study, 12 liters of non-sulfite, filtered wine, which was eligible for labeling as "organic," were obtained from the same local winery.

Determination of Antioxidant Capacity
Antioxidant capacity of the Southern highbush blueberry wine was measured by the Oxygen Radical Absorbance Capacity (ORAC) method.The ORAC values were expressed as mmol Trolox equivalent (TE) per liter.
For the filtered blueberry wine with no sulfite added, the ORAC values were determined using a modified method per Huang et al. (2002).Briefly, 50µL ORAC Phosphate Buffer (75mM, ORAC-PB) and appropriately diluted samples were added to a 96-well black plate (Fisher Scientific, Pittsburg, PA).This was followed by addition of 100µL, 20nM fluorescein working solution to all filled wells.The mixture was incubated at 37 o C for 10 min before the addition of peroxyl radical generator 2,2'azobis (2-amidinopropane) dihydrochloride (140mM, AAPH).The rate of fluorescence decay was monitored over time by calculating the area under the fluorescent decay curve and quantified using a standard curve of TROLOX, using a Spectra Max Gemini XPS microplate reader (Molecular Devices, Sunnyvale, CA).The fluorescence was monitored at 485 nm excitation and 530 nm emissions for 40 min at 1 min intervals.The antioxidant capacities of the extracts were expressed as mmol TE/L of blueberry wine.

Determination of Total Phenols
Total phenols were determined using a modification of Folin-Ciocalteu's method.To a 96 well clear plate (Fisher Scientific, Pittsburg, PA), 12.5µl of 2N Folin-Ciocalteu's phenol reagent was added to 50 µl of deionized distilled water (ddH 2 O) and 12.5µl of wine sample.After 5 min, 7% sodium carbonate (Na 2 CO 3 ) solution (125µl) was added to the mixture and incubated (90 min, 25 o C).The absorbance of the sample was measured at 750 nm versus a reagent blank using a microplate reader.A standard curve for total phenolics was developed using gallic acid.The concentration was expressed as mg of gallic acid equivalents (GAE)/L of wine.

Determination of Total Flavonoids
A standard colorimetric assay (Kim et al., 2003) with slight modifications was used to quantify total flavonoid content.To a 96 well clear plate, 25µl of the wine sample was added to 125 µl of ddH 2 O. Subsequently, 7.5µl of 5% sodium nitrate (NaNO 2) was added to the mixture and allowed to stand for 5 min.Fifteen microliters of 10% aluminum chloride (AlCl 3 ) was added to the mixture and incubated at ambient temperature for an additional 5 min.Following that, 50 µl of sodium hydroxide (1M, NaOH) were added to the mixture and immediately diluted by the addition of 27.5µl of ddH 2 O.The absorbance of the mixture was measured at 510nm against a reagent blank and compared to a catechin standard using a microplate reader.The total flavonoids was expressed as mg of catechin equivalents (CE)/L of wine.

Statistical Analysis and Comparison
For the blueberry wine data obtained in this study, significant differences were analyzed using 1-way ANOVA, with means separated by the Tukey's Studentized Range test, using SAS version 9.0 at a 0.05 significance level.
For the comparison among blueberry wines, grape wines and fruit liquors, a literature search was conducted to gather the reported data on ORAC, total phenols and anthocyanins and tabulate them together with the measured values from this study that were referenced as a benchmark value.A percentage was calculated for the wines or fruit liquors that were above or below the benchmark values of the blueberry wine.

Antioxidant Capacities, Total Phenols, Anthocyanins and Flavonoids of Southern Highbush Blueberry Wine
Similar to the USDA (2010) database for the ORAC values of selected foods, antioxidant capacity of the Southern highbush blueberry wine, with or without sulfite and filtration, was also determined by the ORAC method and expressed in the TROLOX equivalent (TE) values.The key phytonutrients (i.e., total phenols, total anthocyanins and total flavonoids) were measured for the filtered blueberry wine with no sulfite added to provide the benchmark information on the level of phytonutrients of the Southern highbush blueberry wine that is eligible for labeling as organic.The results are shown in Table 1.
The ORAC values of the Southern highbush blueberry wines ranged from 18.54 to 25.48 mmol TE/L.The ORAC values of low sulfite, unfiltered wine appeared to be significantly lower than those of the rest samples (p≤0.05).The average of the ORAC values shown in Table 1 was 22.57±2.92mmol TE/L, which reflected the overall antioxidant capacity of the Southern highbush blueberry wine tested in this study.This ORAC value ranked high compared to most blueberry wines listed in Table 2.
According to the literature, addition of sulfite to non-organic wine has two major purposes: to inhibit microorganisms and to prevent non-enzymatic browning (mainly, Maillard reaction) (Fleet, 1993).Non-enzymatic browning is a problem in wines that leads to a reduction in phenols, such as catechins, which are the major polyphenolic antioxidants in wines (Saucier and Waterhouse, 1999).The rate of Maillard reaction is enhanced by increasing the amount of reducing sugars and/or increasing the temperature (Eriksson et al., 1981).Thus, if blueberry wine, which is rich in reducing sugars (10-12%) and in polyphenols, is exposed to an environment of raised temperature for an extended duration, the Maillard reaction is favorable.As mentioned earlier, sulfite addition can combat this reaction.However, in the event of improper amount of sulfite, or in the case of aforementioned low-sulfite/unfiltered wine, antioxidant capacity might diminish.
Although Table 1 shows the effect of higher sulfite concentration on stabilizing the antioxidant activity of the blueberry wine, sulfite cannot be added to any blueberry wine destined for "organic" labeling, for which the sulfite limit is 10 ppm to reflect the naturally occurring sulfite in wine.The non-sulfite, filtered blueberry wine tested in this study, which could be claimed as organic, had an ORAC value of 23.49 mmol TE/L.
Table 1 also shows the total phenolic, anthocyanin and flavonoid contents of the filtered, non-sulfite Southern highbush blueberry wine.Anthocyanins are the major water-soluble flavonoids in blueberries, giving the red, purple and blue color to many fruits and vegetables (Espín et al., 2007), and considered biologically active compounds exhibiting a wide range of health benefits, e.g., antioxidant (Cao et al., 1997), antifungal (Benkeblia, 2004), and anti-carcinogenic properties (Ames, 1983).Flavonoids are naturally occurring phenols that are present in many plants including blueberries.Flavonoids and phenolics in general are strong protector against heart disease and cancer (Yao et al., 2004).The total phenols andanthocyaninsof the Southern highbush blueberry wine tested in this study are compared with those of grape wines and fruit liquors in section 3.3.

ORAC Comparison between Blueberry Wines and Grape Wines
The data presented in Table 2 reflect the ORAC values of most blueberry and grape wines reported hitherto in the literature.A small number of wines had their antioxidant activities measured by a different method from ORAC, e.g., TRAP in Campos et al. (1996), which were not included in Table 2. Grape wines are divided into red, rose and white wine types.The blueberry wines listed came from highbush or Southern Highbush varieties.
Besides the specific cultivar of Elliot and Weymouth, most blueberry wines were produced from mixed cultivars.For example, Johnson et al. (2011) used 15 cultivars including Berkley, Blue Chip and Blue Haven, while in this study, the mixed cultivars were, as mentioned earlier, Star, Windsor, Emerald, Primadonna and Jewel.
To facilitate the comparison, the average ORAC value of the Southern highbush blueberry wine tested in this study, i.e., 22.57 mmol TE/L, was chosen as a benchmark.For the 20 red wines listed in Table 2, the ORAC values ranged from 5.25 to 39.9 mmol TE/L.Four of the 20 red wines (i.e., 20%) had ORAC values higher than 22.57 mmol TE/L, while 16 of the 20 red wines (i.e., 80%) had lower ORAC values than the Southern highbush blueberry wine.
The ORAC values of the 6 Rose wines listed in Table 2 ranged from 1.52 to 11.2 mmol TE/L, which were all much lower than that the Southern highbush blueberry wine tested in this study.
For the 13 white wine types listed in Table 2, the ORAC values ranged from 0.6 to 5.35 mmol TE/L, which were far below that the Southern highbush blueberry wine.The much lower antioxidant potentials of white wines than red wines or blueberry wines were attributed to the fact that there is no skin or seed contact during the fermentation of white wine, while most antioxidants and phytonutrients are contained in the skins or seeds (Rigo et al., 2000;Yilmaz & Toledo, 2004).
The foregoing comparison suggests that blueberry wine could be more potent than red wine in health enhancement and disease prevention as far as the antioxidant capacity is concerned.

Comparison of Total Phenols and Anthocyanins among Blueberry Wines, Grape Wines and Fruit Liquors
Similar to the ORAC comparison, the total phenolicvalue of the Southern highbush blueberry wine tested in this study, i.e., 929 mg GAE/L, was used as a benchmark for comparison.For the 26 red wines listed in Table 3, the total phenolic values ranged from 700 to 4059 mg GAE/L.A majority of red wines (24/26) had total phenols higher than that of the Southern highbush blueberry wine, with only 2 of 26 falling below.The total phenolic contents of the Rose and white wines were all lower than that of the Southern highbush blueberry wine.Of the 8 fruit liquors, two had a comparable total phenolic value to that the Southern highbush blueberry wine, while the other 6 all fell below.
A limited number of wine's anthocyanin data were reported in literature.Listed in Table 3 are only 10 red wine types for which the total anthocyanin contents were reported.Except for the highbush blueberry cultivar Elliot that had a much lower anthocyanin value (i.e., 14.7 mg C3GE/L), most anthocyanin contents of the blueberry wines and grape wines listed in Table 3 were comparable.

Conclusions
The Oxygen Radical Absorbance Capacity of the Southern highbush blueberry wine ranged from 18.54 to 25.48 mmol TE/L, averaging 22.57±2.92mmol TE/L.About 80% of red wines and 100% of Rose and white wines reported in literature had lower ORAC values than 22.57 mmol TE/L, with only 20% of red wines above this ORAC value.From the perspective of antioxidant activities, blueberry wines could be more potent than most red wines and all Rose or white wines in health enhancement and disease prevention.For total phenolic contents, an overwhelming majority of the red wines was higher than that of the Southern highbush blueberry wine, but all the Rose and white wines were lower than it.Most fruit liquors reported in literature had lower total phenolic contents than the Southern highbush blueberry wine.The anthocyanin contents of blueberry wines were generally comparable to those of the red wines.

Table 1 .
The ORAC values, total phenols (TP), anthocyanins and flavonoids of Southern highbush blueberry wine with and without sulfite addition or filtration Values are mean of six measurements.Values with the same superscripted letters are not significantly different (p≤0.05). Ψ

Table 2 .
Comparison of the ORAC values among the red, Rose, white and blueberry wines

Table 3 .
Total phenol and total anthocyanin comparison amongthe red, Rose, white and blueberry wines and fruit liquors