Journal of Undergraduate Research
Volume 3, Issue 1 - September 2001

The Antioxidant Properties of Florida Grown Blueberries and Blueberry Wine

Kara Sawyer

ABSTRACT

Twelve Florida-grown blueberry cultivars were analyzed at three maturity levels for phytochemical composition contributing to antioxidant capacity. Three of the aforementioned blueberry cultivars were further separated into skin, pulp and seed. Analyses were conducted to show relative differences between cultivars, maturity, and location of antioxidant compounds within the fruit. This information was related to processing procedures for making wine in an effort to extract the maximum amounts of antioxidant compounds. Three processes were then evaluated in the production of blueberry wine. Following fermentation, the wines were stored under regular and accelerated conditions and subsequent phytochemical evaluations were made. Evaluation of the twelve blueberry cultivars in this study showed that the antioxidant compounds found in the blueberry fruit are mostly located within the skin, with a smaller amount in the seeds and insignificant amounts in the pulp. The amount found in the seeds was especially dependant on cultivar. During wine making, maceration of crushed blueberries does not increase the extraction of colored anthocyanins, but does increase the amounts of colorless phenolics. These phenolics contributed to the polymerization of anthocyanins and increased overall antioxidant properties of blueberry wine, while they themselves decrease with time.

INTRODUCTION

Anthocyanins have been of interest in blueberries and grapes because they not only produce color in these fruits and their products, such as in wine, but also add to taste and chemical characteristics (Mazza et al., 1999). If exposed in solution to oxygen, over time anthocyanins will undergo changes in color characteristics and be displaced by more stable polymeric compounds (Mazza, 1995). The rate and degree to which this occurs has to do with many factors, some of which are cultivar, maturity, processing, and sun exposure (Mazza et al., 1999). The purpose of this project is to (1) measure antioxidant compounds in twelve cultivars of Florida grown blueberries with regard to maturity and measure the distribution of these compounds in the fruit, (2) produce blueberry wine using three different processing methods to evaluate changes in antioxidant components that take place during storage.

MATERIALS AND METHODS

Blueberry samples were obtained from a commercial blueberry farm in Alachua County, Florida. Each blueberry cultivar was separated into green, immature and mature berries. Mature Rabbiteye 'Climax", Distinct 91-333 and Highbush 'Emerald' were further manually separated into skin, pulp and seeds for additional analyses. Berries were extracted with a solution of acetone: methanol: water (4:4:2) acidified to pH 2.5 with acetic acid. Samples were homogenized with a Brinkmann homogenizer and filtered to remove insoluble residue.

Wine Production

Late season blueberry cultivar "Powderblue" was used to make the wines for this study.

On hull treatment: Blueberries (25 kg) were crushed with a Pigiadiraspatrice crusher (Sorrento, Italy) and 1.5 kg of sugar was added to reach a brix of 23.4. Berries were inoculated with ~ 0.5 g per gallon of Red Star California Champagne yeast and thoroughly mixed. Fermentation was then conducted at 55ºC for 11 days and at 22ºC for 3 days (to accelerate fermentation) and fermented to dryness. Wines were pressed with a Willmes bladder press (Moffett Co., San Jose, CA) to obtain 4.5 gallons of wine.

Hot press treatments: Two hot pressings of wine were made, hereby called hot press #1 (HP1) and hot press #2 (HP2). Hot press #1 was made with 25 kg of fresh blueberries while hot press #2 was made with 14 kg of fresh blueberries. The blueberries were crushed and hot press #2 was extensively macerated using a Hamilton Beach Scovill grinder with 4 mm extrusion holes. For both treatments, Biopectinase TLQ mashing/macerating enzyme was added at 200 ppm before heating in a steam kettle at 60ºC for about 30 minutes. The mixtures were then pressed with the Willmes bladder press and 1.75 kg of sugar was added to hot press #1 to achieve a brix of 20.0 and 1 kg of sugar was added to hot press #2 to achieve a brix of 22.2. Approximately 1 g per 1 gallon of Red Star California Champagne yeast was added to both treatments to begin fermentation. Fermentation was conducted for 11 days at 55ºC and then for 3 days at 22ºC (to accelerate fermentation) and fermented to dryness. Two mL of a 2.5 ppm sodium azide solution was added to each 300 mL of wine to arrest fermentation and microbiological growth.

Accelerated Shelf-Life

The resultant wines were then stored at two temperatures; 22 or 40ºC to evaluate stability over time. Samples were taken in duplicate every two weeks for 12 weeks for phytochemical analysis.

Chemical Analyses

The total anthocyanin test was done according to the pH shift method of Cheng et al. (1991). Anthocyanins were quantified using the molar extinction coefficient of cyanidin-3-glucoside (29,600) to obtain an adjusted absorbance for anthocyanins

The total soluble phenolics were quantified by the Folin-Ciocalteau assay according to protocols described by Howard et al. (1996) and quantified as chlorogenic acid equivalents following linear regression.

The Oxygen Radical Absorbance Capacity (ORAC) of the blueberries was determined by using a modified version of Cao and Prior (1999) which utilized an Fmax fluorescence microplate reader (Molecular Devices Co., Sunnyvale, CA). Data were expressed in mM Trolox equivalents.

Monomeric and polymeric anthocyanins were measured by a modified method of Mazza (1999) using 400 mL of 5% Na2SO3 . Results were expressed as the percent distribution of monomeric/polymeric anthocyanins.

The color density and hue/tint were measured using the method described by Wrolstad (1976). Diluted wine samples were read at 420, 520, and 700 nm.

Statistical Analyses

For statistical analyses of part (2) of this study, Jmpin version 3.21 by the SAS Institute (Cary, N.C.) was used. Full factorials were run for treatment by time and treatment by temperature with duplicates.

RESULTS AND DISCUSSION

The results of part (1) of this research are summarized in table (1). Differences in anthocyanin, phenolic and antioxidant capacity of the different blueberries tested can be attributed to a range of variables. All of the blueberry cultivars were grown under the same conditions and area, but small variations in cultivar maturing time and access to water could increase or decrease phenolics/antioxidants in the fruit. Blueberry bushes with longer maturing times have more access to sunlight and more time for the development of antioxidant compounds. This is especially true of anthocyanins; none of the green blueberries contained anthocyanins, while the immature blueberries had very low amounts of anthocyanins compared with fully mature blueberries.

Table 1 Results of Blueberry Cultivar and Fractionation Study - Summer 2000
Cultivar	Maturity	Total Anthocyanins mg/kg	Total Phenolics mg/kg	Total Phenolics Error	(ORAC) Trolax Equiv. per gram	ORAC Error
Cultivar Study - Summer 2000
Climax	G	0	8923	73	33.61	5.23
	I	117	7713	34	23.13	5.23
	R	2626	8489	381	41.11	1.79
Emerald	G	0	7710	109	45.19	16.60
	I	151	5801	203	19.32	5.23
	R	1179	5281	51	27.18	3.75
Mature Bonit	G	0	14543	637	63.68	6.49
	I	217	7946	233	29.50	5.23
	R	1940	8742	293	44.91	4.56
91-333	G	0	10608	412	35.16	5.23
	I	262	8502	76	44.05	0.06
	R	2218	9025	247	28.83	5.14
FL90-99	G	0	5860	276	35.63	5.85
	I	259	4846	73	68.05	5.23
	R	2185	6706	134	43.38	11.01
Jewell	G	0	3477	117	26.22	6.24
	I	95	3028	53	23.34	2.19
	R	1606	4425	70	46.63	3.08
Beckyblue	G	0	5654	194	43.14	8.80
	I	406	4335	34	45.25	5.74
	R	1515	5799	140	48.06	4.12
Southern-belle	G	0	4190	165	38.15	18.60
	I	0	3852	64	31.99	5.23
	R	1119	4231	155	37.29	5.40
FL88-53	G	0	5010	228	38.24	2.89
	I	246	4224	77	36.01	4.38
	R	1214	4863	146	46.58	4.02
Sharpblue	G	0	7571	402	42.20	5.23
	I	286	6431	52	36.49	4.40
	R	1217	6170	309	68.31	5.07
HB-A17	G	0	4715	199	29.15	0.70
	I	136	4435	738	29.19	0.26
	R	2296	6575	222	44.47	5.87
Star	G	0	4878	7	20.81	0.12
	I	174	4597	9	26.93	5.23
	R	1310	5290	157	30.04	7.98
Fractionation Study - Summer 2000
Climax	Ripe Skin	5160	17406	1395	47.87	6.28
	Ripe Pulp	0	1109	48	5.14	0.13
	Ripe Seed	0	3508	22	6.11	4.06
Emerald	Ripe Skin	4492	13019	486	37.19	2.73
	Ripe Pulp	0	792	58	10.79	5.23
	Ripe Seed	0	23421	191	36.07	5.23
91-333	Ripe Skin	8034	21930	1055	58.01	8.85
	Ripe Pulp	0	1149	34	5.32	1.92
	Ripe Seed	0	6721	71	21.55	8.77
G=Green, I=Immature, R=Ripe Table (1) also illustrates the differences within the blueberry of the antioxidant compounds. Results indicated that the anthocyanins were only present in the skins of the blueberry fruit, while some phenolics were found in the skins, seed and pulp. Only one cultivar, 'Emerald', had more phenolics in the seed than the skin. Most grapes have significant amounts of antioxidant compounds in the seeds along with the skins (Kandaswami, 1998). Compositionally, the antioxidant capability in blueberry seeds is not as significant as within grapes. Figure 1. Total Soluble Phenolics Figure 2. Total Anthocyanins Treatment by time interactions were found for the attributes of on&endash;hull, hot press #1 and hot press #2 by six 2-week increments and all tests were compared. The treatments were found to be significantly different from one another for the total soluble phenolics test (Figure 1) with significant decreases from the initial occurring at six weeks for all treatments. The total anthocyanin (Figure 2), monomeric anthocyanin (Figure 4), color density (Figure 5) and ORAC tests showed no treatment differences between HP1 and HP2, while the on hull treatment was significantly lower. Total anthocyanin concentrations declined from day 0 to week two and continued to decrease throughout storage for all treatments. Substantial declines in monomeric anthocyanins were inversely related to gains in polymeric anthocyanins (Figure 3) and may be directly related to loss of total phenolics. Week six also showed a significant decline from the original color density, which continued to decline throughout the rest of storage while hue/tint (Figure 6) increased proportionally. ORAC values increased with time, and this may be due to polymerization creating larger, more complex anthocyanins. These larger compounds could be protecting the phycoerythrin protein used in the assay or possibly becoming more specific for the inhibition of the peroxyl radical generated in the ORAC assay even though some hydroxy groups, important to the antioxidant capacity of anthocyanins, may have been lost during polymerization. Figure 3. Polymeric Anthocyanins Figure 4. Monomeric Anthocyanins Figure 5. Color Density Figure 6. Hue/Tint Significant differences were observed when treatments were factored against storage temperature. The total soluble phenolics test showed total phenolics for room temperature to be higher than accelerated conditions by 250 ppm over the entire study. Total anthocyanins and color density showed room temperature to be higher on average than the accelerated samples, while monomeric anthocyanins showed no significant differences with temperature. The results from the ORAC test by temperature showed ORAC values to increase for accelerated storage for HP2 and decrease for accelerated storage for HP1 and on hull. This inverse relationship between hot press #1/on hull and hot press #2 could be attributed to increased polymerization in hot press #2 due to procyanidins, known to exist in the seeds, that would increase overall antioxidant capacity. Overall, the trend shows that longer times and higher temperatures give better ORAC values. In general, significant differences were found between hot pressed wines and the on the hull fermented wine. It was shown that total phenolics, anthocyanins, and antioxidant capacity is markedly affected by both the extraction time and temperature in making blueberry puree (Kalt et al., 2000). This is also true in making blueberry wines. The hot pressed wines had, on average, 72% more phenolics, 50% more anthocyanins, and a 72% more oxygen radical absorbance capacity at the beginning of fermentation as the on the hull wines. The polymerization of anthocyanins in the on hull treatment occurred during fermentation and well before storage while this polymerization didn't occur until storage for the hot press treatments. This may be due to reactions with enzymes contained within the blueberry that were otherwise destroyed in the hot pressing procedure. CONCLUSION Anthocyanins are found completely in the ripe skin of blueberries while colorless phenolics are found mostly in the skin and seed at levels dependant on cultivar and not maturity. For blueberry wine, the maceration of seed in hot pressing didn't change the extraction of total anthocyanins but it did increase total phenolics. These phenolics, though they decreased over time, increased polymerization of anthocyanins and the overall antioxidant capacity of the wine. 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