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J. Agric. Food Chem. 2003, 51, 7162−7169

Effect of High-Oxygen Atmospheres on Blueberry Phenolics,
Anthocyanins, and Antioxidant Capacity
YONGHUA ZHENG,†,‡ CHIEN Y. WANG,*,† SHIOW Y. WANG,§

AND

WEI ZHENG§,#

Produce Quality and Safety Laboratory and Fruit Laboratory, Beltsville Agricultural Research Center,
U.S. Department of Agriculture, Beltsville, Maryland 20705

The influence of high oxygen concentrations on total phenolic, total anthocyanin, individual phenolic
compounds, and antioxidant capacity (measured as oxygen radical absorbance capacity, ORAC) in
highbush blueberry fruit (Vaccinium corymbosum L. cv. Duke) was investigated. Freshly harvested
blueberries were placed in jars ventilated continuously with air or with 40, 60, 80, or 100% O2 at 5 °C
for up to 35 days. Samples were taken initially and at 7-day intervals during storage. Whereas the
quality parameters of titratable acidity, total soluble solids, and surface color were only slightly affected
by the superatmospheric O2 treatments, the antioxidant levels were markedly increased by 60100% O2 treatments as compared with 40% O2 treatment or air control during 35 days of storage.
Elevated O2 between 60 and 100% also promoted increases of total phenolics and total anthocyanins
as well as the individual phenolic compounds analyzed by HPLC. Fruit treated with O2 concentrations
of g60% also exhibited significantly less decay. Data obtained in this study suggest that high-oxygen
treatments may improve the antioxidant capacity of blueberry fruit. Furthermore, antioxidant capacity
may be correlated with total phenolic and anthocyanin contents in blueberries.
KEYWORDS: Blueberry; high-oxygen atmospheres; antioxidant; phenolics; anthocyanins

INTRODUCTION

Fruits and vegetables are rich in phenolic compounds such
as anthocyanins, flavonols, flavones, isoflavones, flavonones,
and catechins. Many of these compounds exhibit a wide range
of biological effects, including antioxidant (1-6), antifungal
(7), and anticarcinogenic properties (8). There is convincing
epidemiological evidence showing that dietary antioxidant
flavonoids may provide protection against coronary heart disease
(9, 10), stroke (11), and lung cancer (12). In recent years there
have been an increasing number of studies that have quantified
the total antioxidant capacity in fruits and vegetables. A high
scavenging activity of berry extracts toward chemically generated active oxygen species has been shown in several studies
(13-15). In a previous study, phenolic compounds from berry
extracts inhibited human low-density lipoprotein (LDL) and
liposome oxidation (14). Thus, high fruit consumption may
significantly reduce the incidence and mortality rates of cancer,
cardiovascular disorders, and other degenerative diseases caused
by oxidative stress (16).
* Author to whom correspondence should be addressed [telephone (301)
504-5981; fax (301) 504-5107; e-mail wangc@ba.ars.usda.gov].
† Produce Quality and Safety Laboratory.
‡ Visiting scientist from the Department of Horticulture, College of Food
Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu
210095, China.
§ Fruit Laboratory.
# Visiting scientist from the Institute of Environmental Science, Zhejiang
University at Yuquan, Hangzhou, Zhejiang 310027, China.

Interest in the role of antioxidants in human health has
promoted research in the field of horticulture and food science
to evaluate fruit and vegetable antioxidants and to determine
how their content and activity can be maintained or even
improved through crop breeding, cultural practices, and postharvest storage and processing. Preharvest factors, such as
genetic background and cultural practices, have the potential
to influence antioxidant capacity in crops. Strawberry fruit from
a hill plasticulture system consistently had higher flavonoid
content and antioxidant capacity than fruit from plants grown
in the row system (17). Postharvest storage can also affect
phenolic compound levels and antioxidant capacity in fruits and
vegetables. In cranberries, storage temperature between 0 and
15 °C increased antioxidant capacity and total anthocyanin and
total phenolic contents. Strawberries and raspberries stored at
temperatures >0 °C also resulted in an increase in antioxidant
capacity (18). Controlled atmosphere (CA) storage of strawberry
fruit did not affect anthocyanin content in external tissues but
decreased anthocyanin content in internal tissues (19). Cv.
Delicious and Granny Smith apples in the first 2 months of
storage at -1 °C had 10-fold increases in the antioxidant
content, whereas antioxidant content significantly decreased
during the following 4 months of storage (20). No significant
changes in flavonoid concentration and antioxidant activity were
found in cv. Jonagold and Elstar apples during long-term storage
under regular and CA conditions (21, 22). A decrease in the
total antioxidant activity in fresh-cut spinach was also observed
during storage, but total flavonoid content remained stable both

10.1021/jf030440k This article not subject to U.S. Copyright. Published 2003 by the American Chemical Society
Published on Web 10/17/2003

To prepare the fruit extracts. The procedures for the ORAC assay on strawberries were modified from the previously described method of Cao et al. Louis. Total Phenolics and Anthocyanins Analysis. PA). One blank. with a gradient profile consisting of A with the following proportions (v/v) of B: 0-1 min. It has also been reported that a freezing process slightly decreased the total phenolic content and produced a decrease of free radical scavenging capacity ranging between 4 and 20% in four cultivars of raspberries (27). and other water-soluble compounds were eluted with 10 mL of 3% formic acid. MATERIALS AND METHODS Chemicals. acids.9 mm. 2′. and three jars were used for each treatment. methanol. Kaempferol. The reaction mixture contained 1. Total Anthocyanins. total anthocyanins.. a*. and water were of HPLC grade and were purchased from Baxter (Muskegon. Samples were taken initially and at 7-day intervals during storage for decay evaluation and other analysis. blueberries have become of special interest to researchers studying antioxidants because of their high phenolic content and antioxidant capacity (3. which was previously activated with methanol followed by water and then 3% aqueous formic acid.5. 42-45 min. shriveled. or 100% O2 (balance N2 in all high-O2 treatments). 40-42 min. The samples were determined using a Waters Corp. Total Titratable Acidity. and stored at -80 °C until analyzed. 4-6% B. AAPH. Total anthocyanin contents of blueberry extracts were measured using the pH differential method (32).2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) was obtained from Wako Chemicals USA Inc.2% formic acid using a Polytron (Brinkmann Instruments. Anthocyanins and other phenolics were then recovered with 2. 20-45% B. MSI. phosphate buffer. The supernatants were combined and transferred to vials. The mobile phase consisted of 5% aqueous formic acid (A) and HPLC grade acetonitrile (B). Pittsburgh. pH was measured with a pH-meter. (Richmond VA). Total Soluble Solids.2 using 0. MI). TSS was determined at 20 °C on a Bausch and Lomb refractometer. NJ) in a water bath at 35 °C. Fruit Decay. particle size ) 4 µm) (Waters). (St. Total phenolic contents in blueberry extracts were determined according to the FolinCiocalteu procedure (31). Westboro. 10-15 min. and pH Determinations. while sugars. MA) HPLC system coupled with a photodiode array detector (Waters 990 series) and equipped with two pumps (600E system controller). 90° ) yellow. Processing also has marked effects on phenolic content and antioxidant capacity in fruits. 45-100% B. 2003 7163 200. The reaction was started by the addition of AAPH. Phosphate buffer was used as a blank. The supernatants (18 mL) from the extractions described above were concentrated to dryness using a Buchler Evapomix (Fort Lee. Duke) were hand-harvested at commercially mature stage from Butler’s orchard. whereas strawberry processing to produce jams decreased the total ellagic acid content by 20% (25) and the flavonoids by 15-20% (26). 80. Fruit decay was visually estimated during the course of the experiment.7. Acetonitrile. and samples were preincubated at 37 °C for 15 min. Twenty berries from each replicate were cut into small slices.0 mL of acidified methanol containing 3% formic acid. and b* values. and a maximum of 10 samples were analyzed at the same time.. 24. and the juice was analyzed for total soluble solids (TSS). and 270° ) blue (30). 60. which provided CIE L*. such as CA. NJ).8-tetramethylchroman-2-carboxylic acid (Trolox) was purchased from Aldrich (Milwaukee. Food Chem. Twenty pieces of fruit from each replicate were wrapped in cheesecloth and squeezed with a hand press. Each sample was repeated three times. 100 µL of 320 mM AAPH. pH. and then used for analysis of total phenolics. one standard. The methanol extract was passed through a 0. Highbush blueberries (Vaccinium corymbosum L. and 100 µL of sample. 35-40 min. Highperformance liquid chromatography (HPLC) was used to separate and determine individual anthocyanins and phenolic compounds in berry tissue samples. Minolta. Surface Color Measurement. Ramsey. three 5-g samples of berries from each replicate were extracted twice with 10 mL of 80% acetone containing 0. Agric. Inc. Plant Materials and Treatments. and ORAC. cv. Sample Preparation for Total Phenolics. However. 100 µL of R-PE (3. As antioxidant content is becoming an increasingly important parameter with respect to fruit and vegetable quality. (33). Results are expressed as milligrams of cyanidin 3-glucoside (C 3-G) equivalents per gram of fresh weight. where 0° ) red-purple. The aim of this work was to investigate the effects of atmospheres containing high O2 on total phenolics.High-Oxygen Effect on Blueberry Antioxidant Capacity in air and in modified-atmosphere packaging (MAP) (23). dissolved in 4 mL of acidified water (3% formic acid). and unripened fruit. TA was determined by diluting each 5 mL aliquot of blueberry juice in 50 mL of distilled water and then titrating to pH 8. 6% B. Maryland. total anthocyanins. Because the stem end of blueberry fruit is the last to develop color (29). stored at -80 °C. and titratable acidity (TA). These values were then used to calculate hue degree (h° ) arctangent[b*/a*]). 28). acetone. The gases were checked regularly with an O2/CO2 analyzer (AMETEK.1 N NaOH. Five hundred grams of sample was placed in each 18-L jar. and quercetin were purchased from Sigma Chemical Co. MD) until the fluorescence of the last reading declined to <5% of the first reading (∼70 min). and then passed through a C18 Sep-Pak cartridge (Waters). Inc. The final volume of 2 mL was used in a 10-mm-wide fluorometer cuvette. and ORAC Assays. PA) and maintained at (2% for the duration of the experiment.0). 4% B. The final results (ORAC value) were calculated and expressed using Trolox equivalents (TE) per gram on a fresh weight basis.7 mL of 75 mM phosphate buffer (pH 7.. on the changes of phenolic compounds and antioxidant capacity in blueberries. and selected for uniform size and color. no significant changes in either total phenolics or free radical scavenging capacity were observed during the following 12 months of frozen storage. The jars were placed at 5 °C and connected to a continuous flow (120 mL/min) of humidified air (control) and 40. little information is available regarding the effects of storage conditions. (Somerville. It has been shown that industrial processing of pomegranates to obtain juice increased their phenolic content and antioxidant capacity (24). 15-35 min. MA).9 mm. (Milford.45 µm membrane filter (Millipore. MO). and chroma (C* ) [a*2 + b*2]1/2). and antioxidant capacity as well as the main phenolic constituents in blueberry fruit during postharvest storage at 5 °C. ORAC Assay. All anthocyanins and their aglycons were obtained from Indofine Chemical Co. 45- . Vol. fruit surface color was measured on this part of 10 fruits from each replicate using a chromameter (CR J. 1-10 min. The flow rate was 1 mL/min. HPLC Analysis of Anthocyanins and Phenolic Compounds. However. R-PE. and 1 µM Trolox (a water-soluble R-tocopherol analogue) was used as a standard during each run. This assay measures the effect of antioxidant components in blueberry extracts to inhibit the decline of R-PE fluorescence induced by a peroxyl radical generator. Westbury. 6-Hydroxy-2. Higher positive b* values indicate a more yellow skin color.4 mg/L). which indicates the intensity or color saturation. Any berries with visible mold growth were considered to be decayed. WI). Negative a* values indicate green and higher positive a* values red color. 18-20% B. The ORAC value refers to the net protection area under the quenching curve of R-PE in the presence of an antioxidant. sorted to eliminate damaged. Other authentic standards were obtained from Sigma and Fisher Scientific (Pittsburgh. mixed. NJ). 51. 6-18% B. Anthocyanins and other phenolics were adsorbed onto the column. NY) for 2 min and then centrifuged at 20000g for 20 min. Samples were injected at ambient temperature (20 °C) into a reversed-phase Nova-Pak C18 column (150 × 3. 180° ) bluish green. it is of great interest to evaluate changes in the antioxidant status during postharvest storage of horticultural crops. (R)-phycoerythrin (R-PE) from Prophydium cruentum. and 20 µL was analyzed by HPLC. No. Recently. and the results are expressed as milligrams of gallic acid equivalent (GAE) per gram of fresh weight. particle size ) 4 µm) with a guard column (Nova-Pak C18. Fluorescence was measured and recorded every 5 min at the emission of 570 nm and the excitation of 540 mm using a Shimadzu RF-Mini 150 recording fluorometer (Columbia. 20 × 3. Fruit decay was expressed as percentage of fruit showing decay symptoms.

45 ± 0.93 ± 0. RESULTS AND DISCUSSION Fruit Decay.00 0. Individual flavonols and anthocyanins were quantified by comparison with an external standard of myricetin.2 9.6 ± 0.1 ± 0.04 0. TSS.94 ± 0.00 3.9 0.. After prolonged storage (28 days and longer).03 3.00 3. Fruit exposed to high-O2 levels tended to have lower pH values and higher TA contents.7 2.1 9. and ethylene concentrations (40). In longan.05 3. air 40% O2 60% O2 80% O2 100% O2 40.2 6. pH.3 ± 0. both in combination with 20% CO2.38 ± 0.93 ± 3.9 1. TA.82 ± 0. b ns ) nonsignificant.67 ± 0.69 ± 0.45 ± 0.02 3.5 ± 0.02 0.00 0.1 ± 0. were more effective in controlling fungal decay than the conventional CA during storage at 8 °C. However.06 3. Statistical Analysis.8 ± 0. recorded with a diode array detector. pH.50 ± 11.13 0.00 0. no significant differences were observed in fruit color of strawberries stored under high-O2 atmospheres compared to air-stored fruit (37.5 ± 0. anthocyanin.03 0.03 3. with only 4.7 ± 0.43 ± 0. However.8 ± 0.55 ± 0.49 ± 0. Blueberry shelf life is limited by fruit decay primarily due to Botrytis cinerea infections at the stem scar.61 ± 0.01 0.49 ± 0.85 ± 0.4 ± 0.08 0.15 0.5 day 21. It is possible that atmospheres containing >40% oxygen are not suitable for the growth of decay microorganisms.8 6.4 ± 0.04 8. 100% B.04 3.2 9.73 ± 0.03 0. Little differences in TA content were also observed among high-O2. It has been shown that exposure of harvested horticultural crops to superatmospheric O2 levels may stimulate.48 ± 0. and flavonoid concentrations in blueberry fruit extract and their antioxidant capacity were evaluated by the Tukey-Kramer multiple-comparison test. no significant differences were observed among all of the treatments on all sampling days. Fruit decay was markedly affected by different high-O2 levels in storage (Table 1). significantly higher values of TSS in three higher level O2-treated fruit were detected in comparison with 40% O2 and air treatments (Table 1).49 ± 0.48 ± 0.90 ± 0. and improved decay control was obtained with increased O2 concentration.04 3.00 3.04 3.8 ± 0.02 3. significantly lower TSS values in high-O2-treated strawberries than in air-stored fruit during the later period of storage at 5 °C were reported in earlier studies (37. or reduce rates of respiration. air 40% O2 60% O2 80% O2 100% O2 20.3 day 14.48 ± 0.85 ± 0. .02 0.2 ± 0.90 ± 0.65 ± 0.02 ± 0. no significant difference was noted in fruit decay between 80% O2 and 100% O2 treatments after 28 days of storage (Table 1). However. No. Differences at p e 0.60 ± 0. air 40% O2 60% O2 80% O2 100% O2 2.2 9. There was a decrease in the measurement of surface color lightness (L value) after harvest in all treatment groups (Table 2). Vol.05 were considered to be significant. increased with storage time. sig ) significant at p e 0.18 ± 0.00 0. and pH during storage.1 9.10 3. 2003 Zheng et al. PH.05 0. The 90% and 100% O2 treatments had significantly less fruit decay than either the 15% CO2 itself or in combination with 40% O2 after 14 days of storage at 5 °C.15 9.52 ± 0.3 4. air 40% O2 60% O2 80% O2 100% O2 0.02 ± 2. Pe´rez and Sanz (38) found that 80% and 100% O2.3 9.71 ± 0.70 ± 0.4 8. maturity stage.50 ± 0.00 0. One hundred percent O2 was the most effective in suppressing fruit decay among all of the treatments. and cyanidin 3-glucoside. The phenolic compounds in fruit extracts were identified by their UV spectra.5 9.51 ± 0.9 39. In strawberry.5 5. CO2. ANOVA of data was performed for this experiment.12 0.01 0.06 0. quercetin.71 ± 0. Comparable L values were found among different highO2 treatment groups and the air control group throughout the experiment.9 0.04 3.03 0.48 ± 0.52 ± 0. On the contrary.03 3. sugars and acids are depleted. TA. 38).1 9.61 ± 0. which gives a better idea of blueberry color.82 ± 0.50 ± 0.05 9.63 ± 0. Differences between means of data were compared by least significant difference (LSD).43 ± 0. The different change patterns of pH. Table 1.73 ± 0.2 9.6 10.2 day 7.7 ± 0.05 significanceb treatment (T) duration (D) T×D decay (%) a Data expressed as mean ± SE of triplicate assays.01 0.14 0. and Total Soluble Solids (TSS) during Storage at 5 °C in Air or High-O2 Atmospheresa pH TA (%) TSS (%) day 0 treatment 0.60 ± 0. fruit decay was also significantly reduced by 70% O2 in comparison with conventional MAP during 40 days of storage at 2 °C (39). 50 min.74 ± 0.41 ± 0.1 3. which indicates the fruit became darker with storage. and O2.05 0.52 ± 0. Atmospheres enriched with g60% O2 were effective in inhibiting blueberry fruit decay during storage.03 8.66 ± 1.2 10.03 3.6 9.1 day 28.27 ± 2. 38).03 0.4 3.05 3.5 ± 0.59 ± 0.04 3. alone or in combination with high CO2.04 0.2 day 35.49% fruit decayed after 35 days of storage. The hue angle. Agric. and TSS in different studies seem to be associated with the different effects of elevated O2 on commodity respiratory rate.5 ± 0.53 ± 0. and data were collected using the Waters 990 3D chromatography data system. the mechanisms by which high-O2 atmospheres inhibit fruit decay is yet unclear. time and temperature of storage. 51.06 0. Similarly.5 ± 0.82 ± 0.39 ± 0.3 8. The effect of high-oxygen treatment and duration on fruit quality (decay.8 ± 0.5 19.44 ± 0. have also been shown to inhibit fungal growth and decay incidence in other studies.89 ± 0. have no effect on.10 ± 0. 24. and by chromatographic comparison with authentic markers (34-36). kaempferol.03 3.12 ± 0.4 ± 0.48 ± 1.01 0. causing corresponding changes in TSS.44 ± 0.07 0.2 18.0 ± 0. no significant differences were observed in TSS among the three higher level O2 treatments.00 0.2 1.0 ± 0.04 3.and air-treated strawberries during 14 days of storage (37). Blueberries stored under air showed slight decay on day 14 but 40% decay on day 35 during storage at 5 °C.06 ± 0.52 sig sig sig ns sig ns ns sig ns sig sig sig LSD0. High-O2 levels g60% tended to maintain higher TSS values than did 40% O2 and air.90 ± 0. The pH of blueberry juice increased slightly during storage.20 ± 2. TA.3 9.9 1.00 3.5 ± 0.2 9.02 3. corresponding to a decrease in TA in all treatments (Table 1).36 ± 1.04 3. and fruit color) and the values of phenolic.03 0.1 2.03 0. and TSS. air 40% O2 60% O2 80% O2 100% O2 6.1 9. Changes in Blueberry Fruit Decay. Titratable Acidity (TA).05.61 ± 0.2 ± 0.08 9.4 ± 0.08 0.54 ± 0.72 ± 0. Wszelaki and Mitcham (37) reported that there was a decrease in fruit decay with an increase in oxygen concentration above 40%. Treatment with 40% O2 had little effect on blueberry decay compared to fruits held in air. Scanning between 250 and 550 nm was performed. High-O2 atmospheres.7164 J.82 ± 0.02 0.03 9. depending on the commodity.02 0. TA.05 3. Food Chem.79 ± 0.1 9. Fruit Color. As the main substrates of respiratory metabolism. Pe´rez and Sanz (38) found significantly higher TA content before day 4 and lower TA content after day 7 in strawberry fruit exposed to 90% O2 + 10% CO2 than in fruit held in air during 9 days of storage at 8 °C.4 10. However.8 ± 0.8 1.01 0.0 ± 0.1 9. However.03 0. Longan fruit kept in 70% O2 also showed significantly lower pH than fruit stored in conventional low-O2 and high-CO2 atmospheres during 40 days of storage (39). TSS decreased during storage in all air-stored blueberry fruit.

3 3.2 ± 0.5 309.5 3.06 ± 1. It was also reported that fruit stored under elevated O2 levels exhibited good antioxidant capacity over the first 4 days of storage.7 330.2 ± 7.1 −1.3 3.46 ± 0.2 3.76 ± 0.3 −2.2 30. indicating more blue color.9 ± 0.2 332.3 1.2 2. it remained steady during the remainder of storage. rabbiteye blueberries (42).21 ± 0.1 −1. total anthocyanins.3 ± 8.96 ± 0. Increases in total phenolic and total anthocyanin content were also observed during postharvest storage in various berry fruits including highbush blueberry cv.1 318.4 330.2 ± 9.2.and .3 −1.4 −2.1 2.8 334.93 ± 0. Total Phenolics.03 ± 0. and ORAC values during the following 3 weeks of storage compared to 40% O2. Comparable hue angle values were found in all treatments during the first 21 days of storage.07 ± 0.0 ± 0.4 ± 0.71 ± 0.5 30. thereafter.3 −1.53 ± 0. total anthocyanins.28 ± 0.0 ± 0.7 ± 0.0 333.80 ± 0.07 ± 0. there were significantly higher levels of total phenolic content in fruits treated with g60% O2 during the following 3 weeks of storage.44 ± 0.66 ± 0.5 ± 0.8 ± 0.76 ± 0.78 ± 0.2 day 14.3 ± 0.01 ± 0.9 ± 0.9 31.2 4. possibly due to O2-promoted oxidation of the main antioxidants including anthocyanins and other phenolic compounds (45).3 ± 5.7 3.1 3.4 ± 4. after 35 days of storage. The changes in total phenolic content.4 ± 0. but this declined with prolonged storage.89 ± 0.6 30.1 ± 0.5 30.2 2.3 2.70 ± 0.56 0. sig ) significant at p e 0.2 3.2 ± 0. who found that.27 ± 0.07 ± 0.1 4. and this increase was accompanied by an increase in total antioxidant capacity.2 1. in comparison with fruits stored in air.4 2.7 ± 0.2 2. an average of 1.05.62 ± 0.2 1.64 ± 0. This was perhaps due to differences in the colors of strawberries and blueberries.0 ± 0. 51. air 40% O2 60% O2 80% O2 100% O2 30.06 ± 0.5 31.9 341.4 309.62 ± 0. Food Chem.7 30.3 2. whereas mature blueberries have more blue color and higher hue angle values.5 1.1 −1.7 ± 0.1 day 21.1 2.9 3.77 ± 0.7 ± 0.9 ± 1.5-fold. However.4 3.1 311.6 308.1 3.6 −1. strawberries (43). The ripe strawberries tend to have more red color and lower hue values because the hue angle starts at the +a* (red) axis.43 12.1 30.7 2. No significant differences in total phenolic content were found during the first 2 weeks of storage in all treatments. The total anthocyanins and ORAC value showed a similar change pattern as did total phenolics during storage in response to high-O2 treatments (Table 3).93 ± 0.6 30.6 −1.71 ± 0.9 ± 12.21 day 7.6 −1.4 0. high-O2 treatments had little effect on total phenolics. and cranberries (44).44 ± 0.6 ± 0.3 301.2 ± 15.80 ± 1.03 ± 0.87 ± 0.3 325.63 ± 0.3 3.2 2.04 ± 0.3 331. On the contrary.3 ± 5.81 ± 0.1 day 35.4 −2.6 30.6 30. strawberries held in 80% O2 + 20% CO2 had significantly higher levels of total anthocyanins during the first 4 days of storage but significantly lower levels of total anthocyanins at the end of storage.5 ± 0.69 0.1 4.5 2. Kalt et al. However.5 1. blueberries stored at 60% O2 and above exhibited significantly higher levels of total phenolics.6 ± 11.5 ± 0.3 30.5 31.2 ± 10.64 ± 0.4 320.27 ± 0.7 30.06 ± 0.85 ± 0.63 ± 0. Pe´rez and Sanz (38) reported significantly lower hue angle values in strawberries held in 80% O2 + 20% CO2 after 2 and 4 days of storage.25 ± 0.3 −2.3 3.30 ± 0.30 ± 0. The total anthocyanin content and ORAC value in fruits treated with g60% O2 increased by averages of 1. The total phenolic content did not differ significantly among the fruits held in atmospheres with >60% O2.6 ± 15.2 −2.1 306.38 ± 0. air 40% O2 60% O2 80% O2 100% O2 30.3 337.05 significanceb treatment (T) duration (D) T×D 0.46 ± 0.42 ± 0.1 1.7 3. air 40% O2 60% O2 80% O2 100% O2 30.7 319.and air-treated fruits at the end of the experiment.24 ± 0. Total Anthocyanins. There was also no difference between 40% O2 treatment and air control. were detected in fruits stored at 60-100% O2 after 28 and 35 days of storage. In the present study.1 3.3 −1.2 ± 17.2 ± 3.2 −1.85 ± 0.86 ± 0.5 31.3 1.1 LSD0. significantly higher hue angle values.0 30.7 −2.7 −2.5 2.4 −1.2 333.40 ± 0. The total phenolic content in control fruit exhibited a slight increase during the first 7 days of storage.7 ± 2.1 ± 15.2 −1. 24.3 4. Whereas the phenolic contents in all high-O2-treated fruit increased gradually throughout the experimental period.4 ± 0.00 ± 0.48 ± 0.73 0.5 −1.9 ± 16.2-fold increase in total phenolic content in high-O2 fruit was observed after 35 days.37 ± 0.9 330. lowbush blueberries (41). Bluecrop (18).7 ± 3. air 40% O2 60% O2 80% O2 100% O2 30.79 ± 0.20 ± 0. No.3 3. Changes in Blueberry Fruit Color during Storage at 5 °C in Air or High-O2 Atmospheresa treatment a L* a* b* chroma value hue degree day 0 32.8 2.2 ± 8. Agric.2 3.9 30.1 2.8 ± 0. This was confirmed by Pe´rez and Sanz (38).30 ± 0.6 2. and ORAC.3 ± 1.5 4.4 30.9 ± 8.3 320.14 ± 0. However.38 ns sig ns ns sig ns ns sig ns ns sig ns sig sig sig Data expressed as mean ± SE of 30 assays.3 31.86 ± 0. air 40% O2 60% O2 80% O2 100% O2 30.98 ± 0. 2003 7165 Table 2.3 3. (18) showed that the contents of total anthocyanins and total phenolics increased substantially in raspberries and strawberries stored at temperatures >0 °C.1 ± 0.4 305.31 ± 0.3 2.1 −2.4 ± 15.8 ± 0.5 4.19 ± 0.. Vol. and ORAC in blueberry fruits over the first 14 days of storage.25 ± 0.5 −1.5 ± 5.3 1.2 2.1 −2.1 340.58 ± 0.1 327.37 ± 0.1 305.6 301.68 ± 0.2 3.53 ± 0.4 −1.High-Oxygen Effect on Blueberry Antioxidant Capacity J.20 ± 0.9 ± 4.7 ± 8.7 day 28.95 ± 0.38 ± 1.49 ± 0.2 2.2 3.6 ± 4.90 ± 0.60 ± 0.36 ± 0.9 ± 9. total anthocyanins.14 ± 0.13 ± 0.5 ± 13.2 30.and 1.60 ± 0. and antioxidant capacity (expressed as an ORAC value) of blueberry fruits stored at different high-O2 atmospheres are shown in Table 3. Fruits stored in g60% O2 exhibited significantly higher total anthocyanins and ORAC value compared to 40% O2. There was an increase in total phenolic content with an increase in O2 concentrations. respectively. b ns ) nonsignificant.1 −1.3 2.15 ± 0.

malvidin 3-glucoside. petunidin 3-galactoside. Table 3. Different hydroxylation and glycosylation may modulate their antioxidative properties (2.883) and that for anthocyanins versus ORAC was 0.5 day 7.151x . respectively (52). b Data expressed as milligrams of gallic acid equivalents per 100 g of fresh weight. myricetin 3-arabino- side. 46). The chlorogenic acid content fluctuated during storage and was not consistently affected by high-O2 treatments. Chlorogenic acid was found to be the major phenolic compound with a high initial concentration of 254.05.4 ± 0.67.7 ± 3. HPLC analysis of blueberry extracts showed that. 51.2 ± 0. followed by dihydroquercetin > kaempferol > quercitrin > chlorogenic acid.827.5 ± 1.83 µmol .951.883 (y ) 10.2 µg/g of fresh weight in blueberry cv.2 17.6 14.8 ± 1. Our results suggest that storage at atmospheres enriched with O2 at g60% will improve the health benefit of blueberry fruit by positively affecting phenolic metabolism to enhance the antioxidant capacity.7.4 17. 2003 Zheng et al.31 sig ns sig sig ns sig sig ns sig a Data expressed as mean ± SE of triplicate assays.01 µg/g of fresh weight at harvest. 47.8 ± 1.18. In general. myricetin 3-arabinoside and quercetin-based flavonol contents in blueberries held in g60% O2 atmospheres increased slightly with storage duration. air-treated fruit (Table 3).826).3 ± 0.1 18. The antioxidant capacities measured by the ORAC assay for quercetin and kaempferol are 3. Quercetin.5 17.8 ± 1.7 ± 0. Malvidin 3-galactoside. Clegg and Morton (53) reported that quercetin had the greatest antioxidant activity. Changes in Blueberry Fruit Total Phenolics. cyanidin 3-galactoside. Vol. Anthocyanins. In the present study. thereafter. r ) 0. 48). The anthocyanin cyanidin possesses a high antioxidant activity (51) and has an antioxidant potential 4 times that of Trolox (48).9 ± 0. It has been shown that phenolic compounds are strong antioxidants (2.and B-rings. sig ) significant at p e 0.9 15.3 ± 2.5 ± 1. Anthocyanins.4′-dihydroxy substitution in the B-ring and conjugation between the A.291x .0 19.6 18. 24.5 ± 0. air 40% O2 60% O2 80% O2 100% O2 324 ± 15 330 ± 9 357 ± 13 358 ± 26 360 ± 12 189 ± 23 181 ± 9 204 ± 12 206 ± 7 207 ± 13 13. petunidin 3-galactoside.3 day 14. total anthocyanins. No.5 ± 2. quercetin 3-galactoside. delphinidin 3-glucoside. It has been shown that anthocyanins are strong antioxidants with free radical scavenging properties attributed to the phenolic hydroxyl groups attached to ring structures. No significant differences in chlorogenic acid were observed among all of the treatments throughout the experiment.826 (y ) 18.1 19. O2 concentration. Flavonols are effective antioxidants (49). The total antioxidant capacity measured by ORAC assay for 11 anthocyanins identified in Sierra blueberries was 12. All of the anthocyanins in fruits held in g60% O2 kept increasing or maintained the same levels during the remainder of storage. reaching much lower levels at the end of the experiment compared to the initial harvest contents. whereas those in blueberry fruit stored in 40% O2 and air decreased steadily with storage time.3 15.05 significancee treatment (T) duration (D) T×D 18 15 3.6 ± 3. r ) 0. all of these anthocyanins except for malvidin 3-arabinoside decreased steadily. air 40% O2 60% O2 80% O2 100% O2 315 ± 18 343 ± 2 362 ± 14 382 ± 27 390 ± 12 160 ± 20 180 ± 25 200 ± 9 206 ± 12 217 ± 9 11. Food Chem.7166 J.5 ± 1.5 ± 0.3 ± 0.5 ± 1.5 12.6 ± 1. The increase of ORAC values observed in this experiment in blueberry fruits subjected to high-O2 treatments may be attributed to the increase of total phenolic as well as total anthocyanin contents. e ns ) nonsignificant.1 ± 3. may have partly contributed to the increased ORAC values in blueberries stored under high-O2 atmospheres (Table 3). has a high antioxidant potential (51).2 treatment LSD0. Duke blueberries contained nine anthocyanins: delphinidin 3-galactoside.. Agric. There were significantly higher levels of myricetin 3-arabinoside and quercetin-based flavonols in fruits stored at 100% O2 than in fruits held in air after 35 days of storage. In general.4 day 35. with malvidin 3-galactoside being the most dominant anthocyanin with an initial content of 554. with 3′.3 18. and delphinidin 3-galactoside were the predominant anthocyanins. air 40% O2 60% O2 80% O2 100% O2 327 ± 14 323 ± 28 351 ± 15 330 ± 7 338 ± 15 188 ± 10 204 ± 8 203 ± 11 360 ± 5 193 ± 11 14.8 day 28. Phenolic Compounds.2 14. 46). and Oxygen Radical Absorbance Capacity (ORAC) during Storage at 5 °C in Air or High-O2 Atmospheresa total phenolicsb (mg/100g) total anthocyaninsc (mg/100 g) ORACd (µmol of TE/g) day 0 313 ± 8 182 ± 9 13.29 and 2. the correlation coefficient for phenolic content and ORAC is higher than that for anthocyanin content and ORAC value (3. 48).5 18.9 ± 1.4 20. and ORAC value may vary depending on the commodity. Previous research shows a linear relationship between total phenolic or anthocyanin content and ORAC in some berry crops (3.4 ± 0.8 day 21. in addition to anthocyanins. 46). especially quercetin-based flavonols. and storage time and temperature. 47. the correlation coefficient for phenolic content (x) and ORAC (y) was 0. and malvidin 3-arabinoside (Table 5).6 20. significantly higher levels of kaempferol were detected in fruits treated with g60% O2 compared to other treatments. Kaempferol contents in all fruits increased steadily during storage. c Data expressed as milligrams of cyanidin 3-glucoside equivalents per 100 g of fresh weight. and kaempferol were detected (Table 4). At the end of storage. Chlorogenic acid was also found in large amounts in other blueberry cultivars (36. quercetin 3-arabinoside. Duke in the present study. Kaempferol has a low antioxidant capacity against peroxyl radicals. The observed higher content of flavonols.8 14.4 15. the nine anthocyanins in air control fruit increased during the first 2-3 weeks of storage.8 15. air 40% O2 60% O2 80% O2 100% O2 323 ± 4 344 ± 8 363 ± 24 378 ± 21 386 ± 49 185 ± 13 183 ± 13 224 ± 22 216 ± 13 228 ± 45 13. In general. malvidin 3-arabinoside. Quercertin and other polyphenols have been shown to play a protective role in carcinogenesis by reducing the bioavailability of carcinogens (50). After 5 weeks of storage.2 ± 2.1 ± 0. air 40% O2 60% O2 80% O2 100% O2 331 ± 15 329 ± 22 354 ± 17 357 ± 24 348 ± 15 199 ± 11 193 ± 38 217 ± 16 218 ± 20 213 ± 8 15. 18. blueberries stored in g60% O2 had significantly higher anthocyanins content than those kept in air and 40% O2. Compounds such as chlorogenic acid. d Data expressed as micromoles of Trolox equivalents per gram of fresh weight. malvidin 3-galactoside. cyanidin 3-glucoside. It seems that the effect of high O2 on total phenolics.0 ± 2. All nine anthocyanins were significantly affected by high-O2 treatments.8 19. Considerable variation was found in flavonols of blueberries stored at different concentrations of high O2. other phenolic compounds were present in significant amounts (Tables 4 and 5). petunidin 3-arabinoside.

1 ± 3.8 241.1 232.2 ± 12.2 ± 18.6 ± 1.6 5.6 165.2 ± 2. b Data of anthocyanidin expressed as micrograms of cyanidin 3-glucoside equivalents per gram of fresh weight.7 ± 6.9 ± 0.5 277.9 10.7 63.2 ± 0.2 311.1 ± 0.6 ± 1.4 ± 0.2 222.8 ± 0.6 169.5 ± 14.7 201.2 ± 16.8 217.6 21. air 40% O2 60% O2 80% O2 100% O2 day 21.4 ± 2.1 ± 1.3 13.9 202.3 ± 8. air 40% O2 60% O2 80% O2 100% O2 LSD0.0 ± 8.5 82.9 ± 1.7 240.3 ± 31.3 ± 1. air 40% O2 60% O2 80% O2 100% O2 day 35.9 ± 15.3 737.9 5.3 ± 2.2 ± 1.8 ± 9.8 ± 0.7 10.1 ± 5.8 14.5 97.3 173.6 ± 1.9 179.4 6.9 ± 0.1 ± 0.0 274.4 20.4 ± 13.3 62.3 ± 0.1 ± 23.2 101.8 ± 6.3 ± 29.4 ± 11.8 ± 5.9 ± 22.8 7.3 ± 4.3 102.8 508.0 1.05.2 ± 23.9 13.6 317.6 ± 0.05 significancee treatment (T) duration (D) T×D chlorogenic acid myricetin 3-arabinosideb quercetin 3-galactosidec quercetin 3-arabinosidec quercetin derivativec kaempferol derivatived 254.2 12.9 762.2 15.7 600.1 ± 3.4 131.5 280.4 303.1 4.2 ± 2.3 111.7 ± 13.0 10.4 ± 1.9 ± 8.3 ± 0.6 184.6 355.0 533.7 ± 4.5 10.8 148.9 125.5 140.91 13.7 ± 10.4 ± 3.3 19.8 8.5 ± 25.8 5.6 195.9 12.8 ns ns ns sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig a Data expressed as mean ± SE of triplicate assays.4 12.1 ± 2.9 256.0 134.6 289.7 ± 28.7 ± 12.9 16.4 131.1 ± 1. Vol.7 112.3 12.6 510.7 ± 0.5 74.2 230.0 170.8 ± 0.5 4.7 ± 2. air 40% O2 60% O2 80% O2 100% O2 day 28.6 ± 12.2 ± 29.2 ± 12.1 321.7 ± 3.5 197.6 588.9 265.9 ± 1.8 89.4 ± 3.8 ± 3.5 77.3 8.2 ± 1.6 85.0 114.4 98.5 21.6 ± 14.7 290.9 280.3 684.7 7.7 ± 0.6 91.7 13.6 13.4 16.3 75.0 116.9 ± 6.7 554.2 ± 1.9 ± 6.0 225. b Data of myricetin 3-arabinoside expressed as micrograms of myricetin equivalents per gram of fresh weight.9 ± 2.9 ± 4.0 10.7 ± 32.1 ± 2.2 120.9 4.5 ± 29.7 10.7 10.3 ± 12.6 11.2 15.5 268.6 ± 0.3 256.6 20.3 12.9 ± 0.9 ± 0.6 14.6 sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig sig a Data expressed as mean ± SE of triplicate assays.7 5.8 ± 13.4 15.9 622.4 ± 4.1 ± 12.3 7.2 ± 1.9 ± 0.2 252.8 223.6 ± 14. 51.8 ± 13.7 ± 0.5 ± 4.1 5.6 632.7 6.7 ± 27.2 90. air 40% O2 60% O2 80% O2 100% O2 day 28.3 13.2 ± 4.2 ± 2.1 22.5 ± 32.4 ± 5.0 ± 1.0 273.8 ± 3.0 ± 2.0 ± 14.7 ± 3.8 146.8 311.2 6.7 ± 0.6 ± 14.1 8.7 10.8 296.0 7.5 214.7 18.3 ± 21.4 138.3 331.2 ± 16.8 9.7 267.2 10.3 ± 17.6 ± 6.8 ± 13.2 229.9 663.8 238.4 97.6 242.4 183.4 ± 19.5 242.0 ± 16.5 ± 20.6 9.4 ± 8.0 ± 1.5 163.6 ± 6.8 ± 6.9 ± 14.0 ± 12.6 62.2 13.5 8.3 117.1 ± 12.9 ± 5.5 ± 2. Food Chem.9 ± 10.5 64.5 11.7 9.9 11.1 ± 21. 24.5 ± 21.7 10.9 ± 35.7 ± 7.2 72.3 ± 13.1 ± 1.3 9.3 7.2 145.3 223.0 274.3 ± 1.3 ± 1.7 ± 19.4 ± 1.9 1.3 ± 1. Data of quercetiin aglycons expressed as micrograms of quercetin equivalents per gram of fresh weight.4 13.3 156.5 ± 0.7 ± 1.5 151.1 349.3 ± 10.8 ± 4.4 97.9 150.9 ± 7.4 ± 3.6 ± 2.7 609.8 189.4 7.4 ± 20.2 10.2 1.1 ± 28.6 10.1 17.2 ± 9.1 ± 35.8 13.8 12.6 ± 2.1 85.1 20.1 72.6 ± 4.1 ± 26.1 ± 11.3 ± 15.6 220.3 254.1 ± 37.1 110.1 7.0 ± 1.0 11.4 19.2 13.5 18.8 ± 3.4 55.2 104.6 ± 13.6 ± 4.0 118.3 183.6 654.1 136.1 20.9 455.0 ± 0.4 ± 0.6 ± 2.3 ± 4.8 ± 0.9 620.3 ± 6.6 2.1 ± 11.3 ± 23.4 ± 6.5 ± 0.0 97.4 ± 0.0 261.4 ± 1.9 634.2 ± 22.0 ± 9.1 16.6 ± 21.2 ± 17.0 177.5 218.1 123.1 ± 0.9 ± 12.9 186.3 ± 15.6 650.5 7.5 12.0 12.9 246.0 20.3 ± 20.0 ± 1.5 20.4 ± 1.1 89.2 113.9 ± 6.4 79.4 ± 7.4 11.1 ± 1.8 234.3 ± 1.0 123.8 ± 3.0 6.0 ± 14.7 190.3 ± 9.3 ± 4.1 ± 2.5 122.8 ± 11.5 251.7 10.8 58.3 ± 12.1 11.1 ± 20.9 317.4 9.2 ± 1.4 59.0 345.8 101.9 ± 1.8 11.6 ± 0.8 24.7 13.1 ± 9.4 89.0 ± 1.4 99.4 ± 20.0 ± 4.5 114.2 78.6 15.4 ± 34.9 ± 0.1 78.8 ± 1.8 ± 1.7 7.3 10.0 ± 9.0 60.4 ± 6.6 ± 6.8 ± 4. air 40% O2 60% O2 80% O2 100% O2 day 35.8 ± 2.2 151.0 ± 2.2 ± 22.4 13.4 ± 28.2 8.0 ± 5.7 ± 1.0 ± 0.1 ± 9.9 100.3 ± 11.5 13.0 ± 5.8 ± 13.3 14. No.7 16.5 ± 6.4b 229.3 ± 29.8 5.1 17.9 140.3 164.5 ± 23. e ns ) nonsignificant.4 87.2 ± 7.1 ± 5.1 ± 5.0 10.7 ± 12.8 ± 13.5 ± 4.7 13.3 13.7 ± 23.8 16.2 101.9 ± 11.3 11.4 ± 1.6 4.4 248.2 ± 3.7 ± 2.3 ± 1. c Table 5.1 90.3 ± 3.2 230.3 264.5 ± 12.1 7.9 100.3 356.2 14.1 309..9 ± 12.3 ± 23.8 ± 27.5 ± 5.4 ± 18.0 112.4 ± 3.0 ± 24.6 ± 13.7 ± 1.4 235.1 ± 16.4 11.3 278.8 ± 0.0 ± 4.9 81. sig ) significant at p e 0.6 ± 24.8 143.7 ± 29.7 280.1 14.3 ± 24. Changes of Main Individual Anthocyanins in Blueberry Fruit during Storage in Air or High-O2 Atmospheresa treatment day 0 day 8.9 170.9 13.6 ± 35.5 15.9 255.1 150.6 ± 16.0 ± 3.1 ± 6.6 281.3 88.2 ± 5.5 ± 2.5 ± 4.5 ± 14.2 ± 1.7 125. air 40% O2 60% O2 80% O2 100% O2 LSD0.5 ± 23.8 10.2 8.8 18. Agric.3 5. 2003 7167 Table 4.1 ± 5.6 246.0 ± 3.1 320.4 109.0 60.6 10.2 ± 8.9 10.9 285.8 ± 10.5 213.9 13.1 ± 0.6 ± 2.3 ± 11.8 ± 7.8 ± 8.6 ± 0.4 ± 4.3 80.0 ± 5.9 69. .7 ± 14.5 13.7 175.1 ± 12.2 ± 22.7 74.8 ± 17.4 7.9 ± 2.0 91.8 ± 0.8 360.4 ± 5.9 284.1 ± 12.7 107.7 ± 3.6 47.2 ± 5.4 ± 0.9 ± 17.9 10.2 ± 0.6 96.7 ± 14.9 720.0 454.7 ± 29.3 ± 1.5 273.5 ± 14.6 ± 0.3 107.1 65.2 ± 12.2 2.6 ± 13.2 6.8 ± 1.2 ± 13.5 313.6 ± 6.2 ± 0.0 14.5 ± 0.4 ± 5.2 ± 2.6 ± 4.1 ± 0.3 ± 25.1 18.7 587.9 ± 0.9 9.1 15.8 ± 2.3 ± 8.6 ± 1.7 13.7 11.7 13.1 ± 34.9 13.8 ± 16.3 247.9 ± 0.2 6.1 ± 17.1 ± 1.9 578.0 10.4 ± 2.5 ± 4.4 ± 1.3 ± 15.05.6 ± 10.3 15.3 87.4 ± 1.6 257.7 ± 4.7 ± 3.7 ± 10.2 5.6 71.1 11.3 ± 5.8 ± 13.7 8.7 681.6 ± 2.0 15.7 ± 30.4 98.9 ± 31.5 12.8 9.5 11.3 248.8 696.5 ± 7.3 129.0 ± 1.8 279.1 93.9 ± 0.1 ± 26.1 ± 16.6 ± 3.8 ± 13.3 ± 54.6 ± 2.4 ± 3.5 6.4 176.5 90.7 ± 4.8 227.1 79.9 ± 5.9 12.4 17.6 ± 3.2 6.9 173.6 321.4 ± 0. air 40% O2 60% O2 80% O2 100% O2 day 21.7 226.9 ± 26.3 95.1 55.9 ± 33.3 625.1 253.4 ± 1.7 ± 2.7 11.2 88.9 53.3 137.8 13.9 167.9 ± 3.1 ± 2.1 ± 6.6 ± 13.4 ± 11.8 0.7 ± 1.7 ± 0.9 ± 1.3 262.1 ± 3.3 44.High-Oxygen Effect on Blueberry Antioxidant Capacity J.4 ± 2.1 7.2 ± 27.6 ± 6.7 ± 3.4 ± 6. d Data of kaempferol derivative expressed as micrograms of kaempferol equivalents per gram of fresh weight.6 ± 27.8 530.1 17.0 ± 3.9 18.2 ± 0. sig ) significant at p e 0.9 ± 4.06 68.5 ± 19.4 ± 0.8 ± 9.2 274.2 8.4 ± 6.5 ± 13.8 18.8 8.2 ± 23.7 ± 20.9 ± 45.0 15. Changes of Chlorogenic Acid and Individual Flavonols in Blueberry Fruit during Storage in Air or High-O2 Atmospheresa treatment day 0 day 8. ns ) nonsignificant.2 17.5 10.1 ± 2.3 ± 5.5 83.6 17.3 ± 11.7 6.2 ± 3.2 63.8 ± 1.8 11.7 ± 21. air 40% O2 60% O2 80% O2 100% O2 day 14.3 17.6 ± 12.2 ± 22.0 ± 0. air 40% O2 60% O2 80% O2 100% O2 day 14.1 ± 4.2 14.6 239.05 significancec treatment (T) duration (D) T×D c delphinidin 3-galactoside b delphinidin 3-glucoside cyanidin 3-galactoside cyanidin 3-glucoside petunidin 3-galactoside petunidin 3-arabinoside malvidin 3-galactoside malvidin 3-glucoside malvidin 3-arabinoside 203.9 ± 0.1 ± 28.3 ± 15.4 76.5 ± 5.3 ± 1.4 259.1 ± 17.1 ± 0.2 11.7 ± 3.6 ± 11.7 ± 3.6 270.0 ± 17.2 ± 15.7 295.9 299.8 ± 2.3 ± 0.2 ± 1.3 251.5 ± 3.0 ± 7.8 ± 20.8 107.7 277.0 297.

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