Vitamin Retention in Eight Fruits and Vegetables: A Comparison of

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Vitamin Retention in Eight Fruits and Vegetables: A Comparison of Refrigerated and Frozen Storage Ali Bouzari, Dirk M. Holstege, and Diane Marie Barrett J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5058793 • Publication Date (Web): 19 Dec 2014 Downloaded from http://pubs.acs.org on December 23, 2014

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Journal of Agricultural and Food Chemistry

Vitamin Retention in Eight Fruits and Vegetables: A Comparison of Refrigerated and Frozen Storage

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Ali Bouzaria, Dirk Holstegeb and Diane M. Barretta a Department of Food Science and Technology, University of California, Davis b University of California, Davis Analytical Lab

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Abstract

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Four vitamins were analyzed in several fruit and vegetable commodities to evaluate the

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differences between fresh and frozen produce. Ascorbic acid, riboflavin, α-tocopherol, and β-

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carotene were evaluated in corn, carrots, broccoli, spinach, peas, green beans, strawberries, and

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blueberries. Samples of each commodity were harvested, processed, and analyzed for nutrient

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content at three storage times per treatment. Ascorbic acid showed no significant difference for 5

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of the 8 commodities and was higher in frozen samples than fresh for the remaining 3

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commodities. Apart from broccoli and peas, which were higher and lower in frozen vs. fresh

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samples, respectively, none of the commodities showed significant differences with respect to

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riboflavin content. Three commodities had higher levels of α-tocopherol in the frozen samples,

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while the remaining commodities showed no significant difference between fresh and frozen. β-

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carotene was not found in significant amounts in blueberries, strawberries, and corn. Peas,

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carrots, and spinach were lower in β-carotene in the frozen samples, while green beans and

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spinach showed no significant difference between the two storage methods. Overall, the vitamin

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content of the frozen commodities was comparable and occasionally higher than their fresh

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counterparts. Beta carotene, however, was found to decrease drastically in some commodities.

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Introduction

ACS Paragon Plus Environment


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Consumption of fruits and vegetables plays an important role in preventing disease and

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maintaining positive overall health (1-4). Ideally, these foods would be consumed immediately

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after harvest, however. Most fresh produce arrives to the consumer several days to weeks after it

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is harvested. During this time, cellular respiration and oxidation can cause substantial nutrient

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degradation (5). In order to halt spoilage and eliminate pathogens, food processing methods such

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as blanching and freezing have been developed (6). While some organoleptic degradation has

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been previously noted in these products, it has been found that the nutritive degradation suffered

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by foods during processing is less substantial than that which occurs over prolonged postharvest

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holding periods of fresh produce (6, 7).

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This study seeks to evaluate the effects of freezing and frozen storage on the vitamin content

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of peas, green beans, broccoli, spinach, corn, carrots, strawberries, and blueberries. Most

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previous studies on this topic were carried out on produce purchased at market. This introduces a

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level of uncertainty with regards to the history of the samples including soil and climate quality

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during growing season, ripeness at harvest, handling, shipping, and storage. In order to minimize

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these sources of uncertainty, all commodities were harvested directly from their source,

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immediately processed, and used for both fresh and frozen storage studies.

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Vitamins are typically categorized as either water or fat soluble. Water soluble ascorbic acid

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and riboflavin, and fat soluble α-tocopherol and β-carotene were used to evaluate vitamin

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degradation.

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Ascorbic acid is one of the most heat labile vitamins. Its relatively low stability makes it an

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ideal indicator of the effects of processing on degradation of nutrients. This is based on the idea

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that if a given process leaves ascorbic acid levels relatively unchanged, it is likely that most other


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nutrients have survived the process as well (6, 8, 9). Degradation of ascorbic acid has been

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shown to vary dramatically among different commodities, and even various cultivars of the same

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commodity can exhibit different trends in ascorbic acid retention (6, 8-10). In fresh produce,

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ascorbic acid begins to degrade quickly soon after the produce is harvested. Refrigeration helps

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to slow this degradation. Frozen storage is effective in preserving ascorbic acid, but the

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blanching process prior to freezing often causes significant degradation in addition to leaching

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into the blanch water (6, 8, 10). Steam blanching results in less leaching of water soluble

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nutrients than water blanching (11).

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Riboflavin can be degraded during thermal processing (12). Riboflavin is light sensitive,

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and thus food products must be stored carefully to avoid exposure. (12) Riboflavin levels in

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processed products that are blanched have been shown to decrease due to leaching into the

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blanch water (13-15). Riboflavin is readily degraded during ambient temperature storage of fresh

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produce, and research has showed that some minor degradation occurs at temperatures

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encountered during frozen storage as well (13, 16).

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Unlike the water-soluble vitamins, alpha tocopherol is not prone to leaching during water-

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based processing steps such as blanching. It has been found in some studies that alpha tocopherol

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content appears to increase to a certain extent during thermal processing, possibly due to

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increased extractability, before declining due to thermal degradation (6, 17). Vitamin E is also

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susceptible to oxidative degradation (18), which can occur in both fresh and frozen storage.

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While fat-soluble vitamin A is not normally found in fruits and vegetables, it can be

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indirectly obtained through consumption of the carotenoid compound β-carotene. β-carotene is a

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metabolic precursor to vitamin A, and in fact many dietary descriptions of plant-based foods



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report a vitamin A correlation that is based on the concentration of β-carotene in the product. β-

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carotene does not leach out of produce during washing and blanching but is very sensitive to

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degradation due to oxidation. This potential for oxidation is dependent on the various processing

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and storage conditions which include exposure to high temperatures, light, and oxygen (6, 19).

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Retention of β-carotene in frozen storage seems to vary by commodity, with studies showing

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decreases to different degrees in β-carotene over a prolonged period of frozen storage (6, 20).

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This is in contrast to fresh storage, where there is reported to be little degradation (13).

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Materials and Methods

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Raw Materials

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Vegetable seeds were donated by the Seminis Vegetable Seed Co., Inc., Woodland, CA. Six

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replicate samples were harvested from different randomly selected points along linear rows for

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each commodity. Commodities were harvested according to the times and locations listed in

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Table 1.

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All commodities were harvested at uniform maturity as determined by both color and

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approximate size, as recommended by the grower. All commodities were transported to the UC

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Davis Food Science & Technology pilot processing plant in refrigerated Styrofoam coolers

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(Lifoam Industries, Hunt Valley, MD) and processed immediately.

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Processing

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Throughout the processing and storage chains, each of the six field replicates was maintained

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as discrete samples. All commodities were given a preliminary rinse with water prior to entering

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the pilot plant to avoid unnecessary contamination of facilities. Commodities were then


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submerged in a flume wash (Food Science and Technology Machine Shop, Davis, CA) filled

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with water and rinsed thoroughly to remove any surface dirt. Some commodities received

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additional processing steps prior to blanching: carrots were diced into 1.5 cm cubes using an

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Urschel G-A Dicer (Urschel Laboratories, Inc., Valparaiso, IN), strawberries had their crowns

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removed by hand, green beans and peas were de-stemmed by hand, broccoli was cut into 3-5cm

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florets by hand, and individual corn kernels were removed from the cob by hand using a Zyliss

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Corn Stripper (Zyliss, Irvine, CA).

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For each field replicate of each commodity, cleaned, prepared samples were randomized and

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separated into two parts. One half of each field replicate was then marked for fresh storage, while

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the other was blanched and frozen. The samples to be blanched were loaded onto the steam

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blanching line (Food Science and Technology Machine Shop, Davis, CA) in stainless steel

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baskets for the specified amount of time and temperature. Following blanching, the samples were

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transferred onto wire mesh racks, and placed immediately into a -32°C walk-in freezer (Estes

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Refrigeration, Inc., Richmond, CA). After 1 hour, the frozen commodity was divided into three

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300g storage samples which were packaged in UltraSource 3 mil polyethelyne pouches

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(UltraSource LLC, Kansas City, MO) and stored at -27.5°C (18°F) for up to 90 days.

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Blueberries and strawberries were not blanched prior to freezing, in accordance with industry

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practices.

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Stability Study

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The fresh half of each field replicate was divided into three 300 g storage samples, which

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were stored in breathable Tuf-R low density polyethylene bags (U.S. Plastic Corp., Lima, OH)

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and stored at 2°C (35.6°F) in a walk-in refrigerator (Estes Refrigeration, Inc., Richmond, CA) for



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up to 10 days. The frozen half of each field replicate was divided into three 300g storage samples

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which were packaged in UltraSource 3 mil polyethelyne pouches (UltraSource LLC, Kansas

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City, MO) and stored at -27.5°C (18°F) for up to 90 days. For each field replicate, one frozen

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pouch and one fresh pouch were analyzed within 24 hours of harvest (day 0) and after each

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storage time: 3 and 10 days for fresh; 10 and 90 days for frozen. Upon completion of each

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storage period, samples were removed from storage and transported in refrigerated coolers to the

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UC Davis Analytical Laboratory facilities for analysis.

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Homogenization and Sample Preparation

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Fresh or frozen samples were blended in a blender (Vita-Prep 3,Vitamix, Cleveland,

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Ohio) with the addition of 6 g deionized water for every 10 g sample.

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Riboflavin

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Extraction

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Homogenized sample (3.2 g, equivalent to 2 g sample) was weighed into 50 mL plastic

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centrifuge tubes. To this, 20 mL 0.1M HCl (Fisher Scientific Co, Pittsburgh, PA) was added, and

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the tubes were capped and shaken for 5 minutes, then incubated at 100°C for 30 minutes. Once

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cool, 2.5 mL 2.5M sodium acetate (Fisher Scientific Co, Pittsburgh, PA) was added, to

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approximate pH of 4.87, and then 100 mg amyloglucosidase (Sigma-Aldrich, St. Louis, MO)

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was added to each sample. The samples were shaken and incubated at 37°C for 15 hours. After

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cooling, 2 mL trichloroacetic acid (Fisher Scientific Co, Pittsburgh, PA) was added to each tube,

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and the samples were heated to 60°C for 15 minutes. After cooling, samples are diluted with

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deionized water to a final volume of 40 mL shaken, and centrifuged for 10 minutes at 4000

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RPM. From the supernatant, 10 mL aliquots were taken, 50 µL of 10 ppm internal standard


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(13C415N2 riboflavin, Sigma-Aldrich, St. Louis, MO) was added and the tubes were vortexed. A

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10 mL sample aliquot was loaded onto an Oasis HLB 3 cc (60 mg) extraction cartridges (Waters

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Corporation, Milford, MA) which was pre-washed with 1 column volume methanol followed by

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1 column volume deionized water. The extraction column was washed with 2.5 mL

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trichloroacetic acid and dried for 5 minutes under vacuum. The column was eluted with 1 mL

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methanol (Fisher Scientific Co, Pittsburgh, PA) into glass test tubes. The extracts were filtered

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through 0.20 µm IC Millex-LG (EMD Millipore Corporation, Billerica, MA) filters into

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autosampler vials. A 15 µL aliquot of sample was then injected onto the HPLC column for

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LC/MS determination.

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Analysis

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The samples were analyzed using high performance liquid chromatography (HPLC) in

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conjunction with mass spectrometry (MS). The apparatus consisted of a Perkin Elmer LC-200

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chromatograph (Perkin Elmer, Waltham, MA) with a Sciex API 2000 mass spectrometer (AB

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SCIEX, Framingham, MA) in the positive in the positive ion ESI mode. The transition ion m/z

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377 to m/z 243 (Riboflavin) and m/z 283 to m/z 249 (13C415N2 riboflavin, internal standard)

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were used. An isocratic mobile phase of 90% methanol with 0.2% acetic acid (Fisher Scientific

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Co, Pittsburgh, PA) and 10% water with 0.2% acetic acid (v/v) at 0.4 mL/min was used on a

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Waters XTerra RP18 column, 3.5 µm pore size, 4.6 x 150 mm (Waters Corporation, Milford,

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MA).

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Ascorbic Acid

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Extraction



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Homogenized sample (6.4 g) was mixed with 13.6 mL of 2% oxalic acid (Fisher

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Scientific Co, Pittsburgh, PA) and homogenized for 30 seconds. From this mixture, 10 mL was

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transferred to a 15 mL centrifuge tube and centrifuged at 10,000 rpm for 10 minutes at 4°C. A

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1.8 ml aliquot was taken, 400 µL of 5% dithiothreitol (Sigma-Aldrich, St. Louis, MO) was

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added, and the sample was filtered through a 0.2 µm filter. The filtered sample was transferred to

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an autosampler vial for HPLC analysis.

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Analysis

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The samples were analyzed using high performance liquid chromatography (HPLC) with

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UV/Vis diode array detection at 261 nm. The apparatus consisted of a Perkin Elmer 200

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quaternary HPLC system (Perkin Elmer, Waltham, MA) with a Perkin Elmer 200 diode array

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detector (Perkin Elmer, Waltham, MA). A Phenomenex Luna C-18 HPLC column (100 mm x

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4.6 mm, 100A) with a C-18 guard column (Phenomenex, Torrance, CA) was used. The mobile

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phase was 95% water and 5% methanol with 5 mM hexadecyltrimethylammonium bromide and

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50 mM potassium dihydrogen phosphate (Sigma-Aldrich, St. Louis, MO) at 1.2 mL/min.

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α-Tocopherol and β-Carotene

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Extraction

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Homogenized sample (1.6 g) was weighed into a 50 mL glass centrifuge tube along with

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5 mL ethanol containing 6% pyrogallol (Sigma-Aldrich, St. Louis, MO) (w/v), and the mixture

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was sonicated for 10 minutes. One mL 50% KOH (Fisher Scientific Co, Pittsburgh, PA)

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(aqueous) was added, the mixture was mixed by vortexing and heated at 70°C for 10 minutes,

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mixed and heated for an additional 10 minutes. The sample was cooled to room temperature and


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5 mL 5% NaCl was added. The sample was extracted with 30 mL extraction solvent (85:15

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hexane:ethyl acetate v/v with 0.05% BHT, Sigma-Aldrich, St. Louis, MO). A 7.5 mL aliquot was

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evaporated to dryness at 40°C under nitrogen using a Zymark TurboVap LV. The extract was

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redissolved in 200 µL ethyl acetate followed by 1.8 mL methanol, mixed and filtered into an

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autosampler vial for HPLC analysis.

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Analysis

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The samples were analyzed using high performance liquid chromatography (HPLC) with

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UV/vis and fluorescence detection. The apparatus consisted of a Perkin Elmer 200 quaternary

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HPLC system (Perkin Elmer, Waltham, MA) with a Perkin Elmer 200 UV/Vis detector (Perkin

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Elmer, Waltham, MA) and a Shimadzu 10A×s flu°°°orescence detector (Shimadzu Scientific

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Instruments, Columbia, MD). Excitation and emission wavelengths of 295 nm and 340 nm were

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used to detect α-tocopherol and an absorbance wavelength of 450 nm was used to detect β-

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carotene. A Phenomenex Kinetex C-18 HPLC column (100 mm x 4.6 mm, 100A) with a C-18

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guard column (Phenomenex, Torrance, CA) and a mobile phase of 9:1 acetonitrile:methanol

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(v/v) (Fisher Scientific Co, Pittsburgh, PA) at 1 mL/min was used.

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Statistical Analysis

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Statistical analysis was performed using JMP statistical software version 9.0.0 (SAS Institute

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Inc., Cary, NJ). A blocked ANOVA was run with storage time point and processing treatment as

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the treatments. Tukey comparisons were used to determine the significance of differences

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between both fresh and frozen treatments as well as storage time points for each commodity and

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nutrient.



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Results and Discussion The concentration of 4 different compounds was evaluated in 8 different commodities stored

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under either refrigeration (fresh) or frozen conditions over three time points (Table 3).

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Ascorbic Acid

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Ascorbic acid was degraded less in frozen stored samples than in fresh (Figure 1). None of

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the eight commodities showed losses during frozen storage. In strawberries, carrots, spinach,

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peas and broccoli, the ascorbic acid content of fresh-stored products was not significantly

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different than that of frozen-stored. In corn, green beans, and blueberries, significantly higher

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levels of ascorbic acid were found in frozen-stored samples when compared to fresh-stored

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samples, which could possibly be attributed to arrested enzymatic activity and slowed oxidative

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degradation of ascorbic acid in the frozen samples (6). Extensive degradation of ascorbic acid in

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fresh-stored produce has been previously reported in vegetables, as compared to their frozen

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counterparts (21, 22).

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Riboflavin

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Riboflavin was well conserved in most frozen samples. Carrots, corn, broccoli, blueberries,

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and green beans all followed the same trend, with fresh containing the same riboflavin content as

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frozen samples (Figure 2a). Of the eight commodities studied, only peas lost riboflavin during

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frozen storage (Figure 2b). The loss of riboflavin in peas is most likely due to oxidative

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degradation of the nutrient. Similar results were found by Gleim et al. (23), who noted large

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decreases in riboflavin in asparagus and spinach.


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Broccoli (Figure 2c) actually had higher riboflavin content in stored frozen vs. fresh samples.

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This contrasts with the majority of the literature, such as Makhlouf et al. (24), who found that,

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while riboflavin content was higher in frozen vegetables than canned, it was not higher in frozen

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vegetables than fresh. Similarly, while Van Duyne et al. (25) found riboflavin to be well retained

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in frozen peas, beans, and spinach, it was not found to be present in any higher amounts in frozen

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produce as compare to fresh.

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Alpha-Tocopherol

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Of all of the nutrients determined in this study, the α-tocopherol content in fruits and

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vegetables benefited the most from blanching, freezing and frozen storage, as compared to fresh.

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When stored fresh, peas, carrots, and corn showed significant decreases in alpha-tocopherol

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content (Figure 3). Fresh green beans had much lower levels of alpha-tocopherol than frozen, but

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the levels of alpha-tocopherol did not decrease over the course of fresh storage. In the remaining

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commodities blueberries, broccoli, green beans, spinach and strawberries, no significant

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difference between fresh and frozen stored samples was observed (Figure 3). Frozen peas and

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green beans exhibited more than two-fold higher levels of alpha-tocopherol, while blueberries,

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spinach, and corn also had significantly higher levels (12-39%) in frozen-stored samples, as

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compared to fresh stored, (Figure 3).

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The observed higher levels of α-tocopherol in some commodities, which is evident

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immediately after blanching and freezing on day 0, may be due to its increased availability after

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steam blanching (26). Alpha-tocopherol levels in fresh broccoli were found to be more than

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twofold higher after heat treatments such as steaming or boiling by previous authors (26). The

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heat treatment administered during blanching could have a similar effect on the commodities in



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this study. Previous authors have not studied the effects of freezing on peas in any detail, but

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lipid oxidation in peas caused by enzymatic or non-enzymatic pathways has been reported to

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consume α-tocopherol, a potent antioxidant (27). Both of these oxidative pathways could have

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been responsible for preferentially lowering levels of alpha-tocopherol in fresh samples. It is

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also unlikely that any leaching of fat-soluble α-tocopherol would occur during blanching in an

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aqueous environment.

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β -Carotene

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β-carotene was not found in any significant amount in blueberries, strawberries, and corn,

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even in fresh samples (

Vitamin Retention in Eight Fruits and Vegetables: A Comparison of

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