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 is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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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

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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 (