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Stability of vitamin C in fruit and vegetable homogenates stored at different temperatures
Accepted Manuscript Title: Stability of vitamin C in fruit and vegetable homogenates stored at different temperatures Author: Katherine M. Phillips McAlister Council-Troche Ryan C. McGinty Amy S. Rasor Maria Teresa Tarrago-Trani PII: DOI: Reference:
S0889-1575(15)00198-2 http://dx.doi.org/doi:10.1016/j.jfca.2015.09.008 YJFCA 2631
To appear in: Received date: Revised date: Accepted date:
19-7-2015 15-9-2015 17-9-2015
Please cite this article as: Phillips, K. M., Council-Troche, M. A., McGinty, R. C., Rasor, A. S., and Tarrago-Trani, M. T.,Stability of vitamin C in fruit and vegetable homogenates stored at different temperatures, Journal of Food Composition and Analysis (2015), http://dx.doi.org/10.1016/j.jfca.2015.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Stability of vitamin C in fruit and vegetable homogenates stored at different temperatures
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Katherine M. Phillipsa*, McAlister Council-Trocheb, Ryan C. McGintya,c, Amy S. Rasora,d, and
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Maria Teresa Tarrago-Trania,e
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Department of Biochemistry, Virginia Tech Blacksburg, VA
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*Corresponding author
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Phone: 540-231-9292; FAX: 540-231-9070; email: [email protected] b
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Department of Biochemistry, 304 Engel Hall (0308), Virginia Tech, Blacksburg, VA24061.
Current affiliation: Polymer Solutions, Inc., Christiansburg, VA, USA;[email protected] c [email protected] d [email protected] e [email protected] Highlights
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strawberries, oranges, and tomatoes; baked potatoes; steamed broccoli and spinach; and
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Vitamin C analyzed in several types of homogenized fruits and vegetables Assayed afterrefrigerated and frozen storage (-20oC, < -55oC)over 1-week period No change in vitamin C at < -55oC Significant losses after refrigeration or freezing at -20oC in some foods Up to 15% loss in non-acidic stored3 days at -20oC (17 mg/100g in raw broccoli) Detailed quality control implemented and described to ensure validity of data
Abstract Vitamin C loss was compared in homogenized raw broccoli, potatoes, spinach,
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pasteurized orange juice after storage under residual nitrogen under refrigeration, and frozen at
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conventional ( 10 to
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Additional foods (cantaloupe, green sweet peppers, collard greens, Clementines) were monitored
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for 3–4 years at < 55oC. Total ascorbic acid was quantified using high-performance liquid
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chromatography and detailed quality control measures. No decrease occurred in any of the foods
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after 7 days at < 55oC. Under refrigeration the largest decreases were in raw spinach and
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broccoli, averaging (mg/100g) 9.5 (29%) and 33.1 (29%), respectively, after 1 day and 31.0 and
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77.0 after 7 days (94% and 68%). With conventional freezing, vitamin C was stable for 7 days in
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most of the products studied; minor losses occurred in raw spinach and broccoli after 1 day but
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were substantial after 3 days (6.9 mg/100g (23%) and 17.0 mg/100g (15%), respectively) and 7
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days (13.1 and 32.0 mg/100g). For homogenates stored long-term at < 55oC, vitamin C loss
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occurred in only cantaloupe, collard greens, and one sample of raw potatoes, all before 50 weeks.
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20oC) and ultra-low (< 55oC) temperatures for 1, 3, and 7 days.
Keywords:Ascorbic acid;Food composition; Food analysis; Food processing; Freezing; Quality
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control; Pureed fruit; Pureed vegetables; Refrigeration; Stability
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Chemical compounds studied in this article
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L-Ascorbic acid (PubChem CID: 54670067); Dehydroascorbic acid (PubChem CID: 210328)
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Abbreviations: AA, ascorbic acid; AO, ascorbate oxidase; APO, ascorbate peroxidase; DDI,
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distilled deionized; DHAA, dehydroascorbic acid; EDTA, Ethylenediaminetetraacetic acid
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disodium salt dihydrate; HPLC, high-performance liquid chromatography; MDD, minimum
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detectable difference; MPA, metaphosphoric acid; RDA; Recommended Dietary Allowance;
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RSD, relative standard deviation; SD, standard deviation; TCEP, tris(2-carboxyethyl) phosphine
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hydrochloride; UV, ultraviolet light
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1. Introduction
Vitamin C is an essential water-soluble vitamin, with a Recommended Dietary
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Allowance (RDA) of 75 mg/day and 90 mg/day for adult women and men, respectively, and 45
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mg/day for children 9 13 years old (Food and Nutrition Board, Institute of Medicine, 2000). L-
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Ascorbic acid [(2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one] (AA) is the
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major naturally occurring compound with vitamin C activity. AA is cofactor for many iron and
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copper hydroxylases involved in key physiological processes such as the production of collagen,
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the synthesis and activation of peptide hormones (norepinephrine, noradrenaline), and the
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synthesis of carnitine (Arrigoni& De Tullio, 2000; Ball, 2006; Davies et al., 1991; Bender, 2003;
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Johnston et al., 2007). AA also has an important role in the intestinal absorption of non-haemiron
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and as a cellular antioxidant, independently or together with the antioxidant action of vitamin E
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(Bender, 2003; Byers & Perry, 1992).Dehydroascorbic acid (DHAA) is formed readily from the
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oxidation of AA in aqueous solutions, and as by-product of cellular reactions of AA or other
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oxidative processes (Tolbert & Ward, 1982). DHAA is present in many foods (Gökmen et al.,
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2000). It is absorbed from the small intestine in humans and reduced to AA intracellularly
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(Deutsch, 2000), and as a result it is bioavailable; thus, the vitamin C content of foods is usually
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considered to be the sum of the AA and DHAA (Wilson, 2002).
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Vegetables and fruits, particularly citrus, green leafy vegetables, broccoli, cauliflower, Brussels sprouts, tomatoes, peppers, and potatoes, are major dietary sources of AA (Eitenmiller
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et al., 2008). For example, the average vitamin C content of one medium baked potato, one
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medium tomato, and one medium navel orange are 16.6 mg, 16.9 mg, and 82.7 mg, respectively
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(U.S. Department of Agriculture, 2014), providing 18 92% of the 90 mg RDA for adult men.
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Epidemiological studies have associated adequate vitamin C intake with decreased mortality, but
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controlled trials are lacking (Enstrom, 2008). Studies that relatenutrient intake to healthusually
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involvecalculating nutrient intake using food composition data combined with known or
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estimated measures of food consumption. Obviously, the accuracy of the calculated intake of a
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particular nutrient depends on how closely its concentration in the food as consumed matches the
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food composition data.The vitamin C content of fruits and vegetables can be substantially
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affected by post-harvest handling and processing (Cocetta et al, 2014; Giannakourou&Taoukis,
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2003; Neves et al., 2015; Rodrigues et al., 2010; Spínola et al., 2013; Tiwari & Cummins, 2013;
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Zee et al., 1991). Natural variability in the vitamin C content of foods as they exist in the food
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supply should be reflected in food composition data. Additionally, once food samples have been
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procured, the accuracy of the analyzed nutrient content depends on preventing changes in
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nutrient during the analytical process itself.
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The primary source of food composition data in the United States is the U.S. Department
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of Agriculture’s (USDA) National Nutrient Database for Standard Reference (SR) (U.S.
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Department of Agriculture, 2014). The USDA’s National Food and Nutrient Analysis Program
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(NFNAP) is an ongoing effort, begun in 1996, to update and improve the quality of food
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composition data in SR (Haytowitz et al., 2008). One of the practical challenges in the NFNAP is
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that a wide range of nutrients must be assayed in each food that is sampled. The cost of
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purchasing, shipping, and preparing the foods for analysis is a significant factor in the total cost
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of the project. Thus, multiple primary samples are often combined to yield composites that are
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statistically representative of the national market for a given food (Pehrsson et al., 2000).
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Documentation of the samples and their handling also must be maintained, with a complete audit
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trail from sample procurement to the release of final data in SR, and archived subsamples of all
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composites are maintained. Therefore, centralized homogenization of samples and distribution of
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subsamples for the various nutrient assays is the only practical approach for the NFNAP.Primary
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food samples (sample units) are procured from retail and wholesale locations and sent to the
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Food Analysis Laboratory Control Center (FALCC) at Virginia Tech (Blacksburg, VA) (Trainer
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et al., 2010) where they are prepared, composited, homogenized, and typically dispensed into
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subsamples in 30- or 60-mL glass jars, sealed under nitrogen, and stored in an ultra-low freezer
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(< 55oC), protected from light, until distributed for analysis along with matrix-matched quality
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control materials (Phillips et al., 2006). Under routine NFNAP processing conditions, a
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minimum of 2 weeks, and often several weeks, elapse between homogenization and analysis.
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The sample processing and storage conditions used in the NFNAP were chosen as what is
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practical to achieve for long term storage of samples of a variety of foods with minimal potential
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for nutrient loss.
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To ensure that the analyzed concentration of vitamin C is representative of the food as
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consumed, the portion of the sample analyzed mustbe representative of the whole food.
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Obtaining a representative portion of a whole foodthat is heterogeneous (e.g., potatoes with skin)
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requires either grinding and extracting a large amount of the whole fooddirectly in the extraction
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solvent (for example, as described by Vanderslice et al., 1990 or Veltman et al., 2000), or
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homogenizing the food and then taking a subsample for analysis. The former may require a large
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volume of solvent to accommodate the minimum sample size necessary to be representative of
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the whole food. Also, in studies involving a large number of samples that must be combined for
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analysis, in which the food must be analyzed for multiple nutrients involving different extraction
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methods and multiple laboratories (such as the NFNAP),it is impossible to directly extract the
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very same whole sample for all assays. Homogenization allows generation of multiple analytical
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subsamples from one larger sample and the ability to store subsamples prior to analysis, yielding
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the greatest flexibility in analysis. However, it is critical to establish that labile nutrients such as
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vitamin C are preserved, to avoid significant errors in food composition data. Once the cell wall
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has been disrupted, the effect of external factors on reactions resulting in irreversible oxidation
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of AA and loss of vitamin C (and other nutrients) is more likely to occur, and failure to stabilize
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AA could result in an assayed vitamin C content of a fruit or vegetable that is quite different
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from what it is as consumed. Spínola et al. (2014) have recently published an excellent review
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that includes sample handling considerations for analysis of vitamin C in foods.
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AA is especially vulnerable to oxidative and enzymatic degradation in raw fruits and
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vegetables in which endogenous enzymes are active (Redmond et al., 2003; Francisco et al.,
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2010; Munyaka et al., 2010a; Wen et al., 2010; Cocetta et al., 2014). Significant and variable
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post-harvest loss of vitamin C in many fruits and vegetables stored under less optimal conditions
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has been reported (e.g., González et al., 2003; Lee & Kader, 2000; Munyaka et al., 2010a; Neves
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et al., 2015; Rybarczyk-Plonska et al., 2014; Szeto et al., 2002; Tiwari & Cummins, 2013;
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Veltman et al., 2000; Zee et al., 1991). González et al. (2003) measured vitamin C in raspberries
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and blackberries stored from 0 to 12 months and found an average decrease of 37% and 31%
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(10.7 and 7.9 mg/100g), respectively. The storage temperature of –24 °C was higher than the–
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60°C used in the NFNAP, and the berries were frozen whole, not homogenized. Vanderslice and
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co-workers (1990) reported on the vitamin C content of somefruits and vegetables and performed
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stability testing on raw broccoli stored under different conditions (refrigerated at –4°C and
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frozen at –40°C, with or without citric acid or metaphosphoric acid). The treatment in the
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Vandersliceet al. (1990) study that is most relevant to NFNAP standard conditions of < 55°C
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under nitrogen was storage at –40°C. In that study, total AAwas constant for 2 weeks (133±7
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mg/100g) and dropped thereafter, reaching 89±25 mg/100g after 2 months (Vanderslice et al.,
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1990). Because pH and other matrix-specific characteristics are known to affect vitamin C
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stability (Musulin& King, 1936; Moser &Bendich, 1990; Wechtersbach&Cigic, 2007), results
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for broccoli might not apply equally to other fruits and vegetables, and the lower temperature and
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nitrogen atmosphere used in NFNAP should impart additional stability. Previously, the stability
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of vitamin C in subsamples of selected raw fruit and vegetable homogenates (clementines,
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collard greens, potatoes) prepared in liquid nitrogen and stored at < 55oC under residual
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nitrogen for one year was established (Phillips et al., 2010).
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Many laboratories do not have access to an ultra-low freezer (< -55oC), and the cost is
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as a matter of practicality,homogenized fruits and vegetables prepared for analysis might be
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exposed to refrigerator or conventional freezer temperatures. The Association of Official
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Analytical Chemists method for analysis of vitamin C in foods (AOAC, 2012) specifies
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homogenization of the food in a blender and subsamplingfor analysis, but there is no indication
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for temperature control. In fact, some literature reports on the vitamin C content of fruits and
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vegetables involve storage of prepared samples at ≥
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(e.g.,Giannakourou&Taoukis, 2003; Kabasakalis et al. 2000; Mahattanatawee et al., 2006;
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Rizzolo et al., 2002; Vanderslice et al., 1990). Whether or not storage at these temperatures is
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necessary for practical reasons, less than optimal conditions could lead to substantial and
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variable deviations in the vitamin C content of the food as assayed versus as consumed.
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higher for storage at ultra-low versus standard freezer temperature (~
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20oC). Therefore,
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20oCprior to analysis
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The objective of this study was to assess the change in the vitamin C concentration in
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different fruits and vegetables that were homogenized while frozen in liquid nitrogen and then
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stored under refrigeration (0 to 5oC), and frozen at conventional ( 10 to
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(< 55oC) temperaturesfor up to one week. Additionally, we report on the stability of vitamin C
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in a variety of similarly homogenized samples of fruits and vegetables stored in an ultra-low
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freezer for >4 years, as a follow-up on results after 1 year storage that were previously reported
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(Phillips et al., 2010).
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2. Materials and Methods
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2.1.1Food selection
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2.1 Storage temperature study
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20oC) and ultra-low
Foods representing good sources of vitamin C, and having characteristics that could
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affect stability of vitamin C (e.g. pH, endogenous enzyme activity) were selected for the study:
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strawberries (Fragaria X ananassa); grape tomatoes (Solanum lycopersicum); baby spinach
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(Spinaciaoleracea), raw and steamed; russet potatoes (Solanum tuberosum), raw and baked;
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broccoli (Brassica oleracea var. italica), raw and steamed; navel oranges (Citrus sinensis); and
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pasteurized orange juice (pulp-free, refrigerated, not from concentrate). Two independent
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experiments were performed for each individual food and were separated by at least three
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months. For each experiment, the product was purchased from a local retail market (Blacksburg,
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VA)[~500 g spinach (2 4 packages); ~1 2kg strawberries, potatoes, grape tomatoes, oranges,
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and broccoli; three 1.89L cartons pulp-free refrigerated, pasteurized,orange juice (not from 8
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concentrate).Navel oranges and broccoli samples wereeach chosen to be similar in colour and
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size and were purchased loose; tomatoes and strawberries, bought in (0.47L (1 pint)containers
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and 908 g (2 lb) containers, respectively, were selected to beof similar size, colour, ripeness and
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expiration date; potatoes chosen for the composite were all approximately the same size and
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colour with as minimal need for trimming of eyes as possible; packages of baby spinach having
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an expiration date of at least a week after the date of purchase and showing no major amount of
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damaged or wilted leaves were selected. Potatoes, strawberries, and tomatoes were purchased
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and stored at room temperature (20°C) until prepared within 24 hours of preparation and
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homogenization (sections 2.1.2, 2.1.3). Oranges, orange juice, broccoli and spinach were
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purchased from the store’s open, refrigerated display case and were stored in a refrigerator
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(1 5°C) until prepared within 24 hours of purchase.
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2.1.2 Food preparation
All food preparation was done under UV-filtered light. Just prior to homogenization,
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foods were cleaned and trimmed as follows, including removal of any damaged portions of the
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produce. Strawberries and tomatoes were rinsed in a stainless steel colander for two minutes with
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distilled deionized (DDI) water and patted dry with a lint-free cloth (Teri® wipe, Kimberly-
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Clark Professional®, Georgia, USA). The top ~2.5 mm of each strawberry, including the leaves,
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were removed with a stainless steel knife before beingcut in half and frozen immediately in
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liquid nitrogen. Rotten, unripe, and split tomatoes were discarded prior to freezing the whole
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fruits in liquid nitrogen. Navel oranges were peeled one at a time, and any significant amount of
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albedo adhering to the fruit flesh was removed. The wedges were immediately frozen in liquid
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nitrogen after separating.
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Raw baby spinach leaves (labelled as triple washed, ready to eat were immediately frozen in liquid nitrogen after discarding any wilted or damaged leaves. Steamed spinach was prepared
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by steaming the leaves for 10 minutes in a 7.57-L stainless steel steamer pot with lid, filled with
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DDI water to a depth of approximately 2.5 cm and brought totheboil prior to adding the
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spinach.After steaming, the steamer basket with the spinach leaves was removed from the pot
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and allowed to cool for 10 minutes before freezing the spinach in liquid nitrogen.
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Individual potatoes were thoroughly scrubbed with a stiff-bristle nylon brush,any eyes
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were removed, and then the potato was rinsed for 2 minutes with DDI water and patted dry with
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a Teri® wipe. Raw, unpeeled potatoes were cut one at a time into ~1.25-cm cubes with a
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stainless steel knifeand frozen immediately in liquid nitrogen.Baked potatoes (unpeeled, whole)
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were prepared by punching six ~0.5 mm wide by ~2.5 mm deep holes, one into each face with a
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stainless steel pick, then baking in a conventional oven for 50 minutes at 218°C (425°F). The
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baked potatoes were allowed to cool at room temperature for 15 minutes, then individually cut
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(unpeeled) into ~1.25 cm cubesand immediately frozen in liquid nitrogen.
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Whole broccoli florets were prepared by paring with a stainless steel knife any leaves and
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removing the smaller stalks of approximately 1 cm or less in diameter from the larger stalk. Any
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yellow, brown or spoiled patches of the florets were removed and the remaining florets were
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trimmed to approximately 1 cmin length.The florets were then rinsed in a stainless steel colander
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for one minute with tap water, followed by two minutes with DDI water. After using a Teri®
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wipe to pat the florets dry, they were cut in half and immediately frozen in liquid nitrogen.
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Florets to be steamed were placed in a 7.57-L stainless steel steamer pot with lid, filled with DDI
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water to a depth of approximately 2.5 cm and brought to theboil prior to addition of the broccoli,
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and steamed for four minutes. The florets were allowed to cool at room temperature for 10
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minutes,cut in half, and thenfrozen in liquid nitrogen.
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2.1.3 Homogenization
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Homogenization was done as previously described and using procedures that have been standard for preparation of fruits and vegetables for analysis in the NFNAP (Haytowitz et al.,
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2008; Trainer et al., 2010) and the previously reported study on stability of vitamin C in
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homogenized fruits and vegetables (Phillips et al., 2010).
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For each experiment, the prepared food that was frozen in liquid nitrogenwas transferred
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to a 6-L stainless steel food processor (Robot Coupe 6L Blixer®; Robot Coupe USA, Jackson,
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MS), blended 10 s at 1500 rpm, then for two additional 30-s periods at 3500 rpm, until a
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homogenous composite (fine powder consistency) resulted.The frozen, ground material was
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transferred to a stainless steel bowl and kept frozen by adding and stirring in additional liquid
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nitrogen as needed.The composite was dispensed among fifty-four 60-mL glass jars with
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Teflon™-lined screw-caplids (~ 10 g/jar, filled approximately half full). The jars were
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wrappedcompletely with aluminium foil to prevent UV degradation from the light in the
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refrigerator. Two individuals worked in tandem to immediately replace the caps of the jars after
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each subsample was dispensed, seal the rims of the lid with 1.9-cm Write-On™ label tape (Bel-
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Art™, New Jersey, USA), and store the samples in their respective storage
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locations(Section2.1.4) before thawing occurred (all tasks completed in under 3 minutes): Nine
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jars were stored in the refrigerator (1 5oC), ninejars were stored in the conventional freezer, and
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ninejars were stored in the ultra-low freezer (< 55oC).Additional subsamples were dispensed
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and stored at < 55oCfor moisture analysis and possible additional assays of vitamin C to
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monitor changes over long term storage. Three replicates (taken from separate subsample jars)
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were extracted immediately, without thawing, for analysis of the initial vitamin C concentration
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as described in Section2.2. Each of the three 1.89-L containers of refrigerated orange juice was shaken vigorously,
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and the entire contents of all containers were stirred together in a stainless steel bowl for two
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minutes using a stainless steel whisk.The temperature of the composite was recorded before
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mixing, after mixing, and after dispensing into to jars, and remained at ~4°C throughout. Ten
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millilitres of the composite were pipetted into each of the subsample jars described above using a
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manual pipette and then blanketed with nitrogen gas before recapping and sealing with tape.
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They were then stored and analyzed as described for the homogenized fruits and vegetables.
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An in-house control composite of cooked/canned fruits and vegetables (spinach,
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babyfood green beans, babyfood carrots, peas, pumpkin, asparagus, tomato puree) was prepared
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using methods previously described (Phillips et al., 2006).
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2.1.4Sample storage appliances
The conventional refrigerator/freezer in which samples were stored was a 0.5 m3 Revco™
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Thermo Fisher Scientific™ (Asheville, NC, USA) model # RCRF192A17. The ultra-low freezer
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was a 0.7 m3Revco model# ULT2586-3-ABA, purchased from Thermo Fisher Scientific™
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(Waltham, MA, USA).( Temperatures were monitored daily. The refrigerator portion of the
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refrigerator/freezer was factory set to 4°C. The freezer portion of the refrigerator/freezer was
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factory set to a minimum temperature of
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built-in auto-defrost feature. Because this was a typical frost-free freezer, the temperature cycle
20°C and maximum temperature of
12°C with a
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was monitored over two four-hour periods to document fluctuations due to the defrost cycle. The
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ultra-low freezer was not frost-free.
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2.1.5Food samples for the long-term stability study
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Homogenates of raw russet potatoes, collard greens (Brassica oleracea var. viridis), and Clementines(Citrus clementina hort. ex Tanaka) prepared and stored at < -55oC withresidual
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nitrogen (from homogenization) and protected from light as previously reported (Phillips et al.,
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2010), were assayed after a total of 127, 187, and 214 weeks (4.1 years; 2007 2012), along with
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control samples, for comparison to results previously reported after 1 year storage (2007 2008)
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Additional homogenates of raw broccoli (florets), steamed broccoli (florets), raw russet potatoes
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(with skin), baked russet potatoes (with skin), raw green sweet bell peppers (Capsicum annuum),
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and cantaloupe (Cucumismelo) were prepared, stored, and assayed in the same manner, after 2,
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31, 75, 129, 135, and 164 weeks (3.2 years; 2008 2012). Samples of a control composite of
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orange juice (pasteurized, pulp free) were also analyzed with these stored homogenates.
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2.2 Analysis of vitamin C
Vitamin C was analyzed as AA after treatment with tris(2-carboxyethyl) phosphine
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hydrochloride(TCEP) to reduce any DHAA, using previously validated and reported
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methodology (Tarrago-Trani et al., 2012). Additional details necessary to provide a full
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description of the reagents, standards and analyses performed in this study are as follows.
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2.2.1 Reagents and standards
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ACS grade L-AA (>99% pure), ACS grade 33.5 36.5% metaphosphoric acid (MPA), and ethylenediaminetetraacetic acid disodium salt dihydrate(EDTA) disodium salt were
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purchased from Sigma-Aldrich (Saint Louis, MO, USA). High-performance liquid
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chromatography (HPLC) grade 85% ortho-phosphoric acid and HPLC grade water were
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purchased from Fisher Scientific (Pittsburgh, PA, USA). TCEP was obtained from Thermo
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Fisher Scientific™ Pierce™ (Rockford, IL, USA).Eight AA calibration standards ranging from
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0.1 12.5 µg/mL were serially diluted from a previously prepared 5 mg/mL AA standard stock
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solution. All AA standard solutions were prepared in extraction buffer (Section 2.2.2), blanketed
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with argon, and stored at< 55oC for up to four months.
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2.2.2Extraction of vitamin C
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All work was done in a temperature controlled (20 22°C) room with UV filtered
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lightbulbs and light-blocking window shades.Extraction buffer (5% MPA/1 mM EDTA/pH 1.5)
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was prepared no more than 5 days in advance of use and kept in the refrigerator. TCEP (5 mM)
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was added on the day of use.
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Each analytical subsample was taken from a separate jar. Each jar was removed from
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storage immediately prior to weighing and kept on ice during the weighing process. Briefly,
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2.0±0.1 g of each frozen homogenized fruit or vegetable (1.0 ± 0.5 g of orange juice) were
323
weighed into 30-mL Teflon® centrifuge tubes (Thermo Fisher Scientific™, cat# 31140030),after
324
thorough mixing to ensure representative subsampling. MPA extraction buffer was added
325
immediately after the analytical subsampling had been completed, and the tube was blanketed
326
with argon and kept on ice until all samples for the analytical batch had been weighed. Prior to
327
mixing, samples stored at < 55°C showed no signs of thawing and had the same dry, free-
328
flowing, powdery consistency as after the original homogenization. Samples stored in the
329
conventional freezer showed a slight lesseningof the powdery consistency but no significant
14
Page 14 of 69
thawing was observed. Samples stored in the refrigerator had completely thawed.No more than
331
~1 minute elapsed from the time asample was removed from storage and the addition of
332
theextraction buffer. Pasteurized orange juice and the control composite (which was aslurry)
333
were weighed after thawing completely in a 30 ± 1.0oC water bath for 20 min and were kept on
334
ice before and during weighing.Extraction buffer (12 mL, except 11 mLfor orange juice)was
335
added immediately to each weighed analytical subsample, and the tube was capped and kept on
336
ice until all samples in the batch had been weighed. After homogenization and initial separation
337
of the supernatant, the pellet was re-extracted twice in the same manner (Tarrago-Trani et al.,
338
2012). All supernatants were combined after centrifugal filtration through a 0.45-µm PVDF filter
339
(cat. #24162; Grace, Columbia, MD, USA) and brought to 50 mL total volume with extraction
340
buffer.Each extract was blanketed with argon, dispensed among8-mL amber glass vials with
341
Teflon® lined caps (Thermo Fisher Scientific™), and frozen at < 55°C until the day of HPLC
342
analysis, which was within 7 8 days for samples extracted on day 0 to 1 2 days for samples
343
extracted on Day 7 of the experiment.
345 346
cr
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344
ip t
330
2.2.3 High-performance liquid chromatography (HPLC) On the day of HPLC analysis, samples that had been frozen and stored in 8-mL vials
347
(Section 2.2.2) were completely thawed for 20 30 minutes in a 20 21°C water bath and
348
vortexed thoroughly for 30 seconds. They were thendiluted with the MPA extraction buffer, if
349
necessary (based on sample size extracted and AA content estimated using values in SR (USDA,
350
2014), or on package labels), to put the final concentration within the range of the calibration
351
curve (0.1 12.5 µg/mL).All samples with the exception of strawberries and the mixed vegetable
352
control composite required a further 1:5 dilution (1:250 total dilution), while the strawberry
15
Page 15 of 69
samples were diluted to a 1:10 dilution (1:500 total dilution) and mixed vegetable samples were
354
analyzed undiluted (1:50 total dilution).After dilution, 400 µL of each extract werefiltered
355
through pre-assembled Mini-Uniprep™ syringe-less 0.45-µm PVDF filtration devices consisting
356
of an amber colouredpolypropylene chamber and plunger filter (Waters Corp., Milford, MA,
357
USA), and then placed in the pre-chilled, 8°C refrigerated HPLC auto-sampler (model 1329A;
358
Agilent, Santa Clara, CA, USA).
cr
ip t
353
An Agilent 1200 HPLC system equipped with an LC quaternary pump (model G1311A)
360
and diode array detector (model G1315D), operated with ChemStation™ (version B.03.02),and
361
fitted with a C18 reversed-phase column with polar end-capping [Phenomenex® Synergi™ 4 µm
362
Hydro-RP (250 mm× 4.6 mm); Phenomenex, Torrance, CA, USA] was employed.The mobile
363
phase was aqueous ortho-phosphoric acid (0.022% w/v; pH 2).Of the filtered extract, 20 µL were
364
auto-injected into the HPLC and eluted under isocratic conditions at 0.7 mL/min with the AA
365
peak eluting at about 7.2–7.5 min and detected at 254 nm. An MPA extraction buffer blank was
366
run between each sample to ensure lack of carryover of AA from sample to sample.
an
M
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Ac ce pt e
367
us
359
The eight AA calibration standards (0.1 - 12.5 µg/mL) were run in triplicate, sequentially
368
from lowest concentration to highest, at the beginning, halfway point, and end of the
369
samplesequence on the HPLC.A calibration curve was prepared based on linear regression of
370
response versus AA concentration.
371
Because of the large number of extracts to be run on the HPLC in each experiment(>80,
372
including calibration standards and blanks), two separate runs were performed over two
373
consecutive days, using the same eight calibration standards and implementing the quality
374
control measures described in Section 2.3.
375
16
Page 16 of 69
376 377
2.3 Quality control Experiments were conducted one at a time on each food, with at least two independent experiments for each food. Careful attention was paid to the manner in which the samples stored
379
at different times and temperatures within each experiment were extracted and run on the HPLC,
380
to avoid confounding of any analytical variability withactual change in sample composition due
381
to the experimental variables, as follows. For each experiment, at each time point, three
382
subsamples of the homogenatefrom each storage temperature were extracted in the same batch,
383
along with a sample of the mixed vegetable control composite(Table 1A).The prepared extracts
384
from the 0, 1, and 3 day time points were held at < 55oC until the samples from 7 day time
385
pointhad been extracted. Then, the extracts from all storage times and temperatures were run
386
together on the HPLC, in two runs, with replicates distributed in a balanced manner between the
387
runs (Table 1B).
M
an
us
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378
Previous studies had established the stability of frozen extracts of the prepared sample
389
extracts stored at < 55oC for up to 4 weeks (K.M. Phillips, Virginia Tech, unpublished data).
390
Thesubsamples of extracts that were prepared immediately after thefood was homogenized, that
391
were stored < 55oC and assayed again after 7 days along with those of samples extracted at the
392
1, 3, and 7 day storage times, served to validate lack of any effect of storage time on vitamin C
393
in the extracts prepared from the homogenized food at different time points. Potential loss of
394
vitamin C in the mixed fruit and vegetable and orange juice control composites during the
395
thawing procedure in the 30°C water bath was confirmed to be absent (K.M. Phillips, Virginia
396
Tech, unpublished data).
Ac ce pt e
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388
397 398
2.4 Data analysis
17
Page 17 of 69
Data were compiled and means and standard deviations were calculated using
399
Microsoft® Excel® 2010 v. 14.0.7151.5001 (Microsoft Corp., Redmond, WA, USA). Statistical
401
analyses were performed using JMP® Pro 11 (SAS Institute Inc., Cary, NC, USA). One-way
402
analysis of variance (ANOVA) and Tukey-Kramer HSD t-test (0.05 significance level)
403
(McDonald, 2008) were performed to compare vitamin C mean values at Time 0 with
404
corresponding vitamin C mean values at different storage times within each storage temperature
405
and experiment. Two-way ANOVA and Tukey-Kramer HSD t-testwere applied to determine
406
within-experiment significant differences (0.05 significance level) between vitamin C means
407
across temperatures, and among storage times for each storage temperature. The critical
408
significance level for one-way and two-way ANOVA was set at 0.05, but differences in means
409
were conservatively estimated as significant for p-values <0.001, nonetheless, to avoid detection
410
of very small changes in the case of data points with very low relative standard deviations
411
(RSD).
cr
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d Ac ce pt e
412
ip t
400
For the samples stored long term at ultra-low temperature, data were evaluated using
413
XLSTAT (version 2012.4.03; Addinsoft™, New York, NY, USA), with ANOVA (α=0.05) and
414
pairwise comparison of means using the Tukey-Kramer HSD t-test, with a 95% confidence
415
interval. The same analysis was applied to the comprehensive in-house data set for the mixed
416
vegetable control material assayed with the samples over the yearsthe study samples were
417
assayed, to account for analytical factors that might have varied over the period, which would
418
not be evident from the data for thecontrols analyzed with the study samples at two time points
419
for which the assays were conducted many months or years apart.For this analysis, data from
420
assay batches in which the control was analyzed in replicate were used. If there was a
421
statistically significant difference in the within-assay means for any pairs of assay batches, the 18
Page 18 of 69
maximum inter-assay difference (in mg/100g) was calculated as percent of the overall assayed
423
mean and rounded up to the nearest 0.5 mg/100g. Then, for each study composite, this
424
percentage of the mean assayed vitamin C concentration at Time 0 was considered the minimum
425
detectable difference (MDD). For values determined at >4 weeks after the initial concentration
426
was determined, if the pairwise comparison of the mean vitamin C concentrations for a given
427
composite at two storage times differed with statistical significance, it was only considered
428
achange in contentif the amount exceeded the calculated MDD.
us an
429
432
3.1 Quality control
433
3.1.1Analytical precision
d
3. Results and Discussion
Ac ce pt e
431
M
430
434
cr
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422
The r2value was ≤0.998 for the linear regression for the HPLC calibration curves in all
435
assays, and the RSDs for within-assay means of each standard level were consistently<0.5%.
436
Figure 1 shows the results for the mixed vegetable control material analyzed with the
437
storage time/temperature experiments throughout the study. The overall precision was excellent
438
(mean, 4.24 mg/100g, 2.0% RSD), and no significant differences were found within each
439
experiment. Within each experiment the median RSD for within-day replicates of the control
440
ranged from 0.1 to 2.1 (median =0.7), and there was no significant difference in the means from
441
the two HPLC runs. These results suggest no analytical bias based on extraction and HPLC assay
442
batch within each experiment outlined in Table 1.
443 19
Page 19 of 69
445 446 447
3.1.2Storage temperatures The results of the daily temperature readings for the refrigerator and freezers used throughout the study are shown in Figure 2. For the refrigerator set at 4oC, the within experiment 7-day means of the daily
ip t
444
temperature readings (oC) (Fig. 2A) ranged from 1.2 to 3.3; medians were from 1.0 to 3.5.
449
Overall, the mean and median temperatures of the refrigerator were both 2.5, with the lowest and
450
highest daily temperatures recorded during any of the experiments being 0.0 and 5.5.
us
451
cr
448
For the frost-free conventional freezer set for a minimum temperature of
20oC and
maximum temperature of
453
readings (oC) (Fig. 2B) ranged from
454
mean and median temperatures of the conventional freezer were both
455
lowest daily temperatures recorded during any of the experiments being
456
actual temperatures reflect normal variation in a typical frost-free freezer at a given set point. As
457
shown in Figure 3, the temperature went through ~60 minute cycles.
21; medians were from
15 to
21. Overall, the
17, with the highest and 7 and
22. These
d
M
14 to
Ac ce pt e
458
12°C,the within experiment 7-day means of the daily temperature
an
452
For the ultra-low freezer set at
60oC, the within experiment 7-day means of the daily
459
temperature readings (oC) (Fig. 2C) ranged from
460
Overall, the mean and median temperatures of the ultra-low freezer were both 68, with the
461
highest and lowest daily temperatures recorded during any of the experiments being
462
and 79.
463
58 to
75; medians were from
58 to
77.
51
The recorded temperatures reflect normal variation that would be expected in typical
464
refrigerators, frost-free conventional freezers, and ultra-low freezers at given setpoints. These
465
data serve as a check on the actual temperatures of storage during the course of each experiment.
466
20
Page 20 of 69
467 468
3.2 Stability of vitamin C in samples stored at different temperatures Figures 4 9 illustrate the relative changes in vitamin C in the fruits and vegetables homogenates prepared in liquid nitrogen and stored with residual nitrogen, in a refrigerator and
470
frozen in conventional and ultra-low freezers.There was no loss of vitamin C in any of the foods
471
after 7-days storage in the ultra-low freezer. However, there were dramatic differences in the
472
stability of vitamin C in different foods stored at refrigerator and conventional freezer
473
temperatures.
us
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469
474
an
475 476
478
3.2.1Conventional freezer
M
477
Vitamin C in strawberries, tomatoes, steamed spinach, baked potatoes, steamed broccoli, navel oranges, and pasteurized orange juice was stable through 7 days in the conventional
480
freezer.After 1 day there was loss of vitamin C in only one of the two experiments for raw
481
spinach and raw broccoli. It is possible that if samples in one experiment happened to be placed
482
in the freezer during the high point of the temperature cycle (Fig. 2) and did not freeze as
483
quickly, loss of vitamin C would be greater. In fact, these two foods were most susceptible to
484
losses with refrigerated storage (Section 3.2.3). After 3 and 7 days there was consistent and
485
substantial loss of vitamin C in these foods: 6.9 mg/100g (23%) in raw spinach and 17.0
486
mg/100g (15%) in raw broccoli after 3 days, and 13.1 mg/100g (42%) in raw spinach and 32.0
487
mg/100g (28%) in raw broccoli after 7 days. Raw potatoes had no loss of vitamin C after 1 day
488
in the conventional freezer. The average losses were 15% and 25% after 3 and 7 days, but
489
represented relatively small amounts (1.6–2.7 mg/100g) based on the low initial concentration.
Ac ce pt e
d
479
21
Page 21 of 69
490 491
3.2.2Refrigerator Only pasteurized orange juice had no loss of vitamin C after 7 days storage in the
493
refrigerator. The most notable lossof vitamin C occurred in raw spinach and raw broccoli (Figs.
494
6A and 8A). After 1 day in the refrigerator,the averagelossin these foods was 9.5 (29%) and 33.1
495
mg/100g (29%), respectively. The losses increased to 31.0 and 77.0 mg/100g after 7 days (94%
496
and 68%, respectively).
us
cr
ip t
492
Tomatoes and raw and baked potatoes also lost a small amount of vitamin C after 1 day
498
(0.7 2.4 mg/100g on average), but after 3 days losses had more than doubled. After 7 days the
499
average loss of vitamin C was substantial in all of these foods: 5.9 mg/100g (69%) in baked
500
potatoes, 7.3 mg/100g (68%) in raw potatoes, and 11.0 mg/100g (35%) in raw tomatoes.
501
There was no loss of vitamin C after 1 day of refrigerated storage of strawberries,
M
an
497
steamed spinach, steamed broccoli, raw navel oranges, and pasteurized orange juice. After 3
503
days, there was some loss (10 17%) in all of these foods except the steamed spinach and the
504
orange juice. After7 days all of these foods except the orange juice had lost a substantial
505
portionof vitamin C, ranging from 14% in steamed spinach and navel oranges (3.5 and 7.9
506
mg/100g, respectively), 20% in strawberries (13.6 mg/100g), and 39% in steamed broccoli (36.3
507
mg/100g).
509 510
Ac ce pt e
508
d
502
3.3 Long term storage at ultra-low temperature The vitamin C contents ofhomogenized raw collard greens, potatoes, and
511
Clementinesafter storage for a total of 214 weeks at < 55oC [previously reported on after 50-
512
weeks storage (Phillips et al., 2010)], and of the additional samples of homogenized raw and
22
Page 22 of 69
steamed broccoli, raw and baked potatoes, raw green sweet bell peppers and cantaloupe for a
514
total of 164 weeks are shown in Figure 10. The results for the in-house control materials
515
analyzed in the corresponding assays are illustrated in Figure 11. The calculated MDD (see
516
Section 2.4) was 5.5% and ranged from 1.0 to 6.5 mg/100g among the samples (shown for each
517
in brackets in the Fig. 10 legend).
In the samples previously reported on (Phillips et al. 2010), raw potatoes and raw collard
cr
518
ip t
513
greens had losses of 10.9 mg/100g (30.4%) and 16.8 mg/100g (14.7%), respectively, after 50
520
weeks. There was no additional lossafter a total of 214 weeks (4.1 years). In the Clementines,
521
which had no detectable loss of vitamin C after 50 weeks, there also was no additional loss after
522
a total of 4.1 years.Of the other foods, only cantaloupe had any loss of vitamin C throughout 164
523
weeks, with all of the 5.7 mg/100g (20.8%) loss occurring before35 weeks.
an
M
524
us
519
Interestingly, in the second sample of raw potatoes (not previously reported onin Phillips et al., 2010), which was monitored for a maximum of 164 weeks storage at ultra-low
526
temperature, there was no loss of vitamin C throughout the 164-week monitoring period (3.2
527
years). This is in contrast to the initial loss seen in the first study of potatoes, which had
528
approximately double the initial vitamin C content of the second sample of raw potatoes. The
529
shortest storage time at which the samples were assayed in the long-term monitoring studies was
530
2 weeks. For the two raw potatoes samples in the 7-day comparison of storage temperatures,
531
there was no loss in either of the samples at 7 days (Fig. 7A). In total these results suggest
532
variability in the stability of vitamin C in different samples of raw potatoes stored at ultra-low
533
temperature, and it would seem reasonable to not to store homogenized raw potatoes to be
534
assayed for vitamin C for longer than 7 days under these conditions.
Ac ce pt e
d
525
535
23
Page 23 of 69
536
4.Discussion
537
4.1
538
Differences in vitamin C loss among foods and samples of the same food The loss of vitamin C in the foods studied could be due to both chemical (non-enzymatic
oxidation) and/or enzymatic processes. AA by virtue of its chemical nature is unstable in
540
aqueous solutions, readily oxidizing to DHAA in the absence of antioxidants (Eitenmiller et al.,
541
2008). Ascorbate oxidase (AO) and ascorbate peroxidase (APO) catalyze oxidation of AA to
542
DHAA (Saari et al., 1995; Shigeoka et al., 2002) using AA as a specific electron donor
543
(Caverzan et al., 2012). Whereas DHAA is absorbed and ultimately utilized as AA, and therefore
544
included as part of the vitamin C (total AA) content of foods, it can undergo further irreversible
545
oxidation to 2,3-diketogulonic acid, which has no vitamin C activity (Nyyssonen et al., 2000).
546
The presence of oxygen and metal ions (especially Cu2+, Ag+, Fe3+), alkaline pH, light, high
547
temperature, and disruption of cellular integrity favour this process and can substantially reduce
548
total AA (Dewanto et al., 2002; Lee & Kader, 2000; Lopez-Sanchez et al., 2015; Wilson et al.,
549
1995;Winkler, 1987).Other oxidases can contribute to AA depletion indirectly by producing
550
reactive oxygen species that can oxidize AA (H2O2, oxygen radicals) (Raseetha et al., 2013;
551
Wang & Jiao, 2000).
cr
us
an
M
d
Ac ce pt e
552
ip t
539
The differences in vitamin C stability among foods and samples of the same food might
553
be explained by the effect of pH, factors affecting enzyme activity, heat denaturation of
554
enzymes, and/or the presence of heat-resistant enzymes and other matrix components either
555
promoting or protecting against irreversible oxidation (Spínola et al., 2013).In the present study,
556
it was clear that stability of vitamin C was greatest in the cooked foods (spinach, potatoes,
557
broccoli) compared to their raw counterparts (Figs 6 8), and in acidic foods (including raw), but
558
the extent of loss varied among foods. It is interesting that in some cases there was a difference
24
Page 24 of 69
in the stability of vitamin C content in the two different samples of a given food, evident for
560
refrigerated storageof strawberries (Fig. 4), tomatoes (Fig. 5), steamed spinach (Fig. 6B), raw
561
and baked potatoes (Fig. 7), and oranges (Fig. 9). In the raw and baked potatoes, steamed
562
spinach, and tomatoes, the initial vitamin C content of the sample differed substantially in the
563
two experiments, and the sample with the highest initial concentration lost a greater amount and
564
percentage of vitamin C.In strawberries and oranges, the initial vitamin C content of the two
565
samples of each food was similar, but loss of vitamin C was notably greater in one [e.g., after 7
566
days refrigeration, 12% vs. 28% for strawberries (Fig. 4) and 8% vs. 19% for oranges (Fig. 9A)].
567
Cooking would be expected to denature endogenous enzymes and mitigate further loss of
568
vitamin C in a homogenized cooked fruit or vegetable, compared to its raw counterpart.This
569
pattern was observed for broccoli, spinach, and potatoes, but the extent of loss varied, in some
570
cases between samples of the same food (Figs 6 8).In a study of the influence of temperature
571
and pH on the activity of AO, Maccarrone et al. (1993) found an optimal temperature of 35°C,
572
complete loss of activity at temperatures above 65°C, and an optimal bell-shaped curve over a
573
pH range from 4 10, with a maximum at pH 6 withascorbate as the substrate.Müftügil(1985)
574
found the activity of peroxidases to be reduced by98.2% in potato cubes after four minutes of
575
blanching in 95°C water; andby 97.6% in spinach after two minutes at 75°C.They also found that
576
gelatinization of starch in potatoes decreased the inactivation of peroxidase.Brewer et al. (1995)
577
showed found that steaming broccoli florets for as little as 4 minutes at 100oC completely
578
inactivated the peroxidase enzymes. However, there are several different peroxidases in fruits
579
and vegetables that have different properties (Aylward&Haisman, 1969; Chang et al., 1988;
580
Delincée et al., 1975), so heat-resistance may vary even within different foods. In fact, Munyaka
581
et al. (2010b) and Leong &Oey (2012) found an increase in total vitamin C in fruits and
Ac ce pt e
d
M
an
us
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ip t
559
25
Page 25 of 69
vegetables that were heated to inactivate AO.Even if AO is denatured (e.g., by heating), studies
583
have shown heat-resistant peroxidases can be activated at lower temperaturesin response to
584
increases in H2O2 caused by oxidative stress, leading to oxidation of AA to DHAA and in turn
585
irremediable loss of DHAA (Deutsch, 1998; Gibriel et al., 1978; O’Kane et al., 1996; Raseetha et
586
al., 2013; Thongsook& Barrett, 2005).
ip t
582
In thepresent study the internal temperature immediately after cooking (for the two
cr
587
experiments for each food) was 71.0°C and 73.0°C for steamed broccoli (Fig. 8B), 75.9°C and
589
80.1°C for steamed spinach (Fig. 6B), and 97.6°C and 99.8°C for the baked potatoes (Fig. 7B). It
590
is it possible that at the shorter heating time and lower temperature relative to the published
591
studies referenced above (Brewer et al.,1995; Müftügil, 1985), enzymes were not inactivated
592
within all cells. Variation in the ability of the steam to penetrate cells in different matrices could
593
have resulted in differences in the degree of enzyme inactivation, explaining the differences in
594
loss of vitamin C among the cooked spinach, broccoli, and potatoes during subsequent storage.
595
Another possibility is variability in the levels of antioxidants and expression of endogenous
596
enzymesin different samples of the same food (e.g., Alvarez-Suarez et al., 2014; Ariza et al.,
597
2015).
an
M
d
Ac ce pt e
598
us
588
Clearly pH also affected stability of vitamin C, with losses being much lower in acidic
599
foods (pH less than ~4.5) (Table 2 and Figure 12).AA has a pKaof 4.17 and pK1 of 4.7 at 10°C
600
(National Center for Biotechnology Information, 2015); thus it would be expected that foods
601
with a pH as high as ~4.5 would retain AA in its reduced form.Once AA is oxidized to DHAA, a
602
higher, more neutral pH is not favourable to the stability of the oxidized form (Davey et al.,
603
2000), leading to its irreversible loss(Deutsch, 1998, 2000; Liao&Seib, 1988; Maccarrone et al.,
604
1993; Parsons& Fry, 2012).It should be noted that this study focused on the stability of AA in
26
Page 26 of 69
food homogenates rather than whole foods;thus it is possible that although there appeared to bea
606
correlation between AA loss and relatively high pH for the homogenized foods in this study (Fig.
607
12), the whole food counterpart may show a higher AA stability because cell wall integrity
608
would not have been compromised.
609
ip t
605
Finally, variability in vitamin C lossin the same food seen in two different experiments
could be influenced by factorsat harvest and during post-harvest handling, which differed in the
611
specific samples of the food in each case. For example, maturity, variety, or growing conditions
612
of a particular sample of a given plant could result in difference in levels of other components
613
(e.g., antioxidants), that can affect the chemical stability of AA and DHAA and/or enzyme
614
activity (Bergquist et al., 2006; Decker, 2003; Farnham et al., 2012; Gil et al., 1999; Lee &
615
Kader, 2000; Kotíková et al., 2011). Substantial degradation of vitamin C could already have
616
occurredin some samples by the time of purchase, for example due to storage time, temperature,
617
or exposure to light (Bergquist et al., 2006; Lee & Kader, 2000; Maccarroneet al., 1993;Tiwari &
618
Cummins, 2013). Another possibility is that some samples had been subjected to non-thermal
619
post-harvest treatments intended to preserve food quality (colour, texture) and/or reduce
620
microbial load that also denatured enzymes involved in degradation of vitamin C. For example,
621
Leong&Oey (2014) showed that pulsed electric field treatment, which has been used to improve
622
the microbial safety of fresh fruits and vegetables (Chauhan &Unni, 2015), affects the
623
thermostability of AO in carrots. Other non-thermal treatments, such as chlorine, irradiation, and
624
ultrasound (Doona et al., 2015), also might affect enzyme activity.
Ac ce pt e
d
M
an
us
cr
610
625 626
4.2 Implications for food composition analysis
27
Page 27 of 69
627
Table 2 summarizes the average loss of vitamin C per typical serving size, for the statistically significant differences relative to original material (Time 0) for each food, and the
629
losses relative to the RDA are illustrated in Figure 13. It is evident that the impact of storing fruit
630
and vegetable homogenates, in which vitamin C is labile, can result in a meaningful difference
631
between the foods as assayed and as consumed.In raw broccoli, losses averaged 24 mg/serving
632
(59% RDA) after 1 day and 55 mg/serving (137% RDA) after 7 days of refrigeration, and 3.1
633
mg/serving (7.7% RDA) after 1 day and 23 mg/serving (55% RDA) after 7 days in a
634
conventional freezer. In steamed broccoli after 3-days storage in the refrigerator there was a
635
small loss of vitamin C after 1 day (3.1 mg/serving, 7.7% RDA) and significant loss after 7 days
636
(23 mg/serving, 57% RDA), but no loss after 7 days in the conventional freezer.Note that if these
637
foods were homogenized for consumption (e.g. babyfood, juices, smoothies),similar losses
638
would be expected, and the amount ofvitamin C consumed could besubstantially lower than
639
what was in the unhomogenized food.
cr
us
an
M
d
It is also worth noting that in this study the fruits and vegetables were frozen in liquid
Ac ce pt e
640
ip t
628
641
nitrogen immediately after cooking, and remained frozen throughout the homogenization
642
process, which would deterenzymatic and oxidative reactions that degrade vitamin C, due to the
643
low temperature and atmosphere devoid of oxygen.Homogenization in liquid nitrogen also
644
allows for powderyconsistency of the frozen homogenate, which can be easily mixed and
645
representatively subsampled for analysis while kept frozen. In contrast, homogenization of a fruit
646
or vegetable without liquid nitrogen results in a slurry, which after freezing must be thawed to
647
obtain a representative subsample for analysis. Processing fruits and vegetables without liquid
648
nitrogen would likely lead to some loss of vitamin C during preparation of samples for analysis,
649
especially in raw broccoli and spinach, in which vitamin C was most labile.
28
Page 28 of 69
650 651 652
5. Conclusions For general food composition work involving analysis of vitamin C in fruits and
654
vegetables, in which samples are homogenized and held before analysis, ultra-low temperature
655
freezer storage (< 55oC) should be standard, and samples should be analyzed within 7
656
days.Storage at this temperature, with residual nitrogen, can be relied on to preserve vitamin C
657
content over a 7-day period for a wide range of homogenized fruits and vegetables, including
658
raw products.However, as previously reported (Phillips et al., 2010) and confirmed in this study,
659
storage for as little as 2 weeks even at ultra-low temperature with residual nitrogen can result in
660
some loss of vitamin C in certain raw fruits and vegetables, including potatoes andcollard greens,
661
and more substantial loss can occur after 1 year. Refrigeration can lead to a substantial decrease
662
in vitamin C within as little as one day, even when samples are stored under residual nitrogen.
663
With conventional freezing ( 10 to
664
homogenized fruits and vegetables.
cr
us
an
M
d
20oC) vitamin C is lost to a variable extent in different
Ac ce pt e
665
ip t
653
Whereas this study was not designed prospectively to specifically study the effect of pH
666
or cooking, vitamin C was stable with conventional freezing in all of the foods that were cooked
667
or had a pH less than ~4.5. Similarly, during long-term storage at ultra-low temperature under
668
residual nitrogen, vitamin C content was found to be stable for 3 4 years in cooked and acidic
669
fruits and vegetables. If ultra-low freezer storage is not available, and foods must be
670
homogenized before extraction for analysis of vitamin C, acidifying the homogenate would be
671
advisable [e.g., as described by Munyaka et al. (2010b) for broccoli]. The losses of vitamin C
29
Page 29 of 69
672
during storage of the homogenized foods prior to analysis (Table 2 and Figure 13) would likely
673
be even greater if homogenized without liquid nitrogen.
674
In light of these results, when comparing data on the vitamin C content for the same food among different studies, attention should be paid to the sample preparation, especially for non-
676
acidic raw fruits and vegetables. Loss of vitamin C during frozen storage is also a concern when
677
considering data for fruits and vegetables that have been freeze-dried, if the samples were cut or
678
homogenized prior to freezing.
cr
us
679
ip t
675
Although the focus of this study was on the effect of storage temperature of fruit and vegetable homogenates prepared for analysis on the accuracy ofthe assayed vitamin C content of
681
a food, the results suggest the potential for substantial loss of vitamin C in fruits and vegetables
682
homogenized for other purposes. Forexample, loss of vitamin C could be even greater in
683
homemade babyfood and fruit/vegetable smoothiesthat are refrigerated or frozen in a
684
conventional freezer, not protected from oxygen, and not consumed immediately after
685
preparation.Further studies would be needed to quantify the potential difference in vitamin C in
686
such products as consumed relative to the original food.
688 689 690 691
M
d
Ac ce pt e
687
an
680
6. Acknowledgments
This work was supported by cooperative agreements 58-1235-2-111, 58-1235-2-113, and 58-1235-3-128 between the USDA Nutrient Data Laboratory and Virginia Tech.
692 693 694
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Wechtersbach, L., Cigic, B. (2007). Reduction of dehydroascorbic acid at low pH. Journal of
1009
Biochemical and Biophysical Methods, 70, 767-772.
cr
1008
1011
us
1010
Wilson, J.X. (2002). The physiological role of dehydroascorbic acid. FEBS Letters 527, 5-9.
an
1012
Wilson, R.J., Beezer, A.E.,Mitchell, J.C. (1995). A kinetic study of the oxidation of L-ascorbic
1014
acid (vitamin C) in solution using an isothermal microcalorimeter.ThermochimicaActa, 264, 27-
1015
40.
d
1016
M
1013
Winkler, B.S. (1987).In vitro oxidation of ascorbic acid and its prevention by
1018
GSH.BiochimicaetBiophysicaActa, 925, 258-264.
1019
Ac ce pt e
1017
1020
Zee, J.A., Carmichael, L.,Codère, D., Poirier, D., Fournier, M. (1991).Effect of storage
1021
conditions on the stability of vitamin C in various fruits and vegetables produced and consumed
1022
in Quebec. Journal of Food Composition and Analysis, 4, 77-86.
1023 1024
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Table and Figure captions
Table 1.Distribution of samples within assays for each experiment, showing measures to avoid
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contribution of analytical variability (A) Extraction batches (B) Sample sequences on HPLC (Calibration standards were run at the beginning and end of each HPLC run, and at the points
us
cr
indicated by dashed lines.) n/a = not applicable.
Table 2.Vitamin C loss in homogenized fruits and vegetables at different temperatures (as
an
amount per typical serving size). The vitamin C loss for each food is the average of the two experiments in which there was a statistically significant difference relative to the initial vitamin
M
C content. Shaded cells indicate conditions for which there was no change in vitamin C content. Bolded entries represent >10% of the 90 mg/100g Recommended Dietary Allowance for adults
Ac ce pt e
in only one of the experiments.
d
(Food and Nutrition Board, Institute of Medicine, 2000).Italicized entries indicate loss occurred
Figure 1.Vitamin C assayed in samples of a mixed vegetable quality control material with each experiment throughout the storage temperature study. Capital letters designating each experiment correspond to letters of label for each experiment, as given in the chart labels in Figures 4 9; n/a = not applicable within this study.
Figure 2.Temperature of refrigerator (A), conventional freezer (B), and ultra-low freezer (C) throughout each experiment. (Capital letters in legend correspond to experiment labels in Figs. 4-
46
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9). Markers of the same colour are for the same food, with a different symbol for each experiment.
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Figure 3.Temperature cycling of frost-free conventional freezer on two days.
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Figure 4.Stability of vitamin C in homogenized raw strawberries stored at different temperatures
us
in two independent experiments (B and J):○ refrigerated conventional freezer
ultra-low freezer. Each data point is the mean ± standard deviation for extraction and analysis
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an
of three subsamples.
Figure 5.Stability of vitamin C in homogenized raw tomatoes stored at different temperatures in
d
two independent experiments (F and M): ○ refrigerated conventional freezer ultra-low freezer.
Ac ce pt e
Each data point is the mean ± standard deviation for extraction and analysis of three subsamples.
Figure 6.Stability of vitamin C in homogenized raw (A) and steamed (B) baby spinach stored at different temperatures in two independent experiments (A and H; C and I, respectively): ○ refrigerated conventional freezer ultra-low freezer. Each data point is the mean ± standard deviation for extraction and analysis of three subsamples.
Figure 7.Stability of vitamin C in homogenized raw (A) and baked (B) potatoes stored at different temperatures in two independent experiments (D and K; E and L, respectively):
47
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○ refrigerated conventional freezer ultra-low freezer. Each data point is the mean ± standard deviation for extraction and analysis of three subsamples.
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Figure 8.Stability of vitamin C in homogenized raw (A) and steamed (B) broccoli florets stored at different temperatures in two independent experiments (P and T; Q and S, respectively):
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○ refrigerated conventional freezer ultra-low freezer. Each data point is the mean ± standard
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deviation for extraction and analysis of three subsamples.
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Figure 9.Stability of vitamin C in homogenized raw navel oranges (A) and purchased pasteurized orange juice (B) stored at different temperatures in two independent experiments (G
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and N; O and W, respectively):○ refrigerated conventional freezer ultra-low freezer. Each data point is the mean ± standard deviation for extraction and analysis of three subsamples.
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Figure 10.Long term stability of vitamin C in homogenized fruits and vegetables stored at
Ac ce pt e
< 55oC under nitrogen.○Markers indicate data for samples previously reported on after 49 weeks storage (Phillips et al., 2010).
Markers indicate data for additional composites not
previously reported on. Dashed lines indicate astatistically significant change (p<0.001) in vitamin C concentration that was greater than the minimum detectable difference (see text, section 2.4), given in brackets in the legend; solid lines indicate no difference. Each data point is the meanfrom extraction and analysis of three subsamples.
Figure 11.Vitamin C assayed in samples of mixed vegetablea and pasteurized orange juice control materials assayed with each analytical batch throughout the study. Solid blue lines
48
Page 48 of 69
bracket the total duration of long term storage of samples previously reported on (Phillips et al., 2010), and solid red lines bracket duration of storage of additional composites (Fig. 10).
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Figure 12.Percent loss of initial vitamin C in homogenized raw broccoli, strawberries, navel oranges, tomatoes, potatoes, and spinach stored under nitrogenrefrigerated (0 5.5oC) for 7 days,
cr
as a function of food pH.The value for each food is the mean of results from two independent
us
experiments, with n=3 replicate analyses at each time point (Figs. 4 9).
an
Figure 13.Average loss of vitamin C per serving (see Table 2) in homogenized foods, relative to Recommended Dietary Allowance (RDA) for adult men (Food and Nutrition Board, Institute of
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Medicine, 2000) in (A) refrigerator and (B) conventional freezer storage at time intervals up to 7
Ac ce pt e
FIGURE 1
d
days. Error bars show the range forresults in two independent experiments.
49
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50
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d
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FIGURE 2
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B.
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A.
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(Fig. 2)
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d
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C.
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d
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us
cr
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FIGURE 3
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cr us
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ce
pt
ed
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FIGURE 4
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ed
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FIGURE 5
55 Page 55 of 69
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FIGURE 6
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ed
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A. Raw spinach
Ac
B. Steamed spinach
56 Page 56 of 69
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FIGURE 7
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pt
ed
M
A. Raw potatoes
Ac
B. Baked potatoes
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FIGURE 8
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pt
ed
M
A. Raw broccoli
Ac
B. Steamed Broccoli
58 Page 58 of 69
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FIGURE 9
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ed
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A. Raw navel oranges
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B. Pasteurized orange juice
59 Page 59 of 69
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d
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FIGURE 10
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d
Not the same material reported on in Figure 1.
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a
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FIGURE 11
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us
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FIGURE 12
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an
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FIGURE 13
TABLE 1
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d
A. Extraction batches
Storage temperature n/a Controlc Refrigerated o
-10 to -20 C o
< -55 C
c
Control Refrigerated o
-10 to -20 C o
< -55 C c
Control Refrigerated o
-10 to -20 C o
< -55 C
Days storage 0 n/a 1
Extraction # of HPLC # of replicates replicates/extracta 3 1 1 3 3 1
Extraction batch A A B
1
3
1
B
1
3
1
B
n/a 3
1 3
3 1
B C
3
3
1
C
3
3
1
C
n/a 7
1 3
3 1
C D
7
3
1
D
7
3
1
D
Total samples extracted/batch 4
Total samples for HPLCb 42
10
10
10
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Controlc
n/a
1
3
D
a
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d
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Per extracted replicate sample. Where number of HPLC replicates>1, the prepared/already diluted extract was split among separate HPLC vials. After each extraction, the diluted samples were placed in HPLC vials so that no dilution was necessary after thawing for analysis b After all extractions (i.e. at the end of the 7 day storage time), samples were run in two HPLC sequences, as shown in Table 1B c In-house control composite of homogenized mixture of cooked/canned fruits and vegetables
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Table 1 (cont’d.) B. HPLC runs (Calibration standards were run at the beginning and end of each HPLC run, and at the points indicated by dashed lines.)
-10 to -20oC o
Replicate#
0 1
A B
1 1
1 2
1
B
1
B
1
n/a
B
1a
a
Control Refrigerated
n/a 3
A C
-10 to -20oC
3
C
< -55 C
3
C
a
Control Refrigerated
3 3
C C
-10 to -20oC
3
o
o
4
5
1a 1
6 7
1
8
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Control
3
an
1
a
1
9
1a 2
10 11
C
2
12
C
2
13
3
a
Control Refrigerated
3 7
C D
1b 1
14 15
-10 to -20oC
7
D
1
16
7
D
1
17
Control Refrigerated
n/a 1
D B
1a 2
18 19
-10 to -20oC
1
B
2
20
1
B
2
21
n/a
B
1b
22
1
B
3
1
1
B
3
2
1
B
3
3
n/a
B
1c
4
Ac ce pt e
< -55 C
o
< -55 C
a
o
< -55 C Control
a
cr
Batch
Position in HPLC sequence
d
< -55 C
Storage time (days)
us
Storage temperature HPLC Run 1: n/a Refrigerated
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Extraction (see Table 1A)
HPLC Run 2: Refrigerated o
-10 to -20 C o
< -55 C Control
a
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Extraction (see Table 1A)
o
< -55 C
Replicate# 3
3
C
3
6
C
3
7
n/a
C
1c
8
a
Control n/a Refrigerated
n/a 0 7
A A D
1b 2 3
9 10 11
-10 to -20oC
7
D
3
7
D
3
Control Refrigerated
n/a 7
D D
1b 2
-10 to -20oC
7
D
a
o
< -55 C
7
D
a
n/a 0
D A
Controla
n/a
A
13
14 15
2
16
2
17
1c 3
18 19
1c
20
d
Control n/a
12
an
< -55 C
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o
us
3
a
Control
a
Batch C
Position in HPLC sequence 5
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-10 to -20oC
Storage time (days) 3
cr
Storage temperature Refrigerated
Ac ce pt e
In-house control composite of homogenized mixture of cooked/canned fruits and vegetables
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1
TABLE 2 Vitamin C loss (mg/serving) [standard deviation] b
Serving a
pH
Food
grams Description
o
mg/serving range Mean
%
166
1 c., sliced
110 - 112
4.2 - 4.9
149
1 c.
41.9 - 50.2
3.6 [ 1.6 ] 8 %
Spinach, raw
5.5 - 6.8
30
1 c.
8.9 - 10.9
2.9 [ 0.1 ] 29 %
Spinach, steamed
6.6 - 7.2
180
1 c.
29.9 - 50.8
6.1
213
1 med. (2.25"-3.25" dia.) 17.5 - 28.5
71
1 c. flowerets
1 med. (2.25"-3.25" dia.)
78.8 - 82.4
Broccoli, steamed
6.3 - 6.5
78
0.5 c., chopped
69.3 - 75.3
Oranges, raw
3.1 - 4.1
140
1 fruit, 2 7/8" dia.
70.1 - 86.7
9.3 - 21.6
Orange juice, pasteurized 3.6 - 4.3
249
1 c.
89.6 - 122
3 days storage
%
Mean
13 %
9%
0.9
6%
23.5 [ 0.2 ] 29 %
1.2 [ 0.2 ]
6.2
8%
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173
6.6 - 6.9
1.1
%
4.3 [ 0.6 ] 20 %
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~6.0
Broccoli, raw
Mean
-60 C
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3.0 -3.5
Tomatoes, raw
Potatoes, baked
o
-20 C
Refrigerated
Strawberries, raw
Potatoes, raw
2
Initial vitamin C
Vitamin C loss (mg/serving) [standard deviation]
b
Serving pH
Food
3.0 -3.5
166
1 c., sliced
Tomatoes
4.2 - 4.9
149
1 c.
Spinach, raw
5.5 - 6.8
30
1 c.
Spinach, steamed
6.6 - 7.2
180
1 c.
110 - 112
19.3
%
Mean
%
%
Mean
17 %
8.9 - 10.9
5.8 [ 0.5 ] 59 %
2.1 [ 2.0 ] 23 %
10.2 [ 3.4 ] 44 %
3.4 [ 1.1 ] 15 %
29.9 - 50.8
6.1
213
1 med. (2.25"-3.25" dia.) 17.5 - 28.5
Potatoes, baked
~6.0
173
1 med. (2.25"-3.25" dia.)
Broccoli, raw
6.6 - 6.9
71
1 c. flowerets
Broccoli, steamed
6.3 - 6.5
78
0.5 c., chopped
69.3 - 75.3
8.9 [ 1.4 ] 12 %
Oranges
3.1 - 4.1
140
1 fruit, 2 7/8" dia.
70.1 - 86.7
8.7
249
o
-60 C
8.3 [ 3.1 ] 18 %
Potatoes, raw
Orange juice, pasteurized 3.6 - 4.3
o
-20 C
41.9 - 50.2
M
Strawberries
mg/serving range Mean
9.3 - 21.6
78.8 - 82.4
d
Ac ce pt e
3
grams Description
Refrigerated
an
a
Initial vitamin C
1 c.
4.8 [ 1.0 ] 35 % 38.6 [ 3.2 ] 48 % 12.1 [ 4.0 ] 15 % 10 %
89.6 - 122
7 days storage
b
Serving
Food
a
pH
grams Description
Vitamin C loss (mg/serving) [standard deviation] Initial vitamin C
o
-20 C
Refrigerated
mg/serving range Mean
%
Strawberries
3.0 -3.5
166
1 c., sliced
110 - 112
22.6 [ 12.6 ] 20 %
Tomatoes
4.2 - 4.9
149
1 c.
41.9 - 50.2
16.4 [ 4.8 ] 35 %
Spinach, raw
5.5 - 6.8
30
1 c.
8.9 - 10.9
9.3 [ 1.2 ] 94 %
Spinach, steamed
6.6 - 7.2
180
1 c.
29.9 - 50.8
6.3 [ 4.5 ] 14 %
6.1
213
1 med. (2.25"-3.25" dia.) 17.5 - 28.5
15.5 [ 4.0 ] 68 %
1 med. (2.25"-3.25" dia.)
10.2 [ 4.5 ] 69 %
Potatoes, raw
Potatoes, baked
9.3 - 21.6
Mean
c
o
-60 C %
Mean
%
3.9 [ 2.8 ] 42 % 5.8 [ 1.9 ] 25 %
~6.0
173
Broccoli, raw
6.6 - 6.9
71
1 c. flowerets
78.8 - 82.4
54.7 [ 3.6 ] 68 % 22.7 [ 2.3 ] 28 %
Broccoli, steamed
6.3 - 6.5
78
0.5 c., chopped
69.3 - 75.3
28.3 [ 4.7 ] 39 %
Oranges
3.1 - 4.1
140
1 fruit, 2 7/8" dia.
70.1 - 86.7
11.1 [ 7.8 ] 14 %
Orange juice, pasteurized 3.6 - 4.3
249
1 c.
89.6 - 122
a
pH of food according U.S. Food and Drug Administration (2015), Albrecht et al. (1990) (raw broccoli), and Hughes et al., (1975) (baked potatoes)
b
4 5 6
c
U.S. Department of Agriculture (2014). Serving size equivalents: 1 c. = 240 mL; 1" = 2.54 cm
Values in italics indicate significance for one but not all experiments
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7 8
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S0889-1575(15)00198-2 http://dx.doi.org/doi:10.1016/j.jfca.2015.09.008 YJFCA 2631
To appear in: Received date: Revised date: Accepted date:
19-7-2015 15-9-2015 17-9-2015
Please cite this article as: Phillips, K. M., Council-Troche, M. A., McGinty, R. C., Rasor, A. S., and Tarrago-Trani, M. T.,Stability of vitamin C in fruit and vegetable homogenates stored at different temperatures, Journal of Food Composition and Analysis (2015), http://dx.doi.org/10.1016/j.jfca.2015.09.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Stability of vitamin C in fruit and vegetable homogenates stored at different temperatures
1 2
Katherine M. Phillipsa*, McAlister Council-Trocheb, Ryan C. McGintya,c, Amy S. Rasora,d, and
4
Maria Teresa Tarrago-Trania,e
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Department of Biochemistry, Virginia Tech Blacksburg, VA
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*Corresponding author
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a
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Phone: 540-231-9292; FAX: 540-231-9070; email: [email protected] b
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Department of Biochemistry, 304 Engel Hall (0308), Virginia Tech, Blacksburg, VA24061.
Current affiliation: Polymer Solutions, Inc., Christiansburg, VA, USA;[email protected] c [email protected] d [email protected] e [email protected] Highlights
33
strawberries, oranges, and tomatoes; baked potatoes; steamed broccoli and spinach; and
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d
12 13 14 15 16 17 18 19• 20• 21• 22• 23• 24• 25 26 27 28 29 30 31 32
Vitamin C analyzed in several types of homogenized fruits and vegetables Assayed afterrefrigerated and frozen storage (-20oC, < -55oC)over 1-week period No change in vitamin C at < -55oC Significant losses after refrigeration or freezing at -20oC in some foods Up to 15% loss in non-acidic stored3 days at -20oC (17 mg/100g in raw broccoli) Detailed quality control implemented and described to ensure validity of data
Abstract Vitamin C loss was compared in homogenized raw broccoli, potatoes, spinach,
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34
pasteurized orange juice after storage under residual nitrogen under refrigeration, and frozen at
35
conventional ( 10 to
36
Additional foods (cantaloupe, green sweet peppers, collard greens, Clementines) were monitored
37
for 3–4 years at < 55oC. Total ascorbic acid was quantified using high-performance liquid
38
chromatography and detailed quality control measures. No decrease occurred in any of the foods
39
after 7 days at < 55oC. Under refrigeration the largest decreases were in raw spinach and
40
broccoli, averaging (mg/100g) 9.5 (29%) and 33.1 (29%), respectively, after 1 day and 31.0 and
41
77.0 after 7 days (94% and 68%). With conventional freezing, vitamin C was stable for 7 days in
42
most of the products studied; minor losses occurred in raw spinach and broccoli after 1 day but
43
were substantial after 3 days (6.9 mg/100g (23%) and 17.0 mg/100g (15%), respectively) and 7
44
days (13.1 and 32.0 mg/100g). For homogenates stored long-term at < 55oC, vitamin C loss
45
occurred in only cantaloupe, collard greens, and one sample of raw potatoes, all before 50 weeks.
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20oC) and ultra-low (< 55oC) temperatures for 1, 3, and 7 days.
Keywords:Ascorbic acid;Food composition; Food analysis; Food processing; Freezing; Quality
48
control; Pureed fruit; Pureed vegetables; Refrigeration; Stability
49
Ac ce pt e
47
50
Chemical compounds studied in this article
51
L-Ascorbic acid (PubChem CID: 54670067); Dehydroascorbic acid (PubChem CID: 210328)
52 53
Abbreviations: AA, ascorbic acid; AO, ascorbate oxidase; APO, ascorbate peroxidase; DDI,
54
distilled deionized; DHAA, dehydroascorbic acid; EDTA, Ethylenediaminetetraacetic acid
55
disodium salt dihydrate; HPLC, high-performance liquid chromatography; MDD, minimum
56
detectable difference; MPA, metaphosphoric acid; RDA; Recommended Dietary Allowance;
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RSD, relative standard deviation; SD, standard deviation; TCEP, tris(2-carboxyethyl) phosphine
58
hydrochloride; UV, ultraviolet light
61
1. Introduction
Vitamin C is an essential water-soluble vitamin, with a Recommended Dietary
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59
Allowance (RDA) of 75 mg/day and 90 mg/day for adult women and men, respectively, and 45
63
mg/day for children 9 13 years old (Food and Nutrition Board, Institute of Medicine, 2000). L-
64
Ascorbic acid [(2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one] (AA) is the
65
major naturally occurring compound with vitamin C activity. AA is cofactor for many iron and
66
copper hydroxylases involved in key physiological processes such as the production of collagen,
67
the synthesis and activation of peptide hormones (norepinephrine, noradrenaline), and the
68
synthesis of carnitine (Arrigoni& De Tullio, 2000; Ball, 2006; Davies et al., 1991; Bender, 2003;
69
Johnston et al., 2007). AA also has an important role in the intestinal absorption of non-haemiron
70
and as a cellular antioxidant, independently or together with the antioxidant action of vitamin E
71
(Bender, 2003; Byers & Perry, 1992).Dehydroascorbic acid (DHAA) is formed readily from the
72
oxidation of AA in aqueous solutions, and as by-product of cellular reactions of AA or other
73
oxidative processes (Tolbert & Ward, 1982). DHAA is present in many foods (Gökmen et al.,
74
2000). It is absorbed from the small intestine in humans and reduced to AA intracellularly
75
(Deutsch, 2000), and as a result it is bioavailable; thus, the vitamin C content of foods is usually
76
considered to be the sum of the AA and DHAA (Wilson, 2002).
77 78
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Vegetables and fruits, particularly citrus, green leafy vegetables, broccoli, cauliflower, Brussels sprouts, tomatoes, peppers, and potatoes, are major dietary sources of AA (Eitenmiller
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et al., 2008). For example, the average vitamin C content of one medium baked potato, one
80
medium tomato, and one medium navel orange are 16.6 mg, 16.9 mg, and 82.7 mg, respectively
81
(U.S. Department of Agriculture, 2014), providing 18 92% of the 90 mg RDA for adult men.
82
Epidemiological studies have associated adequate vitamin C intake with decreased mortality, but
83
controlled trials are lacking (Enstrom, 2008). Studies that relatenutrient intake to healthusually
84
involvecalculating nutrient intake using food composition data combined with known or
85
estimated measures of food consumption. Obviously, the accuracy of the calculated intake of a
86
particular nutrient depends on how closely its concentration in the food as consumed matches the
87
food composition data.The vitamin C content of fruits and vegetables can be substantially
88
affected by post-harvest handling and processing (Cocetta et al, 2014; Giannakourou&Taoukis,
89
2003; Neves et al., 2015; Rodrigues et al., 2010; Spínola et al., 2013; Tiwari & Cummins, 2013;
90
Zee et al., 1991). Natural variability in the vitamin C content of foods as they exist in the food
91
supply should be reflected in food composition data. Additionally, once food samples have been
92
procured, the accuracy of the analyzed nutrient content depends on preventing changes in
93
nutrient during the analytical process itself.
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The primary source of food composition data in the United States is the U.S. Department
95
of Agriculture’s (USDA) National Nutrient Database for Standard Reference (SR) (U.S.
96
Department of Agriculture, 2014). The USDA’s National Food and Nutrient Analysis Program
97
(NFNAP) is an ongoing effort, begun in 1996, to update and improve the quality of food
98
composition data in SR (Haytowitz et al., 2008). One of the practical challenges in the NFNAP is
99
that a wide range of nutrients must be assayed in each food that is sampled. The cost of
100
purchasing, shipping, and preparing the foods for analysis is a significant factor in the total cost
101
of the project. Thus, multiple primary samples are often combined to yield composites that are
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statistically representative of the national market for a given food (Pehrsson et al., 2000).
103
Documentation of the samples and their handling also must be maintained, with a complete audit
104
trail from sample procurement to the release of final data in SR, and archived subsamples of all
105
composites are maintained. Therefore, centralized homogenization of samples and distribution of
106
subsamples for the various nutrient assays is the only practical approach for the NFNAP.Primary
107
food samples (sample units) are procured from retail and wholesale locations and sent to the
108
Food Analysis Laboratory Control Center (FALCC) at Virginia Tech (Blacksburg, VA) (Trainer
109
et al., 2010) where they are prepared, composited, homogenized, and typically dispensed into
110
subsamples in 30- or 60-mL glass jars, sealed under nitrogen, and stored in an ultra-low freezer
111
(< 55oC), protected from light, until distributed for analysis along with matrix-matched quality
112
control materials (Phillips et al., 2006). Under routine NFNAP processing conditions, a
113
minimum of 2 weeks, and often several weeks, elapse between homogenization and analysis.
114
The sample processing and storage conditions used in the NFNAP were chosen as what is
115
practical to achieve for long term storage of samples of a variety of foods with minimal potential
116
for nutrient loss.
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To ensure that the analyzed concentration of vitamin C is representative of the food as
118
consumed, the portion of the sample analyzed mustbe representative of the whole food.
119
Obtaining a representative portion of a whole foodthat is heterogeneous (e.g., potatoes with skin)
120
requires either grinding and extracting a large amount of the whole fooddirectly in the extraction
121
solvent (for example, as described by Vanderslice et al., 1990 or Veltman et al., 2000), or
122
homogenizing the food and then taking a subsample for analysis. The former may require a large
123
volume of solvent to accommodate the minimum sample size necessary to be representative of
124
the whole food. Also, in studies involving a large number of samples that must be combined for
5
Page 5 of 69
analysis, in which the food must be analyzed for multiple nutrients involving different extraction
126
methods and multiple laboratories (such as the NFNAP),it is impossible to directly extract the
127
very same whole sample for all assays. Homogenization allows generation of multiple analytical
128
subsamples from one larger sample and the ability to store subsamples prior to analysis, yielding
129
the greatest flexibility in analysis. However, it is critical to establish that labile nutrients such as
130
vitamin C are preserved, to avoid significant errors in food composition data. Once the cell wall
131
has been disrupted, the effect of external factors on reactions resulting in irreversible oxidation
132
of AA and loss of vitamin C (and other nutrients) is more likely to occur, and failure to stabilize
133
AA could result in an assayed vitamin C content of a fruit or vegetable that is quite different
134
from what it is as consumed. Spínola et al. (2014) have recently published an excellent review
135
that includes sample handling considerations for analysis of vitamin C in foods.
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AA is especially vulnerable to oxidative and enzymatic degradation in raw fruits and
137
vegetables in which endogenous enzymes are active (Redmond et al., 2003; Francisco et al.,
138
2010; Munyaka et al., 2010a; Wen et al., 2010; Cocetta et al., 2014). Significant and variable
139
post-harvest loss of vitamin C in many fruits and vegetables stored under less optimal conditions
140
has been reported (e.g., González et al., 2003; Lee & Kader, 2000; Munyaka et al., 2010a; Neves
141
et al., 2015; Rybarczyk-Plonska et al., 2014; Szeto et al., 2002; Tiwari & Cummins, 2013;
142
Veltman et al., 2000; Zee et al., 1991). González et al. (2003) measured vitamin C in raspberries
143
and blackberries stored from 0 to 12 months and found an average decrease of 37% and 31%
144
(10.7 and 7.9 mg/100g), respectively. The storage temperature of –24 °C was higher than the–
145
60°C used in the NFNAP, and the berries were frozen whole, not homogenized. Vanderslice and
146
co-workers (1990) reported on the vitamin C content of somefruits and vegetables and performed
147
stability testing on raw broccoli stored under different conditions (refrigerated at –4°C and
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Page 6 of 69
frozen at –40°C, with or without citric acid or metaphosphoric acid). The treatment in the
149
Vandersliceet al. (1990) study that is most relevant to NFNAP standard conditions of < 55°C
150
under nitrogen was storage at –40°C. In that study, total AAwas constant for 2 weeks (133±7
151
mg/100g) and dropped thereafter, reaching 89±25 mg/100g after 2 months (Vanderslice et al.,
152
1990). Because pH and other matrix-specific characteristics are known to affect vitamin C
153
stability (Musulin& King, 1936; Moser &Bendich, 1990; Wechtersbach&Cigic, 2007), results
154
for broccoli might not apply equally to other fruits and vegetables, and the lower temperature and
155
nitrogen atmosphere used in NFNAP should impart additional stability. Previously, the stability
156
of vitamin C in subsamples of selected raw fruit and vegetable homogenates (clementines,
157
collard greens, potatoes) prepared in liquid nitrogen and stored at < 55oC under residual
158
nitrogen for one year was established (Phillips et al., 2010).
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Many laboratories do not have access to an ultra-low freezer (< -55oC), and the cost is
161
as a matter of practicality,homogenized fruits and vegetables prepared for analysis might be
162
exposed to refrigerator or conventional freezer temperatures. The Association of Official
163
Analytical Chemists method for analysis of vitamin C in foods (AOAC, 2012) specifies
164
homogenization of the food in a blender and subsamplingfor analysis, but there is no indication
165
for temperature control. In fact, some literature reports on the vitamin C content of fruits and
166
vegetables involve storage of prepared samples at ≥
167
(e.g.,Giannakourou&Taoukis, 2003; Kabasakalis et al. 2000; Mahattanatawee et al., 2006;
168
Rizzolo et al., 2002; Vanderslice et al., 1990). Whether or not storage at these temperatures is
169
necessary for practical reasons, less than optimal conditions could lead to substantial and
170
variable deviations in the vitamin C content of the food as assayed versus as consumed.
d
higher for storage at ultra-low versus standard freezer temperature (~
Ac ce pt e
10 to
20oC). Therefore,
160
20oCprior to analysis
7
Page 7 of 69
171
The objective of this study was to assess the change in the vitamin C concentration in
172
different fruits and vegetables that were homogenized while frozen in liquid nitrogen and then
173
stored under refrigeration (0 to 5oC), and frozen at conventional ( 10 to
174
(< 55oC) temperaturesfor up to one week. Additionally, we report on the stability of vitamin C
175
in a variety of similarly homogenized samples of fruits and vegetables stored in an ultra-low
176
freezer for >4 years, as a follow-up on results after 1 year storage that were previously reported
177
(Phillips et al., 2010).
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2. Materials and Methods
an
179
182
184
2.1.1Food selection
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2.1 Storage temperature study
M
180 181
20oC) and ultra-low
Foods representing good sources of vitamin C, and having characteristics that could
185
affect stability of vitamin C (e.g. pH, endogenous enzyme activity) were selected for the study:
186
strawberries (Fragaria X ananassa); grape tomatoes (Solanum lycopersicum); baby spinach
187
(Spinaciaoleracea), raw and steamed; russet potatoes (Solanum tuberosum), raw and baked;
188
broccoli (Brassica oleracea var. italica), raw and steamed; navel oranges (Citrus sinensis); and
189
pasteurized orange juice (pulp-free, refrigerated, not from concentrate). Two independent
190
experiments were performed for each individual food and were separated by at least three
191
months. For each experiment, the product was purchased from a local retail market (Blacksburg,
192
VA)[~500 g spinach (2 4 packages); ~1 2kg strawberries, potatoes, grape tomatoes, oranges,
193
and broccoli; three 1.89L cartons pulp-free refrigerated, pasteurized,orange juice (not from 8
Page 8 of 69
concentrate).Navel oranges and broccoli samples wereeach chosen to be similar in colour and
195
size and were purchased loose; tomatoes and strawberries, bought in (0.47L (1 pint)containers
196
and 908 g (2 lb) containers, respectively, were selected to beof similar size, colour, ripeness and
197
expiration date; potatoes chosen for the composite were all approximately the same size and
198
colour with as minimal need for trimming of eyes as possible; packages of baby spinach having
199
an expiration date of at least a week after the date of purchase and showing no major amount of
200
damaged or wilted leaves were selected. Potatoes, strawberries, and tomatoes were purchased
201
and stored at room temperature (20°C) until prepared within 24 hours of preparation and
202
homogenization (sections 2.1.2, 2.1.3). Oranges, orange juice, broccoli and spinach were
203
purchased from the store’s open, refrigerated display case and were stored in a refrigerator
204
(1 5°C) until prepared within 24 hours of purchase.
205
207
2.1.2 Food preparation
All food preparation was done under UV-filtered light. Just prior to homogenization,
Ac ce pt e
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208
foods were cleaned and trimmed as follows, including removal of any damaged portions of the
209
produce. Strawberries and tomatoes were rinsed in a stainless steel colander for two minutes with
210
distilled deionized (DDI) water and patted dry with a lint-free cloth (Teri® wipe, Kimberly-
211
Clark Professional®, Georgia, USA). The top ~2.5 mm of each strawberry, including the leaves,
212
were removed with a stainless steel knife before beingcut in half and frozen immediately in
213
liquid nitrogen. Rotten, unripe, and split tomatoes were discarded prior to freezing the whole
214
fruits in liquid nitrogen. Navel oranges were peeled one at a time, and any significant amount of
215
albedo adhering to the fruit flesh was removed. The wedges were immediately frozen in liquid
216
nitrogen after separating.
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Page 9 of 69
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Raw baby spinach leaves (labelled as triple washed, ready to eat were immediately frozen in liquid nitrogen after discarding any wilted or damaged leaves. Steamed spinach was prepared
219
by steaming the leaves for 10 minutes in a 7.57-L stainless steel steamer pot with lid, filled with
220
DDI water to a depth of approximately 2.5 cm and brought totheboil prior to adding the
221
spinach.After steaming, the steamer basket with the spinach leaves was removed from the pot
222
and allowed to cool for 10 minutes before freezing the spinach in liquid nitrogen.
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Individual potatoes were thoroughly scrubbed with a stiff-bristle nylon brush,any eyes
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223
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218
were removed, and then the potato was rinsed for 2 minutes with DDI water and patted dry with
225
a Teri® wipe. Raw, unpeeled potatoes were cut one at a time into ~1.25-cm cubes with a
226
stainless steel knifeand frozen immediately in liquid nitrogen.Baked potatoes (unpeeled, whole)
227
were prepared by punching six ~0.5 mm wide by ~2.5 mm deep holes, one into each face with a
228
stainless steel pick, then baking in a conventional oven for 50 minutes at 218°C (425°F). The
229
baked potatoes were allowed to cool at room temperature for 15 minutes, then individually cut
230
(unpeeled) into ~1.25 cm cubesand immediately frozen in liquid nitrogen.
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231
an
224
Whole broccoli florets were prepared by paring with a stainless steel knife any leaves and
232
removing the smaller stalks of approximately 1 cm or less in diameter from the larger stalk. Any
233
yellow, brown or spoiled patches of the florets were removed and the remaining florets were
234
trimmed to approximately 1 cmin length.The florets were then rinsed in a stainless steel colander
235
for one minute with tap water, followed by two minutes with DDI water. After using a Teri®
236
wipe to pat the florets dry, they were cut in half and immediately frozen in liquid nitrogen.
237
Florets to be steamed were placed in a 7.57-L stainless steel steamer pot with lid, filled with DDI
238
water to a depth of approximately 2.5 cm and brought to theboil prior to addition of the broccoli,
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Page 10 of 69
239
and steamed for four minutes. The florets were allowed to cool at room temperature for 10
240
minutes,cut in half, and thenfrozen in liquid nitrogen.
241
243
2.1.3 Homogenization
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Homogenization was done as previously described and using procedures that have been standard for preparation of fruits and vegetables for analysis in the NFNAP (Haytowitz et al.,
245
2008; Trainer et al., 2010) and the previously reported study on stability of vitamin C in
246
homogenized fruits and vegetables (Phillips et al., 2010).
us
For each experiment, the prepared food that was frozen in liquid nitrogenwas transferred
an
247
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244
to a 6-L stainless steel food processor (Robot Coupe 6L Blixer®; Robot Coupe USA, Jackson,
249
MS), blended 10 s at 1500 rpm, then for two additional 30-s periods at 3500 rpm, until a
250
homogenous composite (fine powder consistency) resulted.The frozen, ground material was
251
transferred to a stainless steel bowl and kept frozen by adding and stirring in additional liquid
252
nitrogen as needed.The composite was dispensed among fifty-four 60-mL glass jars with
253
Teflon™-lined screw-caplids (~ 10 g/jar, filled approximately half full). The jars were
254
wrappedcompletely with aluminium foil to prevent UV degradation from the light in the
255
refrigerator. Two individuals worked in tandem to immediately replace the caps of the jars after
256
each subsample was dispensed, seal the rims of the lid with 1.9-cm Write-On™ label tape (Bel-
257
Art™, New Jersey, USA), and store the samples in their respective storage
258
locations(Section2.1.4) before thawing occurred (all tasks completed in under 3 minutes): Nine
259
jars were stored in the refrigerator (1 5oC), ninejars were stored in the conventional freezer, and
260
ninejars were stored in the ultra-low freezer (< 55oC).Additional subsamples were dispensed
261
and stored at < 55oCfor moisture analysis and possible additional assays of vitamin C to
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Page 11 of 69
262
monitor changes over long term storage. Three replicates (taken from separate subsample jars)
263
were extracted immediately, without thawing, for analysis of the initial vitamin C concentration
264
as described in Section2.2. Each of the three 1.89-L containers of refrigerated orange juice was shaken vigorously,
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and the entire contents of all containers were stirred together in a stainless steel bowl for two
267
minutes using a stainless steel whisk.The temperature of the composite was recorded before
268
mixing, after mixing, and after dispensing into to jars, and remained at ~4°C throughout. Ten
269
millilitres of the composite were pipetted into each of the subsample jars described above using a
270
manual pipette and then blanketed with nitrogen gas before recapping and sealing with tape.
271
They were then stored and analyzed as described for the homogenized fruits and vegetables.
an
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An in-house control composite of cooked/canned fruits and vegetables (spinach,
273
babyfood green beans, babyfood carrots, peas, pumpkin, asparagus, tomato puree) was prepared
274
using methods previously described (Phillips et al., 2006).
276 277
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272
2.1.4Sample storage appliances
The conventional refrigerator/freezer in which samples were stored was a 0.5 m3 Revco™
278
Thermo Fisher Scientific™ (Asheville, NC, USA) model # RCRF192A17. The ultra-low freezer
279
was a 0.7 m3Revco model# ULT2586-3-ABA, purchased from Thermo Fisher Scientific™
280
(Waltham, MA, USA).( Temperatures were monitored daily. The refrigerator portion of the
281
refrigerator/freezer was factory set to 4°C. The freezer portion of the refrigerator/freezer was
282
factory set to a minimum temperature of
283
built-in auto-defrost feature. Because this was a typical frost-free freezer, the temperature cycle
20°C and maximum temperature of
12°C with a
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Page 12 of 69
284
was monitored over two four-hour periods to document fluctuations due to the defrost cycle. The
285
ultra-low freezer was not frost-free.
286
288
2.1.5Food samples for the long-term stability study
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287
Homogenates of raw russet potatoes, collard greens (Brassica oleracea var. viridis), and Clementines(Citrus clementina hort. ex Tanaka) prepared and stored at < -55oC withresidual
290
nitrogen (from homogenization) and protected from light as previously reported (Phillips et al.,
291
2010), were assayed after a total of 127, 187, and 214 weeks (4.1 years; 2007 2012), along with
292
control samples, for comparison to results previously reported after 1 year storage (2007 2008)
293
Additional homogenates of raw broccoli (florets), steamed broccoli (florets), raw russet potatoes
294
(with skin), baked russet potatoes (with skin), raw green sweet bell peppers (Capsicum annuum),
295
and cantaloupe (Cucumismelo) were prepared, stored, and assayed in the same manner, after 2,
296
31, 75, 129, 135, and 164 weeks (3.2 years; 2008 2012). Samples of a control composite of
297
orange juice (pasteurized, pulp free) were also analyzed with these stored homogenates.
299 300
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298
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289
2.2 Analysis of vitamin C
Vitamin C was analyzed as AA after treatment with tris(2-carboxyethyl) phosphine
301
hydrochloride(TCEP) to reduce any DHAA, using previously validated and reported
302
methodology (Tarrago-Trani et al., 2012). Additional details necessary to provide a full
303
description of the reagents, standards and analyses performed in this study are as follows.
304
2.2.1 Reagents and standards
305 306
ACS grade L-AA (>99% pure), ACS grade 33.5 36.5% metaphosphoric acid (MPA), and ethylenediaminetetraacetic acid disodium salt dihydrate(EDTA) disodium salt were
13
Page 13 of 69
purchased from Sigma-Aldrich (Saint Louis, MO, USA). High-performance liquid
308
chromatography (HPLC) grade 85% ortho-phosphoric acid and HPLC grade water were
309
purchased from Fisher Scientific (Pittsburgh, PA, USA). TCEP was obtained from Thermo
310
Fisher Scientific™ Pierce™ (Rockford, IL, USA).Eight AA calibration standards ranging from
311
0.1 12.5 µg/mL were serially diluted from a previously prepared 5 mg/mL AA standard stock
312
solution. All AA standard solutions were prepared in extraction buffer (Section 2.2.2), blanketed
313
with argon, and stored at< 55oC for up to four months.
us
314
2.2.2Extraction of vitamin C
an
315
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307
All work was done in a temperature controlled (20 22°C) room with UV filtered
317
lightbulbs and light-blocking window shades.Extraction buffer (5% MPA/1 mM EDTA/pH 1.5)
318
was prepared no more than 5 days in advance of use and kept in the refrigerator. TCEP (5 mM)
319
was added on the day of use.
d
Each analytical subsample was taken from a separate jar. Each jar was removed from
Ac ce pt e
320
M
316
321
storage immediately prior to weighing and kept on ice during the weighing process. Briefly,
322
2.0±0.1 g of each frozen homogenized fruit or vegetable (1.0 ± 0.5 g of orange juice) were
323
weighed into 30-mL Teflon® centrifuge tubes (Thermo Fisher Scientific™, cat# 31140030),after
324
thorough mixing to ensure representative subsampling. MPA extraction buffer was added
325
immediately after the analytical subsampling had been completed, and the tube was blanketed
326
with argon and kept on ice until all samples for the analytical batch had been weighed. Prior to
327
mixing, samples stored at < 55°C showed no signs of thawing and had the same dry, free-
328
flowing, powdery consistency as after the original homogenization. Samples stored in the
329
conventional freezer showed a slight lesseningof the powdery consistency but no significant
14
Page 14 of 69
thawing was observed. Samples stored in the refrigerator had completely thawed.No more than
331
~1 minute elapsed from the time asample was removed from storage and the addition of
332
theextraction buffer. Pasteurized orange juice and the control composite (which was aslurry)
333
were weighed after thawing completely in a 30 ± 1.0oC water bath for 20 min and were kept on
334
ice before and during weighing.Extraction buffer (12 mL, except 11 mLfor orange juice)was
335
added immediately to each weighed analytical subsample, and the tube was capped and kept on
336
ice until all samples in the batch had been weighed. After homogenization and initial separation
337
of the supernatant, the pellet was re-extracted twice in the same manner (Tarrago-Trani et al.,
338
2012). All supernatants were combined after centrifugal filtration through a 0.45-µm PVDF filter
339
(cat. #24162; Grace, Columbia, MD, USA) and brought to 50 mL total volume with extraction
340
buffer.Each extract was blanketed with argon, dispensed among8-mL amber glass vials with
341
Teflon® lined caps (Thermo Fisher Scientific™), and frozen at < 55°C until the day of HPLC
342
analysis, which was within 7 8 days for samples extracted on day 0 to 1 2 days for samples
343
extracted on Day 7 of the experiment.
345 346
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Ac ce pt e
344
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330
2.2.3 High-performance liquid chromatography (HPLC) On the day of HPLC analysis, samples that had been frozen and stored in 8-mL vials
347
(Section 2.2.2) were completely thawed for 20 30 minutes in a 20 21°C water bath and
348
vortexed thoroughly for 30 seconds. They were thendiluted with the MPA extraction buffer, if
349
necessary (based on sample size extracted and AA content estimated using values in SR (USDA,
350
2014), or on package labels), to put the final concentration within the range of the calibration
351
curve (0.1 12.5 µg/mL).All samples with the exception of strawberries and the mixed vegetable
352
control composite required a further 1:5 dilution (1:250 total dilution), while the strawberry
15
Page 15 of 69
samples were diluted to a 1:10 dilution (1:500 total dilution) and mixed vegetable samples were
354
analyzed undiluted (1:50 total dilution).After dilution, 400 µL of each extract werefiltered
355
through pre-assembled Mini-Uniprep™ syringe-less 0.45-µm PVDF filtration devices consisting
356
of an amber colouredpolypropylene chamber and plunger filter (Waters Corp., Milford, MA,
357
USA), and then placed in the pre-chilled, 8°C refrigerated HPLC auto-sampler (model 1329A;
358
Agilent, Santa Clara, CA, USA).
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353
An Agilent 1200 HPLC system equipped with an LC quaternary pump (model G1311A)
360
and diode array detector (model G1315D), operated with ChemStation™ (version B.03.02),and
361
fitted with a C18 reversed-phase column with polar end-capping [Phenomenex® Synergi™ 4 µm
362
Hydro-RP (250 mm× 4.6 mm); Phenomenex, Torrance, CA, USA] was employed.The mobile
363
phase was aqueous ortho-phosphoric acid (0.022% w/v; pH 2).Of the filtered extract, 20 µL were
364
auto-injected into the HPLC and eluted under isocratic conditions at 0.7 mL/min with the AA
365
peak eluting at about 7.2–7.5 min and detected at 254 nm. An MPA extraction buffer blank was
366
run between each sample to ensure lack of carryover of AA from sample to sample.
an
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367
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359
The eight AA calibration standards (0.1 - 12.5 µg/mL) were run in triplicate, sequentially
368
from lowest concentration to highest, at the beginning, halfway point, and end of the
369
samplesequence on the HPLC.A calibration curve was prepared based on linear regression of
370
response versus AA concentration.
371
Because of the large number of extracts to be run on the HPLC in each experiment(>80,
372
including calibration standards and blanks), two separate runs were performed over two
373
consecutive days, using the same eight calibration standards and implementing the quality
374
control measures described in Section 2.3.
375
16
Page 16 of 69
376 377
2.3 Quality control Experiments were conducted one at a time on each food, with at least two independent experiments for each food. Careful attention was paid to the manner in which the samples stored
379
at different times and temperatures within each experiment were extracted and run on the HPLC,
380
to avoid confounding of any analytical variability withactual change in sample composition due
381
to the experimental variables, as follows. For each experiment, at each time point, three
382
subsamples of the homogenatefrom each storage temperature were extracted in the same batch,
383
along with a sample of the mixed vegetable control composite(Table 1A).The prepared extracts
384
from the 0, 1, and 3 day time points were held at < 55oC until the samples from 7 day time
385
pointhad been extracted. Then, the extracts from all storage times and temperatures were run
386
together on the HPLC, in two runs, with replicates distributed in a balanced manner between the
387
runs (Table 1B).
M
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378
Previous studies had established the stability of frozen extracts of the prepared sample
389
extracts stored at < 55oC for up to 4 weeks (K.M. Phillips, Virginia Tech, unpublished data).
390
Thesubsamples of extracts that were prepared immediately after thefood was homogenized, that
391
were stored < 55oC and assayed again after 7 days along with those of samples extracted at the
392
1, 3, and 7 day storage times, served to validate lack of any effect of storage time on vitamin C
393
in the extracts prepared from the homogenized food at different time points. Potential loss of
394
vitamin C in the mixed fruit and vegetable and orange juice control composites during the
395
thawing procedure in the 30°C water bath was confirmed to be absent (K.M. Phillips, Virginia
396
Tech, unpublished data).
Ac ce pt e
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388
397 398
2.4 Data analysis
17
Page 17 of 69
Data were compiled and means and standard deviations were calculated using
399
Microsoft® Excel® 2010 v. 14.0.7151.5001 (Microsoft Corp., Redmond, WA, USA). Statistical
401
analyses were performed using JMP® Pro 11 (SAS Institute Inc., Cary, NC, USA). One-way
402
analysis of variance (ANOVA) and Tukey-Kramer HSD t-test (0.05 significance level)
403
(McDonald, 2008) were performed to compare vitamin C mean values at Time 0 with
404
corresponding vitamin C mean values at different storage times within each storage temperature
405
and experiment. Two-way ANOVA and Tukey-Kramer HSD t-testwere applied to determine
406
within-experiment significant differences (0.05 significance level) between vitamin C means
407
across temperatures, and among storage times for each storage temperature. The critical
408
significance level for one-way and two-way ANOVA was set at 0.05, but differences in means
409
were conservatively estimated as significant for p-values <0.001, nonetheless, to avoid detection
410
of very small changes in the case of data points with very low relative standard deviations
411
(RSD).
cr
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M
d Ac ce pt e
412
ip t
400
For the samples stored long term at ultra-low temperature, data were evaluated using
413
XLSTAT (version 2012.4.03; Addinsoft™, New York, NY, USA), with ANOVA (α=0.05) and
414
pairwise comparison of means using the Tukey-Kramer HSD t-test, with a 95% confidence
415
interval. The same analysis was applied to the comprehensive in-house data set for the mixed
416
vegetable control material assayed with the samples over the yearsthe study samples were
417
assayed, to account for analytical factors that might have varied over the period, which would
418
not be evident from the data for thecontrols analyzed with the study samples at two time points
419
for which the assays were conducted many months or years apart.For this analysis, data from
420
assay batches in which the control was analyzed in replicate were used. If there was a
421
statistically significant difference in the within-assay means for any pairs of assay batches, the 18
Page 18 of 69
maximum inter-assay difference (in mg/100g) was calculated as percent of the overall assayed
423
mean and rounded up to the nearest 0.5 mg/100g. Then, for each study composite, this
424
percentage of the mean assayed vitamin C concentration at Time 0 was considered the minimum
425
detectable difference (MDD). For values determined at >4 weeks after the initial concentration
426
was determined, if the pairwise comparison of the mean vitamin C concentrations for a given
427
composite at two storage times differed with statistical significance, it was only considered
428
achange in contentif the amount exceeded the calculated MDD.
us an
429
432
3.1 Quality control
433
3.1.1Analytical precision
d
3. Results and Discussion
Ac ce pt e
431
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430
434
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422
The r2value was ≤0.998 for the linear regression for the HPLC calibration curves in all
435
assays, and the RSDs for within-assay means of each standard level were consistently<0.5%.
436
Figure 1 shows the results for the mixed vegetable control material analyzed with the
437
storage time/temperature experiments throughout the study. The overall precision was excellent
438
(mean, 4.24 mg/100g, 2.0% RSD), and no significant differences were found within each
439
experiment. Within each experiment the median RSD for within-day replicates of the control
440
ranged from 0.1 to 2.1 (median =0.7), and there was no significant difference in the means from
441
the two HPLC runs. These results suggest no analytical bias based on extraction and HPLC assay
442
batch within each experiment outlined in Table 1.
443 19
Page 19 of 69
445 446 447
3.1.2Storage temperatures The results of the daily temperature readings for the refrigerator and freezers used throughout the study are shown in Figure 2. For the refrigerator set at 4oC, the within experiment 7-day means of the daily
ip t
444
temperature readings (oC) (Fig. 2A) ranged from 1.2 to 3.3; medians were from 1.0 to 3.5.
449
Overall, the mean and median temperatures of the refrigerator were both 2.5, with the lowest and
450
highest daily temperatures recorded during any of the experiments being 0.0 and 5.5.
us
451
cr
448
For the frost-free conventional freezer set for a minimum temperature of
20oC and
maximum temperature of
453
readings (oC) (Fig. 2B) ranged from
454
mean and median temperatures of the conventional freezer were both
455
lowest daily temperatures recorded during any of the experiments being
456
actual temperatures reflect normal variation in a typical frost-free freezer at a given set point. As
457
shown in Figure 3, the temperature went through ~60 minute cycles.
21; medians were from
15 to
21. Overall, the
17, with the highest and 7 and
22. These
d
M
14 to
Ac ce pt e
458
12°C,the within experiment 7-day means of the daily temperature
an
452
For the ultra-low freezer set at
60oC, the within experiment 7-day means of the daily
459
temperature readings (oC) (Fig. 2C) ranged from
460
Overall, the mean and median temperatures of the ultra-low freezer were both 68, with the
461
highest and lowest daily temperatures recorded during any of the experiments being
462
and 79.
463
58 to
75; medians were from
58 to
77.
51
The recorded temperatures reflect normal variation that would be expected in typical
464
refrigerators, frost-free conventional freezers, and ultra-low freezers at given setpoints. These
465
data serve as a check on the actual temperatures of storage during the course of each experiment.
466
20
Page 20 of 69
467 468
3.2 Stability of vitamin C in samples stored at different temperatures Figures 4 9 illustrate the relative changes in vitamin C in the fruits and vegetables homogenates prepared in liquid nitrogen and stored with residual nitrogen, in a refrigerator and
470
frozen in conventional and ultra-low freezers.There was no loss of vitamin C in any of the foods
471
after 7-days storage in the ultra-low freezer. However, there were dramatic differences in the
472
stability of vitamin C in different foods stored at refrigerator and conventional freezer
473
temperatures.
us
cr
ip t
469
474
an
475 476
478
3.2.1Conventional freezer
M
477
Vitamin C in strawberries, tomatoes, steamed spinach, baked potatoes, steamed broccoli, navel oranges, and pasteurized orange juice was stable through 7 days in the conventional
480
freezer.After 1 day there was loss of vitamin C in only one of the two experiments for raw
481
spinach and raw broccoli. It is possible that if samples in one experiment happened to be placed
482
in the freezer during the high point of the temperature cycle (Fig. 2) and did not freeze as
483
quickly, loss of vitamin C would be greater. In fact, these two foods were most susceptible to
484
losses with refrigerated storage (Section 3.2.3). After 3 and 7 days there was consistent and
485
substantial loss of vitamin C in these foods: 6.9 mg/100g (23%) in raw spinach and 17.0
486
mg/100g (15%) in raw broccoli after 3 days, and 13.1 mg/100g (42%) in raw spinach and 32.0
487
mg/100g (28%) in raw broccoli after 7 days. Raw potatoes had no loss of vitamin C after 1 day
488
in the conventional freezer. The average losses were 15% and 25% after 3 and 7 days, but
489
represented relatively small amounts (1.6–2.7 mg/100g) based on the low initial concentration.
Ac ce pt e
d
479
21
Page 21 of 69
490 491
3.2.2Refrigerator Only pasteurized orange juice had no loss of vitamin C after 7 days storage in the
493
refrigerator. The most notable lossof vitamin C occurred in raw spinach and raw broccoli (Figs.
494
6A and 8A). After 1 day in the refrigerator,the averagelossin these foods was 9.5 (29%) and 33.1
495
mg/100g (29%), respectively. The losses increased to 31.0 and 77.0 mg/100g after 7 days (94%
496
and 68%, respectively).
us
cr
ip t
492
Tomatoes and raw and baked potatoes also lost a small amount of vitamin C after 1 day
498
(0.7 2.4 mg/100g on average), but after 3 days losses had more than doubled. After 7 days the
499
average loss of vitamin C was substantial in all of these foods: 5.9 mg/100g (69%) in baked
500
potatoes, 7.3 mg/100g (68%) in raw potatoes, and 11.0 mg/100g (35%) in raw tomatoes.
501
There was no loss of vitamin C after 1 day of refrigerated storage of strawberries,
M
an
497
steamed spinach, steamed broccoli, raw navel oranges, and pasteurized orange juice. After 3
503
days, there was some loss (10 17%) in all of these foods except the steamed spinach and the
504
orange juice. After7 days all of these foods except the orange juice had lost a substantial
505
portionof vitamin C, ranging from 14% in steamed spinach and navel oranges (3.5 and 7.9
506
mg/100g, respectively), 20% in strawberries (13.6 mg/100g), and 39% in steamed broccoli (36.3
507
mg/100g).
509 510
Ac ce pt e
508
d
502
3.3 Long term storage at ultra-low temperature The vitamin C contents ofhomogenized raw collard greens, potatoes, and
511
Clementinesafter storage for a total of 214 weeks at < 55oC [previously reported on after 50-
512
weeks storage (Phillips et al., 2010)], and of the additional samples of homogenized raw and
22
Page 22 of 69
steamed broccoli, raw and baked potatoes, raw green sweet bell peppers and cantaloupe for a
514
total of 164 weeks are shown in Figure 10. The results for the in-house control materials
515
analyzed in the corresponding assays are illustrated in Figure 11. The calculated MDD (see
516
Section 2.4) was 5.5% and ranged from 1.0 to 6.5 mg/100g among the samples (shown for each
517
in brackets in the Fig. 10 legend).
In the samples previously reported on (Phillips et al. 2010), raw potatoes and raw collard
cr
518
ip t
513
greens had losses of 10.9 mg/100g (30.4%) and 16.8 mg/100g (14.7%), respectively, after 50
520
weeks. There was no additional lossafter a total of 214 weeks (4.1 years). In the Clementines,
521
which had no detectable loss of vitamin C after 50 weeks, there also was no additional loss after
522
a total of 4.1 years.Of the other foods, only cantaloupe had any loss of vitamin C throughout 164
523
weeks, with all of the 5.7 mg/100g (20.8%) loss occurring before35 weeks.
an
M
524
us
519
Interestingly, in the second sample of raw potatoes (not previously reported onin Phillips et al., 2010), which was monitored for a maximum of 164 weeks storage at ultra-low
526
temperature, there was no loss of vitamin C throughout the 164-week monitoring period (3.2
527
years). This is in contrast to the initial loss seen in the first study of potatoes, which had
528
approximately double the initial vitamin C content of the second sample of raw potatoes. The
529
shortest storage time at which the samples were assayed in the long-term monitoring studies was
530
2 weeks. For the two raw potatoes samples in the 7-day comparison of storage temperatures,
531
there was no loss in either of the samples at 7 days (Fig. 7A). In total these results suggest
532
variability in the stability of vitamin C in different samples of raw potatoes stored at ultra-low
533
temperature, and it would seem reasonable to not to store homogenized raw potatoes to be
534
assayed for vitamin C for longer than 7 days under these conditions.
Ac ce pt e
d
525
535
23
Page 23 of 69
536
4.Discussion
537
4.1
538
Differences in vitamin C loss among foods and samples of the same food The loss of vitamin C in the foods studied could be due to both chemical (non-enzymatic
oxidation) and/or enzymatic processes. AA by virtue of its chemical nature is unstable in
540
aqueous solutions, readily oxidizing to DHAA in the absence of antioxidants (Eitenmiller et al.,
541
2008). Ascorbate oxidase (AO) and ascorbate peroxidase (APO) catalyze oxidation of AA to
542
DHAA (Saari et al., 1995; Shigeoka et al., 2002) using AA as a specific electron donor
543
(Caverzan et al., 2012). Whereas DHAA is absorbed and ultimately utilized as AA, and therefore
544
included as part of the vitamin C (total AA) content of foods, it can undergo further irreversible
545
oxidation to 2,3-diketogulonic acid, which has no vitamin C activity (Nyyssonen et al., 2000).
546
The presence of oxygen and metal ions (especially Cu2+, Ag+, Fe3+), alkaline pH, light, high
547
temperature, and disruption of cellular integrity favour this process and can substantially reduce
548
total AA (Dewanto et al., 2002; Lee & Kader, 2000; Lopez-Sanchez et al., 2015; Wilson et al.,
549
1995;Winkler, 1987).Other oxidases can contribute to AA depletion indirectly by producing
550
reactive oxygen species that can oxidize AA (H2O2, oxygen radicals) (Raseetha et al., 2013;
551
Wang & Jiao, 2000).
cr
us
an
M
d
Ac ce pt e
552
ip t
539
The differences in vitamin C stability among foods and samples of the same food might
553
be explained by the effect of pH, factors affecting enzyme activity, heat denaturation of
554
enzymes, and/or the presence of heat-resistant enzymes and other matrix components either
555
promoting or protecting against irreversible oxidation (Spínola et al., 2013).In the present study,
556
it was clear that stability of vitamin C was greatest in the cooked foods (spinach, potatoes,
557
broccoli) compared to their raw counterparts (Figs 6 8), and in acidic foods (including raw), but
558
the extent of loss varied among foods. It is interesting that in some cases there was a difference
24
Page 24 of 69
in the stability of vitamin C content in the two different samples of a given food, evident for
560
refrigerated storageof strawberries (Fig. 4), tomatoes (Fig. 5), steamed spinach (Fig. 6B), raw
561
and baked potatoes (Fig. 7), and oranges (Fig. 9). In the raw and baked potatoes, steamed
562
spinach, and tomatoes, the initial vitamin C content of the sample differed substantially in the
563
two experiments, and the sample with the highest initial concentration lost a greater amount and
564
percentage of vitamin C.In strawberries and oranges, the initial vitamin C content of the two
565
samples of each food was similar, but loss of vitamin C was notably greater in one [e.g., after 7
566
days refrigeration, 12% vs. 28% for strawberries (Fig. 4) and 8% vs. 19% for oranges (Fig. 9A)].
567
Cooking would be expected to denature endogenous enzymes and mitigate further loss of
568
vitamin C in a homogenized cooked fruit or vegetable, compared to its raw counterpart.This
569
pattern was observed for broccoli, spinach, and potatoes, but the extent of loss varied, in some
570
cases between samples of the same food (Figs 6 8).In a study of the influence of temperature
571
and pH on the activity of AO, Maccarrone et al. (1993) found an optimal temperature of 35°C,
572
complete loss of activity at temperatures above 65°C, and an optimal bell-shaped curve over a
573
pH range from 4 10, with a maximum at pH 6 withascorbate as the substrate.Müftügil(1985)
574
found the activity of peroxidases to be reduced by98.2% in potato cubes after four minutes of
575
blanching in 95°C water; andby 97.6% in spinach after two minutes at 75°C.They also found that
576
gelatinization of starch in potatoes decreased the inactivation of peroxidase.Brewer et al. (1995)
577
showed found that steaming broccoli florets for as little as 4 minutes at 100oC completely
578
inactivated the peroxidase enzymes. However, there are several different peroxidases in fruits
579
and vegetables that have different properties (Aylward&Haisman, 1969; Chang et al., 1988;
580
Delincée et al., 1975), so heat-resistance may vary even within different foods. In fact, Munyaka
581
et al. (2010b) and Leong &Oey (2012) found an increase in total vitamin C in fruits and
Ac ce pt e
d
M
an
us
cr
ip t
559
25
Page 25 of 69
vegetables that were heated to inactivate AO.Even if AO is denatured (e.g., by heating), studies
583
have shown heat-resistant peroxidases can be activated at lower temperaturesin response to
584
increases in H2O2 caused by oxidative stress, leading to oxidation of AA to DHAA and in turn
585
irremediable loss of DHAA (Deutsch, 1998; Gibriel et al., 1978; O’Kane et al., 1996; Raseetha et
586
al., 2013; Thongsook& Barrett, 2005).
ip t
582
In thepresent study the internal temperature immediately after cooking (for the two
cr
587
experiments for each food) was 71.0°C and 73.0°C for steamed broccoli (Fig. 8B), 75.9°C and
589
80.1°C for steamed spinach (Fig. 6B), and 97.6°C and 99.8°C for the baked potatoes (Fig. 7B). It
590
is it possible that at the shorter heating time and lower temperature relative to the published
591
studies referenced above (Brewer et al.,1995; Müftügil, 1985), enzymes were not inactivated
592
within all cells. Variation in the ability of the steam to penetrate cells in different matrices could
593
have resulted in differences in the degree of enzyme inactivation, explaining the differences in
594
loss of vitamin C among the cooked spinach, broccoli, and potatoes during subsequent storage.
595
Another possibility is variability in the levels of antioxidants and expression of endogenous
596
enzymesin different samples of the same food (e.g., Alvarez-Suarez et al., 2014; Ariza et al.,
597
2015).
an
M
d
Ac ce pt e
598
us
588
Clearly pH also affected stability of vitamin C, with losses being much lower in acidic
599
foods (pH less than ~4.5) (Table 2 and Figure 12).AA has a pKaof 4.17 and pK1 of 4.7 at 10°C
600
(National Center for Biotechnology Information, 2015); thus it would be expected that foods
601
with a pH as high as ~4.5 would retain AA in its reduced form.Once AA is oxidized to DHAA, a
602
higher, more neutral pH is not favourable to the stability of the oxidized form (Davey et al.,
603
2000), leading to its irreversible loss(Deutsch, 1998, 2000; Liao&Seib, 1988; Maccarrone et al.,
604
1993; Parsons& Fry, 2012).It should be noted that this study focused on the stability of AA in
26
Page 26 of 69
food homogenates rather than whole foods;thus it is possible that although there appeared to bea
606
correlation between AA loss and relatively high pH for the homogenized foods in this study (Fig.
607
12), the whole food counterpart may show a higher AA stability because cell wall integrity
608
would not have been compromised.
609
ip t
605
Finally, variability in vitamin C lossin the same food seen in two different experiments
could be influenced by factorsat harvest and during post-harvest handling, which differed in the
611
specific samples of the food in each case. For example, maturity, variety, or growing conditions
612
of a particular sample of a given plant could result in difference in levels of other components
613
(e.g., antioxidants), that can affect the chemical stability of AA and DHAA and/or enzyme
614
activity (Bergquist et al., 2006; Decker, 2003; Farnham et al., 2012; Gil et al., 1999; Lee &
615
Kader, 2000; Kotíková et al., 2011). Substantial degradation of vitamin C could already have
616
occurredin some samples by the time of purchase, for example due to storage time, temperature,
617
or exposure to light (Bergquist et al., 2006; Lee & Kader, 2000; Maccarroneet al., 1993;Tiwari &
618
Cummins, 2013). Another possibility is that some samples had been subjected to non-thermal
619
post-harvest treatments intended to preserve food quality (colour, texture) and/or reduce
620
microbial load that also denatured enzymes involved in degradation of vitamin C. For example,
621
Leong&Oey (2014) showed that pulsed electric field treatment, which has been used to improve
622
the microbial safety of fresh fruits and vegetables (Chauhan &Unni, 2015), affects the
623
thermostability of AO in carrots. Other non-thermal treatments, such as chlorine, irradiation, and
624
ultrasound (Doona et al., 2015), also might affect enzyme activity.
Ac ce pt e
d
M
an
us
cr
610
625 626
4.2 Implications for food composition analysis
27
Page 27 of 69
627
Table 2 summarizes the average loss of vitamin C per typical serving size, for the statistically significant differences relative to original material (Time 0) for each food, and the
629
losses relative to the RDA are illustrated in Figure 13. It is evident that the impact of storing fruit
630
and vegetable homogenates, in which vitamin C is labile, can result in a meaningful difference
631
between the foods as assayed and as consumed.In raw broccoli, losses averaged 24 mg/serving
632
(59% RDA) after 1 day and 55 mg/serving (137% RDA) after 7 days of refrigeration, and 3.1
633
mg/serving (7.7% RDA) after 1 day and 23 mg/serving (55% RDA) after 7 days in a
634
conventional freezer. In steamed broccoli after 3-days storage in the refrigerator there was a
635
small loss of vitamin C after 1 day (3.1 mg/serving, 7.7% RDA) and significant loss after 7 days
636
(23 mg/serving, 57% RDA), but no loss after 7 days in the conventional freezer.Note that if these
637
foods were homogenized for consumption (e.g. babyfood, juices, smoothies),similar losses
638
would be expected, and the amount ofvitamin C consumed could besubstantially lower than
639
what was in the unhomogenized food.
cr
us
an
M
d
It is also worth noting that in this study the fruits and vegetables were frozen in liquid
Ac ce pt e
640
ip t
628
641
nitrogen immediately after cooking, and remained frozen throughout the homogenization
642
process, which would deterenzymatic and oxidative reactions that degrade vitamin C, due to the
643
low temperature and atmosphere devoid of oxygen.Homogenization in liquid nitrogen also
644
allows for powderyconsistency of the frozen homogenate, which can be easily mixed and
645
representatively subsampled for analysis while kept frozen. In contrast, homogenization of a fruit
646
or vegetable without liquid nitrogen results in a slurry, which after freezing must be thawed to
647
obtain a representative subsample for analysis. Processing fruits and vegetables without liquid
648
nitrogen would likely lead to some loss of vitamin C during preparation of samples for analysis,
649
especially in raw broccoli and spinach, in which vitamin C was most labile.
28
Page 28 of 69
650 651 652
5. Conclusions For general food composition work involving analysis of vitamin C in fruits and
654
vegetables, in which samples are homogenized and held before analysis, ultra-low temperature
655
freezer storage (< 55oC) should be standard, and samples should be analyzed within 7
656
days.Storage at this temperature, with residual nitrogen, can be relied on to preserve vitamin C
657
content over a 7-day period for a wide range of homogenized fruits and vegetables, including
658
raw products.However, as previously reported (Phillips et al., 2010) and confirmed in this study,
659
storage for as little as 2 weeks even at ultra-low temperature with residual nitrogen can result in
660
some loss of vitamin C in certain raw fruits and vegetables, including potatoes andcollard greens,
661
and more substantial loss can occur after 1 year. Refrigeration can lead to a substantial decrease
662
in vitamin C within as little as one day, even when samples are stored under residual nitrogen.
663
With conventional freezing ( 10 to
664
homogenized fruits and vegetables.
cr
us
an
M
d
20oC) vitamin C is lost to a variable extent in different
Ac ce pt e
665
ip t
653
Whereas this study was not designed prospectively to specifically study the effect of pH
666
or cooking, vitamin C was stable with conventional freezing in all of the foods that were cooked
667
or had a pH less than ~4.5. Similarly, during long-term storage at ultra-low temperature under
668
residual nitrogen, vitamin C content was found to be stable for 3 4 years in cooked and acidic
669
fruits and vegetables. If ultra-low freezer storage is not available, and foods must be
670
homogenized before extraction for analysis of vitamin C, acidifying the homogenate would be
671
advisable [e.g., as described by Munyaka et al. (2010b) for broccoli]. The losses of vitamin C
29
Page 29 of 69
672
during storage of the homogenized foods prior to analysis (Table 2 and Figure 13) would likely
673
be even greater if homogenized without liquid nitrogen.
674
In light of these results, when comparing data on the vitamin C content for the same food among different studies, attention should be paid to the sample preparation, especially for non-
676
acidic raw fruits and vegetables. Loss of vitamin C during frozen storage is also a concern when
677
considering data for fruits and vegetables that have been freeze-dried, if the samples were cut or
678
homogenized prior to freezing.
cr
us
679
ip t
675
Although the focus of this study was on the effect of storage temperature of fruit and vegetable homogenates prepared for analysis on the accuracy ofthe assayed vitamin C content of
681
a food, the results suggest the potential for substantial loss of vitamin C in fruits and vegetables
682
homogenized for other purposes. Forexample, loss of vitamin C could be even greater in
683
homemade babyfood and fruit/vegetable smoothiesthat are refrigerated or frozen in a
684
conventional freezer, not protected from oxygen, and not consumed immediately after
685
preparation.Further studies would be needed to quantify the potential difference in vitamin C in
686
such products as consumed relative to the original food.
688 689 690 691
M
d
Ac ce pt e
687
an
680
6. Acknowledgments
This work was supported by cooperative agreements 58-1235-2-111, 58-1235-2-113, and 58-1235-3-128 between the USDA Nutrient Data Laboratory and Virginia Tech.
692 693 694
7. References
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Page 30 of 69
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Albrecht, J.A., Schafer, H.W., Zottola, E.A. (1990). Relationship of total sulfur to initial and
697
retained ascorbic acid in selected cruciferous and noncruciferous vegetables. Journal of Food
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Science, 55, 183.
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Alvarez-Suarez, J.M., Mazzoni , L., Forbes-Hernandez , T.Y., Gasparrini, M., Sabbadini, S.,
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Giampieri, F. (2014). The effects of pre-harvest and post-harvest factors on the nutritional
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Ariza, M., Martinez-Ferri, E., Domínguez, P., Medina-M’inguez, J.J., Miranda, L., Soria, C.
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Arrigoni, O., De Tullio, M.C. (2000). The role of ascorbic acid in cell metabolism: between
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Association of Official Analytical Chemists (AOAC)(2012).Vitamin C (total) in food:
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semiautomatedfluorometric method. Method 984.26. InOfficial Methods of Analysis of the
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Association of Official AnalyticalChemists (19thed). Washington, DC, USA: Association of
716
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Aylward, F., Haisman, D.R. (1969).Oxidation systems in fruits and vegetables. Advances in
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Ball, G.F.M. (2006), Vitamin C. In G.F.M. Ball (Ed.), Vitamins in foods, analysis,
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Bender, D.A. (2003).Vitamin C (ascorbic acid). In D.A. Bender (Ed.), Nutritional
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biochemistry of the vitamins (2nd ed) (pp. 357-384). Cambridge, UK: Cambridge University
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an
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Bergquist, S.Å., Gertsson, U.E., Olsson, M.E. (2006). Influence of growth stage and postharvest
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Brewer, M.S., Begun, S., Bozeman, A. (1995).Microwave and conventional blanching effects on
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Byers, T., Perry, G. (1992). Dietary carotenes, vitamin C, and vitamin E as protective
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antioxidants in human cancers. Annual Review of Nutrition, 12, 139-159.
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Caverzan, A., Passaia, G., Rosa, S. B., Ribeiro, C.W., Lazzarotto, F., Margis-Pinheiro, M.
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(2012). Plant responses to stresses: role of ascorbate peroxidase in the antioxidant protection,
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Genetics and Molecular Biology, 35, 1011-1019.
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Chang, B.S., Park, N.H., Lund, D.B. (1988). Thermal inactivation kinetics of horseradish
746
peroxidase.Journal of Food Science, 53, 920-953.
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Chauhan, O.P., Unni, L.E. (2015). Pulsed electric field (PEF) processing of foods and its
749
combination with electron beam processing. In S. Pillai & S. Shayanfar (Eds.), Electron beam
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pasteurization and complementary food processing technologies (pp. 157-184). Waltham, MA,
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Cocetta, G., Baldassarre, V., Spinardia, A.,Ferrantea A. (2014).Effect of cutting on ascorbic acid
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oxidation and recycling in fresh-cut baby spinach (Spinaciaoleracea L.) leaves. Postharvest
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Davey, M.W., Montagu, M.V., Inzé, D., Sanmartin, M., Kanellis, A., Smirnoff, N., Benzie, I.J.,
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Strain, J.J., Favell, D., Fletcher, J. (2000), Plant L-ascorbic acid: chemistry, function,
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Davies, M.B., Austin, J., & Partridge, D.A. (1991). Vitamin C: its chemistry and
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Decker, E.A. (2003).Natural antioxidants in foods. In R.A. Meyers (Ed.), Encyclopedia of
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Delincée, H., Schaefer, W. (1975). Influence of heat treatments of spinach at temperatures up to
769
100°C on important constituents.Heat inactivation of peroxidase isoenzymes in
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Deutsch, J.C. (1998). Ascorbic acid oxidation by hydrogen peroxide. Analytical Biochemistry,
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Deutsch, J.C. (2000). Review: dehydroascorbic acid. Journal of Chromatography A, 881, 299-
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Table and Figure captions
Table 1.Distribution of samples within assays for each experiment, showing measures to avoid
ip t
contribution of analytical variability (A) Extraction batches (B) Sample sequences on HPLC (Calibration standards were run at the beginning and end of each HPLC run, and at the points
us
cr
indicated by dashed lines.) n/a = not applicable.
Table 2.Vitamin C loss in homogenized fruits and vegetables at different temperatures (as
an
amount per typical serving size). The vitamin C loss for each food is the average of the two experiments in which there was a statistically significant difference relative to the initial vitamin
M
C content. Shaded cells indicate conditions for which there was no change in vitamin C content. Bolded entries represent >10% of the 90 mg/100g Recommended Dietary Allowance for adults
Ac ce pt e
in only one of the experiments.
d
(Food and Nutrition Board, Institute of Medicine, 2000).Italicized entries indicate loss occurred
Figure 1.Vitamin C assayed in samples of a mixed vegetable quality control material with each experiment throughout the storage temperature study. Capital letters designating each experiment correspond to letters of label for each experiment, as given in the chart labels in Figures 4 9; n/a = not applicable within this study.
Figure 2.Temperature of refrigerator (A), conventional freezer (B), and ultra-low freezer (C) throughout each experiment. (Capital letters in legend correspond to experiment labels in Figs. 4-
46
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9). Markers of the same colour are for the same food, with a different symbol for each experiment.
ip t
Figure 3.Temperature cycling of frost-free conventional freezer on two days.
cr
Figure 4.Stability of vitamin C in homogenized raw strawberries stored at different temperatures
us
in two independent experiments (B and J):○ refrigerated conventional freezer
ultra-low freezer. Each data point is the mean ± standard deviation for extraction and analysis
M
an
of three subsamples.
Figure 5.Stability of vitamin C in homogenized raw tomatoes stored at different temperatures in
d
two independent experiments (F and M): ○ refrigerated conventional freezer ultra-low freezer.
Ac ce pt e
Each data point is the mean ± standard deviation for extraction and analysis of three subsamples.
Figure 6.Stability of vitamin C in homogenized raw (A) and steamed (B) baby spinach stored at different temperatures in two independent experiments (A and H; C and I, respectively): ○ refrigerated conventional freezer ultra-low freezer. Each data point is the mean ± standard deviation for extraction and analysis of three subsamples.
Figure 7.Stability of vitamin C in homogenized raw (A) and baked (B) potatoes stored at different temperatures in two independent experiments (D and K; E and L, respectively):
47
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○ refrigerated conventional freezer ultra-low freezer. Each data point is the mean ± standard deviation for extraction and analysis of three subsamples.
ip t
Figure 8.Stability of vitamin C in homogenized raw (A) and steamed (B) broccoli florets stored at different temperatures in two independent experiments (P and T; Q and S, respectively):
cr
○ refrigerated conventional freezer ultra-low freezer. Each data point is the mean ± standard
us
deviation for extraction and analysis of three subsamples.
an
Figure 9.Stability of vitamin C in homogenized raw navel oranges (A) and purchased pasteurized orange juice (B) stored at different temperatures in two independent experiments (G
M
and N; O and W, respectively):○ refrigerated conventional freezer ultra-low freezer. Each data point is the mean ± standard deviation for extraction and analysis of three subsamples.
d
Figure 10.Long term stability of vitamin C in homogenized fruits and vegetables stored at
Ac ce pt e
< 55oC under nitrogen.○Markers indicate data for samples previously reported on after 49 weeks storage (Phillips et al., 2010).
Markers indicate data for additional composites not
previously reported on. Dashed lines indicate astatistically significant change (p<0.001) in vitamin C concentration that was greater than the minimum detectable difference (see text, section 2.4), given in brackets in the legend; solid lines indicate no difference. Each data point is the meanfrom extraction and analysis of three subsamples.
Figure 11.Vitamin C assayed in samples of mixed vegetablea and pasteurized orange juice control materials assayed with each analytical batch throughout the study. Solid blue lines
48
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bracket the total duration of long term storage of samples previously reported on (Phillips et al., 2010), and solid red lines bracket duration of storage of additional composites (Fig. 10).
ip t
Figure 12.Percent loss of initial vitamin C in homogenized raw broccoli, strawberries, navel oranges, tomatoes, potatoes, and spinach stored under nitrogenrefrigerated (0 5.5oC) for 7 days,
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as a function of food pH.The value for each food is the mean of results from two independent
us
experiments, with n=3 replicate analyses at each time point (Figs. 4 9).
an
Figure 13.Average loss of vitamin C per serving (see Table 2) in homogenized foods, relative to Recommended Dietary Allowance (RDA) for adult men (Food and Nutrition Board, Institute of
M
Medicine, 2000) in (A) refrigerator and (B) conventional freezer storage at time intervals up to 7
Ac ce pt e
FIGURE 1
d
days. Error bars show the range forresults in two independent experiments.
49
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50
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d
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FIGURE 2
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B.
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A.
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(Fig. 2)
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C.
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FIGURE 3
53
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FIGURE 4
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FIGURE 5
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FIGURE 6
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A. Raw spinach
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B. Steamed spinach
56 Page 56 of 69
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FIGURE 7
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ed
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A. Raw potatoes
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B. Baked potatoes
57 Page 57 of 69
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FIGURE 8
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A. Raw broccoli
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B. Steamed Broccoli
58 Page 58 of 69
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FIGURE 9
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ed
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A. Raw navel oranges
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B. Pasteurized orange juice
59 Page 59 of 69
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FIGURE 10
60
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d
Not the same material reported on in Figure 1.
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FIGURE 11
61
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FIGURE 12
62
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an
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FIGURE 13
TABLE 1
Ac ce pt e
d
A. Extraction batches
Storage temperature n/a Controlc Refrigerated o
-10 to -20 C o
< -55 C
c
Control Refrigerated o
-10 to -20 C o
< -55 C c
Control Refrigerated o
-10 to -20 C o
< -55 C
Days storage 0 n/a 1
Extraction # of HPLC # of replicates replicates/extracta 3 1 1 3 3 1
Extraction batch A A B
1
3
1
B
1
3
1
B
n/a 3
1 3
3 1
B C
3
3
1
C
3
3
1
C
n/a 7
1 3
3 1
C D
7
3
1
D
7
3
1
D
Total samples extracted/batch 4
Total samples for HPLCb 42
10
10
10
63
Page 63 of 69
Controlc
n/a
1
3
D
a
Ac ce pt e
d
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an
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cr
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Per extracted replicate sample. Where number of HPLC replicates>1, the prepared/already diluted extract was split among separate HPLC vials. After each extraction, the diluted samples were placed in HPLC vials so that no dilution was necessary after thawing for analysis b After all extractions (i.e. at the end of the 7 day storage time), samples were run in two HPLC sequences, as shown in Table 1B c In-house control composite of homogenized mixture of cooked/canned fruits and vegetables
64
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Table 1 (cont’d.) B. HPLC runs (Calibration standards were run at the beginning and end of each HPLC run, and at the points indicated by dashed lines.)
-10 to -20oC o
Replicate#
0 1
A B
1 1
1 2
1
B
1
B
1
n/a
B
1a
a
Control Refrigerated
n/a 3
A C
-10 to -20oC
3
C
< -55 C
3
C
a
Control Refrigerated
3 3
C C
-10 to -20oC
3
o
o
4
5
1a 1
6 7
1
8
M
Control
3
an
1
a
1
9
1a 2
10 11
C
2
12
C
2
13
3
a
Control Refrigerated
3 7
C D
1b 1
14 15
-10 to -20oC
7
D
1
16
7
D
1
17
Control Refrigerated
n/a 1
D B
1a 2
18 19
-10 to -20oC
1
B
2
20
1
B
2
21
n/a
B
1b
22
1
B
3
1
1
B
3
2
1
B
3
3
n/a
B
1c
4
Ac ce pt e
< -55 C
o
< -55 C
a
o
< -55 C Control
a
cr
Batch
Position in HPLC sequence
d
< -55 C
Storage time (days)
us
Storage temperature HPLC Run 1: n/a Refrigerated
ip t
Extraction (see Table 1A)
HPLC Run 2: Refrigerated o
-10 to -20 C o
< -55 C Control
a
65
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Extraction (see Table 1A)
o
< -55 C
Replicate# 3
3
C
3
6
C
3
7
n/a
C
1c
8
a
Control n/a Refrigerated
n/a 0 7
A A D
1b 2 3
9 10 11
-10 to -20oC
7
D
3
7
D
3
Control Refrigerated
n/a 7
D D
1b 2
-10 to -20oC
7
D
a
o
< -55 C
7
D
a
n/a 0
D A
Controla
n/a
A
13
14 15
2
16
2
17
1c 3
18 19
1c
20
d
Control n/a
12
an
< -55 C
M
o
us
3
a
Control
a
Batch C
Position in HPLC sequence 5
ip t
-10 to -20oC
Storage time (days) 3
cr
Storage temperature Refrigerated
Ac ce pt e
In-house control composite of homogenized mixture of cooked/canned fruits and vegetables
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1
TABLE 2 Vitamin C loss (mg/serving) [standard deviation] b
Serving a
pH
Food
grams Description
o
mg/serving range Mean
%
166
1 c., sliced
110 - 112
4.2 - 4.9
149
1 c.
41.9 - 50.2
3.6 [ 1.6 ] 8 %
Spinach, raw
5.5 - 6.8
30
1 c.
8.9 - 10.9
2.9 [ 0.1 ] 29 %
Spinach, steamed
6.6 - 7.2
180
1 c.
29.9 - 50.8
6.1
213
1 med. (2.25"-3.25" dia.) 17.5 - 28.5
71
1 c. flowerets
1 med. (2.25"-3.25" dia.)
78.8 - 82.4
Broccoli, steamed
6.3 - 6.5
78
0.5 c., chopped
69.3 - 75.3
Oranges, raw
3.1 - 4.1
140
1 fruit, 2 7/8" dia.
70.1 - 86.7
9.3 - 21.6
Orange juice, pasteurized 3.6 - 4.3
249
1 c.
89.6 - 122
3 days storage
%
Mean
13 %
9%
0.9
6%
23.5 [ 0.2 ] 29 %
1.2 [ 0.2 ]
6.2
8%
cr
173
6.6 - 6.9
1.1
%
4.3 [ 0.6 ] 20 %
us
~6.0
Broccoli, raw
Mean
-60 C
ip t
3.0 -3.5
Tomatoes, raw
Potatoes, baked
o
-20 C
Refrigerated
Strawberries, raw
Potatoes, raw
2
Initial vitamin C
Vitamin C loss (mg/serving) [standard deviation]
b
Serving pH
Food
3.0 -3.5
166
1 c., sliced
Tomatoes
4.2 - 4.9
149
1 c.
Spinach, raw
5.5 - 6.8
30
1 c.
Spinach, steamed
6.6 - 7.2
180
1 c.
110 - 112
19.3
%
Mean
%
%
Mean
17 %
8.9 - 10.9
5.8 [ 0.5 ] 59 %
2.1 [ 2.0 ] 23 %
10.2 [ 3.4 ] 44 %
3.4 [ 1.1 ] 15 %
29.9 - 50.8
6.1
213
1 med. (2.25"-3.25" dia.) 17.5 - 28.5
Potatoes, baked
~6.0
173
1 med. (2.25"-3.25" dia.)
Broccoli, raw
6.6 - 6.9
71
1 c. flowerets
Broccoli, steamed
6.3 - 6.5
78
0.5 c., chopped
69.3 - 75.3
8.9 [ 1.4 ] 12 %
Oranges
3.1 - 4.1
140
1 fruit, 2 7/8" dia.
70.1 - 86.7
8.7
249
o
-60 C
8.3 [ 3.1 ] 18 %
Potatoes, raw
Orange juice, pasteurized 3.6 - 4.3
o
-20 C
41.9 - 50.2
M
Strawberries
mg/serving range Mean
9.3 - 21.6
78.8 - 82.4
d
Ac ce pt e
3
grams Description
Refrigerated
an
a
Initial vitamin C
1 c.
4.8 [ 1.0 ] 35 % 38.6 [ 3.2 ] 48 % 12.1 [ 4.0 ] 15 % 10 %
89.6 - 122
7 days storage
b
Serving
Food
a
pH
grams Description
Vitamin C loss (mg/serving) [standard deviation] Initial vitamin C
o
-20 C
Refrigerated
mg/serving range Mean
%
Strawberries
3.0 -3.5
166
1 c., sliced
110 - 112
22.6 [ 12.6 ] 20 %
Tomatoes
4.2 - 4.9
149
1 c.
41.9 - 50.2
16.4 [ 4.8 ] 35 %
Spinach, raw
5.5 - 6.8
30
1 c.
8.9 - 10.9
9.3 [ 1.2 ] 94 %
Spinach, steamed
6.6 - 7.2
180
1 c.
29.9 - 50.8
6.3 [ 4.5 ] 14 %
6.1
213
1 med. (2.25"-3.25" dia.) 17.5 - 28.5
15.5 [ 4.0 ] 68 %
1 med. (2.25"-3.25" dia.)
10.2 [ 4.5 ] 69 %
Potatoes, raw
Potatoes, baked
9.3 - 21.6
Mean
c
o
-60 C %
Mean
%
3.9 [ 2.8 ] 42 % 5.8 [ 1.9 ] 25 %
~6.0
173
Broccoli, raw
6.6 - 6.9
71
1 c. flowerets
78.8 - 82.4
54.7 [ 3.6 ] 68 % 22.7 [ 2.3 ] 28 %
Broccoli, steamed
6.3 - 6.5
78
0.5 c., chopped
69.3 - 75.3
28.3 [ 4.7 ] 39 %
Oranges
3.1 - 4.1
140
1 fruit, 2 7/8" dia.
70.1 - 86.7
11.1 [ 7.8 ] 14 %
Orange juice, pasteurized 3.6 - 4.3
249
1 c.
89.6 - 122
a
pH of food according U.S. Food and Drug Administration (2015), Albrecht et al. (1990) (raw broccoli), and Hughes et al., (1975) (baked potatoes)
b
4 5 6
c
U.S. Department of Agriculture (2014). Serving size equivalents: 1 c. = 240 mL; 1" = 2.54 cm
Values in italics indicate significance for one but not all experiments
67
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d
Ac ce pt e us
an
M
cr
ip t
7 8
68
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69
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d
Ac ce pt e us
an
M
cr
ip t