Original Research Article
Stability of vitamin C in fruit and vegetable homogenates stored at different temperatures

https://doi.org/10.1016/j.jfca.2015.09.008Get rights and content

Highlights

  • Vitamin C analyzed in several types of homogenized fruits and vegetables.

  • Assayed after refrigerated and frozen storage (−20 °C, <−55 °C) over 1-week period.

  • No change in vitamin C at <−55 °C.

  • Significant losses after refrigeration or freezing at −20 °C in some foods.

  • Up to 15% loss in non-acidic stored 3 days at −20 °C (17 mg/100 g 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, strawberries, oranges, and tomatoes; baked potatoes; steamed broccoli and spinach; and pasteurized orange juice after storage under residual nitrogen under refrigeration, and frozen at conventional (−10 to −20 °C) and ultra-low (<−55 °C) temperatures for 1, 3, and 7 days. Additional foods (cantaloupe, green sweet peppers, collard greens, clementines) were monitored for 3–4 years at <−55 °C. Total ascorbic acid was quantified using high-performance liquid chromatography and detailed quality control measures. No decrease occurred in any of the foods after 7 days at <−55 °C. Under refrigeration the largest decreases were in raw spinach and broccoli, averaging (mg/100 g) 9.5 (29%) and 33.1 (29%), respectively, after 1 day and 31.0 and 77.0 after 7 days (94% and 68%, respectively). With conventional freezing, vitamin C was stable for 7 days in most of the products studied; minor losses occurred in raw spinach and broccoli after 1 day but were substantial after 3 days, 6.9 mg/100 g (23%) and 17.0 mg/100 g (15%), respectively; and 7 days (13.1 and 32.0 mg/100 g). For homogenates stored long-term at <−55 °C, vitamin C loss occurred in only cantaloupe, collard greens, and one sample of raw potatoes, all before 50 weeks.

Introduction

Vitamin C is an essential water-soluble vitamin, with a Recommended Dietary Allowance (RDA) of 75 mg/day and 90 mg/day for adult women and men, respectively, and 45 mg/day for children 9–13 years old (Food and Nutrition Board, Institute of Medicine, 2000). l-Ascorbic acid [(2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one] (AA) is the major naturally occurring compound with vitamin C activity. AA is cofactor for many iron and copper hydroxylases involved in key physiological processes such as the production of collagen, the synthesis and activation of peptide hormones (norepinephrine, noradrenaline), and the synthesis of carnitine (Arrigoni and De Tullio, 2000, Ball, 2006, Davies et al., 1991, Bender, 2003, Johnston et al., 2007). AA also has an important role in the intestinal absorption of non-haem iron and as a cellular antioxidant, independently or together with the antioxidant action of vitamin E (Bender, 2003, Byers and Perry, 1992). Dehydroascorbic acid (DHAA) is formed readily from the oxidation of AA in aqueous solutions, and as by-product of cellular reactions of AA or other oxidative processes (Tolbert and Ward, 1982). DHAA is present in many foods (Gökmen et al., 2000). It is absorbed from the small intestine in humans and reduced to AA intracellularly (Deutsch, 2000), and as a result it is bioavailable; thus, the vitamin C content of foods is usually considered to be the sum of the AA and DHAA (Wilson, 2002).

Vegetables and fruits, particularly citrus, green leafy vegetables, broccoli, cauliflower, Brussels sprouts, tomatoes, peppers, and potatoes, are major dietary sources of AA (Eitenmiller et al., 2008). For example, the average vitamin C content of one medium baked potato, one medium tomato, and one medium navel orange are 16.6 mg, 16.9 mg, and 82.7 mg, respectively (U.S. Department of Agriculture, 2014), providing 18–92% of the 90 mg RDA for adult men. Epidemiological studies have associated adequate vitamin C intake with decreased mortality, but controlled trials are lacking (Enstrom, 2008). Studies that relate nutrient intake to health usually involve calculating nutrient intake using food composition data combined with known or estimated measures of food consumption. Obviously, the accuracy of the calculated intake of a particular nutrient depends on how closely its concentration in the food as consumed matches the food composition data. The vitamin C content of fruits and vegetables can be substantially affected by post-harvest handling and processing (Cocetta et al., 2014, Giannakourou and Taoukis, 2003, Neves et al., 2015, Rodrigues et al., 2010, Spínola et al., 2013, Tiwari and Cummins, 2013, Zee et al., 1991). Natural variability in the vitamin C content of foods as they exist in the food supply should be reflected in food composition data. Additionally, once food samples have been procured, the accuracy of the analyzed nutrient content depends on preventing changes in nutrient during the analytical process itself.

The primary source of food composition data in the United States is the U.S. Department of Agriculture's (USDA) National Nutrient Database for Standard Reference (SR) (U.S. Department of Agriculture, 2014). The USDA's National Food and Nutrient Analysis Program (NFNAP) is an ongoing effort, begun in 1996, to update and improve the quality of food composition data in SR (Haytowitz et al., 2008). One of the practical challenges in the NFNAP is that a wide range of nutrients must be assayed in each food that is sampled. The cost of purchasing, shipping, and preparing the foods for analysis is a significant factor in the total cost of the project. Thus, multiple primary samples are often combined to yield composites that are statistically representative of the national market for a given food (Pehrsson et al., 2000). Documentation of the samples and their handling also must be maintained, with a complete audit trail from sample procurement to the release of final data in SR, and archived subsamples of all composites are maintained. Therefore, centralized homogenization of samples and distribution of subsamples for the various nutrient assays is the only practical approach for the NFNAP. Primary food samples (sample units) are procured from retail and wholesale locations and sent to the Food Analysis Laboratory Control Center (FALCC) at Virginia Tech (Blacksburg, VA) (Trainer et al., 2010) where they are prepared, composited, homogenized, and typically dispensed into subsamples in 30- or 60-mL glass jars, sealed under nitrogen, and stored in an ultra-low freezer (<−55 °C), protected from light, until distributed for analysis along with matrix-matched quality control materials (Phillips et al., 2006). Under routine NFNAP processing conditions, a minimum of 2 weeks, and often several weeks, elapse between homogenization and analysis. The sample processing and storage conditions used in the NFNAP were chosen as what is practical to achieve for long term storage of samples of a variety of foods with minimal potential for nutrient loss.

To ensure that the analyzed concentration of vitamin C is representative of the food as consumed, the portion of the sample analyzed must be representative of the whole food. Obtaining a representative portion of a whole food that is heterogeneous (e.g., potatoes with skin) requires either grinding and extracting a large amount of the whole food directly in the extraction solvent (for example, as described by Vanderslice et al., 1990 or Veltman et al., 2000), or homogenizing the food and then taking a subsample for analysis. The former may require a large volume of solvent to accommodate the minimum sample size necessary to be representative of the whole food. Also, in studies involving a large number of samples that must be combined for analysis, in which the food must be analyzed for multiple nutrients involving different extraction methods and multiple laboratories (such as the NFNAP), it is impossible to directly extract the very same whole sample for all assays. Homogenization allows generation of multiple analytical subsamples from one larger sample and the ability to store subsamples prior to analysis, yielding the greatest flexibility in analysis. However, it is critical to establish that labile nutrients such as vitamin C are preserved, to avoid significant errors in food composition data. Once the cell wall has been disrupted, the effect of external factors on reactions resulting in irreversible oxidation of AA and loss of vitamin C (and other nutrients) is more likely to occur, and failure to stabilize AA could result in an assayed vitamin C content of a fruit or vegetable that is quite different from what it is as consumed. Spínola et al. (2014) have recently published an excellent review that includes sample handling considerations for analysis of vitamin C in foods.

AA is especially vulnerable to oxidative and enzymatic degradation in raw fruits and vegetables in which endogenous enzymes are active (Redmond et al., 2003, Francisco et al., 2010, Munyaka et al., 2010a, Wen et al., 2010, Cocetta et al., 2014). Significant and variable post-harvest loss of vitamin C in many fruits and vegetables stored under less optimal conditions has been reported (e.g., González et al., 2003, Lee and Kader, 2000, Munyaka et al., 2010a, Neves et al., 2015, Rybarczyk-Plonska et al., 2014, Szeto et al., 2002, Tiwari and Cummins, 2013, Veltman et al., 2000, Zee et al., 1991). González et al. (2003) measured vitamin C in raspberries and blackberries stored from 0 to 12 months and found an average decrease of 37% and 31% (10.7 and 7.9 mg/100 g), respectively. The storage temperature of −24 °C was higher than the −60 °C used in the NFNAP, and the berries were frozen whole, not homogenized. Vanderslice et al. (1990) reported on the vitamin C content of some fruits and vegetables and performed stability testing on raw broccoli stored under different conditions (refrigerated at −4 °C and frozen at −40 °C, with or without citric acid or metaphosphoric acid). The treatment in the Vanderslice et al. (1990) study that is most relevant to NFNAP standard conditions of <−55 °C under nitrogen was storage at −40 °C. In that study, total AA was constant for 2 weeks (133 ± 7 mg/100 g) and dropped thereafter, reaching 89 ± 25 mg/100 g after 2 months (Vanderslice et al., 1990). Because pH and other matrix-specific characteristics are known to affect vitamin C stability (Musulin and King, 1936, Moser and Bendich, 1990, Wechtersbach and Cigic, 2007), results for broccoli might not apply equally to other fruits and vegetables, and the lower temperature and nitrogen atmosphere used in NFNAP should impart additional stability. Previously, the stability of vitamin C in subsamples of selected raw fruit and vegetable homogenates (clementines, collard greens, potatoes) prepared in liquid nitrogen and stored at <−55 °C under residual nitrogen for one year was established (Phillips et al., 2010).

Many laboratories do not have access to an ultra-low freezer (<−55 °C), and the cost is higher for storage at ultra-low versus standard freezer temperature (∼−10 to −20 °C). Therefore, as a matter of practicality, homogenized fruits and vegetables prepared for analysis might be exposed to refrigerator or conventional freezer temperatures. The Association of Official Analytical Chemists method for analysis of vitamin C in foods (AOAC, 2012) specifies homogenization of the food in a blender and subsampling for analysis, but there is no indication for temperature control. In fact, some literature reports on the vitamin C content of fruits and vegetables involve storage of prepared samples at ≥−20 °C prior to analysis (e.g., Giannakourou and Taoukis, 2003, Kabasakalis et al., 2000, Mahattanatawee et al., 2006, Rizzolo et al., 2002, Vanderslice et al., 1990). Whether or not storage at these temperatures is necessary for practical reasons, less than optimal conditions could lead to substantial and variable deviations in the vitamin C content of the food as assayed versus as consumed.

The objective of this study was to assess the change in the vitamin C concentration in different fruits and vegetables that were homogenized while frozen in liquid nitrogen and then stored under refrigeration (0–5 °C), and frozen at conventional (−10 to −20 °C) and ultra-low (<−55 °C) temperatures for up to one week. Additionally, we report on the stability of vitamin C in a variety of similarly homogenized samples of fruits and vegetables stored in an ultra-low freezer for >4 years, as a follow-up on results after 1 year storage that were previously reported (Phillips et al., 2010).

Section snippets

Food selection

Foods representing good sources of vitamin C, and having characteristics that could affect stability of vitamin C (e.g. pH, endogenous enzyme activity) were selected for the study: strawberries (Fragaria X ananassa); grape tomatoes (Solanum lycopersicum); baby spinach (Spinacia oleracea), raw and steamed; russet potatoes (Solanum tuberosum), raw and baked; broccoli (Brassica oleracea var. italica), raw and steamed; navel oranges (Citrus sinensis); and pasteurized orange juice (pulp-free,

Analytical precision

The r2 value was ≥0.998 for the linear regression for the HPLC calibration curves in all assays, and the RSDs for within-assay means of each standard level were consistently <0.5%.

Fig. 1 shows the results for the mixed vegetable control material analyzed with the storage time/temperature experiments throughout the study. The overall precision was excellent (mean, 4.24 mg/100 g, 2.0% RSD), and no significant differences were found within each experiment. Within each experiment the median RSD for

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 aqueous solutions, readily oxidizing to DHAA in the absence of antioxidants (Eitenmiller et al., 2008). Ascorbate oxidase (AO) and ascorbate peroxidase (APO) catalyze oxidation of AA to DHAA (Saari et al., 1995, Shigeoka et al., 2002) using AA as a specific electron donor (Caverzan et al., 2012). Whereas DHAA is

Conclusions

For general food composition work involving analysis of vitamin C in fruits and vegetables, in which samples are homogenized and held before analysis, ultra-low temperature freezer storage (<−55 °C) should be standard, and samples should be analyzed within 7 days. Storage at this temperature, with residual nitrogen, can be relied on to preserve vitamin C content over a 7-day period for a wide range of homogenized fruits and vegetables, including raw products. However, as previously reported (

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.

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