INVESTIGATION OF FACTORS AFFECTING VITAMIN C IN LIQUID
Vitamin C is important for the human body because it is needed in the production of collagen to make connective tissue. Vitamin C also helps the body to absorb iron, helps wounds to heal, helps red blood cell formation and helps to fight infections. Some studies say that vitamin C prevents cancer. A lack of vitamin C can cause a disease called scurvy, iron deficiency and poor wound healing. The healthy diet should include high amounts of vitamin C because the human body cannot make it’s own vitamin C. Oranges are an excellent source of vitamin C. (Gordon, 1995-2005, p.1)(Larsen,1997-2005, p. 1)(General Health Encyclopedia,1998, p. 1)(Royston, 2003, p.19) Orange juice and drinks are common in our diet and some people think they are receiving vitamins in any type of orange drink. I conducted two experiments to compare the content of vitamin C in different kinds of orange juice and drinks.
My hypothesis is that freshly squeezed orange juice will have the most vitamin C compared to the other types of orange juice and drinks, because the fruit is picked fresh, and it is not stored, preserved or exposed to oxygen.
Vitamins help to speed up important reactions in the body. Vitamin C, also known as ascorbic acid, is water soluble, meaning it will dissolve in water and is not stored in the body.
We need to get vitamin C from the foods we eat. (Gordon, 1995-2005, p.1) (Silverstein, 1992, p.8) Vitamin C is found in fruits such as oranges, limes, and grapefruit, and vegetables including tomatoes, green peppers, and potatoes. The recommended amount of vitamin C is 60 to 90 milligrams per day. (Gordon, 1995-2005, p. 2 ) People who smoke need more vitamin C in their diet, because they lose 25 mg. of vitamin C every time they smoke a cigarette. People who are stressed, have infections, take antibiotics, drink lots of alcohol or have been injured need more vitamin C in their diet. (Sullivan, 1997, p. 30) Factors that Affect Vitamin C in Orange Juice Vitamin C is sensitive to light, heat and air. (Alpert, 1997-2004, p. 1) Vitamin C is the most easily destroyed vitamin and it is easily harmed during food preparation. This can happen during chopping, exposure to air, cooking, and boiling. (Joanne Larsen, 1995-2003, p. 1and 2)
There are many factors that will affect how much vitamin C is in orange juice. Townsend (1999) summarized some of these factors. For example, freezing preserves vitamin C. Exposure to oxygen will destroy vitamin C. Oranges will have more vitamin C if they are picked when they are less ripe, if they are early maturing like Navel oranges, if they are grown in soil with low levels of nitrogen or if they are exposed to cooler temperatures while growing. How the juice is made will also affect the amount of vitamin C in it. For instance, frozen orange juice is made from a mix of early ripening and late ripening oranges, so it tends to have higher concentrations of vitamin C. The type of container that the juice is stored in can affect vitamin C levels. For instance, enamel lined cans lose more vitamin C than tin cans. Orange juice in glass jars or cardboard cartons have less. Today cartons have oxygen and light barriers to protect the vitamin C and frozen juices are stored in cardboard cans lined with foil to help keep vitamin C. For the best vitamin C levels, orange juice must be stored at cool temperatures with oxygen barriers. (Townsend, 1999, p. 1 and 2)
When all of the ascorbic acid is used up, that is the end point of the reaction. If there is so much vitamin C that all of the iodine in the starch solution is used up, the solution will become clear because of the dehydroascorbic acid. If there is very little vitamin C, less iodine will be used up and more blue colour will remain. (Thomas, 1999, p. 1)
• Vitamin C Indicator Solution (2% Iodine solution, cornstarch and water)
• Medicine droppers, Test tubes, Test tube holder
• Beverages -12 different kinds of orange juices and drinks:
Indicator Solution Mix one tablespoon of cornstarch and water to make a paste, add 250 millilitres of water and boil. Add 10 drops of this solution to 75 millilitres of hot water, stirring. To this solution, add 12 drops of 2% iodine solution. (USDA Agricultural Research Service, 2005, p. 1)
1.3.1. Experiment One
Add 10 drops of one juice to 5 ml. of indicator solution in a test tube. Stir. Repeat for each beverage. Line the tubes up from lightest to darkest. Record the rank of each beverage from ‘12’ being the lightest (with the most vitamin C) to ‘1’ being the darkest colour (with the least vitamin C). (USDA Agricultural Research Service, 2005, p. 1)
1.3.2. Experiment Two
Add juice one drop at a time to 5 ml. of indicator solution in the test tube until the indicator solution changes from blue to colourless or until the solution no longer changes colour. This is the endpoint. Repeat for each beverage. Observe and count the numbers of drops of orange beverage added to the indicator to cause it to lose its colour or to stop changing colour. Record the number of drops. (Dipaolo, 2002, p. 1)
Ascorbic acid is one of the important water soluble vitamins. It is essential for collagen, carnitine and neurotransmitters biosynthesis.  Most plants and animals synthesize ascorbic acid for their own requirement. However, apes and humans can not synthesize ascorbic acid due to lack of an enzyme gulonolactone oxidase. Hence, ascorbic acid has to be supplemented mainly through fruits, vegetables and tablets. . The current US recommended daily allowance (RDA) for ascorbic acid ranges between 100–120 mg/ per day for adults. Many health benefits have been attributed to ascorbic acid such as antioxidant, antiatherogenic, anti-carcinogenic, immunomodulator and prevents cold etc. Thus, though ascorbic acid was discovered in 17th century, the exact role of this vitamin/nutraceutical in human biology and health is still a mystery in view of many beneficial claims and controversies . Ascorbic acid is a labile molecule; it may be lost from foods during cooking/processing even though it has the ability to preserve foods by virtue of its reducing property. Syntethis ascorbic acid is available in a wide variety of
supplements, tablets, capsules, chewable tablets, crystalline powder, effervescent tablets and liquid form. L-ascorbic acid (C6H8O6) is the trivial name of Vitamin C. The chemical name is 2-oxo-L-threo-hexono-1, 4-lactone-2, 3-endiol, Fig. 1 . Ascorbic acid being a water soluble compound is easily absorbed but it is not stored in the body. The major metabolites of ascorbic acid in human are dehydroascorbic acid, 2, 3-diketogluconic acid and oxalic acid, Fig. 2. The main route of elimination of ascorbic acid and its metabolites is through urine. It is excreted unchanged when high doses of ascorbic acid are consumed. Ascorbic acid is generally non-toxic but at high doses (2-6g/day) it can cause gastrointestinal disturbances or diarrhea [5, 6].
Keeping in view its importance, the analysis of food products and pharmaceuticals containing this vitamin assumes significance. Such attempts to quantify ascorbic acid in these samples have resulted in a large number of methods: titrimetry, voltametry, fluorometry, potentiometry, kinetic-based chemiluminiscence (CL), flow injection analyses and chromatography [7–9].
For determination of ascorbic acid were used two methods: a titrimetric method with potassium brommat-bromide solution in the acid medium  and a conductometric method  based by the calibration curve which was plotted under the fallowing operative conditions: 4°C, 18°C, 30°C and 40°C, the linearity range is 0,008–0,1N ascorbic acid. The methods have been applied to many samples to determine the Vitamin C quantity at different temperature .
All reagents were of analytical-reagent grade and all solutions were prepared using distilled-deionized water. A stock standard aqueous ascorbic acid solution (0,1N) was prepared from L-ascorbic acid (Merck). Other standard ascorbic acid solutions were obtained by appropriate dilutions of the stock solution. For the titrimetric method, the reagents used have been: Na2S2O3 0,1N, KBrO3-KBr 0,05N, K2Cr2O7 0,1N, H2SO4 1N, H2SO4 1:2, KI, starch indicator 1%.
For conductometric procedure was used a Conductivity Meter WTW, LF 340-A/SET, made in Germany.
1.7. CONDUCTOMETRY PROCEDURE
First was plotted the calibration graph (Fig. 3) of different ascorbic acid concentrations (0,008N; 0,01N; 0,03N; 0,05N; 0,08N respectively 0,1N) at the room temperature by reading it at the conductivity meter (ìS/cm).
1.8. TITRIMETRY PROCEDURE
Ascorbic acid, C6H8O6 is cleanly oxidized to dehydroascorbic acid by bromine:
An unmeasured excess of potassium bromide is added to an acidified solution of the sample. The solution is titrated with standard potassium bromated to the first permanent appearance of excess bromine: this excess is then determined iodometrically with standard sodium thiosulfate. The entire titration must be performed without delay to prevent air-oxidation of the ascorbic acid. [2, 10]
1.9. . KINETIC STUDY
For the kinetic study was determinate the rate constant, the half-time and the activation energy for vitamin C.
For rate constant it was applied the formula:
where: k = the rate constant;
t = the interval of time since the reaction has began;
a = the initial concentration at 18°C;
a – x = the concentration at different temperatures (30°C and 40°C).
Arrhenius noted that the k(T) data for many reactions fit the equation:
where A and Ea are constants characteristic of the reaction and R is the gas constant. Ea is the activation energy and A is the pre-exponential factor or the Arrhenius factor. The units of A are the same of those of k. Ea is usuallyexpressed in kcal/mol or kJ/mol.
Taking logs of equation (*), we get:
The nutritional importance of vitamin C (L-ascorbic acid; 2,3- endiol-L-gulonic acid-g-lactone) as an essential water-soluble vitamin is well established. It has long been known that a nutritional deficiency in vitamin C causes scurvy, a disease characterized by bleeding gums, impaired wound healing, anemia, fatigue, and depression, that, without proper care, can eventually be fatal (Davies et al., 1991; Arrigoni and De Tullio, 2000). Ascorbic acid (AA) is a cofactor in numerous physiological reactions, including the post-translational hydroxylation of proline and lysine in collagen and other connective tissue proteins, collagen gene expression, synthesis of norepinephrine and adrenal hormones, activation of many peptide hormones, and synthesis of carnitine (Bender, 2003; Johnston
et al., 2007). Also, due to its redox potential, ascorbic acid facilitates intestinal absorption of iron and functions as a cellular antioxidant alone and coupled to the antioxidant activity of vitamin E (Byers and Perry, 1992; Bender, 2003). Therefore, adequate intake of vitamin C from foods and/or supplements is vital for normal functioning of the human body. Recommended Dietary Allowances (RDA) of 75 mg/day and 90 mg/day have been established for adult women and men, respectively, and 45 mg/day for children 9–12 years old (Food and Nutrition Board, Institute of Medicine, 2000). Recent interest in the
role of dietary antioxidants in general, and of specific food components, requires accurate food composition data to facilitate epidemiological studies and feeding trials relating the intake of vitamin C to physiological effects, and to develop food consumption recommendations.
Vegetables and fruits, particularly citrus fruits, green leafy vegetables, broccoli, cauliflower, Brussels sprouts, tomatoes, peppers, and potatoes, are major food sources of vitamin C (Eitenmiller et al., 2008). However, vitamin C is subject to oxidative and enzymatic degradation to dehydroascorbic acid (DHAA) and also irreversible oxidation via DHAA to diketogulonic acid, and the latter has no vitamin C activity (Nyyssonen et al., 2000). Ascorbic oxidase is the endogenous enzyme involved in this process (Saari
et al., 1995). Various factors, including the presence of oxygen and metal ions (especially Cu2+, Ag+ , Fe3+), alkaline pH, and high temperature affect the vitamin C content of raw produce prior to the point of consumption and result in variation in the actual levels
in different samples of a given product (Lee and Kader, 2000). Light, pH, temperature, oxygen exposure, the presence of oxidizing metals, and oxidizing enzymes can be controlled during the assay itself, but must also be controlled during preparation of samples for analysis, especially if the procedures involve maceration or other disruption of cells which release oxidizing enzymes. Failure to assess stability of vitamin C in raw produce during sample processing and analysis could result in significant errors in analytical results. 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) (USDA, 2008). The USDA National Food and Nutrient Analysis Program (NFNAP) is an ongoing project to update and improve the quality of food composition data in SR (Haytowitz et al., 2008). For the aforementioned reasons, vitamin C in many fruits and vegetables was identified as a key nutrient requiring attention. One of the practical challenges in the NFNAP is that a wide range of nutrients must be assayed in each sample procured, and, furthermore, numerous primary samples must be obtained to represent the national supply of a given food (Pehrsson et al., 2000). The cost of purchasing, shipping, and preparing samples for analysis is a significant factor in the total cost of the project. There is a fundamental need to standardize and document the handling of samples via a complete audit trail from sample procurement to the release of final data in SR, and archived subsamples of all composites must be maintained as well. Therefore, centralized sample preparation is a practical approach for the NFNAP. Primary food samples (sample units) are procured from retail and wholesale locations and are sent to a laboratory [the Food Analysis Laboratory Control Center (FALCC) at Virginia Tech, Blacksburg, VA] where they are prepared, composited, homogenized, and
dispensed into subsamples that are distributed for analysis along with quality control materials (Phillips et al., 2006). Because analytical values are used to estimate nutrient values in the product at point of consumption, it must be ensured that degradation of nutrients does not occur during the preparation process, e.g., homogenization, subsampling, and storage of samples prior to analysis. The degree of nutrient loss during standard storage conditions must be verified for labile nutrients. Under routine NFNAP processing conditions, a minimum of 2 weeks, and often several weeks, elapse between homogenization and analysis. Additionally, it was necessary to determine if vitamin C content of archive samples stored for longer periods would still be representative of the original sample. Previously the stability of folate in raw fruit and vegetable homogenates prepared for NFNAP analysis was established (Phillips et al., 2005). In an initial study of vitamin C in raw produce, results for some products were unexpectedly variable and/or lower than expected (Fig. 1) for some raw fruits, with some values much less than half of the vitamin C concentrations reported in Release 14 of SR (USDA, 2001). Those values were not used to update SR, and reasons for the discrepancies were considered, including stability during sample storage. While it is known that degradation of vitamin C can occur in homogenates of raw produce, literature on the stability of vitamin C in fruits and
vegetables cannot be directly or definitively extended to the NFNAP foods and sample storage protocol. For example, Gonzalez 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, but the storage temperature of 24 8C was higher than the 60 8C used under NFNAP protocols, and the berries were frozen whole, not homogenized. Vanderslice et al. (1990) reported on the vitamin C content of selected fruits and vegetables and performed stability testing on raw broccoli samples stored under different conditions (refrigerated at 4 8C and frozen at 40 8C, 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 (60 8C under nitrogen) was storage at 40 8C. In that
Fig. 1. Preliminary analytical results for vitamin C in selected fresh fruits sampled for NFNAP in 2001–2002, compared to Release 14 of the USDA Nutrient Database for
Standard Reference (SR14) (USDA, 2001). Values plotted are the average for 4 samples, and error bars represent the range.
study, total vitamin C as ascorbic acid plus DHAA 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). However, the lower temperature and nitrogen atmosphere used in NFNAP should impart additional stability. Furthermore, 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 of fruits and vegetables. While homogenization in citric acid or metaphosphoric acid, as reported for broccoli by Vanderslice et al. (1990), might stabilize
vitamin C, this special treatment would be impractical in NFNAP because it would require a separate composite just for this nutrient. The number of samples and composites to be prepared would be effectively doubled. Furthermore, the number of samples to be analyzed exceeds the total that can be assayed in a single batch by the average laboratory, delaying the analysis of some samples.
The objective of this study was to evaluate the retention of vitamin C in frozen homogenates of representative raw fruits and vegetables held over a period of time under the typical processing and storage methods for NFNAP.
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