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INVESTIGATION OF FACTORS
AFFECTING VITAMIN C IN LIQUID
CHAPTER ONE
1.0.
INTRODUCTION
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.
1.1.
HYPOTHESIS
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.
1.2.
BACKGROUND
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)
1.3.
METHOD
Materials
• 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. [1] 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. [2].
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 [3]. 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 [4]. 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].
1.4.
EXPERIMENTAL
For determination of ascorbic acid were
used two methods: a titrimetric method with potassium brommat-bromide solution
in the acid medium [2] and a conductometric method [1] 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 [10].
1.5.
REAGENTS
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%.
1.6.
APARATUS
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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