Vitamin C (also referred to as L-ascorbic acid) is the lactone 2,3-dienol-L-gluconic acid and it belongs to the water-soluble class of vitamins. Ascorbic acid is an odourless, white solid having the chemical formula C6H8O6. Vitamin C is mainly found in fruits and vegetables. In the nutritional content, vitamin C is the L-enantiomic form of ascorbic acid which also encompasses the oxidation product of dehydroascorbic acid with different oxidizing agent. It participates in numerous biochemical reactions, suggesting that vitamin C is important for every body process from bone formation to scar tissue repair (Rickman et al., 2007). The only established role of the vitamin C appears to be in curing or preventing scurvy and it is the major water-soluble antioxidant within the body.
Factors that affect the vitamin C contents of citrus fruits include, production factors and climate conditions, maturity state and position on the tree, type of fruits (species and variety), handling and storage, type of container (Naggy, 1980). Immature fruit has the highest levels and decreases during the ripening process. Early maturing varieties have higher levels than late maturing types. High nitrogen fertilizer rates can lower vitamin C levels in citrus fruits. Proper potassium levels are also needed for good vitamin C level (Padayatty et al., 2003).
Pasteur identified the growth micro organisms such as bacteria and fungi as
the scientific cause of spoilage and decay in the 1860s, other causes include
chemical changes from ripening and senescence (aging) processes occurring in
the fruit. Bacteria and fungi are everywhere in our environment and most foods
provide an excellent substrate (http://www.answers.com/topic/substrate)
for their growth (Manso et al., 2001). Vitamin C bears an obvious structural
similarity with hexose sugars; hence, it is conceivable that the molecule might
serve as a carbon source for respiration or bacterial growth that it might be
fermented (Eddy and Ingram, 1953). Storage conditions of low temperature and
humidity have been found to retard microbial growth; chemical and biological
processes are also slowed down (Manso et al., 2001 actahort.org/books/566/index.htm).
However, once these protective barriers are breached, microbial growth is often
unchecked and rapidly destroys the commodity. The flavour, texture and nutrition
of many fruits and vegetables are reduced before visual appearance of spoilage
(María Gil et al., 2006).
Oxygen is the most destructive ingredient in juice causing degradation of vitamin C. However, one of the major sugar found in orange juice, fructose, can also cause vitamin C breakdown. The higher the fructose content, the greater the loss of vitamin C. Conversely, higher acid level of citric acid and malic acid stabilize vitamin C (Padayatty et al., 2003). Canned juices are often regarded as less nutritious than fresh or frozen products therefore the preference for fresh/preserved fruits in this country. This necessitated this study on the effects of storage on the quality of some common fruits using vitamin C as the reference.
MATERIALS AND METHODS
Sample Collection and Preparation
Fresh fruits of Citrus sinensis (orange), Citrus limon (lemon),
Citrus aurantifolia (lime), Ananus comosus (pineapple), Asimina
triloba (pawpaw) and carrot were purchased from retail outlets in Zaria,
a Northern Nigerian city. The study was carried out in Ahmadu Bello Univeristy,
Zaria-Nigeria between March and June 2007. These fruits were washed thoroughly
with water and the juices were extracted by mechanical pressure. Each type of
juice samples was filtered to remove pulp and seeds and stored in already labelled
All chemicals used were obtained from BDH London, unless otherwise stated
were of analytical grade purity and double distilled water was used.
One percent starch indicator solution was prepared by adding 0.50 g of soluble starch in 50 mL of near-boiling water.
Iodine solution was prepared by dissolving 5.0 g of potassium iodide (KI) and 0.268 g of potassium iodate (KIO3) in 200 mL of water followed by addition of 3 M sulphuric acid. The solution was made up to 500 mL in a graduated cylinder and then transferred to a beaker.
Vitamin C standard solution was prepared by dissolving 0.250 g of vitamin C in 100 mL of water and then diluted to 250 mL with water in a volumetric flask.
Vitamin C Determination by Iodine Titration
Oxidation-reduction method described by Helmenstine (2008)(http://www.chemistry.about.com)
Standardizing Solutions and Titration of Juice Samples
vitamin C solution (25 mL) was transferred into 100 mL conical flask and
10 drops of starch solution was added. This was titrated with the iodine solution
until the first blue colour which persisted for about 20 sec was observed. Juice
samples (25 mL) were titrated exactly the same way as the standard. The initial
and final volume of iodine solution required to produce the colour change at
the endpoint was recorded. Titration was performed in triplicate in all cases.
The samples were cultured on blood agar medium, incubated at 37°C for
24 h, the colonies of the organisms were gram stained, biochemical tests were
carried out to identify the bacteria, according to the method described by Singleton
(1999). The yeast identification was performed with fluoroplate candida agar
according to the method of Manafi and Willinger (1991).
RESULTS AND DISCUSSION
The retention of vitamin C is often used as an estimate for the overall nutrient
retention of food products because it is by far the least stable nutrient; it
is highly sensitive to oxidation and leaching into water-soluble media during
storage (Davey et al., 2000; Franke et al., 2004). It begins to
degrade immediately after harvest and degrades steadily during prolonged storage
(Murcia et al., 2000) and also continues to degrade during prolonged
storage of frozen products (Rickman et al., 2007). Results for the freshly
squeezed fruits shows that the oranges had the highest vitamin C content, followed
by lemons, limes, pineapple, pawpaw and carrot. The values obtained for citrus
fruits are quite lower than values obtained elsewhere (http://www.naturalhub.com/natural_food_guide_fruit_vitamin_c.htm).
This is consistent with reports that, climate, especially temperature affect
vitamin C level. Areas with cool nights produce citrus fruits with higher vitamin
C levels. Hot tropical areas produce fruit with lower levels of vitamin C (Padayatty
et al., 2003). Also, environmental conditions that increase the acidity
of citrus fruits also increase vitamin C levels.
The results have shown that the environment in which juice is stored can affect its vitamin C content significantly (Fig. 1). The pattern of loss in vitamin C showed an initial increase in the first two weeks followed by decrease in orange samples RT. The RC samples decreased initially, followed by an increase and then a decrease. The concentration of vitamin C decreased faster in RC than in RT samples, however, the same pattern was observed throughout the four weeks of storage. The reason for the initial increases is not understood, but Rickman et al. (2007) attributed this to a change in moisture content during the storage of frozen peas.
The trend in the concentration of vitamin C for the lemon samples, over the period of investigation is similar to that observed for oranges. There was an initial decrease, then an increase at two weeks and then a decrease. The difference in the concentration of vitamin C between RT and RC at any particular time is not much. The result also showed that more vitamin C is lost in lemon over this period than in oranges. For the lime sample the pattern of decrease differ slightly for the RC samples. The initial increase in the vitamin C content was not observed for RC samples. However, like the orange and lemon the RC samples lost more vitamin C than the RT samples.
Light exposure was found to promote browning in pineapple juice. Ten percent losses in vitamin C have been reported after 6 days at 5°C in pineapple pieces by María Gil et al. (2006). Pineapple samples showed a different pattern of decrease in vitamin C content compared to the citrus fruits. Here, the RC samples retained more vitamin C than the RT samples after four weeks of storage. The initial increase in the vitamin C content observed in the citrus fruits was not observed with the pineapple sample. This suggests that variation in moisture content cannot be the sole controlling factor leading to the initial increase observed in the citrus fruits. Again no reason can be proffered from this investigation why the retention of vitamin C is more in the RC samples than in the RT samples. Since, vitamin C is unstable in neutral and alkaline environments therefore the longer the exposure, the greater the loss of vitamin C. The increase in pH (Table 1) was related to deterioration of fruit characteristics (María Gil et al., 2006).
The RT pawpaw sample showed a rapid initial decrease in vitamin C content within
the first two weeks. At this period the RC sample showed a steady decrease with
vitamin C content higher than RT. By the third week Vitamin C content in RT
increased above RC after which, both RT and RC decreased very rapidly, with
RC tending towards zero vitamin C content. Finally, in the carrot sample, RT
and RC decreased rapidly at first with RC retaining more vitamin C up to the
second week. After the second week, difference in vitamin C content between
RT and RC became very small, both decreasing till the fourth week.
Variation of vitamin C content of fruits with time and mode of storage
(a) Orange, (b) Lemon, (c) Lime, (d) Pineapple, (e) Paw paw and (f) Carrot
||pH values of the fruit juice with storage time
|RT: Room Temperature; RC: Refrigerated at 4°C
Many chemical reactions contribute to the loss of storage life of vitamin C
and hence chemical deterioration of fruits. The majority of these reactions
are enzymatically driven while others are chemical reactions that occur because
of the senescence (aging) processes. This involves colour, flavour, and odour
changes that result from a chemical reaction between the constituents of the
fruit. Fruit can be a vector and provide a growth medium for many pathogenic
microbes which can produce potent toxins. In this study Bacillus subtilis
and Candida sp. were isolated from both RT and RC of all the fruits used
in this investigation, except in orange where only Candida sp. was isolated.
Bacillus subtilis is not considered a human pathogen; it produces the proteolytic
enzyme subtilisin (a protein-digesting enzyme) and has been implicated in food
poisoning and spoilage (Ryan and Sherris, 1994). Candida albicans sp.
(yeast) has been reported as the causative agent of spoilage of sugary foods,
such as condensed milk, fruit juices and concentrates (Stratford et al.,
2002). The biochemical reactions occurring over the storage period together
with microbial action in all fruit juices resulted in pH changes observed (Table
1). Two-tailed Spearman’s correlation showed that there is a significant
correlation between pH and vitamin C at 95% confidence level for RT samples
of pineapple (r2 = 0.74), pawpaw (r2 = 0.84) and carrot
(r2 = 0.75). For RC samples only pineapple (r2 = 0.77)
and pawpaw (r2 = 0.70) showed a significant correlation. This result
shows that pH is also not the sole controlling factor in the deterioration of
vitamin C in fruit juice with storage life.
This study supports the common perception that fresh is often best for optimal vitamin C content, as long as the fresh product undergoes minimal storage at either room or refrigerated temperatures. Loss of vitamin with time differs from one fruit to the other under similar storage environments. While the refrigerated samples cause significant loss of ascorbic acid in the citrus fruits, this is not so in pineapple, pawpaw and carrot samples. Though pH is significant in the stability of vitamin C, it cannot be said to be the sole controlling factor leading to losses observed in all the fruits investigated.
We express the gratitude to Mallam Mikailu Abdullahi of the Department of Microbiology, National research Institute for Chemical Technology, Zaria-Nigeria, for identifying the microbes.