In Vitro evaluation of erosive potential of frozen and unfrozen fruit juices on primary teeth
ABSTRACT: Dental erosion has gained importance now as a steadily growing public health problem world wide. Fruit juices are an important etiological factor, especially consumption in frozen forms, like fruit lollies and stick. Since this dietary habit is more prevalent among children, primary teeth were selected for this study.
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AIMS: To evaluate pH and titratable acidity of four fruit juices in frozen and unfrozen forms. Calcium dissolution and pattern of etching of enamel by these juices in frozen and unfrozen forms were also determined.
DESIGN: pH of four different juices – apple, orange, musumbi and grapes were determined using a digital pH meter. The titratable acidity of these in frozen and unfrozen forms were determined by adding 0.5 ml of 1N NaOH to these, till a pH=five and pH = seven were reached. Forty eight caries free deciduous anterior teeth specimens were prepared to study the etching pattern by scanning electron microscope [SEM] and calcium dissolution by atomic absorption spectrophotometer.STATISTICAL ANALYSIS: One-Way Repeated Measures ANOVA and pair wise multiple comparison with Bonferroni correction. RESULTS: Increase in titratable acidity, irregular pattern of etching and calcium dissolution were found to be significantly more in the initial thawed frozen fruit juices. Grape juice recorded the lowest pH and more titratable acidity when compared to other juices. CONCLUSION: This study highlights the detrimental effect on teeth by the frequent consumption of fruit juices and their frozen products. The study concluded that sucking on the frozen fruit juice products were more harmful than consuming in unfrozen state because more of erosion occurs in frozen state. Key words: Frozen fruit juices, pH, titratable acidity, erosion, etching, SEM. Introduction: “HEALTHY LIVING “the key to survival has been emphasized by health professionals upon the old and young alike. The “DIETARY FACTOR” is gaining much accolade and health drinks in the form of fruit juices have gained tremendous popularity in this category. Parents regard these as being healthy and nutritious and a good source of Vitamin C.. Experiments conducted on the animals to evaluate the erosive potential of fruit juices showed that these juices were ten times more destructive to the teeth than the whole fruit. Most fruit juices have a low pH and acids which can decalcify the teeth. Hence, the frequent consumption of these fruit drinks is directly related to dental morbidity, especially erosion.Dental erosion has been defined as superficial loss of dental hard tissue by a chemical process not involving bacteria. The aetiopathogenesis can be varied (intrinsic and extrinsic factors),most important are dietary acids. A modern habit among consumers is to freeze and suck out fruit lolly and candies.Sucking on frozen fruit juices has a greater risk for erosion due to slow consumption and longer time required to neutralize it.Sucking of the melting juices from the frozen product is a pleasurable experience along with increase in the initial acidity and buffering capacity.Hence the frequent use of these could produce damaging effects on the teeth. Primary teeth were included in this study,because children are frequently involved in the consumption of frozen fruit juices and deciduous teeth are more susceptible to erosion than permanent teeth due to less mineralisation and immature enamel surface The study was performed to evaluate the pH of four fruit juices in a frozen and unfrozen form. The titratable acidity of these fruit juices to be checked. The total amount of calcium dissolved in fruit juices from the teeth and to study the pattern of etched enamel on teeth.
MATERIALS AND METHODS:
Preparation of juices: Two and half litres of four different fresh fruit juices namely apple, orange, grape and musumbi each were prepared with no added sugars or preservatives. These were allowed to equilibrate to room temperature.
Specimen preparation: Fourty eight freshly extracted caries free deciduous anterior teeth were collected and were used in the study. Extracted teeth were completely coated with nail polish with the exception of a window on the enamel of the labial surface of approximately 2mm X 2mm in diameter.
Method: Two and half litres of apple juice were shaken for 15sec and this was divided into four samples of 600ml each. First sample (600ml) of this was taken and calcium concentration was assessed. From this, 100 ml was separated, pH and titratable acidity were determined.Then another 100 ml was taken and a prepared tooth specimen was dipped in it for two hrs.(For assessment of calcium & etching pattern at room temp).
Remaining 400ml was taken in a bottle, sealed and placed in a recorded (-200c) deep freezer for 24 hrs. The bottle was taken and allowed to defrost. From this, initial 100ml was taken, pH and titratable acidity were determined. Then another 100 ml was taken and a prepared tooth was dipped in it for two hrs( For assessment of calcium & etching pattern immediately after defrosting). Remaining 200 ml was defrosted for two hours. Then 100 ml of this was taken and pH and titratable acidity were determined. Another 100 ml was taken and a prepared tooth was dipped in it for two hrs (For assessment of calcium & etching pattern two hrs after defrosting). This was repeated for remaining three samples (600ml each) of apple juice. The same procedure was done for the other three juices musumbi, orange and grape juice and the values recorded [Table1,2,3]
Determination of pH and titratable acidity: pH was determined by using a pH meter (digital pH meter model –EQ-612).
After determining the pH, the juice was titrated against 1N( Na OH )Sodium hydroxide by adding 0.5ml of the same to the juice, mixed well and this were repeated until pH=five and pH=seven were reached to determine their titratable acidity.
Assessment of calcium content: The teeth were carefully lifted out of the juices and the juices were centrifuged at 3500 rpm for five min. With the help of micropipette [superfit] 200μl of each of these samples of fruit juices were pipetted into the polypropylene tubes. Then 40μl distilled water were rinsed into the polypropylene tubes with the fruit juices. Then concentrated nitric acid (120μl) were added, the lid of the polypropylene tube sealed and the contents of the tube were wet washed, by leaving it at 600c for 12hrs. After cooling, 50μl of one mol/L KCl solution (an ionization suppressant) and 680μl of distilled water were added. Then samples were shaken and the calcium concentrations were determined by the use of a flame (nitrous oxide/acetylene) atomic absorption spectrophotometer. To obtain calcium content of the fruit juices (without teeth), 200 μl of the fruit juices were wet ashed and atomized exactly as mentioned above.
Determination of etching pattern: The teeth specimens were prepared for SEM observation by dehydration followed by sputter coating of 20 nm of gold (JEOL,JFC 1100 E-JAPAN) and examined under a scanning electron microscope (JEOL,JSM-840A-JAPAN).Instant photomicrographs were made at X850,X1000 magnifications. Subsequently the X1000 magnification pictures were selected to compare the pattern of etched enamel surfaces.
Statistical analysis: The data obtained were subjected to statistical analysis by using One-Way Repeated Measures ANOVA and Pair wise multiple comparison with Bonferroni correction
RESULTS: RECORDED pH VALUES OF FOUR FRUIT JUICES (Table 1)
Based on the results obtained in Table 1, the pH values of all the juices were less than 5.5, which is the critical pH, below which enamel dissolves. Grape juice recorded the lowest pH of 2.2 among the juices.
TITRATABLE ACIDITY VALUES OF FOUR FRUIT JUICES (Table 2)
For all the four fruit juices tested, the volume of 0.5 ml 1N NaOH needed to reach pH=five and pH= seven were more for immediate defrosted( initial thawed )juices than the juices at room temperature and that defrosted for two hrs. Hence, the immediate defrosted juices had more titratable acidity compared to the juices tested at other temperatures. Among the juices, grape juice had more titratable acidity.
CALCIUM CONCENTRATION IN FOUR SAMPLES OF FRUIT JUICES MEASURED BY ATOMIC ABSORPTION SPECTROPHOTOMETER ( Table 3)
The amount of calcium dissolution in the juices from the teeth were found to be significantly more in the immediate defrosted juices than the room temperature juices and that defrosted for two hours.
Evaluation of SEM Images :
Representative SEM photomicrographs at magnification X1000.
For teeth immersed in juices at room temperature, relatively smooth and slightly etched enamel surfaces were seen.[Figures 1- 4]
Teeth dipped in juices immediately after defrosting, showed maximal erosion. Among these, apple showed eroded surface with enhanced porosity[figure5],a honeycomb enamel prism pattern noted in musumbi similar to that seen in acid etched enamel[figure 6].Teeth in orange and grapes showed more erosion resembling type II acid etching pattern [figures 7,8].
In case of teeth kept in juices two hrs after defrosting, the changes were similar to those seen at room temperature [figures 9-12].
The awareness of the population about health has lead to an increased consumption of natural food products, especially fruits and fruit juices. But fruit juices contain substantial acids(eg: citric acid in citrus fruits, malic acid in apples, tartaric acid in grapes)which have the potential to cause loss of tooth tissue. Various extrinsic and intrinsic factors contribute to dental erosion like diet, medicaments, occupation, sports, GERD, peptic ulcer, uremia etc.But most significant among these are dietary acids .
Dental erosion due to dietary acids are influenced by a variety of factors like pH, titratable acidity, temperature, concentration, frequency, and exposure time.Many host factors also modify erosion, most important is saliva.Reduced salivary flow rate leads to inadequate oral clearance of dietary acids. Consumption of juices at night increases the erosive potential as salivary flow rate is diminished during this period. .Also different studies on salivary flow rate indicated that young children have lower flow rate, resulting in defective oral clearance. .
Intake of dietary acids decreases pH of the oral environment. Gregory-Head B et al suggested that the pH of the oral cavity affected the solubility of the dental tissues. The critical pH at which the chemical dissolution of enamel occurs is accepted to be 5.5±0.3.In this study, all the four fruit juices namely in the order of grape, orange, musumbi and apple showed a pH below five, thus enhancing the enamel dissolution capacity.
Titratable acidity which denotes the hydrogen ion availability has been acknowledged as a true indicator of erosive potential rather than pH value alone.Studies conducted by Touyz etal have shown that fruit juices have a high intrinsic buffering capacity.Modifying the form in which the fruit drinks are taken (sweets or frozen lollies) is expected to increase erosion. Sucking frozen fruit juices could be more erosive than unfrozen fruit juices because of increase in buffering capacity of initial thawed juice..
The acidity increases on freezing, as the physical state of the residual juice changes. When juice is frozen, water alone is solidified to ice without the solute. The solutes accumulates undiluted (concentrated). When juice is defrosted, the initial(concentrated) melt is more acidic with increased buffering capacity Thus sucking on these could cause a greater fall in oral pH and requires more buffering action to normalize the oral environment pH .As the remaining ice melts and dilutes the solution back to pre-freezed state, the buffering capacity diminishes.
The calcium dissolution potential of these fruit juices was analyzed. It was found to be more in the initial thawed fruit juices than juices tested at other temperatures. This is in accordance with the findings of M.Silove  who suggested that sucking frozen fruit juices can lead to more calcium dissolution than unfrozen juices. Also in our study, lowest calcium concentration was found in grape juice which is in accordance with studies stating that the erosive potential of drinks are associated with low calcium concentration
SEM was done to qualitatively estimate enamel surface alterations. Irregular and pitted enamel surface were observed in teeth exposed to frozen juices than at room temperature and after two hours of defrosting. Different etching patterns as described earlier were observed. [28-30]
Similar studies were done on commercially available fruit juices and they are found to be 6-8 times more erosive than homemade juices. [31-33]The significance of this study was that, it was done in pure fruit juices, in frozen and unfrozen forms and multiple parameters were analyzed. The results showed that even pure fruit juices had erosive potential, especially in frozen forms.
This study was done in vitro conditions, the results cannot be completely extrapolated to in vivo process, as interplay of various oral factors like salivary buffering capacity and flow rate to counteract erosion were not included in the study. 
Recommendations: Although fruit juices are good for health, the frequency, method of drinking and the form in which they are consumed are important factors in dental erosion. Since children are frequently involved in the sucking of frozen fruit juice products, pediatric dentists should strongly discourage them from practicing this form of habit. Advise them to avoid fruit juices at bedtime and always rinse mouth with water after consumption of juices. Another point to consider is the use of additives which alter the pH and titratable acidity, thereby reducing the erosive potential of fruit juices. This requires further study.
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