Comparative Evaluation of Erosive Potential of Various Frozen and Unfrozen Fruit Juices on Primary Teeth Enamel: An In Vitro Study.

BACKGROUND: Changing lifestyle pattern and food habits has a deteriorating effect on dental tissues. Dental erosion is a pathological wear of hard tissues of teeth with increased consumption of acidic and carbonated drinks. Susceptibility to erosion in primary dentition is more compared to permanent dentition due to softer and disordered crystal structure of enamel.
OBJECTIVES: The main aim of the study was to determine and compare the erosive potential of different fruit juices in frozen/unfrozen forms on primary teeth by studying the calcium dissolution.
MATERIALS AND METHODS: pH of four different juices (pure) - apple, orange, citrus limetta (musumbi) and grapes were determined using a digital pH meter. The titratable acidity of these in frozen and unfrozen forms were determined by adding 0.2 ml of 1M NaOH to these to raise to pH=5.5(critical pH) and pH =7(neutral pH). Forty eight caries free deciduous anterior teeth specimens were prepared to study the calcium dissolution by atomic absorption spectrophotometer. The results were analysed for statistical significance using One-way Repeated Measures ANOVA and pair wise multiple comparison with Bonferroni correction.
RESULTS: Total titratable acidity and calcium dissolution were found to be significantly more in the initial thawed fruit juices.
CONCLUSION: Frozen fruit juices had more buffering capacity and erosive potential than unfrozen forms. The study concluded that sucking on frozen fruit juices is more damaging to teeth than unfrozen forms because more of erosion is expected to occur in a frozen state.

INTRODUCTION

Healthy eating habits are one of the most important factors for longevity in life.[1] Children consume fruits in the form of fruit juices rather than cut fruits. There is an ignorance of damaging effects of acid erosion due to fruit juices. Research on animals showed that these fruit juices were 10 times more destructive to the teeth than the whole fruit.[2] Most fruit juices have a low pH and acids that can decalcify the teeth.[3] Frequent consumption of these fruit drinks is directly related to dental morbidity, especially erosion.[4]

Dental erosion is defined as “loss of dental hard tissue by a chemical process that does not involve the influence of bacteria.”[5] Etiopathogenesis can be varied (intrinsic and extrinsic factors) and among them the most important are dietary acids.[6] Erosive potential depends on low pH and buffering capacity of juices. The juices having low pH are strongly buffered with a potential erosive capacity because the acid content of fruit juices influences the buffering capacity. Sucking on frozen fruit juices (popsicles or juice on a stick) has a greater risk for erosion because they are sucked slowly and requires longer time to neutralize.[7] Sucking of the melted juices from the frozen product is a pleasurable experience along with the increase in the initial acidity and buffering capacity.[8] 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 thinner, less mineralized, and immature enamel surface.[9] Various studies have established the effect of fruit juices and teeth erosion, but studies on the effects of fruit juices in frozen and unfrozen forms in dissolving hydroxyapatite of the primary teeth are very rare and have not been researched much. This study aimed to evaluate the pH and titratable acidity of four commonly consumed fruit juices, and their effect on teeth was determined by the total amount of calcium dissolved.

MATERIALS AND METHODS

Two and half liters juices of each of different fruits (pure), namely, apple, orange, grape, and citrus limetta or sweet lime (mosambi) were prepared with no added sugars or preservatives. These were allowed to equilibrate to room temperature. Forty-eight freshly extracted caries and defect-free deciduous anterior teeth were selected, cleaned, and kept in 0.1% thymol solution until use (up to 30 days). Each specimen surface was coated with acid-resistant nail varnish with the exception of a window on the enamel of the labial surface of approximately 2 mm × 2 mm in diameter.

Two and half liters of apple juice was shaken for 15 seconds, and this was divided into four samples of 600 mL each. From the first sample, 100 mL was separated. The pH, titratable acidity (it is a measure of amount of acid present in a solution), and calcium concentration of juice were measured. Prepared tooth specimen was exposed in it for 2 hours (for assessment of calcium at room temperature). Remaining juice was taken in a bottle, sealed, and placed in the deep freezer (−20°C) for 24 hours. The bottle was taken and allowed to defrost. From this, 100 mL was separated for a similar procedure as stated earlier (for assessment of calcium immediately after defrosting). The remaining juice was defrosted for 2 hours. From this, 100 mL was separated for a similar procedure as stated earlier (for assessment of calcium concentration 2 hours after complete defrosting). This was repeated for the remaining three samples (600 mL each) of apple juice. The same procedure was carried out for the other three juices, namely, sweet lime, orange, and grape and the values were recorded.

The initial pH of each fruit juice was measured using a pH meter (digital pH meter model EQ-612). Fruit juices (100 mL) at room temperature, at initial defrosting, and 2 hours after defrosting were placed in a beaker and stirred using a nonheating magnetic stirrer until a stable reading was obtained. After determining the pH, 100 mL of fruit juices were titrated with 1 M NaOH added in 0.2 mL increments until pH reached 5.5 (critical pH) and 7 (neutral pH). This was carried out using a nonheating magnetic stirrer till a stable pH was reached after each increment of NaOH (base) to a chosen end point to measure total titratable acidity.

The teeth exposed to 100 mL of juices were carefully lifted out and the juices were centrifuged at 3500 rpm for 5 minutes. With the help of micropipette (Superfit), 100 mL of each of these samples of fruit juices were pipetted into the polypropylene tubes and then 40 μL of distilled water was rinsed into it. Then concentrated nitric acid (120 μL) was added, the lid of the polypropylene tube was sealed, and the contents of the tube were wet washed. After cooling, 50 μL of 1 mol/L KCl solution (an ionization suppressant) and 680 μL 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 (PerkinElmer, United States). To obtain calcium content of the fruit juices (without teeth), 100 mL of the fruit juices were wet and atomized exactly as mentioned earlier.

RESULTS

Based on the results obtained pH values of all the fruit juices less than 5.5 (critical pH) showed enamel dissolution. Grape juice recorded the lowest pH of 2–3 (pH at normal room temperature 2.56) among the juices [Table 1].

Table 1

Recorded pH values of four fruit juices

Recorded pH	Orange	Grape	Sweet lime	Apple
At room temperature (30°C)	3.52	2.56	3.65	4.50
At initial defrosting	3.12	2.10	3.29	4.19
After defrosting for 2 hours	3.51	2.57	3.65	4.49

After 2 hours, among all the four fruit juices tested, 0.2 mL 1M NaOH was needed to raise the pH to 5.5 and 7 in immediate defrosted (initial thawed) juices than the juices at room temperature and defrosted type. Hence, the immediate defrosted juices had greater titratable acidity compared to the juices at other temperatures [Table 2].

Table 2

Titratable acidity values of four fruit juices

Fruit juices	Titratable acidity (amount of NaOH required) at room temperature up to pH 5.5 and 7	Titratable acidity (amount of NaOH required) at initial defrosting up to pH 5.5 and 7	Titratable acidity (amount of NaOH required) after 2 hours of defrosting up to pH 5.5 and 7
Orange	5.2 mL	6.3 mL	5.1 mL
	6.2 mL	7.7 mL	6.3 mL
Grape	7.2 mL	8.6 mL	7.1 mL
	8.3 mL	9.7 mL	8.2 mL
Sweet lime	4.8 mL	5.9 mL	4.9 mL
	6.0 mL	7.3 mL	6.0 mL
Apple	3.2 mL	4.6 mL	3.0 mL
	4.3 mL	5.2 mL	4.4 mL

The calcium concentration of juices recorded for all the samples without teeth at room temperature and after defrosting for 2 hours did not vary, but at initial defrosting, values were slightly increased with no significance. The amount of calcium dissolution in juices from the teeth were found to be significantly more in the immediate defrosted juices than the room temperature and defrosted for 2 hours [Table 3].

Table 3

Calcium concentration in fruit juices

Fruit juices	Calcium concentration in fruit juices without teeth (mg/100 mL) (A)	Calcium concentration in fruit juices with teeth (mg/100 mL)
		At room temperature (B)	Initial defrosted juices (C)	After defrosting for 2 hours (D)
Apple
Sample 1	21.25	23.05	24.88	23.3
Sample 2	19.44	21.24	22.6	21.36
Sample 3	18.05	19.54	21.32	20.0
Sample 4	18.01	19.71	21.01	19.96
Sweet lime
Sample 1	18.32	20.41	22.01	20.53
Sample 2	19.01	21.01	22.3	21.02
Sample 3	18.46	20.55	22.01	20.65
Sample 4	17.96	20.01	21.40	19.96
Orange
Sample 1	22.35	24.95	26.2	24.55
Sample 2	20.22	22.55	24.2	22.3
Sample 3	21.26	23.52	25.0	23.7
Sample 4	22.00	24.5	25.70	24.2
Grape
Sample 1	13.95	16.75	17.96	16.85
Sample 2	12.75	15.75	16.96	15.4
Sample 3	12.26	15.3	16.5	15.35
Sample 4	13.0	16.0	17.20	15.9

Statistical analysis was carried out using one-way repeated measures analysis of variance and pairwise multiple comparison with Bonferroni correction. There was significant statistical difference in calcium concentration between fruit juices without teeth (A), with teeth at room temperature (B), immediately after defrosting (C), and after defrosting for 2 hours (D). Comparison of calcium concentration in apple, orange, sweet lime, and grape juice showed that there was no statistical difference between teeth at room temperature (B) and after defrosting for 2 hours (D), whereas differences between other groups were significant except for grape juice that showed no relevant statistical difference between immediately defrosted juices (C) and after defrosting for 2 hours (D) [Table 4]. There was significant statistical difference in calcium concentration between fruit juices without teeth (A), with teeth at room temperature (B), immediately after defrosting (C), and after defrosting for 2 hours (D.

Table 4

Comparison of calcium concentration in fruit juices recorded at various temperatures

Calcium	Mean	SD	N	F	Pair	Mean Difference	Pair	Mean difference
Without teeth (A)	19.2	1.5	4	354.21	A and B	1.74	B and C	1.51
With teeth at room temperature (B)	20.9	1.6	4		A and C	3.25	B and D	0.24
Immediately after defrosting (C)	22.4	1.7	4		A and D	1.98	C and D	1.27
After defrosting for 2 hours (D)	21.2	1.6	4

Comparison of calcium concentration in apple, orange, sweet lime, and grape juice showed that there was no statistical difference between teeth at room temperature (B) and after defrosting for 2 hours (D), whereas differences between other groups were significant except for grape juice that showed no relevant statistical difference between immediately defrosted juices (C) and after defrosting for 2 hours (D).

DISCUSSION

Awareness of the population about health has led to an increased consumption of natural food products, especially fruits and fruit juices. But fruit juices contain substantial acids (citric acid in citrus fruits), which have the potential to cause loss of tooth tissue.[101112] Various extrinsic and intrinsic factors such as diet, medicaments, and occupation contribute to dental erosion.[131415]

Dental erosion due to dietary acids are influenced by a variety of factors such as pH, titratable acidity, temperature, concentration, frequency, and exposure time.[16] Many host factors also modify erosion, most important is saliva.[17] 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.[18] The quantity and quality of salivary flow are important in the etiology of dental erosion. Also, different studies on salivary flow rate indicated that young children have lower flow rate, resulting in defective oral clearance and thus further increasing susceptibility to teeth erosion.[19]

Intake of dietary acids decreases the pH of the oral environment. Gregory-Head et al.[20] 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 ± 0.34.[21] In this study, all the four fruit juices, namely, in the order of grape, orange, sweet lime, and apple showed a pH below 5.5, thus enhancing the enamel dissolution capacity.[22]

Titratable acidity, which denotes the hydrogen ion availability, has been acknowledged as a true indicator of erosive potential rather than pH value alone.[23] Studies conducted by Touyz et al. 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.[2425] Sucking frozen fruit juices could be more erosive than unfrozen fruit juices because of increase in buffering capacity of initial thawed juice.[26] 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 solute 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 prefrozen state, the buffering capacity diminishes.

The calcium dissolution potential 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 Silove who suggested that sucking frozen fruit juices can lead to more calcium dissolution than unfrozen juices. Similar studies were conducted on commercially available fruit juices and they were found to be six to eight times more erosive than homemade juices.[272829] The significance of this study was that it was carried out 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 even in frozen forms.

Because this study was conducted under in vitro conditions, the results cannot be completely extrapolated to in vivo process as interplay of various oral factors such as salivary buffering capacity and flow rate to counteract erosion were not included in the study. This study should be interpreted only as a prediction of relative erosive potential of a dietary substance as it cannot fully reproduce the clinical situation.

Research by Edwards et al.[30] concluded that there was significant decrease in oral pH with increased consumption of fruit juices and fruit-based carbonated beverages with decrease in their buffering capacities. Owing to the era of modernization, there has been an increase in the prevalence of erosion in the intake of cola soft drinks. Research have shown a significantly decreased surface hardness (with P < 0.05) not only on enamel but also on dentin and tooth-colored restorative materials. Erosion caused by exposure to orange juices was equivalently significant with decreased surface hardness of enamel (P < 0.05). In contrary to the this study, Lussi et al.[6] suggested that erosive potential of primary teeth was seen to be less susceptible compared to permanent teeth under the influence of beverages such as carbonated beverages and yogurt. In the recent studies, orange juice showed a much higher erosive potential when compared with the apple juice on both enamel and tooth-colored restorative materials.

CONCLUSION

Although fruit juices are good for health, our findings demonstrated that the frequency, method of drinking, and the form in which they are consumed are important factors in dental erosion. Because children are frequently involved in the sucking of frozen fruit juice products, pediatric dentists have a crucial role in educating children about the detrimental effects of these fruit juices and carbonated drinks. “Precaution is always better than cure” awareness regarding the ways to maintain proper oral health is of at most priority in the present era and future. One should be aware of both positive and detrimental effects of fruit juices.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.