Effect of Consuming Tea with Stevia on Salivary pH - An In Vivo Randomised Controlled Trial

Abstract

Purpose:
To assess the effect of consuming tea with stevia on salivary pH.

Materials and methods:
This randomised controlled trial employed a Latin square design. Twenty-four male students aged 20-23 years were randomly allocated to 4 different groups, 3 experimental with tea sweetened by sucrose, jaggery or stevia, and one unsweetened control. Salivary pH assessments were performed at baseline and 1 min, 20 and 60 min after consumption of the respective tea. One-way ANOVA and repeated measures ANOVA followed by Tukey's post-hoc tests were employed to analyse the data.

Results:
One minute after tea consumption, the salivary pH of the sucrose group significantly decreased compared to the stevia group (p = 0.01). There was a significant difference between baseline mean salivary pH and post-interventional mean salivary pH values at all time intervals in the tea + sucrose, tea + jaggery, and plain tea groups (p < 0.01). One hour after consumption of tea, the salivary pH values reached the baseline pH in stevia and plain tea groups, but it remained lower in the sucrose and jaggery groups.

Conclusion:
The results of the present study, in which the salivary pH values returned to baseline pH 1 h after drinking stevia-sweetened tea, suggest stevia's potential as a non-cariogenic sweetener.

Full text

Vol 15, No 4, 2017 315
Effect of Consuming Tea with Stevia on Salivary pH –
An In Vivo Randomised Controlled Trial
Akhil Pallepatia / Puja C. Yavagalb / D. J. Veereshc
Purpose: To assess the effect of consuming tea with stevia on salivary pH.
Materials and Methods: This randomised controlled trial employed a Latin square design. Twenty-four male students
aged 20–23 years were randomly allocated to 4 different groups, 3 experimental with tea sweetened by sucrose, jag-
gery or stevia, and one unsweetened control. Salivary pH assessments were performed at baseline and 1 min, 20 and
60 min after consumption of the respective tea. One-way ANOVA and repeated measures ANOVA followed by Tukey’s
post-hoc tests were employed to analyse the data.
Results: One minute after tea consumption, the salivary pH of the sucrose group significantly decreased compared
to the stevia group (p = 0.01). There was a significant difference between baseline mean salivary pH and post-in-
terventional mean salivary pH values at all time intervals in the tea + sucrose, tea + jaggery, and plain tea groups
(p < 0.01). One hour after consumption of tea, the salivary pH values reached the baseline pH in stevia and plain
tea groups, but it remained lower in the sucrose and jaggery groups.
Conclusion: The results of the present study, in which the salivary pH values returned to baseline pH 1 h after
drinking stevia-sweetened tea, suggest stevia’s potential as a non-cariogenic sweetener.
Key words: salivary pH, Stevia rebaudiana, sucrose, tea
Oral Health Prev Dent 2017; 15: 315–319. Submitted for publication: 03.02.16; accepted for publication: 03.06.16
doi: 10.3290/j.ohpd.a38572
a Postgraduate, Department of Public Health Dentistry, Bapuji Dental College
and Hospital, Davangere, India. Research hypothesis generation, performed
the experiment, wrote manuscript, contributed substantially to discussion.
b Professor, Department of Public Health Dentistry, Bapuji Dental College and
Hospital, Davangere, India. Idea, Hypothesis generation, experimental design,
contributed substantially to discussion, proof read the manuscript.
c Professor, Department of Public Health Dentistry, Bapuji Dental College and
Hospital, Davangere, India. Proof read the manuscript, supervision during the
study period.
Correspondence: Dr. Akhil Pallepati, Department of Public Health Dentistry,
Bapuji Dental College and Hospital, MCC B’ B Block, Davangere 577004, India.
Tel: +91-991-640-1645; Email: akhil.pmrao@gmail.com
T
ea is the second most widely consumed beverage in the
world after water. Regular intake of tea is associated
with improved antioxidant status in vivo, which may contrib-
ute to lowering the risk of coronary heart disease, stroke
and certain types of cancer.12
Drinking tea is associated with low caries experience in
humans and animal models.
12
The polyphenols in tea have
antioxidant properties, and bind to metal ions, preventing
them from participating in peroxidative reactions.14 Saliva
is one of the most important factors in regulating oral
health, with flow rate and composition changing throughout
life and during disease.
12
However, little is known about the
changes in salivary pH after drinking tea.
India is the largest tea-consuming country worldwide. Tea
is consumed as a hot beverage infused either separately or
with a mixture of milk and sugar.
1
The most widely used tea
sweetener is table sugar, which contains 100% sucrose.
Apart from its cariogenic potential, it is associated with many
nutritional and medical problems. After sucrose, jaggery – a
concentrated product of date, cane juice, or palm sap with-
out separation of the molasses and crystals – is the most
common sugar used to sweeten tea in rural India. It is con-
sidered healthiest sugar and has been in use in alternate
forms of medicine like Ayurveda, Siddha and homeopathy.
4
Recently, a sugar derived from the plant Stevia rebaudi-
ana has emerged on the market as a natural and healthy
alternative to table sugar and artificial sweetener. It is edi-
ble, safe and has proven antimicrobial properties in vitro
and in vivo. The stevia plant, commonly known in Sanskrit
as Madhu Patra, belongs to the Asteraceae family. It is ex-
tensively grown in subtropical regions, has been available
for decades, and is widely used as a sweetener in bever-
ages to mask the bitter taste of certain herbal medicinal
plants in several countries, e.g. Brazil, Japan and Para-
guay.
11
Its dry leaves are powdered in a mill and extracts
are usually obtained in ethanol, methanol, ethyl acetate,
chloroform and hexane, using the cold soaking technique.5
It is approved for use in food by the FSSAI (Food Safety and
Standards Authority of India) and is presently recommended

316 Oral Health & Preventive Dentistry
Pallepati et al
for use in soft-drink concentrates, chewing gums, carbon-
ated water and other non-caloric sweetening products, such
as Truvia, Istevia Zero Calorie Sweetener, Rebiana, Nutras-
weet etc.
Drinking tea with sugar and milk is a common dietary
practice in India. Consuming it with sucrose has negative
impact on oral health; hence, replacing sucrose with an ideal
natural sugar substitute would be beneficial to general and
oral health. However, the literature pertaining to possible
anticariogenic effects of stevia is limited. Thus, the purpose
of this in vivo randomised controlled trial was to assess and
compare the salivary pH changes after consumption of tea
containing sugar, jaggery and stevia. The null hypothesis is
that there is no difference in salivary pH changes after con-
sumption of tea containing sugar, jaggery or stevia.
MATERIALS AND METHODS
The study design is shown in Fig 1. An experimental, in
vivo, crossover study (within subjects) with a Latin square
design was planned. The protocol was reviewed and ap-
proved by the institutional review board of Bapuji Dental
College and Hospital, Davangere (Ref No. BDC/
Exam/165/2015-16). This crossover trial conforms to the
CONSORT guidelines for Consolidated Standards of Report-
ing Trials, 2010.
Sample Size and Subjects
The sample size was calculated using the formula8
n = 2 x [Z1-α/2 + Z1-β]2 x SD2
d2
where Z
1-α/2
at 95% confidence interval is 1.96, Z
1-β
at 80%
power is 0.84, SD = standard deviation, and d = clinical
expected difference.
Type I (α) error was fixed at 0.05 and Type II (β) error at 0.2.
The clinically significant, minimum expected difference be-
tween groups was fixed at 1.3 (pH), based on the study done
by Goodson et al.
7
Overall, the sample size was estimated to
be 20, and anticipating a 20% drop-out rate, a final sample
size of 24 was set, with 6 in each group at each phase.
The sample comprised 24 20- to 23-year-old male under-
graduate students of Bapuji Dental College and Hospital,
Davangere, who fulfilled the eligibility criteria and were ran-
domly selected. Subjects who took medications for sys-
temic diseases that affected their salivary flow rate or were
unable to comply with the study appointment schedules
were excluded. The study was conducted on the clinical
premises of the Department of Public Health Dentistry,
Bapuji Dental College and Hospital, Davangere. Ethical
clearance was obtained from the Institutional Review Board
of Bapuji Dental College and Hospital, Davangere. Voluntary
informed consent was obtained from the study participants
prior to the start of the study, after the research details had
been explained to them through a participant information
form. The investigator, participants and statistician were
blinded.
Group Assignment and Description of Interventions
Random assignment of the participants to four interven-
tional groups was done according to the concealed ran-
domisation method by a separate person not involved in
the study, using software for generating random numbers.
A homogeneous purposive sampling technique was em-
ployed in this study.
The four interventional groups were as follows:
y
Group A: tea with sugar (sucrose, 2 teaspoons [10
grams]/cup)
yGroup B: tea with jaggery (2 teaspoons [10 grams]/cup)
yGroup C: tea with stevia powder (0.5 grams/cup)
yGroup D: tea without sugar (negative control)
One teaspoon of sugar sweetness is equivalent to 1/16
sweetness of stevia powder.10
Interventions were conducted for four days, always in the
evening between 3 and 5 pm. Each day, the tea was pre
-
pared for 24 subjects in a tea pan. 100 grams of tea pow-
der (Brooke Bond Red Label; Jalandhar, Punjab, India) was
added to 1 L of boiling water followed by adding 1 L of milk
(Nandini Toned milk; Davangere, Karnataka, India), and
brought to a boil again. 80-ml portions of the prepared tea
were then served to each subject after adding the respec-
tive sweetener. Each subject was instructed to finish drink-
ing the tea within 7 min.
A washout period of 24 h was implemented. In accor-
dance with the crossover Latin square design, all the par-
ticipants were exposed to all four interventions sequentially
at different phases.
Saliva Collection and pH Measurement
All subjects were given clear instructions to refrain from eat-
ing for one hour before collection of unstimulated saliva.
The subjects were instructed to let saliva pool in the floor of
the mouth for at least 1 min and then expectorate into a
sterile disposable cup.
Salivary pH was checked using dental salivary pH indica-
tor strips (GC; Tokyo, Japan). The pH strip was dipped into
the collected saliva, taken out immediately, and observed
for 30 s for colour change. The change in colour was com-
pared with the reference given by the manufacturer and
readings were entered. Salivary pH was recorded at base
-
line and after tea consumption at 1 min, 20 and 60 min.
The same procedure was followed at all four phases of in-
tervention.
Statistical Analysis
The data obtained were systematically entered in a Micro-
soft Excel sheet. Data analysis was performed using SPSS,
version 20 (SPSS; Chicago, IL, USA). The data were nor-
mally distributed, so parametric tests were employed. Re-
peated measures ANOVA was performed to compare the
means of salivary pH within the group at different time inter-
vals. One-way ANOVA was used to compare the means of
salivary pH between the groups. Tukey’s post-hoc test was
applied, as significant differences were found between and
within the groups at different time intervals.

Vol 15, No 4, 2017 317
Pallepati et al
RESULTS
All results are shown in Table 1. There was a significant dif-
ference in mean salivary pH between all groups 1 min after
tea consumption (p = 0.027). At baseline, there was no
statistical difference in mean salivary pH values between
the intervention groups, allowing valid comparisons be-
tween groups post intervention. At 1 min post consumption,
there was a statistically significant difference (p = 0.027)
between mean salivary pH values of the sucrose and stevia
groups. No significant difference in mean salivary pH values
was observed between intervention groups at different time
intervals. There was a highly significant drop in the salivary
pH at 1 min compared to baseline values after consump-
tion of tea in all groups but stevia, where there was no sig-
nificant difference vs baseline in the drop of mean salivary
pH at 1 min (p > 0.05).
From 1 to 20 min, there was a gradual increase in the
mean salivary pH values in all groups. This was significant
in the sucrose and plain tea group (p < 0.05). From 20 min
to 1 h, there was a significant rise in salivary pH in all the
intervention groups (p < 0.05). At the end of 20 min, the
mean salivary pH values remained significantly lower than
baseline values in the sucrose and jaggery groups
(p < 0.05), whereas in stevia and plain tea groups, there
was no significant difference at 1 h after tea consumption
compared to baseline values. At the end of 1 h, the salivary
pH remained significantly lower in the jaggery group com-
pared to baseline pH, whereas in all the interventional
groups, the salivary pH returned to baseline pH.
There were no adverse outcomes or unintended effects
reported during or after the study.
DISCUSSION
The present study assessed and compared the baseline
salivary pH changes after consumption of tea added with
sugar, jaggery, or stevia and plain tea at 1 min, 20 min and
1 h. To the authors’ best knowledge, this is the first study
to compare salivary pH changes between tea with sugar,
jaggery and stevia.
A statistically significant drop in the salivary pH values
was observed in the sucrose group compared to the stevia
1 min after tea consumption. This is in accordance with
Goodson et al,7 where a statistically significant increase in
plaque pH was found in the stevia oral rinse group com-
pared with sucrose oral rinse group.
In the present study, salivary pH decreased in all groups
at 1 min after tea consumption, followed by an increase in
salivary pH at 20 min and 1 h. These findings agree with
those of Kumar et al.9 In that study, the participants were
given 10 ml (20%) of sucrose solution, after which the sali-
vary pH gradually decreased to a certain point and then in-
creased gradually toward the baseline level, but was not
reached even 60 min after sucrose exposure. They sug-
gested that although organisms in the saliva may produce
some acid, the acids arising in the dental plaque or from
bacteria colonising the tongue and other soft tissues might
appear in the saliva, giving rise to a ‘salivary Stephan
curve’.9
In the present study, the mean salivary pH dropped from
baseline to 1 min in the jaggery group, then rose slightly
after 20 min, but did not return to the mean baseline sali-
vary pH value even at the end of 1 h. The pattern of salivary
pH changes in the jaggery group was similar to that of the
sucrose group, as jaggery largely consists of sucrose.
Random allocation
(lottery method)
24-h
washout
Day 1
24-h
washout
Day 2
24-h
washout
Day 3
24-h
washout
Day 4
Ethical clearance obtained from Institutional Review Board of
Bapuji Dental College and Hospital, Davangere
Study plan and hypothesis formulation
Excluded – did not meet
inclusion criteria, declined
to participate
Schematic Representation of Methodology
Male undergraduate students aged 20–23 years of Bapuji
Dental College and Hospital, Davangere city were invited to
participate in the study
24 male undergraduate students who fulfilled the eligibility
criteria comprised the study sample
Tea
(sugar)
Tea
(jaggery)
Tea
(stevia)
Tea
(plain)
Tea
(sugar)
Tea
(jaggery)
Tea
(stevia)
Tea
(plain)
Tea
(sugar)
Tea
(jaggery)
Tea
(stevia)
Tea
(plain)
Tea
(sugar)
Tea
(jaggery)
Tea
(stevia)
Tea
(plain)
Group
A
Group
B
Group
C
Group
D
salivary pH assessments at baseline 1, 20 and 60 minutes
Compilation, analysis and reporting results
Fig 1 Study design.

318 Oral Health & Preventive Dentistry
Pallepati et al
A pilot study was conducted among five subjects who
were not involved in the study to standardise the duration
of tea consumption. They were given 80 ml of tea and the
time taken to consume tea was recorded using a digital
clock. The mean time was 7 min. Hence, 7 min was used in
the present study.
This study had a small sample. Further studies should
be done involving larger sample sizes. Although plaque pH
changes are concurrent with salivary pH changes, it would
have been better if plaque pH had been assessed, as the
process of caries starts at the plaque/tooth interface.
CONCLUSION
The results of the present study, in which the salivary pH
values returned to baseline pH 1 h after drinking stevia-
sweetened tea, suggest stevia’s potential as a complement
to oral care in the form of toothpastes, mouthwashes, and
chewing gums, as well as a non-cariogenic sweetener. It
could thus act as an excellent sugar substitute, replacing
the current artificial sweeteners and refined sugars, thereby
tackling lifestyle-associated diseases such as dental caries,
obesity etc.
ACKNOWLEDGEMENT
We would like to thank Dr. Umesh Wadgave for statistical analyses
and Dr. Varghese Suresh for generating the random number sequence.
These results could not be compared with other studies, as
a literature search revealed no similar studies.
The drop in salivary pH in the stevia group is similar to
the findings of Giongo et al6 and Goodson et al.7 In the
study by Goodson et al,
7
rinses with stevia demonstrated a
mean ± SD pH of 6.92 ± 0.08, which was significantly
higher (p < 0.0001) than that of the sucrose rinse
(5.62 ± 0.13). Both in vitro and in vivo studies have shown
that stevia extracts have antibacterial activity on Streptococ-
cus mutans, Streptococcus sobrinus and Lactobacillus aci
-
dophilus, organisms that are closely related to the genesis
and development of caries. Hence, it could be a potential
sugar substitute to help prevent tooth decay.3
Stephan’s curve describes the changes in dental plaque
pH in response to a carbohydrate challenge over a period of
time. Characteristically, the curve reveals a rapid drop in
plaque pH that is attained after consumption of sugar. It
normally takes at least 20 min for the plaque pH to reach
its resting value.2 In some studies, the salivary pH did not
return to its original baseline level even at the end of 1 h
and remained below the baseline mean salivary pH.9,13
Thus, in this study, salivary pH was checked at 1 min, 20
min and 1 h. However, the washout period of one day was
arbitrarily fixed and used, as the exact time required for the
salivary pH to return to the normal baseline after jaggery- or
stevia-sweetened tea consumption was not known.
Unstimulated whole saliva reflects basal salivary flow
rate, is present for about 14 h a day, and is the secretion
that provides protection to oral tissues.5 Because stimu-
lated saliva cause alterations in salivary pH, unstimulated
saliva collection was preferred. Since there were four inter-
vention groups, in order to minimise and control for be-
tween-subject variations, a Latin square design was se-
lected.
The present study was conducted on male students
aged 20 to 23 years. It was a convenience sample of male
undergraduate students of the same college, residing in the
same boys’ hostel and consuming food from a common
hostel kitchen. Thus, their diets were similar. This mini-
mised the effects of diet, age and sex on variations in sali-
vary pH.
Table 1 Comparison of salivary pH at baseline and different time intervals between intervention groups
Interventional groups Mean (SD) salivary pH
at baseline
Mean (SD) salivary pH
at 1 min
Mean (SD) salivary pH
at 20 min
Mean (SD) salivary pH
at 1 h
Tea with added sugar
(group A)
6.90 (±0.25)ab 6.49 (±0.22)Aacd 6.74 (±0.22)bce 6.86 (±0.23)de
Tea with added jagger y
(group B)
6.94 (±0.21)abc 6.63 (±0.33)a6.67 (±0.26)bd 6.8 (±0.27)cd
Tea with added stevia
(group C)
6.85 (±0.29) 6.75 (±0.35)A6.75 (±0.26)a6.85 (±0.30)a
Plain tea 6.83 (±0.24)a6.62 (±0.22)abc 6.80 (±0.22)bd 6.88 (±0.20)cd
Superscript capital letters indicate significant differences between groups and lowercase letters indicate significant differences within groups.

Vol 15, No 4, 2017 319
Pallepati et al
REFERENCES
1. Ajithkrishnan CG, Thanveer K, Singh RP. An in-vivo evaluation of the ef-
fect of fennel seeds chewing on Sslivary pH. J Oral Health Comm Dent
2014;8:79–81.
2. Azrak B, Willershausen B, Meyer N, Callaway A. Course of changes in sal-
ivary pH-values after intake of different beverages in young children. Oral
Health Prev Dent 2008;6:159–164.
3. Contreras S. Anticariogenic properties and effects on periodontal struc-
tures of Stevia rebaudiana Bertoni. Narrative review. J Oral Res 2013;
2:158–166.
4. Diwedi AK. An empirical study on gur (jaggery) industry. Indian Institute of
Management Working Paper, No. 2010-12-03: Available at http://ssrn.
com/abstract=1783403 or http://dx.doi.org/10.2139/ssrn.1783403
5. Fenoll-Palomares C, Muñoz Montagud JV, Sanchez V, Herreros B, Hernán-
dez V, Mínguez M, Benages A. Unstimulated salivary flow rate, pH and
buffer capacity of saliva in healthy volunteers. Rev Esp Enferm Dig
2004;96:773–783.
6. Giongo FC, Mua B, Parolo CC, Carlén A, Maltz M. Effects of lactose-con-
taining stevioside sweeteners on dental biofilm acidogenicity. Braz Oral
Res 2014;28:1–6.
7. Goodson J, Cugini M, Floros C, Roberts C, Boileau A, Bell M. Effect of a
Truvia™ Rebiana on plaque pH. Abstract presented at the International
Association for Dental Research General Sessions, Barcelona, 14–17
July, 2010.
8. Jekel JF. Epidemiology, biostatistics, and preventive medicine, ed 3. Phil-
adelphia: Saunders/Elsevier, 2007:197–212.
9. Kumar A, Hedge R, Dixit U. Role of plaque in the clearance of salivary su-
crose and its influence on salivary pH. J Indian Soc Pedod Prev Dent
2011;29:310–314.
10. Sahelian R, Gates D. Stevia conversion chart. Available at http://www.
stevia.net/conversion.html, Accessed on 20 Jan 2015.
11. Sattigeri BM. Stevia (Madhu Patra) – A natural low calorie sweetener with
more promising benefits. Int J Phytother Res 2012;2:20–25.
12. Srinidhi PB, Basha S, Naveen Kumar P, Prashant GM, Sushanth VH, Im-
ranulla M. Effect of two different commercially available tea products on
salivary pH: A randomized double blinded concurrent parallel study. Dent
Med Res 2014;2:39–42.
13. Subramaniam P, Kumar K. Change in salivary pH following use of homeo-
pathicmedicines: A preliminary study. Eur J Gen Dent 2013; 2:31–36.
14. Yang CS, Landau JM. Effects of tea consumption on nutrition and health.
J Nutr 2000;130:2409–2412.