Changes in Dental Plaque pH and Blood Glucose Levels

Following the Intake of Selected Drinks and Food

Jouhrah Hussain Khan

[BDS, Pakistan]

A thesis submitted in fulfilment of the requirement for the degree of

Master of Philosophy

Faculty of Medicine and Health

The University of Sydney

March 2020

ii

Dedication

First of all, I would like to dedicate this thesis to my parents: without their prayers, this would

have not been possible.

I would especially like to thank my mother—without her support and love, I would not be the

person I am today.

An exceptional thank you goes to my dearest husband, Dr Hamza Masood, who has been my

strength and support all this time. Thank you for believing in me and always motivating me.

My deepest and most special gratitude is reserved for you. Thank you for always being with

me through all the thick and thin of life.

iii

Statement of Originality

I hereby declare, to the best of my knowledge, that this thesis is my original work except where

references have been made. It has been submitted to the faculty of Medicine and Health, The

University of Sydney, School of Dentistry, Australia. I verify that this thesis has not been

published in any other institution or university.

__________________ Date: ____ / ____ / ____

Jouhrah Hussain Khan

iv

Acknowledgements

I would like to acknowledge a number of people—without their help and guidance, this thesis

would have not been possible.

Professor Joerg Eberhard: I would like to express my deepest gratitude to my supervisor. I

cannot thank him enough for his constant support and guidance. His encouragement gave me

the strength to complete my research and finish my thesis. I was truly blessed to have an

amazing mentor like him, who motivated me at every step of my master’s degree. I will always

treasure this journey.

Professor Jennie Brand-Miller: I would like to thank Professor Brand-Miller for her

continuous support. It has been a privilege and an honour to learn under such a senior and

revered individual in the field.

Dr Fiona Atkinson: I really appreciate her splendid guidance and suggestions she made

throughout this journey. The recommendations she gave really helped me improve my thesis.

Elif Inan Eroglu: Thank you to Elif for her unconditional support and help with finalising my

results section. She is a true example of the expression ‘a friend in need is a friend indeed’.

I would also like to thank the research staff at the Sydney University Glycaemic Index Research

Service for conducting the capillary glucose collection and analysis.

This thesis has been edited by the Elite Editing in accordance with standard D and E of the

Australian standards for Editing Practice.

v

Abstract

Background: Commonly consumed foods have a significant effect on our postprandial blood

glucose levels and dental plaque pH. Growing awareness of the detrimental effect of high-

glycaemic index (GI) foods means that people have become more conscious about their daily

dietary intake. This study was designed to investigate the effect of high-GI foods on

postprandial blood glucose levels and dental plaque pH.

Aim: The study aimed to evaluate the effect of some common carbohydrate food products on

dental plaque pH and their postprandial glucose response.

Material and Methods: Various food products were used in this study: white bread, chickpeas,

pasta, mashed potatoes, breakfast cereals and rice. A glucose drink of 25 g/250 mL was the

standard against which all the other food products were measured. Participants refrained from

any oral hygiene regime the night before and the next morning. Over a period of 2 hours, dental

plaque (0, 12, 22 and 62 minutes) and blood (‒5, 0, 15, 30, 45, 60, 90 and 120 minutes) samples

were collected before and after consumption of the test foods. A micro pH meter was used to

measure the acidity of plaque samples, and an automatic clinical chemistry auto analyser was

used for plasma glucose concentration measurement.

Results: In sub-study 1, a significant difference was found among the glucose 25 g and 50 g

solutions (p = 0.001). At all times, the glucose 25 g solutions displayed a smaller pH drop

compared with the glucose 50 g solutions. In Sub-study 2, for plaque pH, the maximum area

under the curve (AUC) was observed for white bread which was 62.9 ± 19.7 mmol/L, followed

by the glucose 25 g solution, mashed potatoes, pasta and chickpeas. There was a significant

difference between white bread and chickpeas (P = 0.004). For plasma glucose in sub-study 2,

the maximum incremental area under the curve (iAUC) was seen for the glucose 25 g solution

vi

(135.0 ± 52.3 mmol/L), followed by mashed potatoes, white bread, pasta and chickpeas. The

same pattern was observed for the GI values, with mashed potatoes showing the highest GI of

95.9, followed by white bread (77), pasta (46.8) and chickpeas (35.4). In Sub-study 3, Rice B

showed the maximum AUC (59.2 ± 22.2 mmol/L) and the maximum iAUC (110 ± 31.6

mmol/L) for plaque pH and plasma glucose, respectively. Rice B had the highest GI value at

84.2, followed by Bread A (82.3), Cereal A (76.8), Bread B (60.4), Rice A (55.5) and Cereal

B (43.1). In sub-studies 2 and 3, a linear correlation was seen between the AUC of plaque pH

and the GI.

Conclusion: This is the first study that simultaneously measured dental plaque pH and

postprandial blood glucose levels, and it has shown that foods with a high GI, which produces

higher postprandial plasma glucose levels, also produces greater plaque pH drops compared

with low-GI food. The outcome of this study will benefit health care providers by

demonstrating that food high in GI has a greater capacity to initiate dental decay than food with

a low GI.

Keywords: GI (glycaemic index), pH, dental plaque, glucose, food.

vii

Contents

Dedication ................................................................................................................................. ii Statement of Originality ......................................................................................................... iii Acknowledgements ................................................................................................................. iv

Abstract .................................................................................................................................... v

Contents .................................................................................................................................. vii List of Figures ......................................................................................................................... ix

List of Tables ............................................................................................................................ x

Chapter 1: Introduction .......................................................................................................... 1 1.1 Background ...................................................................................................................... 1

1.1.1 Dental caries and oral plaque pH .............................................................................. 1 1.1.2 GI .............................................................................................................................. 2 1.1.3 Potential relationship between plaque pH and GI .................................................... 2

1.2 Objective of Study ........................................................................................................... 3

Chapter 2: Literature Review ................................................................................................ 5 2.1 Caries ............................................................................................................................... 5

2.1.1 Definition .................................................................................................................. 5 2.1.2 Aetiology .................................................................................................................. 5 2.1.3 Signs and symptoms ................................................................................................. 6 2.1.4 Prevention ................................................................................................................. 7 2.1.5 Epidemiology ........................................................................................................... 8 2.1.6 Caries in Rodents ...................................................................................................... 9

2.2 Plaque .............................................................................................................................. 9 2.2.1 Definition .................................................................................................................. 9 2.2.2 Plaque formation ...................................................................................................... 9 2.2.3 Prevention ............................................................................................................... 10

2.3 pH .................................................................................................................................. 10 2.3.1 Definition ................................................................................................................ 10 2.3.2 Measuring plaque pH ............................................................................................. 11

2.4 GI ................................................................................................................................... 13 2.4.1 Definition ................................................................................................................ 13 2.4.2 Classification .......................................................................................................... 13 2.4.3 Measuring the GI .................................................................................................... 13

2.5 Buffering Capacity ........................................................................................................ 14 2.5.1 Definition ................................................................................................................ 14 2.5.2 Types of salivary buffering systems ....................................................................... 14 2.5.3 Measuring buffering capacity ................................................................................. 15

2.6 The Effect of Different Foods on Plaque pH ................................................................. 15 2.6.1 Effect of different breads on plaque pH ................................................................. 15 2.6.2 Effect of different drinks on plaque pH .................................................................. 17 2.6.3 Effect of different snacks on plaque pH ................................................................. 19 2.6.4 Effect of different yoghurts on plaque pH .............................................................. 20 2.6.5 Effect of different starchy food and fruits on plaque pH ........................................ 22

2.7 Hypothesis ..................................................................................................................... 24

viii

Chapter 3: Materials and Methods ...................................................................................... 25 3.1 Ethics Approval ............................................................................................................. 25 3.2 Subject Selection ........................................................................................................... 25 3.3 Measurement Technique ................................................................................................ 26

3.3.1 Measuring plaque pH ............................................................................................. 26 3.3.2 Measuring blood glucose ........................................................................................ 26 3.3.3 Measuring buffering capacity ................................................................................. 27

3.4 Test Food ....................................................................................................................... 28 3.4.1 Sub-study 1: Impact of carbohydrate amount on plaque pH .................................. 28 3.4.2 Sub-study 2: Impact of carbohydrate (GI) on plaque pH ....................................... 29 3.4.3 Sub-study 3: Impact of GI in a food type on plaque pH ........................................ 30

3.5 Statistical Analysis ........................................................................................................ 31

Chapter 4: Results ................................................................................................................. 32 4.1 Sub-study 1: Impact of Carbohydrate Amount on Plaque pH ....................................... 32 4.2 Sub-study 2: Impact of Carbohydrate (GI) on Plaque pH ............................................. 33

4.2.1 pH data .................................................................................................................... 33 4.2.2 Glucose data ........................................................................................................... 36 4.2.3 Correlations between plaque pH and plasma glucose concentration ..................... 40

4.3 Sub-study 3: Impact of GI in a Food Type on Plaque pH ............................................. 41 4.3.1 pH data .................................................................................................................... 41 4.3.2 Glucose data ........................................................................................................... 44 4.3.3 Correlation between plaque pH and GI .................................................................. 47

4.4 Post-hoc power analysis ................................................................................................ 48 4.5 Buffering Capacity ........................................................................................................ 48

Chapter 5: Discussion ............................................................................................................ 50

Chapter 6: Conclusion .......................................................................................................... 57

References .............................................................................................................................. 58

ix

List of Figures

Figure 1: Stephan Curve—pH curve of dental plaque after glucose rinse .............................. 11

Figure 2: Three GI classifications and examples of common carbohydrate foods ................. 13

Figure 3: Ex vivo dental plaque pH in healthy subjects (n = 8) after consumption of 25 g

and 50 g glucose solution (250 mL) ....................................................................... 33

Figure 4: Comparison in plaque pH drop after the consumption of four test foods and

control solution ....................................................................................................... 35

Figure 5: Comparison of all test foods (chickpeas, pasta, mashed potatoes and white

bread) with the standard glucose drink (25 g/250 mL) at four different time

points to determine their effect on plaque pH ........................................................ 36

Figure 6: Plasma glucose response curve for all test foods and the control glucose

solution ................................................................................................................... 39

Figure 7: All the plasma glucose response curves for the test foods (chickpeas, pasta,

mashed potatoes and white bread) compared with the standard glucose drink ...... 40

Figure 8: Correlation between the GI and AUC of plaque pH for all test foods/drinks

included in Sub-study 2 .......................................................................................... 41

Figure 9: Absolute plaque pH values after consumption of 25 g available carbohydrate

portions of the test foods and control glucose solution .......................................... 43

Figure 10: Comparison of all test foods (Bread A, Bread B, Cereal A, Cereal B, Rice A

and Rice B) with the standard glucose drink at four different time points to

determine their effect on plaque pH AUC .............................................................. 44

Figure 11: Plasma glucose response curve for all three test food pairs and the control

glucose solution ...................................................................................................... 46

Figure 12: Comparison of all test food pairs (Rice A, Rice B, Cereal A, Cereal B, Bread

A and Bread B) with the standard glucose drink (25 g available carbohydrates)

at all measured time points to determine their effect on blood glucose levels ....... 47

Figure 13: The relationship between the GI and the AUC of plaque pH for all six test

food pairs included in Sub-study 3 ......................................................................... 48

x

List of Tables

Table 1: Summary of papers on the effect of different breads on human dental plaque ......... 16

Table 2: Summary of papers on the effect of different drinks on human dental plaque ......... 18

Table 3: Summary of papers regarding the effect of different snacks on human dental

plaque ..................................................................................................................... 20

Table 4: Summary of papers on the effect of different yoghurts on human dental plaque ..... 21

Table 5: Summary of papers on the effect of starchy food and fruits on human dental

plaque pH ................................................................................................................ 23

Table 6: Nutritional information of the glucose used .............................................................. 28

Table 7: Nutritional information of test foods used in Sub-study 2 ........................................ 29

Table 8: Nutritional information of test foods used in Sub-study 3 ........................................ 30

Table 9: Mean ± SD absolute plaque pH values following consumption of 25 g and 50 g

glucose solution (250 mL) ...................................................................................... 32

Table 10: Mean ± SD plaque pH values of the test foods/drinks at four different time

points with the corresponding AUC and GI values ................................................ 34

Table 11: Comparison of plaque pH results produced by all the test foods/drinks to

determine their p-values ......................................................................................... 35

Table 12: Plasma glucose concentration (mmol/L) and overall glyceamic response,

quantitified as iAUC (mmol/L.min) ....................................................................... 37

Table 13: Comparison of all test foods/drinks (glucose, white bread, pasta, chickpeas and

mashed potatoes) with their corresponding p-values ............................................. 38

Table 14: Mean ± SD of plaque pH values of the tested foods at four different time points

with their corresponding AUC values and GI values ............................................. 42

Table 15: P-values for the difference in plaque pH across the three pairs of test foods ......... 43

Table 16: Mean plasma glucose concentration ± SD and overall glyceamic response

(iAUC) for all the six paired test foods .................................................................. 45

Table 17: Comparison of the paired test foods (Bread A/ Bread B, Cereal A/ Cereal B

and Rice A/ Rice B) and their P-values .................................................................. 45

Table 18: Salivary buffering capacity of all the subjects who participated in sub-studies 2

and 3 ....................................................................................................................... 49

1

Chapter 1: Introduction

1.1 Background

1.1.1 Dental caries and oral plaque pH

Dental caries, generally known as tooth decay, is the most common non-communicable and

widespread disease worldwide (1), (2). Dental caries is caused by acid-producing bacteria in

the dental plaque as a result of carbohydrate fermentation. These carbohydrates originate from

various food or drink sources. As a result of this fermentation process, the plaque power of

hydrogen (pH) drops, which leads to the demineralisation of the tooth surface. Data from

several studies (3-5) suggest foods that cause a larger drop in plaque pH have a greater

cariogenic potential—that is, a greater ability to cause dental caries. Because of this strong

association, measuring plaque pH has been adopted as the preferred method to determine the

cariogenic potential of different foods and drinks (6).

Over the past several decades, the Stephan Curve has played a very important role in the

research field of caries development and dental plaque pH. In the early 1940s, Robert Stephan

published several papers that introduced the concept of critical pH and also discussed the

production of acids within plaque when exposed to sugar (7, 8). These studies formed the basis

for the understanding of enamel demineralisation as a consequence of sugar metabolism by

bacteria in dental plaque. Stephan also found that multiple factors were involved in caries

development and that carbohydrates in the diet was one of these important factors. He also

found that both monosaccharides and disaccharides rapidly lower plaque pH and induce caries

formation in rats and hamsters (9).

2

1.1.2 GI

The concept of the glycaemic index (GI) was introduced in the scientific literature in 1981 (10).

It ranks different sources of carbohydrate-rich foods according to their effect on blood glucose

levels after they are consumed. Foods with higher GI values produce higher and extended

postprandial blood glucose levels compared with foods that are more slowly digested and

absorbed; these foods produce lower GI values. To determine the GI value of a food, blood

glucose responses are measured at regular intervals over a two-hour period. As stated by

Brouns et al. (11), the postprandial response produced by a certain food is then compared with

the postprandial response produced by a reference food (usually a glucose solution). Ideally,

the quantity of available carbohydrates must be the same in the test food and the reference food

portions.

In recent years measuring GI has gained importance, as low-GI foods have beneficial effects

in terms of managing blood glucose levels in patient with diabetes (12). Evidence shows that

foods with lower GI values have therapeutic and preventive effects on such patients (13, 14).

1.1.3 Potential relationship between plaque pH and GI

To date, only a few studies have explored the potential effect of carbohydrate digestibility, as

measured by postprandial glucose response or GI values, on dental plaque pH. Lingström and

colleagues (10) showed an interesting relationship between the GI and the area under the curve

(AUC) obtained by measuring plaque pH. Test foods were ranked according to their

postprandial glucose response, as assessed by the incremental area under the curve (iAUC). In

2000, a study investigated the plaque pH response of various breads and compared those results

with the GI values of breads obtained from an earlier investigation (15). It was seen that low-

GI breads were not only advantageous metabolically, but they were also less cariogenic,

because they produced a smaller drop in dental plaque pH compared with the higher GI breads.

3

To date, several studies have investigated plaque pH and GI separately and in great detail.

However, no study has undertaken a detailed investigated of GI and dental plaque pH

simultaneously using the same foods and subjects. This means there is a knowledge gap that it

would be helpful to fill to understand the effects of different foods on general and dental health.

1.2 Objective of Study

This research project aimed to simultaneously investigate the changes in dental plaque pH and

GI following the consumption of different foods and drinks. To achieve this, the study was

divided into three sub-studies.

Sub-study1:

This sub-study aimed to establish a dental plaque pH measurement procedure and evaluate the

effect of standardised glucose drinks of 25 g and 50 g as available carbohydrates on the pH of

dental plaque.

Sub-study 2:

This sub-study aimed to determine the effects of four different starchy foods, varying in GI and

consumed in portions of 25 g as available carbohydrates, on dental plaque pH.

Sub-study 3:

This sub-study aimed to measure the effect of three pairs of low- and high-GI carbohydrate

foods, consumed in portions of 25 g (per food item) as available carbohydrates and with a

similar texture and food volume in each food pair, on dental plaque pH.

To date, no previous study has investigated the association between plaque pH and GI

simultaneously. Although extensive research has been carried out on plaque pH and GI

4

separately, no detailed data exists that highlight the relationship between both the variables in

significant detail.

5

Chapter 2: Literature Review

2.1 Caries

2.1.1 Definition

Dental caries is a disease characterised by the demineralisation of tooth hard substances by

acids produced by dental plaque bacteria, which can be described as a community of

microorganisms (biofilm) (16).

2.1.2 Aetiology

Dental caries is one of the most frequent, complex and expensive-to-treat diseases worldwide,

and is one of the most common reasons children are hospitalised in developed countries (2, 17,

18). Its aetiology is multifactorial in nature, and the principal factors involved are the host

(saliva), microflora (plaque), diet and time (19). Therefore, it is very important to determine

the factors involved in caries formation and its prevention.

A recent review on dental caries highlighted the multifaceted nature of the disease (20). It starts

with the formation of a complex biofilm, called dental plaque, on a tooth surface. Bacteria

present in the plaque ferment dietary carbohydrates, especially sucrose, into acids. These acids,

if present for a long time, can cause a drop in plaque pH adjacent to the tooth surface and lead

to demineralisation of the enamel, the hard substance (21). However, dental caries onset and

activity is a complex process, and not every person who consumes carbohydrates will develop

caries. Several modifying risk factors influence the formation of dental caries: the presence of

certain micro-organisms; the consumption of free sugars; developmental defects of enamel;

socioeconomic conditions; and lack of protective procedures, including oral hygiene

maintenance (20).

6

When reviewing caries development and the role of carbohydrates, the classic study conducted

from 1945 to 1954 in the Vipeholm Hospital in Sweden cannot be ignored (22-25). During

phase I (carbohydrate study I) of the Vipeholm study, conducted between 1947 and 1949,

carbohydrate-rich food items were given either in a solution form with little tendency to stick

on the tooth surface or in a sticky consistency between meals (toffees) or at meals (new breads).

In the second phase conducted from 1949 to 1951, the daily amount and type of sugar

consumed by the participants was set similar to what was consumed by the participants in Lund.

The author concluded that the risk of sugar increasing caries activity is greatest when sugar is

consumed between meals, is in a form which increases its tendency to be retained on the tooth

surface or has a transiently high concentrations.

2.1.3 Signs and symptoms

Dental caries develop over time; it may show no symptoms at all or may be very painful. In its

initial stages, it may appear as a chalky white lesion on tooth hard surfaces that is usually due

to the loss of calcium and the accumulation of plaque. If the plaque is not allowed to accumulate

for long and the caries is detected in its early stages, it can be arrested and will progress no

further. If an initial lesion is not treated in time, it may become a surface defect and the chalky

white lesion may turn brown, forming a cavity in the superficial layer of the tooth known as

enamel. At this stage, mild pain and sensitivity can be felt. If not treated, the cavity may

increase in size and the lesion may become softer and further progress into the second layer of

the tooth, known as dentine. If the bacteria invade the dentine, there is increased pain and

sensitivity. The bacteria are capable of further invading the tooth, moving into deeper tissues,

causing pulp damage and possibly leading to abscess formation. Symptoms such as pain,

sensitivity and discomfort may grow with the extent of the invasion.

7

2.1.4 Prevention

Preventing dental caries and treating it once it is formed is one of a dentist’s main concerns.

The past 30 years have seen increasingly rapid advances in the field of dentistry, and recent

developments in this field have heightened the need for new methods and techniques to prevent

caries formation. Some of the most important and common preventive factors that may affect

caries prevention are as follows:

1. Reducing the frequent intake of food high in sucrose is a major preventive strategy.

Dietary sucrose has two major detrimental effects on plaque biofilm that leads to caries

formation. First, frequent consumption of sucrose increases the cariogenic potential of

the plaque biofilm. Second, continuous exposure to sucrose helps the mature plaque

biofilm produce organic acid as a by-product, resulting in an extended duration of

decreased pH (26, 27). The dentist’s aim is to counsel adult patients who have dental

caries to minimise the quantity and frequency of their acidic food consumption and to

consider substituting sucrose-containing foods with sugar-free alternatives.

2. Regularly removing plaque biofilm is an important factor in preventing caries

formation. Tooth brushing, dental flossing and rinsing are the best mechanical and

chemical aids patients can practise daily to maintain good oral hygiene. In addition to

regular toothbrushes, electric toothbrushes and oral irrigation devices may also be used

as oral hygiene tools. Alongside self-initiated oral hygiene, regular professional

cleaning is an effective part of caries reduction (28).

3. Local fluoride application has been proven to prevent dental caries and increase dental

enamel’s resistance to demineralisation. It is available in various forms, such as

varnishes, gels, creams, solutions and tablets (29). Applying fluoride to the teeth of

adults and children with a high risk of caries should be performed periodically.

8

4. Fluoridated community water has proven to be an effective factor in caries reduction.

There has been a 23‒25% reduction in caries formation in adults aged 17‒35 years in

Australia as a result of people’s regular exposure to fluoridated water (30, 31).

5. Saliva plays an important role in caries prevention by neutralising the acidic effect

produced in the plaque biofilm (32). Its buffering capacity assists in remineralisation.

Therefore, patients with low salivary flow rates are usually at high risk of caries

formation. Factors increasing the salivary flow rate include sugar-free and xylitol

chewing gums, non-cariogenic foods that require plenty of chewing, topical lubricants

and the prescription of salivary stimulants by health professionals.

2.1.5 Epidemiology

As stated in the book Oral Health and Dental Care in Australia (33), decay in deciduous teeth

in six-year-old children who visited school dental services decreased from 3.19% in 1978 to

2.58% in 2010. A similar pattern of decreasing caries rates was observed in children aged 12

from 4.79% in 1977 to 0.84% in 2000; this later increased to 1.34 in 2010. In a recent study,

conducted in an Australian population, a substantial increase of dental caries of 26.5% in 10–

11-year-old children was observed from just 2.8% in the two–three-year-old age group (34).

An average decrease of 2.09% was seen in decay formation in adults aged 15 years and above

from 1978‒1988 to 2004‒2006. Overall, dental caries is a prevailing condition in Australian

children, and it needs to be addressed by effective interventions introduced by policymakers

(35). Further research is mandatory to better understand that how sugar intake in our daily diet

changes caries prevalence. Despite the use of water fluoridation, an increase in caries activity

is seen, which is likely caused by a high consumption of food and soft drinks rich in

carbohydrates (36).

9

2.1.6 Caries in Rodents

As in humans, it is not uncommon to find caries in animals. Using animal models to better

understand caries initiation and progression has been in practice for a long time (37-39).

Animal models are considered essential for conducting experiments because they enable

increased control for variability compared with humans and the application of interventions

that are not possible in humans for ethical reasons (40). As in humans, rodents receiving more

sucrose in their diet have shown a higher incidence of carious lesions compared with the control

group (41, 42). A direct relationship between dietary sucrose consumption and caries activity

has been observed in different experimental animals, such as rats and hamsters (43, 44). Studies

have shown that the presence of Streptococcus mutans or Candida albicans and hyposalivation

favours caries formation in humans as well as in rats (38, 41, 45). Findings from a series of

animal experiments in the 1960s (46) led investigators to report that a carbohydrate-free diet

‘prevents the initiation of carious lesions’; however, a stringent restriction of carbohydrates in

humans is impractical because of their widespread occurrence in human foods and their

inclusion in dietary recommendations.

2.2 Plaque

2.2.1 Definition

‘Dental plaque is a complex biofilm that accumulates on the hard tissue (teeth) in the oral

cavity’ (47). It contains water-insoluble glucans, fructans, bacterial and salivary proteins, and

variable quantities of lipids, calcium, phosphorus, magnesium, fluoride and water (48).

2.2.2 Plaque formation

Plaque formation involves a series of events, and it takes up to two weeks for plaque to fully

mature. The process starts with the formation of an acquired dental pellicle, a thin protein-

based layer formed by saliva on the tooth surface (49). Within a few hours, several bacteria

10

attach to the dental pellicle. A clear progression of organisms is seen, with streptococci the

dominant species, followed by a growing number of Actinomyces (47). Eventually, because of

the continued bacterial adhesion, several micro-colonies develop on the tooth surface (27).

More bacterial species become involved, and these micro-colonies continue to grow and form

mature plaque. Dental plaque is not a uniform structure and varies from site to site and tooth

to tooth; therefore, in the same mouth, some areas may form caries and others may not (47).

Despite the large variety and number of bacteria in the mammalian oral cavity, studies have

demonstrated that mutans streptococci (MS) are the most common micro-organisms causing

dental caries (50). MS and lactobacilli produce organic acids causing a low pH in the oral

environment and increasing the risk of tooth cavitation. Species found in the human oral cavity

are predominantly Streptococcus mutans (51). Streptococcus mutans adhere and demineralise

the tooth surface by fermentation of carbohydrates, specifically sucrose and fructose, to organic

acids that lower the plaque pH below the critical pH, thus causing demineralisation (52).

2.2.3 Prevention

To prevent caries formation, plaque build-up must be avoided. Some basic oral hygiene

procedures, such as brushing, interdental flossing and antibacterial mouth rinses, can be used

to help reduce bacterial growth and minimise plaque build-up.

2.3 pH

2.3.1 Definition

The concentration of hydrogen ions in a solution is expressed by the pH, on a scale of 1‒14,

where 7 is the pH value of a neutral solution. Values < 7 are considered acidic and values > 7

are considered basic or alkali.

11

The demineralisation or dissolution of dental enamel is caused by acidogenic bacteria, which

produce an environment in which a rapid decline in the pH level of the tooth biofilm reaches

5.0 or below (20).

2.3.2 Measuring plaque pH

Over the past several decades, the Stephan Curve (7) has been used to demonstrate changes

that occur in dental plaque pH after consumption of various foods or drinks. The Stephan Curve

(Figure 1) can be divided into four stages: the initial phase, or phase A, is where the plaque is

not exposed to any sucrose and gives the baseline pH value; phase B where the preliminary

decline in the pH value is observed; phase C is where the pH value reaches critical pH, which

is below or equal to 5.5; phase D is the final and recovery phase (53). To measure the plaque

pH values, different methods have been used. The three most common are the sampling

method, the microtouch method and the telemetric method (54).

Figure 1: Stephan Curve—pH curve of dental plaque after glucose rinse observed over

a period of 60 minutes (https://creativecommons.org/licenses/by-sa/3.0/deed.en)

12

• The sampling/scraping method (55): a pooled plaque sample is collected each time from

any area on any tooth using a sterile instrument. It is then immediately dispersed in

distilled water. The amount of distilled water varies depending on personal choice. The

plaque sample is then vortexed. Ideally, it should be measured within 20 seconds. The

pH is measured with a pH meter, which is attached to a glass electrode. The electrode

is calibrated before each test is conducted.

• The touch/microtouch method (56): A palladium microtouch electrode with a diameter

of 0.1 mm (a Beetrode) is inserted into an interdental area just apical to the touchpoint

at two defined sites each time, in either the upper or lower jaw. There should not be any

dental restoration present there. The electrode is connected to a pH meter. A salt bridge

is created between the reference electrode and the subject in a potassium chloride

solution. The electrode is calibrated using a standard buffer solution before the start of

each test.

• The telemetric method (57): This system consists of a glass pH electrode with a

diameter of 2 mm. The electrode is mounted on the approximate surface of a natural

tooth crown and incorporated into a removable partial denture. A skin reference

electrode attached to a volunteer’s arm is used. The electrode is calibrated against

standard pH buffers before and after each test.

2.3.3 Organic acids produced by plaque

A number of organic acids are produced by the fermentation process of dental plaque. The

amount and pathway by which they are produced was well explained by Laurence J Walsh in

2006 (58). According to Walsh, large quantities of sucrose are fermented to lactate as one of

the major by-products; however, organic acids such as acetate, formate and pyruvate are also

produced by glucose fermentation. Streptococcus mutans has the capability of altering the

13

pathway of its acid production, depending on the supply of nutrients, and in low cariogenic

environments weaker acids such as acetate are formed.

2.4 GI

2.4.1 Definition

The GI is defined as the postprandial glucose response over a two-hour period after consuming

a food that usually has 50 grams of available carbohydrates compared with a reference food

that has 50 g of carbohydrates—either a glucose solution or white bread (59). In general, foods

containing carbohydrates that break down quickly during digestion have high GI values,

whereas foods that break down more slowly during digestion or contain sugar are inherently

less glycaemic, release glucose more gradually and have low GI values.

2.4.2 Classification

The GI has three main classifications: low, intermediate and high according to a scale of 0‒

100 (Figure 2). The higher a food’s GI value, the higher the blood glucose levels rise after that

food is consumed.

Figure 1: Three GI classifications and examples of common carbohydrate foods

2.4.3 Measuring the GI

A number of different food products have been used as a reference to measure GI. However,

more than 90% of relevant studies have used glucose or white bread as the reference food (11);

low

• 55 or less• gradual increase in

blood glucose levels• e.g.: corn tortilla 46,

orange raw 43, chapatti 52.

intermediate

• 56‒69• moderate increase in

blood glucose levels• e.g.: wheat flake

biscuits 69, pineapple raw 59, museli 57.

high

• 70 or more• fast increase in blood

glucose levels• e.g.: white wheat

bread 75, cornflakes 81, boiled potato 78.

14

pure glucose sugar (assigned a GI value of 100) is the most common choice of reference

food/drink. All the other foods are ranked on a scale of 0‒100 depending on their actual effect

on blood glucose levels compared with the response produced by the reference food. The

methodology used to determine the GI value of a food or drink is described in the International

Standard Organisation (60). Test foods and reference foods must be consumed in equal

available carbohydrate portions: either 25 g or 50 g available carbohydrate loads. The test must

be conducted early in the morning after an overnight fasting period of 10‒14 hours. Participants

are required to avoid any strenuous exercise, alcohol, cigarettes and food rich in fats, and to

consume carbohydrate-rich foods, 24 hours before a test session. Blood samples are collected

either by capillary or via arterialised venous blood samples at taken at regular intervals over a

two-hour period (0, 15, 30, 45, 60, 90 and 120 minutes). Fingertip capillary blood samples are

the gold standard, as capillary blood is most sensitive to acute changes in glycaemia (61). The

iAUC is measured using the trapezoidal rule, ignoring any area below the baseline (11, 60).

2.5 Buffering Capacity

2.5.1 Definition

‘The salivary buffering capacity is defined as the ability of saliva to buffer acids produced by

bacteria’ (62). Salivary buffering capacity is one of the major factors affecting caries formation,

as it neutralises the acidic environment and maintains pH levels above the ‘so-called’ critical

pH level (5.5), thereby preventing demineralisation (63, 64).

2.5.2 Types of salivary buffering systems

Three buffering systems are present in stimulated and unstimulated saliva. These three systems

are the bicarbonate, phosphate and the protein buffer systems (65). The effectiveness of these

three systems depends on the rate at which the saliva is produced (66). Among these three,

bicarbonates are believed to be the major buffering system, and its concentration varies

15

considerably from unstimulated to stimulated saliva. It is higher in stimulated saliva (24 mmol/l

at a flow rate above 2 ml/min) than in unstimulated saliva (5 mmol/1 at a flow rate of 0.3

ml/min) (67). In contrast, phosphates show a higher concentration in unstimulated saliva than

in stimulated saliva. A different variety of proteins with buffering capacities are found in whole

saliva, which may vary in their function and composition depending on the presence of

stimulated and unstimulated saliva (68).

2.5.3 Measuring buffering capacity

In 1959, Ericsson (69) measured the salivary buffering capacity by adding 3.0 mL of

hydrochloric acid (0.005N) to collected saliva samples (1.0 mL). After measuring the pH

values, the buffering capacity was categorised into three ranges: high (³ 5.6), medium (4.1‒

5.5) and low (£ 4). Since then, this test has been used as the standard for measuring salivary

buffering capacity (63). The disadvantage of Ericsson’s method is that it cannot be performed

as a chair-side test in clinical practice. Therefore, commercial methods for measuring saliva

buffering capacity have subsequently been developed. These include colorimetric strip-type

tests, the Saliva-Check Buffer, the Dentobuff Strip method and the CRT Buffer test (70).

2.6 The Effect of Different Foods on Plaque pH

A growing body of literature recognises the effects of different foods on plaque pH. Foods

found to influence plaque pH have been explored previously, and some of these studies are

discussed in this section.

2.6.1 Effect of different breads on plaque pH

Several authors have used various bread products to explore their effect on dental plaque ph.

Different types of breads, ranging from regular soft bread to traditionally baked sangak bread,

have been investigated (54, 71). For various bread types substantial variations in the minimum

16

plaque pH values have been observed, ranging from a minimum plaque value of 4.37 to 6.78

pH (54, 71). However, in most of the studies, bread was associated with a severe drop in plaque

pH (15, 54, 71). The variations may be best explained by factors that include the testing

methodology (54), bread ingredients, baking time and bread preparation methods (71). In

particular, previous studies have not had a consistent amount of carbohydrates in the breads,

with tested portions containing between ~2 to 25 g of available carbohydrates (Table 1). This

makes comparing these results difficult. In light of the publications mentioned above, it can be

concluded that bread possesses moderate to high levels of cariogenic potential, depending on

various factors. Table 1 summarises some of these prior studies.

Table 1: Summary of papers on the effect of different breads on human dental plaque

Author Year Test foods Quantity of available carbohydrates

Method Outcome

Lingstrom, Imfeld (54)

1993 5 g soft bread ‒ Sampling Microtouch Telemetric

Minimum pH by telemetric method = 4.37

pH ¯ after bread consumption

Lingstrom, Liljeberg (15)

2000 Barley kernel bread (BKB) BKB fermented sourdough White wheat bread Syrup-sweetened wheat-rye bread

25 g Microtouch Minimum pH seen in syrup-sweetened wheat‒rye bread = 5.75

pH ¯ after bread consumption

Mortazavi and Noin (71)

2011 10 g traditional sangak 10 g soft, bulky baguette

‒ Microtouch Minimum pH seen in baguette bread = 6.78

pH ¯ after bread consumption

17

2.6.2 Effect of different drinks on plaque pH

Consumption of soft drinks is increasing worldwide. Most of these products can cause dental

and general health problems (72). A growing body of literature recognises the detrimental

effect of drinks, especially carbonated drinks and juices, on dental plaque pH (72-74). In prior

studies involving drinks, substantial variation in the minimum plaque pH has been observed,

with values ranging from a minimum plaque pH of 3.1 to 5.72 (5, 73). However, in most of

these studies, variations were observed for different drinks in terms of their effect on dental

plaque pH (72-74). Most of the drinks produced severe pH decreases, while drinks such as

Horlicks helped increase plaque pH 20 minutes after being consumed (73, 75). This variation

may best be explained by the involvement of factors such as testing methodology (54) and

ingredients. In all these studies, the carbohydrate content in the test drinks varied significantly.

The evidence presented in this section suggests that carbonated drinks and juices should be

avoided, as they have cariogenic potential and can cause general health issues. Table 2

summarises of some of the prior studies conducted on different drinks.

18

Table 2: Summary of papers on the effect of different drinks on human dental plaque

Author Year Test foods Quantity of available carbohydrates

Method Outcome

Birkhed (73)

1984 Fruit juice Fruit drinks Carbonated drinks Sports drinks

‒ Sampling method

Minimum pH 3.1± 0.3 produced by carbonated drinks

All four drinks ¯ plaque pH

Toumba and Duggal (5)

1999 Blackcurrant drink, 7% juice Blackcurrant drink, 10% juice Apple and blackcurrant drink, no added sugar Mixed citrus juice drink

0.49% 0.65% 0.8% 4.5%

Sampling method

Mean minimum plaque pH in the mixed citrus juice drink was 5.72

All four juices ¯ plaque pH

Roos and Donly (4)

2002 15 mL regular Coca-Cola 15 mL Diet Coca-Cola

‒ Microtouch method

Minimum pH seen in regular Coca-Cola with a mean value of 5.5 afger five minutes pH drop after consumption of both drinks

Johansson, Lingstrom (72)

2007 Regular Coca-Cola Light Coca-Cola Fresh orange juice

‒ Microtouch method

Minimum pH seen in Coca-Cola and orange juice with < 5.5. All three drinks lower plaque pH

Mahajan, Kotwal (74)

2014 Orange juice with added sugar Orange juice without sugar Apple juice with added sugar Apple juice without sugar

‒‒ Sampling method

Minimum pH seen in the orange juice with added sugar was 3.5 All drinks, with and without sugar, lower plaque pH

Garg, Karuna (75)

2017 30 g Horlicks 20 g Boost 33 g Complan

‒ Sampling method

Horlicks increase plaque pH Complan and Boost reduce plaque pH

19

2.6.3 Effect of different snacks on plaque pH

Snacks should represent a small portion of food eaten between proper full meals. The amount

of sucrose available in prepacked snacks is usually higher, which makes them taste better, and

they are usually cost-effective (76). Pre-sweetened snacks consumed frequently in between

meals carry a high risk of decreasing plaque pH and have limited nutritional benefits (77).

Sweet and sticky foods are generally considered to have greater cariogenic potential because

they are more difficult to remove from the tooth surface (78). For various snack foods

substantial variation in the minimum plaque pH has been observed, ranging from a minimum

plaque pH value of 4.4 to 5.64 (77). A growing body of literature recognises the cariogenicity

caused by popular snacks such as raisins, chocolates, biscuits and toffees (77, 79, 80). Table 3

summarises some of the studies that have investigated the effect of snacks.

20

Table 3: Summary of papers regarding the effect of different snacks on human dental

plaque

Author Year Test foods Method Outcome

Igarashi, Lee (80)

1988 8.3 g of toffee 3.2 g of sorbitol gum with 2% sodium bicarbonate Chewing gum containing bicarbonate

Telemetric method

Toffee ¯ plaque pH to 4.4

Utreja, Lingstrom (77)

2009 10 g raisins 10 g bran flakes 10 g commercial raisin bran cereal 10 g experimental raisin bran cereal

Microtouch method

Minimum plaque pH seen in commercial raisin bran cereal with 5.64 All test foods ¯ plaque pH

Thaweboon, Suddhasthira (79)

2007 5 g milk chocolate 5 g sweet biscuits 5 g instant noodles 5 g sticky rice with banana

Sampling method

In both the low mutans streptococci and high mutans streptococci groups, milk chocolate showed the maximum drop of 5.36 and 5.33, respectively

2.6.4 Effect of different yoghurts on plaque pH

Sugarless yoghurt is a traditional and commonly consumed Turkish food (81). Yoghurts are

available in raw form and in different flavours. They can be used in regular meals or as snacks.

However, unlike other snacks discussed earlier, sugarless yoghurts or yoghurt with less sucrose

content compared with sugared yoghurt varieties are healthier to eat and have limited

cariogenic potential (81, 82).

21

A very limited variation in the minimum plaque pH of yoghurts has been observed in prior

studies, with values ranging from a minimum plaque pH of 5.54 to 5.72 (81, 82). However, in

most of these studies, variations have still been observed in different yoghurts.

In yoghurts, it is not only the amount of sugar present that makes a difference; other factors,

such as consistency, also play an essential role. Yoghurt with a thick texture decreases plaque

pH for a slightly longer period than yoghurt with a thinner consistency, indicating that

retentiveness plays a more vital role than the amount of sugar (82).

The most important finding to emerge from the above discussion is that it is better to consume

sugar-free or low-sugar yoghurts, as they possess less cariogenic potential than regular

yoghurts. Table 4 summarises some of the prior studies that have investigated different

yoghurts.

Table 4: Summary of papers on the effect of different yoghurts on human dental plaque

Author Year Test foods Method Outcome

Gardiner, Pollard (82)

1997 10 mL Thick and creamy (18.4% sucrose) 10 mL Low fat 1(17.2% sucrose) 10 mL Custard style (16.5% sucrose) 10 mL Low fat 2 (12.2% sucrose) 10 mL Greek style (10.2% sucrose) 10 mL Healthy eating (4.5% sucrose)

Microtouch method

Minimum plaque pH drop seen in thick and creamy yoghurt with 5.54 All yoghurts ¯ plaque pH

Sonmez and Aras (81)

2007 10 g white cheese 10 g sugarless yoghurt

Microtouch method

Minimum pH seen in sugarless yoghurt with 5.72 White cheese ­ plaque pH, whereas yoghurt ¯ plaque pH

22

2.6.5 Effect of different starchy food and fruits on plaque pH

During the past few decades, much research has investigated the effect of starchy food items

on dental plaque pH (83-85). A profound drop in dental plaque pH after the consumption of

starch has been observed in the past as well (86). A likely reason for this drop is the prolonged

retention of starchy food items in the mouth and increased acid production by sucrose in the

presence of starch (87). A study conducted by Lingström in 1993 compared a vast range of

starchy food items to observe their effect on dental plaque pH (88). In this study, it was

concluded that the fall in dental plaque pH by starch depends mainly on the processing

conditions used. Therefore, starches cannot be classified as a single group. When comparing a

group of starchy food items and fruits to observe their effect on dental plaque pH, Pollard found

that oranges and bananas had a more severe demineralisation effect than all the other foods,

including white bread and rice. In another study, three fruits—bananas, oranges and apples—

were compared in three different forms to observe their effect on dental plaque pH (89). The

minimum fall in pH was observed in orange juice (5.28 ± 0.24), but no significant differences

were observed among the different textures of the same fruit. Table 5 summarises the effects

of different starches and fruits on dental plaque pH.

23

Table 5: Summary of papers on the effect of starchy food and fruits on human dental plaque pH

Author Year Test Foods Method Outcome Lingstrom, Birkhed (88)

1993 Spaghetti Macaroni Potato Rice Unsweetened bread Sweetened bread

Microtouch method Sampling method

Microtouch method continuously gave lower pH values than sampling method Minimum plaque pH was observed in sweetened bread for both the methods 6.0 ± 0.41 by the microtouch method and 6.43 ± 0.29 by the sampling method.

Pollard (90) 1995 5 g pasta 5 g banana 5 g Alpen 5 g oranges 5 g Weetabix 5 g white breads 5 g apples 5 g bran flakes 5 g cornflakes 5 g wholemeal bread

Sampling method

All test foods were not significantly different from each other. The maximum AUC was observed with pasta, which was 21.03 ± 9.29

Hussein, Pollard (89)

1996 35 g apple whole 5g banana 35g apple whole 35 g apple homogenised 35 g apple juice 35 g orange whole 35 g orange homogenised 35 g orange washed 35 g banana whole 35 g banana homogenised 35 g banana juice

Sampling method There was no significant difference between extrinsic sugars and intrinsic sugars (different forms of fruits) Maximum plaque pH drop was observed with orange juice at 5.28 ± 0.24

24

2.7 Hypothesis

We hypothesised that food with a high GI reduces dental plaque pH more than food with a

moderate or low GI.

25

Chapter 3: Materials and Methods

3.1 Ethics Approval

The study procedures were approved by the Human Research Ethics Committee at the

University of Sydney (Project number: 2017/801). All participants were given information

sheets about the project, and all participants gave written consent before participating.

3.2 Subject Selection

Participants were recruited via verbal advertisement on the University of Sydney campus and

enrolled in the study based on their inclusion and exclusion criteria. Participants were between

18 to 55 years old, healthy, non-smoking, and without any underlying diseases or conditions.

Exclusion criteria were any food allergies or orthodontic appliances. Individuals who were

willing to participate received a participant information sheet and signed informed consent

forms. Participants were instructed not to brush their teeth or undertake any other oral hygiene

regime the night before or the morning of the test. Consumption of legumes, tofu, lentils and

alcohol was limited 24 hours before the test day. Participants were instructed to refrain from

any unusual physical activity. They were also advised to have a restful sleep of at least six

hours and to stay hydrated before the test. All tests were conducted early in the morning,

between 6:30 am 11:30 am, after participants had undergone a 10‒12-hour overnight fast. All

participants in sub-studies 2 and 3 also gave one saliva sample (~ 1 mL) to measure their

buffering capacity.

26

3.3 Measurement Technique

3.3.1 Measuring plaque pH

Plaque pH was measured using the sampling technique (55). Before measuring plaque pH, the

pH electrode was calibrated against three standard solutions of pH 4.0, 7.0 and 10.0,

respectively. Before each use, the electrode was rinsed with distilled water and dried with tissue

paper. For Sub-study 1, after recording the baseline pH at time point 0, the second plaque

sample was taken two minutes after participants finished the test drink and then 12 and 32

minutes later, respectively. For sub-studies 2 and 3, the baseline pH was taken at time point 0,

the second sample was taken 12 minutes after participants finished the test food with 250 mL

of water and then 22 and 62 minutes later, respectively.

At each time point, a different quadrant of the oral cavity was randomly selected for plaque

sampling. A sterile plastic dental probe (SI551ST Periodontal 3-Piece Examination Kit, MDDI,

Australia) with a metal tip was used to collect supragingival plaque from the mesial, distal,

buccal and palatal/lingual surfaces of the teeth. A sufficient amount of dental plaque—

approximately 1 mm of the tip of the dental probe—was sampled at each time point. The dental

plaque collected was immediately suspended in 25 µL of distilled water in a 2.5 mL tube

(Eppendorf, Sigma-Aldrich, Australia) and vortexed for 20 seconds. Plaque pH was measured

by inserting the electrode into the solution (plaque and water), and each time, the pH was

recorded after 10 seconds. The electrode was washed, dried and calibrated at pH 7 before each

reading was taken.

3.3.2 Measuring blood glucose

Capillary blood samples (> 0.5 mL) were collected before and after consumption of test food

at ‒5, 0, 15, 30, 45, 60, 90 and 120 minutes. Participants warmed the hand to be sampled before

a blood sample was collected. Blood samples were collected by finger-pricking using a sterile,

27

single-use lancet device (Accu-Chek Safe-T-Pro Plus, Roche Diabetes Care GmbH, Germany).

Participants were given up to 12 minutes to consume the test food with 250 mL of water and

were not allowed to eat any other food or drink throughout the test session. Blood samples

were placed into anticoagulant-coated tubes (Eppendorf tubes, grade II; Sigma Chemical

Company, St Louis, MO, USA) containing 10 IU heparin salt. Following this, the tubes were

centrifuged at 10,000 g for one minute at room temperature. The plasma layer was pipetted

into uncoated tubes and stored at ‒30°C until analysis. The glucose concentrations in the

plasma samples were analysed in duplicate using a glucose hexokinase enzymatic assay

(Beckman Coulter Inc., USA) and an automatic centrifugal spectrophotometric clinical

chemistry analyser (Beckman Coulter AU480, Beckman Instruments Inc., USA). The

incremental area under each two-hour plasma glucose response iAUC was determined for each

test session using the average of the two fasting plasma samples as one baseline glucose

concentration and ignoring any area below fasting (60).

3.3.3 Measuring buffering capacity

Saliva buffering capacity was measured using Ericsson’s method (69). Saliva samples were

taken from all the participants in sub-studies 2 and 3.

Each participant was given a Salivette tube (Sarstedt AG & Co, Germany) for saliva collection

first thing in the morning before the start of the pH and blood glucose test. A single saliva

sample (> 1 mL) was collected from each participant. Participants removed the cotton swab

from the tube and chewed on it for five minutes to stimulate saliva production. After that, the

participants returned the swab into the Salivette tube and placed the cap back on the tube. The

tubes were centrifuged at 2000 g for two minutes at 20°C. Clear saliva was then pipetted into

uncoated Eppendorf tubes and stored at ‒30°C. Each saliva sample was then thawed and mixed

by inverting the tube a couple of times prior to measuring buffering capacity. 1.0 mL saliva

28

was mixed with 3.0 mL HCL solution (0.005 M). To prevent foaming, one drop of 2-Octanol

(Sigma-Aldrich 2-Octanol 97% Lot # BCBV5273, China) was added. The solution was then

mixed for 20 minutes to remove the CO2 using a mixing machine (Selby Suspension mixer,

Australia). A pH meter (SevenCompact™ pH/Ion S220, Switzerland) was used to measure the

saliva buffering capacity. Before use, the pH meter was calibrated against a standard buffering

solution. Between taking readings each time, the pH meter was calibrated with pH 7 solution.

All readings were noted when pH meter showed a stabilised reading.

3.4 Test Food

3.4.1 Sub-study 1: Impact of carbohydrate amount on plaque pH

Two different carbohydrate doses, 25 g and 50 g were tested in this study. Table 6 shows the

nutritional value of the glucose powder. The oral glucose solutions were prepared the day

before they were required using sugar (Glucodin Powder, Valeant Pharmaceuticals, Australia)

dissolved in 250 mL water and either 25.7 g of glucose powder (25 g dose) or 51.5 g glucose

powder (50 g dose). Each experiment was conducted in triplicate and the order was randomised

for each participant.

Table 6: Nutritional information of the glucose used

Product Weight (g)

Energy (kJ)

Available carbs (g)

Glucose 25 25.7 436 25 Glucose 50 51.4 873 50

29

3.4.2 Sub-study 2: Impact of carbohydrate (GI) on plaque pH

In Sub-study 2, four different types of starchy carbohydrate food items, representing a range

of GI values, were compared with a reference drink (25.7 g glucose powder dissolved in

250 mL water). The four test food items were 58.5 g of white bread (Wonder White, White

Bread Vitamins & Minerals Sandwich, Goodman Fielder Limited, North Sydney, NSW,

Australia), 34.7 g of dry pasta (San Remo™ 500 g, San Remo Macaroni Co. Pty Ltd, Windsor

Gardens, SA, Australia), 131.6 g of tinned chickpeas (Edgell, Simplot Australia, Pty Ltd,

Mentone, VIC, Australia) and 37.1 g of dry instant mashed potatoes (Continental Deb,

Unilever Australia Ltd, North Rocks, NSW, Australia).

Each test portion contained 25 g of available carbohydrates. The powdered mashed potatoes

were mixed in 161.9 g of boiling water to create the edible portion. The pasta was boiled in

water for 12 minutes, after which the water was drained through a sieve. The water for the

tinned chickpeas was also drained. Table 7 shows the nutritional values for each test food. All

these values were taken from the ingredient tables at the back of the product packs and were

calculated and adjusted according to 25 g of available carbohydrates. The order in which the

test foods were given was random for each participant. Participants were given exactly 12

minutes to consume the test food with a glass of water (250 mL).

Table 7: Nutritional information of test foods used in Sub-study 2

Product Weight (g)

Energy (kJ)

Fat (g)

Available carbs (g)

Sugar (g)

Fibre (g)

Protein (g)

Bread 58.5 585 1.8 25 2.0 4.1 4.3 Pasta 34.7 531 0.6 25 0.8 1.0 4.1

Chickpeas 131.5 751 2.7 25 0.6 7.8 9.4 Mashed potatoes

37.1 602 2.7 25 1.7 ‒ 3.3

30

3.4.3 Sub-study 3: Impact of GI in a food type on plaque pH

In Sub-study 3, three pairs of foods were selected: one pair of rice, one pair of bread and one

pair of breakfast cereals. Within each pair, one food had a lower GI than the other, but both

foods had a similar texture. Each pair featured food products with same quantity of

carbohydrates, but different GI levels. The researcher collecting the dental plaque samples was

not privy to the specific details of the consumed test foods (brand, GI and nutritional values)

until the completion of the study.

The test food portions were adjusted to 25 g of available carbohydrates. The order in which the

test foods were given to the participants was randomised. The rice used in this study was

microwave-cooked: before it was served to participants, it was cooked in the microwave for 60

seconds. The power of the microwave used to cook the rice was 1000 W. The breakfast cereals

and breads were served raw and were not mixed with any other ingredient. Table 8 gives the

nutritional values of each test food. All values were taken from the back of the product pack

and were adjusted according to 25 g of available carbohydrates.

Table 8: Nutritional information of test foods used in Sub-study 3

Product Weight (g)

Energy (kJ

Fat (g)

Available carbs (g)

Sugar (g)

Fibre (g)

Protein (g)

Rice A 69.5 553 1.8 25 0.3 1.7 2.6 Rice B 73.5 574 2.2 25 0.3 0.9 3.2

Bread A 61.8 599 1.5 25 1.8 5.1 4.6 Bread B 60.5 604 1.3 25 1.6 5.6 4.9

Cereal A 32.8 528 1.2 25 4.7 2.3 2.2

Cereal B 38.2 581 1.0 25 4.8 7.0 3.7

31

3.5 Statistical Analysis

A p-value < 0.05 was considered statistically significant. Data were analysed using the

Statistical Package for the Social Sciences (SPSS) version 22 (SPSS Inc., United States). Data

were given as mean ± standard deviation (SD) unless otherwise stated. For Sub-study 1, a

paired sample t-test was conducted to determine the effect of different glucose amounts on

plaque pH at different time points. For Sub-study 2, a paired sample t-test was used to

investigate the effect of different foods on plaque pH and blood glucose levels at different time

points. Data on the AUC for pH and blood glucose were obtained using Excel. For the AUC

of plaque pH and blood glucose for different foods, data were analysed using analysis of

variance (ANOVA). Where ANOVA was significant, a Bonferroni post-hoc analysis was used

for multiple comparisons. For Sub-study 3, a paired sample t-test was conducted to determine

the effect of the same food types with different GI levels on plaque pH and blood glucose levels

at different time points. For the AUC of plaque pH and blood glucose, data were also analysed

using paired sample t-tests. Pearson correlation coefficients were calculated to test the

association between pH and blood glucose. A P-value <0.05 was considered statistically

significant.

32

Chapter 4: Results

4.1 Sub-study 1: Impact of Carbohydrate Amount on Plaque pH

Eight subjects (one male and seven females) participated in this sub-study. Their ages ranged

from 29 years to 55 years. The overall shape of the plaque pH response curve was similar for

the two glucose solutions (25 and 50 g/250 mL) during the 32-minute period (Figure 3). The

maximum plaque pH drop was measured at 12 minutes for all participants (Table 9). The

maximum pH drop was 1.53 pH units for the 25 g glucose solution and 1.50 pH units for the

50 g glucose solution; both values were significantly lower than the baseline values (p = 0.001).

The pH did not return to the baseline values after the total observation period of 32 minutes.

At every time point, the 25 g glucose solution displayed a smaller plaque pH drop compared

with the 50 g glucose solution (P = 0.001).

Table 9: Mean ± SD absolute plaque pH values following consumption of 25 g and 50 g

glucose solution (250 mL) assessed at four different time points (n = 8; with three

technical replicates; p-values are given for both drinks)

Time point (min.)

Glucose 25 g Mean ± SD

Glucose 50 g Mean ± SD

P*

T0 7.7 ± 0.5 7.5 ± 0.4 0.001

T2 6.7 ± 0.2 6.5 ± 0.3 0.001 T12 6.1 ± 0.3 5.9 ± 0.4 0.001

T32 6.9 ± 0.2 6.7 ± 0.3 0.001

Mean ± SD values of eight subjects. P-value from ANOVA with Bonferroni correction.

33

Figure 2: Ex vivo dental plaque pH in healthy subjects (n = 8) after consumption of 25 g

and 50 g glucose solution (250 mL). Curve A (blue line) shows the plaque pH drop after

consumption of glucose 25 g; curve B (green line) shows the plaque pH drop after the

consumption of glucose 50 g (error bars indicate the SD)

4.2 Sub-study 2: Impact of Carbohydrate (GI) on Plaque pH

4.2.1 pH data

Twelve subjects (four males and eight females) participated in this sub-study. Their ages

ranged from 18 years to 30 years. The pH values were measured after they consumed a standard

glucose drink containing 25 g available carbohydrates and four different test foods: white

bread, mashed potatoes, pasta and chickpeas, all with 25 g of available carbohydrate. Table 10

presents the mean ± SD of the plaque pH for all subjects at the baseline and at 12, 22 and 62

mins. Figure 4 shows a maximum pH drop at 22 minutes for all the tested foods and drinks.

34

The white bread showed the most pronounced drop of 1.5 pH values from the baseline at 22

minutes when compared with the other foods. Chickpeas induced the smallest pH drop of 0.7

pH values from the baseline. The AUC for chickpeas of 30.3 ± 13.8 mmol/L was the lowest,

and the AUC for white bread of 62.9 ± 19.7 mmol/L was the highest. A significant difference

in the AUC was calculated between white bread and chickpeas (P = 0.004), and this is shown

in Table 11. The plaque pH values of all the tested foods and drinks returned to the baseline

value after 62 minutes (Figure 4). Figure 5 shows the comparison of all the test foods included

in Sub-study 2 with the control solution (25 g/ 250 mL).

Table 10: Mean ± SD plaque pH values of the test foods/drinks at four different time

points with the corresponding AUC and GI values (n = 12 participants for white bread,

pasta, chickpeas and mashed potatoes, n = 12, with two technical replicates for glucose)

Test food T0 (min.) Mean ± SD

T12 (min.) Mean ± SD

T22 (min.) Mean ± SD

T62 (min.) Mean ± SD

AUC (mmol/L) Mean ± SD

GI

Glucose 7.4 ± 0.3 6.4 ± 0.4 6.2 ± 0.4 7.0 ± 0.3 47.9 ± 22.5 ‒

White bread 7.5 ± 0.3 6.4 ± 0.3 6.0 ± 0.3 6.9 ± 0.1 62.9 ± 19.7 77.0

Pasta 7.3 ± 0.3 6.6 ± 0.2 6.3 ± 0.3 6.9 ± 0.2 41.9 ± 17.4 46.8

Chickpeas 7.4 ± 0.3 7.0 ± 0.3 6.7 ± 0.3 7.0 ± 0.3 30.3 ± 13.8 35.4

Mashed potatoes

7.4 ± 0.4 6.4± 0.7 6.3 ± 0.53 6.9 ± 0.3 47.3 ± 29.2 95.9

GI: glycaemic index. Mean values ± SD of 12 subjects.

35

Table 11: Comparison of plaque pH results produced by all the test foods/drinks to

determine their p-values

Test food p*

Glucose White bread

Pasta Chickpeas Mashed potatoes

Glucose ‒ 0.513 1.000 0.231 1.000

White bread 0.513 ‒ 0.190 0.004 0.792

Pasta 1.000 0.190 ‒ 1.000 1.000

Chickpeas 0.231 0.004 1.000 ‒ 0.553

Mashed potatoes 1.000 0.792 1.000 0.553 ‒

*P-value from ANOVA with Bonferroni correction. aSignificant difference between white bread and chickpeas

(p = 0.004).

Figure 3: Comparison in plaque pH drop after the consumption of four test foods and

control solution: glucose (blue); white bread (green); pasta (brown); chickpeas (purple)

and mashed potatoes (yellow) (for glucose n = 12, with two technical replicates, and for

white bread, pasta, chickpeas and mashed potatoes, n = 12 participants)

36

Figure 4: Comparison of all test foods (chickpeas, pasta, mashed potatoes and white

bread) with the standard glucose drink (25 g/250 mL) at four different time points to

determine their effect on plaque pH (in each figure, the blue line represents the glucose

drink and the green lines represent each test food)

4.2.2 Glucose data

Table 12 shows the results of the glucose concentration measured in 12 participants over a

period of two hours during Sub-study 2. For all foods, plasma blood glucose concentration

peaked at 30 minutes, followed by a drop until 120 minutes (Figure 6). The maximum increase

in postprandial plasma glucose concentration was observed in mashed potatoes, followed by

white bread, pasta and chickpeas. Chickpeas produced the lowest iAUC (47.2 ± 24.4 mmol/L)

and the standard glucose drink showed the highest iAUC (135.0 ± 52.3 mmol/L) followed by

37

mashed potatoes (126.5 ± 46.6 mmol/L). The overall glycaemic responses produced by the

chickpeas and the pasta were significantly lower than the glucose iAUC responses produced

by the glucose solution and the mashed potatoes (Table 12). The glucose response produced

by the chickpeas was significantly lower than the response produced by the white bread (P =

0.026). No other significant differences were observed between the reference food and the four

test foods (Table 13). Figure 7 shows the individual postprandial glucose response comparison

of all the test foods with the standard glucose drink (25 g/250 mL).

Table 12: Plasma glucose concentration (mmol/L) and overall glyceamic response,

quantitified as iAUC (mmol/L.min), after the consumption of all the test foods/drinks at

different time points

Test food T0 (min.) Mean ± SD

T15 (min.) Mean ± SD

T30 (min.) Mean ± SD

T45 (min.) Mean ± SD

T60 (min.) Mean ± SD

T90 (min.) Mean ± SD

T120 (min.) Mean ± SD

iAUC Mean ± SD

Glucose 5.0 ± 0.3

7.8 ± 1.0

8.6 ± 1.0

6.8 ± 1.1

5.1 ± 1.2

4.6 ± 0.6

4.7 ± 0.3

135.0 ± 52.3

White bread

5.1 ± 0.3

6.1 ± 0.3

7.3 ± 0.5

7.0 ± 0.6

5.7 ± 0.6

5.1 ± 0.5

5.0 ± 0.4

98.8 ± 29.4

Pasta 5.1 ± 0.4

6.2 ± 0.7

6.4 ± 0.7

5.8 ± 0.7

5.3 ± 0.5

5.2 ± 0.5

5.1 ± 0.4

61.0 ± 24.1

Chickpeas 5.1 ± 0.3

5.4 ± 0.4

6.0 ± 0.6

6.0 ± 0.4

5.4 ± 0.4

5.1 ± 0.2

5.1 ± 0.3

47.2 ± 24.4

Mashed potatoes

5.1 ± 0.3

7.3 ± 0.6

8.6 ± 0.4

6.9 ± 1.4

5.0 ± 1.3

4.7 ± 0.5

4.8 ± 0.4

126.5 ± 46.6

Mean value ± SD of 12 subjects.

38

Table 13: Comparison of all test foods/drinks (glucose, white bread, pasta, chickpeas

and mashed potatoes) with their corresponding p-values

Test food P*

Glucose White bread

Pasta Chickpeas Mashed potatoes

Glucose ‒ 0.135 < 0.001 < 0.001 1.000

White bread 0.135 ‒ 0.250 0.026 0.977

Pasta < 0.001 0.250 ‒ 1.000 0.002

Chickpeas < 0.001 0.026 1.000 ‒ < 0.001

Mashed potatoes

1.000 0.977 0.002 0.000 ‒

*P-values were calculated by ANOVA with Bonferroni correction.

39

Figure 5: Plasma glucose response curve for all test foods and the control glucose

solution: glucose 25 g/250 mL (blue); pasta (green); chickpeas (brown); white bread

(purple); mashed potatoes (yellow) (maximum rise seen at 30 mins for glucose 25 g

solution followed by mashed potatoes, white bread, pasta and chickpeas, respectively)

40

Figure 6: All the plasma glucose response curves for the test foods (chickpeas, pasta,

mashed potatoes and white bread) compared with the standard glucose drink (25 g

glucose in 250 mL water) over two hours (in each figure, the blue line represents the

glucose drink and the green line represents the test foods)

4.2.3 Correlations between plaque pH and plasma glucose concentration

Figure 8 shows a linear correlation between the AUC of plaque pH and the GI (Table 10) of

all the test foods and drinks included in Sub-study 2. The correlation coefficient was 0.78. A

P-value for the correlation coefficient has not been calculated because of the preliminary and

explorative character of the study and the amount of data available; only a correlation

coefficient was calculated for the test food items.

41

Figure 7: Correlation between the GI and AUC of plaque pH for all test foods/drinks

included in Sub-study 2

4.3 Sub-study 3: Impact of GI in a Food Type on Plaque pH

4.3.1 pH data

Twelve subjects (four males and eight females) with an age range from 21 to 31 years

participated in this sub-study. The pH values were measured after participants consumed the

standard glucose drink (containing 25 g of available carbohydrates) and six test foods: Cereal

A (Cheerios, Uncle Toby’s, Nestle Australia Ltd, North Ryde, NSW, Australia), Cereal B

(Guardian, Kellogg’s Aust. Pty Ltd, Pagewood, NSW, Australia), Bread A (Wonder White,

Goodman Fielder Ltd, North Sydney, SW, Australia), Bread B (white bread, Coles

Supermarkets Pty Ltd, Hawthorn East, VIC, Australia), Rice A (Doongara, Sun Rice,

Ricegrowers Ltd, Leeton, NSW, Australia) and Rice B (Jasmine, Sun Rice, Ricegrowers Ltd,

42

Leeton, NSW, Australia). The consumption of Rice B was followed by a pH values drop of

1.4, whereas Cereal B was followed by a pH values drop of only 0.57 from the baseline to 22

minutes (Table 14). The differences in pH values between food pairs were statistically

significant (Table 15). The pH values started to return to the baseline values after 22 minutes

but did not reach the baseline values after 62 minutes (Figure 9). Figure 10 shows the

comparison of each food pair with the standard glucose drink (25 g/ 250mL).

Table 14: Mean ± SD of plaque pH values of the tested foods at four different time

points with their corresponding AUC values and GI values (n = 12 participants for

Bread A/Bread B, Cereal A/Cereal B and Rice A/Rice B, n = 12; with two technical

replicates for glucose)

Test food T0 (min.) Mean ± SD

T12 (min.) Mean ± SD

T22 (min.) Mean ± SD

T62 (min.) Mean ± SD

AUC (mmol/L) Mean ± SD

GI

Bread A 7.8 ± 0.3 6.9 ± 0.2 6.4 ± 0.3 7.4 ± 0.2 51.7 ±14.6 82.3

Bread B 7.7 ± 0.3 7.0 ± 0.4 6.7 ± 0.5 7.3 ± 0.2 38.4 ± 16.0 60.4

Cereal A 7.8 ± 0.2 6.8 ± 0.3 6.4 ± 0.3 7.3 ± 0.2 51.4 ± 4.71 76.8

Cereal B 7.7 ± 0.4 7.4 ± 0.2 7.2 ± 0.1 7.4 ± 0.1 24.9 ± 5.12 43.1

Rice A 7.7 ± 0.2 7.1 ± 0.3 7.0 ± 0.5 7.3 ± 0.2 31.0 ± 18.1 55.5

Rice B 7.9 ± 0.3 6.9 ± 0.4 6.4 ± 0.3 7.3 ± 0.3 59.2 ± 22.2 84.2

GI, glycaemic index. Mean values ± SD of 12 subjects.

43

Table 15: P-values for the difference in plaque pH across the three pairs of test foods

(Bread A/Bread B, Cereal A/Cereal and Rice A/Rice B)

Test food p*

Bread A 0.032

Bread B

Cereal A 0.006

Cereal B

Rice A 0.001

Rice B

P-value was calculated by ANOVA with Bonferroni correction.

Figure 8: Absolute plaque pH values after consumption of 25 g available carbohydrate

portions of the test foods and control glucose solution: glucose (dark blue); Bread A

(green); Bread B (red); Cereal A (purple); Cereal B (yellow); Rice A (red) and Rice B

(light blue)

44

Figure 9: Comparison of all test foods (Bread A, Bread B, Cereal A, Cereal B, Rice A

and Rice B) with the standard glucose drink at four different time points to determine

their effect on plaque pH AUC (in each figure, the gold line represents the glucose drink

and the blue/green lines represent the test foods)

4.3.2 Glucose data

The plasma glucose concentration reached its highest level after 30 minutes and dropped at

120 minutes (Figure 11). The blood glucose concentration was the highest for Cereal A from

the baseline up until the 30-minute timepoint, whereas Cereal B showed the lowest blood

glucose response at 30 minutes (Table 16). The highest iAUC for the plasma glucose

concentration was observed for Rice B (110.4 ± 31.6 mmol/L). The iAUC for the different

45

food pairs were statistically different (Table 17). Figure 12 shows the comparison of all the test

foods with glucose 25 g/250 mL for the plasma glucose concentration.

Table 16: Mean plasma glucose concentration ± SD and overall glyceamic response

(iAUC) for all the six paired test foods (n = 12 participants)

Test food

T0 (min.) Mean ± SD

T15 (min.) Mean ± SD

T30 (min.) Mean ± SD

T45 (min.) Mean ± SD

T60 (min.) Mean ± SD

T90 (min.) Mean ± SD

T120 (min.) Mean ± SD

iAUC (mmol/L) Mean ±

SD Bread

A 5.0 ± 0.2

6.0 ± 0.5

7.4 ± 0.7

6.9 ± 0.5

5.8 ± 0.8

5.0 ± 0.4

4.9 ± 0.3

104.6 ± 21.7

Bread B

5.0 ± 0.2

5.6 ± 0.3

6.6 ± 0.6

6.3 ± 0.5

5.7 ± 0.4

5.4 ± 0.4

5.1 ± 0.3

79.7 ± 32.1

Cereal A

5.0 ± 0.3

6.5 ± 1.0

7.7 ± 0.9

6.6 ± 0.8

5.3 ± 0.6

5.0 ± 0.6

4.8 ± 0.3

102.3 ± 39.9

Cereal B

5.1 ± 0.2

5.7 ± 0.6

6.2 ± 0.5

5.8 ± 0.5

5.5 ± 0.4

5.3 ± 0.2

5.2 ± 0.2

57.1 ± 24.9

Rice A 5.0 ± 0.3

5.9 ± 0.4

6.8 ± 0.7

5.9 ± 0.4

5.3 ± 0.3

5.3 ± 0.2

5.3 ± 0.2

73.8 ± 27.4

Rice B 5.0 ± 0.2

6.1 ± 0.6

7.4 ± 0.6

6.8 ± 0.7

5.9 ± 0.6

5.4 ± 0.4

5.2 ± 0.2

110.4 ± 31.6

Mean value ± SD of 12 subjects.

Table 17: Comparison of the paired test foods (Bread A/ Bread B, Cereal A/ Cereal B

and Rice A/ Rice B) and their P-values

Test food P*

Bread A 0.008

Bread B

Cereal A 0.001

Cereal B

Rice A < 0.001

Rice B

P-value was calculated by ANOVA with Bonferroni correction.

46

Figure 10: Plasma glucose response curve for all three test food pairs and the control

glucose solution: glucose (dark blue); Rice A (green); Rice B (brown); Cereal A

(purple); Cereal B (yellow); Bread A (red); and Bread B (light blue)

47

Figure 11: Comparison of all test food pairs (Rice A, Rice B, Cereal A, Cereal B, Bread

A and Bread B) with the standard glucose drink (25 g available carbohydrates) at all

measured time points to determine their effect on blood glucose levels (in each figure,

the blue line represents the glucose drink and the green/gold lines represent the test

food)

4.3.3 Correlation between plaque pH and GI

Figure 12 shows a linear correlation between the AUC of plaque pH and the GI (Table 14) of

all the test foods included in Sub-study 3. The correlation coefficient was 0.98. A P-value for

the correlation coefficient has not been calculated because of the preliminary and explorative

character of the study and the number of data available only a correlation coefficient was

calculated for the test food items.

48

Figure 12: The relationship between the GI and the AUC of plaque pH for all six test

food pairs included in Sub-study 3

4.4 Post-hoc power analysis

An exemplary post-hoc power analysis for repeated measures was performed for the AUC data

((mmol/L) Mean ± SD) for the comparison of white bread/chickpeas (sub-study 2) bread A/B,

cereal A/B and rice A/B (sub-study 3). With n = 12 and a probability of an alpha error of 0.05,

a power of 0.9937 (white bread/chickpeas), 0.5292 (bread A/B), 1.000 (cereal A/B) and 0.9018

(rice A/B) was calculated.

4.5 Buffering Capacity

All the subjects who participated in sub-studies 2 and 3 provided a fasting saliva sample to

measure their saliva buffering capacity. Table 18 shows the buffering capacity of all

49

participants, which ranged from very low to normal. Fourteen out of eighteen participants fell

into the very low category.

Table 18: Salivary buffering capacity of all the subjects who participated in sub-studies

2 and 3

Participant no.

pH value Buffering capacity range

1 3.36 Very low

2 2.78 Very low 3 4.70 Normal

4 3.07 Very low 5 2.99 Very low

6 2.88 Very low

7 2.99 Very low 8 3.83 Low

9 2.94 Very low 10 4.09 Low

11 2.92 Very low 12 2.84 Very low

13 2.97 Very low

14 2.72 Very low 15 3.37 Very low

16 2.91 Very low 17 4.24 Low

18 3.25 Very low

50

Chapter 5: Discussion

The objective of this project was to identify the relationship between plaque pH and GI after

the consumption of different starchy food items. It was hypothesised that food items with a

higher GI would show a larger drop in dental plaque pH than food items with a lower GI.

Overall, the study confirmed this hypothesis and showed that high-GI food caused a more

significant drop in plaque pH compared with low-GI food.

In Sub-study 1, it was observed that the amount of glucose sugar (25 or 50 g available

carbohydrates dissolved in 250 mL water) significantly affected the plaque pH curve, with the

maximum drop in plaque pH recorded 12 minutes after the consumption of the glucose drink.

In sub-studies 2 and 3, a standard glucose solution containing 25 grams of available

carbohydrates was selected because test food portions containing 25 grams of available

carbohydrates were more representatives of realistic portion sizes for these foods. Therefore,

the results of the studies demonstrate how starch-based foods may influence dental caries risk

in real-world situations.

In Sub-study 2, four food items containing starch as their primary glucose source were studied:

white bread, mashed potatoes, pasta and chickpeas. Foods selected for this study varied in their

GI, texture and nutritional composition. Chickpeas, which produced the lowest postprandial

glycaemic response, showed the smallest drop in plaque pH compared with the other test foods.

It is likely that their high fibre content (7.8 g/portion) and intact structure slowed down the

digestion of the starch and the release of glucose. A slowly digested food results in lower

postprandial blood glucose levels (91). White bread showed the largest drop in plaque pH and

the second-highest GI, while mashed potatoes showed the highest GI and second-highest drop

in plaque pH values. Both mashed potatoes and white bread are highly processed foods; the

carbohydrates they contain are readily available for digestion and absorption, which contribute

51

to the high GI and larger pH drop compared with the other test foods. Food processing produces

major alterations in the structural and physical properties of the starch, which can result in a

more pronounced drop in plaque pH (83). Another explanation for the pronounced drop in

plaque pH caused by mashed potatoes and white bread is the texture of these foods. Food

texture is an important factor that influences the drop in plaque pH observed following

consumption. Foods with a thick or sticky consistency have the potential to adhere to tooth

surfaces for an extended period, causing greater drops in plaque pH (82). The Vipeholm study

showed that sugar, when consumed in between meals and in a consistency that increases its

retention on the tooth surface, increases caries activity (22). According to this study, bread

consumed once a day did not have as much cariogenic effect as compared to when it was

consumed more frequently during the day. These results are contrary to the present study, as

bread given just once to the participants lowered the plaque pH to a level which could result in

the demineralisation of tooth substance. Stephan, like Vipeholm, observed in one of his papers

published in the early 1940’s that an increased frequency of carbohydrate intake together with

longer duration of its mechanical retention on the tooth surface resulted in more significant

decalcification (92).

In Sub-study 3, three pairs of foods—two types of white bread, two types of white rice and two

types of breakfast cereal—were investigated. To examine the potential influence of GI on

plaque pH, the nutritional composition, texture and portion size were designed to be matched

as closely as possible within each food pair. There were some variations in the fibre quantity

and ingredients within each pair. For example, Cereal A had 2.3 g of fibre, and Cereal B had

7.0 g of fibre per portion. Cereal B’s higher soluble viscous fibre content is one factor that

likely contributed to the low plaque pH drop observed compared with Cereal A. Soluble

viscous fibres help lower GI (93). Low-GI foods release glucose slowly in systemic circulation,

with faster oxidation of fats and slower oxidation of carbohydrates (94). With low-GI foods,

52

slower uptake of nutrients is observed, which results in better metabolism (95). Because of all

these factors, low-GI foods lead to lower postprandial blood glucose levels, which in turn cause

a reduced drop in plaque pH.

The results of this study support our hypothesis that the GI of carbohydrate-containing foods

produces measurable differences in plaque pH drop. Within each food pair, the product with

the known lower GI value caused a smaller plaque pH drop relative to the matched high-GI

food within that pair. One of the potential mechanisms underlying this result is that low-GI

foods have less sucrose than high-GI foods, which produces less acid after fermentation by the

oral bacteria, leading to a reduced drop in plaque pH. Intraoral pH is significantly affected by

an acidic environment that could be produced by extrinsic factors, such as food consumed

during a person’s daily routine (96). Other factors that affect the GI of different foods—for

example, higher quantities of soluble viscous fibres, the presence of proteins and fats, and types

of starch used—also affect dental plaque pH.

The buffering capacity of saliva is another factor that can affect plaque pH and, in turn, caries

formation (97). Buffering capacity is a measure that checks saliva’s ability to counteract the

acidic changes that can influence dental caries development (98). In our studies, 94% of

participants had either low or very low buffering capacity in their fasting saliva sample. The

different levels of buffering capacity achieved greatly depends on the techniques used to

measure buffering capacity (63). We did not include this factor in our statistical analysis

because of the similar intra-individual buffering capacity values and because the saliva samples

were collected on only one occasion. In future studies, we recommend collecting fasting saliva

samples on multiple occasions to rule out whether it affects dental plaque pH in the context of

study being conducted.

53

Our study had a number of methodological strengths. The capillary sampling method used for

measuring postprandial blood glucose levels is the most sensitive to acute changes in glucose

levels (61) and aligns with ISO Standard for determining the GI of foods (60). According to

Lingström et al., (54) who compared three different plaque pH measurement methods—

sampling/scraping, microtouch and telemetric—there are advantages and disadvantages to

each technique. The sampling/scraping method used in our study provides the smallest plaque

pH drop after food is ingested and therefore takes a very conservative approach, potentially

underestimating the true magnitude of acute plaque pH changes in response to starchy foods.

However, participants were all comfortable with this method. The plaque was collected with a

dental probe from tooth surfaces, an act regularly performed by dentists during dental

assessments.

In contrast to other studies, the quantity of digestible carbohydrates was kept constant for all

the foods tested, and the portion sizes used were comparable to the sizes that would be

consumed in real life. Using a controlled amount of digestible carbohydrates across the studies

helped us to elucidate other food factors, such as GI or food texture, that affected the dental

plaque pH changes. The results of our studies suggest that high-GI foods produce a larger

dental plaque pH drop and therefore show greater cariogenic potential. Habitual consumption

of high-GI foods is accompanied by an increased risk of diseases such as type 2 diabetes,

cardiovascular disease and some cancers (99). High-GI foods not only increase the risk of

chronic diseases, but also encourage weight gain and obesity (100). It has been observed in

prior studies that low-GI foods have inhibitory potential regarding insulin resistance syndrome

(12). The most important clinically relevant finding from our study is the correlation between

GI and dental plaque pH. High-GI foods not only have detrimental effects on our general health

but may also have a negative impact on our dental health. Stephan was one of the pioneers who

introduced the potential importance of ‘critical pH’ in 1944, and this was revised by Bowen (8,

54

53). ‘Critical pH’ is defined as a pH that dissolves the enamel in oral fluids (101). Enamel

begins to dissolve rapidly once the pH of dental plaque falls to 5.5 (53). Once consumed,

carbohydrates (mainly sucrose) show a rapid fall in dental plaque pH to 5.0 or below (20). The

reason for this fall is the metabolism of sucrose into acids by oral bacteria, resulting in dental

caries (27). For better general and tooth health, low-GI foods must be part of our daily diet. As

mentioned earlier, GI is a measure of carbohydrate quality. A 2003 report by the World Health

Organization (WHO) on diet, prevention and chronic disease suggested that food plays an

essential role in preventing these diseases, and diet modification must be practised. Foods such

as legumes have been proposed as a suitable choice for patients with diabetes (102). The

addition of nutrients such as novel viscous polysaccharide (103) and ß glucan fibres (104) have

also been shown to play an essential role in reducing the GI of different foods. Replacing higher

GI carbohydrates with lower alternatives would be beneficial to improving dental health and

overall health.

The generalisability of the present results is subject to certain limitations. For measuring the

saliva buffering capacity, only one sample at the end of the study was taken; therefore, for

future studies, repeated sampling of saliva at the time of the experimental procedure is

recommended. This fact, in addition to the storage of the saliva, may be the reason for the

unusually high frequency of ‘low buffering capacity’ of saliva in the current cohort. Because

of limitations in the equipment available, it was not possible to standardise the wet weight of

the dental plaque samples, which reduced our capacity to compare the outcomes of this study

with other research projects. However, we tried to standardize the volume of dental plaque so

that the variability in the amounts of the plaque sample was a random error and only minimally

affected the comparison between the food items tested. The age range of the selected

participants was narrow (18–31 years for sub-studies 2 and 3) and the outcomes of the present

study may not be generalisable to younger or older populations. The low power for the

55

comparison of Bread A and Bread B indicates a high probability of making a type II error

(assuming a difference between the tested foods, while in reality there was no difference). For

future experiments, the power can be further improved by increasing the number of participants

or by reducing the variability in the measurements. This may be achieved by further

standardising the experimental procedures, including a standardised wet weight of the plaque

samples.

Our results have the potential to influence future dental clinical practice guidelines. Starchy

foods are often ignored when dentists are counselling patients about oral care and dental caries

aetiology. Oral health education on dietary intake has traditionally focused on reducing sugary

foods to prevent tooth decay. The results of our studies show that starchy foods, particularly

those that are more rapidly digested in the oral cavity, have a similar or greater potential than

sugary foods to drop plaque pH, and may therefore pose an increased risk of dental caries.

Individuals with high rates of dental caries or poor oral health may benefit from consuming

diets containing carbohydrates that are slowly digested and absorbed, such as low-GI foods, to

help alleviate symptoms or reduce the further incidence of dental caries.

Although this study is unique because it measures the drop in dental plaque pH and increase in

postprandial blood glucose levels simultaneously in great detail, some improvements can still

be made. Further experiments with a larger sample size and participants susceptible to dental

caries would shed more light on this specific field. The studies in this thesis were conducted

with individuals who had good oral hygiene practices and were relatively young and healthy,

but it might be interesting to see how starchy food items potentially influence plaque pH in a

more at-risk population. Alternatively, other than acute intervention studies (like those

included in the report), it could be interesting to conduct a chronic feeding study to identify the

impact on oral health if susceptible individuals followed a low-GI versus a conventional

56

(higher fibre, high-GI) diet. More information on measuring the buffering capacity of saliva

would help us to establish a greater degree of accuracy in this matter. The results of our study

could contribute to shaping dental clinical practices by providing greater insight on the use of

high carbohydrate foods and their effect on decreasing dental plaque pH and causing

demineralisation of the teeth.

57

Chapter 6: Conclusion

This study was designed to determine the effect of food with different GI values on changes in

dental plaque pH. The most significant finding from this study is that high-GI foods produce a

larger drop in dental plaque pH than low-GI food. The second major finding is that, regardless

of their texture, quantity or cooking conditions, high-GI foods will drop dental plaque pH. The

evidence from this study suggests that high-GI foods are not only harmful to health in general

but also have the potential to cause dental caries. This project is the first comprehensive

investigation of the relationship between GI and dental plaque pH. This new understanding

should help health care practitioners to provide information to consumers about the impact of

food high in GI on dental health.

58

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