Nanocellulose as a Negative Calorie and Cholesterol

Controlling Food Additive

Matthew Stewart Michael Fenwick-Nevin Australian Pulp and Paper Institute Chemical Engineering Department Monash University

Executive Summary Lifestyle diseases such as type II diabetes, cardiovascular disease and obesity are growing issues

amongst the global population. Obesity is currently one of the most serious worldwide health issues,

affecting more than a billion people globally. Current treatments for lifestyle diseases include exercise

and dietary modifications such as decreasing portion sizes and decreasing high-calorie food and drinks.

The objective of this research project is to assess the feasibility of creating a nanocellulose based food

additive capable of preventing energy and cholesterol absorption from foods, which not only could

help prevent and treat obesity and other lifestyle diseases, but could also allow individuals to indulge

in foods they normally wouldn’t eat, avoiding weight gain and negative health effects.

In order to achieve the objectives of this project and determine if nanocellulose can be used as a

negative calorie and cholesterol controlling food additive, a systematic methodology was used in order

to determine the interaction of nanocellulose with various food constituents. The figure below

highlights the way in which this project was broken down.

Figure 1: Project Flow Chart

It was hypothesised prior to the commencement of this project that glucose and starch oligomers can

be adsorbed onto nanocellulose fibres. It was also hypothesised that nanocellulose in the diet can

reduce blood cholesterol levels. In order to prove these hypothesis, both experimental work and an

extensive review of literature were conducted.

A literature review relating to glucose interaction with cellulose yielded no results to suggest that

glucose had previously been found to adsorb to cellulose. Experimental work was conducted using

glucometry to measure glucose concentration before and after the addition of nanocellulose. These

experiments confirmed that glucose does not adsorb to nanocellulose.

Cellulose

Energy

Monosaccharides

Glucose

Disaccharides

Sucrose

Polysaccharides

Starch

Fats

Cholesterol Triglycerides

An extensive review of literature found previous works had concluded that sucrose does not bind to

cellulose, and given that this had been previously determined experimental work was not conducted in

order to investigate the adsorption of sucrose to nanocellulose. The conclusion that glucose and

sucrose do not bind to nanocellulose was based on experiments using native forms of nanocellulose,

therefore one recommendation from this project was to conduct future experiments with chemically

modified nanocellulose and test the adsorption of glucose and sucrose to these chemically modified

molecules.

Previously published literature was found that concluded starch adsorbs to the surface of cellulose.

We used the sulphuric acid-phenol method of starch content determination in order to test whether

this result is also achieved with nanocellulose. The results of our experimental work investigating the

adsorption of starch to nanocellulose concluded that starch does bind to nanocellulose, as was

hypothesised. Further experiments were conducted investigating how the action of α-amylase impacts

upon the starch adsorbed to nanocellulose, however unfortunately the results of these experiments

were inconclusive due to the lack of sensitivity of the glucometry method used for sample analysis. As

a result another recommendation for future works is to repeat these experiments, however using a

more sensitive analytical method such as high performance liquid chromatography.

A review of literature found that increasing nanocellulose in the diet results in a reduction of

cholesterol levels in the blood, as well as reducing the likelihood of developing colon cancer. It was

also determined through reviewing literature that fructans can reduce the levels of cholesterol and

triglycerides in the blood. These concepts were only investigated on a theoretical level and therefore

it is recommended that future works confirm experimentally that nanocellulose and fructans can

reduce blood cholesterol and triglyceride levels.

In conducting this research project we were able to achieve our objective of investigating the use of

nanocellulose as a negative calorie and cholesterol controlling food additive. Although not all of our

results supported our hypothesis, many did, and those that didn’t based on these preliminary

experiments, may prove to align better with our hypothesis if further experiments are conducted.

Therefore, overall our project was a success and the use of nanocellulose as a negative calorie and

cholesterol controlling food additive still remains a valid proposal, however requires further

investigation.

Table of Contents Executive Summary ........................................................................................................................................... 1

1. Introduction ................................................................................................................................................... 4

2. Project Definition ........................................................................................................................................... 5

2.1. Problem Statement ................................................................................................................................ 5

2.2. Hypothesis .............................................................................................................................................. 5

2.3. Project Objectives ................................................................................................................................... 5

3. Literature Review .......................................................................................................................................... 7

3.1. Glucose ................................................................................................................................................... 7

3.2. Sucrose ................................................................................................................................................... 7

3.3. Starch ...................................................................................................................................................... 8

3.4. α-Amylase ............................................................................................................................................... 9

3.5. Cellulose ................................................................................................................................................. 9

3.6. Interactions between Starch, Cellulose and Amylase .......................................................................... 10

3.7. Fructans ................................................................................................................................................ 11

3.8. Cholesterol ............................................................................................................................................ 12

3.9. Bile Acids ............................................................................................................................................... 13

3.10. Significance of the Relationship between Bile Acids and Cholesterol ............................................... 13

3.11. Overview of the Human Gastrointestinal System .............................................................................. 13

3.12. Glucometry ......................................................................................................................................... 15

3.13. Starch Quantification Techniques ...................................................................................................... 16

3.14. Kinetics ............................................................................................................................................... 17

4. Methodology ............................................................................................................................................... 18

4.1. Glucose to Nanocellulose Binding Experiments ................................................................................... 18

4.2. Starch to Nanocellulose Binding Experiments ...................................................................................... 21

4.3. Effect of Starch Binding to Nanocellulose on α-Amylase Action Experiments ..................................... 22

4.4. Literature Review ................................................................................................................................. 24

5. Key Results and Findings ............................................................................................................................. 25

5.1. Glucose Binding to Nanocellulose ........................................................................................................ 25

5.2. Sucrose Binding to Nanocellulose ........................................................................................................ 33

5.3. Starch Binding to Nanocellulose ........................................................................................................... 33

5.4. Effect of Starch Binding to Nanocellulose on α-Amylase Action .......................................................... 35

6. Conclusions and Recommendations ............................................................................................................ 36

7. Acknowledgements ..................................................................................................................................... 37

8. References ................................................................................................................................................... 38

1. Introduction Obesity is currently one of the most serious worldwide health issues, affecting more than a billion

people globally (1). It is quickly growing in the younger generations with more than 22 million of

the world’s children under 5 years old being either overweight or obese. There are a variety of

severe health conditions that are associated with being overweight or obese. These include; sleep

apnea, type II diabetes, hypertension, hyperlipidaemia and increased risk of developing

cardiovascular disease (CVD) (2). CVD is a major health risk that has a high mortality rate and has

been the leading cause of death in the United States since the early 1900’s (3). Due to these

factors obesity can have a large impact on expected life span, particularly in younger children,

reducing their life expectancy by up to as many as 20 years (2).

These rising risk factors create the need to implement and devise solutions to combat the growing

obesity pandemic. Current treatments include exercise and dietary modifications such as

decreasing portion sizes and decreasing high-calorie food and drinks (2). The basis of this research

project is to assess the feasibility of creating a nanocellulose based food additive capable of

preventing energy and cholesterol absorption from the food. If this end product is realized, not

only will this help prevent and treat obesity and other diseases associated with being overweight,

but it will also allow individuals to indulge in foods they normally wouldn’t eat, avoiding weight

gain and negative health effects.

2. Project Definition

2.1. Problem Statement

To research the capability of nanocellulose to adsorb carbohydrates (monosaccharides,

disaccharides and polysaccharides), as well as investigating the capability of nanocellulose and

other dietary fibres (e.g. fructans) to regulate cholesterol levels. This research will aid in the

development of nanocellulose as a negative calorie and cholesterol controlling food additive.

2.2. Hypothesis It is hypothesised that glucose and starch oligomers can be adsorbed onto nanocellulose fibres.

The inability of the human gastrointestinal tract to absorb cellulose will cause the glucose and

starch oligomers bound to the nanocellulose to be expelled rather than absorbed, hence removing

potential calories from foods consumed. It is also hypothesised that nanocellulose has the

potential to control the levels of cholesterol related elements in the human digestive tract. The

performance of different nanocellulose varieties is hypothesised to be primarily determined by the

surface area available for binding to glucose, starch and cholesterol.

2.3. Project Objectives The objectives of this project were initially broadly defined as follows:

1. To design and investigate the effectiveness of an experimental system used to measure and

analyse the adsorption of glucose and starch onto nanocellulose.

2. To conduct a review of literature relating to the digestion of carbohydrates and cholesterol in

the human gastrointestinal tract.

As the project progressed, the main objective of the project was refined and can be defined as

follows:

- Through the review of literature and conducting laboratory experiments, investigate the

validity of the concept that nanocellulose can be used as a negative calorie and cholesterol

controlling food additive.

In conducting a literature review relating to the digestion of carbohydrates and cholesterol in the

human digestive system, we aimed to determine multiple characteristics of the system. Of specific

interest were the conditions under which glucose, starch and cholesterol are digested and

absorbed. Such conditions include the temperature, pH, enzymes required and location of

digestion and absorption in the gastrointestinal tract for the different components of ingested

food. We also aimed through our literature review to determine the retention time of glucose,

starch and cholesterol in regions of the gastrointestinal tract such as the stomach and the small

intestine. The primary outcome of achieving the objectives of this literature review were to

provide specific details of the conditions to which glucose, starch and cholesterol are subjected in

the human digestive system. This will allow for further experiments investigating the adsorption

of these molecules to nanocellulose to be analogous to reactions occurring in the human

gastrointestinal tract.

Through conducting laboratory experiments we aimed to gather data that was concordant with

literature and previous experiments, as well as determining and proving on a practical level, that

the concept of nanocellulose being used as a negative calorie food additive is valid. The results of

such experiments, whether proving or disproving the concept, will provide a basis for future

research in this area, saving time and money for future projects. The results of these experiments

will also provide insight into potential changes that could be made to the methodologies used in

this project, allowing for recommendations regarding the specifics of future works relating to

using nanocellulose as a negative calorie food additive.

The figure below illustrates a break down of the various areas that were researched in order to

achieve the objectives of this project as stated above.

Figure 2-3-1: Project Flow Chart

Cellulose

Energy

Monosaccharides

Glucose

Disaccharides

Sucrose

Polysaccharides

Starch

Fats

Cholesterol Triglycerides

3. Literature Review Due to time constraints placed on this project it is difficult to investigate the entirety of the scope

experimentally. Therefore a literature review is necessary to give sufficient background to the

experimental work as well as explore avenues that would be too time intensive to conduct in the

laboratory.

3.1. Glucose

Glucose is essential to human life, providing energy to sustain physiological functions. It is readily

broken down to create ATP, an energy molecule, by either aerobic or anaerobic respiration. The

diet is the crucial source of glucose, where it is either directly absorbed from the food in the small

intestine or synthesised via the gluconeogenesis pathway from precursors obtained from the diet

(4).

Glucose is the only fuel used to any significant extent and is the sole energy source used by the

brain. Glycolysis, the process used to extract the chemical energy contained within the glucose

molecules, is an extremely important function within the body and malfunctions in this process

can lead to a variety of serious diseases. However, when too much glucose is ingested in the diet

it can also lead to the development of disorders like insulin resistance and diseases associated

with severe weight gain (5).

Diabetes has been linked with hyperglycemia, which has seen a recent increase due to the

increasing popularity of high energy density Western-style diets (6).

Although glucose is the building block of carbohydrates, there are also many other sources of

carbohydrates from which the body can gain energy.

3.2. Sucrose

Sucrose is a disaccharide consisting of two monosaccharides, glucose and fructose. These two

monosaccharides are condensed at their glycosidic groups and form a bond known as a glycosidic

bond, forming the disaccharide sucrose. Sucrose is commonly found around the house as table

sugar and is digested in the gastrointestinal tract by the enzyme sucrase, which catalyses the

hydrolysis of the glycosidic linkage of the sucrose molecule, breaking it down into its monomeric

components (7).

Figure 3-2-1: Sucrose, a Disaccharide of Glucose and Fructose (8)

Sucrose was used by Fernandez et al. in an investigation into the retention of fatty acid soaps

during paper recycling. In this experiment sucrose was used as a tracer molecule for a packed bed

experiment. The packed bed was filled with pulp fibres and sucrose was selected as a tracer

molecule due to the fact that it is unable to bind to cellulose (9).

3.3. Starch

Starch consists of two glucose polymers, amylose (a long, unbranched molecule) and amylopectin

(a highly branched molecule) (10). The structures of these polymers are depicted in Figure 3-3-1

and Figure 3-3-2 below. Starch is typically ingested in our diet in the following forms (11):

Cereals: corn, oats and flour

Root vegetables: potatoes, carrots, parsnips

Rice and pasta

Figure 3-3-1: Structure of the Amylopectin Component of Starch (12)

Figure 3-3-2: Structure of the Amylose Component of Starch (12)

There is much energy to be gained from the ingestion of starch in the diet. However, unmodified

it is insoluble and requires the action of certain catalysts before it can be properly utilised by the

body.

3.4. α-Amylase

Complex carbohydrates, including starch, cannot be absorbed in the intestine in their native

states. Nearly all of the carbohydrates ingested must be first hydrolysed into their corresponding

monosaccharides (with the exception of a few disaccharides) before they can be absorbed. There

are hydrolytic enzymes that are present in the saliva and pancreatic secretions that are capable of

achieving this. The most abundant of these is 𝛼-amylase, which breaks links in the starch

molecule 𝛼-(1-4) bonds, in a random fashion to break down the starch constituents of amylose

and amylopectin (13) (14) (11) (10). Salivary 𝛼-amylase hydrolyses approximately 50% of starch in

the diet, however it has an optimum pH of around 7.0, causing it to be inactive once it reaches the

stomach. After the food passes through the stomach, pancreatic 𝛼-amylase is released in a much

larger quantity compared to the salivary 𝛼-amylase, which acts to break down the remaining

starch (11) (13). From previous research, 𝛼-amylase is the most commonly used hydrolytic

enzyme to mimic the break down of starch in vitro, which was the basis of investigating its activity

in this research project (14).

Even though there are many enzymes that facilitate digestion within the gastrointestinal tract,

there are some carbohydrates that are unable to be hydrolysed within the body to gain energy, a

property that this project aims to take advantage of.

3.5. Cellulose Similar to starch, cellulose is a polysaccharide also consisting of glucose monomers and is the most

abundant polysaccharide produced in nature (12). Based on the fact that both starch and

cellulose are formed from the common building block of glucose monomers, we construct our

hypothesis that starch will actively bind to cellulose in the human gastrointestinal tract (15).

Cellulose is the fibre that supports plants, mostly found in cell walls. The glucose monomers that

make up cellulose are configured in such a way that cellulose is resistant to hydrolysis via the

hydrolytic enzymes present in the gastrointestinal tract, unlike starch (16). This means that

cellulose isn’t actually digested, but still plays an important role in providing bulk to stimulate

intestinal motility, preventing constipation (13). This property of cellulose is what allows us to

predict that starch or glucose bound to the cellulose will be excreted rather than digested.

Figure 3-5-1: Structure and Hydrogen Bond Interactions of Cellulose (12)

The previous sections of the literature review outline the underlying roles of starch, cellulose and

𝛼-amylase. We wish to analyse whether interactions between these molecules will affect the

outcomes of the project.

3.6. Interactions between Starch, Cellulose and Amylase It is known that starch has the capacity to bind to cellulose. As starch and cellulose are both

polymers sharing a common monomer in glucose, it is thought that the binding mechanism is due

to surface interaction between the molecules. The amylose contained within starch has a very

similar structure to cellulose, it is characterised by 𝛼-1,4 linkages between glucose units whereas

cellulose is characterised by 𝛽-1,4 linkages between glucose monomers. This results in amylose

being a more flexible molecule, forming natural helical twists which can form a collapsed helix

under certain conditions while cellulose is more rigid and flat, giving strength properties to the

cellulose fibres (17).

As explained previously, starch consists of amylose and amylopectin. Van de Steeg et al. showed

that amylose binds preferentially to cellulose due to its smaller size and more linear structure.

Amylopectin was unable to penetrate the pores of the microcrystalline cellulose as it is a larger,

highly-branched molecule (18).

From experimental analysis of the kinetics of starch adsorption onto cellulose it has been found

that the adsorption mechanism follows Langmuir kinetics further confirming the importance of

surface interaction between the two molecules (19) (18).

As 𝛼-amylase is the main enzyme for breakdown of starch, there is particular focus on this enzyme

in this report. An important factor involved in the interaction of starch and 𝛼-amylase is the

surface area to volume ratio of the starch molecules (as it is much larger than the enzyme) and

therefore particle size (10). Another key variable affecting the starch and 𝛼-amylase interaction is

the degree or order of 𝛼-glucan chains of the starch, with 𝛼-amylase binding most readily to the

exposed/available amorphous 𝛼-glucan chains (20).

An objective of this project is to assess the use of cellulose to prevent the digestion of starch via a

surface binding mechanism. It is currently unknown how the interaction between 𝛼-amylase and

starch will affect the capacity of cellulose to maintain the bound starch on its surface, which will

require further investigation.

Although the experimental scope of this project is to evaluate the use of nanocellulose as a food

additive, in this literature review we have also investigated the use of alternative dietary

substances, which have positive health effects, performing a similar function to that of cellulose.

3.7. Fructans

Fructans are polysaccharides of fructose molecules and can be classified under two main types;

fructooligosaccharides and inulins. Fructooligosaccharides are fructan polymers of shorter chain

length, whilst the inulin types are of longer chain length. Typical foods that contain fructans

include wheat, onion, banana and leek (21).

Fructans are non-digestible carbohydrates and act as dietary fibres; they have been shown to have

positive health benefits, being classified as a prebiotic (22).

Unlike starch, they do not bind to other carbohydrates, however studies on inulin-type fructans in

animals and man have shown that they have the potential to reduce plasma levels of

triacylglycerols (TG). Inulin has also been linked to reducing levels of cholesterol in the

bloodstream. Inulin-type fructans have been shown to have a greater positive effect on the

reduction of TG levels than oligofructose (23).

Because fructans are non-digestible carbohydrates, they pass through the small intestine and

undergo the fermentation process within the large intestine, producing propionic acid. Increased

levels of propionic acid cause a reduction in liver lipogenesis, resulting in a lower hepatic secretion

rate of TG. It is thought that through this mechanism, fructans have the ability to reduce TG and

cholesterol levels as well as through the propionic induced inhibition of cholesterol synthesis and

modifications to bile acid metabolism (23).

The use of fructans were investigated in this report due to their functional characteristics of being

non-digestible, therefore not contributing to dietary energy intake, and their ability to reduce

plasma TG and cholesterol levels. Cholesterol is a focus point of this report due to the strong links

between high blood-cholesterol and serious health issues.

3.8. Cholesterol High blood-cholesterol is an important risk factor for obesity and CVD (3) (2). Cholesterol is

transported around the body via the bloodstream in complexes known as lipoproteins, due to the

fact that cholesterol is a lipid and is therefore insoluble in water. There are two main forms of

lipoproteins, high-density lipoproteins (HDL) and low-density lipoproteins (LDL) (24). LDL’s carry

cholesterol to areas of the body where they are required, while HDL’s transport cholesterol back

to the liver and have a higher proportion of proteins. High LDL cholesterol levels are a health risk

associated with coronary heart disease and CVD, with build-up of LDL cholesterol in arteries being

a major cause of heart attack and stroke (25) (26) (24) (27). Although dietary cholesterol intake

should be regulated, the majority of dietary cholesterol is passed through the gastrointestinal

system with only approximately 20-50% being absorbed (25) (13). High blood-cholesterol levels

are mainly attributed to a high dietary intake of saturated fats, which are then synthesised into

cholesterol (25) (24).

Figure 3-8-1: Structure of the Sterol Cholesterol (13)

The major pathway for excretion of cholesterol from the body takes place in the liver and is via the

bile acid synthesis pathway (16) (26). Of the approximate 1 gram of cholesterol consumed by the

body each day, half of this is degraded to bile acids while biliary cholesterol excretion is

responsible for the loss of the remainder (26).

3.9. Bile Acids Bile acids have several functions (16):

Triglyceride assimilation

Induction of bile flow

Lipid transport

Bile acid synthesis

Water and electrolyte secretion

There are two classes of bile acids in the body; primary and secondary bile acids. Primary bile

acids (cholic acid and chenodeoxycholic acid) are synthesised by hepatocytes in the liver, while

secondary bile acids (deoxycholic acid and lithocholic acid) are formed by bacterial

dehydroxylation of primary bile acids in the intestines (13). Literature exists which suggests that

increased levels of secondary bile acids are a risk factor associated with colon cancer (28).

3.10. Significance of the Relationship between Bile Acids and Cholesterol Experiments have determined that cellulose can aid in binding to secondary bile acids, allowing

them to be excreted as well as accelerating intestinal transit (due to higher faecal weight). This

minimises the time available for primary bile acids to be converted to secondary bile acids, thus

decreasing the risk of developing colon cancer (29).

Previous research has also suggested that added cellulose in the diet increases bile acid

production and excretion and therefore decreased cholesterol levels in the blood as well as

reducing lipid and TG levels (30) (31) (29) (32). For these reasons we have decided to analyse the

relationship between cellulose and cholesterol in conjunction with the glucose and starch

experiments (33).

3.11. Overview of the Human Gastrointestinal System In this research project we will primarily focus on 3 major sections of the gastrointestinal system;

the stomach, the small intestine and the large intestine. However a larger focus will be on the

stomach and small intestine as the absorption of a majority of compounds occurs in the small

intestine (34).

3.11.1. Stomach

The stomach receives and can temporarily store food, its function is to partially digest food and

pump it through to the duodenum region of the small intestine. While food is contained in the

stomach, parietal juice (an acidic solution) and an alkaline solution are released from the mucous

cells of the stomach (16) (13). Hydrochloric acid is released as part of the acidic secretions.

Although hydrochloric acid can aid in breaking down muscle fibres and connective tissue, the main

purpose of its secretion is to lower the pH of the stomach. This allows for the enzymatic activation

of pepsin and more importantly kills harmful organisms, protecting the body from infection (16).

The pH of the stomach changes depending on what stage of the digestion cycle it is in. Typically

when there is food present within the stomach, the secretion of hydrochloric acid will lower the

pH down to around 1-2. However, after the food has been digested the stomach pH may increase

to 3-5. As the stomach is very flexible, the volume usually depends on the amount of material

present. The stomach can have a volume anywhere between 0.5-5 litres (16) (13). The retention

time of material within the stomach is highly dependent on many factors including the type and

amount of food. Food can remain in the stomach anywhere between 1-2 hours after which it is

pumped via muscular contractions into the duodenum. The rate of gastric emptying is dependent

on the volume of materials within the stomach, with a higher stomach volume corresponding to a

higher rate of gastric emptying (16). It should be noted that very few substances are absorbed in

the stomach and it is virtually impermeable to water. Some of the few substances that are

absorbed from the stomach are aspirin and ethyl alcohol (13).

3.11.2. Small Intestine

The small intestine is approximately 3.5 cm in diameter and about 6 metres long, of which the

duodenum occupies the first 25 cm (13). The duodenum is where the mixing of digestive juices

from the liver and the pancreas occurs and is also where bile acids are excreted. However,

intestinal juices are secreted along the entire length of the small intestine. Approximately 95% of

the water which enters the gastrointestinal tract is absorbed in the small intestine, as are glucose

and cholesterol (16) (13). The retention time of the small intestine is slightly longer than that of

the stomach, with typical times ranging between 105-135 minutes (16). Unlike the stomach, the

typical pH range of the small intestine is more neutral to alkaline, rather than acidic. This is

because the action of pancreatic enzymes and lipid absorption require alkaline pH (13).

After the food mass has passed through the small intestine where water and nutrients are

absorbed it continues on and enters the large intestine or colon.

3.11.3. Large Intestine

Compared to the small intestine the large intestine is relatively short, being only 1.5 metres in

length. However, it has a larger diameter of around 6 cm and spans from the end of the small

intestine (ileum) to the rectum where the material that remains is expelled from the body (16)

(13). The main function of the large intestine is to store faecal matter and regulate its release

from the body. The large intestine also acts to absorb water and electrolytes from the chyme, as

well as lubricating the passage for waste material to move through with ease (13). The retention

time of the large intestine is long when compared to that of the stomach and small intestine.

Although food can pass through the stomach and small intestine in less than 12 hours, remnants

of the food can remain in the large intestine for up to a week. However, the majority of it is

expelled by the 4th day after ingestion (13). Although the large intestine is still of interest in this

project, it will not be a major focus as the compounds we are placing emphasis on are not

absorbed in this region of the gastrointestinal tract.

3.12. Glucometry

Various methods exist for the quantification of glucose concentration, with common techniques

including High Performance Liquid Chromatography (HPLC), the Fehling test and enzymatic assays

(35). These methods, though effective in quantifying glucose concentration, are hindered by

disadvantages in that they can be expensive, time-consuming and technically difficult. Ideally

glucose concentration determination in a laboratory setting (as well as in a clinical setting) should

be inexpensive, fast, convenient and reliable.

Blood glucose monitors were developed in order to provide a method of glucometry for diabetics

that achieves an inexpensive, fast and convenient means of determining blood glucose

concentration. In recent studies these glucometers have been used in a laboratory setting in order

to measure glucose concentration in solutions other than blood. The Accu-Chek Performa Blood

Glucose Monitor was used by Sopade and Gidley (36) during their research into the kinetics of

starch digestion, and was also used by Blazek and Gilbert (14) when investigating the enzymatic

digestion of starch by α-amylase.

Two enzymes are commonly used for blood glucose monitors, namely glucose oxidase and glucose

dehydrogenase (35). These enzymes oxidise glucose molecules to gluconic acid and

gluconolactone respectively. The Accu-Chek Performa Blood Glucose Monitor is an example of a

glucometer that uses glucose dehydrogenase enzymes in order to determine the glucose

concentration of a sample. This glucometer uses test strips upon which glucose dehydrogenase is

immobilised. In the presence of the coenzyme pyrroloquinoline quinine, glucose dehydrogenase

oxidises glucose to gluconolactone which creates a DC signal that is converted to a digital display

of the glucose concentration in the solution on the glucometer screen. The Accu-Chek Performa

test strips require approximately 0.6 µL of solution and produce a response in approximately 5

seconds. The glucometer has a glucose concentration range of 0.6-33.3 mM, a temperature range

of 6-44oC and can be used at relative humidity ranging from 10-90% (36).

A comparison of blood glucose monitors in Australia (37) found that the glucometers available in

Australia all show acceptable precision when determining the glucose concentration in blood. The

accuracy and precision of some monitors was found to at times be compromised for glucose

standard solutions, suggesting that researchers need to be careful when using glucometry as a

method of determining glucose concentration in solutions which are not blood-based. A study

which examined the utility of blood glucose monitors in biotechnological applications (35)

determined that when using glucometers it is important to consider the pH, expected

concentration range and presence of other sugars when selecting a glucometer for the

determination of glucose concentration for biotechnological studies. The research of Sopade and

Gidley suggests that the use of glucometry can be extended to analysing the kinetics of starch

digestion (36).

3.13. Starch Quantification Techniques The quantification of the amount of starch in solution has been conducted by various researchers

in the past (38) (39). Determination of the amount of starch that can adsorb to various materials,

such as glass and pulp fibres for example, has been investigated by determining the amount of

free starch in solution before and after introduction of the material to which the starch adsorbs.

The difference in the free starch levels quantifies the amount of starch that is bound to the

material.

Simple sugars, oligosaccharides and polysaccharides (such as starch) produce an orange-yellow

colour when they are exposed to phenol and concentrated sulphuric acid (40). By measuring the

absorbance of starch samples that have been exposed to the phenol-sulphuric acid technique at

490 nm, the concentration of the total starch in the sample can be determined using the Beer-

Lambert Law. This method for determining starch concentration is simple, rapid and sensitive,

using reagents which are stable and relatively cheap. The method requires only one standard

curve and the colours that are produced by the reaction of starch with phenol and sulphuric acid

are stable and permanent (40).

As previously discussed, starch contains two different branched molecules, namely amylose and

amylopectin. The phenol-sulphuric acid technique only quantifies the total starch concentration

of a solution and does not determine the amount of amylose or amylopectin in a sample. The

amount of amylose in solution can be determined by quantifying the iodine binding capacity of the

starch. When amylose in aqueous solution is exposed to iodine it forms a blue coloured helical

complex. By measuring the absorbance of this blue solution the Beer-Lambert Law can be used in

order to determine the concentration of amylose in the solution. By combining the iodine

technique and the phenol-sulphuric acid technique, the amylopectin concentration in solution can

also be determined as this is equal to the difference in concentration of starch and amylose (41).

3.14. Kinetics

The kinetics of adsorption and desorption of materials onto pulp fibres have been investigated by

other researchers. During their research into the adsorption and desorption kinetics of calcium

carbonate onto pulp fibres, Kamiti and Van De Ven modelled these kinetics with a modified

Langmuir equation (42). The same model equation was used by Saint-Cyr, Van De Ven and Garnier

when investigating the kinetics of paper yellowing inhibitors onto pulp fibres (43). The model

equation is shown below:

𝑑𝜃

𝑑𝑡= 𝑘1(𝑛0 − 𝜃) − 𝑘2𝜃 𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 1

In the equation above, θ represents the fractional coverage of fibres by the adsorbed particles, k1

represents the rate of adsorption, k2 represents the rate of desorption and n0 represents the initial

concentration of adsorbing particles, divided by the maximum amount that can deposit in a unit

volume of suspension (42) (43).

The kinetics relating to the binding of starch by α-amylase have also previously been researched.

Warren, Butterworth and Ellis found in their study into the surface structure of starch that the

kinetics of pancreatic α-amylase binding to granular starch molecules could be modelled by

Freundlich enzyme kinetics (10).

4. Methodology In order to achieve the objectives of this research project, the methodology detailed in the

following sections was carried out over a twelve week period.

4.1. Glucose to Nanocellulose Binding Experiments

In order to investigate the potential of glucose to adsorb to nanocellulose, we initially conducted a

literature search in order to determine if previous published works had found evidence of glucose

binding to nanocellulose. After conducting this literature review we carried out a series of

experiments using glucometry in order to measure glucose concentration and determine if glucose

adsorbs to nanocellulose. These experiments are briefly described below.

The glucometer used during these experiments was the Accu-Chek Performa Blood Glucose

Meter. As discussed previously in the Section 3.12, this glucometer uses test strips upon which

glucose dehydrogenase is immobilised. In the presence of the coenzyme pyrroloquinoline quinine,

glucose dehydrogenase oxidises glucose to gluconolactone which creates a DC signal which is then

converted to a digital display of the glucose concentration in the solution on the glucometer

screen. The Accu-Chek Performa test strips require approximately 0.6 µL of solution and produce

a response in approximately 5 seconds. The glucometer has a glucose concentration range of 0.6-

33.3 mM, a temperature range of 6-44oC and can be used at relative humidity ranging from 10-

90%.

Prior to conducting experiments investigating the adsorption capability of glucose onto

nanocellulose we first conducted a calibration experiment in order to find a correlation between

the glucose concentration reported by the glucometer and the known glucose concentration. In

order to conduct this experiment we first created a 15.07 mM glucose solution in 100 mL of

deionised water. We then measured the concentration of this solution with the Accu-Chek

Performa, taking repeated measurements approximately every 5 minutes for a duration of

approximately 30 minutes. The measurements were conducted by dipping the test strip of the

glucometer into the beaker containing the glucose solution. We repeated this experiment using a

2.02 mM glucose solution. The results of these experiments are discussed in Section 5.1.

Due to unexpected results produced from our calibration experiment, we repeated the calibration

experiment changing the methodology slightly each time. In all instances we made a 15 mM

glucose solution in 100 mL of deionised water. The first variation we made to the methodology

was to extract the glucose sample from the beaker using a clean syringe and then transferring the

sample by syringe to the glucometer test strip. This was conducted with the aim of collecting a

sample likely to be more indicative of the entire glucose solution, rather than only testing the

glucose solution at the surface. Unfortunately this did not eliminate the unexpected results

produced by the glucometer, which as explained in more detail in Section 5.1, was reporting a

continuous increase in glucose concentration with time, exceeding the known glucose

concentration.

Another modification to the method involved collecting the glucose solution from different

locations within the beaker of solution. This aimed to eliminate concentration spacial variation.

We also measured the concentration of the glucose solution in the presence and absence of the

magnetic stirrer, immediately after stirring, after waiting for the solution to settle after stirring and

when mixed with Bovine Serum Albumin (BSA). None of these methods were successful in

eliminating the increase in measured glucose concentration with time. We also attempted mixing

the 15 mM glucose solution with blood samples provided by APPI in an attempt to eliminate the

time variation.

Having attempted all of the above variations to the calibration experiment method, a 15 mM

glucose solution was created in 100 mL of deionised water and allowed to mix overnight. The

Accu-Chek Performa was then used to measure the glucose concentration and the increase in

glucose concentration was found to have been eliminated, allowing for a calibration curve to be

created as shown in Section 5.1.

Having successfully conducted the calibration experiment, the capability of glucose to bind to

nanocellulose was tested by again creating a 15 mM glucose solution and allowing it to mix

overnight. Once this mixing was complete, 3.726 g of VTT nanocellulose was added to the 15 mM

glucose solution and mixed overnight. 4 mL of the glucose and nanocellulose solution was then

transferred by syringe to a microtube, allowing for a titre experiment to be carried out.

This experiment involved taking 2 mL out of the “neat” microtube (i.e. the tube containing 4 mL of

the master solution) and adding it to a microtube containing 2 mL of deionised water. Once this 2

mL of the glucose and nanocellulose solution mixed with the water (making a diluted 4 mL

solution) 2 mL of this new solution was extracted and added to another microtube containing 2 mL

of deionised water. This procedure of dilutions was continued until a dilution which was one part

nanocellulose/15 mM glucose solution and 63 parts deionised water was created. The diluted

solutions in the microtubes were then analysed by the glucometer for glucose concentration.

These concentrations were then compared to the measured concentration of the 15 mM glucose

standard solution in order to determine if the addition of 3.726 g of VTT resulted in a reduction in

glucose concentration.

Having conducted the titre experiment with the solution containing 15 mM of glucose and 3.726 g

of VTT nanocellulose, another 15 mM glucose solution was created and 9.117 g of VTT added to it.

Having mixed this solution sufficiently the titre experiment outlined above was repeated for this

solution containing 9.117 g of VTT.

The experiments involving the addition of VTT to 15 mM glucose solutions were repeated, this

time using a 5 mM glucose master solution. To the 5 mM master solutions two different amounts

of VTT (namely 11.425 g and 22.94 g) were added in order to determine if at this lower glucose

concentration, the glucose would bind to the high volume of VTT.

At this point in time it was determined that the moisture content of the nanocellulose could be

impacting on the results, producing false positive readings in relation to the reduction of glucose

concentration. As a result the moisture content of VTT and Daicel nanocellulose, as well as the

moisture content of Tapioca Starch were determined. This was achieved by measuring a known

mass of each substance into a small aluminium plate and then placing the plate in an oven for

approximately two hours. During this time the materials became dried and were reweighed at the

end of the two hours. This allowed the determination of how much moisture the “un-dried” state

of each material contained. The results of this moisture content determination are reported in

Table 5-1-3 in Section 5.1.

Having determined the moisture content of various substances as described above, the titre

experiment previously conducted using the 15 mM and 5 mM master solutions was repeated for a

2 mM glucose master solution. In this instance 7.995 g of Daicel nanocellulose was added to the

glucose solution rather than VTT nanocellulose, due to the lower moisture content of Daicel. As a

control, 7.995 g of Daicel was also added to deionised water. The results of this experiment are

discussed in Section 5.1.

4.2. Starch to Nanocellulose Binding Experiments

From the literature review it is apparent that starch has the capacity to adsorb onto the surface of

cellulose. It was desired to design an experimental methodology to test these findings with

nanocellulose, having a much higher surface area.

As starch is insoluble in water at room temperature, to make a starch solution the starch must be

“cooked” or heated to weaken hydrogen bonds, allowing the starch molecules to swell and take

on water, allowing them to be dissolved. To prepare a stock starch solution for the following

experiments, 9 g of tapioca starch was added to 191 mL of deionised water in a heated vessel. The

temperature of the heated vessel was set at 80°C and a mixer was applied to the water-starch

solution to facilitate dissolution. The solution was left to mix and be heated until the viscosity

visually increased, and then decreased. Following this the solution was mixed for an additional 10

minutes before the heat and mixer were turned off.

Following preparation of the stock starch solution, the exact concentration of this solution needed

to be calculated. This was achieved following the procedure for moisture content determination

outlined in Section 4.1.

The method employed in order to determine the starch concentration was the sulphuric acid-

phenol method. When sulphuric acid and phenol are added to a starch solution, a brown colour is

created when starch is present. The higher the concentration of starch in solution the darker the

colour. Therefore spectrophotometry can be used to determine the concentration of starch in a

solution, however a calibration curve must first be constructed.

To construct the calibration curve for starch concentration using the sulphuric acid – phenol

method, the stock starch solution was used to make 10 different concentrations of starch by

dilution across the range 0-10 g/L. This range was chosen as it was found to have been used in

similar previous experiments. However, when these solutions were tested for absorbance at 490

nm in the spectrophotometer, it was observed that the maximum absorbance was reached at a

starch concentration of approximately 0.5 g/L. Therefore the method for constructing the

calibration curve was repeated for a concentration range of 0-0.5 g/L.

To make up the solutions required for use in the spectrophotometer, the following were added to

a 15 mL centrifuge tube:

2mL starch solution

125μL 80% Phenol solution

5mL 98% Sulphuric acid

These solutions were allowed to sit for 10 minutes before being shaken vigorously and placed in a

water bath at 30°C for 10 minutes. An eppendorf tube was used to transfer approximately 3 mL of

the sulphuric acid – phenol assays into cuvettes to be analysed in the spectrophotometer at 490

nm. From the data gained from the spectrophotometer and the known concentration of starch

solutions a calibration curve was constructed.

To verify starch adsorption onto nanocellulose, four beakers were prepared with each containing

different starch concentrations according to Table 4-2-1 below:

Table 4-2-1: Various Starch Concentrations for Sulphuric Acid - Phenol Method

Beaker Starch Deionised water Daicel nanocellulose Starch concentration

1 0 mL 300 mL 1.207 g 0 g/L

2 0.7 mL 300 mL 1.207 g 0.1 g/L

3 1.4 mL 300 mL 1.207 g 0.2 g/L

4 2.09 mL 300 mL 1.207 g 0.3 g/L

The nanocellulose and water were added first and allowed to mix with a magnetic stirrer for

approximately 45 minutes. Following this the designated amount of stock starch solution was

added to the beakers and mixed for 45 minutes, allowing interaction between the starch and

nanocellulose. To measure the concentration of the free starch (not adsorbed onto

nanocellulose), 10 mL was taken from each beaker and placed into a centrifuge tube. These tubes

were centrifuged for 10 minutes at 5000 rpm, with the nanocellulose bound starch complexes

settling at the bottom of the tubes allowing the supernatant, or free starch, to be separated. 2 mL

of the supernatant was taken from each tube and used as the starch solution in the previously

described sulphuric acid – phenol assay. Absorbances from the spectrophotometer at 490 nm

were recorded and compared with values gained from the calibration curve data in order to

determine the starch concentration.

4.3. Effect of Starch Binding to Nanocellulose on α-Amylase Action Experiments

As mentioned previously in Section 3.6, α-amylase is the enzyme within the human body that is

responsible for the hydrolysis of starch molecules into glucose monomers. It is currently unknown

whether α-amylase has the ability to hydrolyse starch while it is bound to nanocellulose. Also if

this does indeed occur, whether the glucose monomers are released from the nanocellulose

following hydrolysis, negating the positive effects that are the basis of this project. Therefore an

experimental methodology was required to determine whether the action of α-amylase would be

an issue.

Three reactors were used to determine the effect of 𝛼-amylase on starch adsorption onto

nanocellulose, they are described in Table 4-3-1 below:

Table 4-3-1: Reactor Contents Used in α-amylase Experiments

Reactor 43g/L Starch Solution Deionised water Daicel nanocellulose

1 1.4 mL 300 mL 0 g

2 1.4 mL 300 mL 1.207 g

3 1.4 mL 300 mL 2.414 g

Starch, deionised water and nanocellulose were added to Reactors 2 & 3 according to Table 4-3-1

above and were allowed to mix overnight using a magnetic stirrer. When these were thoroughly

mixed, starch and deionised water were added to Reactor 1 and allowed to mix with a magnetic

stirrer for approximately 45 minutes. Less mixing time was required for Reactor 1 as there was no

nanocellulose present, which takes a significant amount of time for the fibres to swell in the

solution and become properly dispersed. After this, the reactors all had the same concentration of

starch, which was calculated to be 0.2 g/L.

Initial glucose measurements were taken from all three reactors using the Accu-Chek Performa

Glucose Meter described in Section 4.1. Then the reactors were placed in a water bath at 37°C to

simulate temperature within the human body. Following this, 100 μL of α-amylase was added to

all three reactors, which was determined to be an appropriate amount from reviewing relevant

literature. Glucose concentration was then measured at regular intervals to measure changes in

glucose concentration arising from starch digestion by α-amylase.

According to the hypothesis it was expected that:

- The glucose concentration in Reactor 1 will increase, as the starch won’t be bound to

any nanocellulose, leaving it free to be hydrolysed by 𝛼-amylase to release glucose

monomers.

- Reactor 2 will either show a slight increase in glucose concentration or no increase in

glucose concentration, as the starch would be bound to the nanocellulose.

- Reactor 3 having the highest concentration of nanocellulose will show the smallest

increase in glucose concentration or no rise at all due to the amount of nanocellulose in

the reactor.

4.4. Literature Review

A main component of this report is a review of current literature to determine the current state of

knowledge surrounding the binding of nanocellulose to carbohydrates and fats, as well as possible

areas for further investigation and continuation of the project. A diverse range of sources was

accessed whilst conducting the literature review with many of the Monash University Library

Databases being used. All references used in this project can be found within Section 8 of this

report.

5. Key Results and Findings Having conducted the literature review as shown in Section 3 and carried out the experimental

work as described in Section 4, we were able to produce a large volume of results and made

multiple findings. The key results and findings from our review of literature and our experimental

work are discussed in the sections below.

5.1. Glucose Binding to Nanocellulose

In order to determine the ability of glucose to bind to nanocellulose, initially an extensive search

of literature was conducted. This research aimed to identify previous works investigating the

binding of glucose monomers to nanocellulose or similar materials, allowing us to conduct similar

experiments in order to test our hypothesis that glucose will bind to cellulose. The search of

literature yielded no results, therefore as far as we could determine this is the first documented

investigation into the adsorption of glucose monomers onto nanocellulose.

Having conducted the search of literature, we designed laboratory scale experiments as described

in Section 4.1 of this report in order to investigate the adsorption of glucose monomers to

nanocellulose. These experiments involved the use of glucometry in order to measure glucose

concentration. Specifically, the Accu-Chek Performa Blood Glucose Meter was used in order to

determine the concentration of glucose in solution. In order to conduct our experiments with the

glucometer we first attempted to create a standard curve. The first standard solution that we

attempted to measure with the glucometer was a glucose solution with a known concentration of

15 mM. Table 5-1-1 below shows the measured glucose concentration using the Accu-Chek

Performa Blood Glucose Monitor.

Table 5-1-1: Measured Glucose Concentration with Time for 15.07mM Glucose Solution

Measurement Time (Minutes) Measured Glucose Concentration (mM)

0 5.9

2 8.3

6 11

10 12.8

15 14.9

20 16.3

25 17.9

30 19.7

33 21.1

Measurements were taken over time with the hope of determining the steady-state measured

concentration. Unfortunately, as can be seen in Table 5-1-1 steady-state was not achieved in the

first 33 minutes of this experiment. In fact it can be seen that the measured concentration

continues to increase, even after having exceeded the expected steady-state value of 15.07 mM.

The results shown in Table 5-1-1 are also illustrated in graphical form in Figure 5-1-1.

Figure 5-1-1: Measured Glucose Concentration vs. Time for 15.07 mM Standard Solution

Due to the unexpected results using the 15.07 mM standard solution, we repeated the

measurements for a 2.02 mM standard glucose solution. In this instance five measurements with

the glucometer were taken for each time point, allowing for an average measured glucose

concentration to be determined. It was expected that the measured glucose concentration would

reach a steady state at approximately 2.02 mM after a given period of time, however as can be

seen in Table 5-1-2 and Figure 5-1-2 as time progressed the measured concentration continued to

increase beyond the expected value. This result was also seen for the 15.07 mM standard solution

as can be seen by referring to Figure 5-1-1.

0

5

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0 5 10 15 20 25 30 35

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Time (mins)

Measured Glucose Concentration vs. Time - 15mM Standard Solution

Table 5-1-2: Measured Glucose Concentration vs. Time (2.02mM Glucose Standard Solution)

Time (min) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Average Standard Deviation

2 Low Low Low Low Low N/A N/A

4 0.7 0.7 0.7 0.7 0.7 0.7 0

7 0.8 0.8 0.9 0.8 0.8 0.8 0.05

10 1.0 1.0 0.9 0.9 1.1 1.0 0.08

15 1.2 1.3 1.3 1.2 1.3 1.3 0.05

20 1.5 1.5 1.6 1.7 1.7 1.6 0.1

25 1.7 1.9 1.7 1.9 1.9 1.8 0.11

30 1.9 2.1 2.1 2.1 2.1 2.1 0.09

37 2.3 2.4 2.4 2.4 2.4 2.4 0.04

50 3.0 2.9 2.9 3.1 3.0 3.0 0.08

The average measured glucose concentration for each time point shown in Table 5-1-2 above is

plotted against time in Figure 5-1-2 below.

Figure 5-1-2: Average Measured Glucose Concentration vs. Time (2.02mM Glucose Standard Solution)

The variation of glucose concentration with time was unexpected, with the trends showing

measured concentration using the Accu-Chek Performa Glucose Monitor increasing well beyond

the known glucose concentration. In an attempt to rectify this unexpected result the supplier of

the glucometer was contacted and previous published works that had used glucometry for glucose

concentration determination were consulted. As a result it was determined that the unexpected

results may have arisen due to the fact that the glucometer was designed for measuring blood

glucose and therefore glucose solutions lacking the other elements of blood (i.e. cells, proteins

etc.) could yield inaccurate results.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

0 10 20 30 40 50 60

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(m

M)

Time (mins)

Average Measured Glucose Concentration vs. Time - 2.02mM Standard Solution

Further to these enquiries, we also trialled many different experiments in an attempt to stop the

variation of measured glucose concentration with time. Such experiments included modifying the

method of transferring the glucose solution to the glucometer, varying the location within the

beaker of glucose solution from which the sample was tested, taking samples with the magnetic

stirrer present and with the stirrer absent and trialling different glucose solution concentrations.

The results of these experiments all consistently displayed a continuous increase in glucose

concentration with time.

In a further attempt to eliminate the increasing measured glucose concentration with time, we

added blood to the glucose solution in order to determine if using the glucometer on a blood

solution would yield results that were consistent with expectations. This addition of blood

stabilised the variation of glucose concentration with time, suggesting that the blood glucose

meter was working as the supplier reported it should. This therefore suggested that as the

supplier stated, the unexpected results being reported by the glucometer may be due to the fact

that the meter is designed for blood samples and not for glucose solutions. This does not however

explain how previous works were successful in using blood glucose meters as a method for

determining glucose concentrations in solution.

It was hypothesised that adding Bovine Serum Albumin (BSA) to glucose solutions could eliminate

the increasing measured concentration by making the solutions closer to the composition of blood

due to the addition of proteins. BSA was therefore added to glucose solutions of varying

concentration and the glucometer was used to measure the glucose concentration over a three

hour period. The results of this experiment found that the addition of BSA did not stop the

increase in measured glucose concentration with time, with a steady-state measured

concentration not being achieved and the known concentration being well exceeded by the

glucometer concentration reading.

It was determined that if the glucose solution was left to mix overnight that a steady state

measured glucose concentration was achieved, albeit well above the known concentration. This

did however allow us to produce a standard curve for the concentration measured with the Accu-

Chek Performa Blood Glucose Meter, as is shown in Figure 5-1-3 below. Figure 5-1-3 shows the

standard curve produced having used the titre experiment outlined in Section 4.1 using a glucose

master solution with a concentration of 15 mM. This figure shows the measured concentration to

be consistently four times higher than the known glucose concentration.

Figure 5-1-3: Glucose Measured Concentration Standard Curve - 15mM Glucose Master Solution

Having determined the standard curve as shown above, we repeated the experiment as is outlined

in Section 4.1, this time making master solutions of 15 mM glucose mixed with 3.726 g and 9.117 g

of VTT nanocellulose. The measured glucose concentration in these nanocellulose/glucose

solutions could then be compared to the standard curve results, which represent the result for the

glucose solution in the absence of nanocellulose. It would therefore be hypothesised that if the

glucose had bound to the nanocellulose that the measured glucose concentration should be lower

in the solution that contains nanocellulose. Figure 5-1-4 on the following page shows the results

of this experiment.

The trends shown in Figure 5-1-4 show that when 3.726 g of VTT was added to the 15 mM glucose

solution, the glucose concentration as measured by the glucometer was very similar to that of the

standard solution (i.e. the solution containing no VTT). This was true for all known glucose

concentrations, except for higher concentrations which recorded a slightly higher measured

glucose concentration when 3.726 g of VTT was added. This result was not expected, as it was

anticipated that the addition of VTT nanocellulose would reduce the measured glucose

concentration. However, due to the lack of precision of the glucometer and the high experimental

error associated with its measurements, the increase in measured glucose concentration noted for

the solution containing 3.726 g of VTT was likely due to experimental error. Therefore it is more

likely that the glucose concentration of this solution is roughly the same as that of the glucose

standard solution. It can be concluded from this result that the addition of 3.726 g of VTT did not

result in a reduction in glucose concentration, hence suggesting that glucose did not bind to the

VTT nanocellulose.

0

5

10

15

20

25

30

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

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(m

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Known Glucose Concentration (mM)

Measured Glucose Concentration on Accu-Chek vs Known Glucose Concentration

Figure 5-1-4: The Effect of Nanocellulose Addition on Measured Glucose Concentration - 15mM Glucose Master Solution

Figure 5-1-4 also shows that the addition of 9.117 g of VTT to the 15 mM master solution results in

a slight reduction in measured glucose concentration for most known concentrations measured.

Upon first inspection this suggests that the addition of such a mass of VTT has resulted in a

reduction in glucose concentration, hence suggesting that glucose has adsorbed onto the VTT.

However as can be seen by referring to Table 5-1-3 below, VTT has a moisture content of 96.44%

and therefore when adding 9.117 g of VTT we are in fact only adding 0.327 g of cellulose and 8.79

g of water. Given that the solution to which the VTT was added only had a volume of 100 mL

(approximately 100 g of water), this addition of 8.79 g of water would be expected to result in a

decrease in the concentration of glucose. Therefore the reduction in measured glucose

concentration seen in Figure 5-1-4 is likely due to the addition of water and therefore cannot be

conclusively linked to the binding of glucose to the added nanocellulose.

Table 5-1-3: Moisture and Solids Content of Starch and Cellulose Materials Used

Tapioca Starch VTT Cellulose Daicel Cellulose

Solid Content 88.04% 3.56% 24.86%

Moisture Content 11.96% 96.44% 75.14%

Figure 5-1-3 and Figure 5-1-4 above represent the results of the experiments using a 15 mM

glucose master solution, the methodologies of which are discussed in Section 4.1. These

experiments were repeated using a lower glucose concentration master solution of 5 mM, in order

to determine if lowering the known glucose concentration would result in measureable binding of

0

5

10

15

20

25

30

35

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0

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Known Glucose Concentration (mM)

Measured Glucose Concentration on Accu-Chek vs Known Glucose Concentration (15mM) - Effect of VTT Nanocellulose

15mM GlucoseStandard Solution

3.726g VTT Added to15mM GlucoseStandard Solution

9.117g VTT Added to15mM GlucoseStandard Solution

glucose to the VTT. The results of these experiments using a 5 mM master solution are shown in

Figure 5-1-5 below.

Figure 5-1-5: The Effect of Nanocellulose Addition on Measured Glucose Concentration - 5mM Glucose Master Solution

The results shown above suggest that adding VTT nanocellulose to the glucose solution resulted in

a reduction in free glucose (the glucose measured by the glucometer). However, this reduction

corresponds to the reduction in glucose concentration expected due to the amount of water

added with the VTT nanocellulose. Adding 22.94 g of “wet” VTT actually translates to adding

approximately 22.12 g of water and only 0.87 g of “dry” VTT. Therefore, as was the case for the 15

mM glucose solution experiments, these results suggest the addition of VTT to a glucose solution

does not result in a decrease in glucose concentration, hence suggesting that glucose does not

adsorb to VTT nanocellulose.

The experiments outlined above for 15 mM and 5 mM glucose master solutions were repeated for

a 2.02 mM glucose solution. For these experiments we added Daicel nanocellulose rather than

VTT due to the lower moisture content of the Daicel, as is highlighted in Table 5-1-3. The resulting

solution was very viscous and when repeating the titre experiment the glucometer was unable to

read the higher concentrations and gave “LO” results for the lower concentrations. This is

highlighted in the Table 5-1-4 on the following page.

0

2

4

6

8

10

12

14

0.0 1.0 2.0 3.0 4.0 5.0 6.0

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Known Glucose Concentration (mM)

Measured Glucose Concentration on Accu-Chek vs Known Glucose Concentration in Water (5mM)

5mM GlucoseStandard Solution

11.425g VTTAdded to 5mMGlucose StandardSolution

22.94g VTT Addedto 5mM GlucoseStandard Solution

Table 5-1-4: The Effect of Nanocellulose Addition on Measured Glucose Concentration - 2mM Glucose Master Solution

Known Glucose Concentration (mM)

Measured Glucose Concentration (mM) - Standard Solution

Measured Glucose Concentration (mM) - 7.995g Daicel Added

2.05 4.92 ERROR

1.03 2.38 ERROR

0.51 1.18 0.72

0.26 0.66 LO

0.13 LO LO

0.06 LO LO

0.03 LO LO

The error reported in the table above was due to the high viscosity of the solution. The figure on

the left below shows the solution of Daicel Nanocellulose and glucose. As can be seen this is a

very viscous solution. It was suggested that the viscosity could be indicative of a binding of the

nanocellulose with the Daicel, however we hypothesised that the high viscosity was merely due to

the swelling of the Daicel. In order to test this we made a solution with the same concentration of

Daicel in water, with no glucose. This solution is seen in the middle figure below. As can be seen

it has the same viscosity as the Daicel and Glucose solution shown in the figure on the left. The

figure on the right allows comparison of the two solutions. Our hypothesis was therefore

validated and it was assumed that the high viscosity is due to the swelling of the Daicel, not due to

the interaction between the Daicel and the glucose.

Figure 5-1-6: A) (Left) Daicel and Glucose Solution. B) (Middle) Daicel Solution. C) (Right) Comparison of Daicel + Glucose Solution and Daicel Solution

Overall, our investigation into the interaction of glucose and nanocellulose found no conclusive

evidence that glucose can adsorb onto cellulose. This is however only true for cellulose and

nanocellulose in unmodified states and further research into the interaction of glucose with

modified cellulose materials needs to be conducted in order to determine if such modifications

could enable glucose to bind to cellulose materials.

5.2. Sucrose Binding to Nanocellulose

From literature it was found that sucrose does not adsorb onto cellulose fibres as it has been used

as an inert tracer in a packed bed of cellulose fibres in previous experimental work (9). Therefore

no experimental work was conducted in this area, saving time to focus on other points. It would

be advantageous to investigate chemical modification of nanocellulose to facilitate adsorption of

sucrose onto the surface.

5.3. Starch Binding to Nanocellulose

By conducting the sulphuric acid – phenol method described in Section 4.2, a calibration curve

relating absorbance and starch concentration was constructed and can be seen in Figure 5-3-1

below.

Figure 5-3-1: Calibration Curve Constructed for the Sulphuric Acid - Phenol Starch Concentration Determination Method Using an Absorbance of 490nm

The calibration curve achieved an R2 value of 0.9834, which comparing to literature was

determined to be within the required error range. From this curve we were able to relate the

absorbance values gained from the UV-Vis spectrophotometer to starch concentration using the

equation y=13.463x+0.1066. Where y is the absorbance at 490 nm and x is the corresponding

concentration of starch in solution. This information allowed the following results to be obtained

to test starch adsorption on nanocellulose.

y = 13.463x + 0.1066R² = 0.9834

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 0.05 0.1 0.15 0.2 0.25 0.3 0.35

Ab

sorb

ance

Starch Concentration (g/L)

Phenol-Sulphuric Acid Determination of Starch Concentration -Standard Curve

Figure 5-3-2: Comparison of Initial Starch Concentration and Starch Concentration after the Addition of Daicel Nanocellulose for Initial Starch Concentrations Ranging from 0-0.3g/L

It can be seen from Figure 5-3-2 above that overall the addition of Daicel nanocellulose related to

a reduction in starch concentration. As the same amount of nanocellulose was added to each

beaker, the greatest effect was in the beaker with the highest initial starch concentration of 0.3

g/L. However a reduction was also realised in beakers 2 & 3, showing that the greatest relative

effect is noticed at higher initial starch concentrations. This suggests that the nanocellulose still

had more binding capacity and was not yet saturated with starch. However the starch was not

reduced to a concentration of zero, which suggests that nanocellulose concentration is also a

factor and a state of equilibrium between bound and free starch is achieved in the solution.

From these results we were successful in confirming the hypothesis that starch could be adsorbed

onto nanocellulose. Following on from this we wished to investigate the action of α-amylase and

whether it would have the capacity to reverse the effects that were established by the results

discussed above.

0

0.05

0.1

0.15

0.2

0.25

0.3

12

34

Star

ch C

on

cen

trat

ion

(g/

L)

Beaker

Starch Adsorption on Nanocellulose

Starch + nanocellulose

Initial Starch Solution

5.4. Effect of Starch Binding to Nanocellulose on α-Amylase Action

As can be seen in Table 5-4-1 below, there were no conclusive results achieved from the regular

glucose concentration readings taken by the Accu-Chek Performa. Before the α-amylase was

added to the reactors they showed a glucose concentration of “LO” on the glucometer, denoting a

reading below the minimum detectable concentration of 0.6 mM.

Table 5-4-1: Measured Glucose Concentrations over Time for Alpha-Amylase Experiment

Time (min) Glucose Concentration (mM)

Reactor 1 Reactor 2 Reactor 3

0 LO LO LO

30 LO LO LO

60 LO LO LO

120 LO LO LO

180 LO LO LO

240 LO LO LO

24 hours LO LO LO

After measuring the glucose concentration at regular intervals up to 24 hours after the α-amylase

was added to the reactors, it was observed that there was no apparent rise in glucose

concentration as all the readings were still “LO”. However, this does not mean that the glucose

concentration didn’t increase, as it is possibly it could have risen from 0.2 mM to 0.5 mM, for

example. However we cannot say with conviction that the glucose concentration increased due to

it being undetected by the glucometer.

It would be advantageous to conduct further investigation in this area as the interactions between

starch, nanocellulose and α-amylase are important in the context of this project.

6. Conclusions and Recommendations Having conducted this research project we have come to multiple conclusions regarding the

potential application of nanocellulose as a negative calorie food additive.

Conducting an extensive review of literature it was determined that no literature exists on the

topic of the adsorption of the monosaccharide glucose to cellulose. The experiments that we

conducted produced results concluding that glucose does not adsorb to nanocellulose.

An extensive review of literature was also conducted regarding the adsorption of sucrose onto

cellulose. It was concluded from the previous works discovered in this investigation of published

literature that the disaccharide sucrose does not bind to nanocellulose.

A large amount of literature was found regarding the binding of starch to cellulose. Our

experiments confirmed our hypothesis that starch adsorbs to the surface of nanocellulose. We

also conducted experiments investigating the effect that α-amylase has on starch bound to

nanocellulose. We were unable to determine if α-amylase was hydrolysing the nanocellulose

bound starch from these experiments, hence these experiments were inconclusive.

A literature review of the effect of nanocellulose on cholesterol levels was also conducted, with

this review suggesting that nanocellulose can regulate blood cholesterol levels. This regulation is

not achieved through the binding of cholesterol to nanocellulose, but rather due to the fact that

increased nanocellulose levels in the diet increases faecal mass, in turn increasing bile acid

production, ultimately resulting in the breakdown of cholesterol and reduction in blood

cholesterol levels. Nanocellulose in the diet can also be linked to the prevention of colon cancer

due to the fact that it binds to secondary bile acids as well as increasing faecal mass, thus reducing

levels of secondary bile acids present in the colon, which has been linked to colon cancer.

Literature relating to the effect of fructans on blood cholesterol levels was also researched as part

of this project. From this review of literature it was concluded that fructans ferment in the colon

producing propionic acid, a chemical that inhibits cholesterol synthesis and lipogenesis in the liver.

Fructans were also found to be able to bind to triglycerides. Therefore it was concluded from this

literature review that fructans have the ability to reduce both cholesterol and triglyceride levels in

the blood.

Based on the conclusions drawn from the experiments conducted and literature reviewed

throughout this project, multiple recommendations for future works have been made.

Firstly, regarding the adsorption of glucose and sucrose onto nanocellulose, it is important to note

that the experiments that have found that glucose and sucrose do not bind to nanocellulose have

only been conducted using nanocellulose in a native form. It is therefore recommended that

nanocellulose be chemically modified in order to alter its functionality, and experiments be

conducted to determine whether glucose and sucrose will bind to this modified nanocellulose.

The experiments regarding the effect that α-amylase action has on starch binding to nanocellulose

produced inconclusive results. These results were deemed to be inconclusive due to the lack of

sensitivity of the glucometry technique used for analysis of samples. It is therefore recommended

that these experiments be repeated, however rather than using glucometry a more sensitive

analytical technique such as high performance liquid chromatography should be employed.

As previously discussed, this project found literature suggesting that nanocellulose and fructans

could, in theory, result in a reduction in cholesterol and triglyceride levels in the blood. Our

project did not however, test this experimentally and it is therefore recommended that future

works experimentally investigate the use of fructans and nanocellulose to reduce cholesterol and

triglyceride levels.

In conducting this research project we were able to achieve our objective of investigating the use

of nanocellulose as a negative calorie and cholesterol controlling food additive. Although not all of

our results supported our hypothesis, many did, and those that didn’t based on these preliminary

experiments, may prove to align better with our hypothesis if further experiments are conducted.

Therefore, overall our project was a success and the use of nanocellulose as a negative calorie and

cholesterol controlling food additive still remains a valid proposal, however requires further

investigation.

7. Acknowledgements We would like to express our gratitude to Professor Gil Garnier, for his guidance and feedback

throughout this project. We would also like to thank Mr Scot Sharman and Dr Heather McLeish for

their continued assistance and advice while in the APPI laboratories.

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19. Cationic Starch Adsorption by Cellulose: I. Nedelcheva, M. P. & Stoilkov, G. V. 1978, Journal of

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20. Binding Interactions of alpha-amylase with Starch Granules: The Influence of Supramolecular

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22. Effects of Fructans-Type Prebiotics on Lipid Metabolism. Delzenne, N. M. & Kok, N. 2001, The

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2005, British Journal or Nutrition, pp. 163-168.

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29. Cholesterol reducing and bile-acid binding properties of taioba (Xanthosoma sagittifolium) leaf

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9. Appendices

9.1. Risk Assessment

Task / Process /

Procedure

Method 1: Risk Assessment

Hazard Type

Consequence &

Likelihood

Risk

Score

Controls Currently

in Place

Controls to be

Implemented By Who By When

In Place

(Sign)

Description of hazard

Inhalation of Sodium

Hydroxide

Chemical

(R37)

Consequence:

Minor

Likelihood:

Occasional

Medium

(D3)

Lab entry restricted to

those wearing

appropriate PPE.

Operate in fume

cupboard & wear safety

mask.

Operator As

required

Skin Contact with Sodium

Hydroxide

Chemical

(R35)

Consequence:

Moderate

Likelihood:

Unlikely

Medium

(C4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE (lab

coat & gloves).

Operator As

required

Eye Contact with Sodium

Hydroxide

Chemical

(R41)

Consequence:

Severe

Likelihood:

Unlikely

High

(B4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE,

especially safety

glasses. Handle

carefully.

Operator As

required

Inhalation of Hydrochloric Acid Chemical

(R37)

Consequence:

Moderate

Likelihood:

Unlikely

Medium

(C4)

Lab entry restricted to

those wearing

appropriate PPE.

Operate in fume

cupboard & wear safety

mask.

Operator As

required

Skin Contact with Hydrochloric

Acid

Chemical

(R35)

Consequence:

Moderate

Likelihood:

Unlikely

Medium

(C4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE (lab

coat & gloves).

Operator As

required

Eye Contact with Hydrochloric

Acid

Chemical

(R41)

Consequence:

Severe

Likelihood:

Unlikely

High

(B4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE.

Handle carefully.

Operator As

required

Inhalation of Phenol Chemical

(R37)

Consequence:

Severe

Likelihood:

Unlikely

High

(B4)

Lab entry restricted to

those wearing

appropriate PPE.

Operate in fume

cupboard & wear safety

mask.

Operator As

required

Skin Contact with Phenol Chemical

(R35)

Consequence:

Severe

Likelihood:

Unlikely

High

(B4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE (lab

coat & gloves).

Operator As

required

Eye Contact with Phenol Chemical

(R41)

Consequence:

Severe

Likelihood:

Unlikely

High

(B4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE.

Handle carefully.

Operator As

required

Inhalation of Concentrated

Sulfuric Acid

Chemical

(R37)

Consequence:

Moderate

Likelihood:

Unlikely

Medium

(C4)

Lab entry restricted to

those wearing

appropriate PPE.

Operate in fume

cupboard & wear safety

mask.

Operator As

required

Skin Contact with

Concentrated Sulfuric Acid

Chemical

(R35)

Consequence:

Severe

Likelihood:

Unlikely

High

(B4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE (lab

coat & gloves).

Operator As

required

Eye Contact with

Concentrated Sulfuric Acid

Chemical

(R41)

Consequence:

Severe

Likelihood:

Unlikely

High

(B4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE.

Handle carefully.

Operator As

required

Inhalation of Nanocellulose Chemical

(R20)

Consequence:

Minor

Likelihood:

Unlikely

Medium

(D4)

Fume cupboard

available.

Wear safety mask,

handle carefully. Operator

As

required

Eye Exposure to

Nanocellulose

Chemical

(R21)

Consequence:

Minor

Likelihood:

Unlikely

Medium

(D4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE,

especially safety

glasses. Handle

carefully.

Operator As

required

Inhalation of D(+)-Glucose

Powder

Chemical

(Irritating if

Inhaled)

Consequence:

Negligible

Likelihood:

Unlikely

Low

(E4)

Fume cupboard

available.

Wear safety mask,

handle carefully. Operator

As

required

Eye Exposure to D(+)-Glucose

Powder

Chemical

(R36)

Consequence:

Negligible

Likelihood:

Unlikely

Low

(E4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE,

especially safety

glasses. Handle

carefully.

Operator As

required

Inhalation of Starch Powder Chemical

(R20)

Consequence:

Minor

Likelihood:

Highly Unlikely

Low

(D5)

Fume cupboard

available.

Wear safety mask,

handle carefully. Operator

As

required

Eye Exposure to Starch

Powder

Chemical

(R36)

Consequence:

Negligible

Likelihood:

Unlikely

Low

(E4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE,

especially safety

glasses. Handle

carefully.

Operator As

required

Inhalation of α-Amylase

Solution

Chemical

(R20)

Consequence:

Minor

Likelihood:

Highly Unlikely

Low

(D5)

Fume cupboard

available.

Wear safety mask,

handle carefully. Operator

As

required

Eye Exposure to α-Amylase

Solution

Chemical

(R36)

Consequence:

Negligible

Likelihood:

Unlikely

Low

(E4)

Lab entry restricted to

those wearing

appropriate PPE.

Ensure wearing

appropriate PPE,

especially safety

glasses. Handle

carefully.

Operator As

required

pH Monitor Physical –

Electrical (P8)

Consequence:

Moderate

Likelihood:

Highly Unlikely

Medium

(C5)

Regular maintenance

checks.

Check equipment

condition before use and

avoid contact with

liquids. Operate only as

directed.

Operator As

required

Magnetic Stirrer Physical –

Electrical (P8)

Consequence:

Moderate

Likelihood:

Highly Unlikely

Medium

(C5)

Regular maintenance

checks.

Check equipment

condition before use and

avoid contact with

liquids. Operate only as

directed.

Operator As

required

Water Bath Physical –

Electrical (P8)

Consequence:

Moderate

Likelihood:

Highly Unlikely

Medium

(C5)

Regular maintenance

checks.

Check equipment

condition before use and

avoid contact with

liquids. Operate only as

directed.

Operator As

required

Computer/Laptop Physical –

Electrical (P8)

Consequence:

Moderate

Likelihood:

Highly Unlikely

Medium

(C5)

Regular maintenance

checks.

Check equipment

condition before use and

avoid contact with

liquids.

Operator As

required

Slipping/Tripping

Physical –

Gravitational

(P3)

Consequence:

Severe

Likelihood:

Highly Unlikely

Medium

(B5)

Regulations on

storage of equipment

and cleanliness of

lab.

Ensure workspace and

lab floor is clear of

obstacles.

Operator As

required

Broken Glass

Physical –

Machinery

(P1)

Consequence:

Moderate

Likelihood:

Unlikely

Medium

(C4)

Dust pan and glass-

specific bin provided.

Handle glassware with

care, notify appropriate

individuals and ensure

glass is completely

cleared.

Operator As

required

Ergonomic Hazards

Associated with Desk Work

Manual

Handling

Consequence:

Minor

Likelihood:

Likely

Medium

(D2)

Adjustable chair and

computer screen.

Ensure working with

correct posture and

taking breaks when

necessary, sit in an

adjustable chair, ensure

computer screen can be

adjusted to optimum-eye

level and that

workspace is tidy and

free of obstacles

affecting posture.

Operator As

required

Repetitive Strain Injury (RSI) Manual

Handling

Consequence:

Negligible

Likelihood:

Occasional

Medium

(E3)

Ergonomically

designed pipettes.

Take breaks when

necessary, avoid

prolonged periods of lab

work, spread work load

between operators.

Operator As

required