FORMULATION AND PHYSICOCHEMICAL ANALYSIS OF A

WATER-RESISTANT GLUE FROM SPOILED MILK CASEIN

MARGRET WANJIRU MWENJA

BTMB/114J/2017

A project proposal submitted to the Department of Pure and Applied

Sciences in partial fulfilment for the award of Bachelor’s degree in

Industrial Microbiology and Biotechnology of Technical University of

Mombasa.

September 2022

i
 DECLARATION AND APPROVAL

I hereby declare that this proposal is my original work and has not been presented for a

degree in any other university.

MARGRET WANJIRU MWENJA

REG NO: BTMB/114/2017

Signature: …………………Date: ……………………

SUPERVISOR:

This proposal has been submitted for review with my approval as supervisor.

DR. CARREN OKERI

DEPARTMENT OF PURE AND APPLIED SCIENCES

Signature …………………………. Date…………………………….

ii
 DEDICATION

I dedicate this research project to my beloved parents for their financial support and

immense moral support during the preparation of this project proposal. I also dedicate it

to the Technical University of Mombasa especially the Department of Pure and Applied

Sciences, all my friends and fellow students in Microbiology field.

iii
 ACKNOWLEDGEMENT

I thank Almighty God for this opportunity and for granting me the gift of good health to date

since the day I enrolled to my current course of study. My gratitude as well goes to my

supervisor for her wisdom and guidance. I appreciate my fellow course-mates for helping

me to this level.

iv
 TABLE OF CONTENTS

DECLARATION AND APPROVAL.......................................................................................ii

DEDICATION..........................................................................................................................iii

ACKNOWLEDGEMENT........................................................................................................iv

ABSTRACT.............................................................................................................................vii

CHAPTER ONE........................................................................................................................1

1.1 Background information...................................................................................................1

1.2 Problem statement............................................................................................................3

1.3. Justification......................................................................................................................3

1.4 Objectives.........................................................................................................................4

1.4.1 General objective.......................................................................................................4

1.4.2 Specific objectives.....................................................................................................4

1.5 Research question........................................................................................................4

CHAPTER TWO.......................................................................................................................5

Literature review........................................................................................................................5

2.1 Milk proteins....................................................................................................................5

2.2 Physicochemical Properties of Milk.................................................................................6

2.3 Caseins..............................................................................................................................7

2.4 Whey.................................................................................................................................8

2.5 Milk Protein Products.......................................................................................................9

2.5.1. Casein Products.........................................................................................................9

2.5.2 Whey Products...........................................................................................................9

2.5.3 Membrane Filtered Milk Protein.............................................................................10

2.6 Milk Protein Polymerization..........................................................................................10

2.7 Milk-Protein-Based Adhesives Applications.................................................................11

2.7.1 Casein-Based Adhesives..........................................................................................11

v
 2.7.2 Whey-Protein-Based Adhesives..............................................................................12

2.8.1Adhesives.....................................................................................................................14

2.8.2Adhesive Testing..........................................................................................................14

CHAPTER THREE..................................................................................................................16

Methodology.........................................................................................................................16

3.1 Study Area......................................................................................................................16

3.2Research Design..............................................................................................................16

3.3 Sample collection...........................................................................................................16

3.4 Formulation of water-resistant casein glue.....................................................................16

3.5 Physicochemical properties testing................................................................................17

3.5.1Tensile testing...........................................................................................................17

3.5.2Shear testing..............................................................................................................17

3.5.3 Peel test....................................................................................................................17

3.5.4 Scrape Test...............................................................................................................18

3.5.5 Pull-Off Adhesion Test............................................................................................18

3.5.6 pH.............................................................................................................................19

3.5.7 Viscosity..................................................................................................................19

3.6 Data analysis...................................................................................................................19

Work plan.................................................................................................................................20

BUDGET..................................................................................................................................21

REFERENCES.......................................................................................................................22

vi
 ABSTRACT

Milk contains a lot of different components with their own functional properties and some of

them, such as casein, have been used in the manufacture of non-food technical products. Milk

proteins like caseins and whey proteins are important protein sources for human nutrition. In

addition, they possess important natural polymers. These protein molecules can be modified

by physical, chemical, and/or enzymatic means. Research on milk-protein-based adhesives is

still ongoing. This study proposes a new applications of milk proteins on a laboratory scale

focused on formulating water resistant glue from fresh and spoiled milk samples in

environmentally safe conditions. It will be followed by analysis of the physicochemical

properties of the casein water resistant glue from, fresh and spoiled milk samples. Data

presentation will be done in tables and pie charts for easy interpretations. This is a promising

route for giving added value to dairy co-products and effluents, which will achieve a great

reduction in dairy industry wastes. This is a promising route in the exploration of the broad

application of milk proteins.

vii
 CHAPTER ONE

1.1 Background information

Natural adhesives are renewable and environmentally friendly. The advantage of casein

adhesives is eco-friendliness in its manufacturability, the absence of harmful chemicals and

odor. The use of water-based adhesives solves the issues related to resource and energy

conservation as well as the protection of the environment as the renewable energy sources are

used and the environment is not polluted: the atmosphere, hydrosphere, and lithosphere

(Karthik & Gopalakrishnan, 2014). Resolving these issues focuses on the creation of new

composite materials from renewable raw materials and the use of modern methods to control

their technological and adhesive properties. In the process of gluing, casein adhesives may

lose the capability to dissolve or soften when heated, so their use when sticking labels leads

to difficulties in washing them away from industrial and household containers if it is

necessary to reuse them (Cherkashina et al., 2020).

In addition, the application of casein adhesives requires special conditions for their

transportation and storage. The need for easily washed adhesives arises when labeling

products packaged in reusable containers, primarily, glass containers. Such adhesives are

subject to the most stringent requirements like high stickiness, adhesion, and water resistance;

at the same time, they should be capable of keeping a label on the glass container, including

under conditions of high humidity (Gierenz& Karm, 2008). The label should be compatible

with the capability of glue to ensure a light separation from the container when laundering it

with harmless liquids, most often aqueous soda solutions or surface-active substances.

Therefore, it is a relevant task to develop a glue adhesive composition with high stickiness for

sticking labels.

viii
Milk and milk components are mostly used in foodstuffs in many different forms.

Nevertheless, milk constituents also find numerous alternative applications in the non-food

area such as in the manufacture of plastic materials, textile fibers, glues or in the production

of ethanol or methane. Some of these technical applications have been well known for a long

time, such as casein-based glues which were used in ancient Egypt, but novel uses are also

proposed for opening new markets for components. Most of the technical applications are

specific to one given milk component in relation to its structural and functional properties.

Separation and extraction techniques may be of importance in the valorization process of

such individual components. Valuable components can be recovered by chemical and/or

physical means in milk and dairy coproducts. Besides, some applications concern the non-

food uses of fractions containing different components together, which greatly simplify

separation steps (Audic et al., 2003).

Casein, the major protein in cow’s milk, has a long history of use in the non-food area. This

Gaussian coil protein has been used since the beginning of the nineteenth century in the

manufacture of a wide range of products including rigid plastics and textile fibers, for

example. Casein is the main protein in bovine milk. Acid and rennet caseins are the two

major types of casein available depending on the coagulation process. Acid casein refers to

precipitated casein, obtained by adjusting skim milk pH to 4.6 with mineral acids such as

hydrochloric or sulphuric acid. Lactic casein is precipitated by acidification due to lactic acid

produced in situ with lactic acid bacteria designed as a starter.

Rennet casein is a protein product that is created when rennet is added to pasteurized skim

milk causing coagulation and precipitation of casein protein. The general composition is 0.5

% fat and 10 % moisture.

ix
This study aims at utilization of fresh and spoiled milk casein in formulation of a water-

resistant glue and determination of its physicochemical properties.

1.2 Problem statement

The demand of dairy products is increasing on daily basis due to increase in consumption of

milk and milk-based products. There are many milk processing industries that have been set

up to counter this high demand. These industries receive high quantities of milk per day from

farmers and process equally a high amount of products. These industries subsequently

generate waste product out of the processing stages which is directly proportional to the

amount of milk being processed. This processing is also accompanied by receiving of milk

that does not meet quality standards from farmers as a result of contaminations during

milking and transportation and in turn it leads its disposal. This affect the industries

production rates and economic development which is also associated with farmers

frustrations since quite a number of the industries do not pay for the spoiled milk which

demoralize the famers production rates too.

In the market today, the available synthetic glues are expensive to formulate and purchase.

They are also not environmentally friendly since they comprise of chemicals as their largest

composition. This calls for an urgent solution that is aimed at utilizing the spoiled milk in

industrial levels to generate water resistant glue that is cheaply available, environmentally

friendly and a natural glue.

1.3. Justification.

With the economic loses encountered when milk from industries spoils there is need to look

for a prime solution that fits and satisfies the farmer and the milk industry equally. This study

is aimed at utilizing casein from the spoiled milk to generate a water-resistant glue that will

be utilized in various parts including paper sticking, timbers sticking, and any other material

x
attachment that will be compatible with it. This will also ease the burden of expensive glues

in the market that are not environmentally friendly and ineffective.

1.4 Objectives

1.4.1 General objective

To formulate a water-resistant glue from animal protein, casein and determine its

physicochemical properties.

1.4.2 Specific objectives

I. To compare casein yields in fresh and spoiled milk.

II. To formulate a water-resistant glue from fresh milk and spoiled milk casein.

III. To determine physicochemical properties of water resistance glue from fresh and

spoiled milk.

I.5 Research question

I what are the casein yields in fresh and spoiled milk?

II. How is fresh and spoiled milk water resistance glues formulated?

III. What are the physicochemical properties of fresh milk and spoiled milk water

resistant glues?

1.6 Limitation

This study will be a small-scale laboratory study due to the length of time available for the

investigation. Candidate samples for glue formulation include waste milk from milk

processing plants and spoilt milk before processing, which is usually disposed of. However,

for the purpose of investigating the potential of milk protein in glue formulation, this study

will use fresh milk samples. Milk proteins are mainly casein and whey. This study will be

limited to use of casein protein, being the highest in bovine milk. Protein crosslinking can be

xi
achieved by physical, enzymatic, and chemical means, but this study will use a chemical

formulation procedure. The success of this study will lead to scaling up, use of a variety of

milk samples and use of other formulation procedures in another study.

CHAPTER TWO

Literature review
2.1 Milk proteins

Bovine milk contains about 3.2% protein, 3.4% fat, and 4.8% lactose, as well as various

minerals and vitamins. It is an important food for most people around the world. Due to its

high nutritional value, milk and its components are widely used in foods in various forms.

Additionally, non-food applications are also a large part of milk utilization, dating back to the

ancient Egyptians in their use of casein as glue till today’s applications, such as in

environmentally safe wood adhesives and paper/label adhesives. Milk proteins are

categorized as caseins and whey proteins, based on different solubility’s at a pH of 4.6, which

are approx. 2.6% and 0.6%, respectively, in bovine milk. The properties of casein and whey

protein (such as the chemistry, structure, functionality, nutrition, etc.) have been extensively

studied over the past few centuries. Casein exists in fresh milk in the form of a “micelle”

structure, which is a complex aggregate of proteins (α-, β-, and κ-casein) and colloidal

phosphate calcium (CCP). Whey proteins are a group of globular proteins, which consist

mainly of β-lacto globulin (β-Lg), α-lactalbumin (α-La), and bovine serum albumin (BSA).

Both caseins and whey proteins exhibit unique polymer properties. As with many other

naturally produced polymers, such as starch, tree gums, and clays, milk protein components

exhibit excellent adhesive properties, and have been used as one of the major natural

adhesive ingredients for thousands of years, until the advent synthetic petroleum-based

polymers. Natural adhesives are safe, renewable, and environmentally safe; in addition, they

xii
possess excellent bonding properties. One of the reasons why caseins or natural polymers

have been steadily replaced by synthetic polymers is the cost, s milk protein is a food of high

nutritional value, and the demands for milk protein for use in food has been increasing

(Krebzdaket al .,2018).

However, the concepts of natural and environmentally safe adhesives are gaining more and

more public attentions. Events where children eat glue/paste, or where people get sick from

formaldehyde emissions, have been reported. Natural and safe polymers should be

reconsidered for use as adhesives in situations such as office and school paper glues, wood

adhesives, and any other high-value-added adhesives. Due to the development of modern

technology, the chemistry, structure, and functionality of milk proteins have been extensively

studied. This new knowledge is helpful in the formulation of more efficient and safe

adhesives. The objective of this article is to review and discuss the application of milk protein

in the formulations of environmentally-safe adhesives, and summarize current and past

examples of milk-protein-based adhesives(Guo & Wang, 2016).

2.2 Physicochemical Properties of Milk

Milk proteins are a heterogeneous mixture of proteins and constitute approximately 3.0%–

3.5% of bovine milk. About 80% of bovine milk protein is called casein, which precipitates at

pH 4.6. The protein remains that are soluble in milk serum at pH 4.6 are called whey protein.

(Goulding et al., 2020). In general, casein and whey proteins have very different properties.

Casein is mostly a random coil with a high proline content, highly phosphorylated for

calcium binding, and exists in large casein micelles; whey protein has ordered secondary

structures with a low proline content, is non-phosphorylated, and has small soluble proteins.

In addition, casein is sensitive to acid and is stable when heated, while whey protein is stable

in acid and is sensitive to heat. (Farkye and Shah, 2014).

xiii
2.3 Caseins

Due to the high level of proline residue, casein molecules are able to form α-helix and β-

sheet, which are essential factors for secondary protein structures. About 85% to 90% of

casein in bovine milk exists in a colloidal form, known as micelles, which are porous,

spherical aggregates with diameters ranging from 50 to 600 nm. Various models of casein

structure have been suggested. It has been suggested that the integrity of the micelle is

attributed to the hydrophobic interactions between casein molecules, between caseins and

calcium phosphate nanoclusters, or between polymeric casein aggregates and casein-calcium

phosphate aggregates (Thorn et al., 2009). The structure of the casein micelle is still elusive,

but the “hairy” layers of κ-casein on the surface of the micellar structure are generally. The

casein micelle is extremely stable in heat treatment, due to the κ-casein layers. Κ-casein is the

only glycosylated casein with oligosaccharides attached to throline residues in the C-terminal,

and the only calcium insensitive casein. The C-terminal, due to the increased hydrophilicity,

is the “hairy” structure that extends into the serum solution, which contributes a stabilizing

force. During the production of cheese or rennet casein, chymosin is added to cut off the κ-

casein tail, and the casein collapses to form a weak gel due to the loss of the inter-micellar

repulsion force That is why sweet whey protein (the whey produced from cheese making)

contains a significant amount of glycomacropeptide (GMP), which is one third of κ-casein

(residues 106–169) and is present in whey protein products. The casein micelle is made up of

94% casein protein and 6% CCP. CCP stabilizes the casein micelle by crosslinking casein

aggregates. During acidification, such as using acid casein, in yogurt making, or in the natural

milk-souring process, CCP is dissolved when the pH drops and causes a gradual dissolution

of micellar integrity, and then casein starts to aggregate or precipitate. The casein micelle is a

very complex polymer aggregate and interacts via hydrophobic and electrostatic interactions

and calcium bridging. A typical casein micelle contains about 10,000 casein molecules.

xiv
(Kumar et al 2002). In the past, bovine casein molecule were usually called random coil

protein because of the high content of proline residues, which prevents the formation of α-

helix or β-sheet structures. Casein has extreme heat stability and shows no evidence of

denaturation to a more disordered structure. The term “rheomorphic” was also suggested for

this type of protein. Caseins, considered rheomorphic proteins, have been adopted by many

researchers. According to de Kruif and Holt’s definition, a rhomorphic protein is “one with

an open conformation and therefore has a considerable degree of side chain, and possibly also

backbone, conformational flexibility (Same et al., 2002).

2.4 Whey

Proteins Whey is the liquid that remains after milk is curdled or strained. In the dairy

industry, whey refers to the co-products during the process of cheese, casein, or yogurt

manufacturing. Cheese whey is currently the most abundant commercially available whey

product. Whey proteins are a group of soluble proteins that are present in the milk serum

phase at a pH of 4.6. Β-Lg is the dominant protein in whey, which accounts for about 50% of

total whey proteins. The molecule is comprised of 15% α-helix (residues 129–143, 65–76 ),

50% β-sheet (residues 1–6, 11–16, 39–45, 80–85, 92–96, 101–107, 117–123, and 145–151),

and 15%–20% reverse turn (residues 7–10, 49–52, 61–64, 88–91, and 112–115) [39]. It has a

very organized secondary structure, which is described as a “cup” or “calyx” shaped three-

dimensional structure. Each monomer has one thiol group (residue 121), and two disulphide

bonds (residues 160–66 and 106–119). The presence of the thiol group and the disulphide

bond gives β-Lg an excellent gelation property via a thiol-disulphide interchange under heat

treatment. Β-Lg can exist in the form of a monomer, dimer, or octamer via the disulphide

bonds, depending on pH, temperature, and ion strength. It exists as a dimer in neutral pH

aqueous solutions. α-La is a globular protein with a two-lube structure. It has 123 amino acid

residues and contains four disulphide bonds, but has no thiol group [8,49]. The absence of the

xv
thiol group gives α-La a better heat stability than β-Lg. Unlike the thermo-gelling property of

β-Lg, α-La forms a molten globule state under mild denaturing conditions. BSA is the third

major proteins in whey. It is found in both mammal blood and milk. The molecular structure

of BSA is much large than that of β-Lg or α-La; it has approximately 580 residues. BSA has

both thiol groups and disulphide bonds (Sawyer et al.,2013).

2.5 Milk Protein Products

2.5.1. Casein Products

Casein products include rennet casein, acid casein, and caseinate. Production starts with skim

milk, and then casein is separated from milk serum by adding rennet (mimicking cheese

making), acid (mimicking natural milk souring), or using membrane filtration. Rennet is an

enzyme complex that comes from the stomach of ruminant mammals. Chymosin is the key

functional enzyme, which hydrolyzes the bond between methionine and phenyalanine in κ-

casein; thus, the casein coagulated into three-dimensional networks. The gel is cut, stirred,

drained, and washed before being dried into a powder (Hammam et al.,2021). Casein can be

precipitated at the pH of its isoelectric point, which is thought to be 4.6. The acidification can

be achieved using mineral acids, e.g., hydrochloric acid or sulphuric acid, or lactic acid

produced by microbiological fermentation. Caseinates (sodium, potassium, or calcium

caseinate) can be obtained by adding alkalis (sodium hydroxide, potassium hydroxide, or

calcium hydroxide) to acid casein (Rollema, 2009)

2.5.2 Whey Products

Whey products were initially viewed as a waste byproduct of cheese for thousands of years of

cheese history. Whey can be basically categorized into acid whey and sweet whey. Sweet

whey is the co-product of rennet cheese production, and the pH is usually around 6.0 or

higher. Acid whey comes from cottage cheese or Greek yogurt and is formed where milk is

xvi
acidified. Acid whey has a pH below 5.0 and has a high lactic acid content; the direct

processing of acid whey is limited, and it is usually considered as the problematic stream.

However, the recent Greek yogurt boom in the USA has driven studies on acid whey

utilization and processing, and acid whey is presently repeating the success of sweet whey

utilization. Liquid sweet whey can be spray-dried into a powder. Sweet whey powder

contains mostly lactose (~80%) and protein (~10%). Membrane technology has been widely

used for further processing of whey products. Whey protein concentrate (WPC) with a

protein level up to 80% (e.g., WPC34 and WPC80) can be produced using ultrafiltration

(UF). Whey protein isolate (WPI) with a protein content around 90%, can be achieved by

combining with microfiltration (MF) in order to remove excess fat. The ash level can be

further reduced by nano-filtration, ion-exchange, or resin to produce deminilized whey

protein products as by Guo(2019).

2.5.3 Membrane Filtered Milk Protein

Membrane filtration technology is able to separate milk protein from the serum phase, based

on the different sizes of molecules. Different membrane pore sizes result in different milk

protein products. Milk protein concentrate or isolate (MPC or MPI) is produced by

ultrafiltration (UF) Both casein and whey proteins are retained by the membrane, but lactose

and minerals permeate. Casein and whey protein can be split by a microfiltration (MF)

membrane, with casein being retained while whey protein, along with lactose and minerals,

can pass through the membrane, which results in micellar casein products. The whey stream

can go through a process similar to the cheese whey process to produce so-called milk serum

protein concentrate and isolates (Khalesi et al., 2021).

2.6 Milk Protein Polymerization

Protein polymerization is essential for milk protein to be used as an adhesive. The casein

micelle itself is a heterogeneous polymer complex, made of different casein molecules.

xvii
Additionally, casein also interacts with whey protein to form casein–whey aggregates. The

thiol group on the protruding κ-casein tail can interact with β-Lg via thiol-disulphide

interchange under heat treatment. On the other hand, β-Lg can interact with α-La and BSA

via the disulfide bonds. Thus, a polymer complex of casein and whey protein can be formed

under heat treatment. In order to obtain a strong adhesive strength or fast setting properties,

protein molecules may need to be crosslinked. Protein crosslinking can be achieved by

physical (irradiation, heat treatment, etc.), enzymatic, or chemical (adding cross linker)

methods. The strength of a protein network is essential to the bonding strength. Most steps in

preparing protein polymer, such as heat treatment, pH adjustment, and the addition of other

ingredients, focus on how to increase protein network strength. Mixing protein polymers with

chemical cross linkers is one of the most common methods to cure protein adhesives.

Thermal crosslinking is widely used in plywood manufacturing. By applying both heat and

pressure, protein polymers, especially heat-sensitive proteins, are denatured and pressed and

are able to form a very firm adhesive film. A hot press is usually used for plywood

manufacturing in order to increase the bonding strength (Holt et al., 2013).

2.7 Milk-Protein-Based Adhesives Applications

2.7.1 Casein-Based Adhesives

The use of casein as glue products can be dated back to ancient Egypt. The first US patent on

using casein products can be traced back to the late-19th century. The early casein adhesive

preparation usually started with sour milk or cheese with decanting whey to remove fat and

water, and then it was subjected to a boiling process to further remove water and to denature

protein. Other ingredients, such like alkali, limewater, or urea, were always added. The casein

micelle is very sensitive to environmental changes, such as pH and ion strength. Acid

precipitation is the easiest way to separate casein from milk, which was widely used in the

early days. With the washed and concentrated acid casein aggregates, alkali is usually added

xviii
to solubilize the casein into viscous, paste like glue. Alkali pH is able to reassemble the

micelle structure of casein that has been collapsed by acidification. Urea and ammonia were

other common ingredients for casein adhesives in the early to lower the viscosity by

decreasing the H-bonds. During an acid rinse, CCPs are solubilized into the serum phase;

thus, the micelle structure collapses, and combined with a heat process, casein can form a

thick, insoluble, adhesive slurry, which resembles white glue. In most cases, casein glues are

available with a casein powder and an alkali, which are ready to be mixed in water prior to

use. (Lambuth, 2006).Many of the casein glues are generally no longer used due to the advent

of synthetic adhesive polymers; however, some are still in use. In general, casein adhesive is

easy to process and provides good bond strength. Casein-based adhesives are still used as a

bottle label adhesive. Bottle-labeling adhesive for beer and soda bottles usually required ice-

proof or water-resistant properties, but also needed to be easily washed off, since bottles may

need to be returnable. Acid-precipitated casein, combined with metallic salts as cross linkers,

has been used for this purpose for about a century. Casein-based wood glue was very

common, due to its excellent water resistance, until synthetic resins were invented. The

commercial products are usually sold as a dry powder, ready to be mixed with water, right

before use, because casein wood glue has a very short pot life. Some casein wood adhesives

are still in use today. One current commercial example is “Casein glue-N” from E Min Ta An

Casein Co. Ltd. (Xinjiang, China), which is used for furniture cross banding and for beer

labels.

2.7.2 Whey-Protein-Based Adhesives

A globular structure is not ideal for adhesive applications; therefore, treatments (such as heat

and solution polarity change) that denature the protein must be applied to globular proteins

for adhesive use. Unlike the long history of casein adhesives, the use of whey protein for

adhesives is relatively new. Tschabold patented an adhesive from whey in 1953, which used

xix
condensed whey. After that, there was very little to be found in the literature until recent

years (Dunky, 2020).WPI has been used as a wood adhesive for both interior and exterior

applications. Similar to casein wood adhesive, whey protein wood adhesive is also a ready-

to-mix adhesive containing a whey polymer aqueous solution and a cross linker. The process

usually starts with a high concentration of WPI solution (20%–40%) polymerized at 60 ◦C or

higher, and is then combined with a synthetic co-polymer, such as polyvinyl acetate (PVAC)

and polyvinyl alcohol (PVA). The polymer suspension is ready to be mixed with a cross

linker, such as phenol-formaldehyde oligomer or polymeric methylene

bisphenyldiisocyanate. Due to the thermo-setting properties of whey protein, desirable

bonding strength can also be obtained by hot pressing the whey protein polymer suspension

without cross linker at 120–140 ◦C (Guo and Wang 2016.)

Office or paper glue is another area where whey protein can be applied due to safety

concerns. Current commercial paper glues are commonly PVAC- and PVA-based.

Polymerized whey protein could be able to provide a desirable bonding strength, but the

control of viscosity during storage, to make the shelf life stable, is a challenge. Polymerized

whey protein suspension can be combined with PVAC or PVA to obtain a good bonding

strength, but the glue suspension is likely to gel during the shelf life; however, polymerized

whey protein with polyvinyl pyrollidone (PVP) as a co-binder has demonstrated very good

bonding strength and a stable shelf life (at least two years). Carbohydrates (like sucrose)

could be used with the polymerized whey protein suspension in order to maintain the

viscosity constant during storage. Whey protein with PVP can also be used for glue stick

applications. β- Lg, α-La, and BSA are all rich in lysyl residues, which have ε-amino groups.

The ε-amino group is very active and can react with cross linkers, such as glutaraldehyde

(GTA). Globular protein and GTA adhesive has been commercially available for use as

surgical glue, under the brand BioGlue®. Tissue adhesive is a new alternate device for

xx
sutures, which could reduce physical pain, prevent fluid leakage, and to shorten operation

times. BioGlue® is an FDA approved tissue adhesive, which contains BSA as a protein

polymer and GTA as a cross linker. BSA is found in both bovine blood and milk. Extensive

in vivo evaluation of BioGlue® has been carried over the past 10 years. GTA can bridge the

protein polymer and the tissue cells via reaction with the free amino groups of the protein

polymers and tissue cell proteins. (Bennett et al., 2022)

2.8.1Adhesives

An adhesive is any substance that creates bond between either the surface of a material and

the adhesive itself or between the surfaces of the surface of two different materials. The

purpose of this bond is to resist any forces that try to separate the objects that have been

bonded together. Common forms that adhesives may take are glue, paste, laminates, cement,

mortar, or the adhesive backing of tapes and seals. Adhesives are an attractive option for

binding because they offer the ability to easily bind different materials together are generally

inexpensive, and efficiently distribute stress over a large area (Lambuth,2006)

2.8.2Adhesive Testing

The purpose of adhesive testing is to determine whether or not a substance is suitable for a

particular application. Many adhesives are subject to a wide range of force and stresses

during their lifetime but will most commonly experience shear, tension, peel, or any

combination of these. Testing adhesives using these forces will reveal its strength which may

be summarized by its tensile strength, shear strength and peel strength which are each

effectively the point at which the adhesive fails due to those forces. During the adhesives

testing one of three failures will occur. The may failing cohesion, where the actual adhesive

is ruptured, or in adhesion, where the adhesive is separated cleanly from the bonded surface,

or a mixture of both adhesion and cohesion.

xxi
xxii
 CHAPTER THREE

Methodology

3.1 Study Area

This research will be conducted at Technical university of Mombasa and Kenya Bureau Of

Standards Nairobi, Kenya.

3.2Research Design

This study will use experimental study design which includes field and laboratory works.

3.3 Sample collection

The sample milk for this study will be collected from KARLO farm in Mtwapa. Five liters of

fresh milk will be collected using a clean container and transported to Technical University of

Mombasa Laboratory which is a certified BSL-3standard laboratory. On reaching the

laboratory, 2.5 liters the milk sample will be store in refrigerator at temperatures of -4 degree

Celsius and another 2.5 liters will be left out on the normal laboratory environment for 24

hours waiting for experimental work. Office glue five star from Lab chem limited will be

used as a control

3.4 Formulation of water-resistant casein glue

Two cups of milk will be warmed up 40 degrees C, two halves of tablespoon of vinegar will

then be added and stirring be done to make it mix. The whey will be allowed to turn almost

clear and separate into large chunks of casein and liquid whey. This separation will happen

very quickly. If stirring is done for a while and this does not happen, more vinegar will be

added. After the whey and casein have separated, they will be strained through towel or

cheese cloth or by clumping together the casein in the mix and just pulling it out of the

saucepan and off the way 1 tablespoon of baking soda will be added the consent. This will

xxiii
react with the vinegar to make the pH less acidic. In this step, 3 to 4 tablespoons of water will

be added and warming it on the heating path. The baking soda will create bubbles and should

be taken care of to avoid it suck up. Mixing of the lump of casein will continue until

itbecomes completely liquid in the water. At this point it will be done and ready for use.This

procedure will be done for both spoiled and fresh milk samples.

3.5 Physicochemical properties testing

There are many different methods for the testing of adhesives but the most popular involve

tensile, shear and peel forces. The tensile testing of adhesives is generally perfumed for two

different situations; the bonding of two rigid substrates and bonding of a flexible and rigid

substrate.The tests will be done in triplicates and means (SD) will be calculated.

3.5.1Tensile testing

Two rigid substrates will be bonded together with above produced glue and a control for

comparison. Both substrates will be gripped properly and pulled apart at a constant rate until

either the adhesive or the substrates fail.

3.5.2Shear testing

The water-resistant glue produced, and a control will be put into a single or double lap joint

and a universal shear testing machine. The glue will be applied to the joint between the two

materials so that one continuous length is formed; two ends of the new sample will then be

placed into their respective grips in the shear tester. The tensile shear tester will then use to

apply a load to the joint until it is ruptured in shear.

3.5.3 Peel test

This will be used for the testing of the bond of the adhesive between a flexible substrate and a

rigid substrate or two flexible substrates. When a flexible material is bonded to a rigid

material either a 90 or 180-degree peel test will be used. These two tests involve the flexible

xxiv
substrate to be bent to the required angle and then pulled away from the rigid substrate

breaking the adhesive bond between the two. If the flexible substrate will be bonded together,

the common test method will be the T peel test method in which the sample is loaded into a

tensile tester in a manner that causes one substrate to point upwards and the other to point

downwards effectively forming a T at the joint. The peel will then pull the two substrates

away from each other destroying the bond at the end of the process (Awalekar et al., 2018).

 3.5.4 Scrape Test

A scrape test is typically perfumed in a laboratory and is limited to testing on smooth, flat

panel surfaces. Adhesion is determined by pushing the coated panels beneath a rounded

stylus or loop that is loaded in increasing amount until the coating is removed from the

substrate surface. A device called a balanced beam scrape-adhesion tester will be used. A

standard method for the application and performance of this test is available in ASTMD2197-

standard test method for adhesion of organic coatings by scrape adhesion as by (Bikerman,

2013)

3.5.5 Pull-Off Adhesion Test

A more quantitative test for adhesion is the pull-off test where a loading fixture, commonly

called a dolly or stub, is affixed by an adhesive to a coating. By use of a portable pull-off

adhesion tester, like the PosiTest AT, a load is increasingly applied until the dolly is pulled

off. The force required to pull the dolly off or the force the dolly withstood, yields the tensile

strength in pounds per square inch (psi) or a mega Pascal’s (MPa). Failure will occur along

the weakest plane within the system comprised of the dolly, adhesive, coating system, and

substrate, and will be exposed by the fracture as by Arukalam et al. (2016).

xxv
 3.5.6 pH.
The pH will be determined by direct immersion of the electrode with a potentiometer (Orion

Research Inc., Boston, MA).

3.5.7 Viscosity
For testing the viscosity of milk glues Brookfield RVT Dial reading viscometer (pictured

left).  This viscometer measures the torque the glue applies to a spindle and then the reading

is converted to centipois.

3.6 Data analysis

The data obtained from physiochemical analyses will be recorded. Student t-test statistical

analysis will be used for comparing the formulated glue and the control data.

xxvi
 Work plan

Month/activity May June July August Sept Nov

Proposal writing

Ethical review

Sample collection

Lab work

Data analysis

Project writing

Submission

Presentation

xxvii
 BUDGET

Description of the item Specific items Specifics Total (ksh)

items cost

Transportation Per person @1000 1,000

Chemicals and reagents  Vinegar (one @1000 1,000

bottle)
@2000 2000
 Sodium hydrogen

carbonate

Cyber cost  Printing@20 per 480*2 960

pg. 100*2 200

 Binding@100 120*2 240

 photocopying@5

Data collection  Bundles 5gb 1000 1,000

 airtime 2000 2,000

Miscellaneous 10% of the total budget 940

Total 10,340

xxviii
 REFERENCES

Alting, A.C., Hamer, R.J., de Kruif, C.G. and Visschers, R.W., 2000. Formation of

disulfide bonds in acid-induced gels of preheated whey protein isolate. Journal of

Agricultural and Food Chemistry, 48(10), pp.5001-5007.

Arukalam, I.O., Oguzie, E.E. and Li, Y., 2016. Fabrication of FDTS-modified PDMS-

ZnOnanocomposite hydrophobic coating with anti-fouling capability for corrosion

protection of Q235 steel. Journal of colloid and interface science, 484, pp.220-228.

Audic, J.L., Chaufer, B. and Daufin, G., 2003. Non-food applications of milk components

and dairy co-products: A review. Le Lait, 83(6), pp.417-438.

Awalekar, Y.J., Takalkar, A.S. and Shinde, S.S., 2018. Investigation of Peel Resistance of

Adhesives Materials: A Review. Proceedings of Engineering and Technology

Innovation, 10, p.19.

Bennett, T.M., Allan, J.F., Garden, J.A. and Shaver, M.P., 2022. Low Formaldehyde

Binders for Mineral Wool Insulation: A Review. Global Challenges, 6(4), p.2100110.

Bikerman, J.J., 2013. The science of adhesive joints. Elsevier.

Brady Jr, R.F. and Singer, I.L., 2000. Mechanical factors favoring release from fouling

release coatings. Biofouling, 15(1-3), pp.73-81.

Chalupa-Krebzdak, S., Long, C.J. and Bohrer, B.M., 2018. Nutrient density and

nutritional value of milk and plant-based milk alternatives. International dairy

journal, 87, pp.84-92.

Cherkashina, A., Rassokha, A., Ryshchenko, I. and Komarova, O., 2020. Development

and research of a label caseine adhesive for packaging the industrial and household

xxix
products. Eastern-European Journal of Enterprise Technologies, 2(6), p.104.

Dunky, M., 2020. Wood adhesives based on natural resources: a critical review Part I.

Protein-based adhesives. Reviews of Adhesion and Adhesives, 8(3), pp.199-332.

Farkye, N.Y. and Shah, N., 2014. Milk proteins. Applied food protein chemistry, pp.427-

458.

Gierenz, G. and Karmann, W. eds., 2008. Adhesives and adhesive tapes. John Wiley &

Sons.

Goulding, D.A., Fox, P.F. and O’Mahony, J.A., 2020. Milk proteins: An overview. Milk

proteins, pp.21-98.

Guo, M. and Wang, G., 2016. Milk protein polymer and its application in

environmentally safe adhesives. Polymers, 8(9), p.324.

Guo, M. ed., 2019. Whey protein production, chemistry, functionality, and applications.

John Wiley & Sons.

Hammam, A.R., Martínez‐Monteagudo, S.I. and Metzger, L.E., 2021. Progress in

micellar casein concentrate: Production and applications. Comprehensive Reviews in

Food Science and Food Safety, 20(5), pp.4426-4449.

Holt, C., Carver, J.A., Ecroyd, H. and Thorn, D.C., 2013. Invited review: Caseins and the

casein micelle: Their biological functions, structures, and behavior in foods. Journal of

dairy science, 96(10), pp.6127-6146.

Huppertz, T. and Chia, L.W., 2021. Milk protein coagulation under gastric conditions: A

review. International Dairy Journal, 113, p.104882.

xxx
Karthik, T., and D. Gopalakrishnan. "Environmental analysis of textile value chain: an

overview." Roadmap to sustainable textiles and clothing (2014): 153-188.

Khalesi, M. and FitzGerald, R.J., 2021. Insolubility in milk protein concentrates: Potential

causes and strategies to minimize its occurrence. Critical Reviews in Food Science and

Nutrition, pp.1-17.

Lambuth, A., 2006. Soybean, Blood, and casein glues. Coatings Materials and Surface

Coatings.

Rollema, H.S. and Muir, D.D., 2009. Casein and related products. Dairy Powders and

concentrated products, pp.235-254.

Sawyer, L., 2013. β-Lactoglobulin. In Advanced dairy chemistry (pp. 211-259). Springer,

Boston, MA.

Syme, C.D., Blanch, E.W., Holt, C., Jakes, R., Goedert, M., Hecht, L. and Barron, L.D.,

2002. A Raman optical activity study of rheomorphism in caseins, synucleins and tau:

New insight into the structure and behaviour of natively unfolded proteins. European

Journal of Biochemistry, 269(1), pp.148-156.

Thorn, D.C., Ecroyd, H. and Carver, J.A., 2009. The two-faced nature of milk casein

proteins: amyloid fibril formation and chaperone-like activity. Australian Journal of

Dairy Technology, 64(1), p.34.

xxxi