ENCAPSULATION OF FISH OIL USING TEXTURIZED WHEY PROTEIN

A project paper

Presented to the Faculty of Graduate School

Of Cornell University

In partial fulfilment of the requirements for the degree of

Master of Professional Studies in Agriculture and Life Sciences

Field of Food Science

By

Natasha Tahilramani

August 2016

ABSTRACT

Whey protein, previously functionalized by a novel super-critical fluid extrusion (SCFX)

process into a texturized whey protein (tWPC), was used to make cold-setting gel and

encapsulate oil (canola and fish oil). The primary purpose was to make a stable emulsion with

protein (tWPC), a natural ingredient instead of synthetic emulsifiers, to encapsulate oil

(especially fish oil) to mask the native oil smell and hence improves its organoleptic properties.

The cold, gel-like emulsions were made at 3 different oil fractions, ɸ = 20, 50, 80 by mixing it

with 80 wt. %, 50% wt. and 20% wt. aqueous tWPC dispersed solution, respectively, at 21°C

(room temperature). The stable emulsions were then treated at different shear rates and

temperatures for characterization and their structure were investigated using optical

microscopy. The results reconfirmed that tWPC has excellent emulsifying properties and could

be profitably used to provide stable and cold setting canola and fish oil emulsions. The

emulsions of both the oils exhibited pseudoplastic behaviour in the temperatures range of 11°C

to 31 °C and also at different shear rates. It was possible to dry the emulsions containing up to

20% wt. oil fraction using vacuum drying and containing it up to 50 wt. % using freeze drying.

Images of the dried samples were obtained by using optical microscopy and they showed that

the structure of the emulsions were maintained.

Keywords: whey protein, super critical fluid extrusion, emulsion, fish oil, shear rate, drying

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BIOGRAPHICAL SKETCH

Natasha Tahilramani was born and brought up in New Delhi, the capital of India. She has

graduated from Amity University in India in 2015, successfully completing her bachelor of

technology in Food Science and Technology.

She has gained her professional experience through a series of internships in manufacturing

facilities. Her interest mainly focuses on food quality management and after graduating in

August she will be working as a quality control specialist at AMES International Inc.

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ACKNOWLEDGEMENTS

I would like to express my utmost deepest appreciation to my mentor in this research as well

as throughout my graduate studies at Cornell University. Prof. Syed Rizvi, has the frame of

mind and demeanour of a genius; he convincingly conveyed a spirit of adventure and

excitement in my research. Without his guidance and persistent help this project would not

have been possible.

I would also like to thank my lab mates: Novita Putri, Richard Hebb, Andrew Melnychenko,

Pranabendu Mitra, Sugirtha Krishnamurth, Ran Zhou, Faiz Shah and Ying Lu for their

constant support.

In addition, I would like to thank my family and friends for their emotional support,

especially to my mother, Ms. Shobha Tahilramani. If it was not for her, I would not be at

Cornell University in the first place. In closing, I am blessed for the opportunity to have

worked in the Rizvi lab and I am confident that my experiences in the lab would help me in

my future endeavour.

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CONTENTS

Biographical Sketch iii

Acknowledgement iv

Chapter 1. Introduction 1

Chapter 2. Literature Review 3

Chapter 3. Material and Methods 6

3.1 Materials 6

3.2 Production of cold, gel like emulsion 6

3.3 Rheological characterization (viscosity determination) of cold, gel like

emulsions 7

3.4 Particle Size Distribution 7

3.5 Optical Microscopy 7

3.6 Vacuum Oven Drying 8

3.7 Freeze Drying 8

Chapter 4: Results and Discussions 10

4.1 Determination of viscosity 10

4.2 Particle size analysis and distribution 14

4.3 Optical microscopically visualization 16

4.4 Vacuum Oven Drying 18

4.5 Freeze Drying 18

Chapter 5. Conclusion 21

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LIST OF FIGURES

Figure 1.Canola oil emulsions: viscosity change over temperature 10

Figure 2. Canola oil emulsions: viscosity change over temperature 11

Figure 3. Canola oil emulsions: viscosity of the emulsion decreases with increase in shear

rate 12

Figure 4. Fish oil emulsions: viscosity of the emulsion decreases with increase in shear

rate 13

Figure 5. Canola oil emulsions: particle size analysis 14

Figure 6. Fish oil emulsions: particle size analysis 15

Figure 7. Canola oil emulsions: optical microscopic images of emulsion stabilized by tWPC

at oil mass fraction at (a) 0.20, (b) 0.50 and (c) 0.80 16

Figure 8. Fish oil emulsions: optical microscopic images of emulsion stabilized by tWPC at

oil mass fraction at (a) 0.20, (b) 0.50 and (c) 0.80 16

Figure 9. Vacuum dried samples. (a) Canola oil: 20% oil fraction, (b) Fish oil: 20% oil

fraction 17

Figure 10. Canola oil emulsions: freeze dried 18

Figure 11. Fish oil emulsions: freeze dried 19

Figure 12. Visualization of freeze dried sample using optical microscopy (a) 20% oil fraction

canola oil (b) 20% oil fraction fish oil 19

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LIST OF TABLES

Table 1. Formulation of emulsions 6

Table 2. Vacuum dried samples analysis 17

Table 3. Freeze dried sample analysis 18

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Chapter 1: Introduction

Whey protein - a liquid by-product of cheese production is the best quality protein among other

proteins and contains all essential amino acids. Whey was earlier discarded as waste but is now

being used to its potential with the advances in processing technology. It is now being

commercially used as an ingredient in varied products like clinical supplements, baby formulas,

sports nutrition products, etc. (Yalcin., 2006). The obvious research questions is: how to make

whey protein more functional? The novel super-critical fluid extrusion (SCFX) processing

texturizes globular proteins by shearing and stretching them into aligned bundles which

enhances adsorption at the oil – water interface. The oil in water gels can also be produced by

heat induction and homogenization together ((Jost, Baechler, & Masson, 1986) or even by

acidification, for example with glucono-δ-lactone (Boutin et al., 2007). Onwulata had proved

that appropriate heat treatment between 75 - 100°C changes functionality of protein which is

not possible through conventional extrusion and hence super-critical fluid extrusion process

was used which controls both temperature and pH conditions mentioned by Boutin in the study

of preparation of gels through acidification. Hence, whey protein was texturized, made

functional using SCFX by partial denaturation and increasing surface hydrophobicity which

favours protein adsorption at the oil in water interface. (Afizah et al., 2014).

Texturized whey protein concentrate (tWPC) which has been functionalized through SCFX

was used in this study with the hypothesis that it is possible to make stable emulsions using

protein instead of commonly used synthetic emulsifiers like lecithin. There is hydrophobic

interactions between a section of the proteins and the oil surface which creates an interfacial

layer which is generally charged. This leads to increase in surface hydrophobicity imparting

cold gel setting characteristics to tWPC which will improve emulsion stability in aqueous

phase. When whey protein is texturized, it develops a different profile as solubility decreases

and water holding capacity, thickening properties, surface hydrophobicity increases. tWPC

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basically makes formation of stable emulsions possible by increasing adsorption at oil-water

interface and forming a modified continuous phase (Manoi., 2009). A number of researchers

have studied the application of emulsifiers in food industry that can produce stable emulsions

but using tWPC is may be more economically sound as it is natively a by-product of cheese

industry. Also, tWPC is a “natural” ingredient which satisfies the latest trend of all natural

amongst consumers. Even though, the term “natural” is not legally regulated, it is the latest

trend which means consumers want simple and clean ingredient list like protein on the label

which everyone understands rather than some synthetic emulsifier on the label like

Polyoxyethylene-20-sorbitan mono-oleate which is nothing less than a jargon for layman.

Emulsions encapsulating oil with tWPC will make it suitable for the food industry to use it not

only in fortification, but also to create food with better organoleptic properties. In recent years,

omega 3 fatty acids (high in fish oil) have developed its importance due to its health benefits

and made it more valuable to incorporate it in food to promote healthy lifestyle of consumers.

Docosahexaenoic acid (DHA) has been attracting researchers as its intake help in preventing

cardiovascular diseases, inflammation (Yan et al., 2013) , lipotoxicity (Frédéric Capel, 2015)

and liver diseases (Fedor et al., 2012). That is why this area of study holds significance.

In this study it was hypothesised that incorporation of tWPC within an aqueous phase will

result into encapsulating oil. The primary objective is to prepare highly nutritious emulsions

using protein and then drying the emulsions to make it easier for handling.

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Chapter 2: Literature Review

Extrusion is one of the most versatile and well established process used in food industry.

Conventional extrusion controls the product attributes by manipulating specific mechanical or

thermal inputs. The temperature goes as high as 130-150°C resulting in loss heat and shear

sensitive flavours, colours, vitamins and proteins. Whereas, in case of supercritical fluid

extrusion (SCFX) the temperature in barrel is maintained at 50-90°C (precisely under 100°C)

such that water just acts as a plasticizer and carbon dioxide (CO2) acts as blowing agent.

Supercritical CO2 injection in the barrel is the key for expansion in SCFX (Rizvi et al., 1995).

Whey Protein is a very useful by-product from dairy industry which can be made more

functional by texturizing globular proteins by shearing and stretching to change the molecular

structure of proteins them with the help of extrusion. Since very high temperature leads to loss

of gel strength, texturizing whey protein with help SCFX (temperature < 100°C) is a better

idea. Anyway, whey protein is already in market and being used in fortification, texturizing it

increases its value (Onwulata et al., 2003) The best property of tWPC is that it has the ability

to form a cold-setting gel (protein ingredients that form a gel at room temperature) without

additional heat input. It acts as an emulsifier as partial denaturation of whey protein during

extrusion produces soluble, aggregated protein with increased surface hydrophobicity, which

favours protein adsorption at the oil–water interface. tWPC produced through SCFX, at higher

temperature (around 90°C) and low pH exhibits the most stable rheological properties (Afizah

et al., 2014).

For the second ingredient which is oil: fish and canola oil are used to prepare stable emulsions.

Even though fish oil has more focus because of its nutritional properties, all the experiments

were carried out with canola oil too to determine efficacy of the process by analysing

comparative data. Like fish oil, canola oil has low amount of saturated fat and high content of

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polyunsaturated fats. Also, canola oil may be the most nutritionally balanced cooking oil of all

major culinary oils. ( O’brien., 2008).

Fish oil, is known as a functional food especially because of the poly unsaturated fatty acid

(PUFA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) (Zatsick et al., 2007). It

is just a challenge to work with them because of its strong odour and weird taste. Also it is not

stable as it can get oxidized very fast. Encapsulation of fish oil with the help of milk proteins

will make the emulsion more stable as whey protein will function as the barrier between oxygen

and fish oil. After the encapsulation is done, it will be converted into a powder base material

through vacuum oven drying subjecting the product to minimum heat required (40-50°C).

According to researchers, spray drying is the most commonly used drying method to dry

emulsions having fish oil which can go up to inlet temperature of 150°C providing oxidative

stability and also providing successful applications in milk and yoghurt for their respective

shelf lives (Wang et al.,2011). Whereas, when influence of drying methods was studied on the

stabilization of fish oil microencapsules, spray granulation proved to be the best method

operated at 70 °C between spray granulation, spray drying and freeze drying. Even though

freeze drying is performed at very low temperatures, it leads to a flake-like, porous and

irregular structure which accelerates oxidation (Anwar et al., 2011). On the contrary, some

researchers have proved freeze drying to deliver finished good with better shelf life and

improved oxidation stability over spray drying targeting niche market (Heinzelmann et al.,

1999).

Encapsulation is technically known as process to entrap active agents within a carrier material.

The main purpose of encapsulation is to stabilize an active ingredient, control its release rate

and convert a liquid formulation into a more viscous semi solid form which makes it easier to

handle. There are a lot of techniques that have been used since 60 years like spray drying, spray

bed drying, fluid bed drying, melt extrusion and the oldest of all is coacervation.(Nedovic et

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al., 2011). Even though encapsulation makes liquid substance easier to handle but the best

technique is to convert the formulation into dried form. Food dehydration refers to removal of

water from foods under controlled conditions to mainly preserve the food product. Drying is

the second part of the research which consists of vacuum drying and freeze drying.

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Chapter 3: Materials and Methods

3.1 Materials

Texturized whey protein was made available through previous student’s research which was

stored in airtight containers dated back to April 7th, 2011. Canola oil was purchased from a

local retailer. Nile red and Fast Green FCF used in optical microscopy were obtained from

Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA).

3.2 Production of cold, gel like emulsion

Aqueous solution containing 20% (w/w) tWPC and deionized water was prepared by stirring

for 2 hours at room temperature, and then stored at 4°C overnight to stabilize the solution

ensuring complete dissolution. Since mold and bacteria can grow in whey protein, sodium azide

(0.04% w/w) was incorporated to the aqueous solution for the prevention of microbial growth.

Emulsions were prepared of different oil fractions (ɸ = 20, 50, 80) to successfully encapsulate

the oil. Hence, the amount of tWPC in aqueous solution was fixed varying the oil fraction. The

emulsion were prepared by mixing the predetermined amount of oil (canola or fish oil) with

correct amount of aqueous tWPC dispersion as explained in Table 1 at 1100rpm for 3 minutes

using a high-speed dispersing and emulsifying unit (IKA-ULTRA-TURRAX® T25 basic,

IKA® Works, Inc., NC, USA). In case of emulsions containing 60% oil, the pre-emulsion

containing 50% oil was first prepared as above. The appropriate amount of oil was then added

to the pre-emulsion at the rate of 10 mL/min and then mixed continuously until the final

emulsion was obtained. The resulting emulsions were stored in airtight containers until they

were analysed.

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Table 1. Formulation of emulsions.

20% oil fraction 50% oil fraction 80% oil fraction

Oil % (w/w) 20 50 80

tWPC % (w/w) 16 10 4

Deionized water % (w/w) 64 40 16

3.3 Rheological characterization (viscosity determination) of cold, gel like emulsions

The rheological properties that could be determined was mainly the viscosity of the emulsion

with the help of Brookfield DV viscometer. Shear rate and shear stress could not be determined

due to limitations of highly viscous product. The impact of temperature (11°C, 21°C, and 31°C)

and rotational speed (20, 50, 100 rpm) of the spindle on the viscosity of emulsion were also

analysed.

3.4 Particle size distribution

Particle size analysis was carried out using 90 Plus particle size analyzer by Brookhaven

Instruments corporation. The basic principle behind particle size analysis is scattering of light

with a dust cut off of 30 nm. For the particle size analyzer to determine the particle size

distribution, scatter of light needed to bump into particles and give out the reading. Hence,

there was a need to dilute the emulsion with deionized water in the ratio of 1:10 (w/w) with the

aim to make the solution translucent for the light to pass through.

3.5 Optical Microscopy

Other than structural visualization, optical microscopy is also used for determining the particle

size. However, it is widely accepted that this method of size analysis cannot adequately

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differentiate between particles of less than I µ diameter owing to the limited resolution of the

light microscope as the particles approach the wavelength of the incident radiation.

The selected oil was stained with Nile Red (1 mM stock solution of Nile Red in high-quality,

anhydrous Dimethyl sulfoxide (DMSO)) to visualize the oil phase and aqueous tWPC

dispersion was stained with Fast Green FCF (0.001%, w/w in deionized water) to visualize the

protein phase. The emulsions were then prepared with stained ingredients so as to increase the

visibility of encapsulated oil phase. The stained emulsion was placed on a glass slide and

observed under optical microscope with magnification of 10x.

3.6 Vacuum oven drying

The emulsion in its liquid phase is harder to be transported or used for fortification than in

dehydrated phase. High quality dehydrated products have a promising potential market. The

samples were placed on petri plate forming a very thin layer which were then placed in a

benchtop vacuum oven was operating with 27 inches of hg vacuum and 50°C temperature.

The drying rate was determined by periodic weighing of the sample. The primary goal was to

dry the emulsion without breaking the emulsion if it is at all possible at such high temperatures

and then to characterize it (Drouzas et al., 1999).

3.7 Freeze Drying

Freeze drying has been considered as the good technique for preservation and drying of

particles. Freezing is the first step of freeze-drying. During this step, the liquid suspension is

cooled, and ice crystals of pure water forms. As the freezing process continues, more and more

water contained in the liquid freezes. This results in increasing concentration of the remaining

liquid. As the liquid suspension becomes more concentrated, its viscosity increases inducing

inhibition of further crystallization. This highly concentrated and viscous liquid solidifies,

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yielding an amorphous, crystalline, or combined amorphous-crystalline phase. The small

percentage of water that remains in the liquid state and does not freeze is called bound water.

(Abdelwahed, et al. 2006)

The emulsions were freeze dried using MillRock Technology model Max 53 at vacuum 29.5

inches of mercury and -40°C temperature. Even though the cost of operating a freeze dryer is

more than that of vacuum oven, researchers have proved that freeze dying is more efficient.

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Chapter 4: Results and Discussions

4.1 Determination of viscosity

The previous work (Manoi, 2009) revealed that tWPC formed a cold-set thickening ability

upon reconstitution with water at 20% (w/w) protein concentration. Once the oil concentrations

in the emulsion system decreased from 80 to 20% (w/w), the final protein and water contents

of emulsions would vary from 4 to 16% (w/w), and 16 to 64% (w/w), respectively. Visual

observations of emulsions after one day of storage at room temperature (25°C) revealed that

all emulsions made from tWPC displayed a self-standing gel with a soft solid-like texture.

It is not fair to the food industry when literature values of viscosity of liquid foods just report

single temperature value. Such data is of limited usefulness in food processing where

temperature and shear rate may be quite different from the conditions of a single viscosity

measurement. The purpose of these rheological measurements was to obtain the needed

viscometric data to characterize the emulsion.

Each sample of the emulsion for both, canola and fish oil emulsions (ɸ = 20, 50, 80) were

analyzed using viscometer with 21°C being the room temperature and also varying the

temperature at 11°C and 31°C using attached water bath. The observations for canola oil

emulsions are reported in Figure 1 and those of fish oil emulsions are reported in Figure 2. The

results shows viscosity of emulsion with 80% oil fraction was much higher than that of 20%

oil fraction which was also confirmed by visual inspection. It can also be determined that

viscosity considerably decreases with increase in temperature. As expected (Saravacos., 1970)

there is an exponential relation between temperature and viscosity. Even though the effect of

temperature was more pronounced in higher total solid content, the pattern of decrease in

viscosity with increase in temperature is followed by both, canola and fish oil emulsions.

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Figure 1.Canola oil emulsions: viscosity change over temperature.

0.00

2000.00

4000.00

6000.00

8000.00

10000.00

12000.00

14000.00

16000.00

11°C 21°C 31°C

Vis

cosi

ty (c

P)

Temperature (°C)

Canola oil Emulsions: Viscosity change with temperature

20 % oil 50% oil 80% oil

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Figure 2. Canola oil emulsions: viscosity change over temperature

Since measurement of shear stress and strain was a limitation with highly viscous fluids,

change in viscosity with exposure to various shear rate is measured by varying rotational

speed of the spindle as higher speed result in more shear on the material. Such an experiment

was carried out by starting with the lowest speed 20rpm and then stepwise increasing the

speed of rotation up to 100 rpm. The emulsion was characterized as pseudoplastic due to

decrease in viscosity with increasing rotational speed that is increasing shear stress. In

rheology, it is also known as shear thinning which basically means viscosity decreases with

increase in shear rate. Similar pattern was observed in both, canola (Figure 3) and fish oil

(Figure 4) emulsions.

0.00

5000.00

10000.00

15000.00

20000.00

25000.00

30000.00

35000.00

40000.00

11°C 21°C 31°C

Vis

cosi

ty (c

P)

Temperature (°C)

Fish oil Emulsions: Viscosity change with temperature

20% oil 50% oil 80% oil

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Figure 3. Canola oil emulsions: viscosity of the emulsion decreases with increase in shear

rate.

0.00

20000.00

40000.00

60000.00

80000.00

100000.00

120000.00

140000.00

160000.00

6.3 s-1 15.7 s-1 31.4 s-1

Vis

cosi

ty (c

P)

Shear rate (s-1)

Canola oil emulsions: Viscosity change with increase in shear rate

20% oil 50% oil 80% oil

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Figure 4. Fish oil emulsions: viscosity of the emulsion decreases with increase in shear rate.

4.2 Particle size analysis and distribution

There is significant impact of particle size upon the physical properties of emulsion reviewed

by other researchers (Groves et al., 1968). It is generally agreed that stability, viscosity, rate of

heat transfer, and optical properties of emulsions are dependent upon the particle size or the

particle-size distribution of the disperse phase. However, there is at present no satisfactory

correlation of the relationship.

Figure 4 and 5 strongly suggests that the fat globule size was larger and when the oil fraction

was higher, which is confirmed by optical microscopic images.

0.00

20000.00

40000.00

60000.00

80000.00

100000.00

120000.00

6.3 s-1 15.7 s-1 31.4 s-1

Vis

cosi

ty (c

P)

Shear rate (s-1)

Fish oil emulsions: Viscosity change with increase in shear rate

20% oil 50% oil 80% oil

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Figure 5. Canola oil emulsions: particle size analysis.

1238.8

1621.3

2802.9

0

500

1000

1500

2000

2500

3000

3500

20% 50% 80%

PART

ICLE

SIZ

E (N

M)

OIL FRACTION %

Canola oil emulsions: Particle size

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Figure 6. Fish oil emulsions: particle size analysis.

4.3 Optical microscopically visualization

The rheological results are well supported by optical microscopic images observed in selected

tWPC-stabilized emulsions at three different oil fractions of 0.80, 0.50, and 0.20 for both,

canola and fish oil emulsions.

150…

2266.5

4544.2

0

1000

2000

3000

4000

5000

6000

20% oil 50% oil 80% oil

PART

ICLE

SIZ

E (N

M)

OIL FRACTION %

Fish oil emulsion: particle size

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(a)

Figure 7. Canola oil emulsions: optical microscopic images of emulsion stabilized by tWPC

at oil mass fraction at (a) 0.20, (b) 0.50 and (c) 0.80.

Figure 8. Fish oil emulsions: optical microscopic images of emulsion stabilized by tWPC at

oil mass fraction at (a) 0.20, (b) 0.50 and (c) 0.80.

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4.4 Vacuum Oven Drying

The drying is carried out till the constant weight is achieved. 80% and 50% oil fraction

emulsion broke down at 50°C within 20 hours in vacuum oven. The dehydrated emulsions were

analyzed as elaborated in Table 2 and visual observations are reported in Figure 9.

Table 2. Vacuum dried samples analysis

Canola oil emulsions Fish oil emulsions

% Oil fraction 20 50 80 20 50 80

Water activity 0.59

Emulsion broke down

0.48

Emulsion broke down Moisture

content (%)

2.67 1.8

Figure 9. Vacuum dried samples. (a) Canola oil: 20% oil fraction, (b) Fish oil: 20% oil fraction

4.5 Freeze drying

After the emulsions broke down in vacuum drying, it was expected that all the oil % fractions

would dry efficiently with the technique of freeze drying. Yet the 80% oil fraction sample for

both, canola and fish oil emulsion broke down. The dehydrated emulsions were analyzed as

elaborated in Table 3 and visual observations are reported in Figure 10. By comparing the

values of Table 2 and 3, the range of water activity and moisture content values are much lower

in the case of freeze drying (in Table 3) than for vacuum drying (Table 2) which proves that

freeze drying works better.

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The data points out that fish oil emulsions dried better than canola oil emulsions by either of

the technique, be it vacuum drying of freeze drying. For eg. In Table 3 the water activity for

20% oil fraction of fish oil is 0.036 which is less than half of 20% oil fraction of canola oil.

This pattern is observed with all the sets of data reported. The same is confirmed by visual

observation in Figure 10 and 11.

Table 3. Freeze dried sample analysis

Canola oil emulsions Fish oil emulsions

% Oil fraction 20 50 80 20 50 80

Water activity 0.094 0.4 Emulsion

broke

down

0.036 0.198

Emulsion

broke

down

Moisture

content (%)

1.01 0.80 0.85 0.531

Figure 10. Canola oil emulsions: freeze dried

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Figure 11. Fish oil emulsions: freeze dried.

Even though optical microscopy is not the best technique for visualization, it gives an idea

that the structure is maintained even after freeze drying the emulsions for 20% oil fraction as

shown in Figure 12.

Figure 12. Visualization of freeze dried sample using optical microscopy (a) 20% oil fraction

canola oil (b) 20% oil fraction fish oil

Chapter 5: Conclusion

It was previously proved that tWPC is an excellent emulsifier and the research concludes that

this fact can be applied to emulsify oils with high unsaturated fatty acids like canola and fish

oil. It is fascinating to note only 4g of tWPC can emulsify 80g of oil and make a stable emulsion

in case of 80% oil fraction. After exposing the samples to increasing shear rate, it is established

that the emulsions exhibit pseudoplastic behaviour in all environmental conditions. It is also

noted that the structure of the emulsions is maintained in all environmental conditions which

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is observed by optical microscopy. Optical microscopy gives a brief idea how the structure is

maintained but previously employed confocal microscopy seems like a better technique for

visualization. It was possible to dry the emulsions using vacuum oven drying and freeze drying.

Not only freeze drying is more efficient than vacuum oven drying, it can also dry up to 50%

oil fraction as compared to 20% oil fraction in case of vacuum oven drying. The research is

focused on 20, 50 and 80% oil fraction and hence drying is limited to such % oil fraction but it

is possible that vacuum drying will be efficient for more than 20% oil fraction but less than

50% oil fraction and in case of freeze drying, it may be possible to dry more than 50% oil

fraction but less than 80% oil fraction. The dried sample analysis also revealed that fish oil

emulsions dry better than canola oil emulsions. However, extensive stability analysis for dried

emulsions would be required before using it for fortification or new product development.

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BIBLOGRAPHY

Afizah, M. N., & Rizvi, S. S. (2014). Functional properties of whey protein concentrate

texturized at acidic pH: effect of extrusion temperature. LWT-Food Science and

Technology, 57(1), 290-298

Anwar, S. H., & Kunz, B. (2011). The influence of drying methods on the stabilization of fish

oil microcapsules: Comparison of spray granulation, spray drying, and freeze drying.

Journal of Food Engineering, 105(2), 367-378.

Abdelwahed, W., Degobert, G., Stainmesse, S., & Fessi, H. (2006). Freeze-drying of

nanoparticles: formulation, process and storage considerations. Advanced drug delivery

reviews, 58(15), 1688-1713.

Boutin, C., Giroux, H. J., Paquin, P., & Britten, M. (2007). Characterization and acid-induced

gelation of butter oil emulsions produced from heated whey protein dispersions.

International dairy journal, 17(6), 696-703

Capel, F., Acquaviva, C., Pitois, E., Laillet, B., Rigaudière, J. P., Jouve, C., ... & Morio, B.

(2015). DHA at nutritional doses restores insulin sensitivity in skeletal muscle by

preventing lipotoxicity and inflammation. The Journal of nutritional biochemistry, 26(9),

949-959.

Drouzas, A. E., Tsami, E., & Saravacos, G. D. (1999). Microwave/vacuum drying of model

fruit gels. Journal of Food Engineering, 39(2), 117-122.

Fedor, D. M., Adkins, Y., Mackey, B. E., & Kelley, D. S. (2012). Docosahexaenoic Acid

Prevents Trans-10, Cis-12–Conjugated Linoleic Acid-Induced Nonalcoholic Fatty Liver

Disease in Mice by Altering Expression of Hepatic Genes Regulating Fatty Acid

Synthesis and Oxidation. Metabolic syndrome and related disorders, 10(3), 175-180.

Groves, M. J., & Freshwater, D. C. (1968). Particle‐size analysis of emulsion systems.

Journal of pharmaceutical sciences, 57(8), 1273-129.

Heinzelmann, K., & Franke, K. (1999). Using freezing and drying techniques of emulsions

for the microencapsulation of fish oil to improve oxidation stability. Colloids and

Surfaces B: Biointerfaces, 12(3), 223-229.

JOST, R., BAECHLER, R., & MASSON, G. (1986). Heat Gelation of Oil‐in‐Water

Emulsions Stabilized by Whey Protein. Journal of Food Science, 51(2), 440-444

Manoi, K., & Rizvi, S. S. (2009). Emulsification mechanisms and characterizations of cold,

gel-like emulsions produced from texturized whey protein concentrate. Food

Hydrocolloids, 23(7), 1837-1847.

Nedovic, V., Kalusevic, A., Manojlovic, V., Levic, S., & Bugarski, B. (2011). An overview

of encapsulation technologies for food applications. Procedia Food Science, 1, 1806-

1815

O'brien, R. D. (2008). Fats and oils: formulating and processing for applications. CRC press

23 | P a g e

Onwulata, C. I., Konstance, R. P., Cooke, P. H., & Farrell, H. M. (2003). Functionality of

extrusion—texturized whey proteins. Journal of dairy science, 86(11), 3775-3782.

Rizvi, S. S. H., Mulvaney, S. J., & Sokhey, A. S. (1995). The combined application of

supercritical fluid and extrusion technology. Trends in Food Science & Technology,

6(7), 232-240.

Yalcin, A. S. (2006). Emerging therapeutic potential of whey proteins and peptides. Current

pharmaceutical design, 12(13), 1637-1643

Zatsick, N. M., & Mayket, P. (2007). Fish oil: getting to the heart of it. The Journal for Nurse

Practitioners, 3(2), 104-109.