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Encapsulation and microwave technologies to preserve/improve quality of foods

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Graduate School ETD Form 9
(Revised 12/07)
PURDUE UNIVERSITY
GRADUATE SCHOOL
Thesis/Dissertation Acceptance
This is to certify that the thesis/dissertation prepared
By Mandar Ranchhod Patel
Entitled
ENCAPSULATION AND MICROWAVE TECHNOLOGIES TO PRESERVE/IMPROVE QUALITY
OF FOODS
For the degree of
Doctor of Philosophy
Is approved by the final examining committee:
M. Fernanda San Martin-Gonzalez
Chair
Tameshia Ballard
Osvaldo Campanella
G. Narsimhan
To the best of my knowledge and as understood by the student in the Research Integrity and
Copyright Disclaimer (Graduate School Form 20), this thesis/dissertation adheres to the provisions of
Purdue University’s “Policy on Integrity in Research” and the use of copyrighted material.
M. Fernanda San Martin-Gonzalez
Approved by Major Professor(s): ____________________________________
____________________________________
Approved by: R. Chandrasekaran
Head of the Graduate Program
06/21/2012
Date
Graduate School Form 20
(Revised 9/10)
PURDUE UNIVERSITY
GRADUATE SCHOOL
Research Integrity and Copyright Disclaimer
Title of Thesis/Dissertation:
ENCAPSULATION AND MICROWAVE TECHNOLOGIES TO PRESERVE/IMPROVE QUALITY
OF FOODS
For the degree of
Doctor
Philosophy
Choose of
your
degree
I certify that in the preparation of this thesis, I have observed the provisions of Purdue University
Executive Memorandum No. C-22, September 6, 1991, Policy on Integrity in Research.*
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I certify that all copyrighted material incorporated into this thesis/dissertation is in compliance with the
United States’ copyright law and that I have received written permission from the copyright owners for
my use of their work, which is beyond the scope of the law. I agree to indemnify and save harmless
Purdue University from any and all claims that may be asserted or that may arise from any copyright
violation.
Mandar Ranchhod Patel
______________________________________
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06/18/2012
______________________________________
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*Located at http://www.purdue.edu/policies/pages/teach_res_outreach/c_22.html
1
ENCAPSULATION AND MICROWAVE TECHNOLOGIES TO
PRESERVE/IMPROVE QUALITY OF FOODS
A Dissertation
Submitted to the Faculty
of
Purdue University
by
Mandar Ranchhod Patel
In Partial Fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
August 2012
Purdue University
West Lafayette, Indiana
UMI Number: 3544324
All rights reserved
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a note will indicate the deletion.
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ii
│
iii
ACKNOWLEDGEMENTS
First and foremost, I would like to express my gratitude towards my major
professor Dr. M. Fernanda San Martin-Gonzalez for showing trust in my abilities and
guiding me throughout my Ph.D. program. Without her guidance, this graduate work
would not have been possible.
I would like to thank my advisory committee members Dr. Tameshia Ballard, Dr.
G. Narsimhan and Dr. Osvaldo Campanella for giving me appropriate directions during
this graduate program and showing great consideration during difficult times. Not to
forget my ex-committee member Dr. David Nivens who always went out of his way to
help me in learning and research.
I am also grateful to Dr. Janaswamy Srinivas, Dr. Bhavesh Patel, Dr. Amandeep
Kaur, Joseph Bledsoe, Weijian (Victor) Wang, Benjamin Paxson, Steve Smith, Anton
Terekhov, my labmates Gulsah Bakir, Veronica Rodriguez-Martinez, Claudia Salazar and
Jose Lopez for assisting me in my research in every possible way.
Life in a land far away from home could not have been easy without all my
friends at Purdue. I thank my friends Deepak, Meghana, Vaishnavi, Preetha, Azalenah,
Shivangi, Rei, Dhananjay, Meenesh, Niraj, Tejaswi, Siddharth, Jason, Abhijit, Shreeram,
Nirupama and many more for giving me reasons to smile.
iv
I can't express my gratitude enough for my parents Mr. Ranchhod and Mrs.
Madhura Patel who have always given me an absolute support all along. Through them, I
experience the Almighty and His unconditional love.
v
TABLE OF CONTENTS
Page
LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES .............................................................................................................x
ABSTRACT ................................................................................................................ xiii
CHAPTER 1. INTRODUCTION ......................................................................................1
1.1
Microwave processing of strawberry puree ....................................................1
1.1.1 Background ...........................................................................................................1
1.1.2 Overall objectives and hypotheses........................................................................2
1.1.3 Significance ..........................................................................................................3
1.2
Encapsulation of ergocalciferol in solid lipid nanoparticles...........................3
1.2.1 Background ...........................................................................................................3
1.2.2 Overall objectives and hypotheses........................................................................5
1.2.3 Significance ..........................................................................................................5
1.3
Dissertation organization ................................................................................6
CHAPTER 2. LITERATURE REVIEW: MICROWAVE HEATING .............................7
2.1
History ……………………………………………………………………. 7
2.2
Microwave heating of foods: Theory .............................................................8
2.2.1 Heating mechanisms .............................................................................................8
2.2.2. Dielectric properties of foods ..............................................................................9
2.3
Microwave heating applications ...................................................................12
2.3.1 Extraction of phytochemicals .............................................................................12
2.3.2 Drying .................................................................................................................14
2.3.3 Baking .................................................................................................................16
2.3.4 Microbial inactivation .........................................................................................17
2.3.5 Enzyme inactivation ...........................................................................................20
2.4
Continuous microwave heating ....................................................................21
2.5
Strawberry.....................................................................................................25
2.6
Anthocyanins ................................................................................................26
CHAPTER 3. LITERATURE REVIEW: ENCAPSULATION OF VITAMIN D2 ........29
3.1
Encapsulation ................................................................................................29
3.2
Solid lipid nanoparticle as a carrier system ..................................................31
vi
Page
3.2.1 Preparation methods for solid lipid nanoparticles ..............................................32
3.2.2
Characterization of solid lipid nanoparticles ............................................35
3.2.3 Solid lipid nanoparticles as carriers for food bioactive compounds ...................37
3.3
Vitamin D .....................................................................................................40
3.3.1 Vitamin D Encapsulation ....................................................................................42
CHAPTER 4. COLOR AND ANTHOCYANIN CONTENT OF STRAWBERRY
PUREE THERMALLY PROCESSED BY A PILOT SCALE CYLINDRICAL
MICROWAVE HEATING SYSTEM (100 KW), TUBULAR AND SCRAPED
SURFACE HEAT EXCHANGERS ................................................................................. 45
4.1
Abstract ……………………………………………………………………45
4.2
Introduction...................................................................................................46
4.3
Materials and Methods .................................................................................47
4.3.1 Continuous flow microwave, scraped surface heat exchanger and tubular heat
exchanger heating of strawberry puree .....................................................................47
4.3.2 Total monomeric anthocyanin content analysis .................................................48
4.3.3 Instrumental color measurement.........................................................................49
4.3.4 Statistical analysis ...............................................................................................49
4.4
4.4 Results and Discussion ...........................................................................50
4.4.1 Total monomeric anthocyanin content ...............................................................50
4.4.2 Instrumental color ...............................................................................................50
4.5
Conclusions...................................................................................................51
CHAPTER 5. EFFECT OF BATCH CONVENTIONAL AND MICROWAVE
HEATING ON RHEOLOGICAL PROPERTIES, ANTHOCYANIN CONTENT AND
COLOR OF STRAWBERRY PUREE ............................................................................. 56
5.1
Abstract ……………………………………………………………………56
5.2
Introduction...................................................................................................57
5.3
Materials and Methods .................................................................................58
5.3.2 Rheological testing .............................................................................................59
5.3.3 Total monomeric anthocyanin content analysis .................................................59
5.3.4 Instrumental color measurement.........................................................................60
5.3.5 Statistical analysis ...............................................................................................60
5.4
Results and Discussion .................................................................................61
5.4.1 Rheology .............................................................................................................61
5.4.2 Total monomeric anthocyanin content ...............................................................62
5.5
Conclusions...................................................................................................63
vii
Page
CHAPTER 6. LABORATORY SCALE (6KW) CONTINUOUS MICROWAVE
HEATING SYSTEM: DESCRIPTION, INSTALLATION AND OPERATION ........... 67
6.1
Introduction...................................................................................................67
6.2
CFMW system (6 kW)..................................................................................67
6.2.1 Description, installation and operation ...............................................................67
6.2.2 Challenges...........................................................................................................69
6.3
Tubular heat exchanger system ....................................................................70
6.3.1 Description, installation and operation ...............................................................70
6.3.2 Challenges...........................................................................................................71
CHAPTER 7. PHYSICOCHEMICAL PROPERTIES OF STRAWBERRY PUREE
OVER A STORAGE PERIOD AS AFFECTED BY 6 KW CONTINUOUS FLOW
MICROWAVE HEATING SYSTEM IN COMPARISON WITH TUBULAR HEAT
EXCHANGER SYSTEM ................................................................................................. 73
7.1
Abstract 73
7.2
Introduction...................................................................................................74
7.3
Materials and Methods .................................................................................76
7.3.1 Puree making ......................................................................................................76
7.3.3 Water holding capacity .......................................................................................78
7.3.4 Rheological testing .............................................................................................78
7.3.5 Total monomeric anthocyanin content analysis .................................................79
7.3.6 Instrumental color measurement.........................................................................79
7.3.7 Turbidity of strawberry serum samples ..............................................................80
7.3.8 Carbohydrate composition determination by proton NMR ................................80
7.3.9 Statistical analysis ...............................................................................................81
7.4
Results and Discussion .................................................................................81
7.4.1 Rheological properties of strawberry puree and selected strawberry sera and
turbidity of selected sera ...........................................................................................81
7.4.2 Water holding capacity .......................................................................................86
7.4.3 Carbohydrate composition by NMR ...................................................................87
7.4.4 Total monomeric anthocyanin content ...............................................................88
7.4.5 Instrumental color ...............................................................................................89
7.5
Conclusions...................................................................................................90
CHAPTER 8. CHARACTERIZATION OF ERGOCALCIFEROL LOADED SOLID
LIPID NANOPARTICLES ............................................................................................ 101
8.1
Abstract 101
8.2
Practical Application ..................................................................................102
8.3
Introduction.................................................................................................103
8.4
Materials and Methods ...............................................................................105
viii
Page
8.4.1 Materials ...........................................................................................................105
8.4.2 Preparation of SLN dispersions ........................................................................105
8.4.3 Turbidity measurement .....................................................................................107
8.4.4 Particle size measurement.................................................................................107
8.4.5 Transmission electron microscopy (TEM) .......................................................107
8.4.6 Differential scanning calorimetry (DSC)..........................................................108
8.4.7 Data analysis .....................................................................................................108
8.5
Results and Discussions ..............................................................................109
8.5.1 Turbidity of SLN dispersions ...........................................................................109
8.5.2 Particle size of SLN dispersions .......................................................................109
8.5.3 Transmission electron microscopy (TEM) of SLN dispersions .......................110
8.5.4 Differential scanning calorimetric (DSC) analysis ...........................................111
8.6
Conclusions.................................................................................................113
CHAPTER 9. OVERALL CONCLUSIONS AND RECOMMENDATIONS FOR
FUTURE WORK ............................................................................................................ 122
9.1
Overall conclusions ....................................................................................122
LIST OF REFERENCES .................................................................................................126
VITA
................................................................................................................153
ix
LIST OF TABLES
Table ..............................................................................................................................Page
Table 5.1 Lightness (L*), redness/greenness (a*), yellowness/blueness (b*), chroma (C*)
and hue angle (⁰h) of strawberry puree samples treated by batch microwave heating
and batch conventional heating................................................................................... 66
Table 7.1 Flow rates and hold times for 6 kW CFMW and tubular H.E. systems. .......... 77
Table 8.1 Mean enthalpy of fusion of the a-subcell polymorph as a function of
ergocalciferol content................................................................................................ 121
x
LIST OF FIGURES
Figure .............................................................................................................................Page
Figure 2.1 Structures of most common anthocyanins present in nature (Modified from
Mateus and De Freitas 2008; Giusti and Jing 2007). .................................................. 27
Figure 3.1 Structure of ergocalciferol ............................................................................... 42
Figure 4.1 Monomeric anthocyanin content of strawberry puree at different storage times
processed by continuous flow microwave system (100 kW), scraped surface heat
exchanger and tubular heat exchanger at 80, 91 and 102ºC. ...................................... 52
Figure 4.2 Lightness (L*) of strawberry puree at different storage times processed by
continuous flow microwave system (100 kW), scraped surface heat exchanger and
tubular heat exchanger at 80, 91 and 102ºC................................................................ 53
Figure 4.3 Chroma (C*) of strawberry puree at different storage times processed by
continuous flow microwave system (100 kW), scraped surface heat exchanger and
tubular heat exchanger at 80, 91 and 102ºC................................................................ 54
Figure 4.4 Hue angle (ºh) of strawberry puree at different storage times processed by
continuous flow microwave system (100 kW), scraped surface heat exchanger and
tubular heat exchanger at 80, 91 and 102ºC................................................................ 55
Figure 5.1 Apparent viscosities (at 100 s-1) of strawberry puree heated by conventional
method and microwave radiation in a batch mode. .................................................... 64
Figure 5.2 Monomeric anthocyanin content of strawberry puree heated by conventional
method and microwave radiation in a batch mode. .................................................... 65
Figure 6.1 Process flow diagram for 6 kW continuous flow microwave processing of
strawberry puree.......................................................................................................... 72
Figure 6.2 Process flow diagram for tubular heat exchanger heating of strawberry
puree............................................................................................................................ 72
xi
Figure .............................................................................................................................Page
Figure 7.1 Apparent viscosity of strawberry puree at 100 s-1 heated by (A) 6 kW
continuous flow microwave system and (B) tubular H.E. system at different
temperature at different storage times......................................................................... 91
Figure 7.2 % Water holding capacity of strawberry puree at 100 s-1 heated by (A) 6 kW
continuous flow microwave system and (B) tubular H.E. system at different
temperature at different storage times......................................................................... 92
Figure 7.3 Apparent viscosity of strawberry sera at 100 s-1 separated from strawberry
puree heated by 6 kW continuous flow microwave system and tubular H.E. system at
different temperature at different storage times. ......................................................... 93
Figure 7.4 Turbidity of strawberry sera separated from strawberry puree heated by 6 kW
continuous flow microwave system and tubular H.E. system at different temperature
at different storage times. ............................................................................................ 94
Figure 7.5 Visual appearance of strawberry puree heated by (A) 6 kW continuous flow
microwave system and (B) tubular H.E. system at 80ºC (right) compared to
corresponding preheated control (left). ....................................................................... 95
Figure 7.6 Representative NMR spectra of strawberry sera separated from puree treated
by 6 kW CFMW/tubular H.E. system ......................................................................... 96
Figure 7.7 Monomeric anthocyanin content of strawberry puree heated by (A) 6 kW
continuous flow microwave system and (B) tubular H.E. system at different
temperature at different storage times......................................................................... 97
Figure 7.8 Lightness (L*) of strawberry puree heated by (A) 6 kW continuous flow
microwave system and (B) tubular H.E. system at different temperature at different
storage times. .............................................................................................................. 98
Figure 7.9 Chroma (C*) of strawberry puree heated by (A) 6 kW continuous flow
microwave system and (B) tubular H.E. system at different temperature at different
storage times. .............................................................................................................. 99
Figure 7.10 Hue angle (⁰h) of strawberry puree heated (A) 6 kW continuous flow
microwave system and (B) tubular H.E. system at different temperature at different
storage times.. ........................................................................................................... 100
xii
Figure .............................................................................................................................Page
Figure 8.1 Decrease in turbidity of SLN dispersions (3/11 dilution) with different %
ergocalciferol loading.. ............................................................................................. 114
Figure 8.2 Particle size of ergocalciferol loaded SLN dispersions ................................. 115
Figure 8.3 TEM images of SLN dispersions with different % ergocalciferol loading. .. 116
Figure 8.4 Differential scanning calorimetry thermogram of freeze dried tripalmitin solid
lipid nanoparticles stabilized by Polysorbate 20 with different % ergocalciferol
concentrations (w/w of dispersed phase). (A) Melting cycle, (B) Crystallization
cycle.. ........................................................................................................................ 117
Figure 8.5 Differential scanning calorimetry thermogram of tripalmitin solid lipid
nanoparticle suspensions stabilized by Polysorbate 20 with different % ergocalciferol
concentrations (w/w of dispersed phase). (A) Melting cycle, (B) Crystallization
cycle.. ........................................................................................................................ 118
Figure 8.6 Enthalpies of fusion and crystallization of ergocalciferol loaded freeze dried
SLN dispersions.. ...................................................................................................... 119
Figure 8.7 Enthalpy of fusion of α-subcell polymorph of ergocalciferol loaded SLN
suspensions.. ............................................................................................................. 120
Appendix Figure ...................................................................................................................
Figure A 1 Representative flow curves for strawberry puree heated by 6 kW
CFMW/tubular H.E. system ..................................................................................... 151
Figure A 2 Flow curves of selected strawberry sera separated from strawberry puree
heated by 6 kW continuous flow microwave system and tubular H.E. system at
different temperature at different storage times. ....................................................... 152
xiii
ABSTRACT
Patel, Mandar R. Ph.D., Purdue University, August 2012. Encapsulation and Microwave
Technologies to Preserve/Improve Quality of Foods. Major Professor: Fernanda San
Martin-Gonzalez.
This dissertation discusses the details of research conducted on two independent
projects. Although independent, these two approaches carry a common thread towards
preserving/improving quality of food products. Summary of these two projects is as
follows.
First project explores microwave heating as an emerging and commercially
applicable thermal processing technique to process perishable fruit products such as
strawberry puree. Strawberry is a fruit best known for its characteristic red color, delicate
flavor and nutritional benefits such as antioxidant capacity. However, high susceptibility
of strawberries and strawberry products to fungal spoilage makes it necessary to process
them for shelf life enhancement. Microwave heating is known to have advantages such
as short come-up times, due to volumetric heating which, can be exploited in inactivating
yeasts and molds responsible for spoilage of strawberry products without compromising
their quality. In this investigation, strawberry puree was thermally processed by
microwave radiation on a batch system at 80ºC and on continuous flow pilot (100 kW)
and laboratory (6 kW) scale systems at 80, 91 and 102ºC at different holding times.
Strawberry puree was also processed by conventional tubular heating methods to
xiv
compare with microwave processing under similar processing conditions. In spite of the
differences in processing conditions and results for batch, small and large continuous
systems, overall, it could be summarized that microwave processing showed higher
retention of anthocyanin content, color and viscosity of strawberry puree as compared to
conventionally heated and untreated puree over a storage period of up to 12 weeks with
no significant differences among three processing temperatures. These findings support
the efficacy of microwave heating in preserving quality of food products when compared
to conventional heating methods as well as its potential commercial applicability due to
the continuous nature of the system.
The second project sets a background for a potential application of solid lipid
nanoparticles (SLN), an encapsulation technique commonly used in pharmaceutical
industry, for encapsulation of food bioactive compounds such as lipophilic vitamins.
Ergocalciferol or vitamin D2 is a fat-soluble compound essential for the development of
bones in human body. It is susceptible to degradation by oxygen and light and hence was
chosen as a model compound for encapsulation in this study. In this research study,
ergocalciferol was encapsulated in tripalmitin solid lipid nanoparticles stabilized by
polysorbate 20. SLN dispersions (5% w/w) with Ergocalciferol concentrations at 0, 5, 10,
15 and 20% (w/w of lipid phase) were prepared by hot high pressure homogenization. As
the proportion of ergocalciferol in the SLN increased from 0 to 20%, size (Z-average
values) of these particles, as measured by dynamic light scattering, gradually decreased
(p≤0.05) from ~120 nm to ~65 nm. Differential scanning calorimetry (DSC) results of
freeze dried SLN samples showed gradual decrease in enthalpies of fusion and
crystallization for the stable β-subcell polymorphic form, whereas for SLN dispersions,
xv
the enthalpy of fusion of unstable α-subcell crystal form increased with increased
ergocalciferol loading. This suggests that increasing ergocalciferol concentration slows
down the crystallization process and results in predominant presence of more unstable
crystal forms. Transmission electron microscopy (TEM) images of ergocalciferol loaded
SLNs showed the presence of spherical as well as rod-shaped nanoparticles. It was also
observed that the turbidity of the SLN dispersions noticeably decreased with increased
ergocalciferol loading, indicating the potential application of ergocalciferol-loaded SLNs
for the fortification of clear beverages.
1
CHAPTER 1. INTRODUCTION
This chapter presents background, objectives, hypotheses and significance of
research conducted on two independent areas: microwave processing of strawberry puree
and encapsulation of ergocalciferol in solid lipid nanoparticles.
1.1
Microwave processing of strawberry puree
1.1.1 Background
Microwave radiation has been used for heating food products in American
households for the last five decades (Buffler 1993; Decareau 1985). Rapid and
volumetric heating, short come-up times, and high penetrating power are some of the
advantages of microwave radiation when used to heat food products (Chavan and Chavan
2010; Sumnu 2001; Huang and Sites 2007). Numerous applications of microwave heating
including extraction of phytochemicals (Lu and others 2010; Wang and Weller 2006; Lu
and others 2010; Wang and Weller 2006; Escribano-Bailon and Santos-Buelga 2003;
Eskilsson and Bjorklund 2000), drying (Mujumdar and Law 2010; Zhang and others
2010; Huang and others 2011; Zhang and others 2010), baking (Ozkoc and others 2009;
Sanchez-Pardo and others 2008; Daglioglu and others 2000; Keskin and others 2005),
inactivation of microorganisms for pasteurization and sterilization (Kozempel and others
2
2000; Shamis and others 2008; Barnabas and others 2010) and enzyme inactivation
(Severini and others 2003; Begum and Brewer 2000; Zheng and Lu 2011) have been
reported. Due to the advantages mentioned above, microwave heating could potentially
be an alternative option to conventional thermal processing methods for pasteurization or
sterilization of food products with minimal degradation of quality.
Strawberry (Fragaria × ananassa) is a globally popular fruit due to its
characteristic color, distinct flavor and nutritional benefits (Hancock 1999; Gossinger and
others 2009; Hartmann and others 2010). Although strawberries are preferably consumed
raw, their highly perishable nature makes them ideal candidates to be processed
(Cordenunsi and others 2005) into various products like puree, jams, jellies, etc.
Conventional thermal processing techniques used for this purpose could degrade the
quality inherent to raw strawberries. Alternate processing methods such as microwave
heating hold potential to address this challenge due the rapid and volumetric nature of its
heating mechanism. Hence, in this research work, the effects of batch and continuous
heating (laboratory and pilot scale) on quality properties of strawberry puree were
investigated.
1.1.2 Overall objectives and hypotheses
First overall objective of this research was to study the effects of microwave
heating on total monomeric anthocyanin content, instrumental color, rheological
properties, water holding capacity and sugar profile of serum. Strawberry puree was
processed using 1) a batch scale domestic microwave (1.8kW, 2450 MHz) oven, 2) a
continuous pilot scale (100 kW, 915 MHz) and 3) continuous lab scale (6 kW, 2450 MHz)
3
cylindrical microwave heating systems. The second objective was to compare these
effects of microwave heating with conventional heating methods at each scale.
The working hypothesis of this research is that the quality of strawberry puree
processed by microwave heating, as evaluated by viscosity, water holding capacity,
anthocyanin content and instrumental color of strawberry puree over a storage period will
be enhanced when compared to untreated and/or conventionally heated puree due to
reduced heating time by microwave technology.
1.1.3 Significance
If the hypothesis holds true i.e. if microwave heating better preserves the quality
attributes of strawberry puree as compared to conventional heating, it will be a feasible
alternative for commercial processors of fruit products. Whether it being a worthwhile
investment for food fruit product manufacturers or not, this research will certainly
provide an additional option for them to consider.
1.2
Encapsulation of ergocalciferol in solid lipid nanoparticles
1.2.1 Background
Encapsulation techniques are widely used in pharmaceutical industry to increase
solubility of active compounds, improve their stability against factors such as oxygen,
light and pH, improve their bioavailability and enhance their controlled release potential
(McClements and others 2007; Wagner and Warthasen 1995; Rieux and others 2006;
4
Wegmüler and others 2004). Production of solid lipid nanoparticles (SLN) is one of such
encapsulation techniques commonly used in the pharmaceutical industry since early
1990s (Pardeike and others 2009; Muller and others 2000). The advantages of using SLN
over conventional emulsions are higher stability of core material and encapsulated
compound, decreased leakage of the active drug compound and controlled release ability
due to reduced mobility of the encapsulated active compound (Bunjes and Siekmann
2006). Since digestion of these lipid nanoparticles is slowed down due to their solid state
(Bonnaire and others 2008), they can be orally administered for controlled release in the
gastrointestinal tract (Muller and others 1996). In recent years, SLN has attracted
attention as an encapsulant for food bioactive compounds (Weiss and others 2008).
Vitamin D is a fat soluble vitamin naturally present in a limited number of foods,
which is an essential factor for the maintenance of plasma levels of calcium and
phosphorus, hence playing an important role in mineralization of bones and
neuromuscular functions (DeLuka and Zierold 1998). Although fortification of certain
food products such as milk with vitamin D has drastically reduced the incidence of
deficiency diseases like rickets and osteomalacia, sporadic cases of rickets have been
reported in children and infants (Hochberg 2003) due to reasons such as limited sun
exposure, use of sunblock, fear of skin cancer and melanoma, limited consumption of
fortified foods and fear of excessive vitamin D intake (Chesney 2003; Ward and others
2007), aging (Need and others 1993), fat malabsorption (Lo and others 1985) and obesity
(Wortsman and others 2003). Due to these reasons, it is necessary to diversify vitamin Dfortified food options. Since, vitamin D is fat-soluble, an encapsulation technique can be
utilized to assist in the fortification of aqueous food products. Ergocalciferol is the plant
5
origin form of vitamin D and is susceptible to degradation by oxygen (Eitenmiller and
others 2008), light and acid conditions (Eitenmiller and others 2008; Grady and Thakker
1980). Susceptibility of ergocalciferol to environmental factors and low solubility in
aqueous food matrices make it a suitable candidate for encapsulation.
1.2.2 Overall objectives and hypotheses
The objective of this study was to characterize tripalmitin solid lipid nanoparticles
loaded with different concentrations of ergocalciferol for their particle size, turbidity,
morphology, and phase transition behavior.
Since this was a characterization study, there was no hypothesis made prior to this
research study.
1.2.3 Significance
Significance of this research study is that the information obtained by
characterizing ergocalciferol loaded solid lipid nanoparticles could be a valuable source
for future studies such as investigating stability of encapsulated ergocalciferol or its
controlled release potential. Possessing knowledge about particle size, polymorphic
structures could be a useful tool to understand and correlate encapsulation efficiency,
stability, bioavailability and release rates of encapsulated active compound.
6
1.3
Dissertation organization
Chapter 1 of this dissertation provides background information and overview of
the two research studies conducted. Chapter 2 reviews available literature on microwave
heating of foods, its mechanisms of action, and reported applications. It also gives basic
information on strawberry fruit and anthocyanins. Chapter 3 discusses literature on
application of solid lipid nanoparticles for encapsulation of food bioactive compounds,
vitamin D, its deficiency and causes of deficiency.
Chapter 4 discusses details of the study conducted with a pilot scale (100 kW)
continuous cylindrical microwave heating system; whereas Chapter 5 presents the study
conducted using a batch microwave system for processing strawberry puree. Chapter 6
describes the installation and operation of a laboratory scale(6 kW) continuous
cylindrical microwave heating system and some of the limitations faced. Chapter 7
reports the study conducted in the laboratory scale (6kW) system for processing
strawberry puree.
Chapter 8 presents the characterization study performed for ergocalciferol loaded
solid lipid nanoparticles.
Chapter 9 concludes findings of this dissertation along with recommendations for
future research followed by references used in the writing of this dissertation.
7
CHAPTER 2. LITERATURE REVIEW: MICROWAVE HEATING
2.1
History
Microwaves encompass a part of the electromagnetic spectrum with frequencies
ranging from 300 MHz to 300 GHz or corresponding wavelengths ranging from 1 m to 1
mm. Most commonly used microwave frequencies are 2450 MHz for smaller scale and
915 MHz for industrial scale applications (Kumar and others 2007; Meda and others
2005; Smith 2010). The earliest application of microwaves dates back to 1934, where
microwaves were used in radio detection and ranging (radar) to detect aircrafts and this
technology was extensively used further in the World War II. After the war, the focus
shifted towards the application of microwaves to heat materials especially foodstuffs. A
patent was filed by Raytheon Corporation in 1945 regarding the application of
microwaves to heat food products followed by development of the first microwave oven
in 1946. By 1947, one restaurant chain in Boston started using microwave oven to thaw
their frozen meals prepared in one centralized facility. However, the use of these bulky,
complex microwave ovens was restricted to restaurants, laboratories and industries due to
their high cost ($1500-2500). In mid-1960s, development of small-sized 1 kW
microwave units working on 110 V and priced below $1000 saw its rapid expansion in
the household market (Buffler 1993; Decareau 1985).
8
2.2
Microwave heating of foods: Theory
2.2.1 Heating mechanisms
There are two mechanisms involved when a food product is heated by the
microwaves. These mechanisms are based on two different types of interaction of
microwaves with the ionic and non-ionic species present in the food.
2.2.1.1
Ionic interaction
Microwaves are electromagnetic waves and hence there is an electric field
associated with these waves. This electric field alternates with the oscillating microwaves.
When a food is exposed to microwaves, ionic species in that material tend to align
themselves with this alternating electric field. These ions having a net positive or a
negative charge are accelerated in one direction and then in the opposite direction in an
alternating fashion due to the alternating nature of the electric field associated with the
microwaves. These accelerated ions possessing kinetic energy collide with neighboring
molecules to produce heat (Buffler 1993; Smith 2010).
2.2.1.2
Dipolar interaction
Water is a major constituent of most of the food products. Two positively charged
hydrogen atoms in a water molecule make a ~105º angle with a negatively charged
oxygen atom to create a ‘dipole’ of opposite charges within a molecule. When a food
product is exposed to microwaves, the dipolar molecules align themselves in the direction
9
of the electric field. When the field changes its direction, the molecules rotate themselves
to align in an opposite direction. These molecules collide with other neighboring
molecules each time they rotate with the electric field. This collision produces heat.
Dipolar interaction is the predominant heating mechanism by microwaves for most of the
foods than the ionic interaction (Meda and others 2005; Buffler 1993; Smith 2010).
2.2.2. Dielectric properties of foods
Similar to the visible spectrum of the electromagnetic radiations, when
microwaves are incident upon the surface of a material, parts of these incident radiations
are reflected, transmitted, refracted and/or absorbed. The extent to which microwaves
would interact with different food materials in different ways is governed by the
dielectric properties of food products. Dielectric properties of materials take into account
only the interaction of electric component of the microwaves with materials and not the
magnetic component. Since most of the food materials are magnetically non-interactive,
only dielectric properties are relevant as far as interaction of microwaves with food
materials is concerned. Dielectric properties are expressed in terms of complex dielectric
constant ε, which mathematically is a combination of the dielectric constant ε’ and an
imaginary term containing dielectric loss factor ε’’ as shown in Equation 1 (Kumar and
others 2007; Meda and others 2005).
... (1)
Where,
,
ε0 = permittivity of free space (8.86 × 10-12 F/m)
10
When microwaves are incident upon the surface of a material, part of them is
reflected. The remaining part penetrates through the bulk of the material. As it passes
through the material, fraction of that penetrated waves is absorbed by the material and the
remaining fraction is transmitted out of the material. Microwaves interact with atoms and
molecules of the material to deviate from their original directional path similar to
refraction of visible light before they are transmitted out of the material. The extent of
this interaction is indicated by the dielectric constant ε', where
, the optical
refractive index of the material. The remaining portion of the penetrated microwave
power is absorbed by the material to convert it into heat by ionic and dipolar interaction
mentioned before. The extent to which the microwave power is absorbed by a material is
dictated by the dielectric loss factor ε''.
The ratio of dielectric loss factor to the dielectric constant is called the ‘loss
tangent’ (Equation 2) which is an indication of the ability of a material to convert
absorbed microwave energy into heat (Meda and others 2005; Berk 2009).
... (2)
Apart from dielectric properties, it is necessary to understand that the heating by
microwaves also depends on how far microwaves can penetrate into different materials.
Different materials have different ‘power penetration depth’, which is defined as the
depth at which the microwave power is decreased to 1/e or 36.8% of the original power
(Buffler 1993; Singh and Heldman 2009).
11
2.2.2.1 Measurement of dielectric properties
As dielectric properties of foods are crucial in their heating by microwaves,
experimental measurement of dielectric properties is important. There are three most
commonly used methods for the measurement of dielectric properties.
i) Transmission line method: This method uses the measurement of amplitude and phase
of the reflected microwaves from a sample to calculate its dielectric constant (ε’) and loss
factor (ε’’). The sample needs to be placed at the end of a short-circuited transmission
line i.e. a waveguide or a coaxial line. For this method, a presumption of the values of
dielectric constant is necessary since the thickness of the sample needs to be
approximately 25% of the wavelength. The coaxial line method is suitable for a wide
range of microwave frequencies where as waveguide method is usually used for a single
wavelength measurement of dielectric properties (Buffler 1993; Regier and Schubert
2005).
ii) Resonance cavity method: In this method, a cavity is made inside a waveguide by
putting two plates with central holes. A sample is placed inside this cavity for the
measurement. The changes in microwave frequency and width of the transmission
characteristics after the sample is placed in the cavity provide necessary information in
order to calculate dielectric properties (Buffler 1993; Regier and Schubert 2005).
iii) Open-ended coaxial probe method: This is the most preferred method for the
dielectric properties measurement compared to other methods because it is easy to use, it
can be used over a wide range of microwave frequencies and is suitable for both solid and
liquid foods (Kumar and others 2007). In this method, an open-ended coaxial probe is
inserted into the sample. The amplitude and the phase of the reflected radiation at the end
12
of this coaxial line are used to measure the dielectric properties of the sample (Buffler
1993; Regier and Schubert 2005).
2.3
Microwave heating applications
Microwave heating of foods has been used and/or investigated for the following
applications.
2.3.1 Extraction of phytochemicals
Dielectric properties of the intracellular materials as well as different polar and
non-polar extraction solvents can be exploited in order to facilitate the extraction of
several plant based compounds using microwave heating. Due to higher penetrating
power of microwave radiation, rapid rupture of plant cell structures is facilitated and that
improves extraction efficiency of many compounds (Lu and others 2010; Wang and
Weller 2006). In addition to rapid heating, other advantages of microwave assisted
extraction (MAE) include significantly lower amount of solvent required (EscribanoBailon and Santos-Buelga 2003) and possibility of extracting from multiple samples at
the same time (Eskilsson and Bjorklund 2000). Solubility of compounds to be extracted
governs the choice of solvent for MAE process. There are two possible ways by which
microwave assists the extraction of compounds distinguished by the polarity of solvents
used. Polar solvent with high dielectric properties get heated rapidly by microwave
radiation and hence accelerates the extraction process. In case of non-polar solvents, the
13
sample matrix gets heated by microwave radiation relatively more as compared to the
surrounding microwave transparent solvent. Due to rapid heating of sample, intracellular
moisture vaporizes and bursts the cell-wall to release and eventually extract the
compounds (Eskilsson and Bjorklund 2000). Both these mechanisms can be utilized at
once by using a mixture of microwave absorbing and transparent solvents to achieve
efficient heating and solubilization of analyte in the solvent at the same time.
Microwave-assisted extraction technique has been employed over a broad
range of materials such as extraction of pesticides, metals, hydrocarbons, pharmaceutical
compounds, cosmetic ingredients and food-based compounds (Eskilsson and Bjorklund
2000). Some recent developments in microwave-assisted extraction of food-based
compounds are as follows. Wang and others (2011) developed a dynamic MAE technique
coupled with an on-line clean up system to detect as low as 0.012 mg/g of caffeine in tea
with a recovery in the range of 88.2% - 99.3%. Liazid and others (2011) developed and
optimized MAE process as a step prior to chromatographic determination of 11
anthocyanins from grape skins and found that extraction at 100⁰C for 5 min in 40%
methanol in water as a solvent to be an optimum condition. The technique has been used
recently for the extraction of phenolic compounds from bean (Sutivisedsak and others
2010), potato peels (Singh and others 2011), foxtail millet (Wang and others 2010) and
peanut skins (Ballard and others 2010).
Cyanuric acid is a potentially toxic adulterant found in pet foods along with
melamine (infamous toxicant in infant formula) responsible for producing lethal insoluble
crystals in kidneys of cat. Microwave-assisted extraction combined with liquid
chromatography - tandem mass spectrometry was used by Han and colleagues (2011) for
14
the determination of cyanuric acid in pet foods. Farhat and others (2010) used a
modification to a typical MAE process without using any solvent or water. They applied
microwave heating for dry diffusion and earth gravity to extract essential oils from dried
caraway seeds. A similar approach was showed by Cendres and others (2011) where they
used hydrodiffusion instead of dry diffusion to extract juice from selected fruits.
2.3.2 Drying
Drying is one of the most common and most energy consuming unit operations
for the preservation of food products (Zhang and others 2010; Duan and others 2010).
Several conventional drying methods such as air drying, vacuum drying and freeze
drying have a major disadvantage of low drying rate during the falling rate period, which
results in quality deterioration of food products due to long exposure at high temperatures
(Zhang and others 2010; Duan and others 2010). Microwave radiation has been used to
assist in drying of foods due to advantages such as increased speed, fast start-up,
relatively uniform heating, better process control, higher energy efficiency and all these
resulting into better quality dehydrated product (Mujumdar and Law 2010; Zhang and
others 2010). However, there are few limitations of the microwave heating itself such as
non-uniform heating due to uneven nature of electromagnetic radiation, focusing, corner
and edge heating and non-uniformity in the composition of material to be heated
(Mujumdar and Law 2010). Also, the loss of moisture as drying progresses reduces the
dielectric loss factor resulting in reduced conversion of microwave energy into heat
15
(Zhang and others 2010). Due to these limitations of microwave heating, it is usually
combined with conventional drying methods to overcome the drawbacks of both methods.
Freeze drying is considered one of the best methods for the dehydration of
thermally sensitive materials in order to preserve their quality (Duan and others 2010). In
microwave-assisted freeze drying (MFD), the heat needed for the sublimation to occur in
freeze drying is provided by the microwave radiation. As microwave heats a material
volumetrically, it increases the rate of freeze drying and hence reduces the energy
consumption (Huang and others 2011; Zhang and others 2010). It has also shown to
retain volatile compounds better than freeze drying (Duan and others 2010). MFD has
been recently studied for drying of meat, jellies, seafood (Duan and others 2010),
potato/banana chips (Huang and others 2011; Jiang and others 2011) and carrots (Yan
and others 2010).
Microwave radiation has also been combined with vacuum drying where moisture
in a product is evaporated using microwave heating under vacuum at relatively lower
temperatures (Zhang and others 2010). Although sublimation (in freeze drying) requires
lesser energy than evaporation, dielectric constant of water is much higher than that of ice
and hence microwave-assisted vacuum drying (MVD) has been shown to be more
efficient than microwave freeze drying (Jiang and others 2011). Some recent studies
involving MVD are drying of products such as carrots (Yan and others 2010; Changrue
2006), potato (Song and others 2009), strawberries (Changrue 2006), grapes (Clary and
others 2007) and rice paddy (Cheenkachorn 2007).
16
2.3.3 Baking
Conventionally, baking is a process where structural changes in food such as
gelatinization of starch, denaturation of proteins, non-enzymatic browning, escape of
water vapor and carbon dioxide resulting in increased volume of the dough take place due
to heat transfer directed from surface to the core of the dough with the formation of a
crust (Chavan and Chavan 2010; Sanchez-Pardo and others 2008; Sumnu 2001).
Although microwave baking has few advantages over conventional baking such as
significantly lower energy and time consumption, lower space requirements and
reduction in losses of nutrients, the consumers have not completely accepted microwavebaked products due to few quality issues (Sumnu 2001; Sanchez-Pardo and others 2008;
Chavan and Chavan 2010). Challenges regarding the quality of microwave baked
products include insufficient height of the baked product, dense or gummy texture,
hardness of the crumb and an undesirable moisture gradient along the height of the baked
product (Sumnu 2001). Desirable changes taking place in a food product during
conventional baking which are mentioned previously are mainly due to the lengthy time
of baking, which allows these changes to occur, whereas quicker microwave heating does
not provide sufficient time for these changes to occur. The most prominent difference in
quality of products baked by these methods is a lack of browning in microwave-baked
products. Unlike conventional oven baking, the temperature inside a microwave oven
does not increase as baking progresses and hence results in surface cooling of the baked
product. This surface cooling prohibits browning reactions such as Maillard browning or
caramelization, which are also responsible for producing distinctive ‘baked’ aroma and
17
flavor (Chavan and Chavan 2010; Sumnu 2001). Sumnu (2001) has summarized
following different approaches in his review to induce browning in microwave-baked
products. Using metalized plastic film laminates in the packaging can absorb microwave
energy and transfer it to the surface of the product to induce localized heating. Adding
salt and coloring compounds can increase the product temperature. Combination of
microwave with conventional baking can give ‘baked’ flavor development in addition to
browning. Impingement of fluid jets on the product surface can enhance surface heating
resulting in crust formation and browning. Finally, direct control of microwave power
and field can help controlling the heating rates and temperature distribution within the
product while baking to imitate conventional baking. Different microwave-baked
products recently studied are bread (Ozkoc and others 2009; Icoz and others 2004;
Demirekler and others 2004), cake (Al-Muhtaseb and others 2010; Sanchez-Pardo and
others 2008; Sakiyan and others 2007), flat dough (Serventi and others 2011), puff pastry
(Daglioglu and others 2000) and cookies (Keskin and others 2005).
2.3.4 Microbial inactivation
Microwave heating has been studied for its efficacy in inactivation of
microorganisms responsible for food borne illnesses as well as spoilage of food products.
Similar to several conventional heating methods, the inactivation of microorganisms by
microwave radiation has been widely thought to be because of its thermal effects (Shamis
and others 2008). However, whether or not there are any nonthermal effects of
microwave radiation responsible for microbial destruction has been a highly debatable
18
issue for several years (Tahir and others 2009; Shamis and others 2008; Kozempel and
others 2000). Main reason for this issue to be controversial is the difficulty in studying
nonthermal effects of microwave radiation unaffected and separated completely from
thermal effects (Kozempel and others 2000). However, attempts have been made to study
the nonthermal effects of microwave inactivation of microorganisms. Kozempel and
others (2000) designed a system of treating different fruit juices inoculated with yeasts
and pathogens by microwave radiation without letting the product to heat up beyond
45˚C using a cooling arrangement. Results of this study were not consistent among
different fruit juices and there were no strong evidences of nonthermal effects. However,
a study conducted by Shamis and others (2008) showed nonthermal destruction of
Escherichia coli and Staphylococcus aureus in raw meats by repeated exposures to
microwave radiation at higher frequency (18 GHz) than commonly used 2.45 GHz.
Barnabas and others (2010) did however used usual 2.45 GHz microwave radiation and
showed nonthermal destruction of pathogenic bacteria on a low dielectric loss substrate.
Although inconclusive, the mechanism of this nonthermal inactivation is discussed to be
to disturb metabolic activities inside the bacterial cell due to dipole alignment of several
intracellular molecules to the alternating microwave field (Barbanas and others 2010).
Microwave heating has found to cause significantly higher degree of protein unfolding
compared to conventional heating (George and others 2008), which could also serve
towards nonthermal destruction of microorganisms.
In spite of whether through thermal or nothermal mechanism, microwave
inactivation of microorganisms has advantages over conventional heating methods. Faster
heating, short come-up times, higher energy efficiency (Chavan and Chavan 2010;
19
Sumnu 2001) and volumetric heating (Huang and Sites 2007) makes microwave
treatment a suitable method for pasteurization or commercial sterilization of food
products where thermal degradation of quality parameters of food products is reduced
(Tajchakavit and others 1998). Numerous studies have been conducted over the years to
explore the effects of microwave heating on microbial inactivation and some of the recent
works are as follows. Huang and Sites (2007) developed an automatically controlled inpackage pasteurization system for ready-to-eat beef frankfurters using microwaves
heating. They observed rates of destruction of Listeria monocytogenes to be 0.41, 0.65
and 0.94 log CFU/pack/min at the surface temperatures of 65, 75 and 85˚C respectively
using microwave heating, which was 30-75% higher than water-immersion heating. In a
very similar study with frankfurters, L. monocytogenes was inactivated up to 3.7 log
CFU/cm2 when heated for 75 s ina domestic microwave oven at 1100 W power level
(Rodriguez-Marval and others 2009). Dabrowski and others (2009) exposed poultry
nuggets inoculated with three strains of Campylobacter to microwave radiation at power
levels of 340, 480 and 760 W for 30, 60, 90, 120 and 180 s. Complete destruction of
Campylobacter spp. was noticed in 90 s for 340 and 480 W power levels whereas 760 W
of microwave power achieved this complete destruction in 60 s. Wang and others (2010)
studied the effects of microwave heating in combination with ultrasound wave on
Alicycloclobacillus spp and found that this combination was more effective than separate
microwave or ultrasound treatments. Granny Smith apple puree treated by microwave
radiation at 652 W for 35 s showed destruction of E. coli O157:H7 and Listeria innocua
to below detectable levels (Picouet and others 2009) whereas blue mold rot (Penicillium
expansum) on pear was partially inactivated by microwave for 3 minutes (Zhang and
20
others 2006). Laguerre and others (2011) investigated microwave heating as a tool to
sterilize infant formula in order to minimize the formation of neo-formed contaminants
(NFC), which are products of Maillard browning reaction and are believed to be harmful
to health. They found microwave heating at high power for a short time to be the best
way to minimize NFC formation.
2.3.5 Enzyme inactivation
Although published research in this specific area is limited in recent years,
microwave heating has been used in inactivation of enzymes. Blanching is a process of
destruction of enzymes such as polyphenol oxidase, which are responsible for enzymatic
browning in fruits and vegetables. Conventional water and steam blanching processes
may however, result in changes in quality attributes such as loss of firmness of texture,
undesirable starch gelatinization, loss of soluble solids (Severini and others 2003),
unwanted changes in color, loss of nutrients and antioxidant properties (Zheng and Lu
2011). Extent of these undesirable changes could be minimized by using microwave
radiation, which is faster and more efficient (Severini and others 2003). Higher retention
of ascorbic acid has been shown by microwave blanching in snow peas (Begum and
Brewer 2000) and green asparagus (Zheng and Lu 2011). In addition to ascorbic acid, βcarotene retention was shown to be higher in other vegetables like spinach, broccoli,
green beans and carrots by microwave blanching with faster inactivation of polyphenol
oxidase (Ramesh and others 2002). Although microwave blanching is not very effective
in minimizing firmness loss, methods such as using calcium chloride and sodium chloride
21
solutions as blanching baths (Severini and others 2001) have helped retaining texture
during microwave blanching.
Apart from polyphenol oxidase, microwave heating has been studied for the
inactivation of phospholipase D in soybean (List and others 1990), peroxidase and
lipoxygenase in broccoli (Orak 2006), myrosinase in canola seeds (Owusu-Ansah and
Marianchuk 1991) and proteolytic enzymes in broad bean seeds (Baraniak and Czuba
2001).
2.4
Continuous microwave heating
In spite of the widespread use of microwaves to heat food products in American
household since 1960s, body of literature available on its use on a continuous system is
small. There have been certain patented attempts to use this technology to heat food
products on a continuous system since mid 1960s (Long and others 1966; Jeppson and
Harper 1967; Kenyon and others 1976) but very few research articles discussing this are
available until late 1990s. Kenyon and others (1971) discuss a system for a continuous
heating of food products packaged in pouches using microwaves. Products such as
chicken and frankfurters were packaged in pouches usually used in conventional retorts
except for the aluminum foil, which is reflectant to microwaves. These pouches were
carried on a conveyer through a high pressure chamber with an applicator area where the
food in pouches was exposed to microwave radiations at 2450 MHz and power up to 10
kW and then cooled down in a water bath. In a continuation to this study (Ayoub and
others 1974), they developed a time-temperature monitoring system in addition to
measurement of temperature distribution within the pre-packed food processed using
22
microwave radiation on a continuous system. However, none of these studies discussed
the effect of this continuous microwave system on the microbial destruction or the quality
of food.
Since 1990s, attempts were made to evaluate the microwave heating of
continuously flowing pumpable foods. Nikdel and MacKellar (1992) designed a
continuous microwave heating system by installing a Teflon tube inside a domestic
microwave oven, through which orange juice was pumped. Different residence times
were obtained by using different lengths of tube and different temperatures were achieved
by using different flow rates of orange juice at 100% power. This microwave treatment
was shown to reduce pectin methyl esterase (PME) activity by 3 logarithmic cycles
compared 2 log reduction by conventional plate heat exchanger with no immediate flavor
changes compared to untreated control (Nikdel and others 1993). In a similar equipment
design with 100% power of 700 W, Tajchakavit and Ramaswamy (1997) studied the
inactivation kinetics of PME in orange juice and concluded that the inactivation was a
first order reaction with decimal reduction times ranging from 38.5 s at 55ºC to 1.32 s at
70ºC at a Z-value of 10.2ºC. They (Tajchakavit and others 1998) also studied the
inactivation of spoilage microorganisms Saccharomyces cerevisiae and Lactobacillus
plantarum in apple juice in this system and found that Z-values for destruction of S.
cerevisiae and L. plantarum were 7 and 4.5ºC respectively by microwaves as compared
to corresponding Z-values of 13.4 and 15.9ºC by conventional batch heating, showing
faster inactivation rates by microwave heating. Valero and others (2000) studied the
effects of continuous-flow microwave and conventional heat exchanger system on pH,
sensory properties, volatile and monosaccharide composition of milk throughout 15 days
23
or storage and observed that the taste and odor were not affected by microwave treatment
after treatment as well as through storage period. However, Sierra and Vidal-Valverde
(2000) compared microwave and conventional tubular heat exchanger system for their
effects on vitamin B1 and B2 contents of milk and found no advantage of microwave
heating over conventional heating.
Above studies with continuous-flow microwave heating were mostly conducted
by modifying domestic microwave ovens but they laid the foundation for modified
designs with microwave powers and frequencies suitable for larger throughputs in the
decade of 2000. Gentry and Roberts (2005) studied the continuous-flow microwave
pasteurization of apple cider at different volume load sizes (0.5 and 1.38 L), input powers
(900-2000 W) and inlet temperatures (3, 21 and 40ºC). They found that larger volume
capacity system was more efficient in terms of absorbing the microwave power and
higher input powers gave higher rates of heating and higher percentage of absorbed
power. As the inlet temperatures of cider were increased, system could be operated at
higher flow rates due to lower heating requirements even at lower heating rates. With this
system they could also achieve 5.13 log reduction of Escherichia coli 25922 at 80ºC. A
research group at the Department of Food Science at North Carolina State University has
done extensive research on continuous flow microwave processing in recent years. In
collaboration with Industrial Microwave Systems, Inc. (Morrisville, NC), they developed
and utilized continuous flow microwave systems at 6 KW and 100 kW power levels for
research and later, for commercial purpose. In a study, Clare and others 2005 compared
sensory, microbiological and biochemical properties of skim milk treated at 137.8ºC for
10 s with microwave in a 60 kW system at 915 MHz frequency (compared to 2450 MHz
24
used in domestic ovens and in studies described previously) and by ultra high
temperature (UHT) systems. While most of the quality attributes were not different for
both heating methods, UHT processed milk showed higher stale, fatty flavor with higher
astringency and were visibly darker than microwave treated milk at the same temperature.
Sabliov and others (2008), evaluated two different Caribbean peanut beverages for their
dielectric properties, heated them in 5 kW microwave systems and observed that due to
their similar dielectric properties of ε'=60 and ε''=23 , both beverages were good
candidates for microwave sterilization and the uniformity in outlet temperature
distribution of these products signified that the microwave treatment could be used for
sterilization of low acid products like peanut beverages. Coronel and others (2005)
aseptically processed sweet potato puree in a 5 kW and larger 60 kW pilot-scale
continuous flow microwave system at 135ºC for a hold time of 30 s and found that the
instrumental color and viscosity of the puree were unaffected even after 90 days room
temperature storage. In a similar study and set-up, they (Steed and others 2008) processed
purple-fleshed sweetpotato puree at 135ºC for 30 s and found that while color of the
puree was unaffected, total phenolics content increased by 5.9% whereas total
monomeric anthocyanin content decreased by 14.5%. They also observed that the gel
strength of the puree increased over storage period but was easily broken. They further
characterized other high viscosity homogeneous products such as carrot and green pea
puree and heterogeneous products such as salsa con queso for their dielectric properties
at 915 MHz within a temperature range and under conventional static and continuous
flow conditions as well to understand their behavior better under continuous flow
conditions (Kumar and others 2007).
25
It takes noticeably longer times to sterilize a high viscosity product such as
sweetpotato puree in a conventional retort. For instance, for a 307 × 409 size can of
sweetpotato puree is at 87ºC initial temperature is required to be retorted for 84 min at
121ºC (Steed and others 2008). This long thermal treatment results in nutritional and
sensory quality degradation of puree. Hence, significantly quicker microwave processing
has been proved successful especially in case of sweetpotato puree. For this reason, it is
the first food product commercially processed by continuous flow microwave system by
Yamco, LLC. (Snow Hill, NC).
2.5
Strawberry
Cultivated strawberry (Fragaria × ananasa) is a fruit grown in temperate regions
of the world and is a part of diets of millions of people across different cultures due to its
delicate flavor and nutritional content (Hancock 1999). Worldwide production of
strawberry in 2010 was 4.4 million tonnes over 240,000 hectares of land (FAOSTAT
2012). Botanically, strawberry is an aggregate fruit and grows on stems in groups of three
and unlike other berries, seeds of strawberry are attached on the skin surface of the fruit
(Sinha 2006; Strik 2007).
Strawberry fruit is comprised of approximately 90% moisture and 10% soluble
solids. Glucose and fructose make up about 80% total soluble sugars contributing
approximately 40% of the total soluble content (Hancock 1999; Talcott 2007). Citric acid
is the predominant organic acid present in strawberry (around 80% of total acids) along
with anticarcinogenic ellagic (Hancock 1999), malic and ascorbic acid (Sinha 2006).
Strawberry flavor is a complex balance among sweetness, acidity and aroma. Flavor is
26
contributed by compounds such as methyl butanoate, ethyl butanoate, methyl hexanoate,
cis-3-hexenyl acetate and linalool (Sinha 2006). Development of red color of strawberry
is a result of gradual production of anthocyanin pigments. Pelargonidin-3-glucoside is the
predominant anthocyanin compound present in strawberry that imparts the characteristic
red color to it (Lopes da Silva and others 2007; Sinha 2006; Hancock 1999). Apart from
providing color, anthocyanins have been shown to have antioxidant capacity and health
benefits such as lowering the risk against cancer and cardiovascular diseases (Giusti and
Jing 2007).
Approximately 75% strawberries in the United States are consumed fresh whereas
remaining 25% are frozen or processed into different products such as juice, concentrates,
puree, jams, jellies, preserves and freeze dried strawberries (Sinha 2006). These
strawberry products are also utilized as ingredients in some other finished food products
such as ice creams, breakfast cereals and granola bars.
2.6
Anthocyanins
Anthocyanins are a group of flavonoid compounds naturally present in and are
responsible for color of fruits, flowers and leaves of many plants. Chemically, they are
glycosides of flavilium or 2-phenylbenzopyrylim salts. Anthocyanin molecules are
composed of anthocyanindin attached to a sugar moiety. Most commonly found
anthocyanidins in nature are cyanindin, pelargonidin, peonidin, malvidin, petunidin and
delphinidin, whereas most common sugar moieties are glucose, galactose, rhamnose,
arabinose and their disaccharide and trisaccharide combinations. Hence, different
27
anthocyanins occur as different combinations of anthocyanidin and sugar moieties. Sugar
moieties can be acylated with phenolic acids (e.g. p-coumaric, ferulic, caffeic or sinapic
acids) or aliphatic acid (such as p-hydroxybenzoic, malonic or acetic acids) giving
relatively more complex acylated anthocyanins (Mateus and De Freitas 2008; Brouillard
1982). Thus, all in all, there have been over 500 different anthocyanins isolated from
plants (Mateus and De Freitas 2008).
Skeletal structure of anthocyanins is as shown in Figure 1. Different combinations
of R1 and R2 give different anthocyanidins whereas different sugar moieties at R3
position give different glycosidic forms. For instance, if R3 is glucose and R1 and R2 are
H, then the name of that particular anthocyanin compound will pelargonidin-3-glucoside.
Anthocyanidin
Pelargonidin
Cyanidin
Delphinidin
Petunidin
Malvidin
Peonidin
R1
H
OH
OH
OH
OCH3
OCH3
R2
H
H
OH
OCH3
OCH3
H
Figure 2.1. Structures of most common anthocyanins present in nature (Modified
from Mateus and De Freitas 2008; Giusti and Jing 2007).
28
Color contributed by an anthocyanin compound is affected by a resonant structure
of that compound, which in turn is governed by the pH of the solution. It has been found
that, four different forms of anthocyanins exist in equilibrium at ambient temperature in
acidic conditions: quinonoidal base, flavylium cation, pseudobase or carbinol and
chalcone. Red or yellow flavylium cation is predominant at pH < 2. As the pH is
increased, red or blue quinonoidal forms are formed due to rapid deprotonation of
flavylium cations. On standing, flavylium cation is converted to colorless carbinol, which
in turn equilibrates to colorless chalcone form (Mazza and Miniati 1993; Brouillard 1982).
Anthocyanins, especially acylated anthocyanins are stable to light and heat but are prone
to oxidative degradation (Mazza and Miniati 1993).
Anthocyanins have been widely applied commercially as natural food colorants in
products such as soft drinks, fruit preserves and jams. There have been many different
methods to extract anthocyanins from plant sources but acidified alcoholic extractions are
widely used. Grapes are traditionally, the most commonly exploited source of
anthocyanins. Many other fruits and berries as well as vegetables such as red cabbage are
also good sources of anthocyanins (Mateus and De Freitas 2008; Emerton 2008).
29
CHAPTER 3. LITERATURE REVIEW: ENCAPSULATION OF VITAMIN D2
3.1
Encapsulation
Encapsulation techniques are extensively used in various fields such as
pharmaceuticals, agrochemicals, foods, perfumes and other industrial chemicals to
protect and deliver various active chemical compounds (Shukla 2006). Approximately 40%
of the pharmaceutical active compounds developed have poor solubility in water. Poor
water solubility creates difficulties in wetting and dissolution of these drugs in
gastrointestinal (GI) tract after oral administration (Hu and others 2004). Most of the food
and beverage systems are aqueous-based and hence the solubility of water-insoluble
colors, flavors and bioactive compounds such as nutrients, antioxidants and
antimicrobials is an issue (MacDougall 2002; McClements and others 2007; Wagner and
Warthasen 1995). The challenge of increasing the solubility and dissolution rates of these
bioactive compounds can be addressed by different encapsulation techniques (Hu and
others 2004). Encapsulation techniques also work towards improving stability of
bioactive compounds against degradation by oxygen, light and pH (McClements and
others 2007; Wagner and Warthasen 1995). Another important function of encapsulation
is to enhance the bioavailability and control the release of bioactive compounds (Rieux
and others 2006; Wegmuler and others 2004).
30
In the pharmaceutical industry, the drugs/bioactive compounds are delivered
through several routes of administration such as oral, dermal, rectal and intravenous,
where they are typically released immediately to raise their concentration in blood to an
effective level. This sudden ‘burst’ release may be enough for therapeutic
effectivenessbut not for a long time and hence results in more frequent dosing to maintain
the effective level. The concerns with drug administration several times a day include
risks of dependence on patients to comply with the periodic dosages and under-controlled
variable drug concentrations (Nitsch and Banakar 1994). It is also necessary to maintain
the drug concentration below the toxic levels (Damitriu and others 1994). For these
reasons, different techniques for encapsulation of bioactive compounds are being
developed to facilitate their controlled release.
The conventional vehicles for the encapsulation techniques can be divided into
two main groups, polymeric and lipidic, based on the carrier materials. The methods for
the preparation of polymeric carriers or nanoparticles involve use of harmful organic
solvents, carcinogenic monomers and crosslinking agents, which are reactive. Complete
removal of these chemicals is a difficult task (Allemann and others 1993). Furthermore,
the polymeric carrier materials and their metabolites can have toxicological effects
(Bunjes and Siekmann 2006). These drawbacks of polymeric carrier systems can be
addressed by using lipid based carrier systems, which involve lipids such as
triacylglycerols, phospholipids and cholesterol. While these lipids have much lower
toxicological risk, there are some drawbacks associated with the lipid carrier systems.
Liposomes have low storage stability and due to their curvaceous shape, they are
thermodynamically unstable (Lentz and others 1987). Plus, liposomes needed for
31
pharmaceutical applications are expensive (Muller and others 2000). In oil in water
emulsion delivery systems, the drug or a bioactive material has a high mobility in the
liquid dispersed phase and because of that they can ‘leak out’ into the aqueous phase.
This may cause a ‘burst’ release in the release medium (Washington and Evans 1995).
Thus, to provide advantages of lipidic carrier systems by overcoming the drawbacks
associated with the liquid state of the emulsion-based lipidic carriers, solid lipid
nanoparticles (SLNs) evolved as an alternative encapsulation vehicle.
3.2
Solid lipid nanoparticle as a carrier system
Solid lipid nanoparticles were emerged in early 1990s as an alternative to
polymeric nanoparticles, liposomes and emulsions (Muller and others 2000; Pardeike and
others 2009). SLNs are prepared by replacing a liquid lipid from an emulsion by a lipid
or a mixture of lipids, which is solid at ambient as well as body temperatures (Lucks and
Muller 1991). SLN suspension consists of solid lipid particles in a size range of 40-1000
nm (Lucks and Muller 1991) dispersed in an aqueous medium and is stabilized by
surfactants. More details of preparation methods for SLN are discussed later in this
review.
The advantages of solid include increased physicochemical stability of both core
material and incorporated compound mainly due to their solid state. Solid state of
particles also results in the decreased leakage of the drug and its interaction with the
emulsifier coat. SLNs have controlled release potential due to the reduced mobility of
encapsulated drug (Bunjes and Siekmann 2006). Due to these advantages, SLNs have
32
been exploited for the controlled release of numerous drugs such as timolol (Cavalli and
others 1992), doxorubicin, idarubicin (Cavalli and others 1992), prednisolone (Zur
Muhlen and Mehnert 1998), cyclosporin (Penkler and others 1999), diazepam (Jenning
and others 2000) and several others. The controlled release of these drugs from the solid
lipid matrix of the nanoparticles is governed by the diffusion of the drug molecule
through the lipid as well as the degradation of the carrier lipid matrix.
Solid lipid nanoparticles can serve as carriers for intravenous, topical and oral
administration of poorly water-soluble drugs. Focusing on oral administration, waterinsoluble drugs can be solubilized in the lipid matrix and can be transported through the
gastro-intestinal tract (GIT) until they are released from the lipid matrix and get absorbed
without precipitation in the GIT (Westesen and others 1997). Since, drugs can be
encapsulated by SLN and delivered orally, food bioactive compounds, which have poor
solubility in water can also be encapsulated solid lipid matrix. These food bioactive
compound-loaded solid lipid nanoparticles will have a huge potential to be incorporated
into water-based foods for the purpose of fortification.
3.2.1 Preparation methods for solid lipid nanoparticles
There are various techniques available by which solid lipid nanoparticles are
manufactured. Few of these techniques are discussed as follows.
33
3.2.1.1
Homogenization techniques
In this method, the SLN dispersions are manufactured using high pressure
homogenization by either melt (hot) or cold homogenization. In melt homogenization, the
lipid phase is melted and the lipophilic drug is dissolved in the melted lipid. This lipid
phase is then dispersed with an aqueous phase containing a surfactant to form a coarse
emulsion (pre-emulsion). This coarse emulsion is high-pressure homogenized to form a
hot nanoemulsion, which is cooled down below its crystallization temperature to solidify
the melted lipids to form solid lipid nanoparticles dispersion (Zimmermann and Muller
2001). In cold homogenization, the drug is dissolved in the melted lipid, which is then
solidified and milled. These lipid particles are then dispersed in the surfactant solution
and high pressure homogenized to form SLN dispersion.
Melt homogenization is the most widely used technique for SLN preparation
because of the advantages such as stable dispersions, good reproducibility and possibility
to use high proportions of lipid. Cold homogenization techniques suits better for very
high melting lipids and heat sensitive incorporated compounds and it also results in
limited partitioning of drugs during homogenization. However, higher energy is
consumed in cold homogenization and it gives relatively coarser dispersions than in hot
homogenization (Bunjes and Siekmann 2006).
3.2.1.2
Precipitation from solvent emulsification/evaporation technique
In this method, a lipophilic material is dissolved in an organic solvent and it is
emulsified with an aqueous phase. When the organic solvent is evaporated, the lipid
34
precipitates to form nanoparticles. This process is characterized by the formation very
small particles. For instance, 25 nm size particles of cholesteryl acetate were formed by
this method (Helgason and others 2008). However, this technique involves use of organic
solvents and the concentration of lipid to be used is restricted and hence the drug loading
(Bunjes and Siekmann 2006).
3.2.1.3
Microemulsion based technique
A warm microemulsion of drug containing melted lipid into an aqueous phase
containing surfactant is brought in contact with cold water under stirring conditions,
where the lipid particles precipitate to form solid lipid nanoparticles (Bunjes and
Siekmann 2006). The advantage of this method is that the particle structure is already
formed in the form of microemulsion droplets and hence no additional homogenization is
required (Sjostrom and Bergenstahl 1992).
Wide variety of solid lipids such as different triglycerides, waxes, paraffin and
fatty acids can be utilized for the preparation of solid lipid nanoparticles. Some
commonly used triglycerides are tripalmitin, tristearin, trilaurin, trimyristin (Boltri and
others 1993) and tricaprin (Mehnert and Mader 2001) whereas commonly used
surfactants are polyxamers 188, 182, 407, polysorbates (Tween) 20, 60 and 80 (Boltri and
others 1993).
35
3.2.2
3.2.2.1
Characterization of solid lipid nanoparticles
Particle size and size distribution
Particle size of solid lipid nanoparticles is very important and is measured very
commonly any study associated with SLNs. The importance of particle size could be
understood in a case where presence of particles bigger than 5 μm may pose serious
problems if SLN dispersion is administered intravenously (Bunjes and Siekmann 2006).
Particle size can be measured by a technique called dynamic light scattering (photon
correlation spectroscopy), where the rate of intensity fluctuation of the light scattered by
the randomly moving (due to Brownian motion) solid lipid nanoparticles can be
correlated to their particle size by Stokes-Einstein equation. Similarly, laser diffraction
technique is used for the particles in upper nanometer or micron size range. However,
both these techniques assume the particles to be spherical for the size calculation and
hence the values provided are overestimated due to non-spherical (platelet-like) shape of
the SLNs (Domb 1995).
...(3)
Where, D = translational diffusion coefficient,
k = Boltzman's constant,
T = temperature,
η = dynamic viscosity,
Rh = hydrodynamic radius
36
3.2.2.2
Crystallinity and Polymorphism
As discussed earlier, the advantages of solid lipid nanoparticles as a controlled
release matrix depends on its solid state. Some lipid matrix may not show immediate
crystallization under certain cooling conditions. Delayed crystallization has been
observed in shorter chain monoacid triglycerides such as tricaprin, trilaurin and
trimyristin (Unruh and others 1999; Westesen and Bunjes 1995). Thus it is important to
observe the status of crystallization of lipid nanoparticles.
Apart from different extents of crystallization, polymorphic transitions are also
observed in solid lipid nanoparticles over time. For SLNs prepared by hot
homogenization of triglycerides have been reported to have α, β’ and β polymorphs
present along with intermediate βi polymorph (Westesen and Bunjes 1995; Schwarz and
Mehnert 1997). Depending on type of lipid, stabilizer composition and particle size it has
been observed that the rate of polymorphic transition is increased in case of nanoparticles
as compared to the bulk lipid (Westesen and others 1993) and in some cases
nanoparticles of certain lipid show some polymorphic transitions, which are not shown
by the bulk lipid (Jenning and others 2000). Shorter chain triglycerides transform more
rapidly from least stable α to most stable β polymorph than longer chain triglycerides so
do the smaller particles than the larger ones (Bunjes and Westesen 2001; Bunjes and
others 2000).
Differential scanning calorimetry (DSC) and X-ray diffraction techniques are
used to study the crystallinity and polymorphism of solid lipid nanoparticles (Schwarz
and Mehnert 1997).
37
3.2.2.3
Morphology
Morphology plays crucial role in correlating shape of the nanoparticles to their
ability to incorporate drugs, stability of the particles and the controlled release. Spherical
particles offer maximum stability to the incorporated drugs due to their minimum surface
area/ volume ratio and hence minimum exposure of the incorporated molecule. Plus
spherical particles require lesser amount of surfactant to cover their surface as compared
to the non-spherical particles. In addition to lipid particles, the micelles of surfactant
present in the dispersion can also act as carriers for the drugs. In such cases, microscopic
examination of morphology would help distinguish them from lipid particles (Bunjes and
Siekmann 2006). Analytical techniques such as transmission electron microscopy (TEM)
and atomic force microscopy (AFM) are used to study the morphology of solid lipid
nanoparticles.
3.2.3 Solid lipid nanoparticles as carriers for food bioactive compounds
As discussed so far, solid lipid nanoparticles are primarily used in pharmaceutical
industry for the delivery of lipophilic drug compounds. Similar encapsulation
methodology can be applied for naturally occurring, lipophilic food-based bioactive
compounds to increase their solubility in aqueous food systems, protect them from
degradation by environmental factors such as temperature, light and oxygen. These
bioactive compounds can be essential micronutrients such as vitamins or non-essential
but health benefitting compounds like phytochemicals. Solid lipid nanoparticles, similar
38
to pharmaceutical drug delivery, can be used for controlled release of these food-based
bioactive compounds and hence have a potential to finally impact their bioavailability.
Although SLN technique has mainly been used as a carrier systems for cosmetic
active ingredients and pharmaceutical drugs for about last two decades (Bunjes and
others 2003), only recently SLN has attracted attention as a carrier systems for the
bioactive food components (Saupe and Rades 2006). Functionality of proteins and
peptides depend on their secondary, tertiary and quaternary structures and hence any
destabilization of their structure may result in the loss of their biological activity or
functionality. Encapsulation techniques such as SLN have been used to protect these
proteins and peptides, the list of which include therapeutic peptides like calcitonin (Weiss
and others 2008), insulin (Garcia-Fuentes and others 2005) and somatostatin (Zhang and
others 2006), protein antigens such as hepatitis B antigen (Reithmeier and others 2001),
and other proteins such as bovine serum albumin (Saraf and others 2006) and lysozyme
(Gualbert and others 2003). However, for this review, encapsulation of some lipophilic
compounds by solid lipid nanoparticles will be discussed briefly as follows.
3.2.3.1.Vitamins
β-carotene and α-tocopherol, due to their antioxidant activity have protective
effects against UV-induced DNA damage resulting in skin cancer. However, these two
compounds are unstable and are prone to photochemical degradation. When these two
compounds were encapsulated in the stearyl ferulate SLN and exposed to pro-oxidants
and light, they were observed to be very stable (Almeida and others 1997). Similar results
39
were obtained for SLNs loaded with all-trans retinol, which is effective against acne and
dermatitis (Trombino and others 2009) and ascorbyl palmitate (Jee and others 2006). In a
study involving encapsulation of retinoids with solid lipid nanoparticles
(Teeranachaideekul and others 2007), it was found that when the polarity of retinoid
compounds was decreased (tretinoin < retinol < retinyl palmitate), their entrapment in
solid lipid increased and good entrapment was correlated with low degree of crystallinity
of the solid lipid.
3.2.3.2 Phenolic Compounds
Encapsulation of curcuminoids (bisdemethoxycurcumin and demethoxycurcumin)
with SLN showed extended release over a period of 12 hours following Higuchi’s square
root model when studied in vitro. Their stability in both forms, lyophilized as well as
when incorporated into a cream was maintained over a period of six months (Jenning and
Gohla 2001). SLN loaded with quercetin showed delayed release in the GIT and five-fold
increase in bioavailability as compared to the quercetin suspension when studied in vivo
in rats (Li and others 2009).
3.2.4 Digestibility of SLN and controlled release of the bioactive compounds
As discussed earlier, the lipids used for the preparation of solid lipid nanoparticles
are of physiological origin and hence their digestibility should be studied similar to that
of the dietary lipids. Bonnaire and others (2008) studied the in vitro digestibility of
tripalmitin solid lipid nanoparticles and they compared it with the digestibility of
40
tripalmitin nanoparticles, which were in a liquid state at 37°C since tripalmitin
nanoparticles are crystallized at around 20°C when cooled down from the molted state
after melt homogenization. They found that the rate as well as the extent of digestion of
lipid was higher in liquid state in emulsion than the solid state in dispersion, which was
>35% digested after 2 hours. This suggests that the solid lipid nanoparticles have a
potential for the prolonged release of the encapsulated bioactive compounds. However,
the combination of type of lipid and surfactant has been shown to show different
sensitivity to pH and electrolyte concentration and may aggregate in gastrointestinal
media in some cases.
3.3
Vitamin D
Vitamin D is a fat soluble vitamin naturally present in a limited number of foods,
which is an essential factor for the maintenance of plasma levels of calcium and
phosphorus and hence plays an important role in mineralization of bones and
neuromuscular functions (DeLuka and Zierold 1998). It is synthesized by animals under
their skin with the help of ultraviolet radiations from the sun. 7-Dehydrocholesterol in
animals is converted to previtamin D3 by exposure of the skin to sunlight, which is then
isomerized to cholecalciferol (vitamin D3). This synthesis due to sunlight exposure
provides most of the vitamin D requirements (Holick 1994). Similarly, ergosterol in plant
tissue is converted to previtamin D2 by light exposure, which is then isomerized to
ergocalciferol (vitamin D2) (Eitenmiller and others 2008). Vitamin D is available in the
41
form of dietary supplement and foods such as milk and margarine are fortified with it (21
CFR; Eitenmiller and others 2008).
Due to its role in calcium absorption, the biologically active form of vitamin D i.e.
25-hydroxy vitamin D (calcidiol) helps in protecting against osteoporosis (Parfitt 1990).
Studies show that vitamin D is effective in preventing colon, breast and prostate cancers
(Davis and others 2007), diabetes (Hyppönen and others 2002; Pittas and others 2006),
hypertension (Krause and others 1998), multiple sclerosis (Munger and others 2006) and
glucose intolerance (Chiu and others 2004).
Deficiency of vitamin D causes rickets in children and osteomalacia in adults
(Kreutler and Czajka-Narins 1986; Shoback and others 2004). Although, fortification of
milk with vitamin D has drastically reduced the occurrences of rickets in the United
States, it is still reported sporadically among African-American infants and children.
Some of the possible reasons given for the vitamin D deficiency are: limited sun exposure
due to clothes, use of sunblock, fear of skin cancer and melanoma (AAP 1994), limited
consumption of fortified foods and fear of excess vitamin D intake (Chesney 2003). The
efficiency of vitamin D synthesis and ability of the kidney to convert it to its active
hormonal form decreases with age (Need and others 1993). A high amount of melanin
pigments in the skin impairs its ability to synthesize vitamin D making people with dark
skin more vulnerable to vitamin D deficiency (Nesby-O’Dell and others 2002). Fat
malabsorption and obesity are also associated with decrease in vitamin D bioavailability
(Lo and others 1985; Wortsman and others 2003).
Ergocalciferol is a white, odorless, crystalline compound soluble in fats and
organic solvents including alcohol (Wilson and others 2004). It is insoluble in water and
42
is oxidized at 5,6 and 7,8 positions in their structure (Figure 2) when comes in contact
with atmospheric oxygen (Eitenmiller and others 2008). Ergocalciferol isomerizes to
isotachysterol and 5,6-trans-isomer in the presence of light or acidic conditions
(Eitenmiller and others 2008; Grady and Thakker 1980). Ergocalciferol can be a suitable
dietary choice in the form of fortified foods for vegans and lacto-vegetarians because of
its plant origin. Poor solubility of ergocalciferol in water and its susceptibility to
degradation makes it a candidate for the encapsulation.
Figure 3.1 Structure of ergocalciferol
3.3.1 Vitamin D Encapsulation
Although numerous process patents on various encapsulation techniques mention
ergocalciferol as a potential compound to be encapsulated (Van Lengerich 2001a,b,c;
Doane and others 1990; Patel and Chen 2003), the published data on encapsulation of
ergocalciferol is scarce. Semo and others (2007) developed a protocol to encapsulate
ergocalciferol into casein micelles. The encapsulation efficiency and ability of casein
micelles to protect ergocalciferol from photochemical degradation by UV exposure was
43
evaluated. Twenty seven percent of the total recovered ergocalciferol (85% of the added)
was encapsulated within the casein micelle as detected after saponification and extraction
of casein micelles and serum in which it was suspended. After ultra-filtration of serum
samples, it was observed that remaining 73% ergocalciferol recovered from serum was
probably bound to the soluble casein in the serum. Encapsulation with casein micelle
provided protection against photochemical degradation of ergocalciferol.
Chitosan/ethylcellulose (CS/EC) complex microcapsule was explored for its
potential for controlled release of ergocalciferol by Shi and Tan (2002). Ergocalciferol
was loaded in CS microspheres, which in turn were coated with EC. When these
microcapsules were suspended in the release medium (artificial gastric and intestine
juice), EC coating swelled and was slowly dissolved in the medium to expose the inner
CS microspheres. The increase in the amount of coating increased the resistance to the
release of ergocalciferol and delayed its release. The release in the artificial gastric juice
was limited but there was a continuous release in the artificial intestinal medium over a
prolonged period of time. Limitation of using CS is that it can swell and form a gel in the
intestine.
Lim and Moss (1983) patented a process of making microcapsules to protect oil
soluble substances especially vitamins. The process involved dispersing one or more
vitamins dissolved in an oil phase, into an aqueous phase containing an alginate of alkali
metal and an alcohol-insoluble polysaccharide. The emulsion thus formed is dropwise
immersed in an alcohol solution of multivalent cations to produce an alginate matrix
containing precipitated polysaccharide and the oil droplets with vitamins in it. Bishop and
others (1998) patented a process for controlled release of activated form of
44
cholecalciferol to target prostatic diseases. The process involves the combination of
activated vitamin D with sustained release matrix and spraying them into granules. These
granules are coated with a water insoluble but slowly water permeable polymeric lacquer.
These granules can be mechanically formed into any shape and can be added into food
systems along with the antioxidants, stabilizers, etc. In another study, cholecalciferol was
encapsulated in a matrix of carbohydrates and hydrogenated carbohydrates using melt
extrusion technology and was found to be protected by environmental influences (Petritz
and others 2006).
45
CHAPTER 4. COLOR AND ANTHOCYANIN CONTENT OF STRAWBERRY
PUREE THERMALLY PROCESSED BY A PILOT SCALE CYLINDRICAL
MICROWAVE HEATING SYSTEM (100 KW), TUBULAR AND SCRAPED
SURFACE HEAT EXCHANGERS
4.1
Abstract
Color is a critical attribute determining the acceptability of foods. Anthocyanins
are pigments responsible for blue/red/ purple colors in fruits and vegetables. In
strawberries, pelargonidin-3-glucoside is the predominant anthocyanin. Processing
temperatures may lead to polymerization of monomeric anthocyanins and loss of color.
The objective of this work was to investigate the effect of heating method on color and
monomeric anthocyanin content of strawberry puree heated by cylindrical microwave
heating system (MW), coiled double tube (TUB) and scraped surface (SS) heat exchanger.
Frozen strawberry puree was thawed for 48h. Flow rate was adjusted to 9.5L/min for an
average holding time 60 s. Product was treated at 80, 91 and 102°C, aseptically packaged
and stored at 4°C. Color was measured using a LabScanXE system (D65, 10°).
Monomeric anthocyanins were determined by the differential pH method and quantified
as pelargonidin-3-glucoside. Results showed that although chroma and hue angle were
better retained at 80ºC, there were no specific trends in differences among three
processing techniques. Similarly, there were no differences observed in monomeric
46
anthocyanin content among three processing techniques as well as three treatment
temperatures. However, anthocyanin content decreased over storage time for all samples
Reason for these inconclusive results were thought to be lack of replicates for the
processing by all three techniques.
4.2
Introduction
Although microwave heating of foodstuff is a widespread concept and an
inseparable part of American kitchens, its application at a commercial level is a rising
phenomenon. Continuous flow microwave processing is a relatively novel technique
(Kumar and others 2007) for pasteurization or commercial sterilization of food products,
where any pumpable food can be thermally processed by microwave radiation having
advantages such as fast (Chavan and Chavan 2010; Sumnu 2001), volumetric heating
(Huang and Sites 2007) and instant come-up time (Chavan and Chavan 2010; Sumnu
2001). Due to these advantages of microwave heating, processed food products suffer
relatively lower quality degradation compared to conventional heating methods
(Tajchakavit and others 1998). Commercial scale continuous flow microwave processing
unit with 60-100 kW power and 915 MHz frequency has been researched with products
such as milk (Clare and others 2005; Kumar and others 2007), and salsa con queso
(Kumar and others 2007) but has been visibly successful with highly viscous vegetable
products such as sweet potato puree (Steed and others 2008; Coronel and others 2005) in
terms of quality preservation ultimately resulting in commercial production of sweet
47
potato puree processed by continuous flow microwave unit (Yamco, LLC, Snow Hill,
NC).
Strawberry (Fragaria × ananasa) fruit and its products are popular due to their
characteristic color and delicate flavor (Hancock 1999; Gossinger and others 2009). It is a
source of vitamin C and anthocyanin pigments, which are also known to have antioxidant
properties (Cordenunsi and others 2005; Hartmann and others 2010). Strawberry is a very
perishable food and is prone to fungal spoilage. Thermal processing techniques used to
inactivate these spoilage microorganisms result in degradation of organoleptic and
nutritional quality of strawberry products (Gossinger and others 2009; Hartmann and
others 2010). Hence, in this study, the effects of 100 kW continuous flow microwave
system on monomeric anthocyanin content and color of strawberry puree were
investigated in comparison with tubular heat exchanger and scraped surface heat
exchanger systems.
4.3
Materials and Methods
4.3.1 Continuous flow microwave, scraped surface heat exchanger and tubular heat
exchanger heating of strawberry puree
Frozen strawberry puree (3+1) was purchased for this study and thawed overnight
prior to processing. Puree was pumped at 9.5 L/min through previously sterilized
cylindrical 100 kW continuous flow microwave system (Industrial Microwave Systems,
Harahan, LA) and heated at 80, 91 and 102ºC with an average holding time of 60 s. After
holding section, processed puree was cooled down to room temperature and aseptically
48
packaged in pre-sterile Scholle aseptic bags (Scholle Packaging, Northlake, IL). For
comparison, strawberry puree was processed through scraped surface heat exchanger (SS)
and tubular heat exchanger (TUB) systems at same processing temperatures and hold
time and packaged aseptically in the same fashion. These aseptically processed and
packaged strawberry puree samples were stored at refrigerated (4ºC), room (22ºC) and
incubated (45ºC) temperatures and analyzed at 0, 2, 4, 8 and 13 weeks. All three thermal
processes were performed only once. All analyses were performed in duplicates.
4.3.2 Total monomeric anthocyanin content analysis
The total monomeric anthocyanin content of strawberry puree samples was
estimated by the pH differential method (Giusti and Wrolstad 2002). Anthocyanins were
extracted from 10 g of strawberry puree samples using 60 mL 0.1% acidified methanol.
The extracts were diluted 6.5 times with pH 1.0 and pH 4.5 buffers. The absorbance of
diluted extracts was measured at 508 nm which is the λvis-max for pelargonidin-3-glucoside,
the predominant anthocyanin in strawberry (Lopes da Silva and others 2007), and at 700
nm to correct for turbidity, using a UV/VIS spectrophotometer (Beckman Coulter,
Fullerton, CA). The monomeric anthocyanin content was calculated as follows.
Monomeric anthocyanin content (mg/kg) = (A×MW×DF×1000)/(ε×1)
Where, A = (Aλvis-max – A700)pH 1.0 – (Aλvis-max – A700)pH 4.5,
MW: molecular weight = 468.8, DF: dilution factor = 17.5,
ε: molar absorptivity = 17330
49
4.3.3 Instrumental color measurement
CIELAB color values for lightness (L*), redness/greenness (a*) and
yellowness/blueness (b*) of strawberry puree samples were recorded using a LabScan
XE colorimeter (0/45o), illuminant D65 and 10o observer (Hunter Associates Laboratory,
Naperville, IL). These values were further used to calculate chroma (C*) and hue angle
(⁰h) for the strawberry puree as follows (McGuire 1992):
⁰
4.3.4 Statistical analysis
Results from all the experiments and measurements were analyzed by statistical
software SAS 9.2 (SAS Institute, Inc., Cary, NC) using analysis of variance (ANOVA)
with a significance level of α=0.05. A three-factor full factorial design was used with
treatment (microwave, scraped surface and tubular H.E.), processing temperatures
(control, 80, 91 and 102ºC), and storage time (0, 2, 4, 8 and 13 weeks) as factors.
Treatment means were compared by Duncan’s least significance difference (LSD) test.
50
4.4
4.4 Results and Discussion
4.4.1 Total monomeric anthocyanin content
Three different thermal processing methods i.e. continuous flow microwave,
scraped surface and tubular heat exchanger systems did not show any difference (p > 0.05)
in monomeric anthocyanin content among them (Figure 4.1). Although these thermal
treatments significantly reduced (p < 0.05) anthocyanin contents compared to control,
differences among three treatment temperatures were not statistically significant. This is
not in agreement with degradation kinetics of anthocyanins discussed in literature, which
shows first order reaction (Kirca and Cemeroglu 2003; Yousefi and others 2012).
Possible explanation for this discrepancy could be a lack of replicates for processing and
hence lack of credibility of these results. Monomeric anthocyanin content decreased over
13 week storage period for each thermal processing method with significant reduction at
each 2 week storage time, which shows that none of these processing methods impacted
the stability of anthocyanin in positive direction over the storage period.
4.4.2 Instrumental color
Lightness (L*), chroma (C*) and hue angle (ºh) all decreased (p < 0.05) over the
storage time for each of three processing techniques and treatment temperatures used for
these processing methods (Figure 4.2-4.4). For all three processing methods, 80ºC
treatment retained chroma and hue angle better than 91 and 102ºC treatments but
differences in lightness were insignificant (p > 0.05). Although, tubular and scraped
51
surface heat exchanger treatments seem to retain lightness and chroma better than
microwave treatments, this trend was not continued over the entire storage period.
Instrumental color values are usually correlated with total anthocyanin content (Skrede
and others 1992) and similar to anthocyanin content, color values showed reduced values
over storage period.
Based on above results, it could be challenging to find correlation between total
monomeric anthocyanin content and instrumental color data. Also, differences between
microwave other two conventional thermal processing methods are inconclusive both for
anthocyanin as well as for color data. In this study, all processing treatments were
performed only once and there were no replicates due to large amounts of puree required
for each run. Without replicates, statistical strength and credibility of these results is
significantly reduced, which could possibly be the main reason behind inconclusive
differences in anthocyanin and instrumental color results.
4.5
Conclusions
Total monomeric anthocyanin content and instrumental color data showed
decreasing trends over storage time but differences among different thermal processing
methods and treatment temperatures were inconclusive. Also, there was no correlation
between anthocyanin content and instrumental color. These inconclusive findings point
towards lack of statistical strength of these results which, is mainly due to the lack of
large amount of strawberry puree required for these big systems and hence inability to
52
perform replicates. Therefore, further investigation is aimed at using smaller systems to
evaluate effect of continuous microwave system on quality parameters of strawberry
puree compared to conventional heating method in a statistically stronger experimental
design.
Monomeric anthocyanin content (mg/kg)
500
450
400
350
300
Control
250
80ᵒC
200
91ᵒC
150
102ᵒC
100
50
0
0
2
4
8 13 0
2
4
8 13 0
2
4
8 13
WK WK WK WK WK WK WK WK WK WK WK WK WK WK WK
Microwave
Scraped Surface
Tubular
Figure 4.1 Monomeric anthocyanin content of strawberry puree at different storage times
processed by continuous flow microwave system (100 kW), scraped surface heat
exchanger and tubular heat exchanger at 80, 91 and 102ºC.
53
50
45
40
Lightness (L*)
35
30
Control
25
80ᵒC
20
91ᵒC
15
102ᵒC
10
5
0
0
1
4
8 13 0
1
4
8 13 0
1
4
8 13
WK WK WK WK WK WK WK WK WK WK WK WK WK WK WK
Microwave
Scraped Surface
Tubular
Figure 4.2 Lightness (L*) of strawberry puree at different storage times processed by
continuous flow microwave system (100 kW), scraped surface heat exchanger and
tubular heat exchanger at 80, 91 and 102ºC.
54
46
Control
44
80ᵒC
91ᵒC
42
102ᵒC
C*
40
38
36
34
32
30
0 WK 1 WK 4 WK 8 WK 13 0 WK 1 WK 4 WK 8 WK 13 0 WK 1 WK 4 WK 8 WK 13
WK
WK
WK
Microwave
Scraped Surface
Tubular
Figure 4.3 Chroma (C*) of strawberry puree at different storage times processed by
continuous flow microwave system (100 kW), scraped surface heat exchanger and
tubular heat exchanger at 80, 91 and 102ºC.
55
34
Control
80ᵒC
91ᵒC
102ᵒC
33
Hue angle (ºh)
32
31
30
29
28
0 WK 1 WK 4 WK 8 WK 13 0 WK 1 WK 4 WK 8 WK 13 0 WK 1 WK 4 WK 8 WK 13
WK
WK
WK
Microwave
Scraped Surface
Tubular
Figure 4.4 Hue angle (ºh) of strawberry puree at different storage times processed by
continuous flow microwave system (100 kW), scraped surface heat exchanger and
tubular heat exchanger at 80, 91 and 102ºC.
56
CHAPTER 5. EFFECT OF BATCH CONVENTIONAL AND MICROWAVE
HEATING ON RHEOLOGICAL PROPERTIES, ANTHOCYANIN CONTENT AND
COLOR OF STRAWBERRY PUREE
Mandar R. Patel, M. Fernanda San Martin-Gonzalez
Published in the proceedings of 45th Annual International Microwave Power Institute
Symposium, New Orleans, LA, June 8-10, 2011.
5.1
Abstract
Fast and volumetric heating properties of microwaves are considered
advantageous over conventional heating methods. In this study, strawberry puree was
heated to 80⁰C conventionally, using a water bath, or by microwave radiation.
Rheological properties, anthocyanin content and instrumental color values of puree
samples were measured. Apparent viscosity (at 100 s-1), anthocyanin content and
lightness (L*) of microwave heated puree were higher than conventionally heated and
untreated control samples. Chroma (C*) and hue angle (⁰h) values of thermally processed
samples were preserved over time compared to untreated puree. Improved viscosity and
anthocyanin extractability due to microwave radiation show potential application in food
product development.
57
5.2
Introduction
Strawberry is a rich source of antioxidants specifically due to its high content of
anthocyanins (Lopes da Silva and others 2007). Anthocyanins, which are the compounds
responsible for the red color of strawberries (Lozano 2006), are known to have health
benefits such as improvement in night vision, antidiabetic effect (Ghosh and Konishi
2007), anticarcinogenic effect (Seeram and others 2006) as well as protective effects
against aging due to their high antioxidant activity (Hannum 2004). Lack of thermal
stability of anthocyanins (Furtado and others 1993) especially under prolonged thermal
treatments (Verbeyst and others 2009) detrimentally affects the natural color of
strawberry and strawberry products. Hence, alternative processing techniques that result
in relatively lower degradation of anthocyanins and overall quality of strawberry need to
be explored.
Microwave radiation has been commonly used in cooking and thawing of food
products across American households for a few decades. Advantages of microwave
heating of foods over conventional methods include short come-up times, faster heating,
higher energy efficiency, convenience and space savings (Sumnu 2001; Chavan and
Chavan 2010). Microwave technology has been used for the extraction of various
compounds from food matrices (Pan and others 2003; Ballard and others 2010), baking
(Sumnu 2001; Chavan and Chavan 2010) and drying (Zhang and others 2010).
Microwave processing has been used in a continuous flow basis to study the kinetics of
microbial (Tajchakavit and others 1998) and enzyme (Tajchakavit and Ramaswamy 1997)
inactivation as well as of nutrient retention (Steed and others 2008; Valero and others
58
2000). However, there is a scarce knowledge about its effect on the rheological properties
of food products. In this study, the effect of batch microwave heating on rheology, color
and anthocyanin content of strawberry puree was studied in comparison with
conventional heating method.
5.3
Materials and Methods
Whole frozen strawberries were purchased from a local store. Strawberries were
thawed and pureed using a hand-held homogenizer. Puree was packaged in freezer bags
and stored at -20⁰C until further processing. All the experiments were performed in
triplicate.
5.3.1 Batch microwave and conventional heating of strawberry puree
Frozen puree was thawed to ambient temperature (22⁰C) prior to processing.
Forty five g of thawed puree was placed in a 100 mL beaker and heated in a microwave
oven (1200 W) adapted with temperature recording system (Fiso Technologies, Inc.,
Quebec, Canada) to 80⁰C and immediately cooled in an ice-water bath. Processed puree
was packed and sealed in 50 mL plastic tubes and stored at 4⁰C until further analyses.
Same amount of thawed puree was heated to reach 80⁰C at the geometric center of the
sample using a heated water bath and immediately cooled down in an ice-water bath,
packed, sealed and stored at refrigerated conditions until further analyses at 0, 2, 4 and 8
weeks.
59
5.3.2 Rheological testing
Flow properties of control and treated strawberry puree samples were performed
using an ARG 2 rheometer (TA Instruments, New Castle, DE) equipped with a computer
software (Rheology Advantage Data Analysis Software v, TA Instruments, New Castle,
DE). Steady state flow curves were obtained using a concentric cylinder geometry. A preshearing conditioning step at 100 s-1 was applied to the strawberry puree for 2 min, after
which apparent viscosity versus shear rate curves were obtained for a shear rate range of
1-1000 s-1 (Nindo and others 2007).
5.3.3 Total monomeric anthocyanin content analysis
The total monomeric anthocyanin content of strawberry puree samples was
estimated by the pH differential method (Giusti and Wrolstad 2002) and as described in
section 4.3.2. Anthocyanins were extracted from 10 g of strawberry puree samples using
50 mL 0.1% acidified methanol. The extracts were diluted 6.5 times with pH 1.0 and pH
4.5 buffers. The absorbance of diluted extracts was measured at 508 nm which is the λvismax
for pelorgonidin-3-glucoside, the predominant anthocyanin in strawberry (Lopes da
Silva and others 2007), and at 700 nm to correct for turbidity, using a UV/VIS
spectrophotometer (Beckman Coulter, Fullerton, CA). The monomeric anthocyanin
content was calculated as follows.
60
Monomeric anthocyanin content (mg/kg) = (A×MW×DF×1000)/(ε×1)
Where, A = (Aλvis-max – A700)pH 1.0 – (Aλvis-max – A700)pH 4.5,
MW: molecular weight = 449.2, DF: dilution factor = 32.5,
ε: molar absorptivity = 26900
5.3.4 Instrumental color measurement
As described in section 4.3.3, CIELAB color values for lightness (L*),
redness/greenness (a*) and yellowness/blueness (b*) of strawberry puree samples were
recorded using a LabScan XE colorimeter (0/45o), illuminant D65 and 10o observer
(Hunter Associates Laboratory, Naperville, IL). These values were further used to
calculate chroma (C*) and hue angle (⁰h) for the strawberry puree as follows (McGuire
1992):
⁰
5.3.5 Statistical analysis
Results from all the experiments and measurements were analyzed by statistical
software SAS 9.2 (SAS Institute, Inc., Cary, NC) using analysis of variance (ANOVA)
with a significance level of α=0.05. Treatment means were compared by Duncan’s least
significance difference (LSD) test.
61
5.4
Results and Discussion
5.4.1 Rheology
Apparent viscosity at a shear rate of 100 s-1 of strawberry puree heated to 80⁰C in
a batch mode by conventional method and by microwave radiation as a function of
refrigerated storage time is shown in Figure 5.1. Apparent viscosity for microwave
treated puree was higher (p<0.05) than both conventionally heated and untreated puree
samples after processing (day 0) and up to two weeks. However, the difference between
the apparent viscosity values for microwave and conventionally heated puree was not
significantly different over a period of four weeks. Apparent viscosity of untreated
control puree samples decreased over time in comparison with both conventionally and
microwave heated puree. Untreated sample showed spoilage before 4 weeks, while
treated samples spoiled sometime between weeks 4 and 8.
One of the objectives of this study was to investigate any possible effect of
microwave radiation on the rheological properties of strawberry puree as compared to
conventional heating method. Rheology data for both heating methods in batch scale
suggested retention of the apparent viscosity of puree by microwave radiation as
compared to conventionally heated and untreated puree. However, this effect was not
significant after two weeks under refrigerated storage and for further storage times. The
batch microwave system used for this study has drawbacks such as poor temperature
control due to non-uniform heating and that could possibly be the reason for this
inconclusive rheology data in the batch mode. Although microwave radiation has been
used in organic synthesis (Bogdal and Prociak 2007), its application to influence food
62
related reactions (Peng and others 2011) has been scarcely studied. Literature discussing
effect of microwave radiation on rheological properties of food products is scarce.
5.4.2 Total monomeric anthocyanin content
Monomeric anthocyanin content of strawberry puree heated to 80⁰C by
microwave radiation in batch mode (Figure 5.2) was higher than conventionally heated
and untreated puree at all storage times.
Microwave radiation has been used by researchers to assist in the extraction of
anthocyanins (Lu and others 2010; Liazid and others 2011). Due to higher penetrating
power of microwave radiation, localized heating produces rapid rupture of plant cell
structures, facilitaing and improving the extraction efficiency of many compounds such
as anthocyanins (Lu and others 2010). This explains higher monomeric anthocyanin
content of microwave heated strawberry puree.
5.4.3 Instrumental color
Lightness (L*), redness/greenness (a*), yellowness/blueness (b*), chroma (C*)
and hue angle (⁰h) of strawberry puree samples treated by batch microwave and
conventional heating are shown in Table 5.1. Lightness (L*) of strawberry puree heated
by microwave radiation in a batch mode was higher (p<0.05) than the conventionally
heated and untreated puree after processing (day 0). After two weeks, lightness of
untreated puree was lower (p<0.05) than both microwave and conventionally heated
63
puree, which would be expected due to the continuous action of polyphenol oxidase
activity. After four weeks, there was no significant difference in lightness values of
samples from both thermal processing methods. There was no difference in chroma (C*)
between any sample after processing (day 0); but after two weeks, thermally treated puree
samples showed higher chroma values than untreated puree. Hue angle showed similar
trend as chroma values over time.
Decrease in lightness (L*) of all puree samples over time indicates gradual
darkening of samples due to enzymatic browning. However, microwave heated samples
showed higher lightness i.e. lower extent of browning compared to conventionally heated
and untreated puree. Chroma and hue angle values of all the samples were not
significantly different after processing but those of untreated samples changed over time
indicating preservation of color and its intensity by both thermal treatments.
5.5
Conclusions
In conclusion, microwave radiation heating can be used as an alternative to
conventional processing to improve retention of quality attributes of fruit purees such as
color and anthocyanin content. The effect of microwave radiation on rheological
properties of food products needs to be validated and warrants further investigation in
terms of possible molecular interactions responsible changes in sample rheology.
64
0.2
Apparent Viscosity at 100 s-1 (Pa.s)
0.18
0.16
0.14
0.12
Untreated
0.1
Conv. 80⁰C
0.08
MW 80⁰C
0.06
0.04
0.02
0
0
2
Time (week)
4
Figure 5.1 Apparent viscosities (at 100 s-1) of strawberry puree heated by conventional
method and microwave radiation in a batch mode.
65
Monomeric Anthocyanin (mg/kg)
340
320
300
280
Untreated
Conv. 80⁰C
260
MW 80⁰C
240
220
200
0
2
Time (Week)
4
Figure 5.2 Monomeric anthocyanin content of strawberry puree heated by conventional
method and microwave radiation in a batch mode.
66
Table 5.1. Lightness (L*), redness/greenness (a*), yellowness/blueness (b*), chroma (C*)
and hue angle (⁰h) of strawberry puree samples treated by batch microwave heating and
batch conventional heating.
Treatment
Untreated: Day 0
Conventional 80⁰C: Day 0
Microwave 80⁰C: Day 0
Untreated: Week 2
Conventional 80⁰C: Week
2
Microwave 80⁰C: Week 2
Untreated: Week 4
Conventional 80⁰C: Week
4
Microwave 80⁰C: Week 4
Untreated: Week 8
Conventional 80⁰C: Week
8
Microwave 80⁰C: Week 8
30.13 b‡
30.59 b
34.15 a
26.09 b
Color Characteristics†
a*
b*
C*
Batch Treatments
40.14 a 26.24 a 47.93 a
37.05 b 23.74 a 44.00 a
39.60 a 25.41 a 47.06 a
36.49 c 22.24 b 42.74 c
33.19 a
32.65 a
32.60 a
31.33 b
29.15 b
38.60 b
45.85 b
32.66 a
30.49 a
40.03 a 26.30 a 47.89 a
Sample Spoiled
33.30 a
29.34 a
28.93 a
38.21 a
38.58 a
32.35 a
32.82 a
L*
24.75 a
24.20 b
24.89 a
45.23 a
45.91 a
⁰h
Sample Spoiled
†Mean of triplicate values for all treatments.
‡ Values with same alphabet within a column (between samples of either batch or
continuous and between only those of same time periods) are statistically not significant
(p>0.05).
67
CHAPTER 6. LABORATORY SCALE (6KW) CONTINUOUS MICROWAVE
HEATING SYSTEM: DESCRIPTION, INSTALLATION AND OPERATION
6.1
Introduction
The purpose of this chapter is to present an overview of efforts taken to install and
operate 6 kW continuous flow microwave (CFMW) processing system and tubular heat
exchanger system for the processing of strawberry puree and challenges and limitations
involved in doing so. Describing limitations faced especially while installing and
operating 6 kW CFMW system is essential not only to understand the processing of
strawberry puree in subsequent chapter but also for future researchers to possibly learn
from these experiences when performing experiments on a similar system.
6.2
CFMW system (6 kW)
6.2.1 Description, installation and operation
A 6 kW CFMW system consisted of a magnetron unit, where microwave
radiation is generated (Figure 6.1). Power of microwave radiation generated could be
controlled using a control knob ranging in scale from 0 to 10, which indicates generated
power from 0 to 100% of total 6 kW capacity. Generated microwave radiation traveled
68
through the 'waveguide', which was a rectangular cavity made up of a metallic material,
which is reflectant to microwaves. Through multiple reflections, microwaves were
focused and impinged on a ceramic 'applicator tube', through which a product to be
processed would flow. Upon top of the waveguide, three tuning knobs were mounted,
which helped to tune the microwave radiation on to the applicator tube to maximize the
absorption of microwave energy by the product flowing through it. Power sensors
equipped with a computer were attached on top of the waveguide to monitor microwave
power going in forward direction as well as unabsorbed microwave power reflected
backwards.
A progressive cavity pump (Seepex) was attached to this system to pump the
product through the applicator tube and the remaining system. All the connections were
made using high pressure hoses connected by sanitary 'triclover' fittings through which a
product would flow. The applicator tube was connected to a 50 cm holding tube insulated
with fiber glass wool followed by a double tube heat exchanger for cooling the product
by tap water. After cooling, the product would flow through a silicon hose inside a
laminar flow hood for aseptic sample collection into sterile glass bottles. The internal
diameter for the entire system was 0.9525 cm. A backpressure valve was attached after
cooling section to adjust the pressure of the product during processing. Temperatures at
three different points: before holding tube, after holding tube and after cooling section
were monitored. An additional double tube heat exchanger was installed within the
system without actually using it, to get same length as in tubular heat exchanger heating
system described later in this chapter.
69
6.2.2 Challenges
Initially, a longer holding tube (15.24 m) was installed to attain longer holding
time. A bypass for cooling water was created to restrict the flow to the double tube heat
exchanger during sterilization of entire system using heated (121ºC) pressurized saline
solution. After double tube heat exchanger (no cooling occurring during system
sterilization), the pressurized saline solution would go through another 15.24 m long tube
used as an 'auxiliary cooling' arrangement to condense before released out of the system
at atmospheric pressure to avoid flashing. However, such a long length of the system was
unable to reach pressure and temperatures required for system sterilization. Above certain
pressure and temperature, the whole system would begin to vibrate, which was a safety
issue. Therefore, the two long tubes were uninstalled and initiative of system sterilization
using high pressure and temperature saline was abandoned. A simpler alternative using
25 ppm iodine solution for system sanitation, as opposed to sterilization, was adopted
prior to processing.
The inability of the progressive cavity pump used to pump the product at a
consistent flow rate was another challenge especially at higher pressures, where flow rate
would fluctuate even at a constant pump setting or sometimes even the flow would
simply stop. This challenge was partially overcome by pumping the product without any
applied back pressure since the highest processing temperature used for strawberry puree
was 102ºC, which could be achieved at atmospheric pressure. Although, achieving
consistent flow rates using the pump was unsuccessful, attempt was made to gain
consistency by using the same pump speed setting each time.
70
When strawberry puree at refrigerated temperature was processed in the
microwave system, fouling and eventual burning of puree inside the applicator tube
occurred due to a huge temperature gradient created by instantaneous heating caused by
microwaves. This challenge was surmounted by preheating the puree in a kettle at 40ºC,
thus decreasing the effective T.
6.3
Tubular heat exchanger system
6.3.1 Description, installation and operation
A tubular heat exchanger system was installed as a 'conventional' thermal
treatment to compare with continuous microwave system (Figure 6.2). Hence, total
volume inside the system (internal diameter and length) were attempted to be similar to
the one used for the 6 kW continuous microwave system. The purpose for having same
internal volume for both systems was for the strawberry puree to experience
approximately same shear while flowing through the system. To compensate for the
length of the applicator tube, it was installed within the system without using as heating
element. All other elements in the system were same as the 6 kW microwave system
except for the heating section.
In this system, heating was accomplished using a double tube heat exchanger,
with pressurized hot water used as the heating medium. Tap water was supplied to a shell
and tube heat exchanger, where it was heated with pressurized steam. An automated PID
(proportional integral derivative) control valve was used to control the temperature of this
71
hot water by controlling the amount of steam to be used to heat it. After performing
preliminary trials, the temperatures of hot water needed to achieve corresponding desired
processing temperatures for strawberry puree were determined and used during the
thermal processing of strawberry puree.
6.3.2 Challenges
Temperature control in the tubular heat exchanger system was better than in the 6
kW microwave system due to PID control. However, since the same progressive cavity
pump was used to pump the product, the challenge of inconsistent flow rates persisted
and similar to the 6 kW microwave system. This issue was addressed by using the same
pump speed setting each time.
72
Figure 6.1 Process flow diagram for 6 kW continuous flow microwave processing of
strawberry puree.
Figure 6.2 Process flow diagram for tubular heat exchanger heating of strawberry puree.
73
CHAPTER 7. PHYSICOCHEMICAL PROPERTIES OF STRAWBERRY PUREE
OVER A STORAGE PERIOD AS AFFECTED BY 6 KW CONTINUOUS FLOW
MICROWAVE HEATING SYSTEM IN COMPARISON WITH TUBULAR HEAT
EXCHANGER SYSTEM
7.1
Abstract
Strawberry is a fruit known for its characteristic red color, distinct flavor and
nutritional benefits such as antioxidant activity. Due to its high susceptibility to fungal
spoilage, strawberry and its products need to be processed to preserve these nutritional
and quality benefits. Thermal processing methods such as microwave heating with
advantages such as rapid, volumetric heating could potentially be used to treat delicate
food products such as strawberry puree. In this study, strawberry puree was processed at
80, 91 and 102ºC with holding times ranging from 2.8 - 3.6 s in a 6 kW continuous flow
microwave system and tubular heat exchanger system and stored at refrigerated
conditions. These puree samples were analyzed for their rheological properties, total
monomeric anthocyanin content, water holding capacity and instrumental color at 0, 2, 4,
6, 8 and 12 week storage time. It was observed that the apparent viscosity of strawberry
puree at 100 s-1 processed by microwave system was maintained throughout 12 weeks
storage period whereas viscosity of puree processed conventionally in a tubular heat
exchanger system along with untreated and preheated (at 40ºC) control decreased
74
significantly over this storage period. Water holding capacity values showed similar trend
as viscosity data possibly suggesting the difference between microwave and conventional
heating in inactivating pectin degrading enzymes and hence protecting molecules such as
pectin, which are known to bind water and influence viscosity. Total monomeric
anthocyanin content of microwave processed puree was higher and relatively more stable
than controls and conventionally heated puree over the storage period correlating well
with increased chroma (C*) values. Although lightness (L*) and hue angle (ºh) values of
all puree samples were higher than untreated control, there was no specific trend
observed over time. Overall, these findings suggest that microwave heating could be a
better thermal processing option than conventional heating method in order to preserve
quality of fruit products such as strawberry puree. Also, due to continuous nature of this
microwave heating system, it has a potential for commercial application.
7.2
Introduction
Strawberry (Fragaria × ananassa) fruit is consumed by millions of people across
the globe due to its delicate flavor and nutritional content (Hancock 1999). Products
containing strawberry puree are especially popular in western Europe (Gossinger and
others 2009). Strawberry is known to be a good source of ascorbic acid and anthocyanins
and has one of the highest antioxidant activities (Cordenunsi and others 2005; Hartmann
and others 2010). These nutritional benefits of strawberry are usually exploited by their
fresh consumption. However, fresh strawberries are highly susceptible to fungal
deterioration and hence they need to be processed to prolong their shelf life (Cordenunsi
75
and others 2005). Processing techniques such as thermal treatments are used to inactivate
microorganisms and more importantly enzymes responsible for the deterioration of fresh
strawberry and strawberry products. However, these conventional pasteurization
techniques result in loss of anthocyanins and hence characteristic color of strawberry
products (Hartmann and others 2010; Gossinger and others 2009). Hence, alternate
treatments such as microwave processing, which could potentially minimize quality
losses in strawberry need to be explored.
Microwave heating is known to be advantageous over conventional heating
methods. Volumetric heating (Huang and Sites 2007), short come-up times, faster heating
and higher energy efficiency (Chavan and Chavan 2010; Sumnu 2001) makes microwave
treatment an apt method for pasteurization or commercial sterilization of food products
with reduced thermal degradation of quality attributes (Tajchakavit and others 1998).
Continuous flow microwave processing is an emerging thermal processing technique for
food products (Kumar and others 2007). In this method, a pumpable product flows
through an 'applicator tube', where it is exposed to microwave power and gets heated very
rapidly prior to entering holding tube to achieve desired lethality. This technique has been
studied with products such as milk (Clare and others 2005), peanut beverages (Sabliov
and others 2008) but has had noticeable success with viscous products such as sweet
potato puree (Coronel and others 2005; Steed and others 2008) and other vegetable
products such as green pea and carrot puree, where nutritional as well as organoleptic
quality of these products was preserved better by continuous flow microwave heating
compared to conventional heating. Although some of these studies used lab-scale (5 kW)
continuous microwave system (Sabliov and others 2008; Coronel and others 2005), focus
76
of most of these studies was on larger commercial level 60-100 kW microwave power
systems, where hundreds of gallons of products need to be consumed per experimental
run restricting the liberty of performing statistically extensive research. For this reason, a
lab-scale continuous flow microwave system at 6 kW power serves as a more practical
option.
In this study, effects of lab-scale 6 kW continuous flow microwave heating
system on rheological properties, water holding capacity, instrumental color, monomeric
anthocyanin content and sugar composition of strawberry puree were investigated in
comparison with tubular heat exchanger system.
7.3
Materials and Methods
7.3.1 Puree making
Frozen whole grade A strawberries were procured from local store. Strawberries
were thawed overnight in a refrigerator. Thawed strawberries were crushed in a liquefier
for about 15 minutes to make puree. Puree was pressed through a separator having 0.58
mm diameter openings several times to separate seeds. Seedless puree was stored in a 20ºC freezer until further processing and subsequent analyses.
7.3.2 Continuous flow microwave and tubular heat exchanger heating
Prior to processing, the entire system was sanitized by re-circulating 25 ppm
iodine solution for 30 minutes. Frozen puree was thawed and pre-heated to 40ºC in a
steam-jacketed kettle. Pre-heated puree was processed through the 6 kW continuous flow
77
microwave (CFMW) heating system and tubular heat exchanger system at temperatures
and holding times described in Table 7.1.
Table 7.1. Flow rates and hold times for 6 kW CFMW and tubular H.E. systems.
Process Temperature
(⁰C)
Flow Rate (mL/min)
Average Hold Time (s)
Tubular
Microwave
Tubular H.E.
Microwave
Untreated Control
371±187
205±91
-
-
Preheated Control
700±90
327±100
-
-
80
585±40
623±115
3.66±0.24
3.52±0.73
91
760±90
687±105
2.84±0.33
3.16±0.49
102
775±129
597±60
2.81±0.45
3.61±0.36
H.E.
After holding section, heated puree was cooled down to ambient temperature in a
double tube heat exchanger using water as a cooling medium. Cooled puree was
aseptically filled into sterile glass bottles under a laminar flow hood, which was
previously sterilized using UV light for 30 minutes. Untreated and preheated puree were
passed through the systems without delivering any heat and collected in a similar fashion
and termed as 'untreated control' and 'preheated control'. Glass bottles containing puree
samples were closed hermetically using sterile caps and stored at 4oC until further
analyses. These tests were performed in triplicates and following analyses were
performed on samples at 0, 2, 4, 6, 8 and 12 weeks.
78
7.3.3 Water holding capacity
Water holding capacity of strawberry puree samples was analyzed using method
described by Takada and Nelson (1983) with few modifications. Approximately 20 g of
puree was centrifuged at 15000×g for 60 minutes at 4ºC. The precipitate was separated
from serum and % water holding capacity (WHC) was calculated as follows:
...(1)
Serum separated in this analysis was stored frozen until further analyses.
7.3.4 Rheological testing
Flow properties of strawberry puree samples were performed as described in
section 5.3.2, using an ARG 2 rheometer (TA Instruments, New Castle, DE) equipped
with a computer software (Rheology Advantage Data Analysis Software v, TA
Instruments, New Castle, DE). Steady state flow curves were obtained using a concentric
cylinder geometry. A pre-shearing conditioning step at 100 s-1 was applied to the
strawberry puree for 2 min, after which apparent viscosity versus shear rate curves were
obtained for a shear rate range of 1-1000 s-1 (Nindo and others 2007). Steady state flow
curves for selected strawberry serum samples (preheated control and 91ºC for both
CFMW and tubular H.E. processed) were obtained using cone (2º angle) and plate
geometry for a shear rate range of 0.1-200 s-1 at 0, 4 and 8 weeks.
79
7.3.5 Total monomeric anthocyanin content analysis
The total monomeric anthocyanin content of strawberry puree samples were
estimated by the pH differential method (Giusti and Wrolstad 2002) as described in
section 4.3.2. Anthocyanins were extracted from 10 g of strawberry puree samples using
50 mL 0.1% acidified methanol. The extracts were diluted 6.5 times with pH 1.0 and pH
4.5 buffers. The absorbance of diluted extracts was measured at 508 nm which is the λvismax
for pelorgonidin-3-glucoside, the predominant anthocyanin in strawberry (Lopes da
Silva and others 2007), and at 700 nm to correct for turbidity, using a UV/VIS
spectrophotometer (Beckman Coulter, Fullerton, CA). The monomeric anthocyanin
content was calculated as follows.
Monomeric anthocyanin content (mg/kg) = (A×MW×DF×1000)/(ε×1)
Where, A = (Aλvis-max – A700)pH 1.0 – (Aλvis-max – A700)pH 4.5,
MW: molecular weight = 468.8, DF: dilution factor = 32.5,
ε: molar absorptivity = 17330
7.3.6 Instrumental color measurement
As described in section 4.3.3, CIELAB color values for lightness (L*),
redness/greenness (a*) and yellowness/blueness (b*) of strawberry puree samples were
recorded using a LabScan XE colorimeter (0/45o), illuminant D65 and 10o observer
(Hunter Associates Laboratory, Naperville, IL). These values were further used to
80
calculate chroma (C*) and hue angle (⁰h) for the strawberry puree as follows (McGuire
1992):
⁰
7.3.7 Turbidity of strawberry serum samples
Turbidity of strawberry serum samples stored for 0, 4 and 8 weeks was analyzed
by measuring their absorbance at 700 nm using a UV/VIS spectrophotometer (Beckman
Coulter, Fullerton, CA).
7.3.8 Carbohydrate composition determination by proton NMR
One dimensional proton nuclear magnetic resonance (H1 NMR) technique was
used to determine carbohydrate composition of selected strawberry sera. One mL of
serum sample was freeze dried and dissolved in 1 mL of deuterium oxide (D2O) three
times to ensure replacement of hydrogen with deuterium in the system. These samples
were dissolved in deuterium oxide for H1 NMR analysis. NMR spectra were obtained
using a Variant Unity INOVA 300 NMR spectrometer (Variant Inc., Palo Alto, CA) at
ambient temperature. Peaks in the spectra were identified using standard components
(glucose, fructose, galacturonic acid and pectin) spectra.
81
7.3.9 Statistical analysis
Results from all the experiments and measurements were analyzed by statistical
software SAS 9.2 (SAS Institute, Inc., Cary, NC) using analysis of variance (ANOVA)
with a significance level of α=0.05. Factorial design with treatment (microwave, tubular),
temperature (untreated, preheated controls, 80, 91 and 102ºC) and storage time (0-12
weeks) as three factors. Treatment means were compared by Duncan’s least significance
difference (LSD) test.
7.4
Results and Discussion
7.4.1 Rheological properties of strawberry puree and selected strawberry sera and
turbidity of selected sera
Although steady state flow curves were obtained for strawberry puree at different
shear rates, focus of this study was to observe changes in viscosity of puree by
microwave heating as opposed to conventional tubular H.E. heating. Hence, for
simplicity of comparison, only apparent viscosity values at 100 s-1 are shown in Figure
7.1 as a representation of overall rheological behavior of puree for a particular treatment
and at a particular storage time. For CFMW processed puree, viscosity of all thermally
treated samples (80, 91 and 102ºC) was higher than (p < 0.05) than that of untreated and
preheated control after the processing up to four weeks. Viscosity of untreated and
preheated samples decreased drastically (~ 70%) up to four weeks after which, all
untreated and preheated samples were spoiled with apparent yeast growth and
82
perceivable fermented aroma and hence were unavailable for further analyses. Regardless
of the heating methods, thermally treated samples showed relatively lower reduction in
apparent viscosity (~25%) over a longer (12 weeks) storage period without any spoilage
plus there were no differences (p > 0.05) among three different treatment temperatures
throughout 12 weeks storage.
For conventionally processed puree, there were no apparent viscosity differences
(p > 0.05) among untreated, preheated controls and thermal treatment temperatures and
there was ~50% viscosity reduction (p < 0.05) for all samples after four weeks storage
time. Similar to CFMW processed puree, untreated and preheated control samples were
spoiled after four weeks whereas for samples treated at 80, 91 and 102ºC, viscosity
remained constant up to 8 weeks after which, they were also spoiled.
Rheological properties of strawberry puree and correlating water holding capacity
results suggest that differences between untreated control and preheated control samples
were insignificant. Similarly, there were no differences among different treatment
temperatures (80, 91 and 102ºC) for a particular processing method (CFMW or
conventional tubular H.E.). Hence, representatives from this pool of samples at different
points of storage time i.e. preheated control and 91ºC samples for both CFMW and
tubular heating from 0, 4 and 8 weeks were selected for rheological analysis of their sera.
Apparent viscosity of serum samples of CFMW processed puree at 100 s-1 was higher (p
< 0.05) than conventionally heated ones (Figure 7.3). Thermally treated samples i.e. 91ºC
samples had higher viscosity than preheated control and viscosity of all serum samples
decreased (p < 0.05) from 0 to 8 weeks. It should also be noted that unlike puree samples,
strawberry sera showed mixed rheological behaviors over time i.e. all serum samples
83
showed shear thinning behavior similar to puree at Day 0 but after 4 and 8 weeks of
storage, all samples showed Newtonian behavior except for microwave treated samples,
which maintained their shear thinning nature even after 8 weeks. Please, refer to Figure
A2 in Appendix to view respective flow curves.
These strawberry sera showed visual differences in their turbidity, which
correlated well with viscosity findings. To quantify these visual differences in turbidity
strawberry serum samples, absorbance at 700 nm was measured (Figure 7.4). Similar to
viscosity values for sera, turbidity of serum of CFMW processed puree was higher (p <
0.05) than conventionally heated ones. Sera of puree heated at 91ºC samples had higher
turbidity than preheated control and turbidity of all serum samples decreased (p < 0.05)
from 0 to 8 weeks.
These viscosity and turbidity results for sera separated from processed strawberry
puree show a direct relationship with viscosity and water holding capacity trends for
strawberry puree. This relationship allows us to hypothesize that the origin of these
viscosity trends in puree could in part at least lie in viscosities of their water soluble
portions i.e. their sera. Further investigation of chemical composition of strawberry sera
could be necessary to potentially explain differences in viscosity of CFMW processed
puree compared to untreated, preheated controls and conventionally heated puree.
Firmness of strawberry fruit is correlated with presence and development of
pectin, hemicelluloses (Huber 1984; Figueroa and others 2010) and polyuronides (Huber
1984) in cell walls during ripening. Softening of fruit during ripening is however mostly
associated with degradation and solubilization of polymeric pectin molecules by enzymes
such as pectin methylesterase (PME) and polygalacturonase (PG) (Ly-Nguyen and others
84
2002; Figueroa and others 2010). Hence, viscosity losses over time for untreated and
preheated control samples and conventionally heated samples can be correlated with
activity of pectolytic enzymes.
When comparing between CFMW and conventionally heated samples, overall
viscosity of microwave treated puree was significantly higher (p < 0.05) than
conventionally heated one. Since separate batches of strawberry puree were made for
CFMW and conventional thermal processing, differences in viscosity could be attributed
to initial inherent viscosity difference between two batches. However, even after
accounting for inbuilt difference between two batches of puree, it should be noted that
each processing had different controls (untreated and preheated) used. Both thermal
processing techniques showed different trends in viscosity changes compared to their
respective controls i.e. viscosity reduction trends were similar for controls and thermal
treatments in case of conventional processing whereas in case of CFMW processing,
thermal treatments maintained viscosity better over 12 weeks storage period as compared
to respective controls. Maintaining viscosity over time could be attributed to efficacy of
inactivation of pectin degrading enzymes. Viscosity reduction in case of conventional
heating and subsequent spoilage of puree after 8 weeks suggests lower extent of enzyme
inactivation compared to CFMW processing. This is in agreement with a very similar
study by Osorio and others (2008) who batch pasteurized strawberry puree
conventionally at 90ºC for 20 s and evaluated its effect on viscosity of puree. They found
that even though viscosity of puree was high after processing, it decreased over storage at
refrigerated temperature. Since, treatment temperatures used in both processing
techniques in current study were same (80, 91 and 102ºC), difference in extent of enzyme
85
inactivation could point towards possible non-thermal inactivation of enzymes by
microwave radiations. However, studies conducted to investigate any non-thermal effect
of microwave radiations for last five decades has been inconclusive (Kozempel and
others 2000). Tajchakavit (1997) attempted to investigate any non-thermal mechanism
behind higher inactivation of PME in orange juice by microwaves by applying full
microwave power (700 W) but keeping juice below 40ºC by simultaneously cooling it.
However, insignificant PME inactivation was shown by this arrangement invalidating
any non-thermal effects. In that same study, another arrangement was made to let the
temperature of juice gradually increase by microwave treatment. It was found that
microwaves showed higher inactivation compared to conventional heating and this
inactivation was higher at higher temperatures (50ºC or above). Hence, they termed this
behavior as "temperature-dependent enhanced thermal effects" by microwave radiations,
where the effect of heat in inactivating enzymes is enhanced by microwave radiation.
Therefore, this enhanced thermal effect phenomenon could possibly be occurring in case
of strawberry puree in the present study.
Turbidity of microwave treated sera was noticeably higher than other samples.
Possible explanation for this could be higher yields of extraction of soluble components
by microwave radiations leading to increased turbidity (Gerald and Roberts 2004), which
could ultimately contribute to higher viscosity of puree. In addition to extraction of
soluble compounds, microwave heating is known to rupture the cell-walls of food
matrices (Eskilsson and Bjorklund 2000). There could be a possibility that this cell-wall
rupture may produce micro or nanosized cell-wall debris, which could potentially exhibit
86
Brownian motion within the syrup. These randomly moving cell-wall particles can scatter
light and result in higher turbidity.
This strawberry serum containing cell-wall particles could be considered a
colloidal dispersion because it is a immiscible mixture of cell-wall debris in aqueous
serum. Viscosity of a colloidal dispersion depends on the size, shape and concentration
(or volume fraction) of dispersed particles. Viscosity increases with volume fraction and
size of dispersed particles. Rod-shaped particles increase the viscosity of the dispersion
more than spherical particles do (Morris 2001). Although in current study, these factors
were not evaluated, their possible effect on viscosity of strawberry puree as well as serum
cannot be ignored.
7.4.2 Water holding capacity
Water holding capacity (WHC) values for strawberry puree samples were
calculated as supporting data for rheological results (Figure 7.2). Trends in WHC values
for strawberry puree were very similar to those in viscosity values. For CFMW processed
puree, all thermally treated samples (80, 91 and 102ºC) had higher WHC values than (p <
0.05) untreated and preheated control after the processing up to four weeks. WHC
reduced by ~34% for untreated and preheated controls whereas for thermal treatments,
the reduction was only ~18% over 12 weeks storage period with insignificant (p > 0.05)
differences among different treatment temperatures. For conventional tubular H.E.
processed puree, surprisingly, WHC values for thermally treated samples were lower (p <
0.05) than untreated and preheated control up to four weeks. Reduction in WHC of
untreated and preheated control was ~20% whereas that in thermally treated samples was
87
~42%. Similar to viscosity values, water holding capacity of strawberry puree treated
with CFMW was higher than that heated conventionally.
As discussed previously, cell walls of strawberry contain pectin, which is mainly
responsible for rheological properties of strawberry fruit and products such as puree in
this case. Pectin is known to bind water (Schroder and others 2004; Vaclavik and
Christian 2007) and that is what contributes to the viscosity of strawberry puree. Hence,
water holding capacity results support trends in viscosity changes where pectolytic
enzymes degrade pectin over time losing its ability to bind water resulting in reduced
water holding capacity and viscosity.
7.4.3 Carbohydrate composition by NMR
Carbohydrate composition as determined by NMR technique showed no
differences among selected strawberry serum samples. Figure 7.6 gives representation of
NMR spectra for strawberry sera. It was hypothesized that the differences in viscosities
of strawberry puree processed by CFMW system and conventional heating could be
explained by any possible changes in carbohydrate composition of the puree. However,
this hypothesis was not supported by NMR results, which show no differences in
carbohydrate composition. Hence, effects of microwave radiations on viscosity of
strawberry puree remain unexplained.
88
7.4.4 Total monomeric anthocyanin content
Monomeric anthocyanin content of strawberry puree processed by CFMW was
higher (p < 0.05) than conventionally heated puree overall (Figure 7.7). For CFMW
processed strawberry puree, there was no difference in anthocyanin content among
untreated, preheated controls and any high temperature treatments and there was no
significant loss of anthocyanin pigments up to 4 weeks after which both control samples
spoiled. However, there was ~25% pigment loss (p < 0.05) in three thermally treated
sample over 12 weeks storage period, 15% of which was in last 4 weeks. For
conventionally heated puree, there was no difference among controls and thermal
treatments similar to CFMW processed puree but there was ~21% pigment loss in first 4
weeks. Thermally treated puree showed significant (p < 0.05) anthocyanin loss at every
two week period until they spoiled after 8 weeks (total ~31% loss) with 91ºC and 102ºC
treatments resulting in higher losses than at 80ºC. Similar levels of anthocyanin loss have
been observed in strawberry nectar (~39%) after pasteurization at 85ºC for 5 minutes as
observed by Klopotek and others (2005). According to Verbeyst and others (2010),
thermal degradation of anthocyanins in strawberry follows first-order kinetics when they
tested it in a range of 80-130ºC. Anthocyanin content of strawberry puree processed by
CFMW was higher (p < 0.05) than conventionally heated puree. Yousefi and others
(2012) also observed similar preservation of anthocyanin pigments by microwave
radiations compared to conventional heating. This could be due to the fact that the higher
penetrating power of microwave radiation facilitates rapid rupture of plant cell structures
89
and that improves extraction efficiency (Lu and others 2010; Wang and Weller 2006) of
compounds like anthocyanins.
7.4.5 Instrumental color
Lightness (L*) (Figure 7.8) of CFMW processed strawberry puree was not
different (p<0.05) than that heated conventionally. For CFMW processed puree, three
thermal treatments in addition to the preheating appeared to have increased (p < 0.05) the
lightness, with no specific trend through storage time. Differences in lightness within
conventionally processed puree similar to CFMW processed ones.
Chroma (C*) values (Figure 7.9) of CFMW processed puree were found to be
higher (p < 0.05) than conventionally heated ones. Chroma values represent the intensity
of the color and hence higher color intensity of microwave processed puree correlates
well with higher anthocyanin content compared to conventionally heated puree. Within
CFMW processed puree, however, no difference was found in chroma values among
different treatment temperatures and both controls and no trend was observed through
storage period. However for conventionally processed puree samples, intensity decreased
(p < 0.05) over storage time with no differences among treatment temperatures and
controls.
Hue (ºh) angle values (Figure 7.10) of microwave processed puree samples were
higher (p < 0.05) than conventionally heated samples. Higher positive hue angle value in
physical terms mean shift from red/purple (0º) to yellow (90º) (McGuire 1992). Within
CFMW processed puree, only untreated control had lower (p < 0.05) hue angle than
90
preheated control and treatment temperatures with no specific trend over the storage
period. Differences in hue angle within conventionally processed puree were similar to
CFMW processed counterparts.
In spite of any statistical differences in instrumental color values, it must be noted
that there was no difference perceived to the naked eyes for any strawberry puree samples
and hence for practical purposes, these statistical differences may or may not be
significant. Also, color perceived by human eyes is a result of intricate blend of light
transmission as well as reflection from the surface of the food whereas instrumental color
is a measure of only reflected light from the surface. Hence, correlation between sensory
and instrumental color perception is not always possible (Saied 2004).
7.5
Conclusions
Continuous flow microwave heating maintained viscosity and water holding
capacity of strawberry puree and serum over a storage period of 12 weeks compared to
untreated, preheated controls and conventionally heating. Although polysaccharide
profiles of strawberry sera were unable to explain these differences, this finding holds
significance in terms of application of microwave radiations in maintaining rheological
properties of fruit purees and provides a processing option to fruit product manufacturers
for potential commercialization. Preservation of color and anthocyanin content by
CFMW compared to conventional heating over 12 week storage period is important in
terms of preserving nutritional as well as sensory quality of fruit products.
91
Apparent viscosity (Pa.s) at 100 s-1
0.25
0.2
Untreated Control
0.15
Preheated Control
80⁰C
0.1
91⁰C
102⁰C
0.05
0
Day 0
2 weeks
4 weeks
6 weeks
8 weeks 12 weeks
(A)
Apparent viscosity at 100 s-1 (Pa.s)
0.25
0.2
Untreated Control
0.15
Preheated Control
80⁰C
0.1
91⁰C
102⁰C
0.05
0
Day 0
2 weeks
4 weeks
6 weeks
8 weeks 12 weeks
(B)
Figure 7.1 Apparent viscosity of strawberry puree at 100 s-1 heated by (A) 6 kW
continuous flow microwave system and (B) tubular H.E. system at different temperature
at different storage times.
92
30
Untreated Control
% Water Holding Capacity
25
Preheated Control
80⁰C
20
91⁰C
15
102⁰C
10
5
0
Day 0
2 weeks
4 weeks
6 weeks
8 weeks
12 weeks
(A)
30
% Water Holding Capacity
25
20
Untreated Control
Preheated Control
15
80⁰C
91⁰C
10
102⁰C
5
0
Day 0
2 weeks
4 weeks
6 weeks
8 weeks
12 weeks
(B)
Figure 7.2 % Water holding capacity of strawberry puree at 100 s-1 heated by (A) 6 kW
continuous flow microwave system and (B) tubular H.E. system at different temperature
at different storage times.
93
Apparent viscosity at 100 s-1 (Pa.s)
0.07
0.06
0.05
0.04
Day 0
0.03
4 weeks
8 weeks
0.02
0.01
0
MW Preheated
Control
MW 91⁰C
Tubular Preheated
Control
Tubular 91⁰C
Figure 7.3 Apparent viscosity of strawberry sera at 100 s-1 separated from strawberry
puree heated by 6 kW continuous flow microwave system and tubular H.E. system at
different temperature at different storage times.
94
1.2
Absorbance at 700 nm
1
0.8
MW Preheated Control
MW 91⁰C
0.6
Tubular Preheated Control
Tubular 91⁰C
0.4
0.2
0
Day 0
4 weeks
8 weeks
Figure 7.4 Turbidity of strawberry sera separated from strawberry puree heated by 6 kW
continuous flow microwave system and tubular H.E. system at different temperature at
different storage times.
95
(A)
(B)
Figure 7.5 Visual appearance of strawberry puree heated by (A) 6 kW continuous flow
microwave system and (B) tubular H.E. system at 80ºC (right) compared to
corresponding preheated control (left).
96
Figure 7.6 Representative NMR spectra of strawberry sera separated from puree treated
by 6 kW CFMW/tubular H.E. system.
97
Monomeric Anthocyanin Content
(mg/kg)
600
500
400
Untreated Control
Preheated Control
300
80⁰C
91⁰C
200
102⁰C
100
0
Day 0
2 weeks
4 weeks
6 weeks
8 weeks 12 weeks
(A)
Monomeric Anthocyanin Content
(mg/kg)
600
500
400
Untreated Control
Preheated Control
300
80⁰C
91⁰C
200
102⁰C
100
0
Day 0
2 weeks 4 weeks 6 weeks 8 weeks 12 weeks
(B)
Figure 7.7 Monomeric anthocyanin content of strawberry puree heated by (A) 6 kW
continuous flow microwave system and (B) tubular H.E. system at different temperature
at different storage times.
98
40
35
Lightness (L*)
30
Untreated
25
Preheated
20
80⁰C
15
91⁰C
102⁰C
10
5
0
Day 0
2 weeks
4 weeks
6 weeks
8 weeks
12 weeks
(A)
40
35
Lightness (L*)
30
Untreated
25
Preheated
20
80⁰C
15
91⁰C
102⁰C
10
5
0
Day 0
2 weeks
4 weeks
6 weeks
8 weeks
12 weeks
(B)
Figure 7.8 Lightness (L*) of strawberry puree heated by (A) 6 kW continuous flow
microwave system and (B) tubular H.E. system at different temperature at different
storage times.
99
60
Chroma (C*)
50
40
Untreated
Preheated
30
80⁰C
91⁰C
20
102⁰C
10
0
Day 0
2 weeks
4 weeks
6 weeks
8 weeks
12 weeks
(A)
60
Chroma (C*)
50
40
Untreated
Preheated
30
80⁰C
91⁰C
20
102⁰C
10
0
Day 0
2 weeks
4 weeks
6 weeks
8 weeks
12 weeks
(B)
Figure 7.9 Chroma (C*) of strawberry puree heated by (A) 6 kW continuous flow
microwave system and (B) tubular H.E. system at different temperature at different
storage times.
100
40
35
Hue Angle (⁰h)
30
Untreated
25
Preheated
20
80⁰C
15
91⁰C
10
102⁰C
5
0
Day 0
2 weeks
4 weeks
6 weeks
8 weeks
12 weeks
(A)
40
35
Hue Angle (⁰h)
30
Untreated
25
Preheated
20
80⁰C
15
91⁰C
102⁰C
10
5
0
Day 0
2 weeks
4 weeks
6 weeks
8 weeks
12 weeks
(B)
Figure 7.10 Hue angle (⁰h) of strawberry puree heated (A) 6 kW continuous flow
microwave system and (B) tubular H.E. system at different temperature at different
storage times.
101
CHAPTER 8. CHARACTERIZATION OF ERGOCALCIFEROL LOADED SOLID
LIPID NANOPARTICLES
Mandar R. Patel and M. Fernanda San Martin-Gonzalez
Published Research Article
Journal of Food Science. 2012. 77(1):N8-N13
8.1
Abstract
The use of solid lipid nanoparticles (SLN) for the encapsulation of drugs is a
technique that has been widely used in the pharmaceutical industry for the last two
decades and has become of increasing interest to food scientists due to its potential for
encapsulation and controlled release. Ergocalciferol (vitamin D2) is a bioactive
compound of which deficiency may lead to rickets in children and osteomalacia in adults.
In this study, ergocalciferol was encapsulated in tripalmitin solid lipid nanoparticles
stabilized by polysorbate 20 (Tween 20). SLN dispersions (5% w/w) were prepared by
hot homogenization technique using a nozzle-type high pressure homogenizer.
Ergocalciferol at 0, 5, 10, 15 and 20% (w/w of lipid) was dissolved in the molten lipid at
80oC, mixed with a 5% (w/w) aqueous solution of polysorbate 20 and homogenized at
138MPa at 80oC. Particle size, thermal properties and microstructure were evaluated by
102
dynamic light scattering, differential scanning calorimetry (DSC) and transmission
electron microscopy (TEM) respectively. As the proportion of ergocalciferol in the SLN
increased from 0 to 20%, the Z-average values of the particles gradually decreased
(p≤0.05) from ~120 nm to ~65 nm. DSC analysis of freeze dried SLN samples showed
gradual decrease in enthalpies of fusion and crystallization for stable β-subcell whereas
for SLN dispersions, the enthalpy of fusion of unstable α-subcell crystal increased with
increased ergocalciferol loading. The TEM images of the ergocalciferol loaded SLN
samples showed the presence of spherical as well as rod-shaped nanoparticles. It was also
observed that the turbidity of the SLN dispersions reduced noticeably with increased
ergocalciferol loading. This finding could be useful in terms of fortification of clear
juices with ergocalciferol.
Keywords: Vitamin D, Ergocalciferol, Solid lipid nanoparticles, encapsulation
8.2
Practical Application
Solid lipid nanoparticles (SLN) were used in this study to encapsulate vitamin D2,
a vitamin important for bone health. It was found that as the concentration of vitamin D2
increased in the lipid phase of SLN dispersion, the clarity of the dispersion increased.
Also, with increased vitamin D2 concentration, the stability of lipid crystal structure was
affected in a way that indicates higher capacity of lipid to incorporate the vitamin
molecules and hence to protect them better from oxygen and light. This vitamin loaded
SLNs may offer alternatives to milk and margarine as a source of vitamin D.
103
8.3
Introduction
Vitamin D deficiency is one of the most common vitamin deficiencies prevailing
globally even in developed countries like the United States and Canada (Ward and others
2007). Deficiency of vitamin D causes rickets in children and osteomalacia in adults,
where the skeletal development is hampered due to impaired absorption of calcium,
which results in weaker and softer bones (Beer and Jones 2005; Ward and others 2007).
Although fortification of certain food products such as milk with vitamin D has
drastically reduced the incidence of these diseases, sporadic cases of rickets have been
reported especially among African-American children and infants (Hochberg 2003). A
high amount of melanin pigments in the skin impairs its ability to synthesize vitamin D
making people with dark skin more vulnerable to vitamin D deficiency (Nesby-O’Dell
and others 2002). Some of the factors contributing to vitamin D deficiency also include:
limited sun exposure due to clothes and northern latitudes, use of sunblock, fear of
contracting skin cancer and melanoma, limited consumption of fortified foods and fear of
excessive vitamin D intake (Chesney 2003; Ward and others 2007). In animals, vitamin
D is synthesized under the skin when exposed to the sunlight, where 7dehydrocholesterol is converted to previtamin D3, which is isomerized to form
cholecalciferol (vitamin D3) (Eitenmiller and others 2008). The efficiency of vitamin D
synthesis and ability of the kidney to convert it to its active hormonal form decreases
with age (Need and others 1993). Fat malabsorption (Lo and others 1985) and obesity
(Wortsman and others 2003) are also associated with decrease in vitamin D
bioavailability. Thus, the necessity of vitamin D cannot be fulfilled by relying solely on
104
its subcutaneous synthesis. Hence, in addition to the two most common products fortified
with vitamin D, milk and margarine (21 CFR; Eitenmiller and others 2008), other
aqueous-based food products also need to be considered for vitamin D fortification. Since,
vitamin D is fat-soluble; an encapsulation technique can be utilized to facilitate its
fortification in water-based products as well as to protect it from environmental factors.
Ergocalciferol is one of the two common forms of vitamin D and is synthesized in plants
in the presence of sunlight (Eitenmiller and others 2008). It is prone to oxidation
(Eitenmiller and others 2008) and is also isomerized to isotachysterol in the presence of
light and under acidic conditions (Eitenmiller and others 2008; Grady and Thakker 1980).
Susceptibility of ergocalciferol to environmental factors makes it a suitable candidate for
encapsulation.
Solid lipid nanoparticles (SLN) are widely used for the encapsulation of drugs in
the pharmaceutical field over the last two decades as an alternative to polymeric
nanoparticles, liposomes and emulsions (Pardeike and others 2009; Müller and others
2000). The advantages of using SLN as delivery vehicle include increased
physicochemical stability of core material and encapsulated compound, decreased
leakage of the active drug and reduced interaction with the emulsifier coat. SLNs exhibit
controlled release potential due to reduced mobility of the encapsulated drug as well as to
its hydrophobic nature which prevents dissolution in aqueous media (Bunjes and
Siekmann 2006). Since solid state of these lipid nanoparticles slows down their digestion
by pancreatic lipase (Bonnaire and others 2008), they can be orally administered for a
controlled release in the gastrointestinal tract (Müller and others 1996). Although SLNs
have been used as a carrier systems for cosmetic active ingredients and pharmaceutical
105
drugs in the past (Saupe and Rades 2006), only recently SLN has attracted attention as a
carrier systems for bioactive food components (Weiss and others 2008). Recently, SLNs
have been used to encapsulate polyphenols such as curcuminoids (Tiyaboonchai and
others 2007) and quercetin (Li and others 2009), proteins and peptides (Almeida and
Souto 2007), lipophilic vitamins such as ascorbyl palmitate (ester of ascorbic acid)
(Teeranachaideekul and others 2007), vitamin A (Jenning and Gohla 2001; Jee and others
2006), β-carotene (Trombino and others 2009) and vitamin E (Trombino and others
2009).
In this study, tripalmitin SLN dispersions were used to encapsulate ergocalciferol
and characterized for turbidity, particle size distribution, morphology and phase transition
behavior.
8.4
Materials and Methods
8.4.1 Materials
Glyceryl tripalmitate (tripalmitin, 85% pure), polyoxyethylene (20) sorbitan
monolaurate (Tween® 20) and ergocalciferol were purchased from Sigma Aldrich (St.
Louis, MO).
8.4.2 Preparation of SLN dispersions
Ergocalciferol loaded SLN dispersions were prepared in triplicates by hot
homogenization technique. Dispersed phase containing ergocalciferol at concentrations 0,
106
5, 10, 15 and 20% (w/w of dispersed phase) were prepared by dissolving 0.000, 0.125,
0.250, 0.375 and 0.500g of ergocalciferol in 2.500, 2.375, 2.250, 2.125 and 2.000g of
molten tripalmitin (80oC). Then, the 2.500g of dispersed phase was mixed with 47.500g
of an 5.26% (w/w) aqueous solution of polysorbate 20 previously heated to 80oC. The
polysorbate solution was prepared by mixing 45.000g of water with 2.500g of
polysorbate 20 for a final concentration in the suspension of 5% (w/w). Thus, a SLN
dispersion consisted of 5% (w/w) lipid phase and 95% (w/w) aqueous phase and
surfactant. A coarse emulsion was prepared by mixing both phases using a hand blender
for one minute. The coarse emulsion was immediately passed through a nozzle-type high
pressure homogenizer (Nano DeBEE, BEE International, South Easton, MA) ten times at
a pressure of 138 MPa at 80°C. Ten passes were selected based on preliminary trials
(results not shown) in which Z-avg values and polydispersity index decreased with
increasing number of passes up to 10 passes beyond which values were not affected any
further. In order to ensure that the whole dispersion was homogenized ten times, the
whole sample was collected after each pass and returned to the feed reservoir for
subsequent processing. At the end of the tenth pass, the hot emulsion was immediately
cooled at ~0°C in an ice/water-bath to obtain ergocalciferol loaded solid tripalmitin
nanoparticle dispersions. An aliquot of each SLN dispersion was freeze dried. The
dispersions and the freeze dried SLNs were stored in a refrigerator at 4°C until further
analyses.
107
8.4.3 Turbidity measurement
SLN dispersions were diluted by mixing 3 mL of dispersion with 8 mL of distilled
water and vortexed for 1 min. Absorbance at 700 nm was read using a UV/VIS
spectrophotometer (DU 720, Beckman Coulter, Fullerton, CA) using distilled water as a
blank.
8.4.4 Particle size measurement
Hydrodynamic radius (Z-average) and polydispersity index was measured by
dynamic light scattering (DLS) using a Zetasizer (Nano ZS90, Malvern Instruments,
Malvern, UK) with a detector placed at 90º angle. The original dispersions were diluted
1:100 using distilled water and vortexed for 30 s prior to particle size measurements. The
measurements were performed at 10°C using a refractive index of 1.54 (for solid
tripalmitin) (Helgason and others 2008).
8.4.5 Transmission electron microscopy (TEM)
Samples were diluted prior to further analysis. Samples with 0 and 5%
ergocalciferol were diluted 1/10 and samples with 10, 15 and 20% ergocalciferol were
diluted 1/20 using distilled water prior to TEM imaging since undiluted emulsions were
too concentrated to yield visible images. The grids were immersed in a drop of sample
and passed through two drops of 2% uranyl acetate. Excess stain solution was absorbed
by filter paper and the grids were air-dried prior to imaging. The samples were imaged
108
using a Philips CM-100 TEM (FEI Company, Hillsboro, OR) operated at 80 kV, spot 3,
200 μm condenser aperture and 50μm objective aperture. Images were captured on
Kodak SO-163 electron image film. Magnifications of 11500 and 21000 х were used for
all samples.
8.4.6 Differential scanning calorimetry (DSC)
Fat polymorphs and their enthalpy of fusion were studied by differential scanning
calorimetry (DSC; Q 2000, TA instruments, New Castle, DE). For SLN dispersions, 8 to
12 mg of sample and for freeze dried SLN particles, 4 to 18 mg of sample was placed in
aluminum pans (Tzero) and lids were hermetically sealed. An empty sealed pan was used
as a reference. The pans were placed in the calorimeter and equilibrated at 20°C. The
pans were heated up to 75°C at a rate of 10°C/min to observe fusion of the SLN particles
and cooled down at the same rate to a final temperature of 5°C to observe the
crystallization of the melted lipid particles.
8.4.7 Data analysis
Experiments were run in triplicate and results from all the experiments and measurements
were analyzed by statistical software SAS 9.2 (SAS Institute, Inc., Cary, NC) using
analysis of variance (ANOVA) with a significance level of 0.05. Treatment means were
compared by Duncan’s least significance difference (LSD) or Tukey’s test.
109
8.5
Results and Discussions
8.5.1 Turbidity of SLN dispersions
Ergocalciferol loaded SLN dispersions prepared by hot-homogenization technique
showed visual differences in turbidity. Turbidity of the SLN dispersions decreased as the
concentration of ergocalciferol in the dispersed lipid phase increased from 0 to 20%
(w/w). To quantify this observation, the turbidity of these dispersions was determined by
measuring absorbance at 700 nm. Results (Figure 8.1) show that the turbidity of the
dispersions decreased (P < 0.05) linearly with ergocalciferol loading. Turbidity is the
extent to which light is scattered by the particles in dispersions. Extent of scattering
depends on the wavelength of light being scattered and the size of the particle, from
which it is scattered. For any wavelength in the visible portion of the electromagnetic
spectrum, the amount of scattering decreases with reduction in particle size when it falls
below 1μm (McClements 1999). Since, the particle size of all the SLN dispersions in this
study was well below 1μm (Figure 2), the trend in turbidity values can be correlated with
the change in particle size as ergocalciferol loading varied.
8.5.2 Particle size of SLN dispersions
The average hydrodynamic diameter (Z-avg) values of ergocalciferol loaded
tripalmitin SLN are shown in Figure 8.2. Similar to the turbidity values, the particles size
of the SLN dispersions also decreased (P < 0.05) linearly from ~125 nm to ~65 nm with
110
increase in the concentration of ergocalciferol in the lipid dispersed phase from 0 to 20%
(w/w). The polydispersity index of all the samples was 0.23 ± 0.01.
A reason behind the decrease in particle size with increased ergocalciferol loading
could be attributed to differences in the viscosities of the dispersed phase. During melthomogenization, the hot emulsion droplets are disrupted due to high energy input before
they crystallize to form solid lipid particles. The viscosity ratio between the dispersed and
continuous phases, plays an important role in the disruption of the dispersed phase
droplet. Reduction in the viscosity ratio (d/c) between the dispersed and continuous
phases reduces the critical Weber number, defined as the value of Weber number above
which higher values result in droplet breakup (Leong and others 2009). Thus, the higher
the viscosity ratio, the larger the particle size will be (McClements 1999). Since, the
continuous phase was maintained constant for all the samples, the decrease in viscosity of
dispersed lipid phase caused by a plasticizing effect of Vitamin D2 could be the reason
behind the change in particle size of the SLN with increased ergocalciferol loading.
8.5.3 Transmission electron microscopy (TEM) of SLN dispersions
The TEM images of ergocalciferol loaded solid lipid nanoparticles are shown in
Figure 8.3. It can be observed from these microscopic images that the lipid particles were
either disk-shaped or rod-shaped. Disk-shaped or spheroidal particles become more
predominant as the ergocalciferol loading in the dispersed phase of the solid lipid
nanoparticles is increased, whereas the number of rod-shaped particles decreased.
However, the polydispersity of the samples remained high in all cases.
111
8.5.4 Differential scanning calorimetric (DSC) analysis
DSC analysis was performed for freeze dried as well as for liquid suspensions for
comparative purposes. DSC data for all freeze dried SLN samples show a fusion peak of
stable β-subcell form at ~63°C and a crystallization peak at ~40°C for all the samples
(Figure 8.4). No peaks for -polymorph were observed in the melting curves of any of
the freeze dried samples due to polymorphic transition caused by freezing and solvent
removal which possibly resulted in expulsion of vitamin D2 from the particles. The
formation of more densely packed lattices during polymorphic transitions is often
accompanied with the expulsion of incorporated molecules (Mehnert and Mader, 2001).
For SLN dispersions (Figure 8.5), two fusion peaks were observed for all the samples,
one at around 43°C indicating least stable α-subcell polymorph (Helgason and others
2009) and another at 53°C suggesting β2 crystal form of tripalmitin (Kellens and others
1990). The crystallization peaks for all the SLN dispersions were observed at around
20°C. For freeze dried SLN, enthalpies of fusion and crystallization decreased with
increase in ergocalciferol loading in the dispersed phase (Figure 8.6). On the other hand,
the enthalpy of fusion of the α-subcell of SLN dispersions increased with ergocalciferol
loading (Figure 8.7). The β1, which is more stable as compared to β2 crystal form
(Kellens and others 1990), showed negligible enthalpy values for all samples in this study
[Figure 8.5 (A)] indicating the predominance of crystal structures having lower stabilities.
The presence of α-subcell and more stable β-subcell polymorphs of ergocalciferol
loaded tripalmitin lipid particles is suggested from the existence of both spherical and
rod-shaped nanoparticles. It can be observed from the TEM images that the spherical α-
112
subcell polymorphs become more predominant as the ergocalciferol loading increases.
This can also be verified from the fact that the enthalpy of crystallization of α-subcell
increased as ergocalciferol loading increased. Also, from the DSC data of freeze dried
SLN, the enthalpies of fusion and crystallization decreases with ergocalciferol loading.
Both of these findings suggest increase in the extent of least stable form (α-subcell) or
decreased in the stability of more stable form (β-subcell) with increasing ergocalciferol
loading. If the crystal structure is more stable, the packing of its molecules within the
lattice is more organized (Lawler and Dimick 2008) and expelling of the encapsulated
compound out of the particle is possible (Helgason and others 2009). The lower stabilities
of the crystal structures in this case suggest enhanced encapsulation efficiency for
ergocalciferol. The addition of ergocalciferol into the lipid phase seems to provide a
stabilizing effect to the  polymorph presumably due to inclusion of the foreign
molecules (ergocalciferol in this case) within the lipid matrix that increase the number of
defects within the lattice, thus delaying polymorphic transitions. A decrease in
crystallinity of tripalmitin and hard fats has been previously reported by Siekmann and
Westesen (1994) after encapsulation of ubidecarenone (Coenzyme Q10). Furthermore,
they reported that when lecithin was used as an emulsifier the rate of polymorphic
transitions increased since the incorporation of the fatty acid chains of lecithin within the
lipid matrix provided an ordering effect in the triglyceride matrix due to structural
similarities between the fatty acid chains from the phospholipids and the triglycerides.
Therefore, in our study it is possible that the increase in  subcell with increasing content
of vitamin D2, a secosteroid, in the SLN is a result of the disruption in the crystal lattice
caused by ergocalciferol molecules embedded into the lipid matrix, similar to what
113
Siekmann and Westesen (1994) observed for ubidecarenone. Values for the H of SLN
dispersions are shown in Table 1. Mean values gradually increased with increasing
content of ergocalciferol. However, at highest ergocalciferol contents, differences in H
values became non-significant, indicating an inability of tripalmitin particles to further
retain Vitamin D2 contents greater than 10-15%.
8.6
Conclusions
Ergocalciferol loaded tripalmitin solid lipid nanoparticles prepared by melthomogenization technique showed linear trends in their turbidity, particle size and
enthalpies of fusion and crystallization. Loading of ergocalciferol in the tripalmitin
dispersed phase decreased the particle size of the nanoparticles linearly, which in turn,
decreased the turbidity of the SLN dispersions, which may hold potential application in
the fortification of clear and cloudy juices with ergocalciferol. The changes in enthalpy
values of different polymorphs of SLN with increased ergocalciferol loading show
potential for enhanced encapsulation efficiency and thus improved stability of the
encapsulated compound. Further studies are required to quantify encapsulation efficiency
and loading, release kinetics and stability during processing and storage.
114
0.9
0.8
Absorbance at 700 nm
0.7
0.6
0.5
0.4
R² = 0.9872
0.3
0.2
0.1
0
0
5
10
15
20
% Ergocalciferol loading (w/w of dispersed phase)
Figure 8.1 Decrease in turbidity of SLN dispersions (3/11 dilution) with different %
ergocalciferol loading.
25
115
140
120
Z-avg (nm)
100
R² = 0.9887
80
60
40
20
0
0
5
10
15
20
% Ergocalciferol loading (w/w of dispersed phase)
Figure 8.2 Particle size of ergocalciferol loaded SLN dispersions.
25
116
Figure 8.3 TEM images of SLN dispersions with different % ergocalciferol loading
117
(A)
(B)
Figure 8.4 Differential scanning calorimetry thermogram of freeze dried tripalmitin solid
lipid nanoparticles stabilized by Polysorbate 20 with different % ergocalciferol
concentrations (w/w of dispersed phase). (A) Melting cycle, (B) Crystallization cycle.
118
(A)
(B)
Figure 8.5 Differential scanning calorimetry thermogram of tripalmitin solid lipid
nanoparticle suspensions stabilized by Polysorbate 20 with different % ergocalciferol
concentrations (w/w of dispersed phase). (A) Melting cycle, (B) Crystallization cycle.
119
110
Enthalpy of
Crystallization
100
Enthalpy of Fusion
Enthalpy (J/g)
90
80
70
R² = 0.974
60
50
R² = 0.9772
40
30
20
0
5
10
15
20
% Ergocalciferol loading (w/w of dispersed phase)
Figure 8.6 Enthalpies of fusion and crystallization of ergocalciferol loaded freeze dried
SLN dispersions.
Enthalpy of fusion of α-subcell form
(J/g)
120
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
% ergocalciferol loading (w/w of dispersed phase)
25
Figure 8.7 Enthalpy of fusion of α-subcell polymorph of ergocalciferol loaded SLN
suspensions.
121
Table 8.1 Mean enthalpy of fusion of the a-subcell polymorph as a function of
ergocalciferol content.
*
Ergocalciferol
H
(% of dispersed phase)
(J/g)
0
1.2093A
0.1240
5
1.5727B
0.0860
10
1.8036BC
0.1189
15
2.0253C
0.1408
20
C
0.1089
1.9325
Standard Deviation
Values with different letters indicate differences significant at =0.05.
122
CHAPTER 9. OVERALL CONCLUSIONS AND RECOMMENDATIONS FOR
FUTURE WORK
9.1
Overall conclusions
In first research project, the effect of batch and continuous microwave systems
(100 kW and 6 kW) on quality of strawberry puree was studied in terms of their effects
on monomeric anthocyanin content, instrumental color, viscosity, water holding capacity
and monosaccharide composition compared to conventional heating methods. For the
pilot scale (100 kW) continuous flow microwave system, monomeric anthocyanin
content and instrumental color values decreased over storage time but there were no
conclusive differences between microwave and other two conventional heating methods,
scraped surface heat exchanger and tubular heat exchanger. The reason for these
inconclusive results was thought to be the statistical weakness due to the lack of
replicates. However, increased viscosity of puree by microwave heating over time as
observed subjectively prompted further investigation of rheological properties in batch
and laboratory scale (6 kW) continuous microwave systems. Batch microwave heating of
strawberry puree in a domestic microwave oven showed higher anthocyanin and color
retention compared to untreated and conventionally heated puree over time. Although
batch microwave heating showed higher viscosity than conventional heating, this trend
123
was not consistent over storage period. Challenges associated with batch microwave
heating such as non-uniform temperature distribution and poor temperature control could
possibly explain inconsistentviscosity results. Laboratory scale (6 kW) continuous flow
microwave heating however, clearly showed higher viscosity, water holding capacity,
anthocyanin content and improved instrumental color values compared to untreated,
preheated controls and conventionally heated puree over 12 weeks storage period.
Carbohydrate composition of sera separated from these strawberry puree samples
however, were not able to explain rheological differences. Although unexplained at
chemical level, these improved rheological properties of strawberry puree along with
enhanced preservation of anthocyanin and instrumental color as delivered by microwave
heating certainly opens doors for its potential commercial applications.
In second research project, triplamitin solid lipid nanoparticles loaded with
ergocalciferol at different concentrations were characterized for their particle size,
morphology and thermal behavior. As the concentration of ergocalciferol in the SLN
increased, the particle size and the turbidity of the SLN dispersions gradually decreased.
There was also a gradual decrease in enthalpies of fusion and crystallization for stable βsubcell in freeze dried SLN particles whereas for SLN dispersions, the enthalpy of fusion
of least stable α-subcell crystal increased with ergocalciferol proportion. The presence of
spherical as well as rod-shaped nanoparticles was seen from transmission electron
microscopy images. These results suggest that as concentration of ergocalciferol in lipid
dispersed phase is increased, thermal stability of the crystal structure is lowered. This
could point towards decreased order in the crystal lattice and hence possibly improved
encapsulation efficiency of ergocalciferol. Improved encapsulation efficiency could help
124
protect the encapsulated compound better from environmental factors such as light and
oxygen. Also, lowering of turbidity of SLN dispersions with increased ergocalciferol
loading can have potential applications in fortification of clear beverages.
9.2 Recommendations for future work
Microwave heating specially on continuous flow system is a novel food
processing technique and there is a substantial scope for this technique to be explored for
its effect on different pumpable food products. Limitations of small (6 kW) continuous
flow microwave discussed in Chapter 6 could be overcome using stronger pump,
automated control over microwave power, desired processing temperature, pressure, flow
rates, backpressure control valve. With such a modified system, wider ranges of
temperatures, pressure and flow rates could be explored to see their effects on various
food products. This will give a wider perspective on microwave processing and its
efficacy in delivering high quality food products. Wider range of knowledge about
microwave processing at different processing conditions and various food products could
help in optimization work as well as developing models. Apart from anthocyanin, color
and rheology, many other nutrients and physicochemical properties could be studied as
affected by microwave radiations. Also, the nature of the mechanism by which the
viscosity of CFMW processed puree was retained over the storage period needs to be
further investigated. This was, perhaps, the most important finding of this project, and
one that could lead to target specific products suitable for microwave heating operations.
Solid lipid nanoparticles (SLN), although used in pharmaceutical industry for two
decades, is finding its place in food applications in recent years. After characterization,
125
ergocalciferol loaded SLN particles could be studied for their protective effect against
environmental factors and for their controlled release potential. These studies could be
conducted in simple SLN dispersions or in complex food matrices for potential
fortification applications. SLNs can also be used to encapsulate other nutrients or
compounds for food applications. One good example of this is SLN loaded with natural
antimicrobials for surface treatment for leafy vegetables and is being currently
investigated in our research group.
126
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APPENDIX
10
1
Viscosity (Pa.s)
1
10
100
1000
Untr. Con. 6 Weeks
Preheat. Con. 6 Weeks
0.1
80⁰C 6 Weeks
91⁰C 6 Weeks
102⁰C 6 Weeks
0.01
0.001
Shear rate (s-1)
151
Figure A1. Representative flow curves for strawberry puree heated by 6 kW CFMW/tubular H.E. system
1
0.01
0.1
1
10
100
1000
MW Pre. Control Day 0
0.1
Viscosity (Pa.s)
MW Pre. Control Week 4
MW 91⁰C Day 0
MW 91⁰C Week 4
0.01
MW 91⁰C Week 8
Tub. Pre. Control Day 0
Tub. Pre. Control Week 4
Tub. 91⁰C Day 0
0.001
Tub. 91⁰C Week 4
Tub. 91⁰C Week 8
0.0001
Shear Rate (s-1)
Figure A2. Flow curves of selected strawberry sera separated from strawberry puree heated by 6 kW continuous flow microwave
152
system and tubular H.E. system at different temperature at different storage times.
153
VITA
153
VITA
Mandar R. Patel
Department of Food Science, Purdue University
Education
B.Tech.,Food Technology and Engineering, 2006, Institute of Chemical Technology
(formerly UDCT), Mumbai, India.
M.S., Food Science, 2008, Mississippi State University, Starkville, Mississippi.
Ph.D., Food Science, 2012, Purdue University, West Lafayette, Indiana
Research Experience
Microwave processing.
Encapsulation.
Food Safety
Cereal products development
Infant formula and beverage for toddlers development
Internships
Kellogg Company, Battle Creek, MI
Mead Johnson Nutrition, Evansville, IN
Coca-Cola Company, Nasik, India
Mafco, Ltd., Pune, India
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