VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY

FACULTY OF CHEMICAL ENGINEERING
DEPARTMENT OF FOOD TECHNOLOGY
OFFICE FOR INTERNATIONAL STUDY PROGRAMS
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UNDERGRADUATE THESIS

INFLUENCE OF PRETREATMENT
CONDITIONS ON β-CAROTENE
ENCAPSULATION USING BY YEAST CELL:
EFFECT OF ACID AND ALKALINE
TREATMENT

ADVISOR: Dr. TA THI MINH NGOC

STUDENTS: NGUYEN THI MINH THU 1752523 CC18HTP1
TRAN HIEN 1752195 CC18HTP1

HO CHI MINH CITY, JUNE 2022

COMMENT OF ADVISOR
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HCMC, ……June, 2022

Advisor’s Signature

Dr. Ta Thi Minh Ngoc

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COMMENT OF REVIEWER
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HCMC, ……June, 2022

Reviewer’s signature

Msc. Le Thi Thuy

iii
ACKNOWLEDGEMENT
First and foremost, we would like to express my sincere gratitude to our
supervisor, Dr. Ta Thi Minh Ngoc for allowing us to work under her supervision,
not only for the valuable guidance but also for continuous support and
encouragement throughout this research work. Besides, Dr. Ngoc provided us with
unlimited support and gave us her precious time and advice enthusiastically
whenever we needed it.
Furthermore, we want to show our appreciation to teachers in HCMUT,
lecturers in the Chemical Engineering faculty, and especially in Food
Technology department. They are the ones who give enthusiasm and dedication to
each lecture including the basis, specialized knowledge, along with the hard and soft
skills for students. Those are the solid foundation for us in the following path.
Special thanks to the researchers and seniors here at the The Food
Microbiology Laboratory and the Food Biochemistry Laboratory who
participated in teaching us some research skills and provided us with continuous
guidance during our work; for their care and guidance during our research work.
Besides, we acknowledge the accompanying of CC18HTP class during our
four years in this school, and the wonderful memories when playing and overcoming
examinations, and assignments together.
We would like to acknowledge our families for their support; without them,
in our life, we couldn’t have achieved this study. We would like to acknowledge our
parents for their continued support and for always being beside us whenever we
face difficulties through this study. They have been a great role model for us and
will always be our heroes.
This thesis was the result of long, hard-working research by three of us.
Although we had tried our best, mistakes were inevitable. We would love to hear the
comments and recommendations from teachers to learn from experience and to
improve in our next research and projects.

iv
ABSTRACT
Microencapsulation techniques are drawing attention in the food industry for their
potential of utilizing natural bioactive compounds. Novel shell materials – yeast cells
have been adopted to overcome some drawbacks of existing ones. However, the
diffusion of active ingredients mechanism is unclear at some points, and the pretreatment
of biomass for improving the encapsulation efficiency is still urgent. In this study, the b-
carotene microencapsulation by Saccharomyces cerevisiae yeast cells was conducted.
The pretreatment conditions such as time intervals, NaOH, and HCl concentrations were
investigated and assessed through the encapsulated b-carotene content and uptake lipid
content. The hydrophobicity and electron donor/ acceptor character on the cell surface
was also examined to study the diffusion system. Our findings showed that the highest
b-carotene of 14.06 ± 1.54 ug/g dry basis was achieved when treated cells solution at
50oC for 3 h. Oil droplets acted as a carrier of the pigment to penetrate across the cell
envelope. The hydrophobicity and electron donor/ acceptor properties proposed as not
correlated with the effectiveness. Nonetheless, the results were lack of
comprehensiveness, thus the analysis of cell wall porosity, zeta potential, and van der
Waals interactions should be carried out in the future.

v
TABLE OF CONTENTS
1. INTRODUCTION ................................................................................. 2
1.1. S. CEREVISIAE YEAST CELL ........................................................... 2
1.1.1.Origin ....................................................................................................... 2
1.1.2.Morphology and physiological properties ............................................... 2
1.1.3.Cell structure ............................................................................................ 2
1.2. MICROENCAPSULATION .................................................................. 7
1.2.1.Definition, Application ............................................................................ 7
1.2.2.Conventional shell materials .................................................................... 8
1.2.3.Yeast cells based microencapsulation ...................................................... 9
1.2.4.Diffusion mechanism ............................................................................... 9
1.2.5.Advantages of yeast cell-based microencapsulation .............................. 13
1.3. B-CAROTENE ...................................................................................... 14
1.3.1.Structure and chemical properties. ......................................................... 14
1.3.2.Biological values .................................................................................... 15
1.4. RESEARCH CONTEXT WORLDWIDE .......................................... 17
1.4.1.Studies of yeast cell-based microcapsules ............................................. 17
1.5. URGENCY OF THE SUBJECT ......................................................... 21
2. MATERIALS AND METHODS........................................................ 22
2.1. MATERIALS ......................................................................................... 22
2.2. CHEMICALS ........................................................................................ 22
2.3. EQUIPMENT & TOOLS ..................................................................... 23
2.4. EXPERIMENTS ................................................................................... 24
2.4.1.Experiment 1: Determination of material composition ......................... 25
2.4.2.Experiment 2: Determination of the pretreatment conditions ................ 25
2.5. ANALYTICAL METHOD ................................................................... 26
2.5.1.Moisture and lipid content analysis ....................................................... 26
2.5.2.Analysis of β-carotene content in biomass ............................................ 26

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2.5.3.Hydrophobicity and electron acceptor/ donor properties analysis ......... 27
2.6. STATISTICAL ANALYSIS ................................................................. 28
3. RESULTS AND DISCUSSION ......................................................... 29
3.1. DETERMINATION OF MATERIAL COMPOSITION .................. 29
3.1.1.The moisture of raw yeast ...................................................................... 29
3.1.2.Lipid content in raw yeast ...................................................................... 29
3.2. DETERMINATION OF PRETREATMENT CONDITIONS.......... 30
3.2.1.Determination of the pretreatment time ................................................. 30
3.2.2.Determination of the pretreatment NaOH concentration ....................... 31
3.2.3.Determination of the pretreatment HCl concentration ........................... 33
3.3. EVALUATION OF THE UPTAKE LIPID OF TREATED BIOMASS
34
3.4. EVALUATION OF THE HYDROPHOBICITY OF CELL
SURFACE AFTER PRETREATMENT .................................................................... 36
3.5. EVALUATION OF THE ELECTRON DONOR/ ACCEPTOR
PROPERTIES OF CELL SURFACE AFTER PRETREATMENT ....................... 38
4. CONCLUSIONS AND PERSPECTIVES ........................................ 40
4.1. CONCLUSIONS .................................................................................... 40
4.2. PERSPECTIVES ................................................................................... 41
5. BIBLIOGRAPHY ............................................................................... 42
6. APPENDIX .......................................................................................... 49
APPENDIX A. ANALYTICAL METHODS .................................................. 49
A1. The experimental procedure ..................................................................... 49
A.2. Lipid content analysis .............................................................................. 50
A.3. β-carotene content analysis ..................................................................... 50
A.4. Hydrophobicity and electron acceptor/donor property analysis ............. 51
APPENDIX B. EXPERIMENTAL RESULTS .............................................. 53
B1. Experimental data to determine the material composition ....................... 53
B2. Experimental data to determine the pretreatment time ............................. 53
B3. Experimental data to determine the pretreatment NaOH concentration .. 55

vii
B4. Experimental data to determine the pretreatment HCl concentration ...... 56
B5. Experimental data to evaluate the hydrophobicity of cell surface ........... 57
B6. Experimental data to evaluate the electron donor and acceptor properties
of cell surface.................................................................................................. 59
APPENDIX C. EXPERIMENTAL EQUIPMENT ........................................ 62

viii
LIST OF TABLES
Table 1. Composition of yeast cell wall S. cerevisiae ........................................................ 4
Table 2. Chemical structure and physical properties of β-carotene ................................. 15
Table 3. Studies of encapsulating the active ingredient in yeast cells S. cerevisiae ........ 18
Table 4. Chemicals used in the research ........................................................................... 22
Table 5. Equipment used in research ................................................................................ 23
Table B1. Chemical composition of yeast cell material ................................................... 53
Table B2. Lipid content of cells after pretreatment .......................................................... 54
Table B3. Lipid content of cells after encapsulation ........................................................ 54
Table B4. β-carotene content of cells after encapsulation ................................................ 54
Table B5. Lipid content of cells after pretreatment with NaOH ...................................... 55
Table B6. Lipid content of NaOH-treated cells after encapsulation ................................ 55
Table B7. β-carotene content of NaOH-treated cells after encapsulation ........................ 55
Table B8. Lipid content of cells after pretreatment with HCl .......................................... 56
Table B9. Lipid content of HCl-treated cells after encapsulation .................................... 56
Table B10. β-carotene content of NaOH-treated cells after encapsulation ...................... 56
Table B11. Hydrophobicity of cell surface in pretreatment time experiment .................. 57
Table B12. Hydrophobicity of cell surface in NaOH experiment .................................... 58
Table B13. Hydrophobicity of cell surface in HCl experiment ........................................ 58
Table B14. Electron donor property of cell surface in pretreatment time experiment ..... 59
Table B15. Electron acceptor property of cell surface in pretreatment time experiment . 59
Table B16. Electron donor property of cell surface in pretreatment NaOH experiment.. 60
Table B17. Electron acceptor property of cell surface in pretreatment NaOH experiment
........................................................................................................................................... 60
Table B18. Electron donor property of cell surface in pretreatment HCl experiment ..... 61
Table B19. Electron donor property of cell surface in pretreatment HCl experiment ..... 61
Table C1. Equipment used in this study ........................................................................... 62

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TABLE OF FIGURES
Figure 1. Structure of the yeast cell.......................................................................... 3
Figure 2. The Fluid Mosaic Model of yeast plasma membrane ............................... 5
Figure 3. Structure and working principle of microencapsulation ........................... 8
Figure 4. The process of diffusional encapsulation of hydrophobes into an intact
yeast cell .................................................................................................................. 10
Figure 5. The scheme shows how the pathway of hydrophobic components crosses
the cell envelope thanks to hydrophobic compounds.............................................. 12
Figure 6. Active dry-yeast material ........................................................................ 22
Figure 7. The research process ............................................................................... 24
Figure 8. The encapsulated β-carotene content at different pretreatment times at
50oC ......................................................................................................................... 30
Figure 9. The β-carotene content of NaOH-treated biomass ................................. 31
Figure 10. Putative molecular organization of the cell wall of S. cerevisiae ......... 32
Figure 11. The β-carotene content of HCl-treated biomass ................................... 33
Figure 12. The uptake lipid content and β-carotene content of different treatment
................................................................................................................................. 35
Figure 13. Hydrophobicity of the cell surface after pretreatment .......................... 36
Figure 14. The electron donor/ acceptor properties of the cell surface .................. 38

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INTRODUCTION

1. INTRODUCTION
1.1. S. CEREVISIAE YEAST CELL
1.1.1. Origin

Saccharomyces cerevisiae yeast is a type of multinuclear monocellular

microorganism of the genus Saccharomyces, class Ascomycetes, fungi kingdom S.
cerevisiae has at least 80 species of the same genus, 600 species of the same family, and
more than 10,000 different strains. This species can be considered the most useful
fungus in human life from thousands of years ago to the present.
S. cerevisiae has an extensive history of uses in the area of food processing. It is
commonly known as baker’s yeast or brewer’s yeast. It has been used for centuries as a
leavening for bread and as a fermenter of alcoholic beverages and wine production (Oca
et al., 2016). They are very common in nature, wild yeast strains have been found on the
surface of plants (leaves, flowers, fruits) or secretions of plants, body surfaces and
intestinal tracts of insects or crustaceans, soils in temperate, tropical or polar regions,
freshwater or saltwater and many other environments (Martini, 2007).

1.1.2. Morphology and physiological properties

S. cerevisiae yeast cells are opaque white or light yellow, spherical, oval-shaped,
elliptical, or egg-shaped. The individual cell size of S. cerevisiae has a large diameter of
5–10 μm or a small diameter of 1–7 μm (Oca et al., 2016). Their shape and size can vary
during the stages of development, and depending on the surrounding conditions.
S. cerevisiae is important in many industries due to its ability to convert sugars
(i.e., glucose, maltose) into ethanol and carbon dioxide (baking, brewing, distillery,
and liquid fuel industries). It breaks down glucose through aerobic respiration in
presence of oxygen. If oxygen is absent, the yeast will go through anaerobic
fermentation. The net result of this process is two adenosine triphosphate (ATP)
molecules, in addition to two by-products; carbon dioxide and ethanol. 98% of glucose
is metabolized during fermentation, while 2% of it is made into cell materials (Oca et
al., 2016). It is not limited to a particular habitat, it can grow in environments where
carbon and nitrogen levels vary. Optimal yeast growth occurs under aerobic conditions,
with an adequate nutrient supply, at temperatures of 28-30°C (O’Kennedy & Reid,
2008).

1.1.3. Cell structure

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INTRODUCTION

S. cerevisiae cells are eukaryotic cells with all of the organelles found in animal
cells, including a nucleus, endogenous mesh, mitochondria, Golgi apparatus, non-cells,
cell skeletons, and many others.
In the G1 phase, the nucleus took up 10.5 percent of the cell weight, the cell wall
took up 17 percent, the vacuole took up 5.8 percent, the cytoplasm took up 64 percent,
and mitochondria took up only 1.7 percent (Yamaguchi et al., 2011). Depending on the
kind and development conditions, yeast chemical qualities include roughly 30–33
percent dry materials, 6.5–9.3 percent nitrogen, 40.6–58.0 percent proteins, 35.0–45.0
percent carbs, 4.0–6.0 percent lipids, 5.0–7.5 percent minerals, and varied levels of
vitamins (Bekatorou et al., 2006).
The two key components that indicate yeast cells' good defense are cell walls
and biofilms.

(Coradello & Tirelli, 2021)

Figure 1. Structure of the yeast cell

1.1.3.1. Cell Wall

The yeast cell is surrounded by a cell wall. On the cell wall, there are many holes,
through which nutrients are absorbed and products of metabolism are released. The cell
wall forms the morphology and ensures the integrity of the yeast during cell growth and
division. It gives the cell mechanical strength to be able to withstand changes in osmotic
pressure in the culture environment.
In yeast, the wall accounts for approximately 15–20% of the cell dry mass. Its
thickness is very variable (70–200 nm) since it increases in response to compression or
osmotic forces; structurally, however, it is a highly polar double-layered matrix (a kind
of hydrogel). Its inner part is mainly composed of branched β-1,3 and β-1,6 glucans
(about 50% of the overall wall) hydrogen-bonded to 3–4% of mostly crystalline chitin

3
INTRODUCTION

(Coradello & Tirelli, 2021). β-1,6-glucan has a high degree of cleft and is water-soluble.
They connect the mannoproteins to the insoluble β-1,3-glucan network.
The outer layer consists of heavily glycosylated mannoproteins emanating from
the cell surface (Klis et al., 2002). Mannans and polypeptide chains showed hydrophilic
and hydrophobic properties, respectively (De Iseppi et al., 2019). The carbohydrate side
chains of the cell surface proteins contain multiple phosphodiester bridges, resulting in
numerous negative charges at the cell surface at physiological pH values (Coradello &
Tirelli, 2021). These side chains are responsible for the hydrophilic properties of the
wall and may be involved in water retention and drought protection. The outer protein
layer accounts for about one-third of the wall's dry weight (Klis et al., 2002).
(Ciamponi, 2011)

Table 1. Composition of yeast cell wall S. cerevisiae

Macromolecule % of cell wall mass

Mannoprotein 30-50

β-(1,6)-glucan 5-10

β-(1,3)-glucan 30-45

Chitin 1,5-6

Cell walls are highly elastic. When yeast cells are transferred to a hypertonic
solution, they quickly shrink, and depending on the osmotic pressure, they can lose more
than 60% of the original volume. This process is reversible, when transferred back to
the original environment, the cells immediately expand to a normal volume. The
flexibility of the cell wall is due to the elasticity of the β1,3-glucan chains (Klis et al.,
2002). In addition, β1,3-glucan along with chitin is also responsible for the mechanical
strength of cells. The lateral protein layer determines the surface characteristics of the
cell such as immunity, charge, hydrophobicity, porosity, and adhesion (Ciamponi,
2011). Furthermore, through their covalent linkage to the β-glucan layer, mannoproteins
contribute to the wall outer porosity (Coradello & Tirelli, 2021). Also, Dadkhodazade
(2018) (Dadkhodazade et al., 2018) concluded that β-glucans and chitin stand for cell
rigidity, and mannoproteins provide cell porosity. Besides, cell permeability can be
increased by implying chemical treatments. Any modifications that affect
mannoproteins and disrupt disulfide bonds and hydrophobic linkages can change
porosity (Dadkhodazade et al., 2018).

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INTRODUCTION

1.1.3.2. Plasma membrane

There are several hypotheses of the plasma membrane model, with which the Fluid
Mosaic model (Figure 2) introduced by S.J. Singer and Garth Nicolson in 1972 being the
most accepted. The cell membrane's structure is quite complicated, with a thickness of
around 5 nm and a majority of lipids and proteins in roughly equal proportions (Stewart,
2017). It is consisted of an equal amount of lipids (primarily glycerophospholipids and
fatty acids, with a smaller amount of sterols such as ergosterol and sphingolipids) and
proteins, and is linked to the cell wall by glycoproteins and glycolipids. It is separated from
the cell wall by the periplasm, which is a non-continuous, enzyme-rich region. The
fundamental function of the membrane is to regulate transit from the cell. (Coradello &
Tirelli, 2021). Therefore, it is the main barrier to permeability for permeable molecules
(Dadkhodazade et al., 2021; De Nobel et al., 1990).

Figure 2. The Fluid Mosaic Model of yeast plasma membrane

Lipid portion:
There are three types of lipids in the yeast cell membrane S. cerevisiae, including
phospholipid, fatty acids, and sterols. Phospholipids are the main component of cell
membrane lipids. They are bipolar, consisting of a polarized head containing choline,
phosphate, and glycerol and a non-polarized tail that contains hydrocarbons derived from
fatty acids. Membrane lipids are arranged into two symmetric parts close together, forming
a double lipid layer and acting as a permeability barrier for most water-soluble molecules.
The hydrophilic head of lipid molecules rotates out of the cell or into the cytoplasm, and
the hydrophobic tails direct in middle. The water heads inside and outside the cell prevent
membrane lipids from escaping from the double layer, but nothing prevents these
molecules from moving and changing positions for each other in the plane of the
membrane. Therefore, cell membranes are highly flexible (Al, 2014).

5
INTRODUCTION

Ergosterol is the major sterol found in yeast. The sterols are placed between
two phospholipid molecules, with the sterols' -OH group facing the phospholipid
circuits' -COOH group. The interaction of sterols and unsaturated acyl circuits raises
the hydrophobicity of the dual lipid layer core and lowers the cell membrane's semi-
permeability(Subczynski & Wisniewska, 2000).
Protein portion:
Based on how it binds to lipid membranes, membrane proteins are divided into
two types: integral proteins and peripheral proteins. Integral proteins make up 70%
of membrane proteins and reside within the bilayer membranes. These proteins float
in the double lipid layer thanks to the hydrophobic bonds. They produce cells that
pass through the cell membrane, whereby molecules and information can enter and
exit the cell. Integral proteins are fibrous in shape and can penetrate the membrane
once or several times, they do not fix in one place but move back and forth.
Peripheral proteins account for around 30% of the membrane protein weight
that appears on the cell membrane's surface or face. Electrostatic or hydrophobic
connections attach peripheral proteins to phospholipids. When exposed to acidic or
alkaline chemicals, they catalyze the hydrolysis of peptide bonds in protein
molecules, damaging the membrane's structure and modifying the cell membrane's
characteristics.
Hydrophobicity of the cell surface:
The hydrophobicity of a cell's surface is a critical property for pathogenic
microorganisms since it impacts the cell's capacity to adhere to its hosts. Cells with a
high hydrophobic surface are also better at fighting macrophages than cells with a
hydrophilic surface (Masuoka & Hazen, 1997).
Membrane lipids determine hydrophobicity, with glycolipids being the most
important as they are located primarily outside the membrane in a single plate.
Glycolipids comprise both hydrophobic and hydrophilic units, such as fatty acid
chains and sphingosine hydrocarbon chains, as well as one or more sugar roots
(Fukui, 1975). When glycolipids on the cell membrane's surface include fatty acid
chains and sphingosine's hydrocarbon chains are predominant, the cell is more
hydrophobic. Cells that show hydrophobicity, on the other hand, are mostly owing to
glycolipids on their surface, which contain sugar radicals.
The acidity of the cell surface:
The electron donating and receiving properties of the cell demonstrate its
acid/base properties. The carboxyl root (COO–) of the integral protein and the
carboxyl root (COO–) of the peripheral protein are both important. Membrane
proteins, on the other hand, bind intimately with membrane lipid chains, and can only

6
INTRODUCTION

be separated via non-polarizing interactions. When peripheral proteins connect to the

membrane mostly
by electrostatic and hydrogen interactions, the majority of these weak bonds are broken
down when washed with pH = 5-6 phosphate cushions. As a result, the surface's
acidity/base is mostly determined by the integral protein's characteristics. The
hydrophilic ends of membrane protein molecules protruding out towards the
membrane's outer and inner surfaces may end up as amino groups (NH4+).
The acid/base characteristics of the cell are also demonstrated by the cell's
adherence to the solvents. The ability to adhere to ethyl acetate (base solvents) reveals
the cell surface's high acidity. Adhesion to chloroform (acidic solvents) on the other
hand, reveals the cell surface's foundation (Muñoz-Provencio et al., 2009).

1.2. MICROENCAPSULATION
1.2.1. Definition, Application

Microencapsulation is the technique of encapsulating a small component called

the core material into a micro-sized wall called the coating or carrier material.
Fragrances, essential oils, lipids, vitamins, colorants, enzymes, probiotics, taste
compounds, and other bioactive substances can all be used as core ingredients.
Microencapsulation in the food industry has several advantages, including lowering core
reactivity to environmental factors, lowering core material transfer rate to the outside
environment, promoting easier handling, controlling core material release, masking core
taste, and finally diluting core material when it is required to be used in very small
amounts (Shahidi & Han, 1993). This technology can assist food manufacturers protect,
isolate, and limit the release of the core substance in the short term.
Microcapsules are currently used in beverages, baked goods, meat, poultry, and
dairy products. The encapsulation of flavors and the conversion of liquid into a powder
allows for many different uses of ingredients, which is one of the greatest potentials of
technology in the food sector (Poshadri, 2010). They can also be employed to administer

7
INTRODUCTION

medications in the pharmaceutical sector or to enhance the shelf life of perfumes in the
perfume industry.

(Peng et al., 2018)

Figure 3. Structure and working principle of microencapsulation

1.2.2. Conventional shell materials

In general, the conventional encapsulation of bioactive agents comprises three

steps:
 formation of a coating wall around the core material,
 ensuring that no undesirable leakage occurs, and
 ensuring the unwanted is kept out.
Spray drying, spray chilling, extrusion coating, extrusion, fluidized–bed coating,
liposome entrapment, coacervation, inclusion complexation, and rotational or
centrifugal suspension separation are some of the methods used. Sugars, cyclodextrins,
maltodextrins, modified starches, gums, proteins and nanoparticles, micelles, and
liposomes have all been utilized as carriers since the stability and release of the core
substance is greatly influenced by the walls' composition. (Gibbs et al., 1999). These
methods and coating materials exhibit a high encapsulation efficiency (EE – the ratio of
the encapsulated amount to the initial input of active compound) and high encapsulation
yield (EY – the ratio of the mass of internal material to the mass of whole microcapsules)
(Pham-Hoang et al., 2013). Nonetheless, they have some disadvantages, such as the
difficulty of creating wall material, chemical residues from harmful solvents employed,
lower sensory value due to poor microparticle uniformity, and the difficulty of matching
internal and external materials. Furthermore, there is no general encapsulation
mechanism. Each approach has its own set of benefits and drawbacks, as well as
regulatory restraints, such as the use of cyclodextrin in some countries. As a result, there
is still a demand for specialized capsules, and many researchers are interested in
addressing the issues or investigating a unique natural carrier material.

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INTRODUCTION

1.2.3. Yeast cells based microencapsulation

Saccharomyces cerevisiae yeast cells demonstrate an exceptional candidate for

the microcapsules’ coating because of their ovoid shape and suitable size which is
approximately 5 micrometers in diameter. the major advantage of a yeast cell is its
convoluted cell envelope: cell wall and plasma membrane. The physically rigid cell wall
is a thick external layer about 100 – 200 nm thick, mainly constituted of polysaccharides
(glucans and mannans) network, a minor percentage of chitin, and a small amount of
entrapped mannoproteins. This structure act as the protector of core substances against
chemical and physical conditions. The thinner plasma membrane (<10 micrometers) is
a typical bilayer unit comprising phospholipids, sterols, and neutral lipids
(triacylglycerol and sterol esters) (Nelson et al., 2006). It affects the selectivity of
compounds in and out of the cells as a semi-permeable membrane.

1.2.4. Diffusion mechanism

Hydrophilic and hydrophobic substances can be encapsulated by yeast cells.

Microencapsulation is accomplished by combining yeast suspension with a solution of
the material to be encapsulated in a water environment (or a mixture of water, solvent,
and emulsifier), shaking for a period of time at a specific temperature. In order to form
oil droplets of the right size for optimal diffusion, the shaking speed must be adjusted.
(Paramera et al., 2014).
Yeast cells die over time as a result of the process. Because the permeability of
hydrophobic substances is primarily based on passive transport, the death rate of cells
has no effect on encapsulation efficiency (Bishop et al., 1998). The release of peptides,
amino acids, nucleotides, and cytoplasmic organelles occurs as bioactive substances
diffuse. (Morris et al., 1986). The release of these molecules frees up space in the
cytoplasm and increases the amount of encapsulated substances in cells. Inactivation of
yeast cells also lowers the core material's reactivity with internal hydrophilic substances.
Chemical, physical, and physiochemical characteristics all influence
encapsulation efficiency. It is also influenced by the cell wall's characteristics and the
makeup of the plasma membrane. Overall, the diffusion of the core material into by
yeast cells has four steps:
 the adhesion to the cell wall surface,
 the penetration across the cell wall,
 the penetration across the plasma membrane, and
 the retention of core materials inside the cytoplasm.

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INTRODUCTION

(Coradello & Tirelli, 2021).

Figure 4. The process of diffusional encapsulation of hydrophobes into an intact yeast cell

1.2.4.1. The adhesion to the cell wall surface

The cell wall is a hydrophilic porous structure composed of

polysaccharide chains (glucans and mannans), chitin, and mannoproteins. When
hydrated, there are pores on the surface that are filled with water. As a result, it is
hydrophilic and readily attaches to hydrophilic substances, making the
encapsulation process easier. At the same time, it performs a critical role in
preventing hydrophobic substances from entering cells. It is thought that increasing
the hydrophobicity of the cell surface by the use of mannoproteins can improve
adhesion capacity and hence favor encapsulation (De Groot et al., 2008).
Mannoprotein digestion may reduce the hydrophobicity of cell walls, resulting in a
lower encapsulation yield (Pham-Hoang et al., 2013). Nevertheless, the cell wall
composition can vary depending on the culture conditions. Furthermore, some
pretreatments, such as heating and/or chemical treatment with emulsifiers, sugars,
salts, and organic solvents, can be employed to make changes (Paramera et al.,
2014).
Electrostatic characteristics, Lewis acid-base interaction, and cell surface
protrusions can all generate hydrophobe adhesion rather than hydrophobicity
(Pham-Hoang et al., 2013).

1.2.4.2. The penetration across the cell wall and cell membrane

Following attachment, cell wall penetration by passive diffusion is the next

stage. Porosity is a key characteristic that influences the rate at which bioactive
chemicals diffuse into and out of cells. Particles with a molecular weight threshold of

10
INTRODUCTION

620–760 Da and an average radius of 0.81-0.89 nm can freely diffuse through the cell
wall (Scherrer et al., 1974). However, if the porosity of the wall is increased, molecules
with a mass of up to 400 kDa can seep through (De Nobel et al., 1991). The porosity
of S. cerevisiae yeast cell walls varies with their growth stages, decreasing as the cell
moves from the log phase to the stationary phase (De Nobel et al., 1991).
Mannoproteins are partially removed to increase the porosity of the cell wall by
treatments. The digestion of these proteins was conducted with enzyme pronase (a
mixture of endo- and exo-proteinases) by Kim et al. 2000 or protease by Salazar et al.,
2007. Reducing agent β-mercaptoethanol was used by (Nelson et al., 2006) to disrupt
disulfide bonds and thioester linkages between mannoproteins. Shi et al. (2010) studied
the use of sodium chloride (NaCl), cetyltrimethylammonium bromide (CTAB), Triton
X-100, sodium dodecyl sulfate (SDS), and ethanol to dissolve mannoprotein thereby
disrupting the structure of the cell wall, facilitating chlorogenic acid (CGA)
(hydrophilic) osmosis into yeast cells. CGA molecules with polarizing functional
groups were able to bind to the polarized end of membrane phospholipids and then
pass through them. Hydrophobic interactions, van der Waals suction, and hydrogen
bonds between the CGA molecule and yeast microcapsule components then work
together to keep them in place (Shi et al., 2010).
Because the surface of yeast S. cerevisiae with low lipid content is low
hydrophobic and contains only a small amount of mannoproteins, hydrophobe
penetration must be dependent on the porosity of the cell wall. Yarrowia lipolytica is
an oleaginous yeast cell with a lipid content of >40% that is grown in a lipid-rich
environment. The cell surface is very hydrophobic, the encapsulated material forms an
affinity with hydrophobic molecules on the cell walls, and diffusion is dependent on
that attraction.

(Pham-Hoang et al., 2013)

11
INTRODUCTION

Figure 5. The scheme shows how the pathway of hydrophobic components crosses the
cell envelope thanks to hydrophobic compounds.
Some evidence proposed that the porosity of the cell wall has little effect on
determining the permeation rate, but mainly on the solubility of hydrophobes in the wall
which is reversely correlated to the hydrophobe partition coefficient (logP) (Ciamponi
et al., 2012). The permeation coefficient (P) has been usually described according to
Overton’s rule which takes into account the partition coefficient (K) between the oily
and aqueous phases: P=KD/I where D is the diffusion coefficient of the molecule in the
membrane, and I, the membrane thickness. In which, K changes more significantly than
D, and it is therefore considered the parameter controlling permeation (Grime et al.,
2008). However, according to (Pradelles et al., 2008), the process is also based on the
positive influence of hydrophobicity and the negative effect of electron donor character
with an insignificant effect of zeta potential.

1.2.4.3. The penetration across the plasma membrane

After passing through the cell wall, active core material comes into contact with
the selective plasma membrane. Small molecules spread considerably faster than large
molecules. (Pham-Hoang et al., 2013).
Furthermore, the higher the lipid solubility, the easier it is for a molecule to
move through a membrane. The hydrophobic chemical can move from a high-
concentration location to a low-concentration area by dissolving in the lipid membrane
and diffusing through it via a passive transportation process (Nelson et al., 2006). The
presence of hydroxyl groups or other polarizing functional groups in hydrophilic
substances creates affinity with the polarized groups of phospholipid membranes,
allowing passage through their membranes (Bishop et al., 1998). As a result, molecule
polarization is a key component that can alter how molecules migrate through the cell
membrane; the more polarized, the better the diffusion.
The solubility of chemicals in the membrane can increase the fluidity of the
phospholipid layer, facilitating diffusion. In the context of (Ngoc Ta et al., 2010), it is
possible to trigger the transfer across the membrane by fluidization. The fluidizing
effects could be enhanced through diverse methods such as heat treatment, plasmolysis,
and alkaline or acidic substances addition.
According to Paramera et al. (2011), the encapsulation yield does not rise
linearly with temperature. There were no significant differences in encapsulation
effectiveness between 25oC and 35oC (which is below the membrane transition phase),
a dramatic increase between 35oC and 45oC (which includes the transition temperature),
and a negligible difference between 45oC and 55oC. When the phase transition
temperature is reached, the membrane transforms from a gel to a liquid-crystal phase,

12
INTRODUCTION

in which the electrons move more freely and the core materials are penetrated more
easily (Paramera et al., 2011).

1.2.4.4. The retention of core materials inside the cytoplasm

The release rate of hydrophobes from the membrane is slower than the pace at
which they penetrate the membrane as well as the rate at which they are displaced inside
the membrane due to their high affinity for the phospholipid bilayer. The transfer of
fatty acids from the inner surface of the membrane into the cytoplasm has been aided by
a set of intracellular proteins capable of interacting with fatty acids (FABP). The length
of the fatty acid circuit determines the kinetics of the release (Nhu N. T., 2018).
The hydrophobic contact, van der Waals force, and hydrogen bonds between
encapsulated chemicals and yeast cell components work together to keep hydrophobes
inside the cell (Paramera et al., 2011).
Lipid bodies are lipid droplets found within cells. They exist as a hydrophobic
component surrounded by a monolayer of membrane. The cellular fat storage is
represented by these little round structures, which reflect a physical limit on the storage
capacity of yeast cells for hydrophobic substances. Furthermore, the presence
of lipid bodies might increase the hydrophobicity of the plasma membrane,
allowing hydrophobic substances to be transferred more easily.
They were first investigated as a reservoir for hydrophobic chemical
encapsulation. Shank patented a method for encapsulating lipophilic compounds
localized in lipid droplets within the cell in 1977 for use in carbonless copy paper.
Following that, many strategies aimed to increase the size and number of lipid droplets.
Compared to the glucose–grown yeast cells, oleate–grown cells possess a higher
amount and bigger size of lipid bodies (Ta et al., 2012). For that reason, the oleaginous
yeast cells with lipid content >40 % typically Yarrowia lipolytica, were often used to
promote efficiency (Dang et al., 2017; Pham-Hoang et al., 2018). Cells can produce fat
globules up to 40-60 % of their dry weight when their cultivation is influenced by limited
nutrient availability.

1.2.5. Advantages of yeast cell-based microencapsulation

There are several benefits of employing yeast cells as core materials since they have:
 relatively homogenous surface,
 simple production with affordable cost,
 biodegradable ability, and
 small size to diffuse into food and beverage.

13
INTRODUCTION

Microencapsulation using yeast cells, in comparison to other approaches, is made

up of easily accessible processes and components. Yeast biomass, water, and core
components are all that is required for the simplest procedure. Bishop et al. (1998)
demonstrated the advantages of yeast cells over liposomes for encapsulating active
chemicals, particularly odor and taste components. The food sector faces an economic
challenge due to the relatively high cost of phospholipids and the complicated method
of manufacturing liposomes. Furthermore, liposomes lack a solid structure that protects
bioactive compounds from thermal degradation. To get around the obstacle, yeast cells
can be used for encapsulation. The double protection has been achieved by thick cell
walls with polysaccharide chains preventing cell breakage, ensuring mechanical
endurance and phospholipid bilayer stabilizing bioactive agents, reducing evaporation
loss, due to light, oxygen, or high temperature (Bishop et al., 1998).
Although this attribute depends on the encapsulated substance and the technique,
yeast cells can achieve a pretty high encapsulation yield. Furthermore, yeast cells can
match the standards of natural origin while also ensuring user safety (Nguyen et al.,
2018). The use of yeast in food items is not subject to any special rules. Furthermore,
this sort of biological material has a number of advantages, including a light color and
low taste intensity, as well as the ability to increase the sensory qualities of products in
some circumstances. As a result, it's ideal for encapsulating perfumes and coloring
agents.

1.3. B-CAROTENE
Carotenoids are a group of natural pigments corresponding to colors from yellow
to red, found in fruits, vegetables and some fish and seafood. Carotenoids give plants
such as carrots, sweet potatoes, and apricots their reddish-violet colors.
Beta-carotene is a type of substance called a carotenoid. This is a group of fat-
soluble pigments, insoluble in water, that can protect cells against photosensitivity and
act as light-absorbing pigments during photosynthesis. Beta-carotene is a provitamin A.
Provitamin A is only found in plants. Vitamin A is also found in foods from animals.
Vitamin A from animal sources is called preformed vitamin A that your body can use
directly. It's found in dairy products, fish oils, eggs, and meat (especially liver).
Vitamin A is available in multivitamins and as a stand-alone supplement. Vitamin
A supplements can contain only beta-carotene, only preformed vitamin A, or a
combination of both types of vitamin A.

1.3.1. Structure and chemical properties.

Beta-carotene, having the chemical formula C40H56, is a member of the isoprene-

carotene group of carotenoids. It is a tetraterpenoid with 40 carbon atoms in the core

14
INTRODUCTION

structure and two -ionone rings in place of conjugated double bonds. -Carotene has a
large absorption peak in the visible spectrum, with a maximum at 450 nm, due to its
extended system of 9 completely conjugated double bonds, giving it an orange to red
color. It is the most common type of carotenoid and a precursor to vitamin A. Two
retinyl groups make up beta-carotene. It's an antioxidant found in leafy green, yellow,
and orange vegetables and fruits.
Table 2. Chemical structure and physical properties of β-carotene

Structure

Structural formula Molecular Mass Melting point

C40H56 536,9 Da 178-179°C

Beta-carotene, being a carotenoid compound, possesses all of the features that

define this class. It has the capacity to absorb light and is soluble in most organic
solvents. In petroleum ether, the absorption maxima of beta-carotene correspond to three
wavelengths: 425, 451, and 478 nm (Rodriguez-Amaya, 2001). Beta-carotene is
moderately heat stable, although it is extremely susceptible to oxidation when exposed
to light, oxygen, or temperature. As a result, it should be kept in an airtight container at
a low temperature and away from light for as long as possible.

1.3.2. Biological values

The provitamin A activity of beta-carotene is due to the presence of two beta-

ionone rings in the molecule. It is metabolized in the body to generate two vitamin A
molecules, either through central cleavage or by breaking the molecule from one side.
Under the action of the enzyme beta-carotene dioxygenase, beta-carotene is converted
to two molecules of retinal, which are subsequently further reduced to retinol in the
intestinal mucosa (vitamin A). The retinol produced is retained as a retinyl ester in the
liver. An overabundance of vitamin A can be hazardous to the body due to this
accumulating process. Not all beta-carotene in meals, however, is converted to vitamin
A. The intestines absorb only about one-third of the beta-carotene in a meal, and roughly
half of it is converted to retinol. The rest is stored as fat in the body (Palozza et al.,
1992).

15
INTRODUCTION

Beta-carotene appears to play an essential role in the prevention of cancer,

particularly breast cancer, according to numerous research concentrating on its
properties. This cyclic compound has 11 conjugated double bonds that can combine with
single-molecule oxygen or free radicals to protect cells from oxidative stress. Beta-
carotene is a potent antioxidant capable of eliminating free radicals produced in the
body, which cause severe cell membrane damage, organ damage, and cancer.
1.3.3 β-carotene degradation
Oxidation of β -carotene can result in a loss of quality (color, rancidity, etc.) as
well as bioactivity. These negative consequences of -carotene oxidation underscore the
importance of a thorough understanding of carotenoid oxidation mechanisms in order
to create solutions that improve the stability of functional food.

1.3.2.1. Autoxidation

Carotenoids have been observed to react with ambient oxygen with relative ease,
notably in systems including pure carotenoids in organic solvents. The induction period
for autoxidation of beta carotene in benzene or tetrachloromethane in the dark at 30°C
under one atmosphere of oxygen or by bubbling oxygen through the solvent was less
than one hour, followed by fast synthesis of oxidation products. Beta carotene was
entirely consumed in 30 hours under these conditions. The autoxidation process
produces epoxides, carbonyl compounds, and unchar-acterized oligomers first, followed
by further oxidation reactions that create secondary short chain carbonyl compounds,
carbon dioxide, and carboxylic acids (Mordi et al.,1993).

1.3.2.2. Thermal Degradation

Thermal treatment of carotenoids in the presence of oxygen results in the

formation of volatile compounds and larger non-volatile components (Bonnie & Choo,
1999). β-carotene reacts with oxygen to form 2,6,6-trimethyl-2-cyclohexene-1-one
through a complex process (Crouzet & Kanasawud, 1992). These products led the
researchers to conclude that under these oxidation conditions, various radical species
are formed and can react with oxygen to form peroxyl radicals, which can undergo
propagation reactions with additional carotenoids (Handelman et al., 1991).

1.3.2.3. Photodegradation

Light exposure degrades carotenoids, and several mechanisms of action have

been proposed. Photooxidation produces species thought to be carotenoid radical cations
(Konovalova et al., 2001; Mortensen, 2002). Laser flash photolysis studies have
produced evidence to suggest that rapid bleaching of β-carotene in some solvents like
chloroform, can occur due to light exciting the molecules, which then instantly react

16
INTRODUCTION

with the solvent to form either a carotenoid-solvent free radical adduct or a β-carotene
radical (due to hydrogen abstraction).

1.3.2.4. Singlet Oxygen

In a mechanism similar to carotenoid excitation described in photodegradation,

light can also excite sensitizers like chlorophylls (JUNG et al., 1991), leading to the
formation of singlet oxygen (O2). Singlet oxygen may then react with neutral
carotenoids to produce excited state carotenoids. Once in an excited state, the carotenoid
may return to the ground state by releasing energy through vibrational and rotational
interactions with the surrounding solvent. This study suggests that the chemical pathway
might involve a direct attack on the double bonds of the carotenoid by singlet oxygen,
which forms biradicals that can eventually lead to carbonyl chain cleavage products
(Garavelli et al., 1998).
1.3.4 Applications
β-carotene is used to enhance or color many foods such as juices, candies,
cheeses, butter, and sauces as well as as an antioxidant and to help prolong the shelf life
of products. In the food additives list, β-carotene has the code E-160A. In addition, it is
also added to foods to increase nutritional value (milk, energy drinks, and juices) or
added to animal feed to improve the quality of milk or eggs.
High purity β-carotene is often used in pharmaceutical products such as
multivitamin tablets for nutritional supplements or for patients with vitamin A
deficiency. It is also often combined with many other ingredients in anticancer drug
formulations.

1.4. RESEARCH CONTEXT WORLDWIDE

1.4.1. Studies of yeast cell-based microcapsules

S. cerevisiae yeast cells have played an important role in the brewing and bread
industry for thousands of years. They were first recognized as capable of encapsulation
when Serozym lab research showed that S. cerevisiae yeast cells after proper treatment
can absorb and retain aromatic substances dissolved in water (Bishop et al., 1998).
Recently, S. cerevisiae yeast cells have been studied to encapsulate many hydrophilic
and hydrophobic active ingredients. In this day and age, a myriad of encapsulation
studies on yeast cells have been carried out. Cells are pretreated by various methods
such as temperature, HCl, NaOH and especially using NaCl to conduct cell plasmolysis
process. The microencapsulation efficiency of the studies varied widely depending on
the pretreatment conditions, encapsulated compounds, and exposure conditions.

17
INTRODUCTION
Table 3. Studies of encapsulating the active ingredient in yeast cells S. cerevisiae

Encapsulatio
Active Ingredients Biomass Pretreatment Condition Author
n Efficiency

CGA: biomass: H20

Chlorogenic acid Treat with 5% of NaCl in 54°C for 24
1:3:50 at 40oC, 4h and 12,6% (Shi et al., 2007)
(CGA) (354,3 Da) hours then dry sublimation
Water- 25MPa
soluble
active -Chemical: NaCl, HCl, NaOH, and
Cow Serum surfactant substances. BSA: biomass: H20
ingredients 10,1% (treat
Albumin (BSA) 0,3:1:20 at 27oC for (Shi et al., 2017)
-Condition: 40°C for 40 hours, 85°C for with NAOH)
(66.5 kDa) 24h
15 minutes

Orange peel
Orange peel oil, Essential oils: oil: 10% (Bishop et al.,
peppermint oil Do not treat living and dead yeast cells biomass: water 1:2:4 at
Peppermint 1998)
(136.2 Da) 40oC for 4h
Essential oil: 40%
oil active
ingredients Limonene (136.2 Pannel et al. (1990)
(Normand et al.,
and Plasmolyse then drying biomass spray European Patent 26,7%
Da) 2005)
fragrance 0242135
compound
Terpenes: limonene The content of
s
(136.2 Da), aromatics in the
(Ciamponi et al.,
carvone (150.2 No treatment exposed environment 4-9%
2012)
Da), linalool (154.3 is up to 30% at 20-
Da) 60oC for 2-8h

18
INTRODUCTION

D-limonene:
D-limonene (136,2 37% DW
Aromatics: biomass
Da), ethyl (Sultana et al.,
Yeast cells are extracted β-glucan 2:1 or 4:1 at 20-50°C Ethyl
hexanoate (144,2 2017)
for 0-8h hexanoate:
Da)
49% DW

Biomass: OEO: water:

ethanol: 14,5%:14,5%:
Oregano essential No treatment, cell autolyzed for 8 and 24h (Dimopoulos et
63,8%: 7,2% in 30 , -
oil (133 kDa) at 52oC al., 2020)
37, 45, 65oC for 48h at
170rpm

Resveratrol: biomass:
Resveratrol (228,3 Treat with 5% of NaCl at 54°C for 24 alcohol: water
High- 4,52% (Shi et al., 2008)
Da) hours then dry sublimation 1:3:50:50 in 40°C for
molecular 4h and 25MPa
active
ingredient Curcumin: biomass
Curcumin (368,4 Treat with 10%,20%, 30%, 40% of NaCl (Paramera et al.,
0,2:10 in alcohol 50% 35,8%
Da) in 55°C for 48 hours or no processing 2011)
in 25-55°C for 48h

a-tocopherol: biomass:
water: ethanol 0,2g:
a-tocopherol (Czerniak &
Treat with 5% of NaCl in 50oC for 24h 0,4g: 15ml: 15ml in Up to 60%
Lipid- (430,71 Da) Jankowski, 2013)
25, 35, 55 and 65oC
soluble for 24h at 250rpm
active
ingredients Cholecalciferol:
Cholecalciferol Treat with 10% of NaCl in 55oC for 48h at biomass (non- From 31,20% (Dadkhodazade et
(384.65 Da) 180rpm plasmolysed and to 76,10% al., 2018)
plasmolysed): ethanol:

19
INTRODUCTION
water 2,5mg: 10g:
12,5mg: 70ml in 40oC
for 12h at 180rpm

20
INTRODUCTION

1.5. URGENCY OF THE SUBJECT

Currently, the side effects of artificial chemicals used in food and agricultural
products are a major concern for consumers and scientists. Artificial coloring, additives,
preservatives used in food processing, and residues of pesticides in vegetables and fruits
can accumulate in the body and cause cancer as well as many other diseases. Therefore, the
demand for naturally sourced antioxidants in the food, pharmaceutical, and cosmetic
industries is increasing more than ever.
Besides, β-carotene has had a lot of applications in these areas. Unfortunately, its
application of it is also limited because the molecule is quite large and hydrophobic. This
pigment is degraded when exposed to light, oxygen, and temperature. Therefore, β-carotene
encapsulation is a way to improve the processing and dissolution of the molecule, to protect
and control its release.
The encapsulation methods commonly used such as spray drying or coagulation also
encounter some inconveniences such as
 the sporadic surface structure,
 the broken shells leading to leakage during the thermal process,
 the selectivity of carrier materials;
 the complicated process, and
 the usage of non-food-standard solvent to wash.
S. cerevisiae yeast cell envelope consists of a cell wall and a semi-permeable
membrane that is effective in protecting intracellular components from the undesired effects
of light, oxygen, etc. Furthermore, studies have shown that the structure of yeast capsules
can resist high temperatures up to 256°C (Paramera et al., 2011). The use of yeast cells as
microcapsule shells has several advantages such as
 homogeneity of surface and size,
 improvement of sensory value,
 well, dispersion in the food system,
 adaptation of food standards, and
 valorization of food by-products (from the brewing industry).
From the references, our team found that S. cerevisiae is a potential carrier material
with the ability to encapsulate both hydrophilic and hydrophobic compounds. However,
there are currently not many studies using microbial cells to encapsulate β-carotene because
its hydrophobicity and large size make it difficult to diffuse into cells. An appropriate
process can increase the microencapsulation efficiency. For the aforementioned reasons,
we proposed the investigation of pretreatment conditions of S. cerevisiae to encapsulate β-
carotene and discover the possible diffusion mechanism.

21
MATERIALS & METHODS

2. MATERIALS AND METHODS

2.1. MATERIALS
 Active dry yeast S. cerevisiae, Mauripan brand of AB Mauri Vietnam Co.,
Ltd. Yeast was vacuum packaged and stored at ambient temperature.
 High purity crystal β-carotene, produced by Sigma-Aldrich, USA, and stored
in a freezer of -20°C.
 Simply soybean oil is produced by Cai Lan Vegetable Oil Co., Ltd.

Figure 6. Active dry-yeast material

2.2. CHEMICALS
Table 4. Chemicals used in the research

No. Chemical Purpose Origin

Determine the content of lipid and β-

1 N-hexane Germany
carotene

As emulsifier of the suspension of yeast and

2 Span 80 China
oil of β-carotene

Determine the content of lipid and β-

3 BHT China
carotene

Determine the content of lipid and β-

4 Ethanol 80oC China
carotene

22
MATERIALS & METHODS

5 HCl Pretreatment condition American

6 NaOH Pretreatment condition China

7 Hexadecane Determine surface properties Germany

8 Decane Determine surface properties China

9 Ethyl acetate Determine surface properties China

10 Chloroform Determine surface properties Thailand

2.3. EQUIPMENT & TOOLS

Table 5. Equipment used in research

No. Equipment Manufacturer Origin

1 UV-Vis spectrophotometer Jinghua China

2 Weigh scale Sartorius Germany

3 Thermostatic shaking tank Memmert Germany

4 Centrifuge Hermle Germany

5 Vortex mixer Hwashin Korea

6 Sample shaker cabinet Labtech Korea

7 Ultrasonic Homogenizer Sonis American

Tools
Beaker Centrifuge tube Glass cuvette
Erlenmeyer flask Thermometer Ray flask
Volumetric flask Glass rod Rubber squeeze
Pipette Micropipette

23
MATERIALS & METHODS

2.4. EXPERIMENTS
determine the moisture content of
Determination the properties the material
of material
determine the lipid content of the
material

investigate different time:

0, 1, 2, 3, 4, 5 h
Determination of
pretreatment conditions on investigate different NaOH
encapsulation efficiency concentrations: 0, 0.05, 0.07, 0.1,
0.12 M

investigate different HCl

concentrations: 0, 0.05, 0.07, 0.1,
0.12 M

determine the hydrophobicity of

cell surface
Evaluation the cell surface
after pre-treatment
determine the electron donor/
acceptor of cell surface

determine the β-carotene content

To evaluate the efficiency of

encapsulation process of determine the moisture content
yeast cell
determine the uptake lipid content

Figure 7. The research process

24
MATERIALS & METHODS

2.4.1. Experiment 1: Determination of material composition

Purpose of experiment: Determine the chemical properties of the material

The object of analysis: inactivated and activated yeast biomass.
Parameters surveyed: lipid and moisture content
Method:
 Determine lipid content by extraction method with n-hexane solvent.
 Determine moisture content by an infrared moisture analyzer.

2.4.2. Experiment 2: Determination of the pretreatment conditions

Purpose of experiment: Assess the effect of the biomass pretreatment under different
conditions on the efficiency of β-carotene encapsulation, thereby determining the
appropriate treatment conditions.
Implementation method:
Dried yeast is activated with distilled water within 5 minutes, collects biomass by
centrifugation at 4000 rpm, for 5 minutes, and washed once again with distilled water at
4oC. Biomass is dispersed into distilled water in certain proportions and incubated under
different conditions. After that, the biomass is recovered by centrifugation at 3000 rpm.
Biomass after treatment is exposed to β-carotene oil in the ratio of 1g wet mass: 10 mL
distilled water: 1g β-carotene oil: 0.1g surfactant (Span 80) and incubate at ambient
temperature for 24 h, with a shaking speed of 200 rpm. Proceed to collect biomass by
centrifuging at 3000 rpm, for 10 minutes and wash three times with distilled water.
Yeast microcapsules are then extracted to measure β-carotene and lipid content with n-
hexane solvents to assess encapsulation effectiveness.

2.4.2.1. Experiment 2.1: Determination of pretreatment time

Fixed parameters:
 Treatment biomass rate: 5 g wet biomass: 100 mL of distilled water
 Processing temperature: 50oC.
Parameter survey: Processing time: 0, 1, 2, 3, 4, and 5 h.

25
MATERIALS & METHODS

2.4.2.2. Experiment 2.2: Determination of pretreatment NaOH concentration

Fixed parameters:
 Treatment biomass rate: 5 g wet biomass: 100 mL of NaOH solution
 Treatment temperature: 50oC.
 Treatment time: 3h
Parameter survey: NaOH concentration: 0, 0.05, 0.07, 0.1, and 0.12 M.

2.4.2.3. Experiment 2.3: Determination of pretreatment HCl concentration

Fixed parameters:
 Treatment biomass rate: 5 g wet biomass: 100 mL of HCl solution
 Treatment temperature: 50oC.
 Treatment time: 3h
Parameter survey: HCl concentration: 0, 0.05, 0.07, 0.1, and 0.12 M.

2.5. ANALYTICAL METHOD

2.5.1. Moisture and lipid content analysis

The moisture content of the biomass was studied by an infrared moisture analyzer
(IR35M – 000230V1, Denver Instrument, Germany).
The lipid content of yeast cells was determined by extraction in a non-polar solvent
(n-hexane) followed by the Bligh-Dyer method. The lipid content was calculated by the
following formula:
𝑚𝑙𝑖𝑝𝑖𝑑
𝐿𝑖𝑝𝑖𝑑 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (𝑔/𝑔) =
𝑚𝑤𝑏
in which:
 𝑚𝑙𝑖𝑝𝑖𝑑 (g) is the mass of extracted lipid
 𝑚𝑤𝑏 (g) is the mass of extracted wet biomass.

2.5.2. Analysis of β-carotene content in biomass

The encapsulated β-carotene content was determined by the UV-Vis absorption

spectroscopy method. This method of analysis is based on a comparison of monochrome

26
MATERIALS & METHODS

radiation absorption (optical density) of the studied solution with the monochrome radiation
absorption of a standard β-carotene solution with a defined concentration.
We used n-hexane solvent to dissolve and extract β carotene from yeast cells. Then
measure the absorption at 450 nm (Rodriguez, 2001) using a UV-Vis absorption
spectrometer.
𝐴450 × 𝑉 × 𝑓 × 10000
𝛽 − 𝑐𝑎𝑟𝑜𝑡𝑒𝑛𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡(µ𝑔⁄𝑔 𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 ) =
2592 × 𝑚
in which,
 𝐴450 is the absorbance of the extract at 450 nm
 𝑉 (ml) is the extraction volume
 𝑓 is the dilution factor
 𝑚 (g) is the mass of the dried biomass
 2592 is the UV-Vis absorption index of β-carotene in n-hexane solvent.

2.5.3. Hydrophobicity and electron acceptor/ donor properties analysis

The MATS (microbial adhesion to solvents) method is based on the comparison

between microbial cell affinity to a polar solvent and a nonpolar solvent by simply
measuring the fraction of cell removal from the aqueous phase in the presence of these
solvents. The polar solvent can be an electron acceptor or an electron donor, but both
solvents must have similar van der Waals surface tension components (Mercier-Bonin et
al., 2004). The following pairs of solvents were used:
 chloroform - an electron acceptor solvent, hexadecane - a nonpolar solvent,
 ethyl acetate - a strong electron donor solvent, and decane - a nonpolar
solvent.
Due to the surface tension properties of these solvents, differences between the
results obtained with chloroform and hexadecane and the results obtained with ethyl acetate
and decane indicated that there were electron donor/electron acceptor interactions at the
bacterial cell surface and revealed hydrophobic and hydrophilic properties (Bellon-
Fontaine et al., 1996).
Hydrophobicity was evaluated by the cell adhesion to hexadecane. The electron
donor character was calculated from the adhesion to chloroform minus that to hexadecane.
The electron acceptor character was calculated from the adhesion to ethyl acetate minus
that to decane.
The percentage of bound cells was subsequently calculated is also the percentage of
cell affinity for each solvent and is determined by measuring absorption at 600 nm
according to the following formula:

27
MATERIALS & METHODS

𝐴𝑠
% 𝑎𝑓𝑓𝑖𝑛𝑖𝑡𝑦 = (1 − ) × 100
𝐴0
in which,
 𝐴0 : absorbance of cell suspension before mixed with the solvent
 𝐴𝑠 : absorbance of cell suspension after mixed with the solvent

2.6. STATISTICAL ANALYSIS

All the experiments were performed in triplicate and the results were expressed as
the mean ± SD of three replicates. Data were analyzed using the one-way analysis of
variance (ANOVA) in Stargraphics Centurion 18. The multiple range test LSD with a
significance level at p-value < 0.05, was applied to the results to test the significant
difference.

28
RESULTS AND DISCUSSION

3. RESULTS AND DISCUSSION

3.1. DETERMINATION OF MATERIAL COMPOSITION
3.1.1. The moisture of raw yeast

The moisture content of raw yeast was 5.64 ± 0.16%. This result was appropriate
with the moisture value of active dry yeast of 4.0 – 8.5% reported by Joseph et al. 2014.
The relatively low value is to ensure the complete deactivation of yeast growth to prevent
instability, and possible contamination and to extend the shelf life to 6 – 12 months at
ambient temperature. However, it is offset by activity loss due to irrecoverable damage to
the cells (Dobbs et al., 1982). After activation, the wet biomass moisture content was
increased to 72.96 ± 0.19%.

3.1.2. Lipid content in raw yeast

Lipid bodies are intracellular lipid droplets. They are present as the hydrophobic
component surrounded by a membrane monolayer. These small round structures constitute
the cellular fat storage, representing a physical limitation on the storage capacity of intact
yeast cells for lipophilic substances. Moreover, the presence of lipid bodies can trigger the
hydrophobicity of plasma membrane leading to a more readily transfer of lipophilic
compounds. They were first studied as a reservoir for the encapsulation of hydrophobic
compounds. In 1977, Shank has patented a technique for encapsulating lipophilic
substances localized in lipid droplets within the cell for the carbonless copy paper
application. Many techniques after that focused to enhance lipid droplets’ size and amount.
Compared to the glucose–grown yeast cells, oleate–grown cells possess a higher
amount and bigger size of lipid bodies (Ta et al., 2012). For that reason, the oleaginous
yeast cells with lipid content >40% typically Yarrowia lipolytica, were often used to
promote efficiency (Dang et al., 2017; Pham-Hoang et al., 2018). Cells can produce fat
globules up to 40-60% of their dry weight when their cultivation is influenced by limited
nutrient availability.
Besides this method, another method called “lipid-extending substances” was used
by utilization of solvents. In which, cells with low lipid content are mixed with lipid
solvents which can handily penetrate cells. Solvents can affect multiple functions to
increase the solubility of active compounds, act as lipid reserves in microbes, and rise the
fluidization of cell membranes (Pham-Hoang et al., 2013).
In this study, the used commercial yeast S.cerevisae had a relatively low lipid
content of 15.4 ± 0.7 mg lipid/g of dry basis. Therefore, the “lipid-extending substances”
method was expected to promote the cell wall porosity, reduce the integrity of the plasma
membrane, then increase the encapsulated β-carotene amount.

29
RESULTS AND DISCUSSION

3.2. DETERMINATION OF PRETREATMENT CONDITIONS

3.2.1. Determination of the pretreatment time

Determination of the pre-treatment at 50oC with different periods of 0, 1, 2, 3, 4, and

5 h was conducted. The changes in β– carotene content in cases are demonstrated in Figure
8.

Figure 8. The encapsulated β-carotene content at different pretreatment times at 50oC

The letters indicate a significant difference (p≤0.05) among intervals.

The results showed that there was a change in the amount of β–carotene encapsulated
when biomass treatment at different intervals, caused by the autolysis process. It is known
that autolysis is the act of native hydrolytic enzymes of the cell that release cytoplasmic
compounds (Alexandre & Guilloux-Benatier, 2006). In food industry, autolysis is used
abundantly and the process is usually performed at 50oC (Dadkhodazade et al., 2021).
Result show that, in untreated cells, the β– carotene content obtained is only 4.20 ± 0.19
μg/g of dry basis and gradually increasing to 14.06 ± 1.54 μg/g of dry basis when the
treatment time lasts up to 3 hours. There was no huge difference in the amount of β–
carotene obtained when treated at intervals between 1 and 5 h or between 2 and 4 h. When
the treatment time lasted 5 h, the β–carotene content decreased slightly to 5.74 ± 0.52 μg/g
of dry basis. The effect of the autolysis process described is in line with the resulting trend
of the (Czerniak et al., 2015) experiment, which mentioned that efficiency increased from
32.6% for the control sample (without autolysis) to 45.3% for samples autolyzed at different
times by addition of ethyl acetate and hydrolysis with Glucanex R200 (yeast lytic enzyme).
Ethyl acetate increased cell membrane permeability and Glucanex R200 did relative
hydrolysis on cell walls (Czerniak et al., 2015).

30
RESULTS AND DISCUSSION

Therefore, the pretreatment time of yeast biomass for β-carotene encapsulation was
selected at 3 hours.

3.2.2. Determination of the pretreatment NaOH concentration

The determination of the pretreatment with NaOH solution with different

concentrations including 0, 0.05, 0.07, 0.1, and 0.12 M at 50 oC for 3 h was conducted.
(Figure 9).
The results show that there were significant differences between the amount of
encapsulated β-carotene in different concentrations of NaOH solution. More specifically,
the pigment content increased from 2.76 ± 0.34 to 12.79 ± 0.57 µg/g of dry basis when the
NaOH concentration increased from 0 to 0.07M. However, as this concentration was
higher, then the content decreased to 8.80 ± 0.06 µg/g of dry basis at 0.12 M.

Figure 9. The β-carotene content of NaOH-treated biomass

The letters indicate a significant difference (p≤0.05) among concentrations.

The treatment with an alkali such as NaOH solution affects the β-glucan network
(Figure 10) by extending its porosity through two possible means:

31
RESULTS AND DISCUSSION

(Klis et al., 2002)

Figure 10. Putative molecular organization of the cell wall of S. cerevisiae

 Eliminating the cell wall proteins: The β1,3-glucan layer forms the inner
layer of the cell wall, whereas the cell wall proteins from the outer layer.
There are two main classes of proteins covalently coupled to the cell wall
network. Firstly, Glycosylphosphatidylinosito cell wall proteins (GPI-CWPs)
are generally indirectly linked to β1,3-glucan through the highly branched
β1,6-glucan moiety or directly connected to the network by an alkaline
linkage. They represent the major component of the outer protein layer and
determine the permeability and adhesion of yeast cells. Secondly,
Plasmodium interspersed repeats cell wall proteins (Pir-CWPs) directly
tethered to β1,3-glucan through the glycopeptide linkage which is alkaline
sensitive. The nature of this linkage is still unknown, but due to its sensitivity
to mild alkali, postulates the involvement of O-linked side chains. This type
of protein is distributed throughout the network. Disruption of them weakens
the cell wall (Klis et al., 2002). Therefore, the release of cell wall proteins can
lead to the formation of pores in the cell wall surface (De Nobel et al., 1989).
The conclusion of Shi et al. (2007) of the treatment with NaOH solution
increased the wall pore size from 4.3 nm (untreated) to 30.6 nm at 0.6 M
NaOH solution also elaborates this hypothesis (Shi et al., 2007).
 Removing the mannoproteins: Cell wall mannoproteins are indirectly
connected by β1,6-glucan through a GPI anchor and directly by β-1,3-glucan
through disulfide bridges that break relatively easily in alkalis (Avramia &
Amariei, 2021). The reduction of intermolecular disulfide bridges causes a
more general opening of the wall (De Nobel et al., 1989).
However, at a high concentration of the alkaline solution, the structure of β-glucan
can be destroyed. According to Klis et al. (2002), the mechanical strength of cell walls
mainly depends on the β-glucan network. Three conformations of glucans have been
reported: The triple helix, the single helix, and the random coil. The biologically active
form of glucan is unclear. Several studies have suggested that the triple-helix is the most

32
RESULTS AND DISCUSSION

biologically active conformation (Kojima et al., 1986; Yanaki et al., 1983). The triple helix
conformer of glucan is formed by three hydrogen bonds to Oxygen in the C-2 position.
These bonds can be broken due to the high temperature or/and high pH (Young & Jacobs,
1998). Consequently, this explains the decrease in encapsulated pigment amount at higher
concentrations of NaOH solution.
In summary, the pre-treatment of 0.07M NaOH solution gave the highest
encapsulated β-carotene content. At lower concentrations, the solution did not dissolve
enough cell wall proteins and at higher concentrations, the solution can lead to the
hydrolysis of the β-glucan network, causing the disruption of cell shape, leading to reduced
encapsulation efficiency.

3.2.3. Determination of the pretreatment HCl concentration

The determination of the pretreatment HCl solution concentrations including 0, 0.05, 0.07,
0.1, and 0.12 M at 50oC for 3 h was conducted (Figure 11).
The results show that there were significant differences between the amount of
encapsulated β-carotene in different concentrations of HCl solution. More specifically, the
pigment content increased from 2.46 ± 0.19 to 6.97 ± 0.52 µg/g of dry basis when the HCl
concentration increased from 0 to 0.07M. However, as this concentration was higher, the
content decreased to 2.91 ± 0.20 µg/g of dry basis at 0.12 M.

Figure 11. The β-carotene content of HCl-treated biomass

The letters indicate a significant difference (p≤0.05) among concentrations.

HCl is a promoter for the yeast cell autolysis as it raises the internal H ions
concentration to the point at which proteases and other lytic enzymes are active
(Sevringhaus et al., 1923). Autolysis has been described as intracellular biopolymer
hydrolysis under the influence of hydrolytic enzymes such as proteinase, ribonucleases, and

33
RESULTS AND DISCUSSION

glucanases (Amer et al., 2021). In which, the cell wall is partially destroyed allowing the
cytoplasm to evacuate outside the cell when the hydrolytic products decrease their
molecular size low enough to cross the pores in the wall. (Amer et al., 2021; Avramia &
Amariei, 2021). As a result, the cytoplasmic space increases, and the cell wall becomes
more porous leading to the beneficial condition for the penetration of β-carotene.
The glucan polymer exists in both alkali-insoluble, acid insoluble, and alkali-soluble
forms. The insoluble glucan fraction helps the cell retain its shape. Me2SO or strong acid
treatments are generally used to convert insoluble glucan into soluble forms (Young &
Jacobs, 1998). The solubility of wall components can lead to the destruction of yeast cells
decreasing the desired efficiency.
The effective promoted encapsulating properties of autolyzed yeast are reported in
works of literature such as hydrophobic flavor compounds (<300 Da) (Dardelle et al.,
2007), chlorogenic acid (354 Da) (Shi et al., 2007), and limonene (136 Da) (Normand et
al., 2005). Nonetheless, in some research, the encapsulation of biomacromolecules such as
BSA (66.5 kDa) (Shi et al., 2017). Thus, the relative mild treatment of yeast with NaCl and
HCl, as plasmolyzer might be applicable to promote the encapsulation of small molecules,
but not effective for the encapsulation of biomacromolecules (Shi et al., 2017). In our case,
β-carotene has a relatively small molecular weight (537 Da) and is effectively encapsulated
in autolyzed yeast cells.
In summary, the pre-treatment of 0.07M HCl solution gave the highest encapsulated
β-carotene content. At proper concentration, it increases the porosity of the cell wall.
Whereas the higher concentrations result in the dissolution of the cell wall.

3.3. EVALUATION OF THE UPTAKE LIPID OF TREATED

BIOMASS
Beta-carotene is a hydrophobic compound, so before making contact, it needs to be
dispersed in oil to form beta-carotene oil mixture, then use a surfactant to form an emulsion
with yeast suspension.

34
RESULTS AND DISCUSSION

Figure 12. The uptake lipid content and β-carotene content of different treatment
The letters indicate a significant difference (p≤0.05) among treatments.

Figure (12) shows lipid and beta-carotene content entering cells after exposure to
beta-carotene oil emulsion of biomass samples pretreated with distilled water at 50°C for 3
h, treated with Treat with HCl and 0.07 M NaOH (50°C for 3 h). In general, the amount of
lipid entering pretreated cells increased significantly compared to raw yeast, from
1.46±0,08 to 5.08±0.46 after pretreatment with distilled water for 3 h. However, the lipid
content entering the cells decreased to 3.97±0.09 when treated with NaOH and 2.51±0.15
when treated with HCl. The reason for the decrease in lipid content when pretreated with
NaOH and HCl at 50°C for 3 h compared with samples treated with distilled water at the
same temperature and time is because NaOH and HCl are promoters for the yeast cell
autolysis during the treatment. For a long time, it breaks down the structure of the cell wall,
thereby leading to a decrease in the amount of lipids entering the cell compared with water
3h treated cell.
From the data shown in the figure, lipids and beta-carotene enter the cells in the same
direction, as lipid content increases, beta-carotene content also increases and vice versa.
There was no significant difference between the beta-carotene content and the lipid content
entering the cells of the samples. This proves that oil acts as a carrier. During the
encapsulation process, oil molecules carrying beta-carotene diffuse through the cell wall
through the pores created by pretreatment, then dissolve into the lipid of the cell membrane
and diffuse into the cell. Beta-carotene oil droplets bind together to form lipid bodies inside
yeast microcapsules.
Pham-Hoang et al. (2018) used confocal microscopy to confirm the presence of beta-
carotene inside Y. lipolytica yeast microcapsules. The results showed that beta-carotene

35
RESULTS AND DISCUSSION

was accumulated in lipid droplets and lipid-rich organelles of the cell. Ciamponi et al.
(2012) reported on the presence of limonene-containing lipid bodies in the yeast
microcapsules S. cerevisiae. The size of the lipid body also depends on the growth phase
of the cell, specifically when using cells in the log phase, the lipid body will have a smaller
size than the cells in the equilibrium phase.

3.4. EVALUATION OF THE HYDROPHOBICITY OF CELL

SURFACE AFTER PRETREATMENT
We used MATS test to evaluate the hydrophobicity of cells to assess the interaction
between cells and oil droplets in the environment and determine the effect of
hydrophobicity on the encapsulation process of β-carotene oil through cell envelope. The
results are shown in Figure 13.

c
b b
a
a

A B

Figure 13. Hydrophobicity of the cell surface after pretreatment

A: the hydrophobicity of cell surface after pretreatment with time; B: the hydrophobicity of cell
surface after pretreatment with HCl; C: the hydrophobicity of cell surface after pretreatment with NaOH

36
RESULTS AND DISCUSSION

The letters indicate a significant difference (p ≤ 0.05) among concentrations.

In general, the proportion of cells involved in bonding with non-polar solvents

(hexadecane in this case) was relatively low. In theory, when the bonding rate with non-
polar solvents is less than 20%, the cell is considered to be non-hydrophobic. As such, the
yeast cells used in those experiments had more hydrophilic properties. These findings are
consistent with results obtained by (Setala et al., 2002).

According to Pham-Hoang et al. (2013), the mannoproteins play an important role

in determining the yeast's hydrophobic character. The digestion of surface mannoproteins
by introducing pronase or protease reduced the hydrophobicity of Candida tropilis and
Yarrowia lipolytica (Kappeli & Fiechter, 1977; Kim et al., 2000). Globally it seems that
treatment makes yeast’s cell wall got thinner since mannoproteins were partially degraded
along with β -1,4 and β -1,6 glucans (Paramera et al., 2011a).

However, in our study, yeast biomass tended to increase hydrophobicity after being
treated, the treatment raised the hydrophobicity from 4.10 ± 0.23 (intact cells) to 9.54 ±
0.32 % (treated with 0.07M HCl solution). The probable reason could be interpreted as
follows: on the one hand, the hydrophobic character is due to the level of cell wall protein
mannosylation and mannoproteins would not be the origin of the cell hydrophobicity
(Masuoka & Hazen, 1997). On the other hand, hydrophobicity also depends on the
hydrophilic fibrils and the level of glycosylation (Danchik & Casadevall, 2021). The
multifactorial nature of hydrophobicity presents a challenge in studying and understanding
the molecular mechanism of this property.

Theoretically, the more hydrophobic surface, the more adhesion of hydrophobes to

the cell wall, and the more b-carotene oil droplets penetrate across the cell envelope (Pham-
Hoang et al., 2013). Nonetheless, the previous result shows that the encapsulated b-carotene
content was higher when biomass was treated with NaOH and HCl compared to solely
treated with temperature and intact cells. Besides, the fashions of the pigment content were
not corresponding to the trends of hydrophobicity among different levels of pretreatment
conditions.

This indicates that the adhesion of Beta-carotene oil droplets did not depend on the
surface hydrophobicity but possibly rather on the porosity, electrostatic properties, Lewis
acid-base interactions, or surface protrusion (Pham-Hoang et al., 2013).

37
RESULTS AND DISCUSSION

3.5. EVALUATION OF THE ELECTRON DONOR/

ACCEPTOR PROPERTIES OF CELL SURFACE AFTER
PRETREATMENT
We used the MATS test to evaluate the electron donor/ acceptor property of cells to assess
the interaction between cells and oil droplets and determine the effect of this property on
the encapsulation process of β-carotene oil through the cell envelope. The results are shown
in Figure 14.

A1 A2

c c
v
b b b b
a a
a a

B1 B2

C1 C2

Figure 14. The electron donor/ acceptor properties of the cell surface

38
RESULTS AND DISCUSSION

A: the electron donor/ acceptor with time; B: the electron donor/ acceptor with HCl; C: the
electron donor/ acceptor with NAOH; 1: stand for electron donor; 2: stand for electron acceptor
The letters indicate a significant difference (p≤0.05) among concentrations.

The “microbial adhesion to solvents” method is based on the comparison between

microbial cell affinity to a polar solvent and a nonpolar solvent by simply measuring the
fraction of cell removal from the aqueous phase in the presence of these solvents. The polar
solvent can be an electron acceptor or an electron donor, but both solvents must have similar
Van der Waals surface tension components. The following pairs of solvents were used:
chloroform – hexadecane and ethyl acetate – decane. Due to the surface tension properties
of these solvents, differences between the results obtained when affinity to chloroform
minus that of hexadecane and the results obtained with ethyl acetate minus decane indicated
that there were electron donor/electron acceptor interactions at the cell surface (Bellon-
Fontaine et al., 1996). If the affinity toward the electron donor solvent exceeds the affinity
for the nonpolar solvent, the cell surface possesses electron acceptor features. Likewise, if
the affinity for the electron acceptor solvent exceeds the affinity toward the nonpolar
solvent, the cell surface can be considered to have electron donor characteristics.
The MATS results demonstrate that cells treated with 0.07 M NaOH solution had
affinity was higher with chloroform (an electron acceptor solvent) than with hexadecane (a
nonpolar solvent), and the difference was excess the discrepancy of ethyl acetate to decane.
It was concluded that these yeast cells were rather electron donors, thus showing basic
character on the surface. Whereas, control yeast and yeast of other pretreatments presented
different surface characteristics. Cells which exhibited a higher affinity with polar solvents
coupled with a more pronounced electron acceptor nature were more acidic.
The data in tables B7 – B25 points out the sporadic fluctuations among time intervals
and concentrations. The findings indicate the independence of encapsulated b-carotene
content of the electron donation and electron reception on the cell surface.

39
CONCLUSIONS AND PERSPECTIVES

4. CONCLUSIONS AND PERSPECTIVES

4.1. CONCLUSIONS
The influence of pretreatment conditions (temperature, HCl treatment, and NaOH
treatment) on cell surface characteristics and Beta-carotene oil encapsulation efficiency was
examined in this work. The following inferences would be based on the findings:
 The investigation of pre-treatment times of 0, 1, 2, 3, 4, and 5 h at 50oC showed that
after 3 h, the encapsulated β-carotene content was highest at 14.06 ± 1.54 (µg/g dry
basis).
 The investigation of NaOH concentrations of 0, 0.05, 0.07, 0.1, and 0.12 M at 50oC in
3 h showed that at 0.07M NaOH solution, the encapsulated β-carotene content was
highest at 12.79 ± 0.57 (µg/g dry basis).
 The investigation of HCl concentrations of 0, 0.05, 0.07, 0.1, 0.12 M at 50oC in 3 h
showed that at 0.07M HCl solution, the encapsulated β-carotene content was highest at
6.97 ± 0.52 (µg/g dry basis).
 The hydrophobicity of all samples was below 20 %, indicating that the cell surface of
S. cerevisiae has hydrophilic nature. The encapsulation efficiency of β-carotene was
not related to the hydrophobicity of the cell surface.
 The acidic nature of the surface was favorable in intact cells and cells treated with solely
temperature and with HCl solution. The basic character was preferable in NaOH-
treated cells. The encapsulation efficiency of β-carotene was not related to the electron
donor/ acceptor feature of the cell surface.
 Oil droplets acted as carriers of hydrophobic compounds penetrating the cell cytoplasm.
The higher the uptake lipid content, the higher the internal β-carotene amount and vice
versa.

40
CONCLUSIONS AND PERSPECTIVES

4.2. PERSPECTIVES
Other cell surface properties such as porosity, zeta potential, and extracted
mannoprotein amount should be evaluated in future experiments to determine the
mechanism of pigment diffusion and the factors that affect encapsulation efficiency, and to
design a process to change these properties to achieve better results. The study concluded
by determining the effects of biomass pretreatment conditions; nevertheless, more research
on incubation conditions is required in order to design the microencapsulation process for
maximum efficiency.

41
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48
APPENDIX

6. APPENDIX
APPENDIX A. ANALYTICAL METHODS
A1. The experimental procedure

Figure A1. The experimental procedure flowchart

49
APPENDIX

A.2. Lipid content analysis

Equipment
 UV – Vis spectroscopy
 Vortex mixer
 Weight scale
 Thermostatic tank
Procedure
 Weigh an empty disk.
 Weight exactly 1 g of wet biomass into the 15 ml centrifuge tube.
 Add 2ml 80o ethanol then incubate in the thermostatic tank at 80oC for 15
min.
 Centrifuge 3000 rpm for 3 min to remove the alcohol.
 Add 1 ml distilled water and 1 g glass bead into the tube.
 Mix for 3 min with the vortex mixer.
 Add 4 ml n-hexane and vortex for 30 s.
 Use a transfer pipette to obtain the supernatant into the disk.
 Add 4 ml n-hexane and vortex for 30 s.
 Use a transfer pipette to obtain the supernatant into the beaker for the second
extraction.
 Wait until n-hexane evaporates and weighs the disk.
 The lipid content was calculated by the following formula:
𝑚1 − 𝑚0
𝐿𝑖𝑝𝑖𝑑 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (𝑔/𝑔) =
𝑚𝑤𝑏
in which:
 𝑚0 (g) is the mass of the empty disk
 𝑚1 (g) is the mass of disk with lipid
 𝑚𝑤𝑏 (g) is the mass of extracted wet biomass.

A.3. β-carotene content analysis

Equipment
 UV – Vis spectroscopy
 Centrifuge
 Vortex mixer
 Weight scale

50
APPENDIX

Procedure
 Weight exactly 1 g of wet biomass into the 15 ml centrifuge tube.
 Add 2 ml of 80 % ethanol, and 1 ml of distilled water with proper mixing.
 Add 100 μl of 10 % BHT solution (in absolute ethanol).
 Add 5 ml of n-hexane into the tube.
 Mix for 5 min by vortex then and keep in the dark condition for 10 min.
 Centrifuge with 2000 rpm in 5 min for phase separation.
 Use a pipette to obtain the aqueous phase into a 25 ml volumetric flask.
 The extraction process was repeated 3 times.
 Add n-hexane to the extract to 25 ml in the volumetric flask.
 Measure the absorbance at 450 nm by a UV – Vis spectroscopy.
 Β-carotene concentration in the microcapsules was calculated using the
following equation:
𝐴450 × 𝑉 × 𝑓 × 10000
𝛽 − 𝑐𝑎𝑟𝑜𝑡𝑒𝑛𝑒 𝑐𝑜𝑛𝑡𝑒𝑛𝑡(µ𝑔⁄𝑔 𝑑𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡 ) =
2592 × 𝑚
in which:
 𝐴450 : the absorbance of the extract at 450 nm
 𝑉 (ml): the extraction volume
 𝑓 : the dilution factor
 𝑚 (g): the mass of the dry basis
 2592: the UV-Vis absorption index of β-carotene in n-hexane solvent.

A.4. Hydrophobicity and electron acceptor/donor property analysis

Equipment & Tools

 UV – Vis spectroscopy
 Micropipette

Procedure

 Prepare the cell suspension in the buffer solution of potassium phosphate 0.1 M, pH
= 7.0 so that the absorbance of the suspension was OD = 0.7 ± 0.02.
 Add 5 ml cell suspension into a tube with a Teflon cap.
 Add 1 ml solvent (hexadecane, chloroform, decane, and ethyl acetate) then slightly
mix 10 times.
 Keep the tube stable for 4 min for phase separation.

51
APPENDIX

 Transfer 2 ml of the mixture into a glass cuvette by a micropipette.

 Measure the absorbance of the mixture at 600 nm.
 The percentage of cell affinity for each solvent as the following equation:
𝐴𝐻
% 𝑎𝑓𝑓𝑖𝑛𝑖𝑡𝑦 = (1 − ) × 100
𝐴0
in which,
 𝐴0 : absorbance of cell suspension before mixed with the solvent
 𝐴𝐻 : absorbance of cell suspension after mixed with the solvent
 Hydrophobicity was evaluated by the cell adhesion to hexadecane
 The electron donor character indicating the Lewis base was calculated from the
adhesion to chloroform minus that to hexadecane.
 The electron acceptor character indicating the Lewis acid was calculated from the
adhesion to ethyl acetate minus that to decane.

52
APPENDIX

APPENDIX B. EXPERIMENTAL RESULTS

B1. Experimental data to determine the material composition
Table B1. Chemical composition of yeast cell material

Inactivated yeast Activated yeast

Moisture Lipid (mg/g Moisture Lipid (mg/g
(%) dry basis) (%) dry basis)
1st 5.79 - 72.75 14.68
2nd 5.48 - 73.03 15.94
3rd 5.66 - 73.10 15.61
Average 5.64 - 72.96 15.41
SD 0.16 - 0.19 0.66

53
APPENDIX

B2. Experimental data to determine the pretreatment time

Table B2. Lipid content of cells after pretreatment (mg/g dry basis)

Interval (h) 0 1 2 3 4 5
1st 14.68 18.62 15.25 13.92 14.10 16.14
2nd 15.94 14.71 16.74 14.20 17.17 13.71
3rd 15.61 16.23 15.19 13.96 15.50 15.62
Average 15.41 16.52 15.73 14.03 15.59 15.16
SD 0.66 1.97 0.88 0.16 1.53 1.28
Anova b c b a b b
*Value with different lowercase letters in the same row are significantly different (p<0.05)

Table B3. Lipid content of cells after encapsulation (mg/g dry basis)

Interval (h) 0 1 2 3 4 5
1st 16.23 21.80 18.87 19.88 19.67 25.74
2nd 17.33 17.50 20.08 19.48 23.77 21.71
3rd 17.05 19.01 18.23 20.11 21.86 24.48
Average 16.87 19.43 19.06 19.83 21.76 23.98
SD 0.58 2.18 0.94 0.32 2.05 2.06
Anova a b b b c d
*Value with different lowercase letters in the same row are significantly different (p<0.05)

Table B4. β-carotene content of cells after encapsulation (µg/g dry basis)

Interval (h) 0 1 2 3 4 5
1st 4.12 6.13 7.12 13.04 6.08 6.02
2nd 4.07 5.79 7.26 13.31 6.37 5.14
3rd 4.41 4.96 7.07 15.84 7.28 6.07
Average 4.20 5.63 7.15 14.06 6.58 5.74
SD 0.19 0.60 0.10 1.54 0.63 0.52
Anova a b c d c b
*Value with different lowercase letters in the same row are significantly different (p<0.05)

54
APPENDIX

B3. Experimental data to determine the pretreatment NaOH concentration

Table B5. Lipid content of cells after pretreatment with NaOH (mg/g dry basis)

Concentration (M) 0 0.05 0.07 0.1 0.12

1st 3.28 5.69 11.36 9.65 7.17
2nd 2.46 5.74 11.18 9.24 7.49
3rd 2.94 5.71 13.56 9.62 7.77
Average 2.89 5.71 12.03 9.50 7.48
SD 0.41 0.03 1.33 0.23 0.30
Anova a b e d c
*Value with different lowercase letters in the same row are significantly different (p<0.05)

Table B6. Lipid content of NaOHl-treated cells after encapsulation (mg/g dry basis)

Concentration (M) 0 0.05 0.07 0.1 0.12

1st 4.57 8.29 15.39 10.90 7.79
2nd 3.93 8.46 15.05 10.75 8.32
3rd 4.40 8.56 17.57 11.12 8.52
Average 4.30 8.43 16.00 10.92 8.21
SD 0.33 0.14 1.37 0.19 0.37
Anova a b d c b
*Value with different lowercase letters in the same row are significantly different (p<0.05)

Table B7. β-carotene content of NaOH-treated cells after encapsulation (µg/g dry basis)

Concentration (M) 0 0.05 0.07 0.1 0.12

1st 2.40 4.92 12.13 11.21 8.10
2nd 2.78 5.28 13.06 10.91 9.17
3rd 3.08 5.56 13.18 11.07 9.13
Average 2.76 5.25 12.79 11.06 8.80
SD 0.34 0.32 0.57 0.15 0.61
Anova a b e d c
*Value with different lowercase letters in the same row are significantly different (p<0.05)

55
APPENDIX

B4. Experimental data to determine the pretreatment HCl concentration

Table B8. Lipid content of cells after pretreatment with HCl (mg/g dry basis)

Concentration (M) 0 0.05 0.07 0.1 0.12

1st 1.70 3.23 5.34 3.62 2.34
2nd 2.15 3.04 5.12 3.60 1.86
3rd 1.56 2.94 4.71 3.32 1.98
Average 1.81 3.07 5.06 3.51 2.06
SD 0.31 0.15 0.32 0.17 0.25
Anova a b d c a
*Value with different lowercase letters in the same row are significantly different (p<0.05)

Table B9. Lipid content of HCl-treated cells after encapsulation (mg/g dry basis)

Concentration (M) 0 0.05 0.07 0.1 0.12

1st 3.42 5.25 7.94 4.85 2.70
2nd 3.73 4.84 7.72 4.87 2.39
3rd 3.03 5.04 7.05 4.63 2.38
Average 3.39 5.05 7.57 4.78 2.49
SD 0.35 0.21 0.47 0.13 0.18
Anova b d e c a
*Value with different lowercase letters in the same row are significantly different (p<0.05)

Table B10. β-carotene content of NaOH-treated cells after encapsulation (µg/g dry basis)

Concentration (M) 0 0.05 0.07 0.1 0.12

1st 2.31 4.42 7.05 5.28 2.98
2nd 2.40 5.45 7.45 4.69 2.69
3rd 2.68 4.48 6.42 4.59 3.06
Average 2.46 4.78 6.97 4.85 2.91
SD 0.19 0.58 0.52 0.37 0.20
Anova a c d c b
*Value with different lowercase letters in the same row are significantly different (p<0.05)

56
APPENDIX

B5. Experimental data to evaluate the hydrophobicity of cell surface

Table B11. Hydrophobicity of cell surface in pretreatment time experiment (%)

0 1 2 3 4 5
Interval (h)
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
A0 0.701 0.701 0.697 0.700 0.699 0.702 0.703 0.698 0.697 0.699 0.703 0.701 0.702 0.702 0.698 0.707 0.706 0.699
AH 0.674 0.672 0.667 0.633 0.634 0.635 0.635 0.633 0.639 0.639 0.644 0.642 0.648 0.645 0.646 0.670 0.664 0.659
%
Hydrophob 3.85 4.14 4.30 9.57 9.30 9.54 9.67 9.31 8.32 8.58 8.39 8.42 7.69 8.12 7.45 5.23 5.95 5.72
icity
Average 4.10 9.47 9.10 8.46 7.75 5.63
SD 0.23 0.15 0.70 0.10 0.34 0.37
Anova a e e d c b
*Value with different lowercase letters in the same row are significantly different (p<0.05)

57
APPENDIX
Table B12. Hydrophobicity of cell surface in NaOH experiment (%)

0 0.05 0.07 0.1 0.12

Concentration (M)
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
A0 0.705 0.702 0.701 0.702 0.698 0.701 0.697 0.702 0.703 0.703 0.701 0.7 0.705 0.699 0.699
AH 0.608 0.612 0.619 0.662 0.589 0.629 0.638 0.637 0.637 0.622 0.592 0.674 0.672 0.638 0.586
% Hydrophobicity 13.76 12.82 11.70 11.40 11.30 10.27 8.46 9.26 9.39 14.37 15.55 15.14 10.35 8.73 9.01
Average 12.76 10.99 9.04 15.02 9.36
SD 1.03 0.62 0.50 0.60 0.87
Anova c b a d a
*Value with different lowercase letters in the same row are significantly different (p<0.05)

Table B13. Hydrophobicity of cell surface in HCl experiment (%)

0 0.05 0.07 0.1 0.12

Concentration (M)
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
A0 0.699 0.701 0.701 0.700 0.702 0.703 0.698 0.697 0.702 0.700 0.698 0.699 0.704 0.701 0.701
AH 0.624 0.628 0.625 0.648 0.648 0.656 0.634 0.629 0.634 0.644 0.646 0.64 0.657 0.654 0.646
% Hydrophobicity 10.73 10.41 10.84 7.43 7.69 6.69 9.17 9.76 9.69 8.00 7.45 8.44 6.68 6.70 7.85
Average 10.66 7.27 9.54 7.96 7.08
SD 0.22 0.52 0.32 0.50 0.67
Anova d ab c b a
*Value with different lowercase letters in the same row are significantly different (p<0.05)

58
APPENDIX
B6. Experimental data to evaluate the electron donor and acceptor properties of cell surface
Table B14. Electron donor property of cell surface in pretreatment time experiment (%)

0 1 2 3 4 5
Interval (h)
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
A0 0.701 0.701 0.697 0.700 0.699 0.702 0.703 0.698 0.697 0.699 0.703 0.701 0.702 0.702 0.698 0.707 0.706 0.699
AC 0.629 0.632 0.628 0.609 0.608 0.606 0.594 0.594 0.597 0.622 0.628 0.628 0.622 0.621 0.619 0.651 0.646 0.642
AH 0.674 0.672 0.667 0.633 0.634 0.635 0.635 0.633 0.639 0.639 0.644 0.642 0.648 0.645 0.646 0.670 0.664 0.659
%AC 10.27 9.84 9.90 13.00 13.02 13.68 15.50 14.90 14.35 11.02 10.67 10.41 11.40 11.54 11.32 7.92 8.50 8.15
%AH 3.85 4.14 4.30 9.57 9.30 9.54 9.67 9.31 8.32 8.58 8.39 8.42 7.69 8.12 7.45 5.23 5.95 5.72
% E. Donor 6.42 5.71 5.60 3.43 3.72 4.13 5.83 5.59 6.03 2.43 2.28 2.00 3.70 3.42 3.87 2.69 2.55 2.43
Average 5.91 3.76 5.82 2.24 3.66 2.56
SD 0.447 0.353 0.220 0.220 0.227 0.128
Anova c b c a b a
*Value with different lowercase letters in the same row are significantly different (p<0.05)

Table B15. Electron acceptor property of cell surface in pretreatment time experiment (%)

0 1 2 3 4 5
Interval (h)
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
A0 0.701 0.701 0.697 0.700 0.699 0.702 0.703 0.698 0.697 0.699 0.703 0.701 0.702 0.702 0.698 0.707 0.706 0.699
AE 0.486 0.486 0.492 0.493 0.482 0.492 0.451 0.452 0.450 0.480 0.494 0.477 0.521 0.510 0.532 0.530 0.518 0.521
AD 0.623 0.621 0.626 0.616 0.610 0.626 0.622 0.628 0.629 0.653 0.656 0.642 0.654 0.643 0.649 0.652 0.645 0.652
%AE 30.67 30.67 29.41 29.57 31.04 29.91 35.85 35.24 35.44 31.33 29.73 31.95 25.78 27.35 23.78 25.04 26.63 25.46
%AD 11.13 11.41 10.19 12.00 12.73 10.83 11.52 10.03 9.76 6.58 6.69 8.42 6.84 8.40 7.02 7.78 8.64 6.72
% E.
Acceptor 19.54 19.26 19.23 17.57 18.31 19.09 24.32 25.21 25.68 24.75 23.04 23.54 18.95 18.95 16.76 17.26 17.99 18.74
Average 19.34 18.32 25.07 23.78 18.22 18.00
SD 0.175 0.759 0.690 0.878 1.261 0.743
Anova c b c a b a
*Value with different lowercase letters in the same row are significantly different (p<0.05)

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APPENDIX
Table B16. Electron donor property of cell surface in pretreatment NaOH experiment (%)

0 0.05 0.07 0.1 0.12

Concentration (M)
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
A0 0.705 0.702 0.701 0.702 0.698 0.701 0.697 0.702 0.703 0.703 0.701 0.700 0.705 0.699 0.699
AC 0.355 0.358 0.352 0.378 0.283 0.371 0.277 0.273 0.261 0.174 0.173 0.184 0.309 0.294 0.289
AH 0.608 0.612 0.619 0.662 0.589 0.629 0.638 0.637 0.637 0.622 0.592 0.674 0.672 0.638 0.586
%AC 49.65 49.00 49.79 46.15 59.46 47.08 60.26 61.11 62.87 75.25 75.32 73.71 56.17 57.94 58.66
%AH 13.76 12.82 11.70 5.70 15.62 10.27 8.46 9.26 9.39 11.52 15.55 3.71 4.68 8.73 16.17
% E. Donor 35.89 36.18 38.09 40.46 43.84 36.80 51.79 51.85 53.49 63.73 59.77 70.00 51.49 49.21 42.49
Average 36.72 40.37 52.38 64.50 47.73
SD 1.20 3.52 0.96 5.16 4.68
Anova a a b d c
*Value with different lowercase letters in the same row are significantly different (p<0.05)

Table B17. Electron acceptor property of cell surface in pretreatment NaOH experiment (%)

0 0.05 0.07 0.1 0.12

Concentration (M)
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
A0 0.705 0.702 0.701 0.702 0.698 0.701 0.697 0.702 0.703 0.703 0.701 0.700 0.705 0.699 0.699
AE 0.595 0.591 0.591 0.593 0.596 0.592 0.657 0.662 0.672 0.664 0.639 0.629 0.620 0.614 0.629
AD 0.606 0.603 0.602 0.613 0.614 0.611 0.563 0.568 0.567 0.437 0.582 0.583 0.525 0.529 0.534
%AE 15.60 15.81 15.69 15.53 14.61 15.55 5.74 5.70 4.41 5.55 8.84 10.14 12.06 12.16 10.01
%AD 14.04 14.10 14.12 12.68 12.03 12.84 19.23 19.09 19.35 37.84 16.98 16.71 25.53 24.32 23.61
% E.Acceptor 1.56 1.71 1.57 2.85 2.58 2.71 -13.49 -13.39 -14.94 -32.29 -8.13 -6.57 -13.48 -12.16 -13.59
Average 1.61 2.71 -13.94 -15.66 -13.08
SD 0.08 0.14 0.87 14.42 0.79
Anova d c e b a
*Value with different lowercase letters in the same row are significantly different (p<0.05)

60
APPENDIX
Table B18. Electron donor property of cell surface in pretreatment HCl experiment (%)

0 0.05 0.07 0.1 0.12

Concentration (M)
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
A0 0.699 0.701 0.701 0.700 0.702 0.703 0.698 0.697 0.702 0.700 0.698 0.699 0.704 0.701 0.701
AC 0.560 0.559 0.566 0.536 0.519 0.523 0.486 0.485 0.488 0.528 0.528 0.521 0.517 0.515 0.498
AH 0.624 0.628 0.625 0.648 0.648 0.656 0.634 0.629 0.634 0.644 0.646 0.640 0.657 0.654 0.646
%AC 19.89 20.26 19.26 23.43 26.07 25.60 30.37 30.42 30.48 24.57 24.36 25.46 26.56 26.53 28.96
%AH 10.73 10.41 10.84 7.43 7.69 6.69 9.17 9.76 9.69 8.00 7.45 8.44 6.68 6.70 7.85
% E. Donor 9.16 9.84 8.42 16.00 18.38 18.92 21.20 20.66 20.80 16.57 16.91 17.02 19.89 19.83 21.11
Average 9.14 17.76 20.89 16.83 20.28
SD 0.71 1.55 0.28 0.23 0.73
Anova a b c b c
*Value with different lowercase letters in the same row are significantly different (p<0.05)

Table B19. Electron donor property of cell surface in pretreatment HCl experiment (%)

0 0.05 0.07 0.1 0.12

Concentration (M)
1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd
A0 0.699 0.701 0.701 0.700 0.702 0.703 0.698 0.697 0.702 0.700 0.698 0.699 0.704 0.701 0.701
AE 0.482 0.482 0.478 0.413 0.412 0.413 0.424 0.417 0.420 0.396 0.394 0.409 0.373 0.378 0.389
AD 0.639 0.648 0.644 0.613 0.611 0.614 0.623 0.628 0.621 0.631 0.644 0.618 0.629 0.624 0.611
%AE 31.04 31.24 31.81 41.00 41.31 41.25 39.26 40.17 40.17 43.43 43.55 41.49 46.79 46.08 44.51
%AD 8.58 7.56 8.13 12.43 12.96 12.66 10.74 9.90 11.54 9.86 7.74 11.59 10.65 10.98 12.84
% E. Acceptor 22.46 23.68 23.68 28.57 28.35 28.59 28.51 30.27 28.63 33.57 35.82 29.90 36.14 35.09 31.67
Average 23.27 28.50 29.14 33.10 34.30
SD 0.70 0.14 0.98 2.99 2.34
Anova a b b c c
*Value with different lowercase letters in the same row are significantly different (p<0.05)

61
APPENDIX

APPENDIX C. EXPERIMENTAL EQUIPMENT

Table C1. Equipment used in this study

Analytical weigh scale Infrared moisture analyzer pH meter

Vortex mixer Thermostatic tank Centrifugate

62
APPENDIX

UV-Vis spectrometer Homogenizer Stirrer

63