DOCTORAL THESIS

Effect of trehalose on the mechanical properties ofdeep-fried foods

LE NGOC DANG TRINH

Program of Food and AgriLife Science, Graduate
School of Integrated Sciences for Life, Hiroshima
University

March 2023

TABLE OF CONTENTS

List of abbreviations

iv

CHAPTER 1

1

Introduction

1

1.1. Background1

1.2. Purpose

5

CHAPTER 2

7

Fundamentals

72.1. Batter

72.2. Trehalose

7

2.3. Fried batter particles

8

2.4. Freeze-dried waxy corn starch solid

92.5. Isothermal mechanical glass transition10

2.6. Texture

13

2.7. Statistical analysis and fitting analysis

13

CHAPTER 3

14

Effect of trehalose content on the mechanical properties of fried batter particles

14

3.1. Introduction

14

iii

3.2. Methods

14

3.2.1. Sample preparation

14

3.2.2. Water sorption

14

3.2.3. Oil content

15

3.2.4. Isothermal mechanical relaxation

15

3.2.5. Texture properties

16

3.3. Results

16

3.3.1. Oil content of fried batter particles

16

3.3.2. Water sorption, water content, and aw of fried particles

17

3.3.3. Isothermal mechanical glass transition of fried batter particles

21

3.3.4. Fracture properties of fried batter particles

23

3.4. Conclusion of this chapter

26

CHAPTER 4

28

Effect of trehalose content on the fracture properties and true density of freeze-dried waxy
starch-trehalose system

28

4.1. Introduction

28

4.2. Methods

28

4.2.1. Sample preparation of freeze-dried porous solid

28

4.2.2. Fracture properties of freeze-dried porous solid

29

4.2.3. Sample preparation of freeze-dried powder

29

4.2.4. True density of freeze-dried powder

30

iii

4.3. Results30

4.3.1. Fracture properties of freeze-dried porous solids

30

4.3.2. True density of freeze-dried powders

32

4.4. Conclusion of this chapter

33

CHAPTER 5

35

General discussion

35

Conclusion

39

References40

Publication

45

Acknowledgement

46

iii

List of abbreviations

Tg: glass transition temperature (oC)aw: water activity (dimensionless)awc: critical water activity (dimensionless)
wc: critical water content (dimensionless)
GAB: Guggenheim, Anderson and de Boer
DM: dry matter
C: coefficient correcting monolayer sorption properties (dimensionless)
K: coefficient correcting multilayer sorption properties (dimensionless)
Wm: monolayer water content (g/100 g-DM)'F: mechanical relaxation force-drop (N)DSC: differential scanning calorimetry
TRA: thermal rheological analysisiv

CHAPTER 1 Introduction

1.1. BackgroundDeep-frying is one of popular cooking methods to produce dry foods having desirable andcharacteristic flavor and texture (Ziaiifar, 2008; Alnadif et al., 2014; Dana & Saguy, 2006;
Van Koerten et al., 2015). Deep-frying is achieved by immersing food stuff into the heatedoil (approximately 150~200 ºC) (Fakas, 1994; Ziaiifar, 2008). In the case of “tempura”,
various physical changes (such as starch gelatinization, water-evaporation, and oil-sorption)
occur competitively and/or synergistically in the batter during the deep-frying (Fakas, 1994;
Ziaiifar, 2008; Bordin et al., 2013; Oke et al., 2018). The fried batter (fry coating) covers thefood stuff of tempura products.
After the deep-frying, tempura is cooled down at room temperature, and the fry coating
becomes glassy state at least at the outer part. The glassy fry coating shows a favorable brittle
texture. When storage time is prolonged, the fry coating changes from brittle texture toductile texture because glass to rubber transition (briefly, glass transition) occurs throughwater sorption originating from the atmosphere moisture and/or from the food stuff. In foodindustry, it is important to maintain the brittle texture of the fry coating (glassy fry coating)as long as possible.
Temperature of which glass transition occurs is denoted as glass transition temperature (Tg).
Typical glass transition behavior of food products is shown in Fig. 1-1. Food product is inglassy (solid-like) state at a lower temperature than the Tg (point A in Fig. 1-1-a). Glassy
porous food shows brittle texture because of its high elasticity. The glassy food becomes1

rubbery (liquid-like) state at a higher temperature than the Tg (A to B in Fig. 1-1-a), and the
texture changes to ductile one because of the reduced elasticity. Glass transition also occurs
even at a constant temperature when water content changes, because the Tg of hydrophilic
amorphous materials decreases with an increase in the water content because of water
plasticizing effect. Thus, the glassy food becomes rubbery state, when the Tg becomes lower
than the holding temperature as the result of water sorption (A to C in Fig. 1-1-a). Water
content of which the Tg becomes 25 ºC (typical room temperature) has been described as
critical water content (wc). In addition, water activity (aw) of which the Tg becomes 25 ºC has
been described as critical water activity (awc). The higher wc and awc, the greater resistanceto the physical deteriorations induced by water sorption. To maintain brittle texture of fry
coating as long as possible (A to C’ in Fig. 1-1-b), it is important to elevate the wc and/or awc
of fry coating (wc to wc’ in Fig. 1-1-b).

2

B

25

(b)

Rubbery state(ductile texture)A

C

Glass transition temperature

Glass transition temperature(a)Glassy state(brittle texture)

25

Rubbery state(ductile texture)A

C’

Glassy state(brittle texture)

wc
Water content

wc
w c’
Water content

Fig. 1-1. Typical glass transition behavior of food products.

Differential scanning calorimetry (DSC) is the most common technique to determine the Tgof amorphous materials. Glass transition can be detected as an endothermic shift in DSC
curve, and Tg is evaluated from the onset point of the shift. In the case of starch-based foods,
it is difficult to determine the clear Tg because a minor endothermic shift is observed in a
broad temperature range. In addition, multiple thermal responses due to heterogeneouscomponents cover glass transition. In this case, thermomechanical approach is more effective(Kawai et al., 2014; Sogabe et al., 2018). In a previous study (Jothi et al., 2018), it wasdemonstrated that the glass transition of fried batter particles (i.e., “agedama” in Japanese)
could be detected by thermal rheological analysis (TRA). In the TRA measurement, the foodsample is compressed at a temperature below expected Tg and heated up to an enough hightemperature. Then, Tg of the sample can be confirmed as a force drop due to the softening3

induced by glass transition. It is known that thermal Tg determined by DSC does not always
agree with the Tg determined by thermomechanical approaches (Boonyai et al., 2007; Kasapis
et al., 2007; Ross et al., 2002). Thus, the Tg determined by thermomechanical approaches isdistinguished as “mechanical Tg”.
It is known that the Tg of amorphous materials can be controlled by the compositions(Balasubramanian et al., 2016; Roos et al., 1996). For example, Tg of amorphous polymers
decreases with an increase in small molecules because of their plasticizing effect (Figueroa
et al., 2016, Carvalho & Mitchell, 2001; Fan et al., 1996). However, the plasticizing effect ofsmall molecules on the mechanical Tg of the fried batter particles does not always obey to theplasticizing behavior. In our previous study (Jothi et al., 2020), it was demonstrated that themechanical Tg of fried batter particles could be elevated by the addition of trehalose(disaccharide). As one of possibilities, it is suggested that trehalose filled the defects in theamorphous amylopectin (the major component of the fried batter particles) and themechanical strength was enhanced (Fig. 1-2). This suggestion is based on “anti-plasticizing
effect” explained in the previous studies (Slade & Levine, 1995; Figueroa et al., 2016).

4

Mechanical strength

B

AC

Plasticizer content

A. Low dense glass

B. High dense glass

C. Rubber

Fig. 1-2. Model drawing of anti-plasticizing effect of small molecules on the polymer.

1.2. Purpose
As explained above, it was demonstrated that the mechanical Tg of fried batter particles
could be elevated by an addition of trehalose. In addition, the fried batter particles containing
trehalose maintained a brittle texture at a higher water content compared to non-additive one.

5

As the mechanism of the physical modification induced by trehalose, anti-plasticizing effectof trehalose on starch is suggested.
To clarify physical modification effect of trehalose on the fried batter particles in moredetail, this study aimed to understand effect of trehalose content on the mechanical propertiesof fried batter particles. In addition, freeze-dried waxy starch (amylopectin) was employed
as a model of fried batter particles. The freeze-dried starch solid has numerous pores formedby ice sublimation and the initial water content can be used to alter the porosity of the solid.
By dehydration, the porous solid becomes glassy state and has similar structure to fried batterparticles. Amylopectin is the major component of fried batter particles, and chemical
reactions and thermal degradation are highly diminished by freeze-drying. Thus, freeze-dried
waxy starch was expected to be an effective model for the better understanding of thephysical modification induced by trehalose addition.
In chapter 1 (this chapter), background and purpose of this study were stated. In chapter 2(Fundamentals), materials and experimental techniques used in this study were explained. In
Chapter 3, effect of trehalose content on the oil content, water content, aw, mechanical glasstransition, and texture properties of fry batter particles was reported. In Chapter 4, effect oftrehalose content on the texture properties and true density of freeze-dried waxy starch wasreported. In chapter 5, general discussion was provided according to the results. In Chapter
6, this study was concluded and outlook for the future was stated.

6

CHAPTER 2 Fundamentals

2.1. Batter
Batter for fry coating of “tempura” is prepared by the mixing of wheat flour and water. For
the brittle texture of fry coating, the development of gluten network during processing should
be suppressed as much as possible. Thus, this study employed wheat flour with a low protein
content (Nisshin Flour Milling Inc., Nagoya, Japan). According to the product information,
the wheat flour contains 8% protein and less than 1.7% lipid. In addition, batter was preparedby mixing wheat flour and chilled water in an iced water bath.

2.2. Trehalose
Trehalose has some advantages as a texture modifier of foods. Firstly, trehalose ischemically stable. Since trehalose is non-reducing disaccharide, non-enzymatic browning
reaction (Maillard reaction) does not occur essentially in food system (Ledl & Schleicher,1990). In addition, trehalose is resistant to hydrolysis. These are effective to diminish color
change to deep brown during frying process. Secondly, trehalose has a high Tg. Anhydrous
Tg of trehalose, sucrose, maltose, and lactose is 113 ºC, 68 ºC, 90 ºC, and 102 ºC, respectively(Kawai et al., 2005; Haque et al., 2006); trehalose is the highest anhydrous Tg among thedisaccharides. This study employed trehalose dihydrate (Hayashibara Co., Ltd., Okayama,
Japan).

7

2.3. Fried batter particles
This study employed fried batter particles (agedama) as a model sample for the fry coatingof tempura according to our previous study (Jothi et al., 2020) with minor modifications. A
micropipette with a 5 mm diameter hole and a digital oil bath with stirrer (EOS-200RD; AS
ONE Co., Osaka, Japan) were used (Fig. 2.1). An enough amount of canola oil was placedin the oil bath, and the temperature was set to 180 °C with the agitation at 250 rpm by usinga magnetic stir bar (diameter 8 mm and length 30 mm). The batter (approximately 4 ml) was
dropped into the oil by the micropipette over a period of 30 to 40 s. The batter was deep-friedat 180 ºC for 4 min. The fried batter particles were scattered on a stainless-steel tray withmesh (70 mm × 70 mm), and they were cooled down at room temperature for approximately
4 min. The samples were kept in a bag until analysis.

8micropipette
5 mmÈ dropchamber(I 208 mm x 123 mm)heat
sensorheaterstir bar
rpm

temp.

Fig. 2.1. Preparation of fried batter particles.

2.4. Freeze-dried waxy corn starch solid
Carbohydrate polymer becomes glassy state by dehydration. The freeze-dried starch solid
has numerous pores formed by ice sublimation. Porosity of the solid can be adjusted by initialwater content. Thus, freeze-dried starch solid can be employed as a model of fried batterparticles. Freeze-dried waxy corn starch solid was prepared according to a previous study(Sogabe et al., 2018) with minor modifications.

9

Waxy corn starch was put into a vial, and distilled water was added to adjust water content.
A magnetic stir bar was put into the vial, and the vial was covered by wrapping with film toprevent water evaporation. The vial was placed in a boiling water bath (98~100 ºC) andheated for 10 min with magnetic stirring to prevent the sedimentation of starch granules at
the initial stage of the heating. The gelatinized waxy starch was cooled at room temperature.
The gelatinized waxy starch was put into cylindrical holes (diameter 20 mm and height 10mm) in a mold and then frozen at -25 ºC for 16 h. The frozen starch was placed on a cooled
stage (-35 ºC) in a freeze-dryer and freeze-drying was carried out at approximately 11 Pa
with an increase in temperature from -35 ºC to 5 ºC over 34 h. The freeze-dried solids withnumerous pores were carefully removed from the mold. In order to remove residual water,
vacuum-drying was carried out at 110 ºC (stage temperature) for 8 h.

Fig. 2.2. Preparation of freeze-dried waxy starch solid.

2.5. Isothermal mechanical glass transition
As explained above, TRA is an effective tool to determine mechanical Tg of food system.
From the effect of water content on the mechanical Tg, water content of which the Tg becomes
25 ºC (typical room temperature) can be evaluated as wc. In addition, the wc can be converted
10

to awc though the effect of aw on the equilibrium water content (i.e., water sorption isotherm).
However, this approach to determine wc and awc needs many experimental data. As more
brief approach to determine wc and awc, isothermal mechanical relaxation measurement was
carried out using a rheometer (CR-150; Sun Scientific Corp., Ltd., Tokyo, Japan) according
to our previous study (Jothi et al., 2020).
Schematic drawing of isothermal mechanical relaxation measurement is shown in Fig. 2-3.
Fried batter particles (14-16 particles) were put into the sample cup (diameter 31.90 mm andheight 9.95 mm) and then placed on the stage of the rheometer at 25 ºC controlled by water
bath circulator (Fig. 2-3-a). The sample was compressed at a constant stress and held in thecompressed condition. In the holding process, force to maintain the initial strain decreases
because of mechanical relaxation. From the force-time curve, the force-drop (ΔF) between
the initial force and the force after the holding was evaluated (Fig. 2-3-b). Glassy sample
shows very little mechanical relaxation (low ΔF value) because of high elasticity. In contrast,
rubbery sample shows great mechanical relaxation (high ΔF value) because of low elasticity.
From effect of aw on the ΔF of samples, aw-range of which mechanical glass transition occurs
can be understood.

11

a)

b)

Glass
RubberFig. 2-3. Schematic drawing of isothermal mechanical relaxation measurement.

122.6. Texture
Texture of glassy porous food has commonly been investigated by rheometer (Jothi et al.,2018; Jothi et al., 2020; Sogabe et al., 2018). From the force-displacement curve, numerical
fracture peaks are detected. One piece of the fried batter particles was compressed with aplate plunger, and fracture force was determined from the first fracture point. In addition, thetotal number of fracture peaks was counted in the displacement range of up to 2.5 mm (Jothi
et al., 2018; Jothi et al., 2020). The fracture force and the number of peaks is expected tocorrespond to the hardness and brittleness, respectively.

2.7. Statistical analysis and fitting analysis
Analysis of variance (ANOVA) with Turkey’s HSD test (p < 0.05) and fitting analysis were
performed using KaleidaGraph software (version 3.6, Hulinks Inc., Tokyo, Japan).

13

CHAPTER 3 Effect of trehalose content on the mechanical properties of fried batterparticles

3.1. Introduction
To maintain the brittle texture of fry coating as long as possible, it is important to elevate
the Tg (wc and/or awc) of fry coating. In our previous study (Jothi et al., 2020), it wasdemonstrated that the mechanical Tg of fried batter particles could be elevated by an additionof trehalose. In addition, the fried batter particles added trehalose maintained a brittle texture
at a higher water content compared to non-additive one. To clarify physical modification
effect of trehalose on the fried batter particles in more detail, effect of trehalose content on
the oil content, water content, aw, mechanical glass transition, and texture properties of friedbatter particles was investigated.

3.2. Methods
3.2.1. Sample preparation
Fried batter particles were prepared as explained earlier (section 2.3). Wheat flour andchilled water were mixed at the weight ratio of 1.0 : 1.6, and trehalose (0, 5, 10, 20%, against
dry wheat flour) was added into there in an iced water bath. The batter was dropped into
heated oil and fried at 180 ºC for 4 min. The fried batter particles were scattered on a stainlesssteel tray with mesh and cooled down at room temperature for approximately 4 min.

3.2.2. Water sorption

14

The samples were vacuum-dried at 110 ºC (stage temperature) for 8 h. The fully driedsamples were equilibrated at 25 ºC for at least 2 weeks in a desiccator under various water
activity (aw) conditions adjusted by the following saturated salts (Greenspan, 1977): LiCl
(aw = 0.113),

CH3COOK(aw = 0.225),

MgCl2 (aw = 0.328),

K2CO3 (aw = 0.432),

Mg(NO3)2 (aw = 0.529), NaBr (aw = 0.576), KI (aw = 0.689), and NaCl (aw = 0.755).
Equilibrium water content (g/100 g-DM) of the samples was determined gravimetrically byvacuum-drying at 110 ºC (stage temperature) for 8 h. The measurements were performed intriplicate and the results were averaged.

3.2.3. Oil content
Oil content of the samples was investigated by the Soxhlet extraction method (AOAC,
2000). The samples were ground, and vacuum-dried at 110 ºC (stage temperature) for 8 h.
The fully dried samples (1 g) were defatted with diethyl ether at 60 ºC for 12 h using Soxhlet
extraction system (Yamato Scientific Co., LTD, Japan). The measurements were performedin triplicate, and the results were averaged.

3.2.4. Isothermal mechanical relaxation
The fried batter particles (14-16 particles) were set into the sample cup and then placed on
the stage of the texture meter at 25 ºC (Fig. 2-3-a). The sample was compressed at 80 N witha plate plunger and held in the compressed condition for 3 min. From the force-time curve,
ΔF (difference in the force between the initial force and the force after 3 min) wasinvestigated. The measurements were performed in triplicate and the results were averaged.

15

3.2.5. Texture properties
One piece of the fried batter particles was put on the sample stage and then compressed witha plate plunger (diameter 30 mm) at 0.5 mm/s. From the force-displacement curve, thefracture force determined from the first fracture point and the total number of fracture peaks
counted in the displacement range of up to 2.5 mm were evaluated (Jothi et al., 2018; Jothi
et al., 2020). The measurements were performed five times, and the results were averaged.

3.3. Results
3.3.1. Oil content of fried batter particles
Effect of trehalose content on the oil content of fried batter particles is shown in Fig. 3-1.
The oil content of 0% and 10% trehalose samples obtained in this study agreed well with thevalues (53% for 0% trehalose sample and 42% for 10% trehalose sample) reported in ourprevious study (Jothi et al., 2020). The oil content of fried batter particles significantly
decreased and then increased slightly with increased trehalose content. Trehalose is a watersoluble carbohydrate, and thus will prevent the oil-sorption of deep-fried foods. TaveraQuiroz et al. (2012) reported that water-soluble materials (methylcellulose-glycerol-sorbitol
mixtures) reduce oil sorption during the frying process. Similar effects of corn gum mixture
and wheat gum mixture (Sahin et al., 2005), wheat cellulose derivatives and wheat-sorbitol
mixtures (Garcıa et al., 2002), and oat bran (Lee & Inglett, 2007) on the oil content of deepfried samples are also reported.

16

70
60

a

b

Oil content (%)

50

d

c

40
30
20

10
0
0

5

10

15

20

Trehalose content (%)
Fig. 3-1. Effect of trehalose on the oil content of fried batter particles. Values are expressed
as mean ± SD. Different characters indicate significant difference (p < 0.05).

3.3.2. Water sorption, water content, and aw of fried batter particles
Effect of aw on the equilibrium water content of fried batter particles held at 25 ºC is shownin Fig. 3-2. The samples showed a sigmoidal water sorption behavior as similar to that forother amorphous starchy foods (Al-Muhtaseb et al., 2002; Kawas & Moreira, 2001). The
solid curves (water sorption isotherm) were obtained by fitting data to the GuggenheimAnderson-de Boer (GAB) model (Eq. 3-1),
W ∙C∙K∙aw
W = (1 – K ∙ a m) ∙(1 + (C – 1)∙K∙a )w(Eq. 3-1)w

17

where W is the equilibrium water content (g/100 g-DM) and Wm, C, and K are constant. The
Wm is the monolayer water content (g/100 g-DM). The C and K are correction factors for thesorption properties of the first layer with respect to the bulk liquid and for the properties ofthe multilayer with respect to the bulk liquid, respectively.
The GAB parameters (Wm, C, and K) are summarized in Table 3-1. The Wm values (2.482.71 g/100 g-DM) agreed well with the values (2.69 g/100 g-DM for 0% trehalose sample
and 2.44 g/100 g-DM for 10% trehalose samples) reported in our previous study (Jothi et al.,2020). The C and K values were slightly higher and lower, respectively, than those of theprevious study. As well-known, the GAB parameters are sensitive to differences inexperimental values; even a small difference in experimental values results in large
differences in GAB parameters (Fongin et al., 2007; Rahman & Al-Belushi, 2006). The GABparameters were used to convert water content to aw.

18

Fig. 3-2. Effect of aw on the equilibrium water content of fried batter particles. Values are
expressed as mean ± SD.

Table 3-1 GAB parameters for the fried batter particles
trehaloseWm%

g/100 g-DM

0

C

K

R2

2.71

10.8

0.95

0.998

5

2.71

12.5

0.94

0.997

10

2.48

41.0

1.00

0.995

20

2.57

24.4

0.95

0.999

2

R : determination coefficient

Effect of the trehalose content on the water content and aw of fried batter particles(unadjusted aw) is shown in Fig. 3-3. The aw of the samples was estimated from the water19hcontent through the water sorption isothermwith GAB parameters. The water content and aw
of 10% and 20% trehalose samples weree significantly lower than that of 0% and 5% trehalose
samples.

0.7

7

Water content (g/100g-DM)

a

5

a

0.6

a

0.5

4
3

0.4

b

b

0.3

b

b

Water activity

a

6

0.2

2
Water content

1

0.1

Water activity (non-adjust)
0

0
0

5

10

15

20

Trehalose content (%)

Fig. 3-3. Effect of trehalose content on the water content and aw of deep-fried samples(unadjusted aw). Values are expressed as mean ± SD. Different characters indicate
significant difference (p < 0.05).

20

3.3.3. Isothermal mechanical glass transition of fried batter particles
Typical isothermal mechanical relaxation curves are shown in Fig. 3-4. The force of thesamples decreased with increasing hold time due to mechanical relaxation. Mechanicalrelaxation nearly reached equilibrium within 3 min, and ∆F was evaluated from theisothermal mechanical relaxation curves. There was very little mechanical relaxation in the
0% trehalose sample having aw 0.113; this suggests that the sample was in a glassy state (highelasticity). On the other hand, the 0% trehalose sample having aw 0.529 showed a large
mechanical relaxation; this suggests that the sample was in a rubbery state (low elasticity).
The mechanical relaxation of 10% trehalose samples was much lower than that of 0%
trehalose samples at each aw.

Fig. 3-4. Isothermal mechanical relaxation curves for fried batter particles.

21

Effect of aw on ∆F of fried batter particles is shown in Fig. 3-5. The ∆F increased linearlywith increasing aw and became much greater above a certain aw. This suggests thatmechanical glass transition occurred at the aw because of water plasticizing. As explained
earlier, the aw of which the mechanical glass transition occurs at 25 ºC is denoted asmechanical awc. The mechanical awc of 0% and 10% trehalose samples is reported to be 0.509
and 0.649, respectively (Jothi et al., 2020). The mechanical awc values are within the turning
range of the aw-dependency of ∆F. Taking this into account, 10% and 20% trehalose samples
are higher mechanical awc than 0% and 5% trehalose samples.
In a comparison of the ∆F values among the glassy samples (open symbols in Fig. 3-5),
10% and 20% trehalose samples had much lower ∆F values than 0% and 5% trehalose
samples. This suggests that 10% and 20% trehalose samples were more rigid glassy material
than 0% and 5% trehalose samples.

22

Fig. 3-5. Effect of aw on ∆F of fried batter particles. Values are expressed as mean ± SD.
Open and closed symbols mean glassy and rubbery samples, respectively.

3.3.4. Fracture properties of fried batter particles
Typical force-displacement curves for 0% and 10% trehalose samples having aw = 0.113
and aw = 0.529 are shown in Fig. 3-6. The 10% trehalose sample having aw 0.113 (glassy
state) showed numerous small fracture peaks (brittle texture). On the other hand, the 0%
trehalose sample having aw 0.113 (glassy state) showed larger and smaller number of peaks
than the 10% trehalose sample. Although the 0% trehalose sample having aw 0.529 (rubbery
state) showed a large fracture peak (ductile texture), the 10% trehalose sample having
aw 0.529 (glassy state) still showed the fracture behavior due to brittle-like texture.

23

Fig. 3-6. Typical force-displacement curves for 0% and 10% trehalose samples having (a)aw 0.113 and (b) aw 0.529.

The effect of aw on the first fracture force of fried batter particles is shown in Fig. 3-7-a.
From Fig. 3-5, the samples indicated by open and closed symbols are expected to be in theglassy and rubbery states, respectively. The first fracture force of the glassy samples
increased linearly with an increase in aw. At each aw in the glassy samples (0.113-0.432),
there were no significant differences between 0% and 5% trehalose samples. The 10%trehalose sample showed a significantly lower value than the other samples (except for the
10% versus 20% trehalose samples having aw 0.225 and aw 0.432). At the rubbery state near24the glassy state, a maximum first fracture force was observed because of the large
displacement due to hard-ductile fracture. At higher aw, the first fracture force largely
decreased with increasing aw because of the structural softening. There were no clear
differences among the rubbery samples.
The effect of aw on the number of fracture peaks for fried batter particles is shown in Fig.
3-7-b. From Fig. 3-5, the samples indicated by open and closed symbols are expected to bein the glassy and rubbery states, respectively. The number of fracture peaks of glassy samples
decreased linearly with increasing aw. At each aw in the glassy state (0.113-0.432), the 10%
and 20% trehalose samples were significantly higher values than the 0% and 5% samples(except for 5%, 10%, and 20% trehalose samples having aw 0.225). There was no significant
difference between 10% and 20% trehalose samples. In addition, there were negligible
differences among the rubbery samples.

25

Fig. 3-7. The effect of aw on the first fracture force of fried batter particles. Values are
expressed as mean ± SD.

3.4. Conclusion of this chapter
The effect of trehalose content on the oil content, water content, aw, mechanical glasstransition, and texture properties of fried batter particles was investigated. From the results,
it was found that 10% trehalose sample was the lowest oil content, water content, and aw. In
addition, 10% trehalose sample was higher mechanical awc, lower first fracture force, andhigher number of fracture peaks than 0% and 5% trehalose samples. There were similareffects on the mechanical awc and fracture properties between 10% and 20% trehalose
samples. This suggests that 10% trehalose is an optimum additive content for the brittle

26

texture of deep-fried foods. The molecular mechanism of the results is discussed in Chapter
5 (General discussion).

27

CHAPTER 4 Effect of trehalose content on the fracture properties and true density offreeze-dried waxy starch-trehalose system

4.1. Introduction
In Chapter 3, the effect of trehalose content on the oil content, water content, aw, mechanicalglass transition, and texture properties of fried batter particles was investigated. To clarify
physical modification effect of trehalose on the fried batter particles in more detail, freezedried waxy starch (amylopectin) was employed as a model of fried batter particles.
Amylopectin is the major component of fried batter particles, and chemical reactions andthermal degradation are highly diminished in the freeze-dried sample. Thus, freeze-dried
waxy starch was expected to be an effective model for the better understanding of thephysical modification induced by trehalose addition. In this chapter, effect of trehalose
content on the texture properties and true density of freeze-dried waxy starch wasinvestigated.

4.2. Methods
4.2.1. Sample preparation for freeze-dried porous solid
The sample was prepared as described in the section of 2.4. In brief, waxy corn starch (5 g,dry basis) was put into a vial, and distilled water was added to produce 90% water content.
For the preparation of the samples containing trehalose, 5%, 10%, and 20% trehalose (perwaxy starch on dry weight basis) was added. Starch in the mixture was gelatinized, and thegelatinized mixture was put into cylindrical holes in a mold. The sample was freeze-dried,28

and porous solids were carefully removed from the mold. The solids were vacuum-dried, andthe fully dried samples were equilibrated at 25 ºC for at least two weeks in a desiccator underaw 0.113, 0.225, and 0.328. Since the purpose of this chapter was to understand the effect oftrehalose content on the fracture properties of glassy trehalose-amylopectin system, thesamples were adjusted to the low aw values.

4.2.2. Fracture properties of freeze-dried porous solid
The fracture properties of freeze-dried porous solids were investigated as described in thesection 2.6. In brief, freeze-dried solid was put on the sample stage and then compressed witha plunger (diameter 5 mm) at 0.5 mm/s. From the force-displacement curve, first fracture
force and the total number of fracture peaks in the displacement range of 2.5 mm were
evaluated. The measurements were performed five times, and the results were averaged.

4.2.3. Sample preparation for freeze-dried powder
The sample was prepared as described in the section 2.4. In brief, waxy corn starch (5 g,dry basis) was placed in a vial, and distilled water was added to produce 99% water content.
For the trehalose-added samples, 5%, 10%, and 20% trehalose (per waxy starch on dry weightbasis) was added. Starch in the mixture was gelatinized, and the gelatinized mixture wasdispensed onto a stainless-steel tray. For the preparation of 100% trehalose sample, a 10%
trehalose aqueous solution was dispensed onto stainless-steel tray. They were freeze-dried
and ground to obtain powder by using a mill. The powder samples were vacuum dried at 60ºC for 6 h.

29

4.2.4. True density of freeze-dried powder
The density of powder samples was determined using a gas pycnometer AccuPyc II 1345
(Shimazu Co., Japan). Helium was used as the displacement gas at an equilibration rate of
0.0345 kPa/min. The fully dried powder (0.05~0.25 mg) was put into the cell (diameter 11.4mm and height 11.66 mm), and a filtered cap was set under nitrogen gas condition. The
measurements were performed in triplicate and the results were average.

4.3. Results
4.3.1. Fracture properties of freeze-dried porous solids
The effect of aw on the first fracture force of the freeze-dried porous solid samples is shownin Fig. 4-1-a. The first fracture force linearly increased with increasing aw, depending on thetrehalose content. At each aw, the 0% trehalose sample was a significantly higher force thanthe other samples. In the contrast, the 10% trehalose sample showed a significantly lower
force than the others (except for 10% versus 20% trehalose having aw 0.113). There was no
significant difference in the values between 5% and 20% trehalose samples.
The effect of aw on the number of fracture peaks of the freeze-dried porous solid samples isshown in Fig. 4-1-b. The number of fracture peaks linearly decreased with an increase in aw,
depending on the trehalose content. At each aw, the 10% trehalose sample was a significantly
larger value than the others (except for 0% versus 10% trehalose having aw 0.113). There
was no significant difference in the values among the 0%, 5%, and 20% trehalose samples(except for 0% and 20% versus 5% having aw 0.225).

30

As stated above, fried batter particles containing 10% trehalose were lower first fracture
force and higher number of fracture peaks. This suggests that the 10% trehalose sample wasmore brittle texture than the others. A similar effect of 10% trehalose addition was also
observed in the freeze-dried porous solid samples. This suggests that molecular interaction
between trehalose and amylopectin is a major factor for the modified texture of fried batterparticles.

Fig. 4-1. The effect of aw on the first fracture force and the number of fracture peaks of thefreeze-dried waxy starch porous solids. Values are expressed as mean ± SD.

31

4.3.2. True density of freeze-dried powders
The effect of trehalose content on the true density of the freeze-dried powder samples isshown in Fig. 4-2. There is no significant difference in the true density between waxy starchand trehalose. Thus, it is expected that waxy starch-trehalose blend powder shows no or littlechange in the true density independent from trehalose content. However, 10% trehalose
sample showed a significantly higher value than the others. This result indicates that 10%
trehalose is embedded into the amylopectin. As stated above, it is suggested that trehalose
fills the defects in the amorphous amylopectin and the mechanical strength is enhanced. Thissuggestion is based on “anti-plasticizing effect” explained in the previous studies (Slade &Levine, 1995; Figueroa et al., 2016). The fact that amylopectin containing 10% trehalose
sample was significantly higher true density strongly supports the suggestion based on theanti-plasticizing effect.

32

1.85

a

1.80

Density (g/ cm3)

1.75
1.70
1.65

bc

1.60

b
bc
c

1.55
1.50
Waxy corn 5% Tre
starch only

10% Tre20% Tre Trehalose
onlyFig. 4-2. Effect of trehalose content on the true density of the freeze-dried powders.
Different characters indicate significant difference (p < 0.05).

4.4. Conclusion of this chapter
Effect of trehalose content on the fracture properties and true density of freeze-dried waxy
starch-trehalose system was investigated. The first fracture force and the number of fracture
peaks showed an almost equivalent behavior to those observed in fried batter particles. In
addition, it was demonstrated that freeze-dried waxy starch containing 10% trehalose sample
was significantly higher true density than the others. This result strongly supports thesuggestion based on the anti-plasticizing effect; trehalose fills the defects in the amorphous

33

amylopectin and the mechanical strength is enhanced. The molecular mechanism of theresults is discussed in Chapter 5 (General discussion).

34

Chapter 5 General discussion

Effect of trehalose content on the oil content, water content, aw, mechanical awc, and fracture
properties of fried batter particles was investigated. From the results, 10% trehalose sample
showed the lowest oil content, water content, aw among the samples. In addition, 10%trehalose sample was higher mechanical awc, lower first fracture force, and higher number offracture peaks than 0% and 5% trehalose samples. The similar effect of 10% trehalose
addition was also observed in the fracture properties of freeze-dried porous solids. These
results are discussed using model drawings as follows.
During deep-frying of batter, gelatinization of wheat starch occurs from approximately 60ºC (Contardo et al., 2016; Yusop et al., 2011) and water evaporation occurs fromapproximately 100 ºC. A major component of wheat flour is amylopectin. Since amylopectin
is a large polymer having many branched segments, oil is trapped in the many gap spaces ofthe branched segments under the dehydrated state.
When gelatinization of wheat starch occurs in the presence of trehalose, the trehalose ispartially incorporated into the gelatinized wheat starch because of the hydrophobic
interaction induced by oil. Thus, the trehalose molecules will act as “internal trehalose” (Fig.
5-1-a). The trehalose except for internal trehalose will locate at the interfacial parts ofamylopectin.
Since internal trehalose can fill the gap spaces of amylopectin, oil uptake is reduced. In
addition, evaporation of water is promoted because water molecules are excluded fromgelatinized wheat starch by internal trehalose. The fact that freeze-dried amylopectin

35containing 10% trehalose was the highest true density suggests that the gap space ofamylopectin is almost saturated by 10% trehalose addition. This corresponds to the lowest
oil content, water content, and aw of fried batter particles containing 10% trehalose. In thecase of 20% trehalose sample, it is thought that the amount of “interfacial trehalose”
increased essentially and generated further oil-spaces and hydration sites in the system. Thus,
20% trehalose sample will have been slightly higher oil content than 10% trehalose sample.
According to the suggestion that internal trehalose fills the gap spaces of amylopectin,
molecular structure of the amylopectin should be physically strengthened. This explains thereason why 10% and 20% trehalose samples were higher mechanical awc than 0% and 5%
trehalose samples.
Since fractures intrinsically occur at the physically weak points in the structure, interfacial
trehalose plays a role of fracture points. Thus, the first fracture force decreased, and thenumber of fracture peaks increased by the addition of trehalose. When excess amount oftrehalose is added in amylopectin, the amount of interfacial trehalose increases, and theinteraction between interfacial trehalose and amylopectin becomes physically strong. Thesesuggestions explain the reason why 10% trehalose is an optimum amount to improve thebrittle texture of fry coating.
Water intrinsically acts as similar to trehalose in amylopectin. However, the first fracture
force increased linearly, and the number of fracture peaks decreased linearly with increasing
aw, even in the glassy state. In addition, ∆F increased linearly with increasing aw. The
different effect between trehalose and water can be explained by the differences in themolecular dynamics. Water is a much lower molecular size and much higher molecular36

mobility than trehalose. Water molecules within the glassy polymer are mobile at ambient
temperature (Roudaut et al., 2004; Watanabe et al., 2019) and small enough to pass throughthe polymeric network. Thus, there will be an unclear distinction between internal andinterfacial water molecules in glassy amylopectin. Water molecules will fill gap spaces in theinternal and interfacial part of amylopectin that trehalose cannot sufficiently fill (Fig. 5-1-b),
and thus fracture points attributed to interactions between interfacial trehalose andamylopectin can be physically strengthened by water. Consequently, the first fracture force
increased linearly, and the number of fracture peaks decreased linearly with increasing aw,
even in the glassy state. In addition, amylopectin has branched segments with varying chain
lengths, and water will enhance the flexibility of the chains from short chain segments tolong chain segments with increasing aw as a local plasticizer. Thus, ∆F increases withincreasing aw under the glassy state. When all chains become flexible by increasing aw, aglass to rubber transition occurs, and the dependence of aw on ∆F becomes more apparent.
Trehalose, on the other hand, is immobile in the glassy matrix because the large molecules
cannot pass through the polymeric network unless the polymer matrix becomes flexible (i.e.,rubbery state).

37

fracture

Fig. 5-1. Model drawings of molecular interactions between trehalose and amylopectin.

38

Conclusion
It is known that the mechanical Tg of fried batter particles could be elevated by an additionof trehalose. In addition, fried batter particles containing trehalose maintained a brittle texture
at a higher water content compared to non-additive one. As the mechanism of the physicalmodification induced by trehalose, anti-plasticizing effect of trehalose on starch is suggested.
This study aimed to understand effect of trehalose content on the mechanical properties offried batter particles. For this purpose, freeze-dried waxy starch (amylopectin) was also
employed as a model of fried batter particles.
Effect of trehalose content on the oil content, water content, aw, mechanical awc, and fracture
properties of fried batter particles was investigated. From the results, 10% trehalose sample
showed the lowest oil content, water content, and aw. In addition, 10% trehalose sample washigher mechanical awc, lower first fracture force, and higher number of fracture peaks than
0% and 5% trehalose samples. The similar effect of 10% trehalose was also confirmed in thefracture properties of freeze-dried porous solids. These results are discussed based on theconcept of internal trehalose and interfacial trehalose.
This study employed fried batter particles as a fry coating model. Fry coating coats wet food
stuff, and thus diffusion of water molecules from the food stuff to the fry coating occurs
quickly. Since deep-fried foods are in non-steady state, there is a gradation of water content
and temperature between inner and outer parts. It is practically important to understand the
kinetic properties of fry coating. These are subjects in further studies.

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Publication
Le, T. N. D., Matsumoto, A., & Kawai, K. (2021). Effects of trehalose content and water
activity on the fracture properties of deep-fried wheat flour particles and freeze-dried porous
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45

Acknowledgement
Foremost, I would like to give my sincere thanks to my supervisor Professor Kiyoshi Kawai,
who gave me a chance to study about food engineering which I was not familiar with. A
deepest gratitude must be given for his kind supports, comments, and encouragement for any
aspects of my study, student life and career path while I am in Japan. He is always willing to
be my help, and his command and experience are as a force for me to manage and overcome
troubles during more than four years. At this point, I also want to thank the advisors,
Professor Yoshio Hagura and Professor Rumi Tominaga for the kind supports. Thank you
for knowledge and experiences you have taught me which have been valuable and helpful
for my thesis project.
As a recipient of scholarships, I would like to express my sincere gratitude to the Ministryof Education, Culture, Sports, Science and Technology Japan (MEXT) as a scholarship donor
for my first year of doctoral course. The fellowship supporting (living expenses and researchexpenses) for the rest of my doctoral student life comes from the Japan Science andTechnology Agency.
A deeply thanks must go for my parents and friends for their advice and attention. Thanks
for being a part of my life to help me improve myself and comfort my home sickness
throughout my life. Thank you for all those that you have taught, sacrificed for me, andalways stood by my side whenever I need a hand.
I am also thankful to Food Engineering lab mates for willingness to give essential supports
on my work and technical problems.

46