Accepted Manuscript

Review on Identification, Underlying Mechanisms and Evaluation of Freezing

Damage

Mohsen Dalvi-Isfahan, Piyush Kumar Jha, Javad Tavakoli, Amir Daraei-

Garmakhany, Epameinondas Xanthakis, Alain Le-Bail

PII: S0260-8774(19)30109-8

DOI: 10.1016/j.jfoodeng.2019.03.011

Reference: JFOE 9551

To appear in: Journal of Food Engineering

Received Date: 01 November 2018

Accepted Date: 15 March 2019

Please cite this article as: Mohsen Dalvi-Isfahan, Piyush Kumar Jha, Javad Tavakoli, Amir Daraei-
Garmakhany, Epameinondas Xanthakis, Alain Le-Bail, Review on Identification, Underlying
Mechanisms and Evaluation of Freezing Damage, Journal of Food Engineering (2019), doi: 10.1016
/j.jfoodeng.2019.03.011

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 ACCEPTED MANUSCRIPT

1 Review on Identification, Underlying Mechanisms and Evaluation of Freezing

2 Damage
3

4 Abbreviated running title

5 Evaluation of deteriorative changes in the freezing process

6

7 Mohsen Dalvi-Isfahan 1, Piyush Kumar Jha 2,3, Javad Tavakoli 1, Amir Daraei-Garmakhany 4
8 Epameinondas Xanthakis 5, Alain Le-Bail 2,3
9

10 1 Faculty of agriculture, Department of Food Science and Technology, Jahrom University,

11 Jahrom, Fars, Iran
12 2 ONIRIS, CS 82225, 44322 Nantes cedex 3, France

13 3 UMR GEPEA CNRS 6144 - ONIRIS, 44322 Nantes cedex 3, France

14 4 Department of Food Science and Technology, Toyserkan Faculty of Industrial

15 Engineering, Bu-Ali Sina University, Hamadan, Iran

16 5 RISE-Agrifood & Bioscience, Box 5401, SE-40229 Gothenburg, Sweden

17
18

19
20

21

22

23

24

25

26 *CORRESPONDING AUTHOR: Mohsen Dalvi-Isfahan

27 Email: Mohsen.Dalvi@gmail.com, Dalvi@jahromu.ac.ir

28 Faculty of agriculture, Department of Food Science and Technology, Jahrom University, Jahrom, Fars,

29 Iran.

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30 Abstract
31 Although freezing is known as the best method of food preservation, physical and chemical
32 changes that occur to the cellular structure during processing and storage may damage the quality
33 of food products. Most freeze damages are associated with ice crystal morphology (size, number,
34 shape and distribution) which in turn affects the microstructure of the frozen food. Therefore, the
35 evaluation of frozen food microstructure provides opportunities for monitoring the ice crystal
36 morphology and also identifies freeze damage at cellular level which can be linked with the final
37 quality of frozen food products. In this review, the most important physical damages that occur
38 during freezing and storage of food matrices are described. In addition, methods for evaluating
39 and observing the morphology of ice crystals and microstructure of frozen food stuffs are
40 comprehensively discussed. An understanding of the freeze damage and their relationship with ice
41 crystal morphology can contribute to the improvement of the freezing process as well as to the
42 frozen product quality.
43 Keywords: Freeze damage, Microscopy, Cryoconcentration, Mechanical damage, Freeze burn,
44 Recrystallization
45 Contents
46 1. Introduction
47 2. Basic physical phenomena in the freezing of tissues
48 2.1. Mechanical damage
49 2.2. Cryoconcentration (Freeze concentration)
50 2.3. Freezer burn
51 2.4. Maturation (Recrystallization)
52 3. Evaluation of Frozen Food Microstructure
53 3.1. Light microscopy
54 3.1.1. Image processing
55 3.1.2. Ice crystal shape
56 3.1.3. Ice crystal size
57 3.2. Electron microscopy
58 3.2.1. SEM/Environmental-SEM/Cryo-SEM
59 3.2.2. Environmental scanning electron microscopy (ESEM)
60 3.2.3. Atomic force microscopy (AFM)
61 3.2.4. Confocal laser scanning microscopy
62 3.2.5. Micro-Slicer Image Processing System (MSIPS)
63 3.3. Micro X-ray computed tomography and image analysis
64 3.4. Hyperspectral imaging, near infrared and Raman spectroscopy
65 3.5. NMR/MRI
66 3.6. DSC (differential scanning calorimetry)
67 3.7. Other quality evaluation techniques
68 4. Conclusion

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69 1. Introduction
70 Most of the food materials are distinguished by high water content and nutrient density which
71 make them prone to deterioration and quality losses. One of the main strategies that can be used
72 to increase shelf life and maintain product quality is freezing (Reid, 1994). During freezing, part
73 of the water content of the foodstuff is converted into ice crystals due to the phase transition. The
74 amount of water that turns into ice depends on the freezing temperature and product composition
75 (Fellows, 2017). Figure 1 shows changes in the ice content at the center of a typical food matrix
76 during freezing as a function of temperature. As it can be seen the majority of ice content is formed
77 at a 5 oC temperature range bellow the initial freezing point. This temperature interval is considered
78 as an important phase of crystallization zone during the freezing process (Le-Bail, Chapleau,
79 Anton-De Lamballerie, and Vignolle, 2008). The food product reaches a critical temperature zone
80 at this early stage of freezing and remains in this zone for a significant duration at which quality
81 degradation of food occurs as a result of exposure to a highly concentrated aqueous solution.
82 Therefore, the faster the food product passes through that critical zone, the lower will be the freeze
83 damage. The span of the critical zone has been defined between TIFP (Initial Freezing Point
84 Temperature) and 7°C below TIFP by (Añón and Calvelo, 1980). The International Institute of
85 refrigeration proposes to consider that a food is frozen if the temperature has been lowered to -
86 10°C or to a temperature for which 80% of the freezable water has been frozen. The freezing rate
87 is the term used to compare the velocity of operation to pass through the critical zone of ice crystal
88 formation and it determines both the number and the size of the formed ice crystals. Generally,
89 faster freezing rate, leads to a greater number of ice crystals with a smaller median size and more
90 uniform size distribution which results in fewer internal changes in structure of frozen food
91 materials (Li et al., 2018).
92 A variety of empirical models has also shown the dependency between freezing rate and ice crystal
93 size in different products such as beef (Bevilacqua et al., 1979), gelatin gels (Woinet et al., 1998;
94 Chevalier et al., 2000), sucrose solutions (Reid, 1980), apple tissues (Bomben and King, 1982),
95 ice cream (Russell et al., 1999). The results of those models showed that the ice crystal size varies
96 inversely with the square root of the freezing rate and this dependence can be fitted by a power
97 law. The change in frozen food quality is caused by a mechanical effect induced by ice crystals
98 formation and also by a biochemical stress induced by the cryoconcentration effect.

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99 The aim of the present study is to identify and investigate the main physical deteriorative and
100 undesirable changes that occur during freezing and seeks to describe the underlying mechanisms.
101 In addition, since most of deterioration changes that occur during freezing are rooted at a
102 microstructural level, studying frozen food microstructure provides the opportunity to monitor ice
103 crystal morphology. In the present article the evaluation methods currently being used for
104 identifying the freeze damage of different food matrices are also comprehensively being discussed.
105 2. Basic physical phenomena in the freezing of tissues
106 Freezing within food products may cause several harmful effects including mechanical damage,
107 cryoconcentration, freeze burn and recrystallization. These phenomena may play a substantial role
108 in the disruption of metabolic systems, dislocation of enzyme systems, cell membrane damage and
109 eventually cell lysis. Hereunder, the explanation of the four most common physical damages,
110 underlying causes and ways to prevent them are being discussed.
111 2.1. Mechanical damage
112 When food materials being subjected to cooling below their freezing point, they undergo a cryo-
113 deformation resulted from various combined actions: (i) expansion of water volume by about 9%
114 while other components of the foodstuff contract as the temperature is decreased. The
115 heterogeneity of the spatial distribution of water in food product lead to non-uniform volume
116 change throughout the food material. (ii) Variations in thermal and mechanical properties with
117 temperature drop between the frozen and the unfrozen part of the food material. For instance, a
118 sharp rise in the Young’s modulus and a gradual reduction in Poisson’s ratio values could be also
119 observed during freezing. (iii) The stress type on surface and core part of food can vary at the
120 different stages of freezing (Dalvi-Isfahan et al., 2017b; Jahanbakhshian et al., 2017; Tremeac et
121 al., 2008; Zartizky and 2011). In the early stages of freezing, expansion of water due to phase
122 change leads to uniform tensile stresses in the unfrozen core and tensile radial stresses and
123 compressive tangential stresses in the outer surface of frozen food (shell). On the other hand, after
124 solidification and expansion, solid ice contracts, the shell frozen body is then exposed to tangential
125 tensile stress instead of compression at the beginning of freezing. This yields in micro-cracks
126 unless the frozen shell is still plastic enough to relieve the stress (Ben Aissa et al., 2008; Pham et
127 al., 2006; Shi et al., 1998) (Figure 2).
128 In order to understand better the mechanism that can contribute on the development of cracks
129 during freezing and propose a new protocol to avoid mechanical failure during freezing,

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130 thermomechanical stress modeling has been a useful tool. In this regard, Pham et al. (2005, 2006)
131 and Tremeac et al. (2007) simulated thermomechanical stress on a spherical and a two layered
132 food with elastic and viscoelastic model respectively. They assessed that total strain is the sum of
133 thermal strain and mechanical strain. Since heat transfer is faster than stress relaxation, a decoupled
134 approach was used with heat transfer computation followed by stress and strain calculations in
135 their models. Their models showed that cracking during freezing is more likely caused by tensile
136 tangential stresses induced by the expansion followed by contraction and vitrification at the
137 surface. The results obtained are in agreement with the radially oriented pattern of cracks that
138 appear in cryogenically frozen food. They concluded that in such case, the strain do not have time
139 to relax; they accumulate and overpass the rupture stress of the food. This issue is known as one
140 of the most problematic topics in the cryogenic method. However, this problem could be alleviated
141 by using precooling or by reduction of the temperature difference between the product and the
142 freezing medium (Zartizky, 2011). The physical properties of the food material including moisture
143 content, young modulus, porosity and density may also affect the susceptibility of food to crack
144 formation. Products with higher densities and lower porosity are generally more vulnerable to
145 freeze-cracking (Kim and Hung, 1994).
146 Another form of mechanical injury induced by freezing can be seen in two layer food material like
147 part baked bread in which some part of crust separate physically from the crumb and will be flaked
148 off during final baking (crust flaking). Crust flaking could be caused by differential strains which
149 induced by different moisture content within the loaf where crust is too rigid and it could not
150 withstand the strain imposed by the crumb (Ben Aissa et al., 2008; Hamdami et al., 2007). A
151 similar problem may occur in other two layer food materials such as an ice cream coated with
152 chocolate or chicken nugget (Jahanbakhshian et al., 2017; Tremeac et al., 2007).
153 2.2. Cryoconcentration (Freeze concentration)
154 Freezing usually occurs outside the cell first, due to a lower concentration of solutes. The formation
155 of ice crystals in the extracellular fluid changes the osmotic balance within the intracellular fluid
156 immediately. If the freezing rate is slow, there will be sufficient time for water to diffuse out of
157 the cell in an attempt to balance the chemical potential between the two compartments resulting in
158 dehydration of the cell and increased solute concentration. The increase of solute concentration
159 can affect the pH and the ionic strength of the unfrozen liquid as well as can cause disruption of
160 an emulsion and even protein denaturation (Fellows, 2017). It is apparent, that both dehydration

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161 and solute concentration have destructive effects on the quality of food and damage to the cell
162 during freezing (Rahman, 2007). Even application of high freezing rate may also significantly
163 reduce the cell viability by creation of intracellular ice crystal and freeze damage to cell membrane
164 (Baier-Schenk et al., 2005a; Ban et al., 2016). This issue should be taken into consideration in food
165 industry for retaining the viability of beneficial microorganisms during freezing process such as
166 frozen starter culture or frozen baker’s yeast in products like probiotic in ice-cream or yeast in
167 frozen dough (Mohammadi et al., 2011; Ribotta et al., 2003; Selomulyo and Zhou, 2007).
168 Therefore, an appropriate method must be chosen with respect to the assurance of a balance
169 between the risk of intracellular ice crystallization and cell dehydration by osmotic effect where
170 crystallization and mass transfer are in competition. For example, the importance of choosing
171 optimal freezing rate on frozen croissant dough was also pointed by the findings of a recent survey
172 conducted by Ban et al. (2016). Their results indicated that the freezing rate is the dominant factor
173 affecting the ice crystal size in dough which in turn affects the yeast viability and dough quality.
174 Additionally, in their results was also mentioned that the final temperature played an essential role
175 in yeast death induced in response to cytoplasmic ice crystal formation.
176 2.3. Freezer burn
177 Other form of dehydration or desiccation related damage that may occur is during frozen storage,
178 especially in unpackaged or poorly packaged (such as damaged packs, poor sealing of pouches,
179 greater air in pouches) food materials, which is known as freezer burn. According to Schmidt &
180 Lee (2009), the term freezer burn encompasses dehydration that can occur on the surface of frozen
181 foods, over time during frozen storage which is associated with degradation in color, texture, and
182 flavor. This phenomenon occurs when water sublimates from the surface of the food product due
183 to the difference between the water vapor pressure (Vp) of frozen food and the surrounding
184 environment (Ashby, James, & Kramer, 1973; Bell, 2001; Fennema, 1973a; Kawai & Hagiwara,
185 2018; Rahman, 2007; Schmidt & Lee, 2009). More precisely, the sublimation of ice from the food
186 surface is governed by the vapour pressure gradient developed between the surface of the food
187 (Vp-ice food), the water vapor in the air (Vp-air), and the ice on the refrigerator coils (Vp-
188 refrigeration coils), where the order of magnitude is Vp-ice food > Vp-air > Vp-refrigeration coils.
189 Since the Vp of ice at the surface of food is greater than the vapor pressure of water in the air
190 (Ashby et al., 1973; Schmidt & Lee, 2009), ice sublimates in order to equilibrate with the vapor
191 pressure of water in the air. In the meantime, water from the air condenses on to the ice at the

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192 refrigeration coils (the coldest part in the freezer) as the Vp-refrigeration coils < Vp-air (Ashy and
193 James 1973; Schmidt & Lee, 2009). The sublimation of water is enhanced by cycles of self-
194 defrosting (particularly in self-defrosting systems) or temperature fluctuation that melts frost
195 from the evaporator coil leading to an increased Vp gradient (Schmidt & Lee, 2009). The presence
196 of head space air (loose packing) also contributes to the freezer burn which is referred as intra-
197 package freezer burn condition manifested by frost or “snow” within the pack (Bell, 2001). Freezer
198 burn causes serious deterioration in quality and value of frozen food through weight loss and
199 increased exposure to oxygen which irreversibly alter color, texture, and flavor of frozen food
200 (Ashby, James, & Kramer, 1973; Bell, 2001; Fennema, 1973a; Kawai & Hagiwara, 2018; Rahman,
201 2007; Schmidt & Lee, 2009). Freezer burn can be avoided by using a variety of techniques such
202 as, glazing, humidification, lowering of storage temperatures, vacuum packaging (reduces the
203 freezer burn by avoiding the food from coming in direct contact with the cold dry air in the freezer
204 and by eliminating air from packaging head space) and packaging in container or film with low
205 water vapor permeability (Barney & Bedford, 2008; Day, 2008; Fennema, 1973a; Zaritzky, 2009).
206 2.4. Maturation (Recrystallization)
207 The food quality degradation during frozen storage continues since there is a change in the number,
208 size, shape, orientation, or perfection of crystals after the completion of the initial solidification.
209 The most commonly used term for this change (in ice crystals morphology) is ‘recrystallization’.
210 The recrystallization of ice in food is marked by (i) an increase in the average size of ice crystals,
211 (ii) a decrease in number of crystals and (iii) a minimization of surface free energy of the entire
212 crystal phase (Chaves & Zaritzky, 2018; Fennema, 1973b; Kawai & Hagiwara, 2018; Martino &
213 Zaritzky, 1988, 1989; Pham & Mawson, 1997; Zaritzky, 2000). The recrystallization occurs
214 because the system tends toward a state of equilibrium wherein free energy is minimized and the
215 chemical potential is equalized among all phases (Fennema, 1973b). For instance, the small size
216 ice crystals (which are thermodynamically unstable) offer a high surface free energy (due to higher
217 surface/volume ratio) to the system. When the free energy is minimized, the total number of
218 crystals decreases at constant ice phase volume, while their mean size increases. Similarly, ice
219 crystals with rough surface (higher surface/volume ratio) will have a higher surface free energy
220 and in an effort to minimize its energy level it attains more compact structure (i.e. rounding off or
221 smoothing of ice surface) with a smaller surface to volume ratio (low surface free energy). In short,
222 recrystallization is directly related to the movement of grain boundary and the interfacial tension

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223 is considered as a driving force of this phenomenon (Chaves & Zaritzky, 2018; Fennema, 1973b;
224 Martino & Zaritzky, 1988, 1989; Pham & Mawson, 1997; Zaritzky, 2000). Different types of
225 recrystallization have been observed during food storage but more attention has been paid to the
226 Ostwald ripening, which is an isothermal process, where small size ice crystals with three or four
227 sides show concave surfaces. In that case the molecules are less surrounded than their neighbors
228 of the other side of the interface and tend to escape because they are energetically more unstable.
229 A consequence of reduced binding energy is a drop in melting point with crystal radius, according
230 to Kelvin equation (or Gibbs-Thomson equation). (Chaves & Zaritzky, 2018; Martino & Zaritzky,
231 1988, 1989; Zaritzky, 2000). Finally, it can be expected that, water molecules tend to detach from
232 small ice crystals, then diffuse through freeze concentrated solution and finally, redeposit on the
233 surfaces of larger crystals in favor of lowering the surface energy of the system. Therefore, all
234 smaller crystals shrink, while larger crystals grow, and overall the average size will increase (Pham
235 and Mawson, 1997; Hagiwara, Hartel, & Matsukawa, 2006). In other words, the main consecutive
236 stages of desorption from surface of small ice crystals, are the diffusion through the bulk matrix,
237 and adsorption by surface attachment on larger ice crystals. The two last stages, and therefore the
238 Ostwald ripening, are considered as limiting steps in the case of concentrated solutions.
239 Lifshitz-Slyozov Wagner theory, known as the LSW has been proposed to explain the Ostwald
240 ripening mechanism for microstructural coarsening in the liquid-phase sintering (Clark, 2014).
241 Figure 3 shows the ice crystal growth rate (change in diameter of ice crystals during storage) as
242 a function of radius of ice crystal, which are determined according to the LSW theory. As it can
243 be seen, the point at which the curve crosses the x axis, shows no changes in radius of ice crystals
244 throughout the frozen storage (da/dt=0), this point represents the mean radius of ice crystal (ac). In
245 other words, there is a single crystal size (ac) that is in equilibrium at that temperature and since
246 all crystals have the same size and exactly equal radius, no change in their radius can be seen over
247 time. Hence, a maximum growth rate it is expected at the point where there is the greatest
248 difference among the ice crystals size. By decreasing the radius of ice crystal from the point
249 da/dt=0, the growth rate showed a decreasing trend (da/dt< 0). This crystal radius – growth rate
250 relationship means that ice crystals with sizes that are smaller than an average radius of the ice
251 crystals in a solution (critical radius) will be shrinking or dissolving over time. On the other hand,
252 an increase of the growth rate can be observed for larger radius values, which corresponds to an
253 increase in size of the larger ice crystals over time (da/dt> 0) (Clark, 2014). The dotted line at the

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254 top part of the figure 3 shows the change in the growth rate of ice crystal as a function of time, the
255 graph illustrates a decreasing trend with the ice crystals radius change upon storage time.
256 Several factors have been identified to affect Ostwald ripening, such as temperature and
257 diffusivity. Temperature is known to affect the kinetics of chemical reactions such as interfacial
258 energy, growth rate coefficients, and equilibrium solubility which are involved in Ostwald
259 ripening process. Rising the temperatures can cause an increase of the reaction velocity and
260 subsequently an enhanced recrystallization could be expected (Madras and McCoy, 2003).
261 Hagiwara et al. (2006) studied the effect of diffusivity (water mobility) on recrystallization rate of
262 freeze-concentrated matrices such as sugar solutions with different sugar types, concentrations,
263 and temperatures. Water mobility was measured via the self-diffusion coefficient of water
264 component obtained by pulse field gradient stimulated echo proton NMR. Results showed that the
265 self-diffusion coefficient of the water component in the freeze-concentrated matrix was a useful
266 parameter to predict and control recrystallization rate.
267 In addition to Ostwald ripening (migratory recrystallization) mentioned above, other types of
268 recrystallization that can be found in food systems include: accretion, iso-mass and melt-refreeze.
269 Recrystallization in ice-cream is mainly influenced by the two latter aforementioned types during
270 frozen storage (Cook and Hartel, 2010; Donhowe and Hartel, 1996a, 1996b). Accretion is the
271 merging of two crystals when they are in contact in the presence of an unfrozen solution, while
272 iso-mass recrystallization involves rounding of edges and other sharp crystal features. Melt-
273 refreeze recrystallization takes place during temperature fluctuations. When the temperature rises,
274 ice crystals partly melt and become smaller but the low supercooling for nucleation during the
275 subsequent freezing cycles does not favor nucleation. As a result, new nuclei are not being formed
276 and simultaneous growth of the existing nuclei takes place. In order to inhibit ice recrystallization,
277 further than the control of the parameters described above (temperature increase and temperature
278 oscillation during storage (Donhowe and Hartel, 1996a, 1996b), and the reduction of water
279 diffusivity (by addition of sweeteners)) (Hagiwara et al. (2006), the addition of stabilizer and ice
280 structuring proteins (ISPs) as a novel food ingredient have been studied (Hagiwara et al., 2011;
281 Harper & Shoemaker, 1983; Kawai & Hagiwara, 2018; Knight & Duman, 1986; Knight, Wen, &
282 Laursen, 1995; MacDonald & Lanier, 1997; Pham & Mawson, 1997; Smith & Schwartzberg,
283 1985; Warren, Hague, Corotto, & Mueller, 1993). The ability of several sweeteners and stabilizers
284 (sucrose solution, corn syrup, high fructose corn syrup (HFCS), and combinations of all these

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285 aforementioned sugars with locust bean gum (LBG)/carrageenan/xanthan/gelatin) in controlling

286 recrystallization in frozen sample have been studied by few researchers (Buyong & Fennema,
287 1988; Harper & Shoemaker, 1983; Miller-livney & Hartel, 1997; Smith & Schwartzberg, 1985).
288 Harper & Shoemaker (1983) found that crystal growth (during recrystallization) was affected by
289 the addition of sweetener, but not by locust gum. Miller-livney & Hartel (1997) reported that the
290 recrystallisation rate, for a given stabilizer, was affected by both sweetener type and storage
291 temperature. For instance, among the ice scream storage conditions of their study, 20 DE (dextrose
292 equivalents) corn syrup and HFCS showed the lowest and the highest rates of recrystallization
293 respectively. When HFCS or sucrose was used in combination of lower temperature (–15 °C) and
294 lower freezing point temperature, as well as under combination of higher temperature (–5.2 °C)
295 and higher freezing point temperature in the presence of 20 DE corn syrup the stabilizers had a
296 pronounced effect on recrystallization. Among the stabilizers used, carrageenan and carrageenan
297 and LBG were most effective in retarding ice crystal growth, but not in all the tested cases. Xanthan
298 gum and gelatin retarded ice recrystallization only at –15 °C and when combined with sucrose or
299 high fructose corn syrup. Smith & Schwartzberg (1985) observed that the recrystallization rate
300 depended on the concentration of sweetener and stabilizer. For instance, the overall mass transfer
301 coefficient decreased roughly from 2 mm/s to 0.5 mm/s when sucrose concentration was increased
302 from 10% to 42%. Similarly, in 10% sucrose solutions, mass transfer coefficient decreased from
303 roughly 2 mm/s to 1 mm/s when added gelatin increased from 0% to 0.5% (Smith & Schwartzberg,
304 1985). Buyong & Fennema (1988) reported that 0.28% gelatin (w/w) did not affect the
305 recrystallization rate of ice in ice cream mix during 2 weeks storage at – 15 ℃. The exact
306 mechanism of action of stabilizers to inhibit ice crystallization process is unknown and some
307 probable mechanisms have been discussed by (Miller-livney & Hartel, 1997, Van Westen & Groot,
308 2018).
309 With respect to the ISPs, natural (antifreeze glycopeptides (AFGP) and peptides (AFP)),
310 genetically engineered and synthetic (analogs of the antifreeze protein or non antifreezing proteins)
311 peptides have been reported capable of minimizing recrystallization by adsorption-inhibition
312 mechanism (Hagiwara et al., 2011; Kawai & Hagiwara, 2018; Knight & Duman, 1986; Knight et
313 al., 1995; MacDonald & Lanier, 1997; Pham & Mawson, 1997; Warren et al., 1993). These
314 peptides are adsorbed to the specific plane of ice crystals and thus inhibit the thermodynamically
315 favored ice crystals to grow, while inhibit the boundary movement of ice grains necessary for ice

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316 coarsening (Hagiwara et al., 2011; Kawai & Hagiwara, 2018; Knight et al., 1995; MacDonald &
317 Lanier, 1997; Pham & Mawson, 1997). However, when salt levels were increased, only peptides
318 with antifreeze activity seemed to be effective. It is worth mentioning that in the case of non
319 antifreezing protein when salts are present, liquid inclusions are formed in which peptides become
320 concentrated. Antifreeze proteins have the freedom to orient themselves, hence they can bind at
321 the liquid-ice interface and inhibit recrystallization by preventing the inclusions from migrating
322 (Hagiwara et al., 2011; Knight et al., 1995; MacDonald & Lanier, 1997; Pham & Mawson, 1997).
323 Hagiwara et al. (2011) reported that supression effect of AFP on recrystallisation rate depended
324 on the concentration AFP being used. The ability of peptides derived from gelatin hydrolysate to
325 inhibit ice crystal growth in a model ice cream mix was also studied by Damodaran (2007) and
326 Damodaran & Wang (2017). Their results showed that the short peptides in gelatin hydrolysate
327 were able to inhibit ice crystal growth in an ice cream mix and the cationic peptides were much
328 more effective than anionic/neutral peptides.
329 3. Evaluation of Frozen Food Microstructure
330 Apart from the freeze damage mechanisms, freezing may change the quality of food products and
331 downgrade their value. These changes are most closely associated with microstructural changes in
332 food materials (Aguilera and Stanley, 1999). Therefore, it is necessary to investigate the
333 microstructure of frozen food and visualize freeze damage at cellular level. Increase the depth of
334 our understanding of the microstructure of frozen food will offer new insight on texture evolution
335 during freezing. Moreover, it can assist on the development of better freezing protocols aiming to
336 minimize the freeze damage of the products. Up to now, apart from various kinds of light and
337 electron microscopic methods, other analytical tools such as non-invasive imaging techniques
338 which include X-ray micro-computed tomography (Mousavi, Miri, Cox, & Fryer, 2005, 2007),
339 nuclear magnetic resonance (NMR) spectroscopy (Hills & Remigereau, 1997), magnetic
340 resonance imaging (MRI) and macrovision imaging (using stereomicroscope) (Chassagne-Berces
341 et al., 2009) are available to study the microstructure of frozen food products. Moreover,
342 another key issue which has been highlighted in recent times is the need for transition from
343 qualitative to quantitative evaluation. This evolution led to the development of advanced image
344 processing software in this field. Hereunder, we will briefly overview some of the main techniques
345 for the evaluation of frozen food microstructure.
346 3.1. Light microscopy

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347 Although, this technique is the most widely used to evaluate the microstructure of frozen food, it
348 is restricted when applied to thick sections, because the images can be blurred by out-of-focus
349 information. Generally, the microstructure of the frozen food with light microscopy is evaluated
350 at room temperature after thawing or after removing almost all the moisture content from the
351 samples. Thereby, there is a need for the initial preparation steps (tissue processing) to fix the
352 original structure to the maximum level and avoid morphological distortions before observation
353 by light microscopy. Common tissue processing methods are freeze-substitution and freeze-drying
354 methods, which often lead to artifacts and long processing times (Chevalier et al., 2000; Kiani and
355 Sun, 2011). Freeze substitution process is performed in two steps, first is the fixation stage and the
356 second is the substitution stage. Fixation step preserves structures and retains the conformational
357 relationships of ice/water phase, by immersing samples into a fixative solution which migrates into
358 the sample and creates crosslinking between and within the molecules of the tissue constituents.
359 In substitution step, the ice crystals/water are removed by substitution with a polar organic solvent.
360 After the evaporation of the organic solvent the matrix can be observed either directly or after
361 embedding. In freeze drying, ice crystals are removed by sublimation under vacuum, producing a
362 porous structure. Subsequent steps are similar to routine procedure followed in freeze substitution
363 (Krishnamurthy, 1999 ).
364 Kono et al. (2012, 2017) used a cryo-adhesive film to observe ice crystal morphology of frozen
365 fish meat. However the main principles of the method are similar to the freeze-substitution
366 technique, including embedding, sectioning, dehydration by alcohol solutions and staining. The
367 use of cryo-adhesive film during the sectioning process helps to collect samples and facilitates the
368 process with subsequently rapid observation of ice crystals by microscope.
369 Other disadvantages of light microscopy include low resolution and magnification and 2D image
370 production (Ishiguro and Horimizu, 2008; Kiani and Sun, 2011). Despite the above limitations,
371 this method has been successfully applied to investigate the microstructure of various food
372 products such as gels (Chevalier et al., 2000; Dalvi-Isfahan et al., 2017; Kiani et al., 2013),
373 vegetables (Van Buggenhout et al., 2006), meat (Bevilacqua et al., 1979; Dalvi-Isfahan et al.,
374 2016), fish meat (Alizadeh et al., 2007; Hafezparast-Moadab et al., 2018) and others (Su et al.,
375 2014). The use of frozen section and cryo-microscopy permits to overcome the artefacts that can
376 develop in the product structure during sample preparation step of ambient stage light microscopy
377 (discussed above) as it can visualize a frozen sample in the native state (i.e. at subzero

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378 temperature). Water-rich tissues such as food materials are hardened by freezing and the ice in the
379 frozen sample may act as the embedding medium. The sections of frozen samples may be cut by
380 using a cryostat, a cooled cabinet that houses an instrument to section frozen samples. Several
381 types of cryostats are commercially available and can be categorized as follows: single compressor
382 (chamber cooling only), double compressor (chamber and object cooling), manual sectioning and
383 motorized sectioning (Scouten, 2010). Subsequently, the sections of interest are stained and
384 examined with a microscope. This technique is much faster than the traditional histology since the
385 fixation step for sample preparation is eliminated. Besides, it also allows dynamic observation of
386 cells during freezing, frozen storage and thawing (McLellan et al., 1991; Wilson, 1991), and thus
387 can be used to estimate the freeze damage in real time (Ninagawa et al., 2016). Islam et al. (2015)
388 successfully employed this technique to investigate the effects of ultrasound-assisted freezing on
389 ice crystal morphology in mushroom. It has also been used to determine the ice crystals as well as
390 ice recrystallization upon storage in frozen desserts (Donhowe, Hartel, & Bradley, 1991; Donhowe
391 & Hartel, 1996b, 1996a). Ninagawa et al., (2016) used a cryomicroscopic system equipped with a
392 high-speed camera and cooling stage (having provision to control cooling rate) for evaluating the
393 structural changes caused by intracellular ice crystal formation in real-time during freezing. Using
394 this method more information about kinetics and mechanism of intracellular ice crystal formation
395 can be obtained (Ninagawa et al., 2016). Kono et al. (2017) also presented a fluorescent cryo-
396 microscopy technique for observations and measurements of the ice crystals formed in frozen rice
397 and gelatin (Kono et al., 2015; Kono et al., 2017). In this method, all components of the food
398 material were stained with a fluorescent color except for ice crystals. Thereby, the ice crystals were
399 observed as black parts in these images.
400 3.1.1. Image processing
401 Image processing software are commonly used in conjunction with light microscopy to quantify
402 the microstructure of observed images. A brief overview of the main steps followed in
403 that process is provided here.
404 1. Calibration: Calibration is a process that convert image scaling (pixel) to metric units.
405 2. Preprocessing: This step offers a wide variety of methods to overcome well known problems
406 arising by lack of contrast, presence of noise, and non-uniform illumination in acquired images
407 (Aguilera and Germain, 2007; Russ, 2004).

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408 3. Segmentation: It is a key step in image processing which is defined as a set of

409 processes which help to identify the objects of interest and separate them from all the other
410 areas of the image. Image segmentation can be performed through different techniques such as
411 thresholding based, region based, gradient based, and classification based approaches (Falcone
412 et al., 2006).
413 4. Object counting/measurement: the subsequent stage in image processing will use the
414 segmented results to estimate and characterize the morphological characteristics of ice crystals
415 (Pechak and Smith, 2007). As previously mentioned, the main morphological characteristics
416 of ice crystals involve; the number, size, shape, and the distribution.
417 5. Data analysis: The final stage consists of summarizing, classifying and presenting the main
418 results. Frequency distributions, percentage distributions, and cumulative frequency
419 distributions are three ways to summarize raw data into a curve that increase interpretability
420 and understanding. Among them, a cumulative-frequency distribution curve is more common.
421 This curve shows the proportion (or percentage) of observations that lies above or below a
422 certain size, and has been used in numerous freezing studies (Dalvi-Isfahan et al., 2017a;
423 Donhowe et al., 1991; Kiani et al., 2013; Zhu et al., 2005). Some morphological characteristics
424 of ice crystals are introduced briefly here.
425 3.1.2. Ice crystal shape
426 Different shape descriptors can be used to define changes in ice crystal shape during storage and
427 processing; but more attention has been paid to fractal dimension, elongation and roundness.
428  Roundness & Elongation
429 The most widely used shape descriptors are roundness and elongation. Roundness is calculated as
430 4πA/p2, where A is the cross-section area (µm2) and p is the perimeter (µm). The roundness is 1.0
431 for a perfect circle. Elongation is also defined as the ratio of major axis length to the minor axis
432 length. Ice crystals that are perfectly symmetrical in all axes, (circle), will have an aspect ratio
433 value of 1.0 and those with values greater than one will have an elongated shape (oval) (Aguilera
434 and Germain, 2007). This shape descriptor was used to study the effects of different assisted
435 freezing methods on ice crystal morphology (Dalvi-Isfahan et al., 2016; Mok et al., 2015; Su et
436 al., 2014)). Zhu et al. (2003) also found that high pressure shift freezing produced ice crystals with
437 higher roundness and less elongation than those produced by the conventional freezing process in
438 salmon fish. Their observations can be related with less damage to fish microstructure and higher

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439 product quality. However, Mock et al., (2015) showed that static magnetic field produced irregular
440 ice crystal shapes compared to control experiments, which was attributed to distortion and
441 breaking of hydrogen bonds induced by magnetic field.
442  Fractal dimension
443 Fractal dimension measures the geometrical complexity of objects and can be measured via
444 different methods like box-counting, particle-counting and Fourier (Tang and Marangoni, 2007).
445 This technique has been developed to characterize changes in surface roughness and tortuosity of
446 ice crystals during processing and storage. Hagiwara et al. (2002) used the area-perimeter method
447 to characterize the fractal dimension and quantify changes in ruggedness of ice crystal size during
448 storage. Their results showed that with increasing storage time, the outline of ice crystals was
449 becoming smoother and that this smoothing process was associated with recrystallization of ice
450 crystal during storage. This descriptor has been also employed to identify the effect of freezing
451 under static electric field on tortuosity of the ice crystals. Results showed that the treated samples
452 had a trend for higher values than the control samples which means slightly higher tortuosity of
453 ice crystals (Dalvi-Isfahan et al., 2016; Xanthakis et al., 2013).
454 3.1.3. Ice crystal size
455 The most common features for evaluation of ice crystal size are: area, perimeter and equivalent
456 diameter. The area of an ice crystal could be computed easily by counting the number
457 of pixels inside the segmented image. The perimeter could also be identified by the length of the
458 segmented image boundary, but the most significant feature for ice crystal size is equivalent
459 circular diameter. This feature is usually being used to characterize a non- circular shaped and
460 defined as the diameter of a circle which has the same area as the identified regions (Aguilera and
461 Germain, 2007). This feature has been used as an index to show efficiency of different assisted
462 freezing methods such as high pressure shift freezing, static electric field, radiofrequency and
463 microwave assisted freezing to improve quality of frozen products (Dalvi-Isfahan et al., 2016;
464 Hafezparast-Moadab et al., 2018; Xanthakis et al., 2014; Zhu et al., 2004).
465 3.2. Electron microscopy
466 Electron microscopy has been widely used to investigate the freeze damage at cellular level in
467 frozen foodstuff and a variety of such microscopes are currently being used to evaluate the
468 microstructure of frozen food. The characteristics of the most important types of electron
469 microscopy that employed on the freezing process are summarized.

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470 3.2.1. SEM/Environmental-SEM/Cryo-SEM

471 SEM has been used for many years to give high-resolution, surface images of biological
472 specimens, but there are still some remaining limitations and challenges in applying this technique
473 for evaluating a freezing process. Those limitations include i) shrinkage and mechanical damage
474 created by the surface tension as water evaporates during sample preparation which can lead to
475 distortion or change of specimen morphology (Rao, 2007). ii) Although, SEM scans the surface of
476 the sample to generate a three-dimensional (3D) image (topography), the inner sector under
477 the surface is not imaged. Therefore, the ability to view hydrated samples with high resolution and
478 observation of internal areas makes Cryo-SEM an appealing technique for viewing the
479 microstructure of frozen tissues (Fazaeli et al., 2012). In such equipment, frozen samples are
480 cooled down, getting broken, metalized and observed. It is expected even to visualize micro cracks
481 in the structure at cellular level which helps to understand better the impact of the freezing rate on
482 freeze damage by Cryo-SEM. Cryo-SEM is a direct method for observing the ice crystals of frozen
483 samples without prior thawing. Another advantage of this method is that it does not require the use
484 of chemical fixation processes. This fact reduces the time required for tissue preparation entailed
485 in chemical fixation as well as possible artefacts associated with drying compared with the typical
486 scanning electron microscopy (Sriamornsak et al., 2008; Sun and Li, 2003). Some results of
487 microstructure analyses from frozen food samples acquired by these microscopy techniques are
488 given below:
489 The microstructural changes in strawberry tissue associated with freezing–thawing conditions
490 have been studied by scanning electron microscopy (SEM) technique after freeze-drying
491 preparation method (Delgado and Rubiolo, 2005) (Figure 4).
492 A similar methodology has been used by Tu et al. (2015), where the microstructure of freeze-dried
493 lotus roots was observed with SEM in order to investigate the effects of three freezing methods,
494 air blast freezing (ABF), immersion freezing (IF) and ultrasound-assisted immersion freezing
495 (UIF). Micrograph images demonstrated that SEM is a useful technique and enables to show the
496 difference between treatments. A cold-stage SEM was employed to monitor the morphology of ice
497 in apple tissues and a sucrose model solution frozen at rates ranging from 450 to 0.031 K/min
498 (Bomben et al., 1983). Zounis et al., (2001) used low temperature scanning electron microscopy
499 (LT-SEM) to overcome the difficulty of studying the damages induced by freezing on
500 microstructure of frozen dough. The effect of ultrasound-assisted immersion freezing on frozen

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501 potato tissues was investigated by Sun and Li (2002). Cryo-SEM of frozen-thawed tissue was used
502 to monitor microstructural evolution. Microscopic analysis showed that the damage to the cells
503 was minimized due to the high freezing rate obtained under high ultrasonic level (15.85 W) and a
504 domination of intracellular fine ice crystals was observed in the frozen matrix.
505 Cryo-SEM has also been employed to study microstructural changes in hardened ice cream and
506 to quantify the ice crystal size in gelatin samples frozen by different treatments (Bolliger et al.,
507 2000; Sriamornsak et al., 2008). The microstructural changes in blueberries after freezing and
508 frozen storage conditions have been also monitored by cryo-SEM microscope observations
509 (Allan-Wojtas et al., 1999).
510 3.2.2. Environmental scanning electron microscopy (ESEM)
511 This instrument has the performance of a conventional SEM but has the advantage that practically
512 any material can be examined in its natural state. The microscope can also offer high-resolution
513 images in a saturated water vapor environment keeping the sample in its original wet state. This
514 technique has been recommended to evaluate the structural damages after thawing (Kiani and Sun,
515 2011). Jalte´ et al. (2009) used ESEM to evaluate potato tissue microstructure pre-treated by pulsed
516 electric fields (PEF) after air-blast freezing and thawing. Results showed that the electroporation
517 of cell membranes induced by pulsed electric fields pre-treatment caused significant structural
518 damage and formation of intercellular voids in freeze–thawed potato in comparison with the
519 control sample. The presence of intercellular voids is indicative of ice crystal formation.
520 3.2.3. Atomic force microscopy (AFM)
521 AFM is a technique that permits nanoscale imaging and force measurement of the product. This
522 method reveals the surface structure of food materials in 3D and can be used to investigate the
523 influence of a process on surface topography. In this regard, Molina et al. (2016) employed this
524 technique to study the physical changes of starch granule when subjected to freezing. Results
525 showed that frozen starch granule had a rougher surface with some protuberances compared to
526 unfrozen native granule. Krok et al. (2000) used non-contact AFM and they were able to observe
527 a significant change in starch granules surface appearance caused by freezing. Kirby et al. (1996)
528 used AFM to visualize the modification in the cell walls of water chestnut due to of freezing-
529 thawing process. Their images showed a minor difference between the fresh and frozen-thawed
530 sample: an increase in the fraction and irregularity of the thinner fibrils were observed in the freeze-
531 thawed sample. However, no gross change in cell wall structure induced by freezing-thawing

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532 process was observed in their case. AFM has also been used to determine the stiffness (mechanical
533 property) of cells and surrounding matrix (Golan et al., 2018; Xu, Li, Cai, Calve, & Neu, 2016).
534 Stiffness mapping revealed significant differences between fresh and frozen-thawed tissues (adult
535 bovine cartilage tissue)as the living cartilage was found to be significantly softer than frozen-
536 thawed one (Xu et al., 2016).

537 3.2.4. Confocal laser scanning microscopy

538 This technique is non-invasive and requires minimum sample preparation which permits optical
539 sectioning to be carried out using a laser beam. It offers also the possibility to examine the internal
540 structure of rather thick samples in three dimensions (Blonk and van Aalst, 1993; Dürrenberger et
541 al., 2001). This method was successfully used to investigate the 3D ice crystal morphology and to
542 monitor microstructural evolution during freezing and thawing of chicken meat (Ishiguro and
543 Horimizu, 2008). A confocal laser scanning microscope (CLSM) equipped with a cooling/heating
544 stage was also applied by Baier-Schenk et al. (2005b) to understand the changes in fermented
545 wheat dough upon freezing and frozen storage. Their results showed that the ice formation and
546 growth occurs preferentially at the gas pore interface and the microstructural changes caused by
547 freeze damage were mostly reversible during thawing.
548 3.2.5. Micro-Slicer Image Processing System (MSIPS)
549 This technique is also able to measure 3D structures and configuration of ice crystals in frozen
550 food. The method can be briefly summarized as follows: The system mechanically cuts the frozen
551 sample (samples are stained by soaking specific fluorophore prior to freezing) by means of a
552 microslicer. The surface of the sample, after each cut, is observed by fluorescence microscope.
553 Then, the 3D images of a sample is reconstructed by volume rendering method based on the image
554 data of the exposed cross-sections obtained by the multi-slicing of the frozen sample (minimum
555 thickness of 1 µm). This technique has been used to observe the ice crystal structures formed in
556 frozen beef and frozen dilute solutions (Do et al., 2004; Ueno et al., 2004).
557 3.3. Micro X-ray computed tomography and image analysis
558 X-ray micro-computed tomography (CT) is a non-invasive technique to study the internal structure
559 of objects by creating three-dimensional images that map the variation of X-ray attenuation
560 coefficient. The samples are imaged either directly in frozen state or can be freeze dried prior to
561 imaging. Freeze drying or lyophilizaion technique removes the moisture from inside of the
562 material by sublimation and leaves a network of voids and pores, hence allowing the imaging at

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563 an ambient temperature. The sample is scanned by an X-ray computed tomography (CT) scanner,
564 and the several hundred images, taken as the sample was rotating, are being used to build 3D
565 images by using an appropriate image analysis software. Although, this method is an accurate way
566 for determination of ice crystal morphology, size distribution, and volume fraction, the X-ray
567 method does not show structural detail and freeze drying can lead to damage of the cell structure
568 by shrinkage (Lim and Barigou, 2004; Schoeman et al., 2016; Vicent et al., 2017). The freeze-
569 thaw cycles are one of the important causes of food deterioration during cold storage. Micro X-
570 ray computed tomography coupled with image analysis has been successfully used to visualize
571 and quantify the ice crystal voids in potato subjected to freeze-thawing (Zhao and Takhar, 2017).
572 3.4. Hyperspectral imaging, near infrared and Raman spectroscopy
573 Hyperspectral imaging is an emerging technique that contains both spatial and spectral information
574 and allows evaluation of the special location quality as every pixel in hyperspectral imaging is
575 associated with a range of wavelength (Dai et al., 2015; Gowen et al., 2009; Shan et al., 2018).
576 This method can differentiate samples rapidly, non-invasively and with great accuracy (Cheng et
577 al., 2014; Dai et al., 2015; Shan et al., 2018; Washburn et al., 2017; Xu & Sun, 2017).
578 Hyperspectral imaging technique have been successfully used to distinguish fresh sample from
579 frozen-thawed sample (Gowen et al., 2009; Shan et al., 2018; Washburn et al., 2017; Zhu et al.,
580 2013). Washburn et al. (2017) used hyperspectral imaging in a frozen state and they were able to
581 differentiate packaged samples subjected to different freezing protocols (i.e. – 40 and – 20 ℃) and
582 stored for both 2 days and 12 months. Moreover, this method clearly showed differences between
583 samples frozen for one and two times (samples frozen, then thawed and followed with second
584 freezing). It was reported that this technique was unable to distinguish samples (in thawed state)
585 frozen by different methods (Washburn et al., 2017). Hyperspectral imaging technique has also
586 shown potential in predicting the firmness of grass carp fish (Cheng et al., 2014) subjected to
587 frozen storage, detecting the freezer burn on salmon surface (Xu & Sun, 2017) and determining
588 the freshness of frozen prawns (Dai et al., 2015).
589 Near infrared and Raman spectroscopy have also been proven their effectiveness in distinguishing
590 fresh and frozen-thawed sample (fish) non-invasively (Reis et al., 2017; Velioglu et al., 2015).
591 Moreover, by using Raman spectroscopy, samples subjected to temperature fluctuations or in other
592 terms freeze-thaw cycles could also be discriminated from the fresh samples as well as on the basis
593 of the freeze-thaw cycles number. These differences were associated with changes in physical

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594 structure, chemical composition of samples as well as biochemical reactions due to the process of
595 freezing-thawing (Cheng et al., 2014; Reis et al., 2017; Velioglu et al., 2015; Zhu et al., 2013).
596 Fourier transform infrared (FT-IR) spectroscopy technique has also the potential to be used as a
597 method for detecting the damage happening to the cell walls (in plant-based product)as a
598 consequence of freeze process. To date, this technique has been used for analyzing cell walls
599 architectures and monitoring cell wall changes due to various factors, such as growth and
600 development processes, genetic modifications, exposition or habituation to cellulose biosynthesis
601 inhibitors and responses to other abiotic or biotic stresses, as well as its biotechnological
602 applications. Besides, it also provides abundant information about cell walls polymers, functional
603 groups, and in muro entanglement (Largo-Gosens et al., 2014).
604 3.5. NMR/MRI
605 These techniques have found important applications in food freezing and some of them are
606 mentioned below. i) NMR and MRI techniques provide the opportunity for elucidating the effects
607 of freezing on food structure and food quality such as beef, lamb, pork meat and trout (Evans et
608 al., 1998). Results showed that the variations in NMR parameters, especially the relaxation time,
609 T2, and the radial diffusion coefficient could be used to describe the structural changes in tissue
610 induced by freezing process (Renou et al., 2003). In another study, freeze damage during storage
611 of cod and mackerel fish was evaluated by MRI. Results indicated that magnetic resonance (MR)
612 parameters such as T1, Tsat1 and MT (Magnetisation Transfer) rate can be used as indicators for
613 determining deleterious effects of frozen storage on fish meat (Nott et al., 1999). ii) MRI has been
614 proven useful in monitoring the development of ice during food freezing and assessing freezing
615 time. In this case, ice crystal formation is marked by an abrupt decrease in spatially located NMR
616 signal strength (Kerr et al., 1998). iii) Since the distribution of the water in food products is affected
617 during freezing/thawing process, NMR transverse (T2) relaxation technique and MRI technique
618 have been also used to follow dynamic changes in the structure and texture of foods during such
619 process (Yano et al., 2002). More details of the implementation and application of these
620 methods are given in Kerr (2006), so this topic will not be extensively discussed in this review.
621 3.6. DSC (differential scanning calorimetry)
622 DSC (differential scanning calorimetry) has also been used recently to determine the freezable
623 water content change in the mangoes due to temperature fluctuations during frozen storage (for
624 one month) (Zhang et al., 2018). In their study, the storage temperature was fluctuated from ― 65

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625 ℃ to ― 28, ― 38, ― 49 and ― 60 ℃ and was compared with the control sample (stored at ― 65
626 ℃ without any temperature fluctuation). Results showed that the increase in freezable water during
627 frozen storage period was lowest for the control sample, while temperature fluctuations of 37 and
628 27 ℃ showed higher freezable water. This higher increase in freezable water was attributed to the
629 larger magnitude of temperature fluctuations that enhanced the ice recrystallization. It is worth
630 mentioning that freezable water in biological tissues may vary, especially for fruits and vegetables
631 where freezable water varies with cultivars, maturity, degree of the ripeness and other factors.
632 Hence, this technique could be used to compare freeze damage imparted by different freezing
633 conditions on the samples from the same unit (i.e. same fruit or vegetables) rather than comparing
634 samples from a different unit. In case of samples acquired from different units, a great variation
635 may occur. Besides care needed to be taken while sample preparation and transfer to the DSC
636 chamber. In this context, longer transfer time at ambient condition may cause some moisture
637 condensation on the sample pan walls (since the sample container is kept at subzero temperature),
638 resulting in an overall increase of pan weight and hence faulty end result. Another approach to use
639 DSC to evaluate freeze damage in food (mainly in meat and fish product) is to compare
640 denaturation enthalpy of fish/meat muscles as freezing treatment can cause protein denaturation
641 probably due to ice crystals formation and cryo-concentration effect (Matos et al., 2011). This
642 technique found a difference in the total denaturation enthalpy (of fish muscles) between fresh,
643 frozen and samples (gilthead seabream fish) subjected to temperature abuse (Matos et al., 2011).
644 3.7. Other quality evaluation techniques
645 Methods like texture analysis, drip loss estimation and chemical-biochemical analysis provide
646 useful information about the microstructure profile. The wellbeing of the original microstructure
647 of samples can be determined by referring to the results obtained from these methods. For instance,
648 higher the damage on the microstructure of a sample greater would be the texture loss, drip loss
649 and change in chemical profile of the sample. In this regards, Chassagne-Berces et al. (2009) and
650 Charoenrein & Owcharoen, 2016 could find a good correlation between the intensity of cell
651 damage (observed using macrovision and CLSM imaging method) and the texture loss, drip loss
652 and change in chemical profile (in terms of change in the sugar composition of the cell wall).
653 Zender optical interferometry and mercury porosimeter and size analyzer capable of analyzing
654 crystals in frozen food can also contribute as a freeze damage assessment method (Bae et al., 1993;
655 Butler, 2001). Other potential methods for freeze damage assessment could be bioelectrical

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656 impedance measurement, dielectric response of sample at microwave frequencies and mass
657 diffusivity method (Alizadeh et al., 2009; Kent et al., 2004; Vidaček et al., 2008).
658 4. Conclusion
659 In this overview, the main physical freeze damage phenomena to the structure of food associated
660 with freezing and frozen storage were described. In summary, it can be mentioned that changes in
661 texture and sensory attributes of food materials via mechanical and biochemical stresses induced
662 by freezing can be correlated with ice crystal morphological characteristics. Since freeze damage
663 greatly affects the microstructure, evaluating the structure of the food matrix at cellular level is
664 essential. Various technologies and analytical tools can then be used to determine the impacts of
665 freeze damage on food microstructure. The application of non-invasive and modern methods for
666 studying ice crystal morphology and food microstructure provides the opportunity to produce high-
667 resolution 3D images with minimal preparation under the native environment of the system. A
668 better understanding of the underlying freeze damage mechanisms, its influence on frozen food
669 microstructure, as well as monitoring and evaluating it at cellular level will undoubtedly lead to
670 improvements in frozen food product quality and better design of efficient industrial processes.
671
672 Acknowledgements
673 Authors wish to thank the Jahrom University for supporting this study.
674 The contributions of Piyush-Kumar Jha and Epameinondas Xanthakis received financial support
675 from the French National Research Agency (ANR) and the Swedish Research Council FORMAS
676 respectively, under the FREEZEWAVE project (SUSFOOD - ERANET, SE: 2014-1925, FR:
677 ANR-14-SUSF-0001).
678
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Figure captions
Figure 1 Percentage of ice formed at different freezing temperatures in a typical food
product with initial freezing temperature of -1°C and assuming an end of freezing
(maximal ice formation) at -20°C.
Figure 2 Scheme presenting the expansion and compression (a) at start of freezing followed
by tensile stress (b) due to ice contraction.
Figure 3 Schematic of change in radius of ice crystals during storage as a function of ice
crystal radius (solid line) or time (dotted line). a, b and c represent food product
with different ice crystals distribution.
Figure 4 Micrographs of scanning electron microscopy. (a) Cellular structure of fresh
strawberry tissue, (b) Strawberry sample frozen at 0.51 m/s and -20oC (Delgado
and Rubiolo, 2005).

Figure 1
Percentage of ice formed at different freezing temperatures in a typical food product with initial
freezing temperature of -1°C and assuming an end of freezing (maximal ice formation) at -20°C.

Figure 2
Scheme presenting the expansion and compression (a) at start of freezing followed by tensile stress
(b) due to ice contraction.
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Figure 3
Schematic of change in radius of ice crystals during storage as a function of ice crystal radius (solid
line) or time (dotted line). a, b and c represent food product with different ice crystals distribution.
𝑲 = 𝟐𝜸𝑪𝑫𝑽𝟐/𝒌𝑻, where γ, C, D, V, k, T are interfacial energy, diffusion coefficient, molar volume,
concentration, constant and temperature respectively.

Figure 4
Micrographs of scanning electron microscopy. (a) Cellular structure of fresh strawberry tissue, (b)
Strawberry sample frozen at 0.51 m/s and -20oC (Delgado and Rubiolo, 2005)
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Highlights

 The main physical deteriorative changes that occur during freezing process are
investigated.
 Various analytical tools to determine the effects of freezing damages on food
microstructure are assessed.
 The ice crystals morphology have great impact on frozen food quality.
 Evaluating frozen food microstructure, may lead to improvements in quality of frozen
product.