Cryobiology 95 (2020) 130–137

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Cryobiology
journal homepage: http://www.elsevier.com/locate/cryo

Quality improvements to mackerel (Scomber japonicus) muscle tissue

frozen using a rapid freezer with the weak oscillating magnetic fields
Kana Okuda a, b, Aiko Kawauchi a, b, Kentaro Yomogida a, *
a
Department of Molecular Cell Biology, Institute for Bioscience, Mukogawa Women’s University, Ikebiraki-cho 6-46, 663-8558, Nishinomiya, Japan
b
Abi Inc., Ohtakanomori-higashi 1-12-1, 270-0138, Nagareyama, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Although freezing is the most popular long-term food preservation method, the formation of ice crystals during
Freezing the freezing process often degrades the quality of the product. Recently, several reports have argued that
Air blast-freezer oscillating magnetic fields (OMFs) may affect ice crystallization. In this paper, we investigated the effects of
Frozen storage
OMFs on fresh mackerel using the Cell Alive System® (CAS®) developed as an additional OMF generator for a
Thawing
Ice water
rapid freezer. Mackerel fillets were frozen with home freezing (HF), air blast freezing without (ABF) or with CAS
Mackerel (ABF-CAS) (ABI Co. Ltd., Chiba, Japan), and stored them for 2 weeks in the frozen storage between 30 � C and
Muscle fiber 35 � C. We analyzed the tissue damages of thawed samples histologically. The OMFs has been shown to
Weak oscillating magnetic fields significantly inhibit tissue damage in mackerel tissue after freezing and thawing (especially, thawing in ice
Cell Alive System® (CAS®) water). And it seems that OMFs suppressed the ice hole counts (p < 0.05), the mean size (p ¼ 0.061), and the
increase of interstitial area% (p < 0.05) after freezing/thawing. We also found that it is necessary to avoid re-
crystallization during thawing to maintain the quality of the frozen product. The use of OMFs with rapid
thawing has the potential to improve cryopreservation in the food industry as well as in the bioscience industry.

1. Introduction their constituent elements thereby avoiding ice crystal formation [11,
36]. However, a specific size of food (e.g. round fish or a chunk of meat)
The population explosion that has occurred in recent years is causing cannot be cooled uniformly, because all existing freezing systems draw
various global food security issues. To supply sufficient food to people heat from the surface. Therefore, rapid freezers with a high cooling
all over the world, food production must increase while food loss and performance, which minimizes the time that the target samples spend in
waste must be reduced. An improvement of food preservation technol­ the CTZ, have been commonly used for foodstuff freezing in the food
ogies is also key to reducing food loss and waste. Freezing is the most industry despite their limitations [18].
popular and universal method of food preservation for the long term and A decade ago, the Cell Alive System® (CAS®), an additional device
is used both in the food industry as well as in ordinary household for the rapid freezer was produced by ABI Inc. (ABI Co., 2007). The
kitchens. Although freezing may well maintain the quality and taste of device can generate weak oscillating magnetic fields (OMFs) in a rapid
food products for a long period of time, the accumulation of micro- freezer. Despite the favorable reports of cryopreservation of small tissues
structural damage caused by ice crystal formation during the freezing using a programmed micro-freezer with CAS® in regenerative medicine
process often leads to degradation of food products [9,18]. To limit the [1,12,17,19,23,27,29,40,41] the inhomogeneous distribution of mag­
damage, research has been conducted with the aim of controlling the netic field intensity in the early models of large CAS® freezers showed
formation of ice crystals during the freezing of biological materials and little effect on food freezing [4,16,30,31,35,38]. Following improve­
foods [3,5,10,13,14,32,39]. The frequency of ice crystallization and the ments in the regulation of magnetic field conditions, the current model
size of ice crystals depends on the latent heat discharge during the has been optimized for frozen food and has acquired international
freezing process [33]. The time spent in the critical temperature zone prominence in the food industry. Nonetheless, the effects of OMFs on the
(CTZ), leading to the release of latent heat, has been shown to be crucial freezing of food products has remained an open question because of the
to prevent the formation of ice crystals [9,28,37]. Indeed, the freezing of general misunderstanding that the CAS® is an additional OMFs gener­
microscopic biological materials in liquid nitrogen can instantly vitrify ator instead of a new freezing device [31] and the lack of analyses

* Corresponding author.
E-mail address: [email protected] (K. Yomogida).

https://doi.org/10.1016/j.cryobiol.2020.05.005
Received 11 July 2019; Received in revised form 8 May 2020; Accepted 8 May 2020
Available online 30 May 2020
0011-2240/© 2020 Elsevier Inc. All rights reserved.
K. Okuda et al. Cryobiology 95 (2020) 130–137

Table 1
Sample processing schedule.
Date of the Number of Number frozen fillets Raw
received (¼ mackerel of fillets
ABF ABF- HF
frozen date) received
CAS

December 5, 15 30 8 8 8 6
2017
December 8, 6 12 4 4 4 0
2017
December 13, 12 24 8 8 8 0
2017
December 14, 12 24 8 8 8 0
2017
total 45 total 90 total total total total
28 28 28 6

Fig. 2. Schematic drawing of the CAS freezer: a) Main components, b) Points at

which magnetic field measurements were performed in freezing trays 1, 5,
and 10.

to our laboratory within 24 h after capture using a refrigerated container

service to ensure maximum freshness, also known as chub mackerel,
Pacific mackerel, or Pacific chub mackerel (Table 1). As soon as the fish
arrived, the heads and offal were removed and they were filleted (weight
of each fillet 180.4 � 33.3 g, thickness 20.6 � 4.1 mm; mean values �
SD) for subsequent experiments (Fig. 1).

2.2. Freezing and frozen-storage conditions

Home freezing (HF): We arranged the samples in one layer on an

aluminum tray in a household-style freezer set at 25 � C (model NR-
C377M-P-, National Co., Ltd., Japan).
Air blast freezing with/without CAS® (ABF-CAS/ABF): An
experimental batch air blast freezer, with and without CAS® was used.
Fig. 1. Schema of a sample. The red frame and red dotted frame indicated
portions isolated for histological analysis. Red dotted frame portion was iso­
The freezer had been designed in consultation with ABI (F201003026,
lated for chemical analyses. Blue diamonds indicate the temperature mea­ ABI Co., Ltd., Chiba, Japan) with the OMFs and control system supplied
surement positions. The samples used for temperature measurement were not and commissioned by ABI. The basic construction of the freezer con­
used for other experiments. sisted of a cooling unit, a freezing cabinet, 2 fans, and 2 control panels.
This cabinet contained a rack with 10 equidistant rails that would
considering the thawing process [4,30]. accommodate up to 10 aluminum trays for food freezing and the mag­
In this study, we selected mackerel (Scomber japonicas) as a target netic field generator. Different freezing conditions, namely air temper­
sample. Mackerel is not only a common fish in Japanese cuisine, but ature (down to 50 � C), airflow (0–100%), and ‘CAS energy’ (0–700),
fresh mackerel is also ideal for eating raw in (e.g. in sushi or sashimi could be set by the control panel (Fig. 2).
(sliced raw fish). However, it is hard to preserve the freshness of The freezer was previously cooled to 30 � C (precooling mode), and
mackerel which negatively affects its value [7,8,25,26]. Moreover, we placed the tray with samples arranged in one layer in the middle of
long-term chilled storage of raw mackerel occasionally leads to prolif­ the cabinet. After the sample was frozen, the air temperature was
eration of Anisakis [34] and the histidine to histamine conversion, both switched to 50 � C (rapid freezing mode) with the airflow 50% (approx.
of which present a significant risk of food poisoning and other symptoms 0.5 m/s). With CAS® freezing, we set the CAS energy at 500, as rec­
including urticaria or anaphylaxis [15]. Therefore, we expect that the ommended by ABI. Without CAS freezing, we turned off the CAS® en­
establishment of an improved frozen storage method that maintains the ergy. The OMF strength was measured using a gaussmeter (EMF-828,
quality of the fish may reduce these risks. To understand the thermo­ Fuso Co. Ltd., Tokyo, Japan). The OMF strength with CAS energy could
dynamic effects of OMFs on the freezing of mackerel, we traced the be controlled in the range of 0.1–1.0 mT. The strength with CAS energy
center temperature change of samples during rapid freezing with and recommended for raw fish by ABI© was controlled at 0.1–0.5 mT. The
without OMFs, as well as during rapid thawing with an agitated mixture freezing process was finished when the center temperature was 50 � C.
of ice and water (ice water) and the popular slow thawing method using After freezing, we immediately vacuum-sealed each sample in a poly­
a household refrigerator. To assess the practical effects of OMFs on food ethylene bag (vacuum pakkun super-roll®, Wide system Co., Ltd.,
freezing, we analyzed the histological properties of the thawed sample Japan) with the unidirectional sheet (Fresh master, Unicharm Co.,
after freezing with and without OMFs. Tokyo, Japan) absorbing drip, and stored them for 2 weeks in the frozen
storage (ABI Co. Ltd., Chiba, Japan) between 30 � C and 35 � C.
All the freezing experiments were performed in triplicate on separate
2. Materials and methods
days. Also, samples were randomly divided into three groups (HF, ABF-
CAS, ABF).
2.1. Samples

After being caught at the south Sanriku coast in December 2017, 45 2.3. Thawing conditions
mackerels (Scomber Japonicus)(weight 790.2 � 119.8 g, length 37.9 �
2.4 cm; mean values � SD) were immediately stored in ice and delivered Ice water thawing: Each sealed frozen sample was thawed in ice

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were not used for further analyses. During sample freezing, the tem­
perature inside of the freezer was also monitored in real time by a
potable data logger (testo184T4, Testo SE & Co. KGaA, Lenzkirch,
Germany).

2.5. Estimation of freezing

Since samples gradually freeze inwardly from the surface into the
center, it is difficult to precisely determine the total freezing time. The
center temperature is reflected outside of the latent heat due to ice
crystallization during freezing. Consequently, we defined “the freezing
time” as a time taken for the center of the sample to reach the setting
temperature from the time that freezing was initiated, and “the time to
freezing temperature” as a time taken from the center temperature
reached to 20 � C from start temperature, because commercially food
has been frozen down to 18 to 20 � C. Also, we defined the crystal­
lization time as a time taken for the center temperature to pass through
the CTZ ( 1 � C to 7 � C) [21].
It has been suggested that the final quality of the frozen sample can
be evaluated by the freezing rate. We calculated the freezing rate from
the time when the center temperature reached 10 � C from 5 � C, as
described [2,20,42].

2.6. Drip loss after thawing

Each sample was weighed using an electric balance (Max2100 g

Mino.5 g e ¼ 0.1 g d ¼ 0.01 g, GX-2000, A&D Co., Ltd., Japan) before
freezing. Each dry sheet (Fresh master, Unicharm Co., Tokyo, Japan)
was weighed before and after use with an electrical balance (Max310 g
Min0.02 g e ¼ 0.01 g d ¼ 0.001 g, GX-300, A&D Co., Ltd., Japan) Each
drip loss was calculated as follows:

Drip loss(%)¼[(WD-W0)/W]�100 (1)

where W0 is the weight of the dry sheet before use (g), WD the weight of
the dripping sheet after use (g) and W the weight of the fresh sample (g).

2.7. Histological analysis

Fig. 3. Time-temperature curves during freezing processes.

To analyze the histological change of samples due to freezing and
(a) The mean center temperature changes in case of the household-style freezer,
thawing conditions, we cut off several small sections of tissue (5 � 5 � 5
and the air blast freezer with or without CAS in the same time scale (n ¼ 3 in
each test). (b) Expanded time-temperature curves of each ABF/ABF-CAS sample mm) from the center of each sample (Fig. 1). The sections of each sample
in (a). (c) The inside temperature changes of the household-style freezer were were fixed in Modified Davidson’s solution (95% absolute ethanol,
monitored during the freezing process. (d) The inside temperature changes of formalin, Acetic acid glacial and water ¼ 33:22:11.5:33.5, also known as
the air blast freezer were monitored the freezing processes. Arrow head in­ Hartmann’s Solution) at 4 � C for 2 h and dehydrated in 70% ethanol at 4
dicates the timing for placing sample in the freezing cabinet. �
C overnight. Samples were subsequently dehydrated in different
ethanol gradients (70% for 2 � 1 h, 80% and 95% for 1 � 1 h, 100% for
water until the center temperature was 0.5 to 0 � C [18]. The temper­ 2 � 1 h) at 4 � C. The dried tissue samples were soaked in resin solution
ature of ice water was constantly monitored using a water-proofed (Methyl methacrylate monomer, Dibutyl phthalate ¼ 3:1, 75% benzoyl
thermocouple-K (0.32 mm diameter 1G, NND Co., Ltd., Japan). To peroxide was added as a 34.3 mg/ml) at 4 � C overnight. We replaced
uniform the ice water temperature, we continued agitation with several resin solution with the fresh resin solution and added 1 vol% N, N-
small submarine motors (ITEM70185, TAMIYA INC., Japan). dimethylaniline to start the polymerization of the resin. Histological
Refrigerator thawing: We placed each sealed frozen sample in the
cold room of the refrigerator (model NR-C32Ep-H, National Co., Ltd., Table 2
Japan) at 4 � C until the center temperature was 0.5 to 0 � C [18]. To Estimation of freezing time.
confirm that each sample was completely thawed, we checked the The The time to freezing Crystallization Freezing
hardness and measured the center temperature (testo 926-Starter set, freezing temperature ( 20 � C) time rate
Testo SE & Co. KGaA, Lenzkirch, Germany) before the analyses. time

(min) (min) (min) (� C/min)

HF 210.0 � 180.0 � 28.3a 80.0 � 0.0a 0.2 � 0.0a

2.4. Temperature monitoring in real time 0.0a
ABF 45.0 � 0.0b 24.1 � 1.6b 12.0 � 2.4b 1.6 � 0.4b
We monitored the center temperature of some samples (Fig. 1) ABF- 46.0 � 0.0b 21.9 � 0.9b 10.9 � 1.9b 2.0 � 0.9b
during freezing and thawing using a thermocouple-type K (0.32 mm CAS

diameter 1G, NND Co., Ltd., Japan) connected to temperature recorder Data are expressed as mean � SD. Different letters for mean values within a
(MSR128-V6, M-System Co., Ltd., Japan) in real time. These samples column represent significant different (p < 0.05).

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Fig. 5. Mean drip losses of the thawed samples. Bars represent mean values �
standard deviation of the samples (n ¼ 5–9).

To evaluate the damage per unit area of each muscle tissue section, the
sample was divided into the muscle fiber area and the interstitial area
and we calculated the area ratio of the interstitial area (%). Moreover,
for the damaged area per unit area of each dark muscle tissue section, we
calculated the mean size of the ice holes (μm^2) and ice hole counts per
area in addition to the interstitial area (%). Each histological unit area
was divided into three areas (intact cytoplasm, ice melting hall, inter­
stitial space) using the Photoshop CC (ver. 20.0.2) and analyzed with
Image J v1.52a (National Institute of Health, Bethesda, MD, USA).

2.9. Statistical analysis

Experimental data were processed by means of variation statistics.

Mean and standard deviation (SD) were used. To compare the para­
metric data in two groups, t-test was used. The statistical analysis was
performed using the Student’s t-test, and differences between the groups
were considered statistically significant when the P value was less than
0.05.

3. Results

3.1. Freezing curve analysis

To understand the freezing performance of each freezer, we moni­

tored the inside temperature of each device during the sample freezing
(Fig. 3c and d). Since the home-freezer only had a weak device to
Fig. 4. Time-temperature curves during thawing processes. (a) The ice water circulate the air, the temperature in the freezer fluctuated between 20
and the sample center temperature changes during the ice water thawing (n ¼ 3
�
C and 30 � C (Fig. 3c). Conversely, the air blast freezer was able to
in each test). (b) The center temperature changes during refrigeration (n ¼ 3 in control the inside temperature precisely during the freezing process
each test). (c) The inside temperature changes of home refrigerator monitored (Fig. 3d) (see Table 2).
during the thawing process. The mean freezing curves (n ¼ 3) of the fillet during the HF, the ABF,
and the ABF-CAS are shown in Fig. 3a. The crystallization time, the time
sections (thickness, 4 μm) of the resin-embedded tissues were prepared to freezing temperature and the freezing time in HF were very extended
(RM2135, Leica Co., Ltd., Japan). The sections were stained with and the freezing rate reflected the internal amount of ice crystals was
hematoxylin-eosin Stain and observed under a light microscope (BZ- only about 0.2 � C/min (Fig. 3a, Table 2). Although there was no sig­
800, KEYENCE Co., Japan). nificant difference between ABF and ABF-CAS in any endpoint (p >
0.05), “the time to freezing temperature” tended to shorten ABF-CAS
2.8. Image analysis than ABF (p ¼ 0.07) (Fig. 3b, Table 2).

To evaluate the damage per unit area of each muscle tissue cross- 3.2. Thawing curves
section, we applied the previous method with slight modifications.
Two sections from each of the tissue samples per fish were used for the The center temperature of the fillets was measured during thawing
histological analyses of muscle tissue and dark muscle tissue from the (Fig. 4). The thawing time in the refrigerator required five to six times as
various conditions. One to two spot areas from samples were randomly long as the thawing in ice water (Fig. 4a and b). Moreover, the thawing
selected at low power and measurements performed at 10x (Muscle time in ice water was significantly different between ABF and ABF-CAS
tissue) or 40x (Dark muscle tissue) magnification to avoid fatty tissue. frozen samples (p < 0.05). The temperature in the refrigerator was kept

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Fig. 6. Hematoxylin eosin stained histological cross sections of the muscle tissue and the dark muscle tissue. The fresh tissue and (a) the ice water thawing or (b) the
refrigerator thawing tissues after 2 weeks of cryopreservation. Each arrow indicates ice crystal holes in the dark muscle fiber and each arrowhead indicates interstitial
space in the muscle tissue. Each scale bar is 100 μm.

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around 5 � C (Fig. 4c).

3.3. The drip loss of each thawed sample

The drip loss was evaluated immediately after the thawing process to
avoid any additional reactions or modifications in volume. Using the
unidirectional sheet, we could measure the only drip by freeze-thaw
excluding the effect of relaxing fluid. Although the difference in the
thawing method had little effect on the drip loss of the sample frozen by
each condition, the drip loss of the ABF-CAS sample was reduced
compared to that of the HF samples (p < 0.05) and to that of ABF (p ¼
0.057) (Fig. 5).

3.4. The OMFs suppressed tissue damages during freezing/thawing

To access freezing and thawing induced tissue damage, we per­

formed image analysis of each freeze-thawed mackerel histological
samples with different freezing/thawing conditions (Fig. 6). The fresh
sample showed a very homogenous muscle fiber distribution, no fis­
sures, with very narrow extracellular space between the muscle tissue,
and the cross-section of the dark muscle tissue had relatively uniform
and regular shapes (Fig. 6, left column).
In ice water thawed samples (Fig. 6a), we observed no obvious
structural differences between the fresh muscle and the ABF-CAS mus­
cle. In contrast, the ABF and HF sample showed extracellular space
expansion and muscle fiber disarrangements. The area ratio of the
interstitial area was not significantly different between the fresh samples
and the ABF-CAS samples (18.3 � 5.1 and 19.2 � 1.8%, p < 0.05), but
the ABF samples (24.0 � 2.6%) and the HF samples (29.5 � 4.8%) were
significantly higher than the ABF-CAS samples (p < 0.01) (Fig. 7a).
Fresh dark muscle tissue did not have holes in the muscle fibers, while
frozen samples did. We presume that these holes are the result of
damage due to ice crystal formation. The interstitial area (%) and ice
hole counts was significantly different between ABF-CAS and other
freezing conditions (p < 0.05). The mean size of the ice holes (μm) in the
ABF-CAS samples was smaller than that in ABF samples (p ¼ 0.061).
However, no significant differences were found between ABF-CAS
and ABF in any of the parameters of the refrigerator thawed muscle
tissue and dark muscle tissue (p > 0.05). The area ratio of the interstitial
area of refrigerator thawed muscle tissue of the ABF-CAS samples (29.2
� 5.5%) was significantly higher than ice water thawed ABF-CAS sam­
ples (p < 0.05) (Fig. 7). As shown in Fig. 6b, ABF-CAS refrigerator
thawed samples showed extracellular space expansion and muscle fiber
disarrangements. The interstitial area (%) and the ice hole counts of the
dark muscle tissue in the ABF-CAS were significantly different between
thawing methods (p < 0.05).

4. Discussion

Since fresh freezing targets such as fish have a specific size and are
composed of many cells and interstitial tissues, it is impossible to
completely freeze them immediately. When the surface layer of a target
sample starts to freeze upon reaching the CTZ, the center temperature
must be in the plateau phase (the ice crystallization time) due to the
latent heat from the outside until the center portion has frozen
completely (Fig. 8). Latent heat is almost completely due to the ice
crystal formation by hydrogen bonding between water molecules. Latent
heat might not only prevent freezing of the inside layer, but also extends
Fig. 7. Image analysis of the histology. (a) The ratio of muscle tissue area and the crystallization time. Indeed, the temperatures of freezing plataue
the interstitial area in each muscle tissue (n ¼ 5). (b) The ratio of the interstitial were different between HF and ABF/ABF-CAS (Fig. 3, Table 2).
area in each dark muscle tissue (n ¼ 6). (c) The mean size of the ice holes in In this study, we examined the effect of CAS® on freezing and the
each dark muscle tissue (n ¼ 6). (d) The ice hole counts per 1800 μm2 in each
effect of different thawing methods on ABF-CAS frozen samples. Since
dark muscle tissue (n ¼ 6).
there are variations in each fish fillet size, uniform gel models were often
used for many freezing temperature-time curve analyses [10,30].
However, the uniform gel model could not accurately reproduce the
temperature change of real fish with complex internal tissue structures

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Fig. 8. Hypothesis of the heat transfer in a certain size of fish meat during freezing and thawing.

[22]. The mackerel fillet used this time was difficult to accurately prevent such tissue damage, an effective thawing method like the ice
analyze the freezing temperature curve due to the variation in shape. water thawing method used in this paper should be selected [6]. Inter­
Therefore, we have confirmed that the use of uniformly sized tuna sec­ estingly, the ice water thawing time of the ABF-CAS sample seemed to be
tions significantly shortened the “the time to freezing temperature” (p < shortened than that of the ABF sample. The ABF-CAS sample might
0.05) and tended to shorten the crystallization time (p ¼ 0.084) by OMFs require less endotherm to melt ice crystals owing to the suppression of
(Data in Brief). ice crystal formation by OMFs. The monitoring temperature valiations
Our data suggests that the OMFs inhibit latent heat by suppressing for each thawing plataue should be due to the individual sample dif­
the hydrogen bonding between water molecules during ABF, as previ­ ference. As mentioned in methods, we confiermed directly that each
ously reported [24]. Indeed, we could only detect very slight tissue center temperature was 0.5 to 0 � C with another thermometer.
damage due to the ice crystal formation in the ABF-CAS samples (Figs. 6 Although the global Japanese food boom affects the value of fresh
and 7). In ice water thawed samples (Fig. 6a), we observed no obvious fish which is intended to be eaten raw, it has been impossible to main­
structural differences between the fresh muscle and the ABF-CAS mus­ tain freshness for a long period of time without the necessary im­
cle. Moreover, in the dark muscle tissue, the interstitial area (%) and ice provements in freezing technologies. As we have described above, the
hole counts were significantly different between ABF-CAS and other optimized OMFs provided by CAS® has the potential to maintain the
freezing conditions (p < 0.05). These results suggest that the OMFs has quality of frozen fresh foods. The prevention of tissue damage due to ice
been inhibited histological damage in mackerel tissue after freezing and crystallization provides high quality frozen fresh food in food engi­
thawing (especially, thawing in ice water). Therefore, the OMFs could neering as well as excellent viability of cells/organs in bioscience.
be suppressed to ice crystal formation in the rapid freezing process.
Alternatively, the OMFs may promote that the conductivity was 5. Conclusion
improved or that the OMF enhanced crystallization throughout the
product. The OMFs has been shown to inhibit histological damage in mackerel
The tissue of vertebrate such as a fish is composed of many cells and tissue after freezing and thawing (especially, thawing in ice water). And
interstitial space. Expansion of the content volume due to the ice crys­ it suggests that OMFs significantly suppressed the ice hole counts (p <
tallization in the space surrounded by biological membranes leads to 0.05) and increase of interstitial area% (p < 0.05) after freezing/thaw­
breaks in the membrane structure allowing biological content to spread ing. Therefore, the OMFs could be suppressed to ice crystal formation in
into the outer space. This damage increases as crystallization time in­ the freezing process. Alternatively, the OMFs may promote that the
creases. Such histological damage in a certain wide tissue area should be conductivity was improved or that the OMF enhanced crystallization
scanned with the light microscope and be evaluated quantitively (Figs. 6 throughout the product.
and 7). Although an electron microscope is a useful tool to investigate Since tissue damage by ice crystallization could be detected very
micro-structure damage, it is not suitable to quantify a certain size of slightly in the ABF-CAS samples, the OMFs might suppress the latent
tissue damage for the extreme micro-field of view. heat generation by inhibiting the hydrogen bonding between water
During the thawing process, an endothermic reaction should occur to molecules. However, to evaluate the effect accurately, it is necessary to
break off the hydrogen bond in ice crystals. If the heat exchange effi­ select an appropriate thawing method that prevents secondary ice
ciency is low, the freshly thawed layer could be occasionally re- crystal damage during thawing. The benefits of the application of the
crystallization by the endothermic reaction from the inner layer OMFs could be two-fold; fresh quality frozen foods for the food industry
(Fig. 8). Our data suggested that inadequate slow thawing increases the and, increased viability of frozen stock cells/organs for bioscience/
probability of repeated re-crystallization and re-thawing of samples medicine.
(Fig. 4b). The severe damage of refrigerator thawed tissues could be due
to the repeating ice crystallization (Figs. 6–7). Unfortunately, various
Author contributions
thawing methods with insufficient consideration drew the different
views on the freezing effects of OMFs in previous researches [4,30]. To
Kana Okuda: conception and design, collection and/or assembly of

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Kawauchi: collection and/or assembly of data, data analysis and inter­
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pretation, and final approval of the manuscript. Kentaro Yomogida: Alaska Fairbanks, 2007, https://doi.org/10.4027/psf.2007.
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