Accepted Manuscript

Preservation of sweet cherry by isochoric (constant volume)

freezing

Cristina Bilbao-Sainz, Amanda Sinrod, Matt Powell-Palm, Lan

Dao, Gary Takeoka, Tina Williams, Delilah Wood, Gideon Ukpai,
Justin Aruda, David F. Bridges, Vivian C. Wu, Boris Rubinsky,
Tara McHugh

PII: S1466-8564(18)31194-9
DOI: https://doi.org/10.1016/j.ifset.2018.10.016
Reference: INNFOO 2080
To appear in: Innovative Food Science and Emerging Technologies
Received date: 17 October 2018
Revised date: 30 October 2018
Accepted date: 31 October 2018

Please cite this article as: Cristina Bilbao-Sainz, Amanda Sinrod, Matt Powell-Palm,
Lan Dao, Gary Takeoka, Tina Williams, Delilah Wood, Gideon Ukpai, Justin Aruda,
David F. Bridges, Vivian C. Wu, Boris Rubinsky, Tara McHugh , Preservation of sweet
cherry by isochoric (constant volume) freezing. Innfoo (2018), https://doi.org/10.1016/
j.ifset.2018.10.016

This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.
 ACCEPTED MANUSCRIPT

PRESERVATION OF SWEET CHERRY BY ISOCHORIC (CONSTANT

VOLUME) FREEZING

Cristina Bilbao-Sainza, Amanda Sinroda, Matt Powell-Palmb, Lan Daoa, Gary Takeokaa, Tina

Williamsc, Delilah Woodc, Gideon Ukpaib, Justin Arudab, David F. Bridgesd, Vivian C. Wud,

T
Boris Rubinskyb, Tara McHugha

IP
CR
a. Healthy Processed Foods Research, U.S. Department of Agriculture, Albany, CA, USA

b. Department of Mechanical Engineering, University of California at Berkeley, Berkeley, CA 94720, USA

c. Bioproducts Research Unit, U.S. Department of Agriculture, Albany, CA, USA

US
d. Produce Safety and Microbiology Research, U.S. Department of Agriculture, Albany, CA, USA
AN
M
ED
PT
CE
AC
 ACCEPTED MANUSCRIPT

ABSTRACT

Due to the perishable nature of fruits and the importance of reducing food waste, an

effective preservation technique is required to prolong the shelf life and maintain the

physical and nutritional properties of seasonal fruits. In this study, we evaluated isochoric

freezing for preserving the quality of sweet cherries. We examined the physical

T
characteristics and nutritional values of thawed cherries frozen to -4ºC or -7ºC in an

IP
isochoric system and compared them with those of fresh cherries, thawed cherries that

CR
were individually quick frozen and thawed cherries frozen to -4ºC or -7ºC in an isobaric

system. We found that isochoric freezing decreased the drip loss and better preserved the

US
color, texture, structure, ascorbic acid, phenolic and antioxidant content of frozen cherries,
AN
thereby proving their potential in frozen fruit applications.
M

Keywords: Cherry; Isochoric freezing; Preservation; Nutritional value

ED
PT
CE
AC
 ACCEPTED MANUSCRIPT

1. INTRODUCTION

The Food and Agriculture Organization (FAO) documented that up to one third of all

food is spoiled or squandered before consumption. This represents a waste of labor, water,

energy, land and other resources that helped to produce the food. For all foods, fruits and

T
vegetables have the highest wastage rates of 40-50%.

IP
One of the most common and well-established food preservation techniques is freezing.

CR
Freezing lowers the rate of deterioration in food quality over time by reducing microbial

and enzymatic activities, oxidation and respiration (Chassagne-Berces, 2010). Thus,

US
freezing results in a longer storage life. However, freezing leads to cellular damage in
AN
most cases. The cause of cell injury depends on the freezing rate. During freezing, ice

first forms outside the cells. For low freezing rates, water inside the cell diffuses through
M

the semipermeable membrane out of the cell to equilibrate the chemical potential between
ED

the interior and the exterior of the cells, which ultimately results in cell dehydration. For

high freezing rates, intracellular freezing will occur. Under both conditions, freezing
PT

exposes the cells to water loss as well as an increase in solutes concentration that can
CE

eventually cause cell damage (Mazur, 1970). Cell damage in biological tissues leads to

irreversible turgor loss, loss of firmness, loss of water holding capacity and increase in
AC

drip loss during thawing. This eventually results in a thawed product with suboptimal

physical properties (Chassagne-Berces et al., 2009) which decreases consumer approval

for frozen foods. The effect is most pronounced in fruits with delicate textures such as

cherries, raspberries, blueberries and strawberries. Nevertheless, many innovative and

advanced freezing methods are being investigated to reduce energy cost and improve
 ACCEPTED MANUSCRIPT

frozen food quality. Some of the new technologies include the use of high-pressure

freezing, antifreeze proteins and bacterial ice nucleation activators. All of these new

technologies focus on producing smaller, less damaging ice crystals.

A new technique that preserves the product at subfreezing temperatures without any ice

damage is called isochoric freezing, which is being developed by Rubinsky and his group

T
(Preciado and Rubinsky, 2010; Rubinsky, 2005; Szobota and Rubinsky, 2006). In

IP
isochoric freezing, the food product is immersed in a solution in osmotic equilibrium with

CR
the product and processed inside a pressure chamber. The volume remains constant

during the freezing process. When the temperature of the isochoric system is decreased to

US
a point where freezing occurs, ice forms and expands in the chamber. This increases the
AN
pressure inside the closed chamber until a thermodynamic equilibrium exists between ice

and water at the set temperature. In this way, the food product can be safely preserved at
M

subfreezing temperatures without being frozen if it remains in the non-frozen volume that
ED

is apart from the space occupied by ice. Le Chatelier’s principle states that as water

expands upon freezing, the high pressure in the system will hinder the further formation
PT

of ice. Therefore, in an isochoric system, the cooling process follows the liquidus curve
CE

in the phase diagram for water. This minimizes the pressure for the given temperature

and its potential harmful effects (Rubinsky, Perez and Carlson, 2005). Another advantage
AC

of isochoric freezing is that the extracellular osmolality of the frozen solution is

comparable to the intracellular osmolality (Rubinsky, Perez and Carlson, 2005; Nastase

et al., 2017) eliminating the aforementioned osmotic damages caused by freezing.

Nastase et al. (2016) has recently introduced a mathematical model to describe the

process of freezing in isochoric thermodynamic systems. The authors demonstrate that

 ACCEPTED MANUSCRIPT

the use of isochoric freezing can likely result in improved quality of frozen foods as well

as substantial energy savings. Also, Nastase et al. (2017) worked with Tilapia muscle

tissues and observed that the muscle tissue frozen at -5 °C in an isochoric system appears

morphologically identical to fresh tissue, with no evidence of dehydration. Encouraged

by these previous findings we set out to evaluate the potential of isochoric freezing in

T
preserving fruit quality.

IP
One potential candidate for isochoric freezing is the sweet cherry (Prunus avium). Sweet

CR
cherries are one of the most popular fruits in the world with a world production of about

2.25 million tons in 2014. They are rich in health-beneficial compounds such as ascorbic

US
acid, anthocyanins, β-carotene and phenolic compounds (Mulagabal et al., 2009;
AN
Chaovanalikit and Wrolstad, 2004; Ferreti et al., 2010). These phytochemicals are

reported to possess antioxidant, anti-inflammatory, anticancer, antidiabetic and antiobese

M

properties (Kris-Etherton et al., 2002). However, the short cherry season and their highly
ED

perishable nature limit the availability of fresh cherries on the market. Therefore, the use

of isochoric freezing to preserve the cherries could lead to increased marketability of

PT

frozen cherries and reduce fruit spoilage.

CE

In this study, we used isochoric freezing to preserve Rainier cherries and characterized

their physical and nutritional properties. We compared isochoric freezing with freezing
AC

under isobaric conditions and individual quick freezing (IQF) currently used in the food

industry.
 ACCEPTED MANUSCRIPT

2. MATERIALS AND METHODS

2.1. Fruit material

Fresh Rainier cherries (6-7 g) were obtained from a local supermarket and local growers

in Marin County (CA). Cherries were stored at 5 °C before subsequent sample

T
processing. The samples were used within one week.

IP
CR
2.2. Isochoric system

The isochoric system consisted of a R1 pressure chamber from High Pressure Equipment

US
Company (Erie, PA, USA). The chamber was made of 4340 alloy steel. It had an inner
AN
diameter of 1 inch, an outer diameter of 3.1/5 inches and an inner length of 6 inches. The

total volume capacity was 66 mL. The constant volume chamber was closed with a screw
M

and metal seal. Sealing was accomplished by a combination of O-ring and separate metal
ED

back-up ring. The back-up ring was designed to expand and contract as pressure

increased or decreased in value. The pressure chamber was connected to an electronic

PT

pressure transducer (Stork Solutions Ltd, Hampshire, UK) that was connected to a laptop.
CE

The data were recorded and displayed with Additel, a data logging and graphical

software. The system was cooled in a recirculating bath (VWR AP 15R-40, Radnor, PA,
AC

US) filled with a water and ethylene glycol (50:50) solution.

2.3. Experimental protocol

Three different methods were used to preserve fresh cherries: preservation for 24 hours at

-4°C or -7°C in an isochoric system, preservation for 24 h at -4°C or -7°C in an isobaric

system and individual quick freezing (IQF). For isochoric treatment, a metal ice
 ACCEPTED MANUSCRIPT

nucleating piece was placed at the bottom of the isochoric chamber to ensure that ice

formation started far from the sample, which was placed at the top of the chamber. The

isochoric chamber was filled with 17.5 Brix sucrose solution and then sealed. The

chamber was completely immersed in a recirculating cooling bath at 0°C and cooled at

0.25 °C/min to -4°C or -7°C. The pressure reached 29.5 MPa at -4°C and 62.1 MPa at -

T
7°C. The cherries remained at the set temperatures for 23 h. After this time, the chamber

IP
was heated back to 0°C at 0.25 °C/min. The chamber was taken from the bath and opened

CR
once it reached room temperature. The principle of operating the isochoric chamber is

US
illustrated in Fig. 1. The isobaric treatment followed the same procedure as the isochoric

treatment except that the cherries were placed in a Ziploc bag and then immersed in the
AN
recirculating bath. After freezing, samples were thawed at room temperature for 30 min.

IQ freezing was performed in a cryogenic freezer using liquid nitrogen (Custom Biogenic
M

Systems, MI, US). Fresh cherries were frozen to ensure freezing throughout the whole
ED

cherry using the following protocol: (a) cooling to -25°C at 25 °C/min, (b) cooling from -
PT

25°C to -40°C at 1 °C/min and (c) cooling to -90°C at 25 °C/min. The total processing

time was 20 min. The frozen fruits were collected and directly stored in a conventional
CE

freezer at -20 °C for 24 h. The mass change of the samples was determined

gravimetrically, Brix of the juice expressed from cherries was determined with a
AC

refractometer and the pH values of cherries were measured using a pH meter (Beckam

Coulter, Indianapolis, US).

 ACCEPTED MANUSCRIPT

2.4. Drip loss Analysis

Three cherries per treatment were cut in half and used to analyze drip loss. Each half

cherry was placed in a 45 mL plastic centrifuge tube suspended on a perforated support.

The samples were centrifuged at 4000 rpm for 10 min. Measurements were done at room

T
temperature. Drip loss of cherry samples was measured as the percentage difference

IP
between the weight of the cherry after freezing and the weight of the cherry after

CR
centrifugation (Eq. 1).

US
% drip loss = (initial wt − final wt)/initial wt × 100 (1)
AN
2.5. Color
M

Rainier cherries have yellow skin blushed with varying levels of red pigment. The color
ED

of the cherries was measured in the yellow areas of the skin after processing and thawing

for 30 minutes. A high-resolution digital camera (Nikon-7000) was used to measure color
PT

by capturing the color image of the cherry fruits under constant lighting. The color was
CE

analyzed with Photoshop, in which the Histogram Window was used to find the average

Lightness, a and b values for an area of 5625 pixels. This was done twice for each fruit.
AC

The values were then converted into the standard L*, a*, b* values using Equations 2, 3

and 4, respectively.

L*=100*Lightness/255 (2)

a*=240a/255-120 (3)
 ACCEPTED MANUSCRIPT

b*=240b/255-120 (4)

L* is the lightness, a* represents the green-red color axis (redness) and b* is the blue-

yellow axis (yellowness). The total chromatic difference with respect to the fresh cherry

sample (ΔE) was calculated using Equation 5.

T
ΔE = (ΔL2+Δa2+Δb2)0.5 (5)

IP
CR
2.6. Mechanical properties

US
Mechanical tests were performed with a Texture Analyzer (Stable Microsystems Ltd.,

TA-XT2i, UK) at 23 °C on the same day after processing. A probe (3 mm diameter

AN
stainless steel cylinder) with a trigger force of 5 N penetrated the sample to a depth of 8
M

mm at a speed of 1 mm/s. It returned to its original height at a speed of 10 mm/s. Five or

six cherries were measured for each treatment and each cherry was punctured twice,
ED

resulting in 10 or 12 measurements. Hardness was calculated as the peak compression

PT

force during cherry penetration. Stress as a function of strain curves were analyzed and

Young’s modulus or modulus of elasticity (E) was obtained from the slope of the curve in
CE

the elastic region. The fracture strain () was the strain at which the cherry failed via
AC

fracture.

2.7. Ascorbic acid determination

Ascorbic acid was extracted from the cherries immediately after processing and thawing

for 30 min by blending edible tissue with cold metaphosphoric and acetic acid (MPA)

solution at a ratio of 1:2.5. The metaphosphoric acid solution consisted of 30 g

 ACCEPTED MANUSCRIPT

metaphosphoric acid, 0.5 g EDTA and 80 mL glacial acetic acid diluted to 1 L with

distilled water. The blended sample was centrifuged for 15 min at 10,000 rpm in a cold

centrifuge (4 °C) and the supernatant was collected. Samples (3 mL) were passed through

solid phase extraction cartridges (Bond Elut C18, 500 mg, 3 mL, Agilent Technologies)

that were preconditioned with 2 mL acetonitrile followed by 3 mL distilled water. All

T
samples were kept on ice and HPLC analysis was performed on the same day as the

IP
extraction. Ascorbic acid was analyzed by injecting 20 μL of the sample into an Agilent

CR
HPLC 1100 series liquid chromatograph (Agilent Technologies, Wilmington, DE, USA).

A 20 mM H2SO4 solution was used as the mobile phase at a flow rate of 0.3 mL/min. A

US
ICSep ICE-ION-300 (300 x 7.8 mm) column and guard column with the same packing
AN
(Transgenomic, Inc., San Jose, CA) were used in conjunction with an Agilent diode array

detector set at 265 nm. Ascorbic acid standards ranging from 25 to 100 mg/mL were used
M

for calibration and sample peaks were identified according to HPLC retention times and
ED

absorbance spectra in comparison with authentic standards. Ascorbic acid determination

was performed in triplicate.

PT
CE

2.8. Total soluble phenolic content

Total soluble phenolic (TSP) content was determined through the Folin–Ciocalteu assay
AC

as described by Singleton and Rossi (1965) on the same day after processing and thawing.

HPLC grade methanol (20 mL) was added to one gram of pitted cherry in a 45 mL

centrifuge tube. Tubes were capped, vortexed for 15 s and then stored at 4 °C overnight.

The next day, the sample was vortexed for 15 s and then clarified by centrifugation

(15,600 rpm, 15 min at 4 °C) using a SORVALL RC 5C Plus centrifuge (Kendro

 ACCEPTED MANUSCRIPT

Laboratory Products, Newtown, CT). A total of 150 μL methanol extract was removed

from the clear supernatant and then diluted with 2400 μL nanopure water. After this,

150 μL of 0.125 mol L−1 Folin-Ciocalteu reagents was added and the sample was

incubated for 3 min at room temperature. The reaction was stopped by adding 300 μL of

0.5 mol L−1 Na2CO3 and the mixture was incubated for an additional 25 min. Absorbance

T
readings at 725 nm were taken using a Shimadzu PharmaSpec UV-1700

IP
spectrophotometer (Shimadzu Scientific Instruments, Inc., Columbia, MD). A blank

CR
prepared with water replacing sample extract was used as control. The total amount of

phenols was determined using a gallic acid standard curve and results were expressed as

US
milligrams of gallic acid equivalent (GAE) per gram of fresh weight of cherry purée.
AN
Three replicates were performed for each sample.
M

2.9. Antioxidant activity analysis

ED

The same methanol extract prepared during TSP analysis was used to determine the

antioxidant activity (AOX) of fresh and treated cherries. Duplicate samples were used for
PT

analysis from each extract. Sample aliquots of 50 μl were taken from the clear

supernatant and reacted with 2950 μL of 2,2-diphenyl-1-picrylhydrazyl (DPPH,

CE

103.2 μM in methanol, and absorbance of ∼1.2 at 515 nm) in a covered shaker at room
AC

temperature. The samples were allowed to react for 20 h. Absorbance at 515 nm was

recorded using the spectrophotometer. The antioxidant activity was calculated by

measuring the decrease in the sample absorbance compared to a methanol sample and

quantified from a standard curve developed for Trolox (0–750 μg/mL). Antioxidant

(AOX) values were expressed as milligrams of trolox equivalent (TE) per gram of fresh

weight. Three replicates were performed for each sample.

 ACCEPTED MANUSCRIPT

2.10. Cryo-SEM observations

Some samples were processed on the same day after processing and thawing to observe

microstructure changes. Immediately after treatment, cherries were cut parallel to the

longitudinal axis using a scalpel. The sample was placed in the SEM sample holder and

T
plunged into subcooled nitrogen (-210 °C). The frozen sample was transferred to the

IP
cryo-stage and then freeze fractured and gold coated. The samples were viewed in a field

CR
emission scanning electron microscope JEOL 7900F (JEOL, Kyoto) using a Quorum

PP3010T cryo system.

US
AN
2.11. Statistical Analysis of Data

Data were analyzed by one-way analysis of variance and Tukey’s multiple comparison
M

tests at 95% confidence level using Minitab version 14.2 statistical software (Minitab Inc.,
ED

State College, PA).

PT

3. RESULTS AND DISCUSSION

CE

3.1. Effects of freezing on mass change, °Brix, pH and drip loss

AC

The mass of cherry samples before and after freezing as well as sensory attributes such as

°Brix (related to sweetness) and pH (related to acidity) are shown in Table 1.

 ACCEPTED MANUSCRIPT

Table 1. Effect of freezing on mass change (%),°Brix, pH and drip loss (%)

Sample ΔM (%) °Brix pH Drip loss (%)

Fresh 22.6 ± 4.5a 3.99 ± 0.14a 23.2 ± 2.1d

Isochoric -4°C 2.99 ± 0.73 18.4 ± 3.6a 4.05 ± 0.15a 32.2 ± 1.6d

Isochoric -7°C 2.27 ± 0.84 17.6 ± 2.7a 3.91 ± 0.13a 50.1 ± 3.5b

T
Isobaric -4°C 0.02 ± 0.03 18.0 ± 1.1a 3.91 ± 0.11a 43.5 ± 0.1c

IP
Isobaric -7°C -0.45 ± 0.56 17.6 ± 2.7a 3.76 ± 0.22a 47.8 ± 1.4b

CR
IQF -0.36 ± 0.33 23.0 ± 0.5a 3.87 ± 0.07a 58.4 ± 1.0a

US
IQF= Individual Quick Freezing

Letters following values in the same column show homogeneous groups at 95% confidence interval (C.I.)
AN
Freezing under isobaric conditions or individual quick freezing did not result in
M

significant mass changes. In comparison, the cherries preserved under isochoric

ED

conditions showed a slight gain in mass (<3%) due to the impregnation of the cherry with

the external sucrose solution. Also, °Brix and pH values for all cherry samples did not
PT

change significantly due to freezing.

CE

Freezing cherries under isochoric conditions resulted in lower percentage of drip loss

than IQ freezing. In fact, the cherries frozen at -4°C showed no significant differences in
AC

drip loss compared to fresh cherries. The increase in drip loss at -7°C might be due to the

higher hydrostatic pressures reached at -7°C since hydrostatic pressure can change cell

permeability in vegetable tissues allowing the movement of water from inside to outside

the cell and can also break the cell structures if the pressure is high enough (Prestamo and

Arroyo, 1998).
 ACCEPTED MANUSCRIPT

Mean drip losses of cherries frozen under isobaric conditions were significantly higher

than drip losses in the fresh samples. This increase in drip loss indicated that some cell

damage occurred during isobaric freezing. In this case, water outside the cells could not

return to the cells during thawing and became drip loss. IQ frozen cherries showed the

greatest values in drip loss. It is well known that rapid freezing resulted in smaller and

T
less damaging ice crystals. However, small crystals are more likely to grow by

IP
recrystallization than large crystals due to their high surface free energy (Mazur, 1970).

CR
Consequently, recrystallization might have occurred in these samples especially if

temperature fluctuated during storage. These large ice crystals can exert sufficient force

US
to rupture cell membranes (Mazur, 1970). During thawing, the water will then leak out of
AN
the matrix and lead to significant drip loss.
M

3.2. Color analysis

ED

Appearance and color is the first important attribute that determines whether a product is

accepted or rejected. Consequently, this is one of the most critical quality attributes for
PT

foods (Barrett et al., 2010). The appearance and color of the fresh and thawed cherries are
CE

shown in Figure 2. Figure 3 shows the L*-a* and a*-b* color space for the fresh and

thawed samples.
AC

Isochoric freezing had a small effect on the L* a* b* values. Isochoric frozen cherries

showed darker color and a slight increase in a* (redness) and b* (yellowness) values.

This was probably due to the sucrose solution filling the porous structure of cherry tissue,

resulting in cherries with a translucent appearance. Samples at -4ºC and -7ºC showed no

significant differences in color, indicating that hydrostatic pressure over the experimental

range did not affect degradation of β-carotene (the major compound that contribute to the
 ACCEPTED MANUSCRIPT

yellow color in Rainier cherries) or phenolic compounds. In fact, a study by McInerney et

al (2007) showed that hydrostatic pressures up to 600 MPa had little influence on β-

carotene content in carrots and broccoli. Also, other authors showed that hydrostatic

pressures up to 600 MPa did not affect phenolic content in fruits (Khan et al., 2018).

In comparison, for isobaric and IQ frozen samples, lightness (L*) and b* values

T
decreased significantly in value, whereas a* values increased in value when compared to

IP
fresh samples. This behavior was more distinct at lower freezing temperature. The

CR
degradation of β-carotene was probably related to enzymatic oxidation caused by native

enzymes such as lipoxygenase (Penicaud et al., 2011). Also, enzymatic browning might

US
have occurred due to the degradation of polyphenols by enzymes such as polyphenol
AN
oxidases and peroxidase (Tian et al., 2004). These degradation reactions were most likely

accelerated due to membrane damage. Membrane damage from freezing most likely
M

assisted enzyme-substrate interactions that led to changes in color of the cherries

ED

In addition, the chromatic difference (ΔE) values confirmed differences in color of the

cherries. Isochoric preservation at -4ºC and -7ºC induced the lowest chromatic difference
PT

(12%), whereas IQ freezing induced the largest chromatic difference (32%). The
CE

chromatic difference for the samples frozen at -4ºC and -7ºC in an isobaric system was

17% and 30%, respectively.

AC

3.3. Mechanical properties

Consumers often purchase a product for the first time based on appearance, but repeat

purchases are driven by quality factors such as texture (Barrett et al., 2010).

Consequently, frozen cherries should have comparable texture to fresh cherries to ensure
 ACCEPTED MANUSCRIPT

greater customer appeal. Cherries preserved under isochoric conditions had the best

mechanical properties (Fig. 4). In fact, cherries preserved at -4°C under isochoric

conditions showed no significant differences in the maximum force, elasticity modulus

and strain fracture values when compared to fresh cherries. Decreasing the temperature

from -4°C to -7°C resulted in a slight decrease in sample firmness and rigidity because

T
high hydrostatic pressures can break cell structures and can cause loss in cell membrane

IP
permeability (Molina-Garcia, 2002).

CR
In comparison, freezing under isobaric conditions and IQ freezing produced the greatest

change in texture properties (Fig. 4). The firmness values of these samples were

US
significantly lower than those of fresh cherries, indicating that the freezing process
AN
weakened the integrity of the cell membranes and walls. The elastic modulus also

decreased significantly in value for these samples, indicating a more elastic behavior
M

associated with a loss in turgidity. The samples did not fracture during the compression
ED

tests since the cherries were not rigid enough to break or rupture. The firmness and

modulus of samples frozen under isobaric conditions or using IQF showed no statistical
PT

(P > 0.05) differences. This indicated that both slow and fast freezing rates produced
CE

considerable sample softening due to cell lysis and subsequent leakage of water and

cellular components. Previous studies had shown similar results. Kong et al. (2017)
AC

observed that thawed cherries stored at -20C for 31 days became extremely mushy in

texture with a complete loss of firmness to touch. Also, Alonso et al. (1995) found that

IQF cherries showed a decrease in firmness and elasticity modulus values. In addition,

Alonso et al., (2005) found that a major contributor to the loss of firmness was due to
 ACCEPTED MANUSCRIPT

leakage of intracellular pectin that resulted in the damage of calcium bridges and the

loosening of cell tissue.

In conclusion, these results indicated that preservation under isochoric conditions was

preferable to IQ freezing and preservation under isobaric conditions since the texture was

comparable to those of fresh Rainier cherries.

T
IP
3.4. Ascorbic acid content

CR
The total ascorbic acid content of fresh Rainier cherries was 5.3  0.8 mg/100 g. Cherries

US
preserved under isochoric conditions retained their ascorbic acid contents (Fig. 5). Also,

an increase in pressure from 30MPa to 62MPa did not have a significant effect on the
AN
ascorbic acid content of the samples. Previous studies had shown that hydrostatic

pressure did not affect low molecular weight food compounds, such as vitamins, since
M

pressure did not affect covalent bonds. In fact, Patras et al. (2009) demonstrated that
ED

pressures up to 600 MPa did not cause any significant changes in ascorbic acid contents
PT

in strawberry and blackberry purées.

In comparison, isobaric freezing and IQF led to significant ascorbic acid losses. This
CE

included 63% loss at -4°C under isobaric freezing, 51% loss at -7°C under isobaric

freezing and 72% loss after the IQF process (Fig. 5). Previous studies had also found loss
AC

in ascorbic acid contents after freezing. Poiana et al. (2010) reported a 12% decrease in

ascorbic acid content immediately after freezing cherries at -18°C. The ascorbic acid

degradation was probably due to enzymatic (via ascorbic acid oxidase) oxidation in the

presence of oxygen. In frozen products, enzymatic reactions are slow, but not completely

eliminated since enzymatic reactions still take place in the presence of non-frozen water.
 ACCEPTED MANUSCRIPT

In contrast to isochoric freezing, the isobaric and IQ freezing amplified these enzymatic

reactions as the freezing within the cherries disrupted the cell membranes, which assisted

enzyme substrate-interactions during thawing.

3.5. Total soluble phenolics (TSP)

Fresh cherries had a TSP value of 0.52  0.03 mg GAE.g−1, which was lower than the

T
reported values of 0.65  0.05 mg GAE.g-1 and 1.42  0.05 mg GAE.g-1 for flesh and

IP
skin, respectively (Chaovanalikit and Wrolstad, 2004). Cherries preserved under

CR
isochoric conditions showed apparent increases in TSP values (Fig. 6). Samples at -4°C

US
showed a 19% increase in TSP value and samples at -7°C showed a 27% increase. The

increase in phenolic compounds at the lower temperature (higher pressure) might be due
AN
to an increase in extractability of phenolics from damaged cells. However, the TSP

content also increased in value at higher temperature (low pressure) where cells remained
M

intact. This might be due to the response of the cherry cells to pressure/temperature
ED

stresses under isochoric conditions. The cells may have synthesized phenolic acids to
PT

protect themselves against these stresses. In this case, higher processing pressures at

lower temperatures could have caused a greater increase in TSP values. More conclusive
CE

experiments need to be performed to validate this theory.

AC

Cherries subjected to isobaric freezing and IQF treatment showed a significant decrease

in TSP content. For isobaric freezing, the TSP decreased 29% in value at -4°C and 18%

in value at -7°C. TSP also significantly decreased in value by 10% after IQ freezing. The

formation of ice might have caused damage to the delicate organelle and membrane

structures of the cells and dislocated the enzyme systems, particularly the

polyphenoloxidase one. This had been shown to be very active in flesh and skins of
 ACCEPTED MANUSCRIPT

cherries (Pifferi and Cultrera, 1974). Polyphenoloxidase activity is temperature-

dependent and consequently, polyphenol degradation can be minimized at lower freezing

temperatures. Several authors had reported a decrease in TSP content of cherries due to

freezing. Poiana et al., (2010) found a 7% decrease in ascorbic acid content immediately

after freezing at -18°C. Chaovanalikit and Wrolstad (2004) reported a 25% decrease in

T
TSP content of Bing cherries frozen under liquid nitrogen and stored for 3 months at -

IP
23°C. Moreover, Kong et al. (2017) reported 50% degradation of soluble phenolics after

CR
storage for one month at -20 °C.

3.6. Antioxidant activity (AOX)

US
AN
Studies show that antioxidant compounds can have positive effects on human health

because of their ability to scavenge free radicals and thus inhibit the oxidative
M

mechanisms that led to degenerative diseases (Kris-Etherton et al., 2002). Cherry is one
ED

fruit with high antioxidant activity, mostly related to anthocyanins, flavonoids and total

phenolics content (Ferretti et al.,2010; Piccolella et al., 2008). The antioxidant activity in
PT

fresh cherries was determined to be 1.9  0.1 mg TE.g−1, which was lower than the
CE

reported values of 3.3  0.1 mg TE.g-1 and 6.8  0.4 mg TE.g-1 in cherry flesh and skin,
AC

respectively (Chaovanalikit and Wrolstad, 2004).

Freezing at -4°C under isochoric conditions did not result in significant changes in

antioxidant activity. However, cherries preserved at -7°C under isochoric conditions

showed an apparent increase in antioxidant activity. This might be related to the increase

in TSP content discussed previously (Fig. 6).

 ACCEPTED MANUSCRIPT

In comparison, cherries subjected to freezing under isobaric conditions showed a slight

reduction in antioxidant activity (Fig. 7). This reduction was not as large as those of

ascorbic acid and total phenolics. One possible explanation could be that antioxidant

activity was correlated with the concentration of all bioactive compounds and some

polyphenolic degradation products still retained antioxidant activity. Previous studies

T
showed comparable results to those in this study. Chaovanalikit and Wrolstad (2004)

IP
reported a slight reduction (0.5%) in the antioxidant activity of cherries frozen under

CR
liquid nitrogen and stored for 3 months at -23°C. Also, Poiana et al. (2010) reported a

relatively small decrease in antioxidant activity in sweet cherries stored at -18°C.

US
AN
3.7. Cryo-SEM images

To better understand the effects of freezing on cherry structure, cryo-SEM images were
M

taken to visualize the tissue structure at the cellular level. The effects of freezing on the
ED

cell structures of thawed cherry parenchyma are shown in Figure 8. For comparison,

fresh cherry tissue is also shown in Fig. 8a.

PT

The cells of fresh cherry appeared compact and turgid. Some intercellular spaces filled
CE

with air could also be observed in the image. Cherries frozen at -4°C under isochoric

conditions (Fig. 8b) had cells that maintained intact membranes and walls. Intercellular
AC

spaces appeared to be filled with the external sugar solution, but the physical structures of

the cherry remained unharmed. The sample frozen at -7°C under isochoric conditions

(Fig. 8c) had tissue with a patchwork appearance that contained areas of well-preserved

cells as well as areas with a vitrified homogenous aspect due to the loss of cell
 ACCEPTED MANUSCRIPT

permeability and compartmentalization. This was probably caused by the different

compressibilities of the cellular materials under pressure (Molina-Garcia, 2002).

In comparison, freezing at -4°C or -7°C under isobaric conditions (Fig. 8d and 8e)

induced large changes in the cellular structures. In these samples, fluid appeared in the

intercellular spaces due to migration of cellular components. Also, some cells appeared

T
loose with large intercellular spaces due to the loss of intracellular pectin. In addition,

IP
dehydration caused buckling and folding of the cell walls. IQ freezing induced major

CR
damage to the tissue (Fig. 8f). In this sample, some cell walls could be identified as

irregular shapes since cell walls appeared as a lighter color. However, the tissue appeared

US
homogenous and it was difficult to differentiate between intra and extra cellular volumes
AN
since both were filled with water and solids due to the release of intracellular content.
M

4. CONCLUSIONS
ED

Rainier cherries frozen at -4C in an isochoric system had similar quality and nutritional

properties to those of fresh cherries. Isochoric freezing at -4C preserved the texture of
PT

cherries and minimized drip loss. The effectiveness of isochoric freezing had been related
CE

to the absence of ice crystals in the fruits during freezing, which reduced the cell damage

in cherry tissue observed under cryo-SEM. Isochoric freezing led to a translucent

AC

appearance in cherries, but they had the most comparable color to fresh cherries. Also,

isochoric freezing at -4C preserved the ascorbic acid content, phenolic compounds and

antioxidant activity in cherries since cell compartmentalization remained intact during

freezing. Reducing the temperature from -4C to -7C in an isochoric system led to a

decrease in firmness, rigidity and water holding capacity of the cherries. This suggested
 ACCEPTED MANUSCRIPT

that cell damage occurred at a discrete pressure threshold. Nonetheless, freezing at -7C

in an isochoric system preserved the quality and nutritional properties of the cherries to a

greater extent than freezing under isobaric conditions or using IQ freezing. In conclusion,

the use of isochoric freezing to extend the shelf life of fruits can lead to more widely

acceptable frozen fruit products with higher quality and a concomitant reduction in fruit

T
waste.

IP
CR
Acknowledgements

US
This work is supported by the USDA National Institute of Food and Agriculture, AFRI

project Proposal #: 2017-05031, Award # 2018-67017-27826 “Preservation of food by

AN
isochoric (constant volume) freezing”.
M

Conflict of interest statement

ED

The authors declare that there are no conflicts of interest.

PT
CE
AC
 ACCEPTED MANUSCRIPT

References

Alonso, J., Rodríguez, T., & Canet, W. J. (1995). Effect of calcium pretreatments on the

texture of frozen cherries. Role of pectinesterase in the changes in the pectic

materials. Journal of Agricultural and Food Chemistry, 43, 1011–1016.

T
Alonso, J., Tortosa, M.E., Canet, W. & Rodriguez, M.T. (2005). Ultrastructural and

IP
changes in pectin composition of sweet cherry from the application of prefreezing

CR
treatments. Journal of Food Science, 70, E526-E530.

Barrett, D. M., Beaulieu, J. C., & Shewfelt, R. (2010). Color, flavor, texture, and

US
nutritional quality of fresh-cut fruits and vegetables: Desirable levels,
AN
instrumental and sensory measurement, and the effects of processing. Critical

Reviews in Food Science and Nutrition, 50:369–389

M

Chaovanalikit, A., & Wrolstad, R. (2004). Total anthocyanins and total phenolics of fresh
ED

and processed cherries and their antioxidant properties. Journal of Food Science,

69(1), FCT67-FCT72.
PT

Chassagne-Berces, S., Poirier, C., Devaux, M.-F., Fonseca, F., Lahaye, M., & Pigorini,
CE

G., (2009). Changes in texture, cellular structure and cell wall composition in

apple tissue as a result of freezing. Food Research International, 42(7), 788-797.

AC

Chassagne-Berces, S., Fonseca, F., Citeau, M., & Marin, M. (2010). Freezing protocol

effect on quality properties of fruit tissue according to the fruit, the variety and the

stage of maturity. LWT–Food Science and Technology, 43, 1441–1449.

Ferretti, G., Bacchetti, T., Belleggia, A., & Neri, D. (2010). Cherry antioxidants: From

farm to table. Molecules, 15(10), 6993-7005.

 ACCEPTED MANUSCRIPT

Khan, M. K., Ahmad, K., Hassan, S., Imran, M., Ahmad, N., & Xu, C. (2018). Effect of

novel technologies on polyphenols during food processing. Innovative Food

Science & Emerging Technologies, 45, 361–381.

Kong, C. H. Z., Hamid, N., Ma, Q., Lu, J., Wang, B.-G., & Sarojini, V. (2017).

Antifreeze peptide pretreatment minimizes freeze-thaw damage to cherries: An

T
in-depth investigation. LWT - Food Science and Technology, 84, 441-448

IP
Kris-Etherton, P. M., Hecker, K. D., Bonanome, A., Coval, S. M., Binkoski, A. E., &

CR
Hilpert, K. F., et al. (2002). Bioactive compounds in foods: Their role in the

prevention of cardiovascular disease and cancer. The American Journal of

Medicine, 113(9), 71-88.

US
AN
Mazur, P. (1970). Cryobiology: The freezing of biological systems, Science, 68, 939-949.

McInerney, J. K., Seccafien, C. A., Stewart, C. M. & Bird, A. R. (2007). Effects of high
M

pressure processing on antioxidant activity, and total carotenoid content and

ED

availability, in vegetables. Innovative Food Science & Emerging Technologies, 8,

543-548.
PT

Molina-Garcia, A. D. (2002). The effect of hydrostatic pressure on biological systems.

CE

Biotechnology and Genetic Engineering Reviews, 19, 3–54.

Mulabagal, V., Lang, G. A., DeWitt, D. L., Dalavoy, S. S., & Nair, M. G. (2009).
AC

Anthocyanin content, lipid peroxidation and cyclooxygenase enzyme inhibitory

activities of sweet and sour cherries. Journal of Agricultural and Food Chemistry,

57, 1239-1246.
 ACCEPTED MANUSCRIPT

Năstase, G., Perez, P. J., Șerban, A., Dobrovicescu, A., Ștefănescu M.-F., & Rubinsky,

B. (2016). Advantages of isochoric freezing for food preservation: a preliminary

analysis. International Communications in Heat and Mass Transfer, 78, 95-100.

Năstase, G., Lyu, C., Ukpai, G., Șerban, A. & Rubinsky, B. (2017). Isochoric and

isobaric freezing of fish muscle. Biochemical and Biophysical Research

T
Communications, 485, 279-283.

IP
Oey, I., Lille, M., Van Loey, A., & Hendrickx, M. (2008). Effect of high-pressure

CR
processing on colour, texture and flavour of fruit-and vegetable-based food

products: A review. Trends in Food Science & Technology, 19 (6), 320-328.

US
Patras, A., Brunton, N. P., Da Pieve, S., & Butler, F. (2009). Impact of high pressure
AN
processing on total antioxidant activity, phenolic, ascorbic acid, anthocyanin

content and colour of strawberry and blackberry purées. Innovative Food Science
M

& Emerging Technologies, 10 (3), 308-313.

ED

Penicaud, C., Achir, N., Dhuique-Mayer, C, Dornier, M., & Bohuon, P. (2011).

Degradation of β-carotene during fruit and vegetable processing or storage:

PT

reaction mechanisms and kinetic aspects: a review. Fruits, 66, 417-440.

CE

Petersen, M., & Poll, L. (1999). The influence of storage on aroma, soluble solids, acid

and colour of sour cherries (Prunus cerasus L.) cv. Stevnsbaer. European Food
AC

Research and Technology, 209 (3-4), 251-256.

Pifferi, P. G., & Cultrera, R. (1974). Enzymatic degradation of anthocyanins: the role of

sweet cherry polyphenol oxidase. Journal of Food Science, 39, 786-791.

Piccolella, S., Fiorentino, A., Pacifico, S., D'Abrosca, B., Uzzo, P., & Monaco, P. (2008).

Antioxidant properties of sour cherries (Prunus cerasus L.): Role of colorless

 ACCEPTED MANUSCRIPT

phytochemicals from the methanolic extract of ripe fruits. Journal of Agricultural

and Food Chemistry, 56(6), 1928-1935.

Poiana, M. A., Moigradean D., & Alexa, E. (2010). Influence of home-scale freezing and

storage on antioxidant properties and color quality of different garden fruits.

Bulgarian Journal of Agricultural Science, 16, 163-171.

T
Preciado, J.A., & Rubinsky, B. (2010). Isochoric preservation: a novel characterization

IP
method, Cryobiology, 60, 23-29.

CR
Prestamo G., & Arroyo, G. (1998). High hydrostatic pressure effects on vegetable

structure. Journal of Food Science, 5, 878-881.

US
Rubinsky, B., Perez, P. A., & Carlson, M. E. (2005). The thermodynamic principles of
AN
isochoric cryopreservation. Cryobiology, 50, 121-138.

Singleton, V.L. & Rossi, J.A. (1965). Colorimetry of total phenolics in grapes and wine
M

with phosphomolybdic–phosphotungstic acid reagents. American Journal of

ED

Enology and Viticulture, 16, 144-158.

Szobota, S. A. & Rubinsky, B. (2006). Analysis of isochoric subcooling. Cryobiology, 53,

PT

139-142.
CE

Tian, S.-P., Jiang, A.-L., Xu, Y., & Wang, Y.-S. (2004). Responses of physiology and

quality of sweet cherry fruit to different atmospheres in storage. Food Chemistry,

AC

87(1), 43-49.
 ACCEPTED MANUSCRIPT

Figure captions

Fig. 1. Schematic of isochoric chamber and generalized freezing process. In a system of constrained liquid

volume with rigid walls and no air pockets, ice expansion will generate hydrostatic pressure, which

depresses the freezing point of the system. The pressure will continue to rise until the freezing point of the

system becomes equal to the system temperature. At this point, liquid and solid phases will exist in

T
equilibrium. Approximately 12% and 23% of the total volume will be frozen at -4°C and -

IP
7°C, respectively.

CR
Fig. 2. Photographs of (a) fresh cherry, (b) thawed cherry frozen to -4ºC in an isochoric system, (c) thawed

US
cherry frozen to -7ºC in an isochoric system, (d) thawed cherry frozen to -4ºC in an isobaric system, (e)

thawed cherry frozen to -7ºC in an isobaric system and (f) thawed cherry from IQF
AN
Fig. 3: L*-a* and a*-b* color space of fresh and thawed cherries frozen under different conditions.
M
ED

Fig. 4. Effects of freezing methods on the (a) maximum force and (b) elasticity modulus of thawed cherries

under different freezing conditions. The same letter indicates no significant differences between treatments
PT

at 95% confidence interval.

CE

Fig. 5. Ascorbic acid contents of fresh and thawed cherries using different freezing methods. The same

letter indicated no significant differences between treatments at 95% confidence interval.

AC

Fig. 6. Total soluble phenolic contents of fresh and thawed cherries using different freezing methods. The

same letter indicated no significant differences between treatments at 95% confidence interval.

Fig. 7. Effects of freezing methods on the antioxidant activity of thawed cherries under different freezing

conditions. The same letter indicates no significant differences between treatments at 95% confidence

interval.
 ACCEPTED MANUSCRIPT

Fig. 8. Cryo-SEM images of cherry parenchyma tissue in (a) fresh cherry, (b) thawed cherry frozen to -4ºC

in an isochoric system, (c) thawed cherry frozen to -7ºC in an isochoric system, (d) thawed cherry frozen to

-4ºC in an isobaric system, (e) thawed cherry frozen to -7ºC in an isobaric system, (f) thawed cherry from

IQF

T
IP
CR
US
AN
M
ED
PT
CE
AC
 ACCEPTED MANUSCRIPT

Highlights

 Isochoric freezing can preserve cherries at subfreezing temperatures without any

ice

T
IP
 Cherry quality is better preserved with isochoric freezing than conventional

CR
freezing


US
Quality of the cherries remain intact when frozen at -4C in an isochoric system
AN
M
ED
PT
CE
AC
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8