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A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing
INNFOO-01228; No of Pages 8 Innovative Food Science and Emerging Technologies xxx (2014) xxx–xxx
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A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing Thunyaboon Arunyanart a, Ubonrat Siripatrawan a,⁎, Yoshio Makino b, Seiichi Oshita b a
Department of Food Technology, Faculty of Science, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand Laboratory of Bioprocess Engineering, Department of Biological and Environmental Engineering, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
b
a r t i c l e
i n f o
Article history: Received 10 June 2014 Accepted 2 September 2014 Available online xxxx Editor Proof Receive Date 30 October 2014 Keywords: Xenon Gas hydrate Food processing Preservation technique Freezing Nuclear magnetic resonance
a b s t r a c t Freezing usually causes cell and tissue damage in frozen fruits. This study attempted to use a combined method of xenon hydrate formation and freezing (CXF) for the preservation of apple parenchyma tissue and to compare it with the freezing alone process (FAP). CXF included two steps. The first step was to initiate a certain amount of xenon hydrate by introducing the apple parenchyma tissue to the xenon gas at 1.0 MPa and 1 °C for 0, 1, 2, 3, 4, 5, 6 and 7 d. It was found that the amount of xenon hydrate in apple parenchyma tissue increased with storage time and 2 d was optimum to obtain the certain amount of xenon hydrate. In the second step, the sample with optimum xenon hydrate formation was frozen at −20 °C. The results showed that CXF was more effective in maintaining firmness, turgor pressure, and cell membrane integrity of the apple parenchyma tissue than FAP. A typical restricted diffusion phenomenon which indicates that water molecules are maintained in the apple parenchyma cells was found in the CXF samples, while the FAP samples showed an unrestricted diffusion phenomenon. In addition, firmness, turgor pressure, cell membrane integrity, and restricted diffusion phenomenon of the CXF samples were similar to those of the fresh samples. The CXF could preserve the apple parenchyma tissue because of the bulk water inside the cells and the water surrounding the cells which transformed to ice crystals is limited. Thus, cell and tissue damage due to the formation of ice crystals was reduced. The obtained results indicated that the CXF is effective for the preservation of the apple parenchyma tissue. Industrial relevance: There has been an attempt to improve the quality of frozen fruit by using innovative techniques, in opposition to simply freezing. This present work proposed xenon hydrate formation for the reduction of bulk water before freezing in order to reduce freezing damage due to a large amount of ice crystal formation. The combined method of xenon hydrate formation and freezing has been proved to be able to reduce cell membrane damage usually occurring in frozen fruit. Thus this new technique has potential to be used for improving the quality of frozen fruit. The xenon hydrate formation is considered as an innovative technique for the preservation of fruit, which is expected to be useful for the frozen food industry. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Freezing is an excellent method for preservation and storage of fruits in the food industry because it preserves color, flavor and nutrition values of fruits. During freezing process, bulk water in fruit tissue is transformed to ice crystals. The ice crystals expand the tissue matrix leading to cell volume changes, resulting in cellular structural damage (De Ancos, Sánchez-Moreno, De Pascual-Teresa, & Cano, 2012; Silva, Gonçalves, & Brandoão, 2008). Freezing damage of cellular structure due to ice crystals usually occurs in plant tissues because of the semirigid nature of cells which leads to loss of tissue firmness, and a subsequent loss of textural quality in frozen fruits (Chassagne-Berces et al., 2009; Khan & Vincent, 1996; Zaritzky, 2012). ⁎ Corresponding author. Tel.: +66 22185536; fax: +66 22544314. E-mail address: [email protected] (U. Siripatrawan).
An attempt to improve quality of frozen fruits using novel preservation technique has been investigated. Osmotic dehydrofreezing technique has been successfully used in freezing of fruits with minimal damage to cellular integrity and texture. This technique aims to reduce the water content in food materials by immersion in aqueous solution before freezing (Ando, Kajiwara, Oshita, & Suzuki, 2012; Li & Sun, 2002; Silva et al., 2008). The disadvantage of an osmotic dehydrofreezing technique is that it is a type of an aqueous solution that affects the sensory characteristics of products (Dixon & Jen, 1977). Furthermore, leaching of food materials in the aqueous solution can result in loss of the nutritional content of products (Blanda et al., 2009). Recently, xenon hydrate formation has been introduced as an innovative technique for the preservation of agricultural products. Xenon gas is used for the preservation of agricultural products due to its non-polar nature and no reaction with biological materials (Purwanto, Oshita, Seo, & Kawagoe, 2001). Gas hydrates, composed of water and gas, are
http://dx.doi.org/10.1016/j.ifset.2014.09.008 1466-8564/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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crystalline solids or ice-like crystals of which gas molecules are trapped in water lattice that are composed of hydrogen-bonded network of water molecules. Gas hydrate formation is a time-dependent process. The process of gas hydrate formation from water and gas comprises four steps. In the first step, labile ring structures of water pentamer (H2O)5 and water hexamer (H2O)6 are formed from water molecules. The labile ring structure can be formed when the water becomes supercooled water of which optimally at low temperature and under pressure. In the next step, structured water is initiated when labile clusters (hydrogen bond network) of water molecules form around the dissolved gas molecules. After that, the labile clusters agglomerate. In the final step, the labile clusters grow and form rigid gas hydrate (Sloan & Koh, 2008). Wang, Ando, Kawagoe, Makino, and Oshita (2009) found that barley coleoptile cells preserved by xenon hydrate formation at low temperature showed significantly higher viability than those preserved by freezing. However, the viability of barley coleoptile cells under xenon hydrate formation decreased with storage time. Ando et al. (2011) reported that texture characteristics such as elasticity of the onion tissue after xenon hydrate formation-decomposition decreased with the amount of xenon hydrate. They hypothesized that the cell membrane of onion tissue was damaged due to the growth of xenon hydrate with storage time. The results of using xenon hydrate formation for the preservation of agricultural products had a limitation because increasing the amount of xenon hydrate with storage time leads to cell and tissue damage. Reduction of freezing damage caused by ice crystal is a key factor in the preservation of frozen fruits because fruits contain a large amount of water (Silva et al., 2008), their cellular structure could be destroyed by ice crystal during freezing. A procedure to reduce the amount of bulk water before transformation to ice crystal could reduce freezing damage. This present work proposed xenon hydrate formation in the reduction of bulk water before freezing in order to reduce freezing damage due to ice crystal formation. There has been no research using xenon hydrate formation for the preservation of fruit tissue. The xenon hydrate formation is considered as an innovative technique for the preservation of fruit, which is expected to be useful for the frozen food industry. Therefore, the objective of this research was to use the combined method of xenon hydrate formation and freezing (CXF) for the preservation of apple parenchyma tissue in comparison with the conventional freezing alone process (FAP). 2. Materials and methods 2.1. Sample preparation The apple parenchyma tissue was used as studying structural cell components because of its low metabolic activity (Snaar & Van As, 1992) and its macroscopic flesh homogeneity. Fresh apples (Malus pumila cv. Fuji) at commercial maturity (total soluble solids at 20 °C were in the range of 13–14 °Brix) were purchased from a local supermarket in Tokyo, Japan. The apple was cut into a 4 mm × 4 mm × 10 mm tissue block, which was taken equidistantly from the outer cortex and the core in the parenchyma region (Fig. 1) and dipped into 1.0% citric acid solution for 10 min in order to prevent enzymatic browning (De Ancos et al., 2012). 2.2. Combined method of xenon hydrate formation and freezing vs freezing alone process In this study, two methods including CXF and FAP were compared. The fresh sample was used as reference for each measurement. The schematic of the experimental setup was shown in Fig. 2. For the CXF method, the optimal xenon hydrate formation was first determined and followed by freezing the xenon hydrate processed sample at −20 °C. Xenon hydrate formation was conducted by placing an apple tissue block into a nuclear magnetic resonance (NMR) glass tube, having
Fig. 1. Apple parenchyma tissue block.
an outer diameter of 10 mm, and xenon gas (99.995%, Iwatani Co., Tokyo, Japan) was introduced under pressure of 1.0 MPa at 1 °C for 2 d using a pressurizing unit, as shown in Fig. 3. The tube was then kept in a medical chest freezer (SANYO, MDF-435, Tokyo, Japan) at −20 °C for 7 d. From the preliminary studies, xenon hydrate formation was conducted by introducing xenon gas under pressure of 1.0 MPa at 1 °C for 0, 1, 2, 3, 4, 5, 6 and 7 d and the formation of xenon hydrate was monitored daily by using a NMR measurement of solid echo. It was found that the optimal xenon hydrate formation in apple parenchyma tissue is 2 d. When stored longer than 2 d, cell membrane damage caused by a larger amount of xenon hydrate was evidenced as measured by using a self-diffusion coefficient of water molecules. After the optimal xenon hydrate formation was obtained, the sample was stored at − 20 °C for 7 d. For FAP, the apple tissue block was placed in a NMR glass tube and was frozen in a medical chest freezer at −20 °C for 7 d. After storage, both CXF and FAP samples were thawed at 20 °C for 1 h before the measurements of texture, water content in cellular structure, and self-diffusion coefficient. For the CXF samples, xenon hydrate was decomposed by releasing xenon gas from the NMR glass tube together with thawing of ice crystal. 2.3. NMR measurement of the amount of xenon hydrate Solid echo NMR was used to measure the amount of xenon hydrate in apple parenchyma tissue sample. A 25-MHz pulsed NMR spectrometer (JNM-MU25A, JEOL Ltd., Tokyo, Japan) with an attached temperature control unit (NM-VT/MU25, JEOL Ltd., Tokyo, Japan) was used. In order to measure the free induction decay (FID) by NMR spectrometer, dimension of apple tissue block of 4 mm × 4 mm × 10 mm was required to get stable NMR signals. To measure the spin–spin relaxation time of protons (T2) in the solid component of the sample, a solid echo pulse sequence was applied following the method of Ando, Suzuki, Kawagoe, Makino, and Oshita (2009) and Gribnau (1992). The sequence of a solid echo pulse consists of 90°–τ–90°–τ–echo, where 90° is the flip angle induced by the radio frequency pulse and τ is time between pulses in a pulse sequence. The signal intensity was recorded as free induction decay. The pulse interval for eliminating the background effect from the empty sample tube was determined to be 8 μs. The repetition time and scan number were 30 s and 16 times, respectively. The amount of xenon hydrate in apple parenchyma tissue was determined from the FID as the solid ratio (the number of molecules of solid components to the
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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Fig. 2. The schematic of the experimental setup. (CXF = combined method of xenon hydrate formation and freezing, FAP = freezing alone process).
combined number for all forms of water molecules) according to the method of Gribnau (1992). When a mixture of solid and liquid is measured, the FID normally shows two different regions. Since the intensity of the FID signal is directly proportional to the number of proton spins, the ratio of proton spins can be quantified in different phases. Thus, a solid ratio can be determined. The experiment was performed under 1.0 MPa xenon pressure at 1 °C.
2.4. Texture measurement A Texture analyzer (RE2-33005S, YAMADEN Ltd., Tokyo, Japan) with a 3 mm diameter cylindrical probe was used to determine the texture of the samples. The texture was measured at a horizontal position of the samples. The penetration test was set as 20 mm into samples at 1 mm/s. Firmness value was the maximum force of the force–deformation curve and express as newtons (N). The measurements were conducted with five replications for each treatment. 2.5. NMR measurement of water content Relaxation time NMR was used to indicate the water content in the cellular structure of the samples. A 25-MHz pulsed NMR spectrometer with an attached temperature control unit was used to measure the spin–spin relaxation time of protons at 20 °C. The transverse or spin– spin relaxation time was measured using Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence. The sequence of a CPMG consists of 90°–(τ– 180°–τ– echo)n, where n is the number of times the cycle is repeated. 2.6. NMR measurement of self-diffusion coefficient
Fig. 3. Unit for transmission pressurized xenon to the NMR sample tube.
Stimulated echo NMR was used to measure self-diffusion coefficient (D) of water molecules in the samples. Self-diffusion coefficient of water molecules in the apple parenchyma tissue was measured following the modified method as described by Ando, Fukuoka, Miyawaki, Watanabe, and Suzuki (2009). Each sample was set on 25 MHz pulse NMR spectrometer equipped with pulse field gradient accessory. The experiment was done by the stimulated echo pulse sequence (90°–τ–90°–τ–90°–τ– echo) with the magnitude of pulse field gradient, varying from 21.0 to 49.2 Gauss/cm, at a selected diffusion time. Degree of the restriction shows capacity of water diffusion through cell membrane in the tissue (Ando, Fukuoka, Miyawaki, Watanabe, & Suzuki, 2009; Tanner, 1978). The experiment was performed at 20 °C under atmospheric pressure.
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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When restricted diffusion was measured by using pulse field gradient nuclear magnetic resonance (PFG-NMR), the permeability of cell membrane was evaluated from the relationship between self-diffusion coefficient and diffusion time by using Eq. 1 (Anisimov, Sorokina, & Dautova, 1998).
1=D∞ ¼ 1=D0 þ 1=ðP aÞ
ð1Þ
where D∝ is the self-diffusion coefficient at maximum diffusion time (m2/s), D0 is the self-diffusion coefficient at minimum diffusion time (m2/s), P is the permeability of cell membrane (m/s), and a is the fixed cell diameter parameter (μm) which is observed by using an optical microscope. 2.7. Statistical analysis
the tissue was stored for 2, 4 and 7 d under xenon gas pressure of 1.0 MPa at 1 °C. It was found that xenon hydrate started to form after storage for 2 d. The amount of xenon hydrate in the apple parenchyma tissue was determined by the NMR technique. Fig. 6a shows free induction decay of protons in apple parenchyma tissue under xenon gas pressure of 1.0 MPa at 1 °C. The decay curve of samples after 0 and 1 d storage shows a linear decay, which presents the relaxation time of the water (liquid) component in the apple parenchyma tissue. When the samples were stored from 2 to 7 d, the decay curves showed a two-component pattern that indicates solid and water phases in the apple parenchyma tissue after xenon hydrate formation. The solid ratio in the sample increased with storage time. The percentage of solid ratio, as shown in Fig 6b, was calculated from Eq. 2 (Gribnau, 1992). solid ratio ð%Þ ¼ ðS−LÞ=S 100
A completely randomized design (CRD) was used as an experimental design. The difference between means was determined at p ≤ 0.05 using the Duncan's new multiple range test. All statistical analyses were performed using SPSS for Windows. 3. Results and discussion 3.1. Amount of xenon hydrate formation in apple parenchyma tissue The parenchyma cell of apple is in a polygonal shape as shown in Fig. 4, with an average cell diameter of 206 μm. The parenchyma cells of apple were observed at 20 °C with an optical microscope (Leica DML, Wetzlar, Germany). The main compartment of the parenchyma cell consists of vacuole, cytoplasm, middle lamella and intercellular space. Vacuole, the largest part of cell volume, is related to turgor pressure of the plant cell. Cytoplasm locates between cell wall and vacuole. Middle lamella combines the region of parenchyma cells to form the parenchyma tissue. Intercellular space is the separated region of each parenchyma cell which contains air or water (Brecht, Ritenour, Haard, & Chism, 2008; De Ancos et al., 2012; Mohsenin, 1986). In this study, the xenon hydrate formation was conducted under pressure of 1.0 MPa at 1 °C because at this condition the small diameter hydrate crystals were formed. It has been suggested that the small diameter of hydrate crystals could reduce cell membrane damage (Wang et al., 2009). Fig. 5 illustrates fresh apple parenchyma tissue and xenon hydrate processed apple parenchyma tissue during storage of 2, 4 and 7 d under 1.0 MPa xenon pressure at 1 °C. The appearance of xenon hydrate is ice-like crystals that gradually grow with storage time. The xenon hydrate was observed on the apple parenchyma tissue surface when
ð2Þ
where S is the total signal intensity of solid and liquid, and L is the signal intensity of liquid. Fig. 7 shows xenon hydrate (%) in apple parenchyma tissue calculated from the percentage of solid ratio. In this study, the percentage of solid ratio in samples indicated the amount of xenon hydrate in the samples. The xenon hydrate in apple parenchyma tissue significantly increased (p ≤ 0.05) with storage time. Approximately 20% of bulk water in the apple parenchyma tissue was transformed to xenon hydrate when the sample was stored under xenon gas pressure of 1.0 MPa at 1 °C for 2 d. The highest amount of xenon hydrate was found to be approximately 32% when the samples were stored up to 6 d or longer. From the preliminary results, cell membrane of apple parenchyma tissue was destroyed by increasing of amount of xenon hydrate from 3 to 7 d (approximately from 25% to 32%), which is indicated by selfdiffusion coefficient of water molecules. These results are in good agreement with those of Ando et al. (2011) who found that an increase in the amount of xenon hydrate during storage under xenon hydrate formation destroyed onion tissues. Since 20% of xenon hydrate was found to be optimum to avoid cell damage, the condition of storage under xenon gas pressure of 1.0 MPa for 2 d was used for the CXF. According to Bishnoi and Natarajan (1996), gas hydrates are formed by certain gases such as CH4, Xe, N2 and O2 when contacted with water under optimal temperature and pressure conditions. It has been reported that temperature and pressure optimal for xenon–water clathrate or xenon hydrate formation of water range between 0–12 °C and 0.15– 0.50 MPa, respectively (Ewing & Ionescu, 1974). In this present work, it was suggested that the portion of the bulk water inside and surrounding the cells (intercellular space and middle lamella) of the apple parenchyma tissue started to transform from water to xenon hydrate when
Fig. 4. The parenchyma cells of apple (Malus pumila cv. Fuji) under an optical microscope at 10× magnification.
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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Fig. 5. Xenon hydrate formation in apple parenchyma tissue under 1.0 MPa at 1 °C and stored for 2, 4, and 7 d. (0 d is the sample exposed to xenon gas under pressure of 1.0 MPa at 1 °C for 1 h.) The arrows indicate xenon hydrate.
the samples were stored under xenon pressure of 1.0 MPa at 1 °C for 2 d. As shown in Fig. 5, xenon hydrate in apple parenchyma tissue increased with storage time. The water in apple parenchyma tissue contains solutes (e.g. sugars and organic acids), which is considered to be an aqueous solution. The elevated pressure of xenon was required for the formation of xenon hydrate. This reason is in good agreement with that of Purwanto et al. (2001) who investigated the concentration of liquid foods by the use of xenon hydrate and reported that the hydrate forming pressures for the coffee solution of 14.7 wt.% were higher
than those required for 5.1 wt.% concentration and distilled water, respectively. 3.2. Firmness of apple parenchyma tissue The firmness of fresh, CXF, and FAP samples is shown in Fig. 8. The firmness of samples from FAP significantly decreased (p ≤ 0.05) when compared to that of fresh samples. The decreasing of firmness was probably because FAP causes cell membrane damage due to the formation of
Fig. 6. Free induction decay of sample with xenon hydrate formation stored for 0–7 d (a), and percentage of solid ratio in the sample calculated from free induction decay of sample (b).
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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and −196 °C affected the texture of apple tissue due to cell membrane rupture and tissue breakage. The firmness of CXF samples slightly decreased (p ≤ 0.05) when compared to that of fresh samples. On the other hand, the firmness of FAP samples (24% loss) were found to be significantly lower (p ≤ 0.05) than that of CXF (9% loss) and fresh samples, respectively. It has been suggested that the xenon hydrate formation can reduce bulk water inside and surrounding the cells (intercellular space and middle lamella), and subsequently limits the available water transformed to ice crystals during freezing process. Thus, cell and tissue damage due to formation of intracellular and extracellular ice crystals was reduced. 3.3. NMR relaxation time of apple parenchyma tissue
Fig. 7. Percentage of xenon hydrate in apple parenchyma tissue under 1.0 MPa at 1 °C measured by NMR spectroscopy with a solid echo pulse sequence. The error bars indicate standard deviation (n = 2). Different letters indicate significant difference (p ≤ 0.05) when analyzed by Duncan's new multiple range test.
ice crystals, leading to disruption of cellular structure (De Ancos et al., 2012; Silva et al., 2008), and loss of turgor pressure (Brown, 1977) in fruit tissues. The FAP caused loss of firmness in the samples because ice crystals formed in the extracellular areas (intercellular space and middle lamella) destroyed the cell membrane. Since the cell membrane was damaged, during thawing water migrated out of the cell due to osmotic pressure (Zaritzky, 2012). In general, freezing of food tissue can lead to extracellular ice crystals because the water surrounding the cell is frozen before the cell contents due to the fact that cytoplasm is more concentrated than liquid in the extracellular areas and, thermodynamically, the component with the largest volume nucleates first (Dumont, Marechal, & Gervais, 2004; Zaritzky, 2012). Thus, food tissue damage is mainly caused by extracellular ice crystals. The loss of firmness and the decrease in strength of the frozen-thawed apple tissue have been reported to be due to the loss of turgor pressure (Sterling, 1968). Chassagne-Berces et al. (2009) reported the effects of three different freezing processes (at −20 °C, −80 °C and −196 °C) on mechanical properties of apple tissue and found that freezing at −20 °C
Fig. 8. Firmness of apple parenchyma tissue in fresh, CXF, and FAP samples. The error bars indicate standard deviation (n = 5). Different letters indicate significant difference (p ≤ 0.05) when analyzed by Duncan's new multiple range test.
The spin–spin relaxation time of protons can be used to identify three relaxation components pertaining to water in vacuole, cytoplasm, and intercellular space of apple parenchyma tissue (Snaar & Van As, 1992). In this experiment, two components of T2 were obtained from the apple parenchyma tissue including T2a (long relaxation time) indicating the vacuole water and T2b (short relaxation time) indicating the cytoplasm and intercellular water. As shown in Fig. 9, T2a value of FAP samples significantly decreased (p ≤ 0.05) when compared to that of fresh samples. This indicated that during the freezing process vacuolar membrane was damaged by intracellular ice crystal, leading to vacuole rupture and leakage of vacuole water. This phenomenon causes loss of turgor pressure and affects the texture of fruit tissues. It is well known that cell turgor pressure is related to the water content in vacuole (Waldron, Smith, Parr, Ng, & Parker, 1997) and the loss of turgor pressure causes a major change in textural characteristics of fruit and vegetable (Brown, 1977). The T2a value of CXF samples was higher (p ≤ 0.05) than that of FAP samples because water content in vacuole of CXF samples is more than that of FAP samples. The CXF could reduce vacuole rupture and water leakage from vacuole because the formation of intracellular ice crystal was limited. The T2a value corresponded with firmness. Therefore, the CXF could maintain turgor pressure and firmness of apple parenchyma tissue better than FAP. T2b value of FAP samples significantly decreased (p ≤ 0.05) when compared to that of fresh samples (Fig. 9). This indicated that cell membrane was probably damaged by extracellular ice crystals, causing a leakage of water out of the cell. The T2b value of CXF samples slightly decreased (p ≤ 0.05) when compared to that of fresh samples. The result showed that cytoplasm and intercellular water content in cells of CXF samples is more than those of the FAP samples. It was suggested that the CXF could maintain cellular structure and water holding capacity in the apple parenchyma tissue because transformation of water surrounding the cells to extracellular ice crystals was limited. As shown in Fig. 9, T2a value is much higher than T2b value indicating that water content in vacuole is more than that in cytoplasm and intercellular. Vacuole occupies the largest part of the plant cell and controls turgor pressure of the cell (De Ancos et al., 2012). Thus, this research T2a was used for monitoring the firmness of fruit tissue. In addition, it should be noted that the NMR technique for measuring spin–spin relaxation time of protons (e.g. T2a) could be used as a rapid method for monitoring of the freezing damage in fruit tissues because loss of firmness is a major deterioration of frozen fruits. This technique is a qualitative analysis for the determination of freezing damage in fruit tissues without a complicated sample preparation. Determination of freezing damaged in fruit tissues by using the NMR technique is currently reported in other research. For example, Hills and Remigereau (1997) used spin–spin relaxation time of protons to indicate loss of cell membrane integrity in frozen-thawed apple tissue. The spin–spin relaxation time was also used to reflect the rupture of navel juice sacs due to a frozen-thawed process (Gambhir, Choi, Slaughter, Thomson, & McCarthy, 2005).
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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Fig. 9. Spin–spin relaxation time of protons of apple parenchyma tissue in fresh, CXF, and FAP samples. T2a and T2b are the long and short relaxation times. The error bars indicate standard deviation (n = 2). Different letters indicate significant difference (p ≤ 0.05) when analyzed by Duncan's new multiple range test.
3.4. Self-diffusion coefficient of water molecules and permeability of cell membrane in the apple parenchyma tissue Self-diffusion coefficient shows molecular mobility of water that can be studied using the PFG-NMR technique. This coefficient is related to the average distance the molecules travel, thus providing direct information on the material transport properties (Brosio, 2009). The selfdiffusion coefficient of water molecules of all samples is shown in Fig. 10. Self-diffusion coefficient of fresh samples significantly decreased (p ≤ 0.05) with increasing diffusion time and approach a constant value, whereas the relative self-diffusion coefficient vs diffusion time shows an exponential behavior. A typical restricted diffusion phenomenon is shown in fresh samples. Typically, the apparent self-diffusion coefficient of water molecules in plant tissues is not constant, but decreases with diffusion time because water molecules are in the limited space of plant cell (Ando, Fukuoka, Miyawaki, Watanabe, & Suzuki, 2009). The restricted diffusion phenomenon indicates that water molecules cannot move freely due to barrier properties of the cell membrane which is semi-permeable (Meerwall & Ferguson, 1981; Tanner, 1978). Like fresh apple parenchyma tissue, CXF sample maintained a restricted
Fig. 10. Self-diffusion coefficient of water molecules of apple parenchyma tissue in fresh, CXF, and FAP samples. The error bars indicate standard deviation (n = 2).
diffusion phenomenon to be at minimum. In contrast, the self-diffusion coefficient of FAP samples was higher (p ≤ 0.05) than that of fresh samples, indicating an unrestricted diffusion phenomenon. It can be explained that the cell membrane of FAP samples was damaged probably due to the formation of extracellular ice crystals. To evaluate cell damage in the apple parenchyma tissue, the change in permeability of cell membrane was calculated by using Eq. 1. To calculate permeability of cell membrane, the self-diffusion coefficient value (D∝ at 500 ms and D0 at 20 ms from PFG-NMR measurement) and cell diameter (a = 206 μm from optical microscopic observation) are used, as known parameters. Fig. 11 shows permeability of cell membrane of fresh, CXF, and FAP samples. The permeability of cell membrane of the FAP samples (P = 4.94 × 10−6 m/s) significantly increased (p ≤ 0.05) when compared with that of the fresh samples (P = 0.91 × 10−6 m/s). This is probably because extracellular ice crystal formation during FAP destroyed the semi-permeability of cell membrane. For the CXF samples, the permeability of cell membrane (P = 1.78 × 10− 6 m/s) slightly increased when compared with that of the fresh samples. It was indicated that the CXF could maintain the semi-permeability of cell membrane in the samples because cell membrane damage was reduced due to the limit of water surrounding the cells to form extracellular ice crystals.
Fig. 11. Permeability of cell membrane of apple parenchyma tissue in fresh, CXF, and FAP samples. The error bars indicate standard deviation (n = 2). Different letters indicate significant difference (p ≤ 0.05) when analyzed by Duncan's new multiple range test.
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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The results from the self-diffusion coefficient and the permeability of cell membrane are corresponded with the T2b value suggesting that FAP destroyed the cell membrane of the apple parenchyma tissue while CXF could reduce cell membrane damage and maintain the permeability of cell membrane in the apple parenchyma tissue. This is probably because xenon hydrate formation in the apple parenchyma tissue, as evidenced by NMR measurement of solid echo, could limit the transformation of water inside and surrounding cells into ice crystals. 4. Conclusions This experiment demonstrates that the CXF could preserve the apple parenchyma tissue. It was effective in maintaining the texture of the apple parenchyma tissue and lowering the loss of firmness when compared to FAP. The CXF could maintain turgor pressure that was correlated with firmness, as shown in vacuole water (T2a value). It could also maintain cell membrane integrity, as shown in the cytoplasm and intercellular water (T2b value). In this study, it was suggested that T2a can be used for the detection of freezing damage. Furthermore, using the CXF was effective in maintaining a typical restricted diffusion phenomenon and permeability of cell membrane in the apple parenchyma tissue. The CXF could more effectively maintain apple parenchyma tissue than FAP. It was apparent that the textural quality and cellular integrity of CXF samples were as good as those of fresh samples. Therefore, this research suggested that the CXF can be used as an innovative technique for the preservation of frozen fruit products. Acknowledgements This research was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (grant no. PHD/ 0019/2552), the 90th Anniversary of the Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) (grant no. F-31GS-ES13) and the Mayekawa Houonkai Foundation. References Ando, H., Fukuoka, M., Miyawaki, O., Watanabe, M., & Suzuki, T. (2009). PFG-NMR study for evaluation freezing damage to onion tissue. Bioscience, Biotechnology, and Biochemistry, 73, 1257–1261. Ando, H., Kajiwara, K., Oshita, S., & Suzuki, T. (2012). The effect of osmotic dehydrofreezing on the role of the cell membrane in carrot texture softening after freeze-thawing. Journal of Food Engineering, 108, 473–479. Ando, H., Suzuki, T., Kajiwara, K., Kawagoe, Y., Makino, Y., & Oshita, S. (2011). Effects on Xe hydrate formation for texture in vegetable tissue. Proceedings of the 11th International Congress on Engineering and Food (ICEF 11), 22–26 May 2011, Athens, Greece. Ando, H., Suzuki, T., Kawagoe, Y., Makino, Y., & Oshita, S. (2009). Detection of Xenon gas hydrate formation in onion tissue for the application to chilling temperature storage. Proceedings of the 5th CIGR Section VI International Symposium on Food Processing, Monitoring Technology in Bioprocesses and Food Quality Management, 31 August–2 September 2009, Potsdam, Germany. Anisimov, A.V., Sorokina, N.Y., & Dautova, N.R. (1998). Water diffusion in biological porous systems: a NMR approach. Magnetic Resonance Imaging, 16, 565–568. Bishnoi, P.R., & Natarajan, V. (1996). Formation and decomposition of gas hydrates. Fluid Phase Equilibria, 117, 168–177.
Blanda, G., Cerretani, L., Cardinali, A., Barbieri, S., Bendini, A., & Lercker, G. (2009). Osmotic dehydrofreezing of strawberries: polyphenolic content, volatile profile and consumer acceptance. LWT—Food Science and Technology, 42, 30–36. Brecht, J.K., Ritenour, M.A., Haard, N.F., & Chism, G.W. (2008). Postharvest physiology of edible plant tissues. In S. Damodaran, K.L. Parkin, & O.R. Fennema (Eds.), Fennema's food chemistry (pp. 975–1049) (4th ed.). Boca Raton: CRC Press. Brosio, E. (2009). Introduction to the nuclear magnetic resonance spectroscopy. In E. Brosio (Ed.), Basic NMR in foods characterization (pp. 1–8). Kerala: Research Signpost. Brown, M.S. (1977). Texture of frozen fruits and vegetables. Journal of Texture Studies, 7, 391–404. Chassagne-Berces, S., Poirier, C., Devaux, M. -F., Fonseca, F., Lahaye, M., Pigorini, G., et al. (2009). Changes in texture, cellular structure and cell wall composition in apple tissue as a result of freezing. Food Research International, 42, 788–797. De Ancos, B., Sánchez-Moreno, C., De Pascual-Teresa, S., & Cano, M.P. (2012). Freezing preservation of fruits. In N.K. Sinha, J.S. Sidhu, J. Barta, J.S.B. Wu, & M.P. Cano (Eds.), Handbook of fruits and fruit processing (pp. 103–119) (2nd ed.). Iowa: WileyBlackwell. Dixon, G.M., & Jen, J.J. (1977). Changes of sugars and acids of osmovacuum-dried apple slices. Journal of Food Science, 42, 1126–1127. Dumont, F., Marechal, P.A., & Gervais, P. (2004). Cell size and water permeability as determining factors for cell viability after freezing at different cooling rates. Applied and Environmental Microbiology, 70, 268–272. Ewing, G.J., & Ionescu, L.G. (1974). Dissociation pressure and other thermodynamic properties of xenon–water clathrate. Journal of Chemical and Engineering Data, 19, 367–369. Gambhir, P.N., Choi, Y.J., Slaughter, D.C., Thomson, J.F., & McCarthy, M.J. (2005). Proton spin–spin relaxation time of peel and flesh of navel orange varieties exposed to freezing temperature. Journal of the Science of Food and Agriculture, 85, 2482–2486. Gribnau, M.C.M. (1992). Determination of solid/liquid ratios of fats and oils by lowresolution pulsed NMR. Trends in Food Science & Technology, 3, 186–190. Hills, B.P., & Remigereau, B. (1997). NMR studies of changes in subcellular water compartmentation in parenchyma apple tissue during drying and freezing. International Journal of Food Science and Technology, 32, 51–61. Khan, A.A., & Vincent, J.F.V. (1996). Mechanical damage induced by controlled freezing in apple and potato. Journal of Texture Studies, 27, 143–157. Li, W., & Sun, D. -W. (2002). Novel methods for rapid freezing and thawing of foods–a review. Journal of Food Engineering, 54, 175–182. Meerwall, E.V., & Ferguson (1981). Interpreting pulsed-gradient spin-echo diffusion experiments with permeable membranes. The Journal of Chemical Physics, 74, 6956–6959. Mohsenin, N.N. (1986). Physical properties of plant and animal materials (2nd ed.). Amsterdam: Gordon and Breach Publishers. Purwanto, Y.A., Oshita, S., Seo, Y., & Kawagoe, Y. (2001). Concentration of liquid foods by the use of gas hydrate. Journal of Food Engineering, 47, 133–138. Silva, C.L.M., Gonçalves, E.S., & Brandoão, T.R.S. (2008). Freezing of fruits and vegetables. In J.A. Evans (Ed.), Frozen food science and technology (pp. 165–183). Oxford: Blackwell Publishing. Sloan, E.D., & Koh, C.A. (2008). Clathrate hydrates of natural gases (3rd ed.). Florida: CRC press. Snaar, J.E.M., & Van As, H. (1992). Probing water compartments and membrane permeability in plant cells by 1H NMR relaxation measurements. Biophysics Journal, 63, 1654–1658. Sterling, C. (1968). Effect of low temperature on structure and firmness of apple tissue. Journal of Food Science, 33, 577–580. Tanner, J.E. (1978). Transient diffusion in a system partitioned by permeable barriers. Application to NMR measurements with a pulsed field gradient. The Journal of Chemical Physics, 69, 1748–1754. Waldron, K.W., Smith, A.C., Parr, A.J., Ng, A., & Parker, M.L. (1997). New approaches to understanding and controlling cell separation in relation to fruit and vegetable texture. Trends in Food Science and Technology, 8, 213–221. Wang, L., Ando, H., Kawagoe, Y., Makino, Y., & Oshita, S. (2009). Basic study of the storage of agricultural produce by using xenon hydrate. Cryobiology, 59, 370–418. Zaritzky, N. (2012). Physical–chemical principles in freezing. In D. -W. Sun (Ed.), Handbook of frozen food processing and packaging (pp. 3–37) (2nd ed.). Boca Raton: CRC press.
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A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing Thunyaboon Arunyanart a, Ubonrat Siripatrawan a,⁎, Yoshio Makino b, Seiichi Oshita b a
Department of Food Technology, Faculty of Science, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand Laboratory of Bioprocess Engineering, Department of Biological and Environmental Engineering, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
b
a r t i c l e
i n f o
Article history: Received 10 June 2014 Accepted 2 September 2014 Available online xxxx Editor Proof Receive Date 30 October 2014 Keywords: Xenon Gas hydrate Food processing Preservation technique Freezing Nuclear magnetic resonance
a b s t r a c t Freezing usually causes cell and tissue damage in frozen fruits. This study attempted to use a combined method of xenon hydrate formation and freezing (CXF) for the preservation of apple parenchyma tissue and to compare it with the freezing alone process (FAP). CXF included two steps. The first step was to initiate a certain amount of xenon hydrate by introducing the apple parenchyma tissue to the xenon gas at 1.0 MPa and 1 °C for 0, 1, 2, 3, 4, 5, 6 and 7 d. It was found that the amount of xenon hydrate in apple parenchyma tissue increased with storage time and 2 d was optimum to obtain the certain amount of xenon hydrate. In the second step, the sample with optimum xenon hydrate formation was frozen at −20 °C. The results showed that CXF was more effective in maintaining firmness, turgor pressure, and cell membrane integrity of the apple parenchyma tissue than FAP. A typical restricted diffusion phenomenon which indicates that water molecules are maintained in the apple parenchyma cells was found in the CXF samples, while the FAP samples showed an unrestricted diffusion phenomenon. In addition, firmness, turgor pressure, cell membrane integrity, and restricted diffusion phenomenon of the CXF samples were similar to those of the fresh samples. The CXF could preserve the apple parenchyma tissue because of the bulk water inside the cells and the water surrounding the cells which transformed to ice crystals is limited. Thus, cell and tissue damage due to the formation of ice crystals was reduced. The obtained results indicated that the CXF is effective for the preservation of the apple parenchyma tissue. Industrial relevance: There has been an attempt to improve the quality of frozen fruit by using innovative techniques, in opposition to simply freezing. This present work proposed xenon hydrate formation for the reduction of bulk water before freezing in order to reduce freezing damage due to a large amount of ice crystal formation. The combined method of xenon hydrate formation and freezing has been proved to be able to reduce cell membrane damage usually occurring in frozen fruit. Thus this new technique has potential to be used for improving the quality of frozen fruit. The xenon hydrate formation is considered as an innovative technique for the preservation of fruit, which is expected to be useful for the frozen food industry. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Freezing is an excellent method for preservation and storage of fruits in the food industry because it preserves color, flavor and nutrition values of fruits. During freezing process, bulk water in fruit tissue is transformed to ice crystals. The ice crystals expand the tissue matrix leading to cell volume changes, resulting in cellular structural damage (De Ancos, Sánchez-Moreno, De Pascual-Teresa, & Cano, 2012; Silva, Gonçalves, & Brandoão, 2008). Freezing damage of cellular structure due to ice crystals usually occurs in plant tissues because of the semirigid nature of cells which leads to loss of tissue firmness, and a subsequent loss of textural quality in frozen fruits (Chassagne-Berces et al., 2009; Khan & Vincent, 1996; Zaritzky, 2012). ⁎ Corresponding author. Tel.: +66 22185536; fax: +66 22544314. E-mail address: [email protected] (U. Siripatrawan).
An attempt to improve quality of frozen fruits using novel preservation technique has been investigated. Osmotic dehydrofreezing technique has been successfully used in freezing of fruits with minimal damage to cellular integrity and texture. This technique aims to reduce the water content in food materials by immersion in aqueous solution before freezing (Ando, Kajiwara, Oshita, & Suzuki, 2012; Li & Sun, 2002; Silva et al., 2008). The disadvantage of an osmotic dehydrofreezing technique is that it is a type of an aqueous solution that affects the sensory characteristics of products (Dixon & Jen, 1977). Furthermore, leaching of food materials in the aqueous solution can result in loss of the nutritional content of products (Blanda et al., 2009). Recently, xenon hydrate formation has been introduced as an innovative technique for the preservation of agricultural products. Xenon gas is used for the preservation of agricultural products due to its non-polar nature and no reaction with biological materials (Purwanto, Oshita, Seo, & Kawagoe, 2001). Gas hydrates, composed of water and gas, are
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Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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crystalline solids or ice-like crystals of which gas molecules are trapped in water lattice that are composed of hydrogen-bonded network of water molecules. Gas hydrate formation is a time-dependent process. The process of gas hydrate formation from water and gas comprises four steps. In the first step, labile ring structures of water pentamer (H2O)5 and water hexamer (H2O)6 are formed from water molecules. The labile ring structure can be formed when the water becomes supercooled water of which optimally at low temperature and under pressure. In the next step, structured water is initiated when labile clusters (hydrogen bond network) of water molecules form around the dissolved gas molecules. After that, the labile clusters agglomerate. In the final step, the labile clusters grow and form rigid gas hydrate (Sloan & Koh, 2008). Wang, Ando, Kawagoe, Makino, and Oshita (2009) found that barley coleoptile cells preserved by xenon hydrate formation at low temperature showed significantly higher viability than those preserved by freezing. However, the viability of barley coleoptile cells under xenon hydrate formation decreased with storage time. Ando et al. (2011) reported that texture characteristics such as elasticity of the onion tissue after xenon hydrate formation-decomposition decreased with the amount of xenon hydrate. They hypothesized that the cell membrane of onion tissue was damaged due to the growth of xenon hydrate with storage time. The results of using xenon hydrate formation for the preservation of agricultural products had a limitation because increasing the amount of xenon hydrate with storage time leads to cell and tissue damage. Reduction of freezing damage caused by ice crystal is a key factor in the preservation of frozen fruits because fruits contain a large amount of water (Silva et al., 2008), their cellular structure could be destroyed by ice crystal during freezing. A procedure to reduce the amount of bulk water before transformation to ice crystal could reduce freezing damage. This present work proposed xenon hydrate formation in the reduction of bulk water before freezing in order to reduce freezing damage due to ice crystal formation. There has been no research using xenon hydrate formation for the preservation of fruit tissue. The xenon hydrate formation is considered as an innovative technique for the preservation of fruit, which is expected to be useful for the frozen food industry. Therefore, the objective of this research was to use the combined method of xenon hydrate formation and freezing (CXF) for the preservation of apple parenchyma tissue in comparison with the conventional freezing alone process (FAP). 2. Materials and methods 2.1. Sample preparation The apple parenchyma tissue was used as studying structural cell components because of its low metabolic activity (Snaar & Van As, 1992) and its macroscopic flesh homogeneity. Fresh apples (Malus pumila cv. Fuji) at commercial maturity (total soluble solids at 20 °C were in the range of 13–14 °Brix) were purchased from a local supermarket in Tokyo, Japan. The apple was cut into a 4 mm × 4 mm × 10 mm tissue block, which was taken equidistantly from the outer cortex and the core in the parenchyma region (Fig. 1) and dipped into 1.0% citric acid solution for 10 min in order to prevent enzymatic browning (De Ancos et al., 2012). 2.2. Combined method of xenon hydrate formation and freezing vs freezing alone process In this study, two methods including CXF and FAP were compared. The fresh sample was used as reference for each measurement. The schematic of the experimental setup was shown in Fig. 2. For the CXF method, the optimal xenon hydrate formation was first determined and followed by freezing the xenon hydrate processed sample at −20 °C. Xenon hydrate formation was conducted by placing an apple tissue block into a nuclear magnetic resonance (NMR) glass tube, having
Fig. 1. Apple parenchyma tissue block.
an outer diameter of 10 mm, and xenon gas (99.995%, Iwatani Co., Tokyo, Japan) was introduced under pressure of 1.0 MPa at 1 °C for 2 d using a pressurizing unit, as shown in Fig. 3. The tube was then kept in a medical chest freezer (SANYO, MDF-435, Tokyo, Japan) at −20 °C for 7 d. From the preliminary studies, xenon hydrate formation was conducted by introducing xenon gas under pressure of 1.0 MPa at 1 °C for 0, 1, 2, 3, 4, 5, 6 and 7 d and the formation of xenon hydrate was monitored daily by using a NMR measurement of solid echo. It was found that the optimal xenon hydrate formation in apple parenchyma tissue is 2 d. When stored longer than 2 d, cell membrane damage caused by a larger amount of xenon hydrate was evidenced as measured by using a self-diffusion coefficient of water molecules. After the optimal xenon hydrate formation was obtained, the sample was stored at − 20 °C for 7 d. For FAP, the apple tissue block was placed in a NMR glass tube and was frozen in a medical chest freezer at −20 °C for 7 d. After storage, both CXF and FAP samples were thawed at 20 °C for 1 h before the measurements of texture, water content in cellular structure, and self-diffusion coefficient. For the CXF samples, xenon hydrate was decomposed by releasing xenon gas from the NMR glass tube together with thawing of ice crystal. 2.3. NMR measurement of the amount of xenon hydrate Solid echo NMR was used to measure the amount of xenon hydrate in apple parenchyma tissue sample. A 25-MHz pulsed NMR spectrometer (JNM-MU25A, JEOL Ltd., Tokyo, Japan) with an attached temperature control unit (NM-VT/MU25, JEOL Ltd., Tokyo, Japan) was used. In order to measure the free induction decay (FID) by NMR spectrometer, dimension of apple tissue block of 4 mm × 4 mm × 10 mm was required to get stable NMR signals. To measure the spin–spin relaxation time of protons (T2) in the solid component of the sample, a solid echo pulse sequence was applied following the method of Ando, Suzuki, Kawagoe, Makino, and Oshita (2009) and Gribnau (1992). The sequence of a solid echo pulse consists of 90°–τ–90°–τ–echo, where 90° is the flip angle induced by the radio frequency pulse and τ is time between pulses in a pulse sequence. The signal intensity was recorded as free induction decay. The pulse interval for eliminating the background effect from the empty sample tube was determined to be 8 μs. The repetition time and scan number were 30 s and 16 times, respectively. The amount of xenon hydrate in apple parenchyma tissue was determined from the FID as the solid ratio (the number of molecules of solid components to the
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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Fig. 2. The schematic of the experimental setup. (CXF = combined method of xenon hydrate formation and freezing, FAP = freezing alone process).
combined number for all forms of water molecules) according to the method of Gribnau (1992). When a mixture of solid and liquid is measured, the FID normally shows two different regions. Since the intensity of the FID signal is directly proportional to the number of proton spins, the ratio of proton spins can be quantified in different phases. Thus, a solid ratio can be determined. The experiment was performed under 1.0 MPa xenon pressure at 1 °C.
2.4. Texture measurement A Texture analyzer (RE2-33005S, YAMADEN Ltd., Tokyo, Japan) with a 3 mm diameter cylindrical probe was used to determine the texture of the samples. The texture was measured at a horizontal position of the samples. The penetration test was set as 20 mm into samples at 1 mm/s. Firmness value was the maximum force of the force–deformation curve and express as newtons (N). The measurements were conducted with five replications for each treatment. 2.5. NMR measurement of water content Relaxation time NMR was used to indicate the water content in the cellular structure of the samples. A 25-MHz pulsed NMR spectrometer with an attached temperature control unit was used to measure the spin–spin relaxation time of protons at 20 °C. The transverse or spin– spin relaxation time was measured using Carr–Purcell–Meiboom–Gill (CPMG) pulse sequence. The sequence of a CPMG consists of 90°–(τ– 180°–τ– echo)n, where n is the number of times the cycle is repeated. 2.6. NMR measurement of self-diffusion coefficient
Fig. 3. Unit for transmission pressurized xenon to the NMR sample tube.
Stimulated echo NMR was used to measure self-diffusion coefficient (D) of water molecules in the samples. Self-diffusion coefficient of water molecules in the apple parenchyma tissue was measured following the modified method as described by Ando, Fukuoka, Miyawaki, Watanabe, and Suzuki (2009). Each sample was set on 25 MHz pulse NMR spectrometer equipped with pulse field gradient accessory. The experiment was done by the stimulated echo pulse sequence (90°–τ–90°–τ–90°–τ– echo) with the magnitude of pulse field gradient, varying from 21.0 to 49.2 Gauss/cm, at a selected diffusion time. Degree of the restriction shows capacity of water diffusion through cell membrane in the tissue (Ando, Fukuoka, Miyawaki, Watanabe, & Suzuki, 2009; Tanner, 1978). The experiment was performed at 20 °C under atmospheric pressure.
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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When restricted diffusion was measured by using pulse field gradient nuclear magnetic resonance (PFG-NMR), the permeability of cell membrane was evaluated from the relationship between self-diffusion coefficient and diffusion time by using Eq. 1 (Anisimov, Sorokina, & Dautova, 1998).
1=D∞ ¼ 1=D0 þ 1=ðP aÞ
ð1Þ
where D∝ is the self-diffusion coefficient at maximum diffusion time (m2/s), D0 is the self-diffusion coefficient at minimum diffusion time (m2/s), P is the permeability of cell membrane (m/s), and a is the fixed cell diameter parameter (μm) which is observed by using an optical microscope. 2.7. Statistical analysis
the tissue was stored for 2, 4 and 7 d under xenon gas pressure of 1.0 MPa at 1 °C. It was found that xenon hydrate started to form after storage for 2 d. The amount of xenon hydrate in the apple parenchyma tissue was determined by the NMR technique. Fig. 6a shows free induction decay of protons in apple parenchyma tissue under xenon gas pressure of 1.0 MPa at 1 °C. The decay curve of samples after 0 and 1 d storage shows a linear decay, which presents the relaxation time of the water (liquid) component in the apple parenchyma tissue. When the samples were stored from 2 to 7 d, the decay curves showed a two-component pattern that indicates solid and water phases in the apple parenchyma tissue after xenon hydrate formation. The solid ratio in the sample increased with storage time. The percentage of solid ratio, as shown in Fig 6b, was calculated from Eq. 2 (Gribnau, 1992). solid ratio ð%Þ ¼ ðS−LÞ=S 100
A completely randomized design (CRD) was used as an experimental design. The difference between means was determined at p ≤ 0.05 using the Duncan's new multiple range test. All statistical analyses were performed using SPSS for Windows. 3. Results and discussion 3.1. Amount of xenon hydrate formation in apple parenchyma tissue The parenchyma cell of apple is in a polygonal shape as shown in Fig. 4, with an average cell diameter of 206 μm. The parenchyma cells of apple were observed at 20 °C with an optical microscope (Leica DML, Wetzlar, Germany). The main compartment of the parenchyma cell consists of vacuole, cytoplasm, middle lamella and intercellular space. Vacuole, the largest part of cell volume, is related to turgor pressure of the plant cell. Cytoplasm locates between cell wall and vacuole. Middle lamella combines the region of parenchyma cells to form the parenchyma tissue. Intercellular space is the separated region of each parenchyma cell which contains air or water (Brecht, Ritenour, Haard, & Chism, 2008; De Ancos et al., 2012; Mohsenin, 1986). In this study, the xenon hydrate formation was conducted under pressure of 1.0 MPa at 1 °C because at this condition the small diameter hydrate crystals were formed. It has been suggested that the small diameter of hydrate crystals could reduce cell membrane damage (Wang et al., 2009). Fig. 5 illustrates fresh apple parenchyma tissue and xenon hydrate processed apple parenchyma tissue during storage of 2, 4 and 7 d under 1.0 MPa xenon pressure at 1 °C. The appearance of xenon hydrate is ice-like crystals that gradually grow with storage time. The xenon hydrate was observed on the apple parenchyma tissue surface when
ð2Þ
where S is the total signal intensity of solid and liquid, and L is the signal intensity of liquid. Fig. 7 shows xenon hydrate (%) in apple parenchyma tissue calculated from the percentage of solid ratio. In this study, the percentage of solid ratio in samples indicated the amount of xenon hydrate in the samples. The xenon hydrate in apple parenchyma tissue significantly increased (p ≤ 0.05) with storage time. Approximately 20% of bulk water in the apple parenchyma tissue was transformed to xenon hydrate when the sample was stored under xenon gas pressure of 1.0 MPa at 1 °C for 2 d. The highest amount of xenon hydrate was found to be approximately 32% when the samples were stored up to 6 d or longer. From the preliminary results, cell membrane of apple parenchyma tissue was destroyed by increasing of amount of xenon hydrate from 3 to 7 d (approximately from 25% to 32%), which is indicated by selfdiffusion coefficient of water molecules. These results are in good agreement with those of Ando et al. (2011) who found that an increase in the amount of xenon hydrate during storage under xenon hydrate formation destroyed onion tissues. Since 20% of xenon hydrate was found to be optimum to avoid cell damage, the condition of storage under xenon gas pressure of 1.0 MPa for 2 d was used for the CXF. According to Bishnoi and Natarajan (1996), gas hydrates are formed by certain gases such as CH4, Xe, N2 and O2 when contacted with water under optimal temperature and pressure conditions. It has been reported that temperature and pressure optimal for xenon–water clathrate or xenon hydrate formation of water range between 0–12 °C and 0.15– 0.50 MPa, respectively (Ewing & Ionescu, 1974). In this present work, it was suggested that the portion of the bulk water inside and surrounding the cells (intercellular space and middle lamella) of the apple parenchyma tissue started to transform from water to xenon hydrate when
Fig. 4. The parenchyma cells of apple (Malus pumila cv. Fuji) under an optical microscope at 10× magnification.
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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Fig. 5. Xenon hydrate formation in apple parenchyma tissue under 1.0 MPa at 1 °C and stored for 2, 4, and 7 d. (0 d is the sample exposed to xenon gas under pressure of 1.0 MPa at 1 °C for 1 h.) The arrows indicate xenon hydrate.
the samples were stored under xenon pressure of 1.0 MPa at 1 °C for 2 d. As shown in Fig. 5, xenon hydrate in apple parenchyma tissue increased with storage time. The water in apple parenchyma tissue contains solutes (e.g. sugars and organic acids), which is considered to be an aqueous solution. The elevated pressure of xenon was required for the formation of xenon hydrate. This reason is in good agreement with that of Purwanto et al. (2001) who investigated the concentration of liquid foods by the use of xenon hydrate and reported that the hydrate forming pressures for the coffee solution of 14.7 wt.% were higher
than those required for 5.1 wt.% concentration and distilled water, respectively. 3.2. Firmness of apple parenchyma tissue The firmness of fresh, CXF, and FAP samples is shown in Fig. 8. The firmness of samples from FAP significantly decreased (p ≤ 0.05) when compared to that of fresh samples. The decreasing of firmness was probably because FAP causes cell membrane damage due to the formation of
Fig. 6. Free induction decay of sample with xenon hydrate formation stored for 0–7 d (a), and percentage of solid ratio in the sample calculated from free induction decay of sample (b).
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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and −196 °C affected the texture of apple tissue due to cell membrane rupture and tissue breakage. The firmness of CXF samples slightly decreased (p ≤ 0.05) when compared to that of fresh samples. On the other hand, the firmness of FAP samples (24% loss) were found to be significantly lower (p ≤ 0.05) than that of CXF (9% loss) and fresh samples, respectively. It has been suggested that the xenon hydrate formation can reduce bulk water inside and surrounding the cells (intercellular space and middle lamella), and subsequently limits the available water transformed to ice crystals during freezing process. Thus, cell and tissue damage due to formation of intracellular and extracellular ice crystals was reduced. 3.3. NMR relaxation time of apple parenchyma tissue
Fig. 7. Percentage of xenon hydrate in apple parenchyma tissue under 1.0 MPa at 1 °C measured by NMR spectroscopy with a solid echo pulse sequence. The error bars indicate standard deviation (n = 2). Different letters indicate significant difference (p ≤ 0.05) when analyzed by Duncan's new multiple range test.
ice crystals, leading to disruption of cellular structure (De Ancos et al., 2012; Silva et al., 2008), and loss of turgor pressure (Brown, 1977) in fruit tissues. The FAP caused loss of firmness in the samples because ice crystals formed in the extracellular areas (intercellular space and middle lamella) destroyed the cell membrane. Since the cell membrane was damaged, during thawing water migrated out of the cell due to osmotic pressure (Zaritzky, 2012). In general, freezing of food tissue can lead to extracellular ice crystals because the water surrounding the cell is frozen before the cell contents due to the fact that cytoplasm is more concentrated than liquid in the extracellular areas and, thermodynamically, the component with the largest volume nucleates first (Dumont, Marechal, & Gervais, 2004; Zaritzky, 2012). Thus, food tissue damage is mainly caused by extracellular ice crystals. The loss of firmness and the decrease in strength of the frozen-thawed apple tissue have been reported to be due to the loss of turgor pressure (Sterling, 1968). Chassagne-Berces et al. (2009) reported the effects of three different freezing processes (at −20 °C, −80 °C and −196 °C) on mechanical properties of apple tissue and found that freezing at −20 °C
Fig. 8. Firmness of apple parenchyma tissue in fresh, CXF, and FAP samples. The error bars indicate standard deviation (n = 5). Different letters indicate significant difference (p ≤ 0.05) when analyzed by Duncan's new multiple range test.
The spin–spin relaxation time of protons can be used to identify three relaxation components pertaining to water in vacuole, cytoplasm, and intercellular space of apple parenchyma tissue (Snaar & Van As, 1992). In this experiment, two components of T2 were obtained from the apple parenchyma tissue including T2a (long relaxation time) indicating the vacuole water and T2b (short relaxation time) indicating the cytoplasm and intercellular water. As shown in Fig. 9, T2a value of FAP samples significantly decreased (p ≤ 0.05) when compared to that of fresh samples. This indicated that during the freezing process vacuolar membrane was damaged by intracellular ice crystal, leading to vacuole rupture and leakage of vacuole water. This phenomenon causes loss of turgor pressure and affects the texture of fruit tissues. It is well known that cell turgor pressure is related to the water content in vacuole (Waldron, Smith, Parr, Ng, & Parker, 1997) and the loss of turgor pressure causes a major change in textural characteristics of fruit and vegetable (Brown, 1977). The T2a value of CXF samples was higher (p ≤ 0.05) than that of FAP samples because water content in vacuole of CXF samples is more than that of FAP samples. The CXF could reduce vacuole rupture and water leakage from vacuole because the formation of intracellular ice crystal was limited. The T2a value corresponded with firmness. Therefore, the CXF could maintain turgor pressure and firmness of apple parenchyma tissue better than FAP. T2b value of FAP samples significantly decreased (p ≤ 0.05) when compared to that of fresh samples (Fig. 9). This indicated that cell membrane was probably damaged by extracellular ice crystals, causing a leakage of water out of the cell. The T2b value of CXF samples slightly decreased (p ≤ 0.05) when compared to that of fresh samples. The result showed that cytoplasm and intercellular water content in cells of CXF samples is more than those of the FAP samples. It was suggested that the CXF could maintain cellular structure and water holding capacity in the apple parenchyma tissue because transformation of water surrounding the cells to extracellular ice crystals was limited. As shown in Fig. 9, T2a value is much higher than T2b value indicating that water content in vacuole is more than that in cytoplasm and intercellular. Vacuole occupies the largest part of the plant cell and controls turgor pressure of the cell (De Ancos et al., 2012). Thus, this research T2a was used for monitoring the firmness of fruit tissue. In addition, it should be noted that the NMR technique for measuring spin–spin relaxation time of protons (e.g. T2a) could be used as a rapid method for monitoring of the freezing damage in fruit tissues because loss of firmness is a major deterioration of frozen fruits. This technique is a qualitative analysis for the determination of freezing damage in fruit tissues without a complicated sample preparation. Determination of freezing damaged in fruit tissues by using the NMR technique is currently reported in other research. For example, Hills and Remigereau (1997) used spin–spin relaxation time of protons to indicate loss of cell membrane integrity in frozen-thawed apple tissue. The spin–spin relaxation time was also used to reflect the rupture of navel juice sacs due to a frozen-thawed process (Gambhir, Choi, Slaughter, Thomson, & McCarthy, 2005).
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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Fig. 9. Spin–spin relaxation time of protons of apple parenchyma tissue in fresh, CXF, and FAP samples. T2a and T2b are the long and short relaxation times. The error bars indicate standard deviation (n = 2). Different letters indicate significant difference (p ≤ 0.05) when analyzed by Duncan's new multiple range test.
3.4. Self-diffusion coefficient of water molecules and permeability of cell membrane in the apple parenchyma tissue Self-diffusion coefficient shows molecular mobility of water that can be studied using the PFG-NMR technique. This coefficient is related to the average distance the molecules travel, thus providing direct information on the material transport properties (Brosio, 2009). The selfdiffusion coefficient of water molecules of all samples is shown in Fig. 10. Self-diffusion coefficient of fresh samples significantly decreased (p ≤ 0.05) with increasing diffusion time and approach a constant value, whereas the relative self-diffusion coefficient vs diffusion time shows an exponential behavior. A typical restricted diffusion phenomenon is shown in fresh samples. Typically, the apparent self-diffusion coefficient of water molecules in plant tissues is not constant, but decreases with diffusion time because water molecules are in the limited space of plant cell (Ando, Fukuoka, Miyawaki, Watanabe, & Suzuki, 2009). The restricted diffusion phenomenon indicates that water molecules cannot move freely due to barrier properties of the cell membrane which is semi-permeable (Meerwall & Ferguson, 1981; Tanner, 1978). Like fresh apple parenchyma tissue, CXF sample maintained a restricted
Fig. 10. Self-diffusion coefficient of water molecules of apple parenchyma tissue in fresh, CXF, and FAP samples. The error bars indicate standard deviation (n = 2).
diffusion phenomenon to be at minimum. In contrast, the self-diffusion coefficient of FAP samples was higher (p ≤ 0.05) than that of fresh samples, indicating an unrestricted diffusion phenomenon. It can be explained that the cell membrane of FAP samples was damaged probably due to the formation of extracellular ice crystals. To evaluate cell damage in the apple parenchyma tissue, the change in permeability of cell membrane was calculated by using Eq. 1. To calculate permeability of cell membrane, the self-diffusion coefficient value (D∝ at 500 ms and D0 at 20 ms from PFG-NMR measurement) and cell diameter (a = 206 μm from optical microscopic observation) are used, as known parameters. Fig. 11 shows permeability of cell membrane of fresh, CXF, and FAP samples. The permeability of cell membrane of the FAP samples (P = 4.94 × 10−6 m/s) significantly increased (p ≤ 0.05) when compared with that of the fresh samples (P = 0.91 × 10−6 m/s). This is probably because extracellular ice crystal formation during FAP destroyed the semi-permeability of cell membrane. For the CXF samples, the permeability of cell membrane (P = 1.78 × 10− 6 m/s) slightly increased when compared with that of the fresh samples. It was indicated that the CXF could maintain the semi-permeability of cell membrane in the samples because cell membrane damage was reduced due to the limit of water surrounding the cells to form extracellular ice crystals.
Fig. 11. Permeability of cell membrane of apple parenchyma tissue in fresh, CXF, and FAP samples. The error bars indicate standard deviation (n = 2). Different letters indicate significant difference (p ≤ 0.05) when analyzed by Duncan's new multiple range test.
Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008
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The results from the self-diffusion coefficient and the permeability of cell membrane are corresponded with the T2b value suggesting that FAP destroyed the cell membrane of the apple parenchyma tissue while CXF could reduce cell membrane damage and maintain the permeability of cell membrane in the apple parenchyma tissue. This is probably because xenon hydrate formation in the apple parenchyma tissue, as evidenced by NMR measurement of solid echo, could limit the transformation of water inside and surrounding cells into ice crystals. 4. Conclusions This experiment demonstrates that the CXF could preserve the apple parenchyma tissue. It was effective in maintaining the texture of the apple parenchyma tissue and lowering the loss of firmness when compared to FAP. The CXF could maintain turgor pressure that was correlated with firmness, as shown in vacuole water (T2a value). It could also maintain cell membrane integrity, as shown in the cytoplasm and intercellular water (T2b value). In this study, it was suggested that T2a can be used for the detection of freezing damage. Furthermore, using the CXF was effective in maintaining a typical restricted diffusion phenomenon and permeability of cell membrane in the apple parenchyma tissue. The CXF could more effectively maintain apple parenchyma tissue than FAP. It was apparent that the textural quality and cellular integrity of CXF samples were as good as those of fresh samples. Therefore, this research suggested that the CXF can be used as an innovative technique for the preservation of frozen fruit products. Acknowledgements This research was supported by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (grant no. PHD/ 0019/2552), the 90th Anniversary of the Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) (grant no. F-31GS-ES13) and the Mayekawa Houonkai Foundation. References Ando, H., Fukuoka, M., Miyawaki, O., Watanabe, M., & Suzuki, T. (2009). PFG-NMR study for evaluation freezing damage to onion tissue. Bioscience, Biotechnology, and Biochemistry, 73, 1257–1261. Ando, H., Kajiwara, K., Oshita, S., & Suzuki, T. (2012). The effect of osmotic dehydrofreezing on the role of the cell membrane in carrot texture softening after freeze-thawing. Journal of Food Engineering, 108, 473–479. Ando, H., Suzuki, T., Kajiwara, K., Kawagoe, Y., Makino, Y., & Oshita, S. (2011). Effects on Xe hydrate formation for texture in vegetable tissue. Proceedings of the 11th International Congress on Engineering and Food (ICEF 11), 22–26 May 2011, Athens, Greece. Ando, H., Suzuki, T., Kawagoe, Y., Makino, Y., & Oshita, S. (2009). Detection of Xenon gas hydrate formation in onion tissue for the application to chilling temperature storage. Proceedings of the 5th CIGR Section VI International Symposium on Food Processing, Monitoring Technology in Bioprocesses and Food Quality Management, 31 August–2 September 2009, Potsdam, Germany. Anisimov, A.V., Sorokina, N.Y., & Dautova, N.R. (1998). Water diffusion in biological porous systems: a NMR approach. Magnetic Resonance Imaging, 16, 565–568. Bishnoi, P.R., & Natarajan, V. (1996). Formation and decomposition of gas hydrates. Fluid Phase Equilibria, 117, 168–177.
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Please cite this article as: Arunyanart, T., et al., A new approach for the preservation of apple tissue by using a combined method of xenon hydrate formation and freezing, Innovative Food Science and Emerging Technologies (2014), http://dx.doi.org/10.1016/j.ifset.2014.09.008