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3.4. Chemical and structural composition of cell wall
The pectic substances are brought into solution more easily than other cell wall polymers and are more chemically reactive. This leads to the frequent observation that processes that result in textural changes, such as ripening, storage and cooking, are accompanied by significant changes in the characteristics of the pectic substances (Buren, 1979).
Reid in 1986 (Reid, 1986) reported that the changes in the cell wall, in particular, changes in the pectic fraction of the cell wall complex during freezing-thawing contributed to some extent in softening of Aiko strawberries. Among all pectin fractions, the water-soluble pectin fraction (WSP) showed the most dramatic change upon freezing and frozen storage compared to unfrozen sample. WSP decreased upon freezing and frozen storage, paralleling a decrease in the firmness value. Moreover, based on the compositional studies of the WSP, the researchers suggested that some of the changes were associated with the pectin rhamno- galacturonan backbone. In another study, Simandjuntak et al. (1996) reported that the total
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30 sugar composition of cell wall polysaccharides (CWP) of muskmelon decreases with the frozen storage prolongation indicating the modification and solubilisation of pectins and hemicellulose fraction of CWP. Moreover, during the frozen storage period of 10 months, the highest change in pectin content happened during the first five months of frozen storage and this was attributed to the maximum ice crystals growth during the initial period of frozen storage. However, no quantitative data on ice crystals growth during frozen storage period was reported by them.
Recently, Chassagne-Berces et al. (2009) studied the impact of three different protocols (−20 °C, −80 °C using nitrogen gas convection and −196 °C by immersion in liquid nitrogen) on the sugar composition of cell walls. They observed a reduction in the proportion of cell wall neutral sugars and uronic acids of apple during freezing and thawing for all freezing protocols. Among the three freezing protocols being studied, slow freezing (at −20 °C) created more damages than the other freezing conditions (−80 °C and dipped in liquid nitrogen). The main sugars, the amounts of which were modified after freezing at −20 °C, concern arabinose representative of the pectin and mannose, which is a cellulosic–
hemicellulosic sugar. The reduction in the arabinose content as a consequence of freezing- thawing could be due to the loss of arabinan from the rhamnogalacturonan I domains of pectin, which is supposed to participate in the cell wall mechanical characteristics by forming an interaction with the cellulose (Zykwinska, Ralet, Garnier, & Thibault, 2005). The modification of pectins and hemicelluloses was considered responsible for the collapse of the cell walls, resulting in cell separation (larger intercellular spaces) and development of softer texture in samples frozen at −20 °C.
3.5. The main advantage of these methods is that they can allow tracking of the freeze damage at a molecular level. On the other hand, some limitations that arise can be that the sample preparation for this technique can be time-consuming and requires skilled
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31 people to perform the tests. Another drawback that can be mentioned is that cell wall polysaccharide composition in fruits and vegetables depends on various factors such as cultivars, maturity, degree of the ripeness of the sample and other factors. Hence, this method can be used to compare freeze damage imparted by different freezing conditions on the samples from the same fruit rather than comparing samples from different fruits.
In the case of samples from different fruits a great variation may occur. Impedance Measurement
Measurement of electrical impedance can be a simple, rapid and inexpensive method of detecting freeze-thaw damage in fruits and vegetables (Zhang & Willison, 1991, 1992a,b).
This technique is based on the fact that the undamaged tissues have resistance and capacitance.
On the contrary, fully damaged plant tissue has no capacitance (Figure 9). Capacitance corresponds to intercellular domains, whereas intracellular domains are rather resistive domains. Capacitance components will increase in electrical impedance with increasing frequency whereas resistive domain’s impedance will not be function of the frequency.
Usually, the impedance measured before freezing is compared to that measured after thawing to determine the extent of freeze damage tissues suffer during the processing. Upon knowing the conductivity of initial intact and treated (freeze-thawed) samples at low and high frequencies, one can calculate the cell disintegration index ( ) in Eq.3. This disintegration index characterizes the proportion of damaged (permeabilized) cells within the plant product (for intact cells, is 0; for total cell disintegration, is 1) (Knorr & Angersbach, 1998;
Angersbach et al., 1999; Parniakov et al., 2015; Wiktor et al., 2015).
( )
( ; (3)
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32 where and are the electrical conductivity of samples in low frequency range (1–5 kHz), for intact and treated (freeze-thawed) samples, respectively. While, and are electrical conductivities of samples in high frequency range (3–50 MHz), for intact and treated samples.
Greenham in 1966 (Greenham, 1966) was the first to study the freezing injury caused to four varieties of alfalfa (Medicago saliva L.) by the means of impedance measurement. He found that the impedance value decreased due to the freezing-thawing process. Later in 1991, Zhang and Willison developed a new system for tissue impedance measurement and implemented it to detect injuries in frozen potato, carrot, and cabbage tissues in the years 1992 and 1993 (Zhang & Willison, 1991; Zhang & Willison, 1992a,b; Zhang & Willison 1993). They extracted the tissue impedance value from directly measured impedance (DMI) by performing multiple measurements at different inter-electrode spacing. Their data can be considered more relevant than Greenham’s as the impedance value proposed by him included both tissue impedance and electrode impedance. They reported that the impedance value of the frozen- thawed sample was lesser than the intact cell as a result of membrane injuries happening during the freezing-thawing process. Hayden et al. (1972) found this technique potentially useful in determining the frost resistance among four Solanum clones without causing any appreciable damage to the plant under test.
To the best of our knowledge, this technique has not been used to compare the impact of different freezing rates which are known to control the freeze damage in plant tissues, thus, it is worthy, the effectiveness of this method in assessing the freeze damage to be further explored.
Since this method has not been used for distinguishing the freezing techniques, hence the authors believe that it is too early to comment on the advantages and disadvantages of this technique.
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33 3.6. Solute diffusivity test
Mass transfer during osmotic treatment occurs through semi-permeable cell membranes present in biological materials, which imparts dominant resistance to the mass transfer process. Depending on the processing conditions, the state of cell membrane can change from being partially to totally permeable and this can lead to significant changes in tissue architecture. The increase in cell membrane permeability increases the mass transfer rate of solutes and water during osmotic treatment process (Rastogi, Eshtiaghi, & Knorr, 1999;
Alizadeh, Chapleau, de-Lamballerie, & Le-Bail, 2009). The diffusivity of solutes also depends on other factors, such as: the temperature and concentration of the osmotic solution, the size and geometry of the material, the solution-to-material mass ratio, and the level of agitation of the solution (Rastogi, Raghavarao, Niranjan, & Knorr, 2002). If all these factors are kept constant then the diffusity of solutes into the product can be related to cell damage that the product suffers during the processing steps. Since freezing has a detrimental effect on a cellular system (ruptures the cell membranes), the diffusion methods can be used to quantify freeze damage in fruits and vegetables at a global level. Figure 10 presents the relationship between the freeze damage and solute mass diffusivity and shows that mass diffusivity values can be calculated by fitting the dry matter vs. time data to diffusion models (based on sample configuration). Until now, only Alizadeh et al. (2009) used this method to compare different freezing parameters. They found that the frozen/thawed sample (Atlantic salmon) had a higher mass diffusivity than the untreated sample. Moreover, different mass diffusivity rates were observed for samples treated with different freezing conditions. For example, high pressure shift frozen sample had a higher mass diffusivity than samples frozen at 20 C in static freezer. High pressure-induced permealisation was considered as a candidate factor for the higher mass diffusivity in the high pressure shift frozen sample.
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34 Similar to impedancemetry, this method has also not been used for distinguishing the freezing techniques; hence, it is believed too early to comment on the advantages and disadvantages of this technique.