P2: SFK BLBS102-Simpson March 21, 2012 240 13:11 Trim: 276mm X 219mm Printer Name: Yet to Come Part 2: Biotechnology and Enzymology PG and Expansin Control, juice Control, paste 8000 7000 Number of particles 6000 5000 4000 3000 2000 1000 1660 1255 948.2 716.9 541.9 409.6 309.6 234.1 176.8 133.7 Particle diameter (µm) Supressed PG/Exp1, juice Supressed PG/Exp1, paste 8000 7000 6000 5000 4000 3000 2000 1000 1660 1255 948.2 716.9 541.9 409.6 309.6 234.1 176.8 133.7 The influence of the suppression of the expression of two cellwall-related proteins in the same transgenic line has been reported (Kalamaki et al 2003a, Powell et al 2003) Tomato lines with simultaneous suppression of the expression of both PG and the ripening-associated expansin have been produced and their chemical and physical properties characterized (Kalamaki et al 2003a, Powell et al 2003) Expansins are proteins that lack wall hydrolytic activity and are proposed to act by disrupting the hydrogen bonding between cellulose microfibrils and the crosslinking glycan (xyloglucan) matrix (see Fig 12.1) Expansins are involved in cell wall disassembly during ripening (Brummell et al 1999a) Their role in ripening is proposed to be that of loosening the wall and increasing the accessibility of wall polymers to hydrolytic enzymes (Rose and Bennett 1999) In tomato, the product of one expansin gene (Exp1) is accumulated exclusively during tomato fruit ripening (Rose et al 1997, Brummell et al 1999b) Juices and pastes prepared from fruit with modified levels of Exp1 exhibit enhanced viscosity attributes (Kalamaki et al 2003b) Double transgenic lines were generated by crossing single transgenic homozygous lines Fruit was harvested at the mature green stage and allowed to ripen at 20◦ C, and texture was determined as the force required to compress the blossom end of the tomato by mm Fruit from the double suppressed line were firmer than controls and single transgenic lines at all ripening stages The same results were observed in fruit ripened on vine At the red ripe stage, fruit from the double suppressed line were 20% firmer than controls The flow properties of juice prepared from control, single transgenic, and double transgenic lines at the mature green/breaker, pink, and red ripe stages were evaluated using a Bostwick consistometer Average juice viscosity decreased as ripening progressed in all genotypes Juice prepared from fruit of the suppressed PG and the double suppressed line at the red ripe stage exhibited higher viscosity than control (Powell et al 2003) In another experiment, an elite processing variety was used as the wild-type background to suppress the expression of PG, Exp1 and both PG and Exp1 in the same line (Kalamaki et al 2003a) Juice and paste were prepared from control, single suppressed and double suppressed fruit, and their flow properties were characterized In paste diluted to Brix, an increase in Bostwick consistency of about 18% was observed for the PG-suppressed and the Exp1-suppressed genotypes Diluted paste from the double suppressed genotype exhibited the highest viscosity; however, this increase in consistency of the double transgenic line was only by an additional 4% compared to the single transgenic lines Analysis of particle size distributions at Brix in juices and pastes of the different genotypes indicated that suppression of PG or Exp1 results in small increases in the number of particles below a diameter of 250 µm (Fig 12.5) Suppression of Exp1 also results in the appearance of some larger particles, above 1400 µm in diameter The presence of these larger fragments shows that suppression of Exp1 affects the way the cells and cell walls rupture during processing Biochemical characterization of cell wall polymers did not show Number of particles P1: SFK/UKS BLBS102-c12 Particle diameter (µm) Figure 12.5 Particle size determination in juice and paste from control and suppressed PG/Exp1 fruit (Based on data presented by Kalamaki et al 2003a.) large differences in the amounts of extractable polyuronide or neutral sugar in sequential cell wall extracts of juices and diluted concentrates of the various genotypes Only minor size differences appear in individual polymer sizes in sequential extracts In suppressed PG genotypes, larger pectin sizes were observed in the CDTA extract The most dramatic difference was in the carbonate extracts where the double suppressed-line contained larger pectin polymers in all paste concentration levels relative to other genotypes (Kalamaki 2003) Concurrent suppression of PG and Exp1 in the same transgenic line showed an overall increase in viscosity compared to control, but only a 4% additional increase compared with the single transgenic lines Particle size distribution in juices and pastes from the double transgenic line are more poly-disperse (Fig 12.5), and particles appear to be more rigid In this line, an increase in the size of P1: SFK/UKS BLBS102-c12 P2: SFK BLBS102-Simpson March 21, 2012 13:11 Trim: 276mm X 219mm Printer Name: Yet to Come 241 12 Pectic Enzymes in Tomatoes carbonate-soluble pectin polymers was observed This size increase could suggest differences in the degree of polymer cross-linking in the particles, resulting in altered particle properties By modifying cell wall metabolism during ripening, the processing qualities of the resulting juices and pastes were influenced Hence, reducing the activity of these cell wall enzymes may be a route to the selection of improved processing tomato varieties PG and PME The activities of both PG and PME were suppressed in the same line, and juices were evaluated and compared to single transgenics and controls (Errington et al 1998) Small differences in Bostwick consistency were observed in hot-break juices with the suppressed PG juices having the highest viscosity In cold-break juices from PG-suppressed fruit, a time-dependent increase in viscosity was observed Since a similar increase was not observed in the double transgenic line, it was concluded that in order for the increase in viscosity of cold-break juice to occur, both absence of PG and continued action of PME is required Absence of PG activity will lead to larger size of pectin, whereas continuing demethylesterification of pectin chains by PME will increase calcium associations between pectin chains, leading to gel formation and thus improved viscosity In conclusion, simultaneous transgenic suppression of PG and PME expression did not result in an additive effect result in changes in the microstructure of fruit cell wall that are manifested as changes in viscosity (Xu et al 1986) Since the integrity of cell wall polymers in the juice is imperative, plant pectic enzymes usually have to be inactivated during processing in order to diminish their activity and prevent pectin degradation Pectin degradation could lead to increased softening in, for example, pickled vegetable production or peach canning, and loss of viscosity in processed tomato products (Crelier et al 2001) Although several enzymes are reported to act on cell wall polymers, the main depolymerizing enzyme is PG Therefore, inactivation of PG activity during tomato processing is essential for viscosity retention However, there are cases where residual activity of other pectic enzymes is desirable, as for example, with PME Retention of PME activity with concurrent and complete inactivation of PG could lead to products with higher viscosity (Errington et al 1998, Crelier et al 2001) Selective inactivation of PG is not possible with conventional heat treatment, since PME is rather easily inactivated by heat at ambient pressure, while PG requires much more severe heat treatment for complete inactivation PME is inactivated by heating at 82.2◦ C for 15 seconds at ambient pressure, while for PG inactivation, a temperature of 104.4◦ C for 15 seconds is required in canned tomato pulp (Luh and Daoud 1971) In order to achieve this selective inactivation, a combination of heating and high hydrostatic pressure can be used during processing or alternatively, the expression of a particular enzyme can be suppressed using genetic engineering in fruit Thermal Inactivation TOMATO PROCESSING The majority of tomatoes are consumed in a processed form, such as juice, paste, pizza and pasta sauce, and various diced or sliced products Most of the products are concentrated to different degrees and stored in a concentrated form until ready to use Industrial concentrates are then diluted to reach the desired final product consistency Textural properties of tomato fruit are important contributors to the overall quality in both fresh market and processing tomatoes (Barrett et al 1998) In some processed products, the most important quality attribute is viscosity (Alviar and Reid 1990) It was recognized early that the structure most closely associated with viscosity is the cell wall (Whittenberger and Nutting 1957) Both the concentration and type of cell wall polymers in the serum fraction and the pulp (particle fraction) are important contributors to viscosity In serum, the amount and size of the soluble cell wall polymers influence serum viscosity (Beresovsky et al 1995), whereas in pulp, the size distribution, the shape, and the degree of deformability of cell wall fragments influence viscosity (Den Ouden and Van Vliet 1997) Viscosity is influenced partly by factors that dictate the chemical composition and physical structure of the juice such us fruit variety, cultivation conditions, and the ripening stage of the fruit at harvest However, it is also influenced by processing factors such as break temperature (Xu et al 1986), finisher screen size (Den Ouden and Van Vliet 1997), mechanical shearing during manufacture, and degree of concentration (Marsh et al 1978) These processes Thermal processing (that is, exposure of the food to elevated temperatures for relatively short times) has been used for almost 200 years to produce shelf-stable products by inactivating microbial cells, spores, and enzymes in a precisely defined and controlled procedure Kinetic description of the destructive effects of heat on both desirable and undesirable attributes is essential for proper thermal process design At constant temperature and pressure conditions, PME thermal inactivation follows first-order kinetics (Crelier et al 2001, Fachin et al 2002, Stoforos et al 2002) dA − = kA, k = f (T , P , ) (1) dt where A is the enzyme activity at time t, k the reaction rate constant, and P and T the pressure and temperature process conditions Ignoring the pressure dependence, Equation leads to A = Ao e−kT t (2) where Ao is the initial enzyme activity and the reaction rate constant kT , function of temperature, is adequately described by Arrhenius kinetics through Equation kT = kTref exp − Ea R 1 − T Tref (3) where kT ref is the reaction rate constant at a constant reference temperature T ref , Ea is the activation energy, and R is the universal gas constant (8.314 J/(mol · K)) P2: SFK BLBS102-Simpson March 21, 2012 13:11 Trim: 276mm X 219mm 242 Printer Name: Yet to Come Part 2: Biotechnology and Enzymology 0.05 T (°C) 105 100 95 90 85 80 75 70 65 60 55 k (min-1) PG -4.0 PG 0.04 -3.0 lnk (k in s-1) P1: SFK/UKS BLBS102-c12 PME 0.02 -5.0 0.01 -6.0 0.00 -7.0 PME 0.03 100 200 300 -8.0 -9.0 0.0026 400 500 600 700 800 P (MPa) 0.0027 0.0028 0.0029 1/T (K-1) 0.0030 0.0031 Figure 12.7 Schematic representation of the effect of processing pressure on PG and PME inactivation rates during high-pressure treatment (at 60◦ C) Figure 12.6 Effect of processing temperature on PG and PME thermal inactivation rates at ambient pressure (Based on data presented by Crelier et al 2001.) PG thermal inactivation follows first-order kinetics (Crelier et al 2001, Fachin et al 2003) as suggested by Equation above, or a fractional conversion model (Equation 4), which suggests a residual enzyme activity at the end of the treatment A = A∞ + (Ao − A∞ )ekT (4) where A∞ is the residual enzyme activity after prolonged heating From the data presented by Crelier et al (2001), the effect of processing temperature on crude tomato juice PG and PME thermal inactivation rates at ambient pressure is illustrated in Figure 12.6 The higher resistance of PG, compared with PME, to thermal inactivation is evident (Fig 12.6) Furthermore, PG inactivation is less sensitive to temperature changes, compared with PME inactivation (as can be seen by comparing the slopes of the corresponding curves in Fig 12.6) Values for the activation energies (Ea , see Equation 3) equal to 134.5±15.7 kJ/mol for the case of PG and 350.1±6.0 kJ/mol for the case of PME thermal inactivation have been reported (Crelier et al 2001) It must be noted that the literature values presented here are restricted to the system used in the particular study and are mainly reported here for illustrative purposes Thus, for example, the origin and the environment (e.g., pH) of the enzyme can influence the heat resistance (k or D—the decimal reduction time—values) of the enzyme as well as the temperature sensitivity (Ea or z—the temperature difference required for 90% change in D—values) of the enzyme thermal inactivation rates High Pressure Inactivation High hydrostatic pressure processing of foods (i.e., processing at elevated pressures (up to 1000 MPa) and low to moderate temperatures (usually less than 100◦ C)) has been introduced as an alternative nonthermal technology that causes inactivation of microorganisms and denaturation of several enzymes with minimal destructive effects on the quality and the organolep- tic characteristics of the product The improved product quality attained during high-pressure processing of foods, and the potential for production of a variety of novel foods, in particular, desirable characteristics, have made the high pressure technology attractive (Farr 1990, Knorr 1993) As far as high pressure enzyme inactivation goes, PG is easily inactivated at moderate pressure and temperatures (Crelier et al 2001, Shook et al 2001, Fachin et al 2003), while PME inactivation at elevated pressures reveals an antagonistic (protective) effect between pressure and temperature (Crelier et al 2001, Shook et al 2001, Fachin et al 2002, Stoforos et al 2002) Depending on the processing temperature, PME inactivation rate at ambient pressure (0.1 MPa) is high, rapidly decreases as pressure increases, practically vanishes at pressures of 100–500 MPa, and thereafter starts increasing again, as illustrated on Figure 12.7 High pressure inactivation kinetics for both PG and PME follow first-order kinetics (Crelier et al 2001, Fachin et al 2002, Stoforos et al 2002, 2003) Values for the reaction rate constants, k, for high pressure inactivation of PME and PG as a function of processing temperature, at selected conditions, are given in Table 12.1 (Crelier et al 2001) Through the activation volume concept (Johnson and Eyring 1970), the pressure effects on the reaction rate constants can be expressed as: kp = kPref exp − Va (p − Pref ) R T (5) where kPref is the reaction rate constant at a constant reference pressure, Pref , and V a is the activation volume Models to describe the combined effect of pressure and temperature on tomato PME or PG inactivation have been presented in the literature (Crelier et al 2001, Stoforos et al 2002, Fachin et al 2003) On the basis of the literature data (Crelier et al 2001), a schematic representation of high pressure inactivation of tomato PG and PME is presented in Figure 12.7 From data like these, one can see the possibilities of selective inactivation P1: SFK/UKS BLBS102-c12 P2: SFK BLBS102-Simpson March 21, 2012 13:11 Trim: 276mm X 219mm Printer Name: Yet to Come 243 12 Pectic Enzymes in Tomatoes Table 12.1 First-Order Reaction Rate Constants, k (sec−1 ), for High Pressure PME and PG Inactivation as a Function of Processing Temperature (Based on Data Presented by Crelier et al 2001) First-Order Reaction Rate Constants, k (sec−1 ) PME P (MPa) 0.1 100 300 400 500 600 800 PG 60◦ C 65◦ C 70◦ C 75◦ C 161 · 10−6 7.58 · 10−6 9.70 · 10−6 872 · 10−6 163 · 10−6 112 · 10−6 6250 · 10−6 883 · 10−6 51.4 · 10−6 43.8 · 10−6 1500 · 10−6 44.3 · 10−6 2330 · 10−6 70.2 · 10−6 2920 · 10−6 3680 · 10−6 574 · 10−6 18.3 · 10−6 38.6 · 10−6 103 · 10−6 52.5 · 10−6 2710 · 10−6 of the one or the other enzyme by appropriately optimizing the processing conditions (pressure, temperature, and time) FUTURE PERSPECTIVES Texture in ripe tomato fruit is largely dictated by cell wall disassembly during the ripening process Cell wall polysaccharides are depolymerized, and their composition is changed as ripening progresses The coordinated and synergistic activities of many proteins are responsible for fruit softening, and although expansins and PGs are among the more abundantly expressed proteins in ripening tomato fruit, the modification of their expression has not been sufficient to account for all of the cell wall changes associated with softening or for the overall extent of fruit softening The texture of fresh fruit directly influences the rheological characteristics of processed tomato products Viscosity is influenced by modifications of the cell wall architecture but also by changes that may affect polysaccharide mobility and interactions of different components in juices and pastes Since cell wall metabolism in ripening fruit is a complex process and many enzymes have been identified, which contribute to this process, further insight to the effect of these enzymes on fruit texture and processing attributes can be gained by the simultaneous suppression or over-expression of combinations of enzymes Of course, the quest is on for identification of enzymes acting on RGI, RGII, and XGA of plant pectins similar to those found in fungi and bacteria (Wong 2008) Moreover, as our knowledge on cell wall biosynthesis and deconstruction increases, the ability to genetically engineer pectin structure in target plants would further improve fruit texture and processing characteristics (Ramakrishna et al 2003, Vicente et al 2007, Matas et al 2009) Manipulation of the expression of multiple ripening-associated genes and/or transcription factors that regulate ripening (Vrebalov et al 2009) in transgenic tomato lines will further shed light to the ripening physiology, postharvest shelf life and processing qualities of tomato fruit Finally, the interest exists and efforts are made to design optimal processes for selective enzyme inactivation and thus for production 30◦ C 193 · 10−6 3890 · 10−6 40◦ C 351 · 10−6 4090 · 10−6 1720 · 10−6 50◦ C 60◦ C 701 · 10−6 3620 · 10−6 4810 · 10−6 52.6 · 10−6 309 · 10−6 2510 · 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High Pressure Effects on Cellular Processes Academic Press, New York, pp 1–44 ... (MPa) 0.1 100 300 400 500 600 80 0 PG 60◦ C 65◦ C 70◦ C 75◦ C 161 · 10−6 7. 58 · 10−6 9.70 · 10−6 87 2 · 10−6 163 · 10−6 112 · 10−6 6250 · 10−6 88 3 · 10−6 51. 4 · 10−6 43 .8 · 10−6 1500 · 10−6 44.3 ·... Bennett AB 1 988 In vitro synthesis and processing of tomato fruit polygalacturonase Plant Physiol 86 : 1057– 1063 DellaPenna D et al 1 989 Transcriptional analysis of polygalacturonase and other ripening... nor, and nr tomato fruit Plant Physiol 90: 1372–1377 DellaPenna D et al 1990 Polygalacturonase isoenzymes and pectin depolymerization in transgenic rin tomato fruit Plant Physiol 94: 188 2– 188 6