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2 Novel processing: technology and effects on biomaterials

2 Novel processing: technology and effects on biomaterials

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248˘˘ï»¿



Indrawati Oey



mass molecules such as vitamins, pigments, and volatile compounds

in contrast to that of high-molecular-mass molecules such as proteins/

enzymes in which various covalent and non-covalent interactions stabilize

its complex three-dimensional structure architecture. However, different processes can occur simultaneously during HP processing (100–1000

MPa/–20 to 60°C); that is, (1) cell wall and membrane disruption enabling

contact between enzymes and their substrates, (2) enhancement and retardation of enzymatic and chemical reactions, (3) microorganism inactivation, and (4) modification of biopolymers including protein denaturation,

enzyme inactivation, and gel formation (Oey et al., 2008).

Pressure effects on enzymatic or chemical reactions and physical

changes (e.g., protein denaturation, phase transition) depend on the resulting total volume changes during processing. According to the Le€Chatelier

principle, pressure shifts the reaction equilibrium to the state having

the smallest volume. As a consequence, the aforementioned reactions

and changes can only be enhanced if pressure decreases the total reaction volume (a negative change of partial molar volume between initial

and final state at constant temperature). Since pressure favors reactions

accompanied with a volume decrease and vice versa, it indirectly implies

that mechanism and kinetics of enzymatic and chemical reactions during

HP processing could differ from those occurring at atmospheric pressure.

Hereto, a better understanding of pressure effect on biomaterials is still a

great challenge.



10.2.2â•…High-intensity pulsed electric field (PEF) processing

The basic construction of a PEF unit consists of a pulsed generator, treatment chamber(s) (e.g., cylindrical or rectangular chamber, electrodes),

temperature and pulse monitoring systems, and a fluid handling system for food product in case of continuous mode. PEF processing can

be conducted both in batch and continuous systems (Min et al., 2007).

Circuitry of pulsed power supply system influences the resulting shape

of the pulses such as (monopolar) rectangular/square-wave, bipolar of

rectangular, exponential decay, damped oscillating shape etc (de Haan,

2007). Similar to other processing technologies, PEF treatment also has

problems with process uniformity; for example of temperature, electrical current. In this case, the design of the treatment inside the chamber

plays an important role in the distribution of temperature inside the PEF

chamber.

In literature, electric field strength (expressed in kV/cm) and total treatment time/energy are usually referred to as important process parameters

to identify the intensity of PEF processing. The duration of PEF treatment

normally ranges from micro- to milliseconds shorter than HP processing.



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Chapter ten:â•… Effect of novel food processing



249



The calculation of total treatment time or energy varies depending on

pulse number, pulse frequency, pulse delay, pulse width, pulse shape,

and so on. However, it should be taken into account that other process

parameters such as pulse polarity, pulse shape, pulse frequency, etc.

involved in the processing could also affect the stability of biomaterials.

These aforementioned process parameters are dependent on the design

of PEF equipment and experiment. PEF equipment and the concomitant

process parameters are not yet well standardized. Equipment specifications (such as pulsed power supply covering voltage rating, power rating, pulse duration and pulse repetition rate, geometry of the treatment

chamber, circuitry, etc.) and the details of experimental setup are not fully

documented. Therefore, appropriate comparison and evaluation between

studies found in literature are limited.

The feasibility of using PEF technology for food applications has been

studied at temperatures above subzero (mostly from moderate to elevated

temperatures). Alteration of the cell membrane electropermeabilization is

one of the key elements of PEF treatment. Depending on the process intensity, the occurrence of reversible and irreversible pore formation and cell

disintegration could (1) affect the cell vitality such as resulting in inactivation of microorganisms (Min et al. 2007); (2) induce stress response reactions at low intensity; and (3) affect permeabilization of plant and animal

tissues enhancing enzymatic reaction and improving mechanical separation, extractability, or mass transport. Furthermore, several studies have

shown that PEF affects the stability of enzymes (for a detailed discussion,

see Section 10.4) and preheating might be incorporated to optimize the

PEF effects on biomaterials. Hereto, PEF applications open up a lot of new

possibilities, particularly for handling raw materials and microbial decontaminants (Knorr et al., 2008).



10.3â•…Effect of HP processing on fruit

and vegetable enzymes

10.3.1â•…Understanding of HP processing effect on stability

of enzyme as a protein molecule

Pressure-temperature stability of proteins reveals an elliptical contour

(Suzuki, 1960; Brandts et al., 1970; Hawley, 1971; Zipp and Kauzmann,

1973), as schematically illustrated in Figure€10.1. It depicts the possibility

of protein denaturation by low temperature (cold inactivation), elevated

pressure, high temperature (heat inactivation), or a combination of these

factors (Mozhaev et al., 1994; Hayashi et al., 1998).



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Pressure inactivation



Pressure

(MPa)



Cold inactivation



273K; 0.1MPa



Heat inactivation



Figure 10.1╇ Schematic pressure-temperature diagram of protein stability (P: pressure, T: temperature, k: reaction rate).



Regarding the hierarchy of protein structure, four structural levels

involving different bonds and interactions can be distinguished. The primary structure of protein is limitedly affected under pressure since pressure has a limited effect on covalent bonds (Cheftel, 1991; Heremans, 1992;

Mozhaev et al., 1994). Hydrogen bonds, which are responsible for maintaining the secondary, tertiary, and quaternary structure levels of a protein, are

rather stable toward pressure, and very high pressure levels (> 700 MPa)

can disrupt these bonds, affecting the secondary structure. The changes

in secondary structure inevitably lead to irreversible protein denaturation (Balny and Masson, 1993). In contrast to the unaltered primary and

secondary structural levels due to pressure, the tertiary and quaternary

structure of proteins is lost due to pressure (Heremans, 1993; Mozhaev et

al., 1996) predominantly caused by (1) a disturbance of hydrophobic and

electrostatic interactions beyond 150–200 MPa (Cheftel, 1991; Balny and

Masson, 1993); (2) a dissociation of oligomeric enzymes into subunits at

about 150–200 MPa (Balny and Masson, 1993); (3) imperfect packing of

atoms at the subunit interface together with the disruption of hydrophobic and electrostatic interactions in the inter-subunit area leading to large

volume changes (Cheftel, 1991; Mozhaev et al., 1994); and (4) unfolding of

dissociated subunits at high pressure (Silva and Weber, 1993).

Reversible protein denaturation could occur at low pressure (< 200 MPa)

(Cheftel, 1991; Heremans, 1992; Masson, 1992). A slow refolding process

and both conformational drift and hysteric behaviors occur after pressure release. Pressure beyond 300 MPa could result in irreversible effects

including chemical modifications or unfolding of single-chain proteins



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Chapter ten:â•… Effect of novel food processing



251



and protein denaturations. Changes of protein structure under pressure

are also governed by the Le Chatelier principle in which pressure favors

reactions accompanied by negative volume changes. It is also suggested

that elevating pressure level increases the degree of protein molecule

ordering (referred to the principle of microscopic ordering) (Cheftel, 1991;

Heremans, 1992; Masson, 1992; Mozhaev et al., 1994).

At moderate temperature and atmospheric pressure, most enzymes are

stable for example between 20 and 45°C. Elevating pressure or temperature

respectively at constant temperature or pressure might enhance enzyme

inactivation. In literature, this phenomenon is generally termed synergistic

pressure and temperature effect (Figure€10.1). In this case, the rate constants

(k values) of enzyme inactivation increase with elevating pressure at constant temperature or with elevating temperature at constant pressure.

At high temperatures (close to the temperatures resulting in thermal inactivation of enzymes at atmospheric pressure), elevating pressure (mostly <200 MPa) could retard the thermal inactivation of enzymes

(lower k values when pressure is increased, Figure€10.1). Such antagonistic

effect of pressure on thermal inactivation can be explained by the fact that

at atmospheric pressure, elevating temperature (related to heat inactivation) affects non-covalent as well as covalent bonds (above 70°C), resulting in aggregated or incorrectly folded enzymes and chemically altered

enzymes, respectively. An enzyme will lose its activity if the active site

becomes inaccessible or disassemble due to protein unfolding. From a

thermodynamic point of view, enzyme denaturation leads to a very large

change in entropy (caused by a less ordered conformational structure)

exceeding the absolute value of the enthalpy change and making the

change in Gibbs free energy negative in which the denaturation is favorable. Since elevating pressure can increase the degree of protein molecule

ordering, it could reassemble the appropriate protein structure, especially

the active site leading to partial/complete recovery of enzyme activity.

Some enzymes could undergo significant denaturation during freezing and thawing; however, many are unaffected. At subzero and low temperatures, elevating pressure could affect the enzyme stability resulting

in enzyme activity loss such as lipoxygenase (Indrawati et al., 1999, 2000a,

2000b, 2001; Van Buggenhout et al., 2006). In cases of cold denaturation/

inactivation at atmospheric pressure, the inverse thermodynamic explanation at high temperature (i.e., a negative entropy change corresponding

to a higher ordered protein molecule) cannot solely explain the phenomenon because the interactions between protein molecules and adjacent

water must be taken into consideration (Meersman et al., 2008). At low

temperature, water molecules may form a shell or a layer around adjacent

nonpolar molecules resulting in different nonpolar entities and loss of the

ability to interact among each other. As a consequence, low temperature

promotes an exposure of nonpolar side chains to water and hydrophobic



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Indrawati Oey



associations become less stable (Privalov, 1990; Da Poian et€al., 1995; Silva

et al., 1996). The nonpolar interactions are more affected by pressure

because they are more compressible (Weber, 1995). It explains the additive effect of high pressure and low temperature to reduce the entropy

(Silva et al., 1996). As a consequence, temperature decrease under pressure

enhances the enzyme inactivation. Such antagonistic effects of pressure

and low temperature on enzyme inactivation (Figure€ 10.1) have been

noticed for lipoxygenase (Indrawati et al., 1999, 2000a, 2000b, 2001) and

myrosinase (Van Eylen et al., 2007, 2008a, 2008b).



10.3.2â•…Effect of HP processing on stability of fruit

and vegetable enzymes

It is a challenge to thoroughly elucidate pressure effects on enzyme stability in fruit and vegetables because (1) enzymes in fruit and vegetables

are present in a complex system as illustrated in Figure€10.2 and (2) at the

same time pressure enhances/retards the chemical and enzymatic reactions depending on the reaction volume. Hence, mechanistic and kinetic

studies on enzyme stability at different molecular levels and different

complexities of food matrix have been conducted. To decrease the complexity of enzyme system or to eliminate the presence of other endogenous

(bio)compounds, fruit and vegetable enzymes are (partially) purified and

afterwards dissolved in controlled buffer medium (e.g., certain pH, buffer, ion strength) or in fruit/vegetable juices (Table€10.1). Pressure effects

on endogenous enzymes have been studied in fruit and vegetables with

different intensities of matrix disruption such as in juices, purees, or in

intact food matrices (Table€ 10.2). The latter allows an overall evaluation



: enzyme



: enzyme-inhibitor interaction

: enzyme-substrate interaction

: reaction product

: cofactor/coenzyme

: substrate



: enzyme inhibitor/inactivator



Figure 10.2╇ Schematic illustration of complex enzyme systems in fruit and

vegetables.



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Enzymes

Lipoxygenase (LOX)



Enzyme and buffer

solution/medium



Processing

condition



Partially purified tomato

LOX; MOPS/KOH

(10 mM; pH 6.8)



10 to 60°C; 100 to

650 MPa; various

treatment time up

to 60 min.



Irreversible inactivation

T≥20°C, synergistic effect of

increases in pressure and

temperature on inactivation

T<20°C, lowering temperature

enhanced pressure inactivation

First-order inactivation kinetics

Antagonistic effect of pressure

(<550 MPa) on thermal inactivation

(50 and 60°C)



Rodrigo et al.

(2006a)



Commercial purified

soybean LOX; Tris HCl

buffer (0.4 mg/mL

10 mM; pH 9)



−15 to 68°C; 0.1 up

to 650 MPa;

various treatment

time



Irreversible inactivation

T≥30°C, synergistic effect of

increases in pressure and

temperature on inactivation

T<30°C, lowering temperature

enhanced pressure inactivation

First-order inactivation kinetics

Antagonistic effect of low pressure

(<200 MPa) on thermal inactivation

(65°C)



Indrawati et al.

(1999)



a



Enzyme stabilityb



References



Chapter ten:õ Effect of novel food processing



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Table€10.1╇ Effect of Combined High Pressure and Temperature Processing on the Stability of (Partially) Purified Fruit

and Vegetables Enzymes in Buffer Solution or in Fruit/Vegetable Juices



(continued)



253



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Enzymes

Myrosinase (MYR)



Pectinmethylesterase

(PME)



Enzyme and buffer

solution/medium



Processing

condition



Partially purified broccoli

MYR; phosphate buffer

(0.1 M; pH 6.55)



20°C; 350 to

500 MPa and

35°C; 150 to 450

MPa; various

treatment time up

to 80 min.



Irreversible inactivation

At 35°C, antagonistic effect of low

pressure (<350 MPa) on thermal

inactivation

Inactivation kinetics described by

consecutive step model



Ludikhuyze et

al. (1999)



Partially purified mustard

seed MYR; broccoli juice

(pH adjusted to 6.5)



40 to 60°C; up to

700 MPa; max.

treatment time=2 h



Very pressure stable

No inactivation at 55°C and 600

MPa for 2 h.



Van Eylen et al.

(2008a)



Commercial purified

orange peel PME; clear

apple juice



25°C; 200–400 MPa;

various treatment

time intervals up

to 180 min.



400 MPa/25°C/25 min.: highest

enzyme inactivation (1 log unit

reduction)



Riahi and

Ramaswamy

(2003)



Tomato PME purified with

cation exchange

chromatography; Na

acetate buffer (40 mM;

pH 4.4)



25°C; up to 8500

MPa; 17 min.

(including 2 min.

equilibration time)



Pressure stable

850 MPa/25°C/17 min.: 50%

inactivation

Tomato varieties gave no influence of

PME pressure stability



Rodrigo et al.

(2006b)



Tomato PME purified with

affinity chromatography;

citrate buffer (50 mM; pH

4.4)



25°C and 66°C; 550

to 700 MPa; various

treatment time



Pressure stable

Antagonistic effect of pressure

on thermal inactivation



Fachin et al.

(2002a)



Enzyme stabilityb



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Table€10.1╇ Effect of Combined High Pressure and Temperature Processing on the Stability of (Partially) Purified Fruit

and Vegetables Enzymes in Buffer Solution or in Fruit/Vegetable Juices (Continued)

References



Indrawati Oey



3/31/10 4:32:53 PM



10°C; 850 to

1000 MPa; various

treatment time up

to 600 min.



Very pressure stable

Pressure labile and stable fractions

observed

10% pressure stable fraction

Only pressure labile fraction

inactivated

Elevating pressure enhanced the

inactivation



Ly-Nguyen

et al. (2002a)



White grapefruit PME

purified with affinity

chromatography; Tris

buffer (20 mM; pH 7)



10 to 62°C; 100 to

800 MPa; various

treatment time



Pressure labile and stable fractions

observed

20% pressure stable fraction

Only pressure labile fraction

inactivated

Synergistic effect of increases in

pressure and temperature on

inactivation

Antagonistic effect of low pressure

(up to 200 MPa) on thermal

inactivation



Guiavarc’h

et al. (2005)



Green pepper PME crude

extract and purified with

affinity chromatography;

citrate buffer (pH 5.6)



25 to 60°C; 400 to

800 MPa; 15 min.



Pressure stable

Purified PME in citrate buffer was

more pressure stable than PME in

crude extract



Castro et al.

(2005)



Chapter ten:â•… Effect of novel food processing



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Strawberry PME purified

with affinity

chromatography; Tris-HCl

buffer (20 mM; pH 7)



(continued)



255



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Enzymes



Enzyme and buffer

solution/medium



Processing

condition



Enzyme stabilityb



256˘˘ï»¿



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Table€10.1╇ Effect of Combined High Pressure and Temperature Processing on the Stability of (Partially) Purified Fruit

and Vegetables Enzymes in Buffer Solution or in Fruit/Vegetable Juices (Continued)

References



10 to 62°C, 100 to

800 MPa, various

treatment time



Pressure labile and stable fractions

observed

Effective to inactivate pressure labile

fraction

At 10 to 30°C, pressure stable

fraction could be inactivated at

800 MPa

Synergistic effect of increases in

pressure and temperature on

inactivation

Antagonistic effect of low pressure

(up to 350 MPa) on thermal

inactivation (>54°C)



Castro et al.

(2006a)



Carrot PME purified with

affinity chromatography;

Tris buffer (20 mM; pH 7)



10°C; 600 to 700

MPa; various

treatment time up

to 20 h



Pressure labile and stable fractions

observed

5–10% pressure stable fraction

Only pressure labile fraction

effectively inactivated

Synergistic effect of increases in

pressure and temperature on

inactivation



Ly-Nguyen

et al. (2002b)



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Indrawati Oey



Green pepper PME

purified with affinity

chromatography; citrate

buffer (pH 5.6)



10 to 65°C; 100 to

825 MPa; various

treatment time



Pressure labile and stable fractions

observed

5–6% pressure stable fraction

Only pressure labile fraction

effectively inactivated

Synergistic effect of increases in

pressure and temperature on

inactivation

Antagonistic effect of low pressure

(up to 300 MPa) on thermal

inactivation (>50°C)



Ly-Nguyen

et al. (2003)



Banana PME purified with

affinity chromatography;

Tris buffer (20 mM; pH 7)



10°C; 600 to

700 MPa; various

treatment time



Pressure labile and stable fractions

observed

8% pressure stable fraction

Only pressure labile fraction

effectively inactivated

Synergistic effect of increases in

pressure and temperature on

inactivation



Ly-Nguyen

et€al. (2002c)



Plums PME purified with

affinity chromatography



25°C; 650 to

800 MPa; various

treatment time



First-order kinetic inactivation



Nunes et al.

(2006)



Chapter ten:õ Effect of novel food processing



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Carrot PME purified with

affinity chromatography;

Tris buffer (20 mM; pH 7)



(continued)



257



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Enzymes



Peroxidase (POD)



Enzyme and buffer

solution/medium



Processing

condition



Enzyme stabilityb



258˘˘ï»¿



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Table€10.1╇ Effect of Combined High Pressure and Temperature Processing on the Stability of (Partially) Purified Fruit

and Vegetables Enzymes in Buffer Solution or in Fruit/Vegetable Juices (Continued)

References



Tomato PME purified by

affinity chromatography

and followed by cation

exchange

chromatography; citrate

buffer (0.1 M; pH 6)



20 and 40°C; 100 to

800 MPa; various

treatment time up

to 30 min.



Pressure labile isozyme found

First-order kinetic inactivation

600 MPa/40°C/6 min: one log unit

of inactivation

600 MPa/20°C/18 min.: one log unit

of inactivation



Plaza et al.

(2007)



Partially purified kiwi

POD



10 to 50°C; 200 to

500 MPa; various

treatment time up

to 30 min.



Fang et al.

(2008)



Commercial purified

horseradish POD; Tris

buffer (50 mM; pH7) with

H2O2 (50 mM) and

guaiacol (0.23 M)



25 and 40°C; 100 to

500 MPa; various

treatment time up

to 5 min.



Different isozymes had different

resistance towards pressure

At 30 and 50°C, synergistic effect of

increases in pressure and

temperature on inactivation

At constant pressure and 50°C,

prolonging treatment time

remarkably enhanced the

inactivation

600 MPa/50°C/30 min.: max. 70%

inactivation

Irreversible inactivation



Garcia et al.

(2002)



Indrawati Oey



3/31/10 4:32:54 PM



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