High-Pressure Processing for Modification of Food Biopolymers

High-Pressure Processing for Modification of Food Biopolymers

11

K. Olsen, V. Orlien University of Copenhagen, Frederiksberg, Denmark

1. Introduction Over the last two decades, high-pressure (HP) treatment of food products has increasingly been implemented in the food industry as an alternative, cold pasteurization technique superior to thermal treatment. HP is an excellent method to extend the microbial shelf-life of raw food (juice, smoothie, and shellfish), processed and convenience food products (guacamole, sauce, spreads), and ready-to-eat (RTE) meat products (sausages, sliced ham, salami) (Camus, 2010; Hiperbaric, 2015). HP can be performed at ambient or lower temperatures without affecting covalent bonds, and does therefore not destroy flavor compounds and vitamins to the same extent as thermal treatment (Schindler et al., 2010). The isostatic principle ensures that pressure is transmitted instantaneously and uniformly throughout the product, thereby no gradient is formed and advantages are that the product gets a uniform treatment and the processing time is reduced considerably (Camus, 2010). This chapter establishes the high-pressure modification of food biopolymers regarding functional properties in relation to structure and texture of foods with an emphasis on the underlying mechanisms. It is not a comprehensive review of all research studies on HP effects on foods, but is based on the most important studies defined by investigations that contribute to explaining the molecular mechanisms behind the functionality. It is well-known that HP affects the noncovalent hydrophobic and electrostatic interactions and hydrogen bonds whenever the resulting effect (destabilizing or stabilizing) is accompanied with a reduced molecular volume. Thereby, HP offers opportunities to modify structure and interactions within or between food biopolymers (Boonyaratanakornkit et al., 2002). In this way it is possible to modify or change the texturizing, stabilizing, or emulsifying properties of proteins, starch, and hydrocolloids.

2. Protein Pressure-induced changes in protein structure and function are expected to vary considerably depending on the type and concentration of protein and on the intensity and duration of the pressure treatment. In general, most globular proteins denature

Innovative Food Processing Technologies. http://dx.doi.org/10.1016/B978-0-08-100294-0.00011-0 Copyright © 2016 Elsevier Ltd. All rights reserved.

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under pressure due to disruption of the molecular forces responsible for maintaining the native conformation of the protein molecule.

2.1

Milk

Milk is a complex system of various proteins and minerals, and the chemical and physical mechanisms governing maintenance of the system are not fully understood. It is known that HP of milk and milk systems induces several physicochemical changes of both the proteins and the mineral balance. High-pressure effects on milk have been studied extensively with the goal of understanding the pressure-induced changes in various milk systems toward developing new dairy processes and products, and knowledge has been highlighted in many reviews (Devi et al., 2013; Considine et al., 2007; Huppertz et al., 2002, 2006; L
opez-Fandi~no, 2006). Information about the stability of the pressure-induced changes in milk proteins is important for an understanding of the functional properties of HP-modified milk proteins and for practical applications of HP technology in the dairy industry.

2.1.1

Whey Proteins

The most important whey proteins are a-lactalbumin (a-la) and b-lactoglobulin (b-lg) in the context of functional proteins in milk. b-Lactoglobulin is a compactly folded globular protein and at milk’s pH of 6.8 b-lg is mainly a dimer. b-lg is very pressure-sensitive and its denaturation process, which is inherently linked to its functionality, has been studied in detail by various researchers. The following step-by-step summary of the pressure-induced denaturation process provides the background for the functionality section. The first step in the denaturation process is dependent on the initial pH of the b-lg solution, and will, thus, control the specific mechanism of the denaturation. Dufour et al. (1994) suggested that the HP (0.1e350 MPa) transition involved three states at natural pH; the first state is the native b-lg, which transforms the denatured b-lg under pressure (second state) and the last state is the denatured b-lg after pressure release. Moreover, they found that the pH of the b-lg solutions determined the conformational changes (at a lower pH of 3.0, b-lg was less affected by pressure) and related this finding to electrostatic interactions and hydration of the protein. Tanaka et al. (1996) showed that the pressure-induced irreversible denaturation of b-lg is caused by the reaction of free thiols (sulfur/hydrogen: SeH). Later, Valente-Mesquita et al. (1998) expanded the one-step model (native to denaturation) and suggested that the dissociation and unfolding under pressure facilitated the formation of nonnative disulfide bonds (sulfur/sulfur bond: SeS) resulting in incorrectly folded, expanded conformations relative to native dimers. In 1999, the one-step model was further expanded to a three-step model: pressure melting followed by reversible unfolding, followed by irreversible aggregation and gel formation due to thioledisulfide exchange (Stapelfeldt and Skibsted, 1999). Since then, the denaturation process has been investigated in detail by several researchers. Fig. 11.1 shows a more detailed representation/sequence of the denaturation: in Fig. 11.1A/B, the pressure-induced denaturation of b-lg

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(B)

P > 150 MPa

P > 50 MPa

Stabilising interaction 100%

(C)

S-S

50% EI + HB HI P > 300 MPa

Pressure duration

0

(E) S S

S

(D)

P > 500 MPa

S S S

Figure 11.1 Pressure-induced denaturation and aggregation of b-lg. (A) Native dimer with water hydration on surface. (B) Monomers. (C) Molten globules and disulfide-bonded dimers. (D) Denaturation. (E) Aggregation or gelation. The time evolution diagram, which shows the stabilizing interactions; SeS is disulfide bonds, EI is electrostatic interaction, HB is hydrogen bonds, and HI is hydrophobic interaction, in a pressure-induced WPI gel is redrawn from Keim and Hinrichs (2004). Based on Orlien, V., Olsen, K., Skibsted, L.H., 2007. In situ measurements of pH changes in b-lactoglobulin solutions under high hydrostatic pressure. Journal of Agricultural and Food Chemistry 55, 4422e4428; Anema, S.G., Stockmann, R., Lowe, E.K., 2005c. Denaturation of b-lactoglobulin in pressure-treated skim milk. Journal of Agricultural and Food Chemistry 53, 7783e7791; Considine, T., Singh, H., Patel, H.A., Creamer, L.K., 2005. Influence of binding of sodium dodecyl sulfate, all-trans-retinol, and 8-anilino-1-naphthalenesulfonate on the highpressure-induced unfolding and aggregation of b-lactoglobulin B. Journal of Agricultural and Food Chemistry 53, 8010e8018.

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begins with dissociation of the native dimer, which increases the accessible surface area of the molecules, resulting in an increase in hydration of the protein molecules. In Fig. 11.1B/C, the change in the water shell around the protein affects the spatial distribution of charges and thereby the structural rearrangement of the protein/solvent system. Dissociation of ion pairs will facilitate motion of the side chains and polypeptide backbone, which increases the conformational fluctuations of the protein and provides pathways for water to penetrate into the hydrophobic interior of the native protein. These fluctuations are enhanced by pressure due to increased water exchange between the protein interior and bulk solvent. As a result of water penetration and the accompanying electrostriction (which is the contraction of solvent water due to alignment of dipolar water molecules in the electric field of an exposed charge) of water in the protein interior leads to conformational transitions of the protein structure to adopt the conformation of a molten globule, a compact, partially folded conformation without specific tertiary structure. Dependent on the pressure condition, a continuum of molecular molten globule states is suggested to exist (Aouzelleg et al., 2004). The increased hydration of the protein interior leads to two opposing forces and the various molten globule conformations may be formed due to their interaction or counteraction: (1) the pressure-induced hydration of the polypeptide leads to decreases in the protein compressibility and flexibility due to electrostriction and loss of void volume resulting in decreased mobility of the polypeptide and (2) hydration reduces the number of intramolecular hydrogen bonds and promotes the formation of intermolecular hydrogen bonds with water, thus causing increased conformational fluctuation of peripheral protein segments. In addition, it is suggested that nonnative disulfide bonds may be important for stabilizing the molten globular structure, which possesses a unique hydrophobic character (Yang et al., 2001). In Fig. 11.1C/D, increasing pressure further disrupts the forces stabilizing the molten globular structure leading to a denatured protein. Cooperative effects involving the caseins, a-la, and b-lg with thioledisulfide interchange and calcium, have been suggested to result in a higher degree of denaturation in milk and whey solution compared to a phosphate buffer (Mazri et al., 2012). The importance of the protein/solvent system interaction of the denaturation was also stressed by Belloque et al. (2007). They found that b-lg was more resistant to unfold under acidic than natural conditions and at acidic pH the denatured protein was able to gain its native structure after pressure treatment. In Fig. 11.1D/E, the pressureinduced denaturation of b-lg ends with aggregation into large polymers of proteins or formation of a gel network. Belloque et al. (2000) showed that the aggregates are large polymers formed by disulfide crosslinking due to the SeH/SeS exchange between the external SeS bond at C66eC160 and the accessible SeH group at the C121 of the denatured protein. Computer simulation of the pressure-induced aggregation showed that in the initial aggregation process (within 5 min) only dimers and trimers are formed due to the SeH/SeS interaction (Reznikov et al., 2011). In a comprehensive quantitative work by Anema et al. (2005c) it was observed that the mechanistic character of the whole denaturation process changes at around 300 MPa. At lower pressures of about 200 MPa the aggregation reactions are ratedetermining, while at higher pressures of about 600 MPa it is the denaturation reaction which is the rate-determining step.

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Apparently, the SeH/SeS exchange reaction is of utmost importance in the denaturation process of b-lg. However, regarding pressure-induced denaturation of a-la it is the four intramolecular disulfide bonds, the strong calcium-binding site, and lack of a free thiol group that makes this protein much more resistant to pressure. This rigid structure makes a-la stable after HP treatment at 800 MPa, and is still resistant to denaturation at pressures up to 400 MPa in milk systems (Jegouic et al., 1996). For this reason, a-la does not aggregate or form gels under pressures as high as 800 MPa (He et al., 2013). Yet, it possesses some “pseudo”-functional properties in milk systems, where it is capable of participating in b-lg networks (to be discussed later).

2.1.2

Casein and Casein Micelles

Like b-lg, the casein micelles were the subject of numerous studies concerning highpressure effects due to their pronounced sensitivity to pressure. Fig. 11.2 provides a schematic summary of the different effects on the casein micelles during and after pressure treatment. Though the exact nature of the micelle is unknown, at natural pH and room temperature, the integrity of casein micelles in milk is optimally balanced by hydrophobic interactions and electrostatic interactions, mainly through micellar clusters of calcium phosphate. It is obvious that the pressure effects on the micelles reflect the changes in the intramicellar hydrophobic interactions and the attractive and repulsive electrostatic forces controlled by the charge of the caseins and the state of colloidal calcium phosphate (CCP). In Fig. 11.2A/B, the initial step is the incorporation of water molecules into the micellar structure leading to disruption of the hydrophobic interactions (Desobry-Banon et al., 1994; Gaucheron et al., 1997). These hydrophobic interactions destabilize more easily compared to the electrostatic-based interactions (eg, hydrogen-bonded water or CCP). In Fig. 11.2B/C, following the disruption of hydrophobic interactions, the dissolution of CCP is favored upon increased pressure, resulting in the release of calcium and phosphorus from the micelles and an increase in calcium and phosphorus has been reported by several scientists (eg, Regnault et al., 2006; L
opez-Fandi~no et al., 1998). The overall dissolution of CCP is a result of individual effects on the balance between CCP, hydrated calcium phosphate, and dissociated ions. HP-induced dissociation of the phosphate salts and self-ionization of water in serum result in a pH decrease that favors dissolution of CCP and an increased concentration of phosphate ions that favors binding of calcium (Orlien et al., 2010). It has been found that the amount of free calcium ions does not increase upon pressurization (L
opez-Fandi~no et al., 1998) most likely due to binding to soluble proteins (Regnault et al., 2006). However, increasing the amount of calcium ions in a caseinate system, more colloidal calcium remained after HP treatment (200 MPa), hence underlining the importance of calcium in the pressure-induced dissociation of micelles (Anema et al., 1997). Concurrently with the CCP dissolution, the casein proteins are released from the micelles (L
opez-Fandi~ no et al., 1998; Regnault et al., 2006; Anema, 2007). The mechanism, rate, and extent of the disruption of the interaction between caseins and CCP depend on the starting point of the micellar forces in the initial state of the casein micelles

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(A) = Casein (all types) = CCP = Ca2+ = Water hydraƟon layer = electrostaƟc interacƟon

P < 50 MPa

(B)

P < 200 MPa

(C)

P > 300 MPa (E)

250 MPa < P < 300 MPa

(D)

(F)

Figure 11.2 Pressure-induced dissociation of casein micelles. (A) Native micelle. (B) Hydration of micelle and disruption of hydrophobic interactions. (C) Solubilization of CCP and dissociation into sub-micelles. (D) Re-association. (E) Dissociation. (F) Pressure release. Based on Orlien, V., Knudsen, J.C., Colon, M., Skibsted, L.H., 2006a. Dynamics of casein micelles in skim milk during and after high pressure treatment. Food Chemistry 98,

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(Gebhardt et al., 2005; Orlien et al., 2010). The barostability of the micelles depends on temperature and pH; at low temperature the pressure-unstable state dominates, while at high temperature the micelles were more stable (Gebhardt et al., 2005), and at low pH the micelles were fairly unstable, while at high pH the strong, stable micelles dominated (Orlien et al., 2010). As an effect of dissolution of caseins and CCP, the micelles dissociate into smaller submicelles. In Fig. 11.2C/D/E, depending on the pressure conditions, level, duration, and temperature, two different effects on the submicelles can be observed (Anema, 2007). In Fig. 11.2C/D, treatment at 250e300 MPa for prolonged times changes the casein micelle sizes markedly both during and after pressure treatment. This pressure range leaves the partially dissociated casein micelles and the dissociated caseins particularly prone to reassociate, resulting in formation of casein micelle sizes larger than those observed in untreated milk (Orlien et al., 2006a). This explains the broad size distribution of the casein micelles observed by several scientists (Anema et al., 2005b; Huppertz et al., 2004; Orlien et al., 2006a). In Fig. 11.2C/E, at pressures above 300 MPa the dissociation of casein micelles dominates and the casein micelles remain dissociated without major changes (Orlien et al., 2006a). It is further suggested that the micelles decompose completely into casein monomers (Gebhardt et al., 2006). In Fig. 11.2D/E/F, after pressure release the soluble caseins, calcium and phosphorus and the submicelles will assemble into new micellar-like structures. However, such decompression-assembled micelles are formed under different conditions than the native micelles and are unlikely to be similar in size and structure.

2.1.3

Dairy Products

The conformational changes of b-lg and the disruption of the casein micelles are necessary for utilizing, improving, or changing their functional properties. One of the functional properties of milk proteins is their ability to form a gel network. Not surprisingly, for pure b-lg the pressure-induced gelation is closely related to both concentration and pressure level. Thus, increased hardness was observed with increasing concentration (10e18%) and/or pressure (200e800 MPa, 10 min), though the lowest concentration of 10% needed the highest pressure at 800 MPa to form a soft gel (Kanno et al., 1998a). In addition, it was shown that the gel formation was driven by the different forces: hydrophobic interactions, hydrogen bonding, and SeS linkage. The different microstructure of the gels may express different dominating forces in the gel, as the b-lg gels at alkaline pH resembled a honeycomb structure, whereas the acidic b-lg gels resembled a coral structure (Kanno et al., 1998a). Surely, the

=

513e521; Orlien, V., Boserup, L., Olsen, K., 2010. Casein micelle dissociation in skim milk during high-pressure treatment: effects of pressure, pH, and temperature. Journal of Dairy Science 93, 12e18; Gebhardt, R., Doster, W., Friedrich, J., Kulozik, U., 2006. Size distribution of pressure-decomposed casein micelles studied by dynamic light scattering and AFM. European Biophysics Journal 35, 503e509.

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pressure dependency on gelation is reflected in the pressure dependency on the denaturation process of b-lg (Fig. 11.1). Olsen et al. (1999) found that treatment of a 5% b-lg solution at 450 MPa (30 min) led to the formation of a gel (going through all steps to E, Fig. 11.1), while low (150 MPa) and moderate pressures (300 MPa) only led to a range of oligomers and polymers in the solution (ending at step C/D, Fig. 11.1). The differences in the two threshold concentration for gelation of b-lg can be ascribed to the dynamics of the aggregation process, as Olsen et al. (1999) observed a variation in the distribution of the pressure-formed polymers at the low and moderate pressure and upon storage after HP treatment. Thus, the high concentrated solution will experience unfolding of b-lg and aggregation to some extent, but the pressurization time of 10 min is not sufficient to form large, stable aggregates forming a hard gel. On the other hand, the low-concentrated sample had sufficient time for the denatured b-lg to rearrange, resulting in increased amounts of polymeric b-lg, forming a gel. The concentration/pressure/time combination exhibits a specific critical region for the aggregation stability where small variations will have a pronounced effect on the degree of aggregation. Introducing a-la to the b-lg solution resulted in increasing pressureinduced oligomerization of a-la and b-lg (upon increasing the weight fraction of b-lg) due to the thioledisulfide exchange based on the free SeH in cysteine 121 in b-lg (Jegouic et al., 1997). The importance of a-la and low-molecular-weight compounds in the aggregation and gelation of b-lg was also observed by Ipsen et al. (2002), who found that the whey protein gels were stronger than the pure b-lg gels. Dumay et al. (1998) found a time-dependent strengthening of the proteineprotein interactions in b-lg isolate (including small amounts of a-la) gels. They suggested that initially after pressure (24 h) the gel is stabilized by weak hydrophobic interactions and some SeS bonds, but during prolonged storage (45 h after pressure release) the stabilization of the gel network was dominated by the disulfide bonds. The necessity of intermolecular disulfide bonds in the pressure-induced gelation was confirmed by He et al. (2013) as a-la (15%) only gelled in the presence of 5% b-lg. Interestingly, the microstructure of the two gels differed according to the dominant protein, where the b-lg isolate gel was porous and coral-like (Dumay et al., 1998), but the a-la gel had a homogeneous, fine-stranded structure (He et al., 2013). The pressure-induced gelation of b-lg was suggested to originate from random aggregation reactions between attractive sites of primary aggregates, which further aggregated during storage, resulting in increased rigidity of the gel (Dumay et al., 1998). The homogeneous structure of the a-la gel was explained by the lack of electrostatic complexes between a-la and b-lg, together with a tight association of the water molecules to the protein matrix (He et al., 2013). Introducing other compounds to a b-lg system had a negative effect on gelation, thus, the concentration of whey protein concentrate (WPC) must be higher than whey protein isolate (WPI) in order to form a gel at the same pressure level (Kanno et al., 1998b). The difference was not only ascribed to the lower protein concentration in the WPC system but also to the effects of other ingredients like lactose, lipids, and inorganic materials on the pressure-induced gelation, especially the baroprotective impact of lactose (Kanno et al., 1998b). The pressure-induced denaturation process of b-lg (Fig. 11.1) is dictating the impact of pressure on the gelation properties as

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the accessibility of the free thiol at C121 is crucial for the thioledisulfide exchange, which is the most important interaction when establishing the gel network. Fertsch et al. (2003) concluded that pressure-induced gelling of WPI takes place during pressurization, where the disulfide bonds are structuring and stabilizing the gel network, thus longer holding times provide firmer gels due to the increased degree of protein denaturation following higher amount of disulfide bonds. A semiquantitative extraction study by Keim and Hinrichs (2004) showed the evolution of the different stabilizing interactions in a 15% whey protein gel during pressurization at 600 MPa. The pictorial representation in Fig. 11.1 shows the change from a weak interaction domain to the strong disulfide bonding domain upon increasing pressure hold time. The time-dependent development of the different gel-stabilizing interactions is almost similar to the pressure dependent denaturation process of b-lg. The role and importance of the different stabilizing interactions in pressure-induced whey protein gels is emphasized by the impact of changing pH before pressurization. Van Camp et al. (1997) and He et al. (2010) showed that the pH of the WPC and WPI solutions, respectively, had a pronounced effect on the gel firmness, although the HP treatment was well above the 300 MPa needed for b-lg denaturation and gelation. At low pH (3 and 4) no gel was formed after treatment at 400 MPa for 30 min due to the repulsive forces among the positively charged proteins (Van Camp et al., 1997). Thus, acidic whey solutions will remain in the weak interaction domain because on the one hand these noncovalent interactions are not capable of inducing gel formation and on the other hand the reactivity of the free SeH is very limited, which affects the SeS crosslinking. Increasing pH changes the stabilizing domain and promotes the intermolecular SeS bonds because the reactivity of the SeH increases and results in stronger gels (Van Camp et al., 1997; Famelart et al., 1998; He et al., 2010). Similarly, the pressure dependency of the gelation is reflected in the pressure dependency of the dissociation process of the casein micelles (Fig. 11.2). Gelation of solutions of micellar casein powder was found to depend on the pressure release rate after HP treatment at 600 MPa (30 min, 30
C) (Merel-Rausch et al., 2007). Upon increasing pressure up to 600 MPa the micelles dissociate according to step A/E in Fig. 11.2, and then step E becomes determining for the gelation. Merel-Rausch et al. (2007) found that a fast pressure release (600 MPa/min) is necessary for the formation of a firm and fine (microstructure) gel, because the formed submicelles (under pressure) obtained a larger diameter and volume during pressure release, which enabled aggregation and formation of a gel. On the other hand, slow pressure release (20 MPa/min) did not affect the submicelles and did not change the solution characteristic. Changing the liquid base of WPI solutions from water to milk had a great effect on the gel formation, most likely due to the contribution of the caseins to the gel network stabilization (Orlien et al., 2006b). Thus, fortifying milk with 15% WPI increased the elastic modulus by a factor of five compared to water with 15% WPI after treatment at 400 MPa for 30 min (Orlien et al., 2006b). In this respect both the b-lg denaturation and casein micelle dissociation processes become important. The pressure-induced gelation of b-lg occurs under pressure, where the intermolecular disulfide bonds are established, and at the same time the casein micelles will dissociate or re-associate

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depending on pressure level and duration. The formation of submicelles and the concomitant mineral (Ca and P) release is shown to be necessary for the gelation process (Famelart et al., 1998; Keenan et al., 2001). Moreover, the release of the casein proteins from the micelles may also play an important role in the gel-forming ability. Patel et al. (2006) showed that denatured b-lg could form disulfide bonds with k-casein and as2-casein, thereby incorporating b-lg into aggregates. The increased gel strength of the 15% WPI in milk can result from a combination of formation of casein aggregates and complexation with b-lg (Huppertz et al., 2004). This is supported by the observation that the firmness of casein gels (based on casein micelle powder without whey proteins) was lower than the whey protein gels (Fertsch et al., 2003). It was explained that the caseins only provided noncovalent interactions resulting in a soft network, especially at slow pressure release (Fertsch et al., 2003). The investigations of set yogurt made from HP-treated (P > 600 MPa) milk or HPtreated milk followed by heat treatment varies regarding the improvement in gel strength, but confirms that the microstructure of the gels was considerably different from those obtained with traditional heat treatment (Needs et al., 2000; Harte et al., 2002, 2003; Penna et al., 2007). Harte et al. (2002) found a similar yield stress, while Needs et al. (2000) reported a higher storage modulus of their HP yogurts compared to heat yogurts, and both Harte et al. (2003) and Penna et al. (2007) observed improved texture and viscosity of their HP þ heat yogurts. In all cases, the difference in microstructure was a consequence of the micelle dissociation under pressure, which provided new ways of proteineprotein interactions similar to the above-discussed gelation of neutral milk gels. The fermentation step lowers the pH of the milk system and results in small compact aggregates or submicelles. The denatured b-lg will form disulfide bonds with the k-casein on the surface of the submicelles, and thereby restrict the further aggregation. The consequence is an interconnected gel network consisting of smooth, spherical, uniform particles. For a visual overview we refer to the schematic diagrams in Harte et al. (2002) and Penna et al. (2007). All yogurts, made from either HP-treated milk or HP- þ heat-treated milk, had a much better water-holding capacity compared to traditional yogurts (based on heat-treated milk), therefore reducing syneresis considerably. The intermolecular crosslinking between whey proteins and caseins was further improved when transglutaminase treatment was performed under or after pressurization of the milk resulting in increased gel firmness (Anema et al. (2005a); Tsevdon et al., 2013). However, stirred yogurt had the same rheological properties whether it was prepared from HP-treated milk or not, suggesting that the improved network is destroyed in the stirred gels (Knudsen et al., 2006; Udabage et al., 2010). Generally, the mechanisms of the HP effect on rennet coagulation are the same as for the HP effect on milk gelation, and will not be described here (see, eg, Considine et al., 2007; L
opez-Fandi~ no, 2006). On one hand, the pressureinduced micelle dissociation and release of k-casein facilitates the action of rennet, thus reducing the rennet coagulation time (RCT) though only at HP < 300 MPa, whereas at HP > 400 MPa the association between b-lg and submicelles will restrict the accessibility, thus increasing the RCT. On the other hand, an increase in cheese yield was explained by the pressure-induced incorporation of whey proteins into the curd.

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2.2

301

Meat

High-pressure treatment of meat and meat products was at first studied to examine the effect on microorganisms, and HP has since been documented to improve the microbiological safety of the final product. It was then observed that pressure affects protein structures leading to changes such as dissociation, denaturation, aggregation, and gelation, thereby affecting molecular functionality. Heat treatment is commonly used to obtain such changes, but the resulting appearance and rheological properties of the meat products are different from what can be obtained by pressure. Therefore, HP became of increasing scientific interest and investigation of the pressure-induced modification of meat proteins, with the aim of understanding the mechanisms underlying the pressure-modification and how to utilize pressure for meat product manufacturing, intensified. In addition, numerous reviews of the high-pressure effect on meat emerged (Buckow et al., 2013; Ma and Ledward, 2013; Bajovic et al., 2012; Simonin et al., 2012; Sun and Holley, 2009; Colmenero, 2002). The HP technology gave the meat product industry potential to produce new, innovative, safe, highquality, convenience, and RTE meat products. However, the ability to control and direct pressure-induced changes in the meat and obtain a high-quality product are fundamental for a successful implementation of HP technology in the meat industry. As discussed before for diary proteins, pressure induces structural changes of protein molecules due to different states of unfolding and denaturation and this pressuremodification of the protein structure results in changed reactivity of the proteins. Meat or muscles are composed of collagen, sarcoplasmic proteins, and myofibrillar contractile proteins. The connective tissue, collagen, is highly structured and is not affected by high pressure alone (Suzuki et al., 2006).

2.2.1

Sarcoplasmic Proteins

The sarcoplasmic proteins, which account in average for one-third of the total muscle proteins, are mainly heme pigments and enzymes. The color of meat is defined by the meat pigments and meat structural proteins and is one of the main meat product attributes that influence consumer’s acceptance. Overall, meat color is very much affected by high pressure, and the different myoglobin species regarding conformational state, the state of iron, and the ligand bound to the heme has recently been discussed in the context of HP-induced discoloration of pork (Bak et al., 2012), but is per se not related to functionality. Modification of proteins by oxidation may result in changes of the protein backbone and/or the amino acid side chains, like denaturation, peptide bond cleavage, and formation of new intermolecular covalent bonds, and in this way affect functionality. The thiol group is the most functional group in a protein as the easily oxidized SeH will lead to the formation of intra- or intermolecular disulfide bonds, thereby establishing a gel-like network in the meat and affecting meat texture. The extent of protein oxidation in meat as evaluated by measurement of the loss of sulfhydryl groups is a new approach, but the impact on meat texture has not been clearly established yet. Both the sarcoplasmic proteins, mainly the enzymes, and the myofibrillar proteins, mainly myosin with several cysteine residues, are prone to thiol

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oxidation. During pressurization, the proteins and enzymes may be released, thereby making surface thiols accessible, or proteins may be denatured, thereby making interior thiols exposed, in effect providing reactive SeH available for disulfide crosslinking. However, there are conflicting results of the effect of HP on the content of thiols in meat and further insight into the mechanism and consequences of meat protein oxidation is needed (Grossi et al., 2014). Pressure-modification of proteins is initiated by the destabilization and/or disruption of the hydrophobic and electrostatic interactions of the quaternary and tertiary structure resulting in a partial or full protein unfolding or denaturation, thereby exposing various reactive domains. Once the domains are accessible for other constituents, new hydrophobic interactions, hydrogen bonding, or disulfide bonds are possible and responsible for protein aggregation. Such changes in the protein conformation will affect molecular sizes and thereby protein solubility due to denaturation following covalent linking (decreased solubility) or degradation into lowermolecular-weight compounds (increased solubility). However, only a few investigations have shown that HP treatment reduces the solubility of the sarcoplasmic proteins (Grossi et al., 2014). In this context, analyzing the solubilized proteins by electrophoresis (PAGE) will reveal which and to what extent proteins have been affected by the HP treatment. Changes in the PAGE band intensities of sarcoplasmic proteins have been shown for pork (Grossi et al., 2014), beef (Marcos et al., 2010; Sikes et al., 2010), and turkey (Chan et al., 2011). One explanation is that the proteins have denatured following aggregation, resulting in large, insoluble aggregates resistant to extraction. Another explanation for the disappearance of the band can be the hydrolysis by the enzymes. The hydrolysis of sarcoplasmic proteins has been reported to account for the disappearance of bands (Picariello et al., 2006). It is shown that HP treatment can increase the activity of cathepsins by releasing them from lysosomes (Homma et al., 1994; Kubo et al., 2002; Grossi et al., 2012a). Moreover, it was found that HP induced cathepsin B and L activity during storage, which in turn caused the degradation of higher-molecular-weight proteins/peptides and the formation of new smaller peptides (Grossi et al., 2012a). The pressure effect on the water-soluble sarcoplasmic proteins has not been investigated in detail and has not been related to functionality of the proteins and thereby texture of meat.

2.2.2

Myofibrillar Proteins

Though the myofibrillar proteins are structural proteins and as such more stable than the sarcoplasmic proteins, they relate more directly to meat texture in terms of functionality in meat products, and have been investigated in more detail regarding pressure effects. The myofibrillar proteins, which account for around two-thirds on average of total muscle proteins, consist mainly of myosin heavy chains (HC), actin, a-actinin, tropomyosin, troponin-T, and myosin light chains (LC), and are the foundation of the meat structure. High-pressure treatment affects myofibrillar proteins in the same way as the sarcoplasmic proteins by decreasing their solubility. The decreased solubility of the myofibrillar proteins upon increasing the pressure level has been reported for beef

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(Chapleau and Lamballerie-Anton, 2003; Lee et al., 2007; Speroni et al., 2014), pork (Grossi et al., 2012a,b; Tintchev et al., 2013), turkey (Chan et al., 2011), and chicken (Iwasaki et al., 2006). In addition, it was also observed that the solubility increased at low or moderate pressure (Iwasaki et al., 2006; Lee et al., 2007; Tintchev et al., 2013; Speroni et al., 2014). In these latter studies, protein suspensions or meat batters were pressurized, and it is likely that the protein extraction or mincing prior to HP processing had already disrupted molecular interactions, thereby inducing greater solubility. HP processing of minced meat may act differently on the proteins, in effect leading to different structure modifications from the ones in HP-treated whole muscle meat. Still, some overall principles can be deduced. The decrease in protein solubility is caused by protein denaturation following formation of larger insoluble protein aggregates that cannot be extracted: (1) the protein denatures due to the rupture of noncovalent interactions within the protein molecules; and (2) new exposed areas of the denatured protein facilitate formation of new intra- and/or intermolecular bonds such as hydrogen bonding, hydrophobic or electrostatic interactions, and disulfide crosslinking, resulting in aggregates. Based on nonreducing and reducing electrophoretic analysis it has been suggested that the aggregation of myosin-HC is due to intermolecular disulfide bonds (Angsupanich et al., 1999; Chattong and Apichartsrangkoon, 2009). Recently, loss of solubility was examined in detail by different reagents targeting the disruption of specific molecular interactions in order to assess which type of bonds are responsible for the aggregation mechanism and to elucidate the nature of these new proteineprotein interactions (Grossi et al., 2016). Thus, it was suggested that formation of hydrogen bonds had a major role in protein aggregation during pressurization, while disulfide crosslinks and hydrophobic interactions may not be responsible for the loss of protein solubility in HP-treated meat. This result supports the suggestions by Angsupanich et al. (1999) and Ma and Ledward (2004) that pressure-induced myosin aggregations are stabilized by hydrogen bonds. The myofibrillar proteins are involved in the process of muscle contraction and their roles differ extremely depending on their structure. Myosin is a heterogeneous hexamer composed of two heavy chains and four light chains and actin is a globular protein that tends to form microfilaments in cells. Pressurization of meat above 200 MPa leads to disruption of the I-band, M-line, and Z-line (which are the different locations of the thin and thick filaments in the filamentous structure (Zubay, 1993)) and dissociation of the thin and thick filaments occurs (Suzuki et al., 1990; Iwasaki et al., 2006). Such pressure-induced rupture of the filamentous structure, concomitant with fragmentation of the myofibrils with increasing pressure up to 200 MPa, caused solubilization of the myofibrillar proteins (Iwasaki et al., 2006). Electrophoretic analysis has shown that the decreased solubility of the myofibrillar proteins, upon increasing pressure above 200 MPa, was a result of the pressure impact of the individual proteins (Angsupanich et al., 1999; Tinchev et al., 2013; Speroni et al., 2014; Grossi et al., 2016). The electrophoretic profiling showed that the myofibrillar proteins were modified by pressure-induced denaturation, and immediately followed by either degradation or aggregation. In this way, the disappearing of bands or newly formed bands is attributed to either degradation of larger proteins into small subfragments or aggregation of low-

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molecular-weight proteins. Myosin is the most abundant protein in the myofibrils and is recognized to be of utmost importance for the quality of meat products because of its ability to bind meat proteins together and to hold water. The myosin molecule has two identical globular head regions, which are composed of various fragments that connect with the light chains and the heavy chains as tails. Thus, myosin has been the main interest concerning pressure effects on myofibrillar proteins. Yamamoto et al. (1993) suggested that monomer myosin initially associated by head-to-head intra- or intermolecular interactions followed by aggregation into a so-called daisywheel (the heads are connected together in the middle and the tails are pointing outwards) oligomer, with the tails remaining intact. However, Tintchev et al. (2013) showed that pressure at 200 and 300 MPa resulted in dissociation of myosin into the smaller-molecular-weight fragments named N-terminal and C-terminal from the subfragment 1 (Iwasaki and Yamamoto, 2002). Based on this, Tintchev et al. (2013) proposed the aggregation mechanism; this initial (up to 300 MPa) disruption of the myosin molecules is followed by a hydrophobic packing into daisy-wheels, which further (350e600 MPa) form larger aggregates with other unidentified proteins into a protein network. Another suggestion was based on the observation of a decrease in the myosin-HC content, while the myosin-LC content did not change, thus the pressure-induced aggregation involved the dissociation of myosin heavy and light chains followed by aggregation of the heavy chains (Speroni et al., 2014). These suggestions emphasize that the exact configuration of the aggregates formed and the underlying proteineprotein interactions are still unknown. Recently, a Westernblotting analysis, which targets selected myofibrillar proteins, thereby monitoring their individual and specific behavior at different HP levels, showed that myosin and actin lose their native solubility at HP treatment above 400 MPa, while a-actinin and troponin-T are less affected by pressure (Grossi et al., 2016). This supports the findings of other authors that myosin, especially the globular head (which is composed of several low-molecular-weight fragments) (Iwasaki and Yamamoto, 2003), is the most pressure-sensitive, and it dissociates under pressure, followed by the formation of aggregates (Iwasaki et al., 2006; Tintchev et al., 2013; Yamamoto et al., 1993). From the target electrophoretic analysis it is suggested that the pressure-induced aggregation is mainly caused by hydrogen bond formation (Grossi et al., 2016).

2.2.3

Meat Products

Two types of meat products can be considered; whole meat muscle and comminuted meat products, and the desired HP impacts on the respective product texture differ considerably. Generally, HP has been investigated as a tool to tenderize whole meat pieces, thereby aiming at solubilizing proteins, or as a tool to produce meat gels, thereby aiming at binding proteins together. Regarding HP-tenderization, the reader is referred to Chapter 12 (Application of high hydrostatic pressure for meat tenderization) for in-depth discussions on this subject, which will not be further discussed in this chapter. In light of the microbial shelf-life improvement, HP may also be an interesting process for functional improvement in the meat industry. Salt is commonly used in

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cooked meat products (whole muscles or comminuted meat) for its technological improvements (texturizing and water binding) and taste. Thus, the combined use of salt and HP was investigated in order to assess the quality characteristics (Duranton et al., 2012). Pressurization (500 MPa) and subsequent cooking (to a core temperature of 70
C) of pork biceps femoris significantly increased the shear force compared to cooked-only meat. The pressure-induced hardening due to the protein aggregation was thus further amplified by the second processing step (cooking), but addition of salt (1.5 or 3%) counteracted the hardening effect and made the HP þ cooked products comparable to the cooked ones (Duranton et al., 2012). The microstructure analysis revealed different microstructures of all the samples (cooked vs. HP þ cooked vs. salt þ HP þ cooked), where the combination of salt, HP, and cooking led to a completely disrupted structure. Water-holding capacity (WHC) was reduced markedly after HP and cooking treatments, but improved to the same extent by adding salt (Duranton et al., 2012). It was suggested that excessive protein denaturation due to both pressure and heat caused the decrease in protein ability to bind water. In contrast to the decrease in WHC, it was observed that HP (600 MPa) of brine enhanced pork semitendinosus reduced the drip loss considerably (Grossi et al., 2014). The differences may be explained by the different measure of water binding, where WHC is a measure of the total water released by an applied force, while drip loss is a measure of the simple water exudation due to the treatment, and different molecular forces may influence both types of water releases. Generally, the effect of combined salt and pressure on WHC or drip loss in whole meat is dependent on pressure level, duration, temperature, and ionic strength and the swelling and/or disruption of the myofibrils is the critical factor (Grossi et al., 2014). The manufacturing of meat gels like sausages is based on production of meat batters followed by heat treatment in order to induce gelation. Moreover, a relatively high content of salt is needed to facilitate protein solubility and water binding, and to obtain the desired firm texture. The ability of HP to induce protein solubility and aggregation and improve WHC has, thus, been studied for the possibility of producing low-salt sausages. In contrast to whole-muscle meat products, the meat is minced upon preparation of the meat batter, thereby already introducing a mechanical disruption of the filaments. Still, HP processing provides different gelling properties of the proteins compared to heat processing. The pressure-induced denaturation of the myofibrillar proteins was found to be the main reason for the pressure-induced (350 MPa, 6 min, 20
C) hardening of pork meat batters without salt and phosphates (Villamonte et al., 2013). Their thermal analysis (differential scanning calorimetry) showed that apparently it was the type of denaturation and not the amount of denatured proteins that affected the texture, especially the denaturation of myosin and actin together with the formation of a new protein structure. This observation agrees with the suggestions that at pressure above 350 MPa myosin and actin lose their native solubility due to formation of larger aggregates. However, the addition of salt and phosphate to the meat batter had no effect on the improvement of the texture, and was explained by the electrostatic interaction and hydrogen bonding between proteins and salt molecules in effect impairing the proteineprotein interactions forming the gel network (Villamonte et al., 2013). Yet, the salt acted synergistically with HP and improved the WHC

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(Villamonte et al., 2013). Tintchev et al. (2013) proposed a mechanism for the pressure-induced watereprotein interaction resulting in improved water binding in frankfurter sausage batters. Basically, the pressure-induced unfolding of the proteins increases the intermolecular space and the active side chains leading to more accessible charged groups for interaction with the water molecules (Tintchev et al., 2013). Based on the detailed study on pressureetemperature (PeT) effect on meat proteins together with other reports, Tintchev et al. (2013) summarized the structure modification and functional properties in a PeT diagram (Fig. 11.3). Tintchev et al. (2013) found that the maximal protein solubilization occurred at 200 MPa at 40
C (depending on treatment time) and with respect to meat batter structure, the HP effect on protein solubility is the key factor (depicted as the solubilization line from 5 to w50
C and the hatched area compiled from other studies). On the one hand, HP increases the solubility of muscle proteins and thus improves the functional properties of certain myofibrillar proteins. On the other hand, HP may also induce protein denaturation and aggregation, resulting in loss of solubility of the main myofibrillar proteins. Thus, using pressure for structure modification of meat proteins is a weighing between protein solubilization and aggregation. NaCl and phosphates affect the solubility of myofibrillar meat proteins (like myosin and actin), thereby affecting their ability to form a cohesive protein matrix, and are used to produce products based on meat batters like sausages. To aid or improve acceptable meat binding when reducing the salt content, other functional ingredients are needed. Based on the suggestion from Tintchev (Fig. 11.3) the pressure needs to be above 500 MPa (and possible 70
C) to be industrially relevant. The available industrial HP equipment operates up to 600 MPa but at much lower temperatures than the suggested 70
C (usually at room temperature or below, eg, tap water). Though, heating of the pressure-transmitting

Figure 11.3 Hypothetic PeT ranges of myosin solubilization, aggregation, and gelation after HPP of 240 s. Reproduced from Tintchev, F., Bindrich, U., Toepfl, S., Strijowski, U., Heinz, V., Knorr, D., 2013. High hydrostatic pressure/temperature modeling of frankfurter batters. Meat Science 94, 376e387.

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water may be feasible, it will most likely not be economically attractive as heating of water is an energy-consuming process. However, with the aid of natural compounds, the pressurization temperature may be lowered. Grossi et al. (2012b) found that addition of carrot fiber or potato starch to the meat emulsion made it possible to lower the salt content to 1.2% (and no phosphates) in pressurized (400, 600, or 800 MPa at 5 or 40
C for 5 min) pork sausages. An important finding regarding the salt reduction was that the use of starch or fiber had more impact on textural properties than the level of salt and, moreover, the water-binding capacity was improved. The effect of HP (400, 600, and 800 MPa) and starch or fiber enhanced protein binding and resulted in increased hardness (with starch being better than fiber), and mild heating (40
C) during pressurization acts synergistically, improving even further the meat sausage characteristics (Young’s modulus and strain at fracture) (Grossi et al., 2012b). Clearly, the investigated pressures are above the pressures for protein solubilization and in the interval for following aggregation and gelation, but the effect of HP on the starch and fiber seems decoupled from the protein network. This observation can be explained by the fact that insoluble fibers favor water-binding properties because water binds to insoluble polysaccharides by hydrogen, ionic, and/or hydrophobic interactions. It was previously described that aggregation was a result of H-bonding or hydrophobic packing, therefore, the possible interaction sites with the proteins are largely occupied with water molecules. Starch gelatinizes under pressure in two steps, first a hydration of the amorphous parts of the starch granules occurs followed by the swelling and distortion of the crystalline region, in effect filling up in the meat matrix. A similar effect can be ascribed to the result on the effect of HP on chicken meat with b-glucan (Omana et al., 2011). They found that when using temperature-assisted HP processing (400 and 600 MPa at 40 or 60
C for 30 min) it is possible to substitute some of the NaCl (from 2.5% to 1.0%) and obtain the same hardness (Omana et al., 2011). b-Glucan is a polysaccharide with a strong water-binding capacity through H-bonds, and it is suggested that the texturing property is based on b-glucan and water filling up the cavities in the protein network, rather than participating directly in the network. Chattong et al. (2007) also found that incorporation of nonmeat ingredients (locust bean gum, carboxymethylcellulose, and xanthan gum) had no effect on the gelling properties of meat proteins after a severe HP treatment (600 MPa at 50
C for 40 min) of ostrich meat sausages. Trespalacios and Pla (2007) showed that the combined use of microbial transglutaminase, egg proteins, HP (700 or 900 MPa at 40
C for 30 min) and with reduced salt and without phosphates produced chicken meat batter gels with substantially more enhanced textural properties than without enzyme or heat-only treated. In contrast to the latter studies, the improved hardness was a direct improvement of the protein network, and it was explained that pressure denatured ovalbumin making it accessible to transglutaminase, thereby making SeS crosslink to myosin available. However, for the three latter investigations it is noted that HP processing of 30 min is economically not feasible. With the exponential growth in industrial HP equipment solely for preservation processing in various food industries (Campus, 2010) it is foreseen that the structure modification application by HP will gain a foothold on the market during the next decade. Overall, it is evident that high-pressure processing has great potential as a tool to produce low-salt meat

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products with pressure level being the key parameter for the formation of a pressureinduced meat gel network.

3.

Conclusion and Future Trends

For the two major food industries, dairy and meat, high-pressure technology offers a nonthermal treatment to modify proteins for new product development. Using pressure for structure modification of dairy and meat products is a delicate balance between destroying and building intra- and intermolecular forces in order to affect the structure/texture properties of the proteins in the expected way. For milk systems, the disulfide crosslinking through thioledisulfide exchange is the most important interaction for establishing and/or stabilizing a milk gel, thus the denaturation of b-lactoglobulin becomes the controlling and adjustable factor for the HP gelation properties. For meat systems, the HP impact on the protein myosin determines the resulting meat texture. However, the exact interacting force in establishing and/or stabilizing the meat protein network is not yet completely identified, and both hydrogen bonding and the hydrophobic package have been suggested to lead to the formation of larger aggregates into a protein network; though it has been proven that first the proteins are solubilized, followed by aggregation when meat is subjected to pressure. Although the underlying mechanism of the relationship between pressuremodification and functional properties may not be well-established, this review shows that considerable scientific knowledge exists and the numerous studies on product properties demonstrate that the potential use of high-pressure processing in industrial applications for the purpose of protein, and therefore, product structure modification, is only a matter of time.

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opez-Fandi~no, R., 2006. High pressure-induced changes in milk proteins and possible applications in dairy technology. International Dairy Journal 16, 1119e1131. L
opez-Fandi~no, R., Fuente, M.A.D.L., Ramos, M., Olano, A., 1998. Distribution of minerals and proteins between the soluble and colloidal phases of pressurized milks from species. Journal of Dairy Research 65, 69e78. Ma, H.-J., Ledward, D.A., 2004. High pressure/thermal treatment effects on the texture of beef muscle. Meat Science 68, 347e355. Ma, H.-J., Ledward, D.A., 2013. High pressure processing of fresh meat. Meat Science 95, 897e903. Marcos, B., Kerry, J.P., Mullen, A.M., 2010. High pressure induced changes on sarcoplasmic protein fraction and quality indicators. Meat Science 85, 115e120.

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