temperature modeling of frankfurter batters

Meat Science 94 (2013) 376–387

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High hydrostatic pressure/temperature modeling of frankfurter batters Filip Tintchev a, b, c,⁎, Ute Bindrich a, Stefan Toepfl a, Ulf Strijowski a, Volker Heinz a, Dietrich Knorr b a b c

German Institute of Food Technology, Prof.-von-Klitzing-Str. 7, D-49610 Quakenbrück, Germany Berlin Institute of Technology, Department of Food Biotechnology and Food Process Engineering, Königin-Luise-Straße 22, D-14195 Berlin, Germany McAirlaid's GmbH & Co. KG., Zum Eichberg 2, D-37339 Berlingerode, Germany

a r t i c l e

i n f o

Article history: Received 25 May 2012 Received in revised form 12 February 2013 Accepted 20 February 2013 Keywords: High pressure/temperature processing Pressurizing gradient (PG) Pork batter modification Water holding capacity Protein solubilization Salt reduction

a b s t r a c t The impact of high pressure/temperature treatment on structure modification and functional sensory properties of frankfurter batter was investigated. The degree of solubilization of meat proteins, particularly of myosin, was identified as a key process with significant effect on the batter's structural properties. The maximal solubilization level was at 200 MPa/40 °C IT for all formulations which was found to be treatment time dependent. The impact of the pressurizing gradient — PG = 40 MPa/s and PG = 2.5 MPa/s was investigated and estimated to have a significant effect on the protein network and functional properties, respectively. These were improved at low PG (2.5 MPa/s) as a phenomenon of secondary network formation parallel to the main matrix. Batter secondary-structure characteristics were found to be ionic-strength dependent. According to SDS-PAGE analysis, the major role in the solubilization, aggregation and gelation processes occurring in the aqueous phase was due to the myosin S-1 and S-2, N-terminal, C-terminals, the MLC and actin during the high pressure/temperature treatment. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Almost 100 years after the pioneering work of Hite (1899), the first industrial application of high pressure processing (HPP) was realized. The scientific interest of HPP in the last two decades has increased and has led to more industrial applications in various fields of the food industry (Tonello, 2010). The great increase in industrial applications since 2000 demonstrates the efficiency and the competitiveness of HP-food processing. Parallel to the industrialization of HPP, intensive further equipment development (seal improvement) and energy cost optimization (volume increase up to 420 L, parallel working vessels— Tandem, NC-Hyperbaric) have been made. Presently, there are over 150 HPP installations worldwide in use with a total food production of 250,413 t, and 90,315 t of which are meat products (calculated for 2009, (Tonello, 2010)). The mild character of HPP provides a great opportunity for developing innovative food products of superior nutritional and sensory quality. The advantages of HPP of foods compared to conventional thermal treatments, such as preservation at moderate temperature, retaining the fresh character of the foods, treatment of packed products, and immediate pressure effect inside the matrix without any delay, promise a lot of future application opportunities. In general, industrial high pressure processing (HPP) meat applications are performed in order to extend shelf life at low and medium temperatures, where inactivation of vegetative pathogen microorganisms at pressures above 400 MPa is possible (Buckow & Heinz, 2008).

⁎ Corresponding author at: Göttingerstr. 24, D-37115 Duderstadt, Germany. E-mail address: [email protected] (F. Tintchev). 0309-1740/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.meatsci.2013.02.012

In addition to microorganism inactivation, pressure affects the processes of meat protein denaturation, solubilization, aggregation and gelation (Cheftel & Culioli, 1997; Iwasaki, Noshiroya, Saitoh, Okano, & Yamamoto, 2006; Jimenez Colmenero, 2002; Sikes, Tobin, & Tume, 2009; Yamamoto, Yoshikawa, & Okada, 1993). Pressure induced gels provide generally smoother, more glossy, less firm, and more elastic gels with improved water holding capacity, compared to thermally induced gels (Cheftel & Culioli, 1997; Jimenez Colmenero, 2002). HP-pre-treatment before conventional thermal processing is also reported to improve the functionality of sausage batters (Sikes et al., 2009; Suzuki & Macfarlane, 1984). Suzuki and Macfarlane (1984) and Sikes et al. (2009) report the effect of processing pressure on batter structure (gelation) different from that of thermal processing. This provides the great potential of modifying meat-processed products such as batter, by affecting their functional properties by HP and creating innovative products. Today, innovations can be a significant determining factor in one's ability to take advantage of the market. Frankfurters originated more than 100 years ago and now enjoy worldwide popularity. They belong to the group of classical raw-cooked meat products. The stage after comminuting and mixing of the mass (raw process phase) is known as a batter. Frankfurter sausage batter can be defined as poly-dispersed systems consisting of a liquid continuous phase (water and soluble proteins, ions), a dispersed liquid phase (fat droplets) and a dispersed solid phase (non-solvated muscle fiber particles, connective tissue, spices). With respect to rheological properties, sausage batters show visco-elastic behavior that may result in effects, such as the Weissenberg-effect and

F. Tintchev et al. / Meat Science 94 (2013) 376–387

swelling. Conventionally, denaturation processes, e.g., transforming batter to sausage, are induced by heating to a core temperature of approx. 72 °C. Denaturation processes in the batter matrix can also be caused by application of high hydrostatic pressures (HP) or combined high pressure–temperature (HP–T) treatment. During the course of this work, essential mechanisms of HP and HP–T texture and functional property modification of frankfurter batters have been studied. Systematic studies to identify the mechanisms of solubilization, structure modification, protein disruption, aggregation and gelation depending on the temperature, treatment time, pressurizing gradient and salt content have been performed. Detailed studies of meat batter protein solubilization under different process and formulation variations have been carried out. Determination of the maximum solubilization level of particular meat proteins and protein subunits, as well as their participation in aggregation and gelation processes, was of great importance for modifying sausage structure. Possibilities of salt reduction after high pressure processing without any negative effects on the sausage batter structure and sensory properties have been tested. The hypothetic mechanisms of the high pressure effect on the meat batter proteins, and the mechanism of high pressure aggregation and gelation in the aqueous phase have been developed. 2. Materials and methods 2.1. Material Sausage batter was made using a standardized method in order to obtain reproducible material properties. A frankfurter type formulation has been used. Ingredients for a 5 kg batch are: 1.25 kg pork meat II (neck), 1 kg grade pork III (shank meat), 1.6 kg grade pork IV (back fat), 1.15 kg ice, 100 g pickling salt (99.6% NaCl and 0.4% NaNO2 from Suprase, Hengelo, Netherlands), and 15 g diphosphate (AVO-Werke August Beisse GmbH). Barrow pork meat supplied from the Diekmann G.H. slaughterhouse in Essen, Oldenburg, Germany was used 24 h post-slaughter. It was chilled to 4 °C before grinding. Subsequently, the meat was cut into small pieces. For grinding a meat grinder Model Mado (Typ MEW 603) with two knives, one kidney plate and two grinder plates was used (with hole size 16 mm and 3 mm). The temperature after the grinding process was 9–11 °C. Grinding was followed by chopping where the lean meat was placed into a six knife bowl cutter model Alexander N20 (without vacuum) with salt and phosphate for the whole batch. The mixture was chopped for 30 s without ice (dry chopping). Afterwards ice was added (575 g) and the chopping continued at fast bowl chopper speed until reaching a mixture temperature of 3 °C. In the next step fat (pre-minced and chilled) was added and chopped at high speed until a batter temperature of 12.5 °C was reached. The remaining ice (575 g) was added and chopped to a final batter temperature of 12.5 °C. In the closing step batter was filled into Nalo cellulose casings, caliber 50 mm (Kalle GmbH, Wiesbaden, Germany) using a filling machine (piston stuffer). The maximum capacity of the casings was filled to avoid surface wrinkles as well as air pockets in the final product. This was very important for homogeneous product properties. The final pH of the standardized batter was 6.0–6.2. Four different salt contents in the batter formulations were used: - The standard frankfurter batter formulation with 2% NaCl and 0.3% phosphate. - A reduced NaCl batter formulation with 1% NaCl and 0.3% phosphate. - A reduced NaCl batter formulation with 0.5% NaCl and 0.3% phosphate. - A reduced NaCl and phosphate formulation with 1% NaCl and 0.15% phosphate. To guarantee reproducibility, the uniformity of the sausage batter initial state had to be tested for several days after slaughter to determine when a batch of raw material can be processed to produce sausage

377

batters and how long after preparation a batter can be considered stable with respect to rheological properties. For this purpose, sausage batter rheological properties were assessed using an oscillation test which is very sensitive for the detection of structure changes resulting in changes to rheological properties. It was found that the raw material starts to change the properties of the batter 4 days after slaughter and a batch of sausage batter should be used not 45 min before and more than 4 h after finishing. Outside this timeframe spontaneous structural processes occur which influence the material properties. The impact of storage duration was found to depend on the ion concentration (NaCl and phosphate content), and in fact, a significant relation between batter firmness and salt concentration was found. Batters with reduced NaCl (0.5%) were found to be significantly firmer than conventional batter (with 2% NaCl). A possible explanation for this effect is the influence of the ionic strength, affecting protein solubility and firmness. A significant change of batter rheological behavior (firmness profiles) during storage and specifically to the different batter formulations was detected. An increase in firmness and network building capacity with increasing storage time was observed. After 4 h of storage an abrupt firmness increase for the low sodium gel (0.5% NaCl, 0.3% phosphate) was detected. Similar rheological behavior has been reported (Euring, Grupa, Bernhard, & Pietsch, 2009; Hammer, 2001). According to the firmness behavior during storage, work areas with a similar initial parameter (firmness) for each formulations were defined. Work areas were defined by analyzing the firmness behavior of eight different batches (n = 8). 2.2. Methods 2.2.1. Processing High-pressure processing of sausage batter was carried out in an industrial scale HP system (NC-Hyperbaric, Spain) which is able to realize a pressurization gradient of 2.5 MPa/s as well as in pilot equipment (Uhde, Germany) with a pressurization gradient of 40 MPa/s. NC-Hyperbaric equipment with a vessel volume of 55 L, allows pressurization of 350 kg/h. The maximum pressure of 600 MPa is built up by water as a pressure transmitting medium. The Uhde pilot system possesses a vessel volume of 3 L. Pressurization up to 700 MPa is effected by a plunger moving forward directly into the vessel decreasing the vessel volume. Water is also used as the pressure transmitting medium. Sausage batter samples were high-pressure treated in the range of 0.1–600 MPa and up to 60 °C initial temperature for different treatment times. Unpressurized samples were used as controls. 2.2.2. Temperature treatment Prior to the high pressure treatment batter samples were immersed in a thermostatic water bath (±0.1 °C) at 10–80 °C. Temperature was monitored at the center of the sample via a Type E thermocouple. The treatment was stopped when the meat center differed b 0.2 °C from the bath temperature (within 17 to 20 min). After reaching the initial temperature the samples were immersed in a Teflon liner filled with preheated water. After closing, the Teflon liner was placed in the high pressure vessel and the high pressure treatment was performed. 2.2.3. Process temperature Measuring of the temperature profile inside the vessel during high pressure processing with both equipments was not possible. Therefore the temperature parameter in this study is presented as initial temperature (IT) and calculated process temperature (PT). Process temperature was calculated based on experimental data reported in the literature. To reach a uniform and constant temperature during the holding time for high pressure treatment, a Teflon liner was used. Compression of water leads to adiabatic heating of 2 °C and 5 °C/100 MPa at low and high (T > 80 °C) initial temperatures,

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respectively (Buckow, 2006). Adiabatic heating of ~ 3 °C for water, ~ 3.2 °C for salmon, ~ 4.5 for chicken fat, ~ 6.3 for beef fat, ~ 8.7 for olive oil, and ~ 9.1 for soy oil per 100 MPa compression at 25 °C initial temperature is reported (Ting, Balasubramaniam, & Raghubeer, 2002). Process temperature of the frankfurter batter was calculated based on the dependence observed by Maslak (TU-München, Germany, personal communication, 2010). Adiabatic heating in the sausage matrix (white sausage, Bavarian type) was studied by Maslak in 2007 at the Berlin University of Technology. Temperature profiles in white Bavarian sausage (in the sausage core) and in water (pressure transmitting medium) during HPP at different initial temperatures were measured. He found an adiabatic heating dependence, similar to that reported by Buckow (2006), namely that the temperature increase due to adiabatic heating is higher when HPP is performed at higher initial temperatures. Adiabatic heating of 3 °C/100 MPa for 5 °C initial temperature, 3.33 °C/100 MPa for 15 °C initial temperature and 3.48 °C/100 MPa for 20 °C initial temperature was estimated. According to Maslak's measurements, the adiabatic heating was dependent on the initial temperature, with an increase of about 0.33 °C per 10 °C increase in initial temperature. Therefore a batter sample with an initial temperature of 50 °C heats due to adiabatic heating of 4.5 °C/100 MPa, a process temperature of 77 °C on reaching 600 MPa. 2.2.4. Analytical methods 2.2.4.1. Batter network firmness. The oscillatory test is an appropriate non-destructive technique to the characterize firmness of sausage batter structures. Storage modulus, G′, represents the strength which is necessary to reversibly stretch internal structural elements (F). The storage modulus-frequency function was measured (AR 2000, TA Instruments) using parallel-plate geometry, which can be fitted to an empirical power law function. The visco-elastic behavior of the model (frankfurters) batter corresponds to the Maxwell model, which contains a Hookean spring in series with a Newtonian dashpot. Changes of rheological properties are results of structure modifications and/or changes of the physico-chemical state of the internal network. The storage modulus/frequency function was evaluated according to the power law, where the coefficients are used to characterize changes of rheological properties. G′ ¼ k⋅f

n

ð1Þ

where G′ is the storage modulus, f is the frequency, and k and n are empirical parameters. The parameter k corresponds to the firmness of the visco-elastic material while n represents the viscous component in the undestroyed structure. 2.2.4.2. Batter network structure. Sausage batter structure was investigated by Scanning Electron Microscopy (SEM). Volume elements of edge length of 1.5 mm were frozen in super-cooled liquid N2 and broken. Free water was removed by sublimation. Sample surfaces were sputtered with gold and images were obtained by cryo-SEM (JEOL JSM-6460, Japan) at − 180 °C. 2.2.4.3. Water holding capacity. The ability of sausage to retain water (sometimes called serum) was determined on the basis of the amount of drip loss resulting from samples of 1 g placed between double layers of filter paper when treated at 19.62 kPa for 5 min (Hamm, 1972). The percentage drip losses were calculated according to the equation Drip loss ð% Þ ¼

W 1 −W 2 100: W1

ð2Þ

2.2.4.4. Qualitative and quantitative analysis of batter protein solubility 2.2.4.5. Preparation of the soluble proteins. Sausage batter (28 g) was diluted with bi-distilled H2O (5 mL and 15 mL) in order to achieve a total protein content of 1%–5% (w/w). After preparation, the batter dispersion was allowed to equilibrate under mild agitation for 1 h at 20 °C. After equilibration, samples were centrifuged at 18,000 g for 20 min at 10 °C (Sorvall Ultra centrifuge; OTD Combi, Du Pont Company, Wilmington Delaware, USA). Up to three distinct phases could be recovered after centrifugation. The aqueous phase was carefully removed, chilled, and stored for SDS-PAGE and HPLC analyses. 2.2.4.6. HPLC analysis. High performance liquid chromatography HPLC analysis was performed using a Waters 2695 Alliance Separations Module equipped with a Waters 2996 photodiode array detector (WATERS, Milford MA, USA). The protein content was determined by Size-Exclusion HPLC. An isocratic flow with aqueous phosphate buffer (0.15 M, pH 6.8) was used. Analyses were carried out at room temperature at a flow rate of 0.5 mL/min using a Superdex 200 10/300 column (10 ∗ 300 mm, 13 μ, GE HEALTHCARE, Munich, Germany). Peak detection was performed using UV at 214 nm. 2.2.4.7. SDS-PAGE analysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to qualify soluble proteins. Commercially available gradient gels (4 to 20%) were used for all experiments (BioRad, München, Germany). The BioRad Mini Protean electrophoresis cell (BioRad, München, Germany) at voltages of 100 mA and 60 mA was used. A buffer solution of 12.1 g Tris, 7.5 g glycine, 1 g SDS per 1000 mL bi-distilled H2O was used. Samples were pre-treated according to the Laemmli method (Laemmli, 1970). The gels were stained with 80 mL Coomassie blue-R350 and distained in a 200 mL solution containing methanol/acetic acid/bi-distilled H2O (30:10:60, v/v/v). The molecular weight of each band was estimated by comparing the migration distance with that obtained for proteins of known molecular weight; a SigmaMarker™ Wide Molecular Weight Range Standard (MW 6500-205,000 D Sigma-Aldrich, Steinheim, Germany) was run alongside the other samples. After fixation and drying, each gel was scanned and analyzed with image-analysis software (UN-SCAN-IT gel, Version 6.1, Gel and Graph Digitizing Software, Silk Scientific Inc., Utah, USA). 2.2.4.8. Statistical analysis. The data were analyzed using Origin 7.0 (OriginLab, Northampton, MA, USA). Statistical analysis was performed by analysis of variance (ANOVA) and Tukey test. The results are expressed as mean ± SD, and difference was considered to be statistically significant at P b 0.05. 3. Results and discussion 3.1. Rheological characterization of pressure and heat induced structure formation according to the salt content An investigation of the effect of pressurization gradient (PG) on gel firmness of batters with diverse ion concentrations (combinations of NaCl and phosphate) was performed. Pressure levels at 300 and 600 MPa at 10 °C, 30 °C and 40 °C (IT) and a holding time of 240 s were used. Pressurization was carried out at low PG—2.5 MPa/s and high PG—40 MPa/s. To determine the high pressure effect, an initial temperature of 10 °C (IT) was used. The combined pressure/temperature treatments were carried out at 30 °C and 40 °C (IT). As expected, batter firmness increased with increasing pressure, additionally firmness also increased with increasing initial temperature. Maximum batter firmness for all formulations combinations was at 600 MPa and 40 °C IT (Fig. 1, Table 1). Temperature was observed to amplify the pressure induced effects.

0.28bcdf 0.26abf 0.87ab 0.21cd 1.78f 116bcdf ± ± ± ± ± ± 9.71 10.52 10.71 9.23 7.95 9.42 2.97a 2.9a 1.06a 0.09a 0.78a 1.71a ± ± ± ± ± ± 18.18 17.87 18.71 20.32 20.77 21.22 1.19bef 0.5adfg 0.31a 1.17bd 0.48bg 0.84bd ± ± ± ± ± ± 10.38 13.31 16.09 11.45 12.1 11.25 0.57c 1.25d 1.04ce 0.24cd 0.21de 0.39cd ± ± ± ± ± ± 14.23 11.6 13.24 12.83 11.81 12.54 0.43ab 0.96ab 0.86abd 0.31ab 0.4bcde 4.77cdeg ± ± ± ± ± ± 8.68 8.94 13.02 9.66 18.70 24.14 0.58ef 0.1g 0.89df 1.1gh 2.00 2.71h ± ± ± ± ± ± 35.87 51.81 41.75 51.89 62.76 47.01 0.02bch 0.11dfh 0.03ei 0.210ei 0.92g 0.89 ± ± ± ± ± ± All values are means ± SD (n = 3). Means followed by the same letters within each column are significantly different, P ≤ 0.05.

6.33 7.58 11.74 11.95 18.00 22.44 0.34b 0.02ceg 0.01dg 0.14cg 0.95fh 0.91h ± ± ± ± ± ± 19.6 41.5 52.5 29.3 53 65 PG = 40 MPa/s 300 10 300 30 300 40 600 10 600 30 600 40

4.85 7.82 9.46 8.81 13.35 14.05

0.73a 0.86ab 1.68ac 0.65ad 1.31ad 0.43e 0.3ae ± ± ± ± ± ± ± 12.56 11.60 11.37 10.78 10.34 14.24 13.73 0.5a 0.57b 0.63b 0.08b 0.98bc 0.77b 0.36c ± ± ± ± ± ± ± 21.16 11.21 12.42 11.49 9.8 12.49 8 1.57a 0.28a 0.17bc 1.03bd 0.74cde 0.43be 0.53 ± ± ± ± ± ± ± 15.03 13.46 11.9 11.56 9.52 10.93 5.9 0.74a 1.03b 0.85c 0.65ac 0.01ab 0.92d 0.77 ± ± ± ± ± ± ± 16.89 18.6 13.89 14.83 17.9 10.42 6.9 0.3a 0.1ab 0.34c 0.56bcd 0.43ce 2.7f 2.01fg ± ± ± ± ± ± ± 11.25 13.51 20.28 18.28 20.35 29.46 28.80 0.73a 0.32b 1.9c 0.61c 1.17c 0.99d 1.48e ± ± ± ± ± ± ± 11.75 14.84 21.00 22.84 24.25 40.89 30.71 0.04a 0.43b 0.49bc 0.03d 0.8de 0.14def 0.12g ± ± ± ± ± ± ± 2.33 5.13 5.39 8.80 10.18 8.44 16.85 0.06a 0.04b 0.00c 0.15cd 0.10be 0.14f 1.15 ± ± ± ± ± ± ± 1.53 4.10 7.30 8.62 5.90 11.71 19.63 19.6 41.5 52.5 29.3 53 65

Drip loss (%)

2% NaCl 0.3% phosphate 1% NaCl 0.3% phosphate

Calculated process temperature PT (°C)

379

PG = 2.5 MPa/s 0.1 10 300 10 300 30 300 40 600 10 600 30 600 40

Water holding capacity was characterized by the drip loss parameter for the pressure levels of 300 MPa and 600 MPa and initial temperatures up to 40 °C (Fig. 2, Table 1). A decrease of drip loss with increasing pressure and temperature was detected, with a minimum observed at 600 MPa and 40 °C IT. After HP treatment at low PG a positive effect on drip loss reduction for all formulations was seen. The improvement of the WHC at low PG was more marked for the reduced NaCl formulation (0.5% NaCl) probably as a result of some structural changes provoked through low PG. This improvement correlated with the hardness decrease discussed in the previous section.

Initial temperature IT (°C)

3.2. High hydrostatic pressure/temperature impact on the water holding capacity depending on salt ion content

Pressure (MPa)

The PG effect on batter firmness at low (PG = 2.5 MPa/s) and high (PG = 40 MPa/s) pressurization rates was compared (Fig. 1, Table 1). Batters with 2% NaCl, 0.3% phosphate in their formulations at low PG were significantly firmer at 600 MPa and 40 °C than those at high PG. Conversely, batters with reduced 0.5% NaCl were significantly firmer at high PG for all pressure/temperature combinations, with a maximum at 600 MPa and 30 °C. The effect of PG was most clearly seen in the reduced 0.5% NaCl content batter, where the in-salting effect did not play a major role in the batter functional properties as that of HP and PG. The hardness for this formulation was significantly reduced at low PG but was still significantly firmer than the formulations with higher NaCl content. The difference between batter hardness of 2% and 1% NaCl at high PG was more significant for the treatments at high PG and high initial temperature (30 °C and 40 °C) than that at low PG.

Table 1 Influence of salt level and pressure/temperature application on firmness (k) and drip loss of frankfurter batter.

Fig. 1. Gel firmness of frankfurter pork batters for different NaCl contents as a function of temperature after pressurizing at a) PG = 2.5 MPa/s and b) PG = 40 MPa/s to the desired set pressure for a 240 s holding time. The temperature parameter is presented as initial (x-axis) and process temperature.

0.5% NaCl 0.3% phosphate

1% NaCl 0.15% phosphate Firmness parameter k (kPa)

2% NaCl 0.3% phosphate

1% NaCl 0.3% phosphate

0.5% NaCl 0.3% phosphate

1% NaCl 0.15% phosphate

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Fig. 2. Influence of reduced NaCl on WHC expressed as drip loss by combined HP–T treatment, plotted as a function of temperature after pressurizing at a) PG = 2.5 MPa/s and b) PG = 40 MPa/s to the desired set pressure for a 240 s holding time. The temperature parameter is presented as initial (x-axis) and process temperature.

Drip losses of the 0.5 NaCl batter were not significantly higher for most treatments compared to the formulations with higher NaCl contents (2% and 1%) at low PG. In contrast drip losses in the batters treated at high PG were significantly higher than those in all other formulations. 3.3. Effect of combined high pressure/temperature treatment, PG and salt content on batter structure visualized by SEM To investigate the mechanism of structure modifications of batters induced by pressure, temperature, PG and salt content, scanning electron micrographs were taken (Fig. 3). Two control samples (untreated batter), one with 2% NaCl and 0.3% phosphate, and the other with 0.5% NaCl and 0.3% phosphate, were compared to the pressure treated samples. SEM-micrographs of two pressure/temperature treatments were performed according to the maximum impact on the firmness and drip loss (Section 3.2), namely the pressure/temperature treatments at 300 MPa and 10 °C (low P–T-impact) and 600 MPa/40 °C (high P–T-impact). Micrographs of samples treated with both pressurizing gradients were taken, with PG = 40 shown in the first and third columns of Fig. 3, and PG = 2.5 in the second and fourth columns. Compared to the control samples of the 0.5% and 2% NaCl batters in Fig. 3a and b, some differences in the protein matrix were observed. The matrix of the reduced-NaCl batter was found to be rougher than that of the conventional batter. Furthermore, the batter network contained a lower number of gaps (expressing sublimated water) and higher structure density. However the matrix of the conventional salted batter (2% NaCl) exhibited a good structure, where more gaps were homogeneously arranged (Fig. 3b). Swelling of the batter matrix was observed

at 300 MPa/10 °C IT for the 240 s treatment time (Fig. 3c–f). However the matrix treated at low PG was more swollen (Fig. 3d, f) compared to the matrix treated at high PG (Fig. 3c, e). The improvement of the matrix swelling of the conventional formulation (2% NaCl) (Fig. 3e, f) was thought to be a result from higher amounts of immobilized water (see the gap in Fig. 3l) compared to the batter with reduced NaCl content (0.5% NaCl) (Fig. 3c, d). This phenomenon can be explained by the higher immobilizing properties of the protein matrix caused by the combined salt and pressure impact. The SEM-observed structure modifications correspond and explain the behavior of the firmness and drip loss parameters, such as how the functional properties of the 0.5% NaCl batter at low PG was significantly improved after HP–T treatment. The process of gelation was clearly visible at 600 MPa/40 °C IT for the 240 s treatment (Fig. 3g–j). The formation of a secondary network parallel to the main matrix was observed (Fig. 3j). This was composed of a network of fine strands, thought to be a result of networking of small aggregate conformations. Therefore the low PG impact was expressed in a noticeably extended secondary network, which was not visible in batters treated with high PG. This overlay network surface extension enhances the possibility for more water molecules to be immobilized in the protein network and consequently for more protein/water interactions to occur. This parallel network is suggested to be formed by soluble proteins, which migrated in the aqueous phase due to the combined salt/pressure effect. The denaturation of the soluble proteins in the aqueous phase resulting in a 3-D structure has often been reported in the literature concerning HPP (Chapleau, Mangavel, Compoint, & Lamballerie-Anton, 2003). Formation of a secondary network was not found in the batter matrix treated with PG of 40 MPa/s (Fig. 3g), which was rougher compared to the low-PG matrix for the same salt content (Fig. 3h). These PG-dependent observations correlate with the significant firmness and WHC differences discussed in Sections 3.1 and 3.2. In order to improve structure firmness and WHC, combined P–T treatments at 600 MPa and higher initial temperatures in the range of 40 °C, 50 °C and 60 °C for 4 min were performed and compared to the conventional thermally treated batter. Simulating industrial conditions of sausage production, the industrial NC-Hyperbaric (Burgos, Spain) equipment with low PG of 2.5 MPa/s was used. As shown in Fig. 4 the temperature increase was found to have a great impact on the batter matrix. After treatment at 600 MPa/ 40 °C/ min, the parallel secondary matrix was significantly swollen. Furthermore changes in the batter matrix continued at temperatures above 50 °C IT. The structure of batter treated with 50 °C IT was found to be comparable to the conventional (thermal) batter structure. This observation can be explained by the similar process temperature of 77 °C in the batter matrix during HHP/T treatment. However, the pressure treated matrix was a little more voluminous than the thermally treated one. Moreover, the batter matrix at 60 °C IT was significantly more voluminous compared to the thermally treated batter. The thickness (swollenness) of the gels indicates WHC improvement, resulting in a smoother and more elastic structure compared to the thermally treated one (Cheftel & Culioli, 1997). Based on this result, it could be concluded that significant gelation improvement would occur with increasing temperature at higher pressure levels. Besides the WHC improvement, a firmness increase was also seen due to increasing temperature (Sections 3.1 and 3.2), possibly caused by increased protein/protein interactions and formation of a secondary matrix surface. High pressure processing with moderate or high IT due to adiabatic heating can affect the gelation and denaturation processes. However the pressure/temperature treated gels differ to the thermally treated ones. Adiabatic heating has the ability to increase uniform temperature in the whole batter matrix in a short treatment time. This feature of the high pressure processing could be used industrially for modifying batter structures at higher temperatures in a very short processing time.

F. Tintchev et al. / Meat Science 94 (2013) 376–387

381

IW

a PG= 40 MPa/s

300 MPa/ 10o C IT 19.6o C PT

PG= 2.5 MPa/s

c

d

g

h

PG= 2.5 MPa/s

PG= 40 MPa/s

e

f

Solubilization increase

Max.

Gelation

600 MPa/ 40°C IT 65°C PT

b

Denaturation

Control

FD

Solubilization increase

2% NaCl 0.3% phosphate

0.5% NaCl 0.3% phosphate

j

i Induction of secondary matrix

Gelation PG impact Fig. 3. SEM micrographs of frankfurter batter structure of control samples at 0.5% NaCl, 0.3 phosphate (a); and 2% NaCl, 0.3 phosphate (b); and treated at two pressure–temperature combinations 300 MPa/10 °C IT (c–f), and 600 MPa/40 °C IT (g–j) for 240 s. Impact of PG = 40 MPa/s is shown in rows I and III, and 2.5 MPa/s in rows II and IV. Micrographs are presented at 5 μm bar of length magnification. The initial temperatures of 10 °C and 40 °C correspond to calculated process temperatures of 19.6 °C and 65 °C. FD—fat droplets. IW—immobilized water.

3.4. Definition of solubilization and denaturation pressure treatment intervals; comparison to thermal treatment studied by SDS-PAGE In order to define solubilization and denaturation levels of frankfurter batters, pressure/temperature (100–600 MPa/10 °C IT) and thermal (55 °C–75 °C) treatments for 5 min holding time were performed (Fig. 5). Solubility and denaturation were analyzed by SDS-PAGE which detected proteins with molecular weights in the range of 6.5– 205 kDa. The band densities after HPP were found to be more intensive compared to those after thermal treatment, indicating a significant increase of soluble protein fraction (Fig. 5). The most important protein for batter matrix formation is myosin. Myosin can also be split into two functional fragments, light meromyosin with molecular weight of 150 kDa and heavy meromyosin with MW of 350 kDa. Furthermore, meromyosin can be split into S-1 (115 kDa) (sub-fragment 1) and S-2 (60 kDa) (sub-fragment 2-S-2). S-1 is especially important for the functionality of myosin in processed meat products due to its excellent binding capacity (Borejdo, 2002; Borejdo & Assulin, 1980). It is composed of three domains with molecular weights of 27 kDa (N-terminal), 50 kDa and 20 kDa (C-terminal) (Iwasaki, Yamamoto, & Rikimaru, 2002). Maximum solubilization of both S-1 domains with molecular weight of 50 kDa and at 27 kDa, as well as S-2, was found at 200–300 MPa (Fig. 6). Similar solubilization behavior

with a maximum at 200 MPa was detected for actin (43 kDa). Pressures above 300 MPa led to a density decrease, indicating denaturation. A significant effect on protein solubilization was observed after HPP at the lowest pressure level (100 MPa) even at low temperature and short treatment time, compared to the control sample. No myosin heavy chains were found in the supernatant after any of the treatments. Therefore, it was concluded that mechanical (cutting, grinding) and chemical (in-salting) impacts damage myosin heavy chains during batter preparation. To isolate the pressure effect on the solubilization, measurements at an initial temperature of 10 °C for 5 min are presented. A clear tendency of increasing solubilization of proteins with increasing pressure and temperature was observed (Fig. 6B, D). The solubilization maximum for tropomyosin, S-2, MLC-1, and actin was found after HPP at 300 MPa. However, the maximum solubilization for the MLC-2, N-terminal, and C-terminals at 50 kDa and 20 kDa was at 200 MPa. The pressure/temperature effect on the solubilization process of reduced NaCl batters (1% NaCl, 0.3 phosphate) is presented in Fig. 6C, D. Generally the band profiles of this formulation were identical to the conventional ones, but their densities were lower compared to those for 2% NaCl. However in all treatment combinations, solubilization increases with increasing pressure and temperature as found in the 2% NaCl-batter.

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600 MPa/40°C IT/4 min 65°C PT

Control

600 MPa/ 50°C IT/ 4 min 77°C PT

conventional thermal 600 MPa/60°C IT/4 min treatments (76°C/ 30 min) 88.8°C PT Fig. 4. SEM micrographs of frankfurter batter structure (2% NaCl, 0.3 phosphate) treated at 600 MPa/40 °C/4 min, 50 °C, 60 °C IT and PG = 2.5 MPa/s in comparison to control and thermally treated samples.

3.5. Qualitative and quantitative analysis of batter protein solubility studied by HPLC analysis Quantitative and qualitative analysis of the batter aqueous phase using SDS-PAGE analysis was limited to the molecular weight range of 6.4 kDa–205 kDa. Therefore HPLC analysis was performed to observe protein changes outside this range. It is difficult to identify particular proteins using HPLC. However it is useful for obtaining information about the changes of protein sub-fragments smaller than 6.4 kDa, which result after tissue disintegration and pressure-induced protein damage. Another parameter obtained with this method is the aggregation

Tropomyosin Myosin heavy chains

range, where the soluble proteins, their sub-fragments, and subunits start to aggregate. HPLC profiles of the soluble protein fraction, after high pressure processing (100–600 MPa) at 40 °C IT and 5 min treatment time are shown in Fig. 7. A clear trend in the behavior of the protein units and subunits was observed. After pressure treatment at 100 MPa, a large number of subunits were observed below 6.4 kDa, which were undetectable by SDS-PAGE. In this range the total content of protein subunits reached its maximum at 200 MPa. Therefore, this pressure level was defined as the maximum solubilization level, where the protein molecules were maximally damaged (solubilized

Solubilization maximum of myosin components

Denauration increase of myosin compnents and actin

MW (kDa)

α- actinin

205 116 97 66 55

S2

45

50 kDa

36

Actin

Control 55°C 65°C 70°C 75°C

100

29 24 20 14,2 6,5

200

300

Troponin T Tropomyosin

MLC Tr.-C MLC 2

N- terminal

400

500 600 MPa / 10°C IT for 5 min treatment

C- terminal

Fig. 5. SDS-PAGE pattern of solubilized fraction (diluted with 5 mL bi-distilled H2O) pressure and temperature treated sausage batter after 5 min treatment time (2% NaCl, 0.3 phosphate). Temperature is presented as IT. The process temperature PT for the different pressure levels are: 100 MPa/13.2 °C PT; 200 MPa/16.4 °C PT; 300 MPa/19.6 °C PT; 400 MPa/22.8 °C PT; 500 MPa/26.04 °C PT; and 600 MPa/29.3 °C PT.

F. Tintchev et al. / Meat Science 94 (2013) 376–387

MW (kDa)

MW (kDa)

2% NaCl, 0.3 Phosphate

100 MPa, 10°C (13.2°C PT) 200 MPa, 10°C (16.4°C PT) 300 MPa, 10°C (19.6°C PT) Control

205 116 97

S-2

66 55 45 36 29 24 20 14.5 6.5

50 kDa Actin TN-T TM N. ter.-

MLC- 1 C-ter MLC-2

2 % NaCl, 0.3 Phosphate

205 116 97 66 55 45 36 29 24 20 14.5 6.5

S-2 Actin TM N- ter. MLC- 1 C- ter. MLC- 2

MPa

100

MPa MPa

10°C IT/ 5min

Control 10°C 10°C 10°C 5 min 5 min 5min

a

MW (kDa)

Control 100 MPa, 10°C, (13.2°C PT) 200 MPa, 10°C, (16.4°C PT) 300 MPa, 10°C, (19.6°C PT)

116 97

Control 40°C 40°C 40°C 5 min 5min 5min

40°C IT/ 5 min

205

Control 100 MPa, 40°C, (44.2°C PT) 200 MPa, 40°C, (48.4°C PT) 300 MPa, 40°C, (52.5°C PT)

116 97

S-2

S-2

66 55 45

66 55 45 36 29 24

50 kDa Actin TM

36 29 24 20

N- ter.

50 kDa Actin TM N-ter. MLC- 1

MLC- 1

14.5 6.5

C- ter. 100

Marker

200

5 min 5 min 5min

C- ter. 100

300

Marker

MPa MPa MPa Control 10°C 10°C 10°C

MLC- 2

20 14.5 6.5

MLC- 2

c

300

MPa MPa MPa

Marker

b

200

MW (kDa) 1 % NaCl, 0.3 Phosphate

1 % NaCl, 0.3 Phosphate

205

100 MPa, 40°C, (44.2°C PT) 200 MPa, 40°C, (48.4°C PT) 300 MPa, 40°C, (52.5°C PT) Control

50 kDa

100 200 300 Marker

383

10°C IT/ 5 min

d

200 300

MPa MPa MPa Control

40°C 40°C 40°C 5min 5min 5min

40°C IT/ 5min

Fig. 6. A) SDS-PAGE pattern and quantification surface peaks of solubilized fraction (diluted with 15 mL bi-distilled H2O) depending on pressure increase at a) 10 °C and B) 40 °C IT, for 5 min holding time (2% NaCl, 0.3 phosphate) and at C) 0 °C and D) 40 °C IT for 5 min holding time (1% NaCl, 0.3 phosphate).

or disrupted). This was confirmed by observations of the peaks at the next pressure level—300 MPa, where the amount of small soluble protein sub-fragments (under 6.4 kDa) was significantly reduced. Furthermore, this reduction was combined with an increase of protein fragmentation, detected as a shift in the protein peaks to higher molecular weights (above 240 kDa). This led to the conclusion that at 300 MPa, growed aggregations of small molecular protein sub-fragments in to bigger conformations. These observations correlate with the results reported by Yamamoto et al. (1993). They found an aggregation process at 210 MPa. However they did not perform tests at higher pressure levels. 3.6. Drip loss P–T landscapes of frankfurter batter of conventional formulation (2% NaCl; 0.3 phosphate) depending on PG In order to obtain the impact of PG on drip loss, P–T models were obtained by empirically fitting of the Cosine series bivariate order 4 equation (Table Curve 3D v3 Statistical Package, Systat Software Inc., Richmond, CA, USA). Frankfurter batters have been HP– temperature treated with low and high PG for the whole range up to 600 MPa in process temperature combinations up to 60 °C for 300 s (Fig. 8). The significant improvement of WHC (drip loss reduction) was clearly visible for all low PG-treatment combinations. Its maximum was between 100 and 200 MPa for temperatures higher than 40 °C for both PG parameters. This phenomenon can be explained by the maximum meat protein solubilization (according

to the previous sections), which at higher temperatures results in improvement of the batter matrix and WHC. In this case the gel formation was dominated by the temperature. Similar rheological properties but dominated by the pressure were seen above 400 MPa at lower temperatures (0–40 °C PT). The comparison of the PG effect of drip loss showed a significant improvement for the batters treated at low PG, especially at the higher pressures. These observations lead to the conclusion that PG-parameter is of great importance for modifying batter properties. According to the results discussed in Sections 3.3, 3.4, and 3.5 this positive effect is based on the longer processing time between 100 and 300 MPa during the pressurizing process, where more proteins have the possibility to migrate into the liquid phase, affecting positively the protein matrix as the pressure reaches higher levels. The effects over the whole range of the process parameters were required to determine the optimal batter structure. Therefore, according to the results in the current work, as well to other reports (Yamamoto et al., 1993, Cheftel & Culioli, 1997, Chapleau et al., 2003, Iwasaki & Yamamoto, 2003, Jimenez Colmenero, 2002), a hypothetical mechanism of water binding and solubilization caused by water– protein interactions was developed. This was based on the ion-binding mechanism of Honikel (1986), combining the effect of HPP and salts (Fig. 9). By the conventional “in salting” mechanism, water binding and solubilization are positively affected through the ionic strength increase. HPP seems to induce synergistically the

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SDS- PAGE area

12.270

6.5 kDa

20.00

25.00

45.391

40.517

42.403

39.953

35.00

40.00

45.00

50.00

45.282

46.974

32.990 33.211

29.823

23.963 25.185 25.857

42.170

0.10

40.723

17.356

0.20

19.606 20.467

15.025

AU

0.30

60.00

100 MPa/ 40 °C IT 44.2 °C PT

0.50 0.40

55.00

Minutes 38.532

0.60

35.171 36.231

30.00

0.00 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

55.00

60.00

Minutes 44.382

56.633

54.265

29.068

34.047

48.536

52.066

0.05

46.909

0.10

200 MPa/ 40 °C IT 48-3°C PT

37.763 38.373

0.15

27.522

0.20

23.585

0.25

20.149

AU

0.30

17.484

0.35

16.176

0.40

Aggrigation increase

0.45

32.078

0.50

Max

Denaturation increase

15.070

Increase of small molecular weight fractions

10.006

49.057

5.00

27.917

0.00

28.355

25.104

20.955

23.455

33.344

Control

16.385

AU

205 kDa 0.65 0.60 0.55 0.50 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00

0.00 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

55.00

60.00

300 MPa/ 40 °C IT 52.51°C PT

0.20

42.082

35.134

28.105

0.30

20.009

16.451

11.457 11.871

0.40

AU

21.112

0.50

39.458

0.60

32.793

23.390

Minutes

0.10 0.00 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

50.00

45.00

55.00

60.00

Minutes 32.686

0.60 0.50

41.242 42.538

0.10

39.404

35.411

25.167

11.907 12.288

0.20

23.217

0.30

21.025

16.202

AU

0.40

28.124

600 MPa/ 40 °C IT 65°C PT

Max

0.00 0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00

55.00

60.00

Minutes

Fig. 7. HPLC profiles of batter soluble proteins after different high pressure treatments at 40 °C initial temperature and 5 min treatment.

water–protein interactions. It is proposed that the HPP-salt impact depends on a combination of processes occurring during HPP, as described below.

The process of myosin denaturation started at about 100 MPa and finished at about 300 MPa (Section 3.4). The myosin unfolding results in quaternary and tertiary structure disruption as well as

F. Tintchev et al. / Meat Science 94 (2013) 376–387

a

385

b

PG Impact

Fig. 8. Drip loss P–T landscapes of frankfurter batters according to the conventional formulation (2% NaCl, 0.3% phosphate) depending on PG (PG = 40 MPa/s − a(R2 = 0.90); PG =2.5 MPa/s − d(R2 = 0.64)) and treatment time of 300 s. Calculated process temperature was used in preparing P–T landscapes.

small changes in the secondary structure (Chapleau et al., 2003). Therefore, an increase of free side chains (more charged groups) occurs, leading to intensification of water–protein interactions and improved binding. Opening of the protein molecule provoked by the repulsing effect of phosphates is proposed to be promoted through penetration, by adding more water (−ΔV) into the same volume during pressurization. The level of molecule opening is assumed to be pressure dependent, as shown in Fig. 9. Parallel to the HP-induced process of myosin denaturation, dissociation of the actomyosin complex occurs. The process of actomyosin complex dissociation is caused by phosphate in the conventional batter preparation. The process of ionic strength increase, caused by water dissociation during HPP, could additionally enhance the solubilization effect on the meat proteins. As a result, hydrogen and hydroxide ions can be attracted to the protein charged groups (Fig. 9). HPP induced molecule disruption of meat proteins, and particularly of myosin, observed by SDS-PAGE and HPLC played a key role affecting the protein solubilization and increasing the amount of protein (especially

the amount of sub-fragments and subunits from the primary meat proteins) in the aqueous phase. The present study has shown the dependence of the pork batter functional properties on PG during the high pressure processing. A relationship between process parameters, salt content and functional properties was investigated. It was found that protein solubilization, in particular myosin and myosin sub-fragments was additionally induced by high pressure–temperature processing. The changes in the myosin molecules, occurring in the treated batter could be summarized in the following hypothetical mechanism (Fig. 10). After chemical, mechanical and pressure/temperature strain during processing no intact myosin molecules were found in the supernatant. Instead a disruption of the myosin molecules to myosin fragments was detected. The amount of three S-1 myosin domains (N-terminal and C-terminal—50 kDa and 20 kDa), the regulatory and essential light chains, as well as actin, increased. This maximum was in the range of 150–250 MPa. Due to the high hydrophobic character of the myosin head domains, agglomeration begins to occur. A transformation of the protein sub-fragments to a more stable energy level, known as hydrophobic packing occurs

Cl

+

Cl +

-

2 H2O ⇔ H3O + + OH −

+

H3 0+

H3 0+

-

-

+

+ Na Cl

HPP

Increase of intermolecular space after HPP

+

-

Intermolecular space before HPP

H30+

OH-

Disruption of the meat protein molecule

+ Cl

- Na + OH-

+

+ Na +

-+ Cl

Fig. 9. Effect of high pressure and salts (NaCl and PP—polyphosphate) on the mechanism of water binding of meat proteins.

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Daisy -wheel agglomerate

HPP (100-300 MPa) NaCl, Phosphate Grinding, Chopping

S-2

S-2

Myosin molecule disintegration

Hydrophobic packing

Further pressure increase 350-600 MPa

- 50 kDa Domain - N-terminal - Actin - C-terminal - Regulatory light chains - Essential light chains - Protein fragments smaller than 6 kDa (not defined)

Increase of swelling Gel formation Fig. 10. Hypothetical mechanism of secondary structure formation during high pressure–temperature treatment based on Yamamoto's mechanism of hydrophobic packing and formation of daisy-wheel conformations.

caused by the pressure-induced volume decrease in this pressure range. The aggregation mechanism is supposed to be similar to the daisy-wheel formation of Yamamoto et al. (1993). The difference to Yamamoto's mechanism was based on the fact that agglomerations of smaller protein (S-1 sub-fragments) proceeded, as the integrity of the myosin molecule was significantly disrupted. These aggregations, caused mostly by hydrophobic interactions, could be the first stage of the secondary matrix network formation. The regulatory and the essential light chains as well as actin, S-2 sub-fragment and other unidentified proteins participate in the further formation of that matrix. Actin is also supposed to affect the gel properties as it is responsible for the viscous element of the system. The exact configuration of these aggregates and the detailed protein–protein interactions are unknown. With increasing pressure the agglomerations developed themselves (induced from the swelling increase) in a protein network, which under certain conditions form gel structures. Pressures above 400 MPa lead to more unfolded structures due to breaking of hydrophobic interactions. This is in agreement with observations of Chapleau et al. (2003) showing this starts at pressures above 300 MPa. The resulting structure is more spread (efficient packing), so that more water may be immobilized by capillary forces. According to Iwasaki and Yamamoto (2003) pressure induced gelation of myosin occurs only by head to head interactions. Tail-to-tail interactions, occurring in heat-induced gelation are not involved. However their suggestions were made by experimentation at pressures up to 400 MPa. Therefore tail-to-tail interactions as well as tail entangling at higher pressures, and especially at higher temperatures (above 40 °C IT), should not be excluded. The processes of batter protein solubilization, aggregation, denaturation and gel formation after 240 s (industrial relevant preservation treatment time) can be summarized in a P–T-diagram (Fig. 11). The range of myosin solubilization (or molecule disruption) can be schematically defined to occur in the range of 0–300 MPa (Sections 3.4 and 3.5) with a maximum at 200 MPa/40 °C IT. The solubilization range corresponds to the myosin denaturation area of 0–200 MPa (Section 3.4). After disruption of the myosin molecule due to denaturation, solubilization or disintegration the aggregation process started at about 300 MPa (Sections 3.4 and 3.5). This correlates with the results of Iwasaki et al. (2002). However the aggregation process continues up to higher pressure levels, where gelation begins.

This, according to the results discussed in Sections 3.3 and 3.4, started at pressures above 400 MPa. Gel formation is possible at lower pressures and higher temperatures (above 60 °C). According to the P–T landscapes of WHC pressure induced gels with good functional properties can be obtained at low pressures and higher temperatures (above 60 °C PT). Meat protein denaturation was found to increase linearly with increasing pressure and temperature, resulting in a firmness increase (Section 3). Pressure/temperature formation of frankfurter sausages after 240 s holding time with improved functional properties and with microorganism inactivation can be defined to be above 500 MPa and initial temperatures above 40 °C. These processing parameters could be industrially relevant. The processes of solubilization, aggregation and gelation are more developed, resulting in improved sausage structure, at low PG. Therefore a slow compression rate (low PG) should be preferred for industrial applications. Based on the results and the mechanisms of solubilization, aggregation and water binding a comparison of the different batter formulations under HP–T treatment can be made.

Fig. 11. Hypothetic P–T ranges of myosin solubilization, aggregation and gelation after HPP of 240 s, summarizing this work and other reports.

F. Tintchev et al. / Meat Science 94 (2013) 376–387

The difference between the formulations with 2% and 1% NaCl and 0.3% phosphate was found to be minimal as no significant difference, in the functional properties for the most of the treatments, especially at low PG was found. Batters with reduced salt content (1%, 0.5% and 1.5% NaCl) were found to be firmer and maximal for the 0.5% NaCl formulation, compared to the conventional formulation. Batter firmness correlated with poor WHC. PG impact on the firmness reduction and increase of WHC can be related to the additional soluble proteins in the batter matrix. Therefore the in-salting effect can be significantly compensated for through HPP (Fig. 9). According to the SDS-PAGE analysis HP–T treatment caused an increase in the myosin head sub-fragments. These are known to be very hydrophobic. Hydrophobic interactions are very significant in food products in relation to structure and function (Skaara & Regenstein, 1990). A high correlation between the surface hydrophobicity of food proteins, their surface activity, and emulsifying properties has been reported (Kato & Nakai, 1980). 4. Conclusion High pressure processing can be used not only for preservation but also for structure modification of meat products. Besides the typical process parameters such as process temperature, pressure level, and holding time, the pressure gradient has a significant effect on the functional properties of pork meat batters. Improvement of protein solubilization and gelation can occur through proper adjustment of PG. This can lead to a reduction of salt content by 50% and improvement of such functional properties as WHC. Therefore a better understanding of all process parameters required for optimal preservation-and functional properties of the sausage batter is needed. References Borejdo, J. (2002). Mapping of hydrophobic sites on the surface of myosin and its fragments. Biochemistry, 22(5), 1182–1187. Borejdo, J., & Assulin, O. (1980). Binding of heavy meromyosin and subfragment-1 to thin filaments in myofibrils and single muscle fibers. Biochemistry, 19(21), 4913–4921. Buckow, R. (2006). Pressure and temperature effects on the enzymatic conversion of biopolymers. Unpublished PhD Thesis, Berlin University of Technology.

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