alginate association

Meat Science 83 (2009) 209–217

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Influence of adding Sea Spaghetti seaweed and replacing the animal fat with olive oil or a konjac gel on pork meat batter gelation. Potential protein/alginate association F. Fernández-Martín *, I. López-López, S. Cofrades, F. Jiménez Colmenero Instituto del Frío (CSIC), Ciudad Universitaria, E-28040-Madrid, Spain

a r t i c l e

i n f o

Article history: Received 26 November 2008 Received in revised form 24 April 2009 Accepted 28 April 2009

Keywords: Pork meat Sea Spaghetti seaweed Olive oil Konjac gel Protein/alginate association Mixed gelation Glass transition DSC MDSC DRTA TPA

a b s t r a c t Standard and modulated differential scanning calorimetry (DSC, MDSC) and dynamic rheological thermal analysis (DRTA) were used to in situ simulate the batter gelation process. Texture profile analysis (TPA) and conventional quality evaluations were applied to processed products. Sea Spaghetti seaweed addition was highly effective at reinforcing water/oil retention capacity, hardness and elastic modulus in all formulations. Olive oil substituting half pork fat yielded a presumably healthier product with slightly better characteristics than control. A konjac-starch mixed gel replacing 70% of pork fat produced a similar product to control but with nearly 10% more water. DSC revealed the currently unknown phenomenon that Sea Spaghetti alginates apparently prevented thermal denaturation of a considerable protein fraction. MDSC confirmed that this mainly concerned non-reversing effects, and displayed glass transition temperatures in the range of 55–65 °C. DRTA and TPA indicated however much stronger alginate-type gels. It is tentatively postulated that salt-soluble proteins associate athermally with seaweed alginates on heating to constitute a separate phase in a thermal composite-gelling process. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Edible seaweeds contain various bioactive compounds with potential health benefits and their use as functional ingredients opens up new prospects for food processing, meat product formulations included (Cofrades, López-López, Solas, Bravo, & JiménezColmenero, 2008; Fleurence, 1999). Seaweeds basically contain high proportions of polysaccharides along with various other potentially beneficial compounds such as good-quality protein and essential fatty acids, particularly long-chain n-3 polyunsaturated fatty acids (PUFAs). Alginates are the most abundant ionic polysaccharides present in brown seaweeds, Sea Spaghetti (Himanthalia elongata) among them. They are unbranched copolymers composed of b-D-mannuronic acid (M) and a-L-guluronic acid (G) residues linked by (1 ? 4) glycosidic bonds in homopolymer blocks. In the presence of monovalent cations, alginates do not form gels and can be used as thickening agents in foods. In the presence of divalent cations, alginates form gels, with Ca2+ forming a typical egg box structure, that can be used in foods as texture modifiers. This effect increases as the ratio of guluronic to mannu* Corresponding author. Tel.: +34 91549 2300; fax: +34 9149 3627. E-mail address: [email protected] (F. Fernández-Martín). 0309-1740/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2009.04.020

ronic units grows larger (Sime, 1990). Little work has been done on the uronic acid composition for H. elongate; Jones (1956) speculated that mannuronic may mainly be its alginic acid, and Sánchez-Machado, López-Cervantes, López-Hernández, PaseiroLosada, and Simal-Lozano (2004) have reported an M/G ratio of 3.6 in a sample from the same commercial source as that used in this work. Partial replacing the animal fat present in a meat product with vegetable or marine lipids is used in formulating healthier products. Olive oil is the vegetable lipid that has received most attention because of its beneficial biological attributes, mainly as an unsaturated oil and a source of PUFAs (Bloukas & Paneras, 1993; Bloukas, Paneras, & Fournitzis, 1997; Pappa, Bloukas, & Arvanitoyannis, 2000). Konjac glucomannan (KGM) is a neutral polysaccharide extracted from the Amorphophallus konjac tuber. Its main chain consists essentially of binary copolymer blocks composed of b-D-mannosyl (M) and b-D-glucosyl (G) units linked by (1 ? 4) glycosidic bonds (M/G  1.6/1), mainly linear but with some slight branching by (1 ? 3) linkages. KGM is partially acetylated (1 of every 19 units), which confers extremely high hygroscopic and water sorption capacities. The refined tuber flour is used in foods and medicines as dietary fiber (no caloric value), affording notable

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physiological effects as well as exceptional technical properties. KGM does not form gels in pure water but rather yields highly viscous pseudoplastic solutions that can be used as thickening agents. However, gelation may take place in the presence of alkaline coagulants (e.g., calcium hydroxide) after deacetylation, yielding thermoirreversible and highly heat-stable gels. KGM can also become a gel by synergistic interaction with other plant/algal hydrocolloids (starch, carrageenan, furcellaran, gellan gum). The physicochemical and textural properties of these kinds of gels make KGM an ideal fat replacer, and has thus been formulated to produce different types of low-fat comminuted meat products (Chin, Keeton, Miller, Longnecker, & Lamkey, 2000; Kao & Li, 2006; Osburn & Keeton, 1994). The object of this study was to evaluate the thermal behaviour of meat batter formulations during thermal processing in response to adding the brown seaweed Sea Spaghetti (alginates, dietary fiber) and partially replacing the pork backfat with olive oil or a konjac-flour (glucomannan) gel, and to compare the effect of adding combinations of these ingredients with an unmodified control formulation. Thermal analysis was performed using standard and modulated differential scanning calorimetry (DSC, MDSC) and dynamic rheological thermal analysis (DRTA) to simulate the thermal gelation process. Quality evaluation criteria were provided by texture profile analysis (TPA) and emulsion stability determinations on the thermally processed systems. 2. Materials and methods 2.1. Meat formulations Post-rigor pork meat (pH = 5.9) and fresh backfat (80% fat) were purchased from a local market. Sea Spaghetti seaweed (H. elongata) powder was supplied by Algamar C.B. (Redondela, Pontevedra, Spain). Composition was 51% total dietary fiber, 5% protein, 30% ash (dry matter) (Cofrades, López-López, Solas, Bravo, & Jiménez-Colmenero, 2008). The additives for the basic meat systems were analytical grade salts; NaCl (1.5%) from Panreac Química S.A. (Barcelona, Spain) and sodium tripolyphosphate (TPP) (0.3%) from Manuel Riesgo S.A. (Madrid, Spain). Other additives were commercial ‘‘extra virgin” olive oil from Carbonell S.L. (Cordoba, Spain), konjac-flour (83% glucomannan, 120 mesh) from Trades S.A. (Barcelona, Spain), pre-gelled cornstarch (Amigel) from Julio Criado S.L. (Madrid, Spain), and Ca(OH)2 from Panreac Química S.A. (Barcelona, Spain). Six different product formulations (Table 1) were prepared with a targeted final meat protein content of 12% and one of two fat levels, 10% or 3%, the latter with 10% higher humidity. Control C was made only from the raw pork-materials (meat (M) and backfat), water, and the conventional salts. Around 60% of the pork backfat ingredient was replaced by 5% olive oil to yield a half and half mixture of animal fat and vegetable oil in O and OS. Batters K and KS were very low-fat (3%) formulations with 90.6% of the pork backfat replaced by 8.6% of a previously prepared mixed konjac-cornstarch gel. To make the gel, konjac flour (5%) was homogenized in water (67%) and mixed with pre-gelled cornstarch powder (3%) pre-

viously dispersed in 15% of water, 1% Ca(OH)2 solution (10%) was then added with gentle stirring at room temperature as reported by Osburn and Keeton (2004), and the admixture cast and allowed to stand in appropriate containers at 2 °C to gel, yielding a hard, solid gum. An amount of 3.4% Sea Spaghetti seaweed powder, offset by reducing the water added by about 10%, was incorporated to S, OS and KS formulations. The general procedure for preparing the batters (Cofrades et al., 2008) was: 10 kg of raw (different muscles) pork meat and 1.5 kg of pork backfat were separately passed through a grinder (Marina, Granollers, Spain) with a 0.6 cm plate, divided into portions (500 g) which were hermetically vacuum packaged into plastic bags, frozen and stored at 20 °C until use (within 3 weeks). Frozen blocks were thawed (overnight at 4 °C) immediately before producing a batch (Table 1) of the six formulations, 650 g each. The thawed raw pork materials were further comminuted in a chilled (2 °C) cutter (model UM5 Universal Machine, Stephan u. Söhne GmbH and Co., Hameln, Germany) for 1 min. The salts (NaCl plus TPP) were dissolved in the water and the seaweed powder dispersed and the mixture added to the ground meat and mixed again for 1 min. The pork backfat, oil or konjac gel (ground and homogenized at 2 °C for 30 s) were added at the same time and mixed for 1 min. Lastly, the resulting meat batter formulation was homogenized under vacuum for 2 min. Mixing time was standardized to 5 min, and the final batter temperature was always less than 12 °C. The batters were stuffed into propylene tubes (2.7 cm inner diameter, 11.5 cm length; Falcon conical centrifuge tubes, BD Biosciences, San Jose, CA, USA) and stored at 2 °C until the next day, when they underwent heat processing (70 °C in a non-stirred water bath for 30 min, previously established to get the cooking temperature to the batters thermal centre). Two batches were prepared and studied separately. A third batch was produced for MDSC determinations. 2.2. Chemical analysis Moisture and ash contents of the processed batters were determined (AOAC, 2000) in triplicate. Protein content was measured in triplicate using a LECO FP-2000 nitrogen determinator (Leco Corporation, St. Joseph, MI, USA). Fat content was determined in duplicate according to Bligh and Dyer (1959). The pH was read six times on a homogenate comprising 10 g of sample in 100 mL of distilled water using an Orion Research 720A pH-meter (Instrumentación Analítica S.A., Madrid, Spain). The number of determinations refers to each formulation and batch. 2.3. Emulsion stability After 22 h in the cold room, the raw batters were centrifuged at 2500 g and 3 °C for 15 min (Multifuge 3L-R, Kendro Laboratory Products GmbH, Hanau, Germany) to eliminate any air bubbles, and the filled tubes were then hermetically closed and heat-processed. The tubes were immediately taken off from the heating bath, opened and left standing upside down at room temperature

Table 1 Raw ingredients (%) and nomenclature for the different formulations: C, control; S, Sea Spaghetti seaweed; O, olive oil; OS, olive oil plus seaweed; K, konjac gel; KS, konjac plus seaweed. Formulation

Pork meat

Pork backfat

Seaweed powder

NaCl

TTP

Olive oil

Konjac gel

Water

C S O OS K KS

54.6 54.6 55.5 55.5 54.6 54.6

9.6 9.6 3.9 3.9 0.9 0.9

 3.4  3.4  3.4

1.5 1.5 1.5 1.5 1.5 1.5

0.3 0.3 0.3 0.3 0.3 0.3

  5.0 5.0  

    8.6 8.6

34.1 30.6 33.8 30.4 34.1 30.6

TTP = Sodium tripolyphosphate.

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for 50 min to release any exudate onto a plate. The exudate was called total fluid released (TR) and expressed as % of the initial sample weight. Emulsion stability was assessed on the total fluid released and its components, such that the larger the TR, the lower the emulsion stability. The water released component (WR, % of the initial sample weight) was determined from the dry matter content of TR after heating at 105 °C for 16 h. The fat released component (FR, % of the initial sample weight) ignored any minor protein or salt components and was taken as the difference between TR and WR. Five determinations (five tubes) were made on each formulation per batch.

the factors was significant (p < 0.05), least-squares differences were used to compare the mean values and the Tukey-HSD test used to identify statistically significant differences.

2.4. Texture profile analysis (TPA)

3.1. Batter composition

TPA was performed using a TA-XT2 Texture Analyzer (Texture Technologies Corp., Scarsdale, NY, USA). Five determinations on each heat-processed formulation and batch were carried out at room temperature by axially compressing a probe (2.7 cm diameter, 3.0 cm height; taken from the central zones of the five batter cylinders) to 40% of its original height with a 250 N load cell applied at a constant crosshead speed of 0.8 mm/s. The parameters derived from the force-deformation curves in a two-cycle test (Bourne, 1978) were: hardness, Hd (N); cohesiveness, Ch (dimensionless); springiness, Sp (mm); chewiness, Cw (Nmm).

Table 2 sets out the proximate composition of the processed meat batters. Protein content ranged from 11.9 to 13.1%. Formulations C, S, O, and OS had fat levels of 10–11% (half fat/half oil in O and OS) and moistures of 71–72%, compared with around 3% fat and 80% moisture in the two low-fat formulations K and KS. Formulations that contained Sea Spaghetti (S, OS, and KS) had the highest ash levels, consistent with the seaweed mineral content. The pH of the meat batters (6.6) did not differ significantly (p P 0.05).

3. Results and discussion Analysis of results between batches showed that in all cases, the respective two mean values for a given property were not significantly different (p P 0.05). The following tables of properties display the corresponding mean and pooled standard deviation values calculated from both batches in each formulation.

3.2. Emulsion stability 2.5. Standard and modulated differential scanning calorimetry (DSC and MDSC) Thermal behaviour of meat systems was monitored by DSC using a previously calibrated differential scanning calorimeter (Q1000 differential scanning calorimeter, TA Instruments, New Castle, DE, USA). Sample weight was around 15 mg (±0.002) determined by an electronic balance (Sartorius ME235S, Goettingen, Germany). Samples were encapsulated in hermetically sealed aluminum pans. The samples (4–6 per formulation and batch) were scanned at 10 °C/min from 5 to 90 °C under a dry nitrogen purge at 50 mL/min. Second scans were recorded after cooling (10 °C/min) the samples down to 5 °C to check for residual/new effects. Additionally, modulated DSC by ‘‘heating only method”, with a modulation of ±0.48 °C every 60 s and 3 °C/min heating, was performed on certain samples (M, C and S). The water content of each individually encapsulated (pinhole in the lid) sample was determined by desiccation at 105 °C to normalize thermal data for dry matter. Temperature, T (°C), and enthalpy of transition, H (J/gdm), data are reported as mean values with their standard deviations.

Table 3 summarizes the water and fat-binding properties as evaluated by the fluids released (TR, WR, and FR) by the processed batters. The emulsion stability data for C and O were similar, with lower (p < 0.05) water and fat-retaining properties and significantly higher TR values (10%) than the rest of the formulations. On the whole, adding different oils (olive, peanut, high-oleic acid sunflower, sunflower, soy bean) to meat batters has been observed to have little or no effect on meat emulsion stability (Ambrosiadis, Vareltzis, & Georgakis, 1996; Hong, Lee, & Min, 2004; Marquez, Ahmed, West, & Johnson, 1989; Park, Rhee, Keeton, & Rhee, 1989). Sea Spaghetti yielded significantly (p < 0.05) lower TR, WR, and FR values (S and OS, Table 3), i.e., the highest emulsion stabilities, OS being slightly (not significantly) lower than S. Similar results were reported by Cofrades et al. (2008) for a meat model system containing Sea Spaghetti seaweed (5%). The likely reason for this is the well-known ability of (Sea Spaghetti) alginates (the polysaccharides that comprise the main active component in the dietary fiber from the brown seaweeds) to bind water and strongly adsorb oils and thus restrict their molecular mobilities.

2.6. Dynamic rheological thermal analysis (DRTA) Dynamic rheology was monitored using a Bohlin CSR-10 Rheometer (Bohlin Instruments Inc., Cranbury, NJ, USA), provided with a Bohlin temperature control unit, in small deformation oscillatory mode. The raw batter was placed between flat parallel (40 mm diameter) plates (1 mm gap) with its perimeter coated with a thin layer of silicone oil to prevent dehydration. After equilibration at 20 ± 1 °C, they were sheared at a fixed frequency of 0.5 Hz with a strain of 0.02 (dimensionless) while the temperature was increased to 70 °C at 1 °C/min. Changes in the dynamic storage (elastic) modulus, G0 (Pa), were monitored throughout the simulated gelling process. The thermo-rheograms presented are the mean of at least two replications per formulation and batch.

Table 2 Proximate analysis (%) and pH of the heat-processed meat systems. C, control; S, Sea Spaghetti seaweed; O, olive oil; OS, olive oil plus seaweed; K, konjac gel; KS, konjac plus seaweed. System

Moisture

Protein

Fat

Ashes

pH

C

72.69a (1.17) 71.59a (0.23) 71.66a (0.22) 71.52a (0.49) 81.02b (0.70) 78.80c (0.63) 0.38

13.05a (0.38) 12.18bc (0.06) 12.69ab (0.07) 11.91c (0.23) 12.62ab (0.13) 12.63ab (0.15) 0.11

11.45ab (1.02) 10.10a (0.40) 11.82b (0.50) 10.42ab (0.52) 3.23c (0.09) 3.32c (0.14) 0.31

2.20a (0.03) 3.50b (0.02) 2.16a (0.14) 3.48b (0.01) 2.31a (0.13) 3.51b (0.03) 0.05

6.59a (0.23) 6.60a (0.02) 6.68a (0.10) 6.74a (0.11) 6.52a (0.08) 6.60a (0.05) 0.05

S O OS K KS

2.7. Statistical analysis SEM

The effect of the batches and of the different formulations was analyzed using the Statgraphics Plus 5.1 (STSC Inc. Rockville, MD, USA) package with a one-way ANOVA. When the effect of any of

Figures between brackets represent (±) standard deviations. SEM = Standard error of the mean. Different letters in the same column indicate significant differences (p < 0.05).

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Table 3 Emulsion stability of the heat-processed meat systems measured as percentage of initial weight: TR, total fluid released; WR, water released; FR, fat released. C, control; S, Sea Spaghetti seaweed; O, olive oil; OS, olive oil plus seaweed; K, konjac gel; KS, konjac plus seaweed. System

TR (%)

WR (%)

FR (%)

C

10.00a (0.59) 0.34b (0.10) 9.29a (0.24) 0.09b (0.13) 7.07c (0.70) 2.37d (0.31) 0.18

9.48a (0.55) 0.31b (0.09) 8.82a (0.23) 0.08b (0.12) 6.73c (0.66) 2.20d (0.29) 0.17

0.52a (0.03) 0.03b (0.01) 0.47c (0.01) 0.01b (0.01) 0.34d (0.03) 0.17e (0.02) 0.01

S O OS K KS SEM

Figures between brackets represent (±) standard deviations. SEM: Standard error of the mean. Different letters in the same column indicate significant differences (p < 0.05).

Low-fat formulations made with the mixed konjac-glucomannan gel (K and KS) achieved better emulsion stabilities than control C, with significantly (p < 0.05) lower TR, WR, and FR values (Table 3), even though the K-type formulations had the highest (+10%) water contents (K > KS) but the lowest (3%) fat compositions (Table 2). The higher water retention in the K-type formulations could be due to the fact that the pre-made konjac gel wholly retained its own compositional water without sharing any with the meat matrix during batter emulsification (Lin & Huang, 2003). As expected, alginates from Sea Spaghetti in KS further improved emulsion stability, with significantly (p < 0.05) lower TR, WR, and FR values compared with K (Table 3).

cantly (p P 0.05). Chewiness evolved in a very similar way to Hardness but its reduction in K relative to control C was statistically significant (p < 0.05). Sea spaghetti seaweed was the main single contributor to hardness. Olive oil was relatively the biggest single contributor to springiness, a property related to the amount of recovery after the deformation force was removed. Konjacstarch mixed gel was the principal single factor responsible for the reduction of hardness and chewiness (product of Hd, Sp, and Ch, associated with chew count and chew rate). Adding different sorts of fiber (soy, wheat, cereal, fruit) to comminuted meat emulsions has generally been found to augment hardness in cooked products (Cofrades, Guerra, Carballo, Fernández-Martín, & Jiménez-Colmenero, 2000). This was also the case for several brown seaweeds, Sea Spaghetti included, in a meat model system (Cofrades et al., 2008). The type and amount of fat have been reported to play an important role in the thermal gelation of meat batters (Barreto, Carballo, Fernández-Martín, & Jiménez Colmenero F., 1996). Replacing animal fat with olive oil in comminuted meat products has been observed to result in harder/firmer fermented sausages and low-fat frankfurters (Bloukas & Paneras, 1993; Bloukas et al., 1997; Paneras & Bloukas, 1994). However, Lurueña-Martinez, Vivar-Quintana, and Revilla (2004) recorded lower hardness and chewiness in low-fat frankfurters when olive oil was used in combination with locust bean/xanthan gum used to replace and reduce the amount of pork fat. In line with this, TPA values were somewhat lower in K than in C, although differences were not statistically significant in all cases. Under the batter preparation conditions employed, the konjac gel was dispersed in the raw protein matrix, which may have given rise to local weaknesses in the three-dimensional matrix formation on subsequent gelation. The TPA results for S and O were in good agreement with the preceding observations. TPA behaviour for the ‘‘combined” formulations (OS and KS) was intensified considerably relative to their ‘‘single” counterparts (O and K), obviously due to the key role played by the seaweed alginates.

3.3. Texture profile analysis (TPA) 3.4. Differential scanning calorimetry (DSC and MDSC) The additives affected the textural parameters of the meat systems to differing extents (Table 4) relative to control C. Hardness (Hd) was significantly (p < 0.05) increased by Sea Spaghetti (S, OS, and KS) and not significantly (p P 0.05) decreased by replacing half the animal fat with olive oil (O) or seven-tenths with the konjac gel (K). As expected, springiness (Sp) was modified slightly (not significantly; p P 0.05), decreasing with Sea Spaghetti and/or konjac gel (S, K, and KS) and increasing with olive oil (O and OS). Cohesiveness was significantly (p < 0.05) reduced with respect to control C in all cases except in O where Ch increased, not signifi-

Fig. 1 depicts the typical DSC traces normalized for dry matter for the different meat systems. Plots disclose two distinct temperature regions, one for the thermal response of the fat/oil phase, 10– 50 °C (zone F), and one for the protein matrix, 40–90 °C (zone P). 3.4.1. Lipids Fig. 1a, solid line, presents a typical DSC curve for raw ground pork meat (M) with a subtle (low fat content) endothermic pork fat melting event in the range of 20–45 °C, centered around 30–

Table 4 Texture profile analysis of the heat-processed meat systems. C, control; S, Sea Spaghetti seaweed; O, olive oil; OS, olive oil plus seaweed; K, konjac gel; KS, konjac plus seaweed. System

Hardness Hd (N)

Springiness Sp (mm)

Cohesiveness Ch (dimensionless)

Chewiness Cw (Nmm)

C

26.81a (0.79) 48.79b (0.97) 24.41a (0.89) 45.93b (2.32) 22.87a (0.80) 33.99c (3.35) 0.90

10.55ab (0.10) 10.42a (0.10) 10.64ab (0.06) 10.68b (0.15) 10.45a (0.03) 10.45a (0.13) 0.05

0.480a (0.001) 0.460b (0.002) 0.486a (0.005) 0.465b (0.003) 0.466b (0.002) 0.467b (0.001) 0.002

135.82a (4.68) 234.02b (4.98) 126.18ac (3.50) 228.14b (6.03) 111.17c (3.92) 165.78d (5.88) 4.74

S O OS K KS SEM

Figures between brackets represent (±) standard deviations. SEM = Standard error of the mean. Different letters in the same column indicate significant differences (p < 0.05).

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a

b Heat Flow (Endo Down)

Heat Flow (Endo Down)

F

P

I II

III

20

30

OS KS

O

K C

P zone

0.05 W/gdm

10

S

0.2 W/gporkfat

40

50

60

70

80

90

15

20

25

30

35

40

45

50

Temperature (o)

Temperature (ºC)

c

F zone

Heat Flow (Endo Down)

S OS KS K O

C

0.1 W/gprotein 40

50

60

70

80

90

Temperature (ºC) Fig. 1. (a) Normalized DSC trace for ground pork meat (M): F, fat melting zone; P, protein denaturation zone; I, myosin region; II, myosin, collagen, and sarcoplasmic protein region; III, actin region. (b) Normalized DSC traces for the fat melting process in the different meat formulations. (c) Normalized DSC traces for the protein denaturation process in the different meat formulations. C, control; S, Sea Spaghetti seaweed; O, olive oil; OS, olive oil plus seaweed; K, konjac gel; KS, konjac plus seaweed. Solid lines, first scans; dashes, second scans. Bars indicate ordinate scales.

35 °C (solid and dash lines) and an enthalpy of melting of 1.12 J/ gdm (29 J/gfat). Fig. 1b displays typical DSC traces of first (solid lines) and second (dashes) scans of unaltered pork backfat (C and S) and backfat replaced by olive oil (O and OS) or the konjac gel (K and KS). As expected, fat melting endotherms appeared to start at lower temperatures than the initial ones recorded and extended in a series of overlapping events nearly up to protein zone P. Essentially, first melting yielded a maximum effect in the form of a pronounced, complex event at peak temperatures of around 26, 27.5, and 29 °C corresponding to the medium melting triglycerides, lengthened by a wider, shallower event up to around 50 °C caused by the high melting species (curve C, solid line). Second scans always exhibited milder melting processes, because the thermal conditioning did not allow for extensive crystallization (curve C, dashes). Olive oil yielded softer lipid phases (curves O and OS), with some significant (p < 0.05) downward shifts (ca. 2 °C) in the onset and peak temperatures (solubilization effect), and correspondingly significant (p < 0.05) reductions in melting enthalpy values (Table 5, column 3). Sea Spaghetti raised significantly (p < 0.05) the onset temperature by about 4 °C in S (curve S), presumably because the liquid lipid-phase was adsorbed by the seaweed fiber, making the coexisting solid lipid-phase somewhat harder, i.e., melting at higher temperatures by freeing it from the liquid phase influence. This was also true for OS (curve OS), where the ‘‘softening” effect of the olive oil was to a large extent counteracted by the ‘‘hardening” effect of the seaweed fiber alginates (Table 5, column 2). As expected, konjac gel had no practical effect on

pork fat melting. Experimental DH (J/gdm) data associated with lipid phase thermal transitions (Table 5, column 3) showed nearly linear variation with pork fat content. This was clearly displayed by data normalized to the animal-fat content of the formulation (Table 5, column 4), based on the bulk composition values (Table 2) and the corresponding nominal reductions in animal fat (Table 1). The normalized enthalpy DH (J/gporkfat) data obtained for the six formulations showed no significant (p P 0.05) differences, yielding a consistent average (n = 6) value (±standard deviation) of 31.0 ± 1.3 (J/gporkfat) for pork fat melting, which would seem to be a reasonable value in view of the incomplete fat crystallization/melting undergone by the samples (Barreto et al., 1996). 3.4.2. Proteins A complex endotherm appeared in the protein zone P of raw meat M (Fig. 1a), with an onset temperature of 51 °C and an enthalpy of transition of 13.7 J/gdm. This thermal denaturation of the myofibrillar proteins comprised several overlapping endothermic events typically grouped in three regions, with peak temperatures around 58 °C related to myosin (region I), 62 and 70 °C related to myosin, collagen, and the sarcoplasmic proteins (region II), and a more distant peak at 79 °C related to actin (region III). The data are in good agreement with published results for the thermal denaturation of mammalian skeletal proteins, particularly those from pork (Fernández-Martín & Jiménez-Colmenero, 1998), the only peculiarity being a slightly smaller first myosin peak and consequently a slightly lower enthalpy of transition, probably related

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Table 5 Temperature (T, °C) and enthalpy of transition (DH, J/g) data for fat melting and protein denaturation in the different batter formulations. C, control; S, Sea Spaghetti seaweed; O, olive oil; OS, olive oil plus seaweed; K, konjac gel; KS, konjac plus seaweed. System

Fat melting Tonset

C S O OS K KS SEM

a

21.5 (0.19) 25.4b (0.41) 19.3c (0.08) 24.8b (0.24) 21.5a (0.13) 24.3b (0.25) 0.16

Protein denaturation

DH (J/gdm)# a

12.5 (0.55) 11.1a (0.62) 6.8b (0.42) 5.7b (0.39) 5.8b (0.61) 6.5b (0.50) 0.52

DH (J/gporkfat) a

29.8 (0.67) 31.3a (0.52) 32.9a (0.61) 31.1a (0.75) 31.4a (0.49) 29.3a (0.58) 0.64

Tonset a

62.6 (0.50) 64.5b (0.57) 58.0c (0.60) 61.7a (0.49) 60.8a (0.38) 62.4a (0.43) 0.50

Tpeak a

71.8 (0.41) 71.7a (0.48) 71.4a (0.52) 70.6a (0.55) 70.4a (0.45) 72.0a (0.62) 0.51

DH (J/gdm)

#

a

3.9 (0.19) 2.2b (0.14) 3.8a (0.17) 2.8b (0.31) 5.5c (0.16) 4.3a (0.24) 0.21

DH (J/gprotein) 8.3a (0.31) 5.2b (0.25) 8.6a (0.32) 6.9c (0.36) 8.7a (0.29) 7.2ac (0.35) 0.32

*

Referred to the main peak. Straight baseline integration between 18 and 47.5 °C (fat) or between 47.5 and 87.5 °C (protein). Figures between brackets represent (±) standard deviations. SEM = Standard error of the mean. Different letters in the same column indicate significant differences (p < 0.05).

#

to some sort of minor meat aging (pH lowering) effect (Stabursvik, Kristen, & Frøystein, 1980). Fig. 1c depicts the substantial change in the DSC pattern in the meat batters with respect to the raw ground meat. It is known that the salts (NaCl, TPP) added to meat comminution can increase the myofibrillar-protein’s thermal instability, by making the myosin and actin, converge to the same denaturation temperature. The M complex protein denaturation profile evolved into practically a single endotherm (shoulder for the most thermolabile myosin fractions at 52 °C) with a peak at around 71–72 °C, the cooking temperature (curve C, solid line) (Fernández-Martín, Fernández, Carballo, & Jiménez Colmenero, 1997; Quinn, Raymond, & Harwalkar, 1980). The second scans (curve C, dashes) always produced flat traces, indicating that protein denaturation was irreversible. All the batters exhibited similar DSC patterns, with similar onset (Table 5, column 5) and peak (column 6) temperatures. However, onset temperature varied significantly (p < 0.05) in S increasing by ca. 2 °C, and in O decreasing by ca. 4 °C. Furthermore, the changes in curve morphology (curve S; curves OS and KS to a lesser extent) implied that the degree of denaturation (partial area integration) at Tonset for (S), 64.5 °C was around 7% in S, but ranged from 20% to 30% in the rest of the formulations. It was as if a considerable fraction of the muscle proteins (around 37% if all the proteins are assumed to have the same denaturation enthalpy) had been removed from the thermal denaturation process in formulation S (around 17% and 13% for OS and KS respectively). However, second scans (curve S, dashes) failed to disclose any endothermic event. Experimental enthalpy values (Table 5, column 7) for the alginate-containing batters were considerably lower than expected, particularly in formulation S. This was clearly evident in the enthalpy data normalized to protein content (Table 2), as shown in Table 5 (column 8) where DH(S) was significantly (p < 0.05) the lowest value, followed by the statistically different DH(OS), and then by DH(KS) which occupied an intermediate position with a value not significantly (p P 0.05) different from C, O and K formulations. When the S-type data were excluded, a consistent average (n = 3) value for the denaturation enthalpy (±standard deviation) of myofibrillar proteins of 8.5 ± 0.2 (J/gprotein) was obtained. To the best of our knowledge this surprising role of Sea Spaghetti (highly mannuronic) alginates in apparently preventing protein thermal denaturation has not been reported previously. Fig. 2 shows corresponding modulated differential scanning calorimetry (MDSC) traces of total heat flow (T curves) and its reversing (R curves) and non-reversing (N curves) components for M (Fig. 2a), C (Fig. 2b) and S (Fig. 2c) systems. Well resolved traces yielded tem-

peratures and total enthalpy changes (Table 6, T columns) agreeing well with previous DSC data (Table 5). Reversing traces R may be related with weakly endothermic protein–protein aggregations by electrostatic and hydrophobic bonding. System M exhibited the same but greatly smoothed pattern as T, with a distinct peak centered at 77 °C (Fig. 2a, curve R). This was related to actin and it logically disappeared (vide supra) in corresponding traces for C (Fig. 2b, curve R) and S (Fig. 2c, curve R) formulations. As expected, reversing effects (Table 6, R columns) were the smaller component in all cases, with C and S batters displaying not significantly (p P 0.05) lower values than raw meat M. It seems that the protein denaturation involved in meat comminution with salts particularly affected the heat-stable proteins, which could easily be inferred from the changes in the DSC traces of raw meat M when processed to batters C or S. Reversing effects were close for C and S, which was likely due to essentially the same sort of thermal protein–protein aggregation irrespective of the non-meat ingredient added. Non-reversing traces N displayed the effects associated with the irreversible protein thermal denaturation of the systems, and obviously were the larger component (Table 6, N columns). Worth noting is the fact that the Non-reversing to Reversing ratio (N/R: column 7/column 5) amounted to around 4.9, 3.7 and 2.1 for M, C and S respectively, i.e., S gave nearly half the C ratio. MDSC thus complemented the DSC results in that Sea Spaghetti seaweed alginates apparently essentially prevented myofibrillar proteins from non-reversing (protein denaturation/aggregation) thermal transitions. Interestingly, second scans recorded subtle glass transitions in the reversing MDSC traces of the fresh gelled systems (Figs. 2a–c, R dashes). Glass transition temperatures (Tg, °C), labeled in respective Fig. 2 and shown in Table 6 (column 8). Ground meat M displayed a glass transition at a significantly (p < 0.05) higher temperature than control C: M retained much of the original myosystem structure with water relatively hindered in mobility; meat comminution and the presence of lipids plasticized batter C. Sea Spaghetti seaweed caused a statistically significant (p < 0.05) increase in the glass transition temperature for S with respect to control C, probably because the molecular mobilities of water and lipids were largely reduced. 3.5. Dynamic rheological thermal analysis (DRTA) Fig. 3 depicts the dynamic storage modulus (G0 ) for the different meat systems during thermal gelation. The main rheological changes taking place in control C during heating occurred above 40 °C, with a gradual increase in G0 related to the formation of a

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a

T 55

60

65

70

R

0.05 W/gprotein

40

50

N

Heat Flow (Endo Down)

Heat Flow (Endo Down)

N

50

b

F zone

T

48

50

52

54

56

58

60

R

Tg 0.05 W/gprotein

Tg 60

70

80

90

40

50

60

Temperature (ºC)

80

90

c

F zone

Heat Flow (Endo Down)

70

Temperature (ºC)

N

55

60

65

70

75

T R

0.05 W/ g protein

40

Tg

50

60

70

80

90

Temperature (ºC) Fig. 2. Normalized MDSC traces for: (a), ground pork meat M; (b), control C; (c), Sea Spaghetthi seaweed S. T lines, total heat flow; R lines, reversing component; N lines, nonreversing component. Solid lines, first scans; dashes, second scans. Bars indicate ordinate scales. Labels Tg and arrows indicate glass transition temperatures. Insets display enlarged glass transition zones.

Table 6 Total enthalpy of transition (DH, J/g), its reversing and non-reversing components for protein denaturation, and glass transition temperatures (Tg,, °C) in the different meat systems. M, ground pork meat; C, control; S, Sea Spaghetti seaweed. System

Total (T)

M C S SEM

Glass Transition– Tg (°C)

Protein denaturation (DH) Reversing (R)

Non-reversing (N)

(J/gdm)

(J/gprotein)

(J/gdm)

(J/gprotein)

(J/gdm)

(J/gprotein)

13.2a (0.06) 4.0b (0.24) 2.3c (0.12) 0.16

13.7a (0.06) 8.5b (0.48) 5.4c (0.21) 0.30

2.2a (0.07) 0.9b (0.21) 0.7b (0.16) 0.16

2.3a (0.09) 1.8a (0.50) 1.7a (0.29) 0.34

10.9a (0.18) 3.2b (0.29) 1.6c (0.21) 0.23

11.3a (0.20) 6.7b (0.31) 3.6c (0.23) 0.25

63.5a (0.58) 55.4b (0.61) 63.6a (0.45) 0.55

– Second scans. Figures between brackets represent (±) standard deviations. SEM = Standard error of the mean. Different letters in the same column indicate significant differences (p < 0.05).

rather stiff elastic matrix. This rheological pattern was typical of the thermal gelling of myofibrillar proteins with added salt (Section 3.4.2). The pattern was consistent with the DSC trace for C in Fig. 1c, indicating that structure formation was not yet complete up to 70 °C and that G0 was still rising. Adding the non-meat ingredients brought about certain differences in the thermo-rheological profiles of the formulations, namely, the onset temperature of the G0 on T profile during heat processing (between 30 and 40 °C), the slope of the main increase (up to 50–55 °C), and lastly the asymp-

totic value for G0 . Adding konjac gel or olive oil singly to replace the pork backfat noticeably lowered the onset temperature in K and O while yielding nearly the same (K) or distinctly higher (O) values for the slope and the final elastic modulus of the gelled samples with respect to gel C. In contrast, adding Sea Spaghetti alone greatly increased the values of all three parameters, resulting in a steeper and more highly altered curve compared to C. The combined seaweed powder and olive oil or konjac gel (OS and KS) had values close to but slightly lower than those for S, oil and konjac

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40

S OS KS

35

G' (kPa)

30

O

25 C K

20 15 10 5 0 10

20

30

40

50

Temperature

(oC)

60

70

Fig. 3. Dynamic storage modulus (G’, Pa) on temperature (T, °C) for the different meat formulations. Five-pointed stars, control C; Triangles, Sea Spaghetti seaweed S; Circles, olive oil O; Rhombi, olive oil plus seaweed OS; Squares, konjac gel K; Inverted triangles, konjac plus seaweed KS.

gel components impairing somewhat the effects induced by Sea Spaghetti. This was also consistent with the greater hardness of S and related formulations OS and KS as evidenced by the TPA parameter Hd, caused mainly by the texturizing effects of the seaweed alginates. Therefore DRTA and TPA behaviour of the S-type formulations appears to conflict with the DSC and MDSC results, i.e., that nearly one third the proteins might have been left out of the usual meat batters structuring process. We speculate on this as follows. By experience, it is assumed that the ionic strength in control C may have yielded a salt-soluble protein fraction of 20–25%. However, formulation S may have a considerably higher solvent capacity because of the substantial mineral content (Table 2) of the added seaweed, hence 30–35% could be an acceptable estimate for the salt-soluble fraction in this formulation. On the other hand, the mainly mannuronic configuration of the Sea Spaghetti alginates makes them particularly prone (against Ca2+ gelation) to the formation of electrostatic associations with proteins. According to Dumay, Laligant, Zasypkin, and Cheftel (1999), most of the alginate carboxylic groups will be negatively charged at pH 7.0 and form electrostatic association with b-lactoglobulin. On the other hand, it is generally accepted that electrostatic interactions occur between the anionic groups of a polysaccharide (seaweed polyanionic alginates) and the positive charged groups of proteins (Tolstoguzov, 1986), and that they are virtually formed athermally (Imeson, Ledward, & Mitchell, 1977). On this basis, it is tentatively postulated that the salt-soluble proteins first are adsorbed on the swollen seaweed alginates and then undergo, on heating conformational changes conducive to the formation of mannuronic-protein complexes, essentially electrostatic and athermal in nature. The upshot is that this protein fraction is DSC/MDSC silent in the S systems. The reduced capacity of seaweed alginates for this in combined formulations OS and KS (nearly half the S alone; Section 3.4.2) could mostly be due to strong oil adsorption by alginates and possible steric hindrances throughout the systems by oil-in-water emulsions and finely dispersed konjac-gel. This seaweed alginate-myofibrillar protein association could lead to a phase separated from the main protein–protein matrix, strongly contributing to the increased mechanical and viscoelastic properties of a mixed gel in the S-type formulations. Further studies are needed to test this hypothesis. 4. Conclusions Alginates from Sea Spaghetti seaweed were effective structuring agents which led to firmer batter matrices and harder fat

phases, gel S giving the highest overall score. Olive oil produced softening effects in the fat phases but did not alter protein denaturation, and gel O scored similarly to but somewhat higher than control C. Konjac-gel resulted in neutral behaviour concerning both fat melting and protein denaturation, the low-fat gel K showing similar but slightly lower scores than control C although containing more water. Combined formulations OS and KS benefited greatly from alginate functionality and behaved much like formulation S, yielding final products with stronger properties with respect to control C. DSC and MDSC revealed the previously unreported phenomenon that a considerable fraction of proteins were apparently prevented from thermal denaturation in the usual protein-self association process. DRTA and TPA provided conflicting views by revealing S-type stronger thermal gels. An electrostatic and athermal association between high-mannuronic alginates and salt-soluble myofibrillar proteins is tentatively proposed, which would lead to a separate phase from the usual protein–protein aggregation, so constituting a composite gel. Results suggested that, additionally to health benefits (fat reduction, inclusion of fiber and/or some bioactive components) and economic considerations, the use of these non-meat ingredients provide valuable information for formulating novel protein mixed gels suitable in the manufacture of comminuted or restructured meat-like products. Acknowledgments This research was supported under projects AGL2005-07204CO2 and CARNISENUSA CSD2007-00016. IL-L thanks Ministerio de Educación y Ciencia for a predoctoral fellowship. Seaweed kind supply is also thanked to Algamar S.A. References AOAC. (2000). Official methods of analysis of AOAC international (17th ed.). Association of Official Analytical Chemistry, Maryland, USA. Ambrosiadis, J., Vareltzis, K. P., & Georgakis, S. A. (1996). Physical, chemical and sensory characteristics of cooked meat emulsion style products containing vegetable oils. International Journal of Food Science and Technology, 31(2), 189–194. Barreto, G., Carballo, J., Fernández-Martín, F., & Jiménez Colmenero, F. (1996). Thermal gelation of meat batters as a function of type and level of fat and protein content. Zeistschrift für Lebensmittel Untersuchung und Forschung, 202(3), 211–214. Bligh, E. G., & Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911–917. Bloukas, J. G., & Paneras, E. D. (1993). Substituting olive oil for pork backfat affects quality of low-fat frankfurters. Journal of Food Science, 58(4), 705–709. Bloukas, J. G., Paneras, E. D., & Fournitzis, G. C. (1997). Effect of replacing pork backfat with olive oil on processing and quality characteristics of fermented sausages. Meat Science, 45(2), 133–144. Bourne, M. C. (1978). Texture Profile Analysis. Food Technology, 32(7), 62–65. Chin, K. B., Keeton, J. T., Miller, R. K., Longnecker, M. T., & Lamkey, J. W. (2000). Evaluation of konjac blends and soy protein isolate as fat replacement in low-fat bologna. Journal of Food Science, 65(5), 756–763. Cofrades, S., Guerra, M. A., Carballo, J., Fernández-Martín, F., & Jiménez-Colmenero, F. J. (2000). Plasma protein and soy fiber content effect on bologna sausage properties as influenced by fat level. Journal of Food Science, 65(2), 281–287. Cofrades, S., López-López, I., Solas, M. T., Bravo, L., & Jiménez-Colmenero, F. (2008). Influence of different types and proportions of added edible seaweeds on characteristics of low-salt gel/emulsion meat systems. Meat Science, 79(4), 767–776. Dumay, E., Laligant, A., Zasypkin, D., & Cheftel, J. C. (1999). Pressure- and heatinduced gelation of mixed b-lactoglobulin/polysaccharides solutions: Scanning electron microscopy of gels. Food Hydrocolloids, 13(4), 339–351. Fernández-Martín, F., Fernández, P., Carballo, J., & Jiménez Colmenero, F. (1997). Pressure/heat combinations on pork meat batters: Protein thermal behavior and product rheological properties. Journal of Agricultural and Food Chemistry, 45(11), 4440–4445. Fernández-Martín, F., & Jiménez-Colmenero, F. (1998). Pressure/temperature processing of low- and high-fat frankfurters. Denaturation effects on the proteins. In Proceeding of 44th international congress on meat science and technology (Vol. II, pp. 546–547), 30 August–4 September 1998, Barcelona, Spain. Fleurence, J. (1999). Seaweed proteins: Biochemical, nutritional aspects and potential uses. Trends in Food Science & Technology, 10(1), 25–28.

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