Effect of Amidated Low-Methoxyl Pectin on Physicochemical Characteristics of Jumbo Squid ( Dosidicus gigas ) Mantle Muscle Gels

Jumbo squid (Dosidicus gigas) is a cephalopod that plays a valuable role in the pelagic ecosystem of the Gulf of California (1). Most of its capture is exported to Asian and European countries, either fresh-frozen or cooked-salted (daruma), with almost no added value. Its commercial att ractiveness lies in its low cost, high processing yield (up to 75 % aft er gutt ing), low fat, and muscle whiteness. Several eff orts have been att empted to use its muscle as a raw material for various gel-type products (2–4). Nevertheless, its muscle forms weak gels with low water ISSN 1330-9862 preliminary communication


Introduction
Jumbo squid (Dosidicus gigas) is a cephalopod that plays a valuable role in the pelagic ecosystem of the Gulf of California (1).Most of its capture is exported to Asian and European countries, either fresh-frozen or cooked-holding capacity (WHC) (5,6).Thus, there is a need to fi nd a gel-improving additive if this muscle is going to be used as a raw material for these products.
Various types of fi bre, such as glucomannan, wheat and oat fi bre, cellulose, carrageenan, and pea fi bre, have been studied to improve the gelling and WHC characteristics of a range of fi sh and beef muscles, modifying the functional, as well as technological properties of the fi nal product (7)(8)(9).When added to gel formulations (sometimes used as a fat replacer), the fi bre has a profound effect on the physicochemical interactions and structural changes of the protein system (10), generating an improved product, without aff ecting its edible quality (8).Furthermore, fi bre consumption has been associated with a reduction in the risk of coronary heart disease, stroke, hypertension, diabetes, obesity development, and some gastrointestinal diseases (11).
Pectin is abundantly found in vegetable tissues and commonly used as a food ingredient for several applications (12).It is a complex polysaccharide consisting mainly of esterifi ed α-(14)-d-galacturonic acid units.Some of the carboxyl groups are partially esterifi ed by methoxy groups (-OCH 3 ) at the C6 position and/or acetyl groups (-O-C(O)CH 3 ) at the O2 and/or O3 positions, depending on the plant species (13).Additionally, its carboxylic groups can be amidated by a relatively simple chemical modifi cation, obtaining amidated low-methoxyl pectin (ALMP), a compound that imparts certain functionality to gel products.Although pectin and its derivatives are used abundantly in the food industry, no reports have been made on their use to improve jumbo squid muscle gelation.Hence, the main objective of the present research is to assess the addition of ALMP, as a commercial natural fi bre, added at 0.5, 1.0, 1.5, 2.0 and 3.0 %, on the physicochemical and functional characteristics of jumbo squid (D. gigas) mantle muscle gels.

Sample
The raw mantle muscle of jumbo squid (Dosidicus gigas) was obtained in a frozen state from a local fi shery located in Hermosillo, Sonora, Mexico, in the summer of 2013.The specimens were captured in the Gulf of California.The mantles were thawed at 2-4 °C overnight, cleaned from the internal and external collagenous tunics, and finally cut into 2 cm×2 cm sections.The samples were kept at 2-4 °C before analysis.

Gel preparation
Sols were prepared by combining 4.0 g of NaCl with 196 g of muscle using a Cuisinart DLC-8 food processor (Cuisinart Inc., Greenwich, CT, USA) applied with three bursts of 30-second intervals until the temperature reached 5 °C.Then, the fi nal moisture of the sol-gels was adjusted to 80 % with sucrose, as described by Cortés--Ruiz et al. (18).GRINDSTED ® LA 410 commercial citrus pectin (ALMP; Danisco USA Inc., New Century, KS, USA) with 29-33 % degree of esterifi cation and 17-21 % degree of amidation) was added to the sol-gels at 0.5, 1.0, 1.5, 2.0 and 3.0 %.The sol-gels were vacuum packed in polyethylene bags that were then placed in stainless steel tubes (3.5 cm i.d.×7.5 cm length) and heated in a water bath at 90 °C for 30 min to form gels. Aft er that, the tubes were immediately immersed in an ice-water bath and held overnight at 2-4 °C before analysis.

Gel-forming ability
The gel-forming ability of samples was evaluated by texture profi le analysis (TPA), Warner-Bratzler shear force (WBSF) and the folding test.TPA was conducted on cylindrical samples of uniform dimensions (1 cm diameter×1 cm height) obtained from each gel using a sharp-edged plastic tube sampler.Gels were left at ambient temperature (25 °C) for 30 min before analysis.Texture was analysed using a Texture Lab Pro texturometer (Food Technology Corp., Sterling, VA, USA) using a 2.5-cm diameter compression plunger att ached to a 10 N load cell.The samples were compressed twice to 75 % of the original gel sample height at a speed of 1 mm/s to obtain the gel hardness, springiness and cohesiveness according to Bourne (19).
As described by Zhang and Mitt al (20), the WBSF was analysed on tempered (as per the TPA analysis) rectangular gels (1 cm height×1 cm width×3 cm length) using a Warner-Bratzler blade att ached to a texturometer (Food Technology Corp.) with a 10 N load cell and operating at a speed of 2 mm/s at 30 mm displacement.The maximum force (highest peak) resulting from the analysis was recorded as the shear force.
The folding test involved folding the gel slices (3.0 mm thickness×30.0mm diameter) between the thumb and index fi nger, as described previously (21).The gel samples were graded according to their cracking degree during folding, as follows: grade AA=extremely elastic (no cracks on folding into quarters), grade A=moderately elastic (no cracks on folding in half, but cracks on folding into quarters), grade B=slightly elastic (some cracks on folding in half), grade C=non-elastic (breaks into pieces on folding in half), and grade D=poor (breaks into pieces with fi nger pressure).Five slices per gel per replicate were analysed.

Water holding capacity
The water holding capacity (WHC) was evaluated using the technique outlined by Jiang et al. (22), whereby 5 g of each gel were centrifuged at 3000×g for 20 min at 4 °C (Beckman J2-21 centrifuge; Beckman Instrument Inc., Palo Alto, CA, USA).WHC was expressed as the percentage of retained water relative to the amount of water present in the sample before centrifugation.

Tristimulus colourimetry of gels
A Konica-Minolta CR-400 Tristimulus colourimeter (Konica Minolta Sensing, Inc., Tokyo, Japan) was used to objectively measure CIE L* (brightness), a* (redness/ greenness) and b* (yellowness/blueness) parameters of the cast gels, as mentioned by Lanier (23).Data were obtained from six diff erent measurement sites on the gel.Whiteness index (WI) was calculated from the L*, a* and b* values using the following equation:

Diff erential scanning calorimetry
Changes in protein thermal stability of sol-gels with added fi bre (2.5 % NaCl and 80 % moisture) were measured using a DSC7 Perkin Elmer calorimeter (Perkin Elmer de México S.A., Monterrey, México).Approximately 50 mg of sample were loaded into the stainless steel pan and sealed hermetically.An empty pan was used as a reference.A calibration for temperature accuracy using indium was conducted.Samples were heated from 20 to 90 °C at a constant rate of 5 °C/min.Thermograms were analysed with the Pyris™ v. 4.02 soft ware (24) to obtain the onset temperature (t onset ), the temperature at maximum heat fl ow (t max ), and heat enthalpy (ΔH).

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
The potential eff ect of added fi bre on protein interactions during heat-induced gel network formation was evaluated by SDS-PAGE, performed according to Laemmli (25) with some modifi cations (26).Aliquots of 2 g (gel) were blended with 18 mL of dissolved solution (5 % SDS, 0.1 % β-mercaptoethanol) using an Ultra-Turrax ® T 25 D S1 homogeniser (IKA Works Inc., Wilmington, NC, USA).Fresh mantle muscle was also treated with the dissolved solution.The homogenates were incubated at 80 °C per 1 h for maximal solubilisation and protein extraction, and centrifuged at 3000×g (Beckman J2-21 centrifuge) for 15 min.The protein amount of the supernatants was measured by the biuret method.The samples (0.2 % protein) were then mixed (at a 1:1 volume ratio) with SDS-PAGE sample buff er (4 % SDS, 20 % glycerol, 10 % β-mercaptoethanol and 0.125 M Tris, pH=6.8) and dissolved by heating in boiling water for 3 min.Aliquots of 30 μg of protein per well were loaded onto an acrylamide gel (10 % resolving, 4 % stacking) with dimensions of 80 mm×60 mm×1.5 mm (width×height×thickness).For protein markers, a broad-range molecular mass protein standard (Bio-Rad Laboratories, Richmond, CA, USA) was used.Aft er electrophoresis, the gel was stained with 0.125 % (mass per volume) Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories) in 40 % (by volume) methanol and 7 % (by volume) acetic acid, and destained with 50 % (by volume) methanol and 10 % (by volume) acetic acid.Methanol and acetic acid were purchased from Sigma--Aldrich Inc. (St. Louis, MO, USA).

Experimental design
Data were analysed by one-way analysis of variance using NCSS 97 statistical soft ware (27).When diff erences among mean values were found at 5 % signifi cance level, a Tukey-Kramer mean comparison was conducted.All analyses were conducted in triplicate for each sampling (N=3).

Eff ect of ALMP on gel forming ability of muscle
Protein gelation involves interactions of protein molecules under controlled conditions to form a three-dimensional network, which can entrap water.This particular system can be improved, when needed, by adding various types of compounds, such as ALMP.Table 1 shows the eff ect of ALMP addition on the TPA of jumbo squid muscle gels.In general, fi bre addition improved the gel texture, increasing (p<0.05)hardness, cohesiveness and springiness values.Particularly, 0.5 % fi bre showed a statistically signifi cant eff ect (p<0.05) on gel hardness compared to the control.The hardness values of samples with intermediate fi bre content (1.0-2.0 %) were higher than the control (p<0.05) but lower than 0.5 % fi bre addition.However, 1.5 and 2.0 % treatments were not signifi cantly diff erent (p≥0.05).Samples with the highest fi bre mass fraction (3.0 %) also had the highest hardness (p<0.05)value.Further analysis must be conducted to elucidate which gel hardness data have corresponding sensorial attractiveness.Similar to the gel hardness data, fi bre addition mostly increased the gel cohesiveness.However, no signifi cant diff erences (p≥0.05) were found between the treatments and the control, except at 2.0 % fi bre addition, which increased gel hardness by 21 % when compared to the control.
Fibre addition also practically maintained or increased (p≥0.05)gel springiness.Although 0.5 % addition increased this parameter, it was statistically similar (p≥0.05) to the control.Comparable results on hardness, springiness and cohesiveness behaviour were documented when ALMP (25 % amidation) was added to striped mullet (Mugil cephalus) restructured fi sh products (29).ALMP, being an anionic carbohydrate, can interact with the cationic groups of protein and to a lesser extent with hydrogen bonds, increasing the functional and mechanical properties of protein systems (30,31).However, its addition can also aff ect the gelling process, depending on concentration and raw matt er (32).Overall, the TPA results showed that ALMP addition benefi tt ed the gelation characteristics of jumbo squid muscle proteins by increasing the measured parameters.
WBSF measures the maximum force encountered by the blade cutt ing action on the sample (19), mimicking the bite.In the present study, WBSF values tended to increase as fi bre was added (p≥0.05).Although 0.5 % fi bre addition reduced this value, the decrease was not signifi cantly (p≥0.05)diff erent to the control.At the highest amount of added fi bre (3.0 %), this parameter increased by 36 % (p<0.05).Consequently.fi bre addition increased the WBSF, in concurrence with the gel hardness values.Finally, the ALMP positively infl uenced the jumbo squid muscle gel characteristics, based on the folding test, showing gels with A-B and A-AA scores when 0.5-1.0 % and 1.5-3.0% fi bre were added, respectively.The texture results indicate that adding ALMP to jumbo squid muscle sol-gel promotes protein-ALMP interactions, increasing the gel mechanical characteristics.Although the addition of ALMP at any mass fraction improved jumbo squid muscle protein gel functionality, further research is required to confi rm which fi bre mass fraction is the most adequate, in terms of functionality and cost benefi t of a gel-type product.

Eff ect of ALMP on WHC of gels
One characteristic of jumbo squid gels, besides their soft texture, is their poor WHC (6), showing syneresis during their handling.Besides carboxyl and amino groups, ALMP is relatively hydrophilic, possessing many hydroxyl groups.This interaction with water is the main reason these compounds are extensively used in the food industry (33).Fig. 1 shows the WHC of the jumbo squid gels with various ALMP mass fractions added.Fibre addition (0.5 %) increased (p<0.05) the capacity of gels to hold water (18.5 %, from (75.5±3.1) to (89.5±1.8)%, control vs. 0.5 % ALMP, respectively).Further addition (up to 2.0 %) maintained (p≥0.05) a similar water retention ((89.0±3.1),(87.9±2.4) and (87.7±4.5)% in samples with 1.0, 1.5 and 2.0 % ALMP, respectively).However, maximum water retention ((94.7±1.7)%) was observed when 3.0 % fi bre was added, increasing the WHC by 25.5 %, com-pared to the control.These results suggest that a high level of carbohydrate-water interaction occurred in the samples, without aff ecting gel structure formation.Similar results were observed by Uresti et al. (30) and Rodríguez et al. (34), when they added ALMP of similar characteristics (27-33 % degree of esterifi cation) to restructured fi sh products.

Colour att ributes
Acceptance of a new product relies mainly on its colour, being the consumer's fi rst perception.At 2.0 and 3.0 % addition, ALMP aff ected (p<0.05) the lightness (L*) of gels (Fig. 2), reducing this value from 86.0 (control) to 84.5 (1.7 % reduction) and 81.6 (5.1 % reduction), respectively.This is a signifi cant change given that the human eye can detect a diff erence of a unit (35).Although small diff erences (p<0.05) were detected in the a* and b* parameters with the fi bre addition, all treatments rendered very pale samples when these values (L*, a* and b*) were set in the tridimensional colour solid (36).However, the whiteness index, and L*, a* and b* integrating parameters were used to observe changes (Fig. 2) due to fi bre addition.According to the L* values, diff erences (p<0.05) were observed only when 2.0 and 3.0 % ALMP were added.The original fi bre was yellow, thus, the observed changes were associated with the increasing concentration of fi bre used, reducing the gel L* value and whiteness index, and increasing the b* parameter (more yellow) (Fig. 2).

Thermal behaviour of ALMP-squid protein complexes
Heat-set gelation of muscle proteins requires irreversible denaturation.This endothermic transition can be detected by diff erential scanning calorimetry (DSC).Generally, when a hydrocolloid is mixed with protein, it affects its thermal behaviour (37,38).Thus, DSC was used to elucidate the ALMP eff ect on jumbo squid protein thermal behaviour.In the present study, the control (sol-gel without added fi bre) and all treatments showed two endothermic peaks (Table 2), at around 46 and 71 °C, corresponding to myosin and actin, respectively (39).Fibre addition did not change (p≥0.05) the t onset of either of the endotherms, however, t max of both endotherms increased (p<0.05) with higher temperatures and increasing fi bre addition (i.e. by 1.3 °C at 3.0 % fi bre and 2.1 °C at 1.0 % fibre of endotherms 1 and 2, respectively).On the contrary, a signifi cant change (p<0.05) in net heat energy (ΔH) during thermal protein denaturation was observed only of endotherm 2 (actin), requiring more energy for denaturation as fi bre was added.The shift in jumbo squid protein thermal behaviour might be ascribed to the rupture of hydrogen bonds (40), formed by a protein-carbohydrate interaction because this type of fi bre is a high-molecular--mass complex polysaccharide, with considerable number of hydroxyl (-OH) groups (41).

Electrophoretic profi le
Fig. 3 shows the electropherogram of the jumbo squid mantle muscle gels with various mass fractions of added ALMP.The main myofi brillar proteins found were myosin heavy chain, heavy meromyosin, paramyosin (the characteristic protein only found in invertebrates) and actin.In the present study, gel functionality could be improved by: (i) the presence of protein-protein covalent crosslinking, promoted by intrinsic muscle transglutaminase (42,43), (ii) by the promotion of protein-carbohydrate noncovalent bonds or (iii) by a mixture of both.The electrophoretic patt ern confi rmed that the ALMP-induced changes in gelation and calorimetry were not a result of

Conclusions
The present study demonstrated that amidated low--methoxyl pectin (ALMP; 0.5-3.0%) is an excellent ingredient to improve jumbo squid mantle muscle protein functionality, increasing texture and water retention characteristics of the produced gels.However, further research is needed to confi rm which fi bre mass fraction is most adequate in terms of functionality and cost benefi t for a gel-type product.

Table 1 .
Gel-forming ability of jumbo squid mantle muscle gels formulated with various mass fractions of amidated low-methoxyl pectin (ALMP) c AA-A Values are presented as mean±standard deviation of six repetitions per sampling (N=3).Mean values in the same column with diff erent lett ers are signifi cantly (p<0.05)diff erent

Table 2 .
Calorimetric behaviour of jumbo squid mantle muscle gels formulated with various mass fractions of amidated low-methoxyl pectin (ALMP)Values are the mean±standard deviation of six repetitions per sampling (N=3).Mean values in the same column with diff erent lett ers are signifi cantly (p<0.05)diff erent.t onset =onset temperature, t max =temperature of maximum transition, ∆H=enthalpy of transition.The endothermic peaks 1 and 2 represent the endothermic transitions of myosin and actin denaturation, respectively covalent interactions between fi bre and/or proteins because no new high-molecular-mass bands appeared on the gel.