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Physical, chemical and rheological parameters of pressurized swai-fish (Pangasius hypophthalmus) emulsion incorporating fermented soybeans
FBP-534; No. of Pages 8
ARTICLE IN PRESS food and bioproducts processing x x x ( 2 0 1 4 ) xxx–xxx
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Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp
Physical, chemical and rheological parameters of pressurized swai-fish (Pangasius hypophthalmus) emulsion incorporating fermented soybeans Jiranat Techarang a , Arunee Apichartsrangkoon b,∗ a
Division of Food Science and Technology, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand b Postharvest Technology Research Institute, Chiang Mai University, Chiang Mai 50200, Thailand
a b s t r a c t The objective of this study was to investigate the effect of ultra-high pressure on the degree of denaturation of swai-fish emulsion. Accordingly, 90% swai-fish muscle was emulsified with 10% fermented soybeans (as a treatment) and was subjected to high pressure at 400, 500, and 600 MPa for 20 and 40 min at 25 ◦ C. After that, their viscoelastic, physical, chemical and microbiological properties were examined in comparison with those of pressurized 100% swai-fish emulsion (as a control). The results showed that increase of pressure significantly augmented water holding capacity, gel strength and whiteness index of both controls and treatments, while DSC thermograms demonstrated that swai-fish protein was denatured at pressure 400 MPa. However, to eliminate most undesirable microorganism needed higher pressure level. The viscoelastic characterization showed that all storage and loss moduli increased according to the pressure severity which suggested that more cross-link densities were formed at higher levels of pressurization. Furthermore, the electrophoregrams had specified such linking as hydrophobic and disulphide interactions. Some evidences of interaction between fish and soy proteins also indicated by the electrophoregrams. © 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Viscoelastic characterization; Ultra-high pressure; Fermented soybeans; Swai-fish emulsion; SDS-PAGE electrophoregrams
1.
Introduction
Iridescent shark (Pangasius hypophthalmus) or “swai” as its Thai name is classified under the species of shark catfish (family Pangasiidae) (NaNakorn and Moeikum, 2009). Because of its tender flesh and little small bones, swai gains popularity among consumers who appreciate fish filets. Since swai provides various desirable qualities such as low cholesterol levels (21–39 mg/100 g), absence of fishy odor, good flavor after being cooked (Orban et al., 2008) and reasonable price, this fresh water fish has gained not only high economical values but also an excellent source of protein. To develop a high-quality swai flesh emulsion, plant protein such as fermented soybeans thua nao or natto and miso could be simply fortified. Generally, thua nao contains significant amounts of essential amino acids and some phytoestrogen like isoflavones (Dajanta et al., 2009, 2011). Upon fermentation
by Bacillus subtilis TN51, cooked soybeans increased in total phenolic contents, total free amino acids and essential amino acids by 21%, 644% and 583%, respectively, while total isoflavones including glucosides (daidzin, glycin, and genistin) and aglycones (daidzein, glycitein, and genistein) increased roughly 43% and 300%, respectively. To improve flavor and strong odor of thua nao, miso was also incorporated in the swai-fish emulsion. Yamabe et al. (2007) found that the fermentation of rice-koji miso after 6 months, glycosides decreased from 86.4% to 44.9% but aglycones increased from 9.6% to 53.3%. Protein of fish emulsion is commonly coagulated by heat. Ultrahigh pressure is an alternative technique to enable denaturation of fish protein, which is different from heat. Apichartsrangkoon (2003) reported that pressurized soy-protein gels appeared more shining, more elastic, smoother and softer structure than those heated samples, because pressure enhanced differently conformational structure
∗
Corresponding author. Tel.: +66 53944031; fax: +66 53944031. E-mail addresses: [email protected], [email protected] (A. Apichartsrangkoon). Received 1 April 2014; Received in revised form 28 July 2014; Accepted 2 September 2014 http://dx.doi.org/10.1016/j.fbp.2014.09.002 0960-3085/© 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Techarang, J., Apichartsrangkoon, A., Physical, chemical and rheological parameters of pressurized swai-fish (Pangasius hypophthalmus) emulsion incorporating fermented soybeans. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.002
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of protein. In addition, Supavititpatana and Apichartsrangkoon (2007) found that a pressure of 700 MPa brought about a complete denaturation of ostrich-meat protein with good water holding capacity. Chattong and Apichartsrangkoon (2009) applied pressure 200, 400 and 600 MPa with 40 and 50 ◦ C for 40 and 60 min for setting ostrich-meat sausage and found that the products responded as weak viscoelastic gel with the predominance of an elastic component. One of the principle concepts for characterizing the viscoelastic behavior of food emulsion is the application of oscillatory stress sweep. Apichartsrangkoon and Ledward (2002) investigated the viscoelastic behavior of pressurized gluten–soy mixtures, and found that the loss tangents of the gels were less than one indicating a solid-like behavior, probably due to the increase of cross-linking with increasing severity of the treatments. Saowapark et al. (2008) examined the viscoelastic properties of pressurized tofu gels and found that these gels showed stronger or had a greater elasticity than those of the equivalent heated gels. In this study, the effects of high pressure on dynamic viscoelastic, physical, chemical and microbiological properties of swai-fish emulsion incorporating fermented soybeans (90% swai muscle, 7% thua nao, and 3% miso) compared with 100% swai-fish emulsion as a control were investigated.
2.
Materials and methods
2.1.
Materials
pressurized fish emulsions. Accordingly, the instrument was calibrated using indium. A weight of 10 ± 3 mg emulsion was heated from 30 to 100 ◦ C at a scanning rate of 10 ◦ C/min using an empty sealed pan as a reference. All samples were rescanned to assess reversibility and at least 10 runs per sample were determined.
2.4.
Determination of water holding capacity
2.4.1.
Released water
A proportion of released water of the product was the weight of sample left after blotting water from the surface following the method described by Funami et al. (1998). Sample with casing was weighed (A) and after removing the casing, the surface water of both product (B) and casing (C) was wiped with filter paper (Whatman No. 2, 90 mm in diameter) then reweighed (B, C). The proportion of released water was calculated according to Eq. (1): Released water =
2.4.2.
(A − B) − C A−C
(1)
Expressible water
The swai-fish muscle with proximate compositions of 75.50% moisture, 15.88% protein and 9.69% fat (AOAC, 2000) was purchased from a market in Chiangmai, Thailand. An alkaline fermented soybean (thua nao) was produced following a modified method of Dajanta et al. (2011) using Bacillus subtilis TISTR001 as a pure starter culture. The proximate compositions of the prepared thua nao were 36.63% moisture, 39.75% protein, and 16.68% fat (AOAC, 2000). Rice-koji miso (Ken Co. Ltd., Japan) with proximate compositions of 35.21% moisture, 27.04% protein and 11.07% fat (AOAC, 2000) was purchased from a market in Chiangmai, Thailand.
A proportion of expressible water of the product was measured as described by Funami et al. (1998). The samples were cut into 15 mm thickness then placed between double layers of filter paper (Whatman No. 2, 90 mm in diameter) and subsequently subjected to compression using a Texture Analyzer TA-XT Plus (Stable Micro Systems Ltd., Surrey, UK) with a cylindrical aluminum probe (50 mm in diameter). The measurement was performed with a 50 kg load cell at a cross-head speed of 3 mm/s to 70% strain for 60 s. The proportion of expressible water was calculated as the ratio of apparent expressible water to the total moisture content measured by the method of AOAC (2000). The proportion of expressible water was calculated according to Eq. (2):
2.2.
Expressible water =
Preparation of pressurized fish emulsions
The preparation of swai batters was divided for the control using 100% (w/w) chopped swai-muscle and for the treatment using 90% (w/w) chopped swai-muscle plus 7% (w/w) thua nao and 3% (w/w) miso of which this formula was selected earlier. The control or treatment was blended for 6 min with 1% (w/w) sodium chloride and 0.3% (w/w) sodium tripolyphosphate using a blender (Model HR1372, Philips Electronics Thailand Limited). Subsequently, all fish emulsions were stuffed in collagen casing (2.3 cm in diameter, Food EQ Ltd., Thailand) prior to pressurization. The stuffed emulsions were then subjected to ultra-high pressure at 400, 500, and 600 MPa for 20 and 40 min at 25 ◦ C. The high pressure rig was a model ‘Food Lab’ 900 (Stansted Fluid Power, Essex, UK) with increasing pressure rate of about 330 MPa/min. The pressure transmitting medium was a mixture of castor oil and 98% ethanol (Chemical & Lab Supplies, Thailand) at the ratio of 20:80 (v/v). After pressurization, the products were kept at 4 ◦ C until use.
2.3. Analysis of thermal denaturation of swai-fish emulsion Differential scanning calorimeter (Perkin-Elmer Diamond DSC, USA) was used to examine thermal denaturation of swai proteins (myosin and actin) in the unpressurized and
App TM
(2)
where App = apparent expressible water, TM = total moisture content of fish emulsion. Apparent expressible water =
W1 − W2 W1
(3)
where W1 = weight before compression, W2 = weight after compression. Water holding capacity was calculated by combining values of released water plus expressible water subtracted from 1 (×100).
2.5.
Determination of gel strength
Gel strength of the fish emulsions was determined using a Texture Analyzer TA-XT Plus (Stable Micro Systems Ltd., Surrey, UK) with a 50 kg load cell. The samples without casing were cut into pieces of 23 mm in diameter × 20 mm length. For this purpose, shear force was determined using a Warner Blatzler blade moved perpendicularly toward the samples with a distance of 40 mm using 10 mm/s cross-head speed. The maximum peak of shear force was recorded. Gel strength was then calculated by multiplying shear force (N) with the distance of shearing (m) (Supavititpatana and Apichartsrangkoon, 2007).
Please cite this article in press as: Techarang, J., Apichartsrangkoon, A., Physical, chemical and rheological parameters of pressurized swai-fish (Pangasius hypophthalmus) emulsion incorporating fermented soybeans. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.002
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G' and G" (Pa)
food and bioproducts processing x x x ( 2 0 1 4 ) xxx–xxx
3
1000
G' of treatment (600 MPa/ 40 min) G" of treatment (600 MPa/ 40 min) G' of control (400 MPa/ 20 min) G" of control (400 MPa/ 20 min)
100
0.2
2
20
200
Oscillatory stress (Pa) Fig. 1 – Stress amplitude sweep (0.1–500 Pa) at a frequency of 1 Hz of the controls (400 MPa/20 min) and the treatments (600 MPa/40 min).
2.6.
Color measurement
A colorimeter (Minolta Chroma Meter, CR-300, Japan) was used with a white plate standard to measure the color of the pressurized emulsions. Color parameters [L (lightness), a* (redness), and b* (yellowness)] were then measured and used to calculate whiteness index (WI = 100 − [(100 − L*)2 + a*2 + b*2 ]1/2 (Sánchez-Alonso et al., 2007).
2-mercaptoethanol/100 mL water and boiled for 10 min for reducing protein (Apichartsrangkoon and Ledward, 2002).
2.9.
Standard plate count of pressurized fish emulsions was performed following the AOAC method no. 966.23 (AOAC, 2000).
2.10. 2.7.
Dynamic viscoelastic characterization
Dynamic viscoelastic behavior of the fish emulsions was measured following a method of Chattong and Apichartsrangkoon (2009) with some modification. Intentionally, a controlled stress rheometer (Advance Rheometer AR2000, TA Instruments-Waters LLC, New Castle, USA) equipped with a 25 mm parallel plate measuring system with 1 mm gap width was used. Stress sweeps were initially performed at a frequency of 1 Hz for all samples in order to ensure that all measurements were carried out within a linear viscoelastic region. Samples of the typical plots are shown in Fig. 1. Based on the finding, stress amplitude of 10 Pa was chosen for further frequency-sweep measurement. The edges of samples were covered with light mineral oil (Sigma–Aldrich Co., Ltd., Gillingham, UK) to prevent from drying out. Oscillatory frequency-sweeps were carried out at 25 ◦ C with a frequency range of 0.1–10 Hz. Consequently, storage (G ) and loss (G ) moduli as well as loss tangent were obtained.
2.8.
Electrophoretic analysis
Sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed following a method of Chattong and Apichartsrangkoon (2009) using an electrotransfer unit (Bio-Rad Laboratories Ltd., Waukegan, Illinois, USA). A weight of 0.05 g sample was dissolved in 1 mL of 2 M Tris–HCl (pH 6.8) buffer containing 40 g SDS/100 mL water and 2 g bromophenol blue/100 mL water. Subsequently, 10 L of extracted sample was loaded to each well of 7.5% gel. High range standards (BioRad Laboratories Ltd., Hercules, California, USA) were used to compare the molecular weights. The gels with protein bands were stained with 1 g Coomassie Blue R-250 (USB Corporation, UK)/100 mL water. The samples were dissolved in 2 mL
Microbiological examination
Statistical analysis
Statistical design was 3 × 2 factorial experiment in CRD consisted of pressure 400, 500, 600 MPa and holding times 20 and 40 min. Means of the controls and treatments of each examination were subjected to analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) was applied to analyze the differences among variables (P ≤ 0.05). All data were analyzed using SPSS 17 software (SPSS Inc., Chicago, USA).
3.
Results and discussions
3.1.
Examination of thermal denaturation
Fig. 2 shows DSC thermograms of 100% swai-fish emulsion (control), emulsion of 90% swai-fish plus 7% thua nao and 3% miso (treatment), pressurized 100% swai-fish emulsion at 400 MPa for 20 min and rescanned thermogram of the control. Two discernible peaks of the control was found at the transition temperatures 56.09 (H = 0.25 J/g) and 68.09 ◦ C (H = 0.18 J/g), respectively, which corresponded to the transition temperatures of myosin and actin, while the transition temperatures of the endothermic peaks for the treatment were 55.51 ◦ C (H = 0.17 J/g) and 66.96 ◦ C (H = 0.13 J/g), respectively (Fig. 2). These suggested that incorporating fermented soy proteins in the fish emulsion induced slight shift of the transition temperatures, presumably due to the effect of interaction between fish and soy proteins which was better clarified by the gel electrophoregrams in Fig. 4. After rescanning, all endothermic peaks had disappeared, presumably due to a complete denaturation of both proteins from earlier scanning. Other findings for example, Matos et al. (2011) reported that myosin and actin denaturation peaks of gilthead-sea bream (Sparus aurata) muscle were in the range of 47–50 ◦ C and 73–75 ◦ C; in addition, the endothermic peaks of Japanese-sting fish (Sebastes inermis) dorsal muscle were 40.9 ◦ C for myosin and 61.1 ◦ C for actin (Takeshi et al., 1999).
Please cite this article in press as: Techarang, J., Apichartsrangkoon, A., Physical, chemical and rheological parameters of pressurized swai-fish (Pangasius hypophthalmus) emulsion incorporating fermented soybeans. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.002
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Fig. 2 – DSC thermograms of unpressurized and pressurized samples at 400 MPa for 20 min obtained at a scanning rate of 10 ◦ C/min. Nonetheless, the endothermic peaks of myosin and actin for pressurized (400 MPa/20 min) control and treatment were not found, which might be due to the impacts of pressure triggering a complete denaturation. This result was matched with the work of Angsupanich and Ledward (1998) showing that myosin, actin and most of the sarcoplasmic proteins of cod muscle were denatured by low levels of pressure 100–300 MPa.
3.2.
Determination of water holding capacity
Table 1 shows that water holding capacities (WHC) of the controls and treatments were significantly increased (P ≤ 0.05) with increasing pressure levels regardless of the holding time. Moreover, the effect of pressure levels was more prominent beyond 500 MPa indicating by significantly higher (P ≤ 0.05) WHC of both controls and treatments compared to those of lower pressurized samples. This suggested that the firm structure had been set at 500 MPa. However, WHC of the controls and treatments were not different, in other words, composition of the emulsion had no effect on WHC. Angsupanich et al. (1998) stated that during pressurization, high pressure induced a partial unfolding of myosin and promoted non-covalent interactions between the subunits such as disulphide, hydrophobic, hydrogen, and ionic bonds. On releasing pressure, a flexible network which could entrap water was formed. Similarly, Chattong and Apichartsrangkoon (2009) and Supavititpatana and Apichartsrangkoon (2007) pressurized
ostrich-meat sausages and found that the levels of pressure significantly influenced WHC of the products. Apart from pressure, the salt content also had some effects on WHC of the pork emulsion (Ma et al., 2012).
3.3.
Determination of gel strength
Similar to WHC, the levels of pressure significantly took an influence (P ≤ 0.05) on the gel strength of the controls and treatments, whereas pressurized samples at 600 MPa received the highest gel strength. On the other hand, the holding time showed less effect on gel strength (Table 1). However, on a precise clarification of each pressure level, pressure 600 MPa/20 min onward had settled texture of the controls and treatments. When comparing between the controls and treatments, gel strength of the treatments was slightly higher than those of the controls, presumably due to the impacts of the interactions among fish, thua nao and miso proteins which could be more specified by electrophoregrams in Fig. 4. Previous study elucidated that pressurized ostrich-meat sausage at 300–700 MPa with temperature 40 and 60 ◦ C for 40 and 60 min exhibited rise of gel strength according to the treatment severities (Supavititpatana and Apichartsrangkoon, 2007). Chen et al. (2010) studied the effects of pressure levels (0–500 MPa) and holding time (10–40 min) on textural properties of duck muscle gels containing 1% curdlan and noticed that the hardness, springiness, cohesiveness and chewiness of the gels
Table 1 – Physical properties of pressurized fish emulsions for 100% fish muscle (controls) and 90% fish muscle plus 7% thua nao and 3% miso (treatments). High pressure conditions
Water holding capacity (%) Controls
400 MPa 20 min 400 MPa 40 min 500 MPa 20 min 500 MPa 40 min 600 MPa 20 min 600 MPa 40 min
87 88 91 91 92 92
± ± ± ± ± ±
1.1b 2.7b 1.7a 1.7a 0.9a 1.4a
Treatments 86 87 89 90 91 91
± ± ± ± ± ±
1.3c 1.7c 1.8b 0.8ab 0.8a 1.4a
Gel strength (N m) Controls 0.6 0.7 0.7 0.7 0.8 0.8
± ± ± ± ± ±
0.1b 0.1b 0.1b 0.1b 0.0a 0.0a
Treatments 0.7 0.7 0.7 0.8 0.9 0.9
± ± ± ± ± ±
0.1b 0.1b 0.1b 0.1ab 0.1a 0.1a
Whiteness index Controls 80 81 82 83 84 84
± ± ± ± ± ±
0.7c 0.6bc 0.7b 1.4ab 0.5a 1.0a
Treatments 67 68 71 71 72 72
± ± ± ± ± ±
1.0b 1.0b 1.0a 1.0a 0.4a 1.0a
Means within each column followed by the same letters have no significant difference (P > 0.05). Each parameter is an average of three replications.
Please cite this article in press as: Techarang, J., Apichartsrangkoon, A., Physical, chemical and rheological parameters of pressurized swai-fish (Pangasius hypophthalmus) emulsion incorporating fermented soybeans. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.002
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a) Controls G'/ 400 MPa/ 20 min (k=0.16)
G' and G" (Pa)
G"/ 400 MPa/ 20 min (k=0.20) G'/ 400 MPa/ 40 min (k=0.16) G"/ 400 MPa/ 40 min (k=0.19)
1000
G'/ 500 MPa/ 20 min (k=0.15) G"/ 500 MPa/ 20 min (k=0.17) G'/ 500 MPa/ 40 min (k=0.15) G"/ 500 MPa/ 40 min (k=0.16) G'/ 600 MPa/ 20 min (k=0.14) G"/ 600 MPa/ 20 min (k=0.15) G'/ 600 MPa/ 40 min (k=0.13) G"/ 600MPa/ 40 min (k=0.15)
100
1
0.1
10
Frequency (Hz)
b) Treatments G' 400 MPa/ 20 min (k=0.15)
G' and G" (Hz)
G" 400 MPa/ 20 min (k=0.16) G' 400 MPa/ 40 min (k=0.15) G" 400 MPa/ 40 min (k=0.15)
1000
G' 500 MPa/ 20 min (k=0.15) G" 500 MPa/ 20 min (k=0.14) G' 500 MPa/ 40 min (k=0.15) G" 500 MPa/ 40 min (k=0.14) G' 600 MPa/ 20 min (k=0.14) G" 600 MPa/ 20 min (k=0.13) 100
G' 600 MPa/ 40 min (k=0.14) 1
0.1
10
G" 600 MPa/ 40 min (k=0.13)
Frequency (Hz) Fig. 3 – Typical plots of storage (filled symbols) and loss (unfilled symbols) moduli of pressurized samples at 400, 500 and 600 MPa for 20 and 40 min; k = slopes of each actual plot of the controls (a) and the treatments (b). were increased (P ≤ 0.05) above 300 MPa while the holding time had no significant influence.
3.4.
pressure, while the holding time had also no influence on this attribute. It was noteworthy that the WI of pressurized controls and treatments lay in the range of 80–84 and 67–72, respectively, whereas the WI for unpressurized (raw muscle) control and treatment were 69 and 62, respectively. The sharp increase of WI after pressurization triggered off the loss of translucency from unpressurized state to more opaquewhite-cooked color, indicating that the fish protein had been undergone denaturation by pressure 400 MPa onward.
Color measurement
Different colors of the swai-fish emulsions had been identified by whiteness index (WI) calculated from L, a* and b* parameters. Table 1 shows that WI of the controls and treatments significantly increased (P ≤ 0.05) with the increase of
Table 2 – The loss tangent measured at a frequency of 1 Hz and standard plate count of pressurized fish emulsions for 100% fish muscle (controls) and 90% fish muscle plus 7% thua nao and 3% miso (treatments). Loss tangent (G /G )
High pressure conditions
Controls 400 MPa 20 min 400 MPa 40 min 500 MPa 20 min 500 MPa 40 min 600 MPa 20 min 600 MPa 40 min
0.25 0.25 0.24 0.22 0.22 0.22
± ± ± ± ± ±
0.01a 0.01a 0.00a 0.01b 0.01b 0.01b
Standard plate count (log CFU/g)
Treatments 0.28 0.25 0.24 0.24 0.23 0.23
± ± ± ± ± ±
0.01a 0.01b 0.00c 0.01cd 0.01d 0.00d
Controls 3.0 ± 2.4 ± 2.0 ± 1.4 ± <1e <1e
0.1a 0.1b 0.1c 0.1d
Treatments 3.9 3.9 3.8 3.7 3.6 3.6
± ± ± ± ± ±
0.0a 0.2a 0.0b 0.0c 0.0d 0.0d
Means within each column followed by the same letters have no significant difference (P > 0.05). Each parameter is an average of three replications.
Please cite this article in press as: Techarang, J., Apichartsrangkoon, A., Physical, chemical and rheological parameters of pressurized swai-fish (Pangasius hypophthalmus) emulsion incorporating fermented soybeans. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.002
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However, this finding agreed with the DSC thermograms in 3.1. Previously, Angsupanich and Ledward (1998) stated that pressure of 200 MPa enabled the denaturation of cod protein by changing L values with an opaque appearance compared to that of unpressurized cod muscle. To sum up, as comparison between the control and treatment batches, the treatments displayed markedly lower WI than those of the controls, presumably due to the addition of fermented soybeans (thua nao and miso) in the treatments which were visualized darker than the fish muscle.
3.5.
Dynamic viscoelastic characterization
Fig. 3 shows the plots of elastic or storage (G ) and viscous or loss (G ) moduli against frequency 0.1–10 Hz. The plots of G were greater than G over most of the measured frequency range and the ratio of G to G of each plot was nearly 10, indicating a rigid-gel-like structure for which all gels gave the frequency profiles expected for weak viscoelastic systems (Saowapark et al., 2008). Such profiles were consistent with the
behavior of a material formed by the specified cross linking of the denatured proteins such as hydrophobic and disulphide interaction as described in Section 3.6. All plots of storage and loss moduli for the controls and treatments were frequency dependent, but the slopes were lower for those treated at higher levels indicated by the reduction of slopes or k values (Fig. 3). Moreover, higher G and G of fish emulsions treated at a higher pressure and a holding time suggested that the higher pressure and the holding time gave rise to stronger gel system with more cross-link densities (Apichartsrangkoon and Ledward, 2002), consequently, these agreed with the gel strength in Table 1. Similarly, loss tangent (G /G ) at 1 Hz in Table 2 of the controls and treatments significantly reduced (P ≤ 0.05) with the increase of pressure and holding time, which were complied with the slopes. In addition, the pressure of 500 MPa/40 min was a critical level of these alleviations, meaning that this pressure enabled setting of firm viscoelastic structure. Apichartsrangkoon (2002) also studied the viscoelastic properties of heated gluten/soy protein gels and found that lower loss tangent or less
Fig. 4 – SDS-PAGE electrophoregrams of the controls (a) and reduced with 2-mercaptoethanol (c); the treatments (b) and reduced with 2-mercaptoethanol (d). Please cite this article in press as: Techarang, J., Apichartsrangkoon, A., Physical, chemical and rheological parameters of pressurized swai-fish (Pangasius hypophthalmus) emulsion incorporating fermented soybeans. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.002
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frequency dependence of the shear moduli indicated a stronger gel structure with more cross-link densities. It was noticed that the treatments with the addition of thua nao and miso (Fig. 3b) brought about a higher overall moduli than those of the controls (100% swai muscle, Fig. 3a), which could be less moisture in the treatments (∼80%) than the controls (∼83%). Nonetheless, this finding also agreed with the gel strength (Table 1).
inactivate most of the undesirable microbes with the presence of less than 10 CFU/g left. Stewart et al. (2000) revealed that pressure 404 MPa/70 ◦ C/15 min could reduce B. subtilis 168 by 5 log cycles at pH 6.0–7.0, while the pressurization with the same condition at 25 ◦ C could inactivate this bacteria by less than 0.5 log cycle. In addition, Gao et al. (2006) stated that pressure 576.0 MPa/87 ◦ C/13 min gave rise to inactivate spores of B. subtilis As.1.1731 in milk buffers by 6 log cycles.
3.6.
4.
Electrophoretic analysis
Pressure took an effect on protein denaturation, i.e., the disruption of hydrophobic and electrostatic interactions, enhancing hydrogen bonding, and promoting formation of disulphide bonds, consequently, the proteins were denatured and aggregated leading to the improved gelation or protein structure modification (Galazka et al., 2000). In this context, the extent of swai-fish protein denaturation had been shown by SDS-PAGE electrophoregrams in Fig. 4. The electrophoregrams in Fig. 4 displayed that hydrophobic interactions of myosin (200 kDa) or actin (45 kDa) for the pressurized controls (Fig. 4a) and treatments (Fig. 4b) (lanes 2–7) were dissociated in the presence of SDS by dissolving band densities of the myosin but increasing band densities of the lower molecular weight protein (between 66 and 97 kDa and lower than 45 kDa) as compared to the unpressurized samples (lane 1). It was noticed in particular that all myosins at 200 kDa were present as thick bands (Fig. 4a and b) presumably not being dissolved by SDS. After the addition of a reducing agent (2mercaptoethanol), these bands were thinner (Fig. 4c and d) which could be due to the dissociation of disulphide bond. All these phenomena indicated that the pressure denaturation of swai-fish proteins might be dealt with hydrophobic and disulphide interaction. In addition, the distinct proteinbands occurred especially between the molecular weight of 66 kDa and 97 kDa of the treatment (Fig. 4b and d), while the electrophoregrams of the controls (Fig. 4a and c) had bands with much less density. These suggested that upon pressurization, soy proteins present in the treatments had shown some degree of interaction with fish protein in the specified bands. A previous finding by Chattong and Apichartsrangkoon (2009) illustrated that the structural changes of pressurized ostrichmeat emulsion might be associated with the hydrophobic and disulphide interactions.
3.7.
Microbiological examination
Table 2 shows standard plate counts of the pressurized controls and treatments. Treatments with the addition of thua nao and miso exhibited very much higher microbial counts than those of the controls. It was obvious that thua nao was fermented by spore-forming bacteria (Bacillus subtilis TISTR001) which could resist pressure up to 600 MPa, hence the existence of these high viable counts after pressurization could be some survival B. subtilis. However, the residuals of survival B. subtilis in the pressurized emulsion were roughly 10–1000 CFU/g, which could enhance the emulsion with slimy appearance during storage. The effects of pressure levels and two holding time on the standard plate counts of the controls showed significant difference (P ≤ 0.05), but the holding time was less influent on the treatments. These meant that higher pressure level with longer holding time would efficiently reduce the undesirable microbes except B. subtilis. To sum up, the pressure beyond 600 MPa would satisfactorily
Conclusion
The DSC thermograms revealed that pressure 400 MPa/20 min was able to denature fish protein, while the levels of pressure significantly affected other parameters including water holding capacity, gel strength and whiteness index of both controls (100% swai-fish emulsion) and treatments (emulsion of 90% swai fish plus 7% thua nao and 3% miso) regardless the holding time. The increase of these parameters was in accordance with raising pressure. The viscoelastic characterization illustrated that the storage modulus was greater than the loss modulus over most of the measured frequency range. All storage and loss moduli were frequency dependent, whereas the loss tangent at 1 Hz significantly decreased with the increase of pressure and time. These indicated that more cross-link densities or solid-like structure occurred with the products treated at higher levels. electrophoregrams elucidated that the pressure induced denaturation of swai-fish proteins with the association of hydrophobic and disulphide interactions. Some evidences of interaction between fish and soy proteins also indicated by the electrophoregrams. Results of microbiological examination showed that pressure beyond 600 MPa was efficiently eliminated undesirable microbes, however, this pressure could not completely inactivate spore-forming bacteria such as B. subtilis in fermented thua nao.
Acknowledgements The authors wish to thank National Research Council of Thailand and Chiang Mai University, Chiang Mai, Thailand for their financial support. We also would like to express our appreciation to Associate Professor Dr. Saranya Savetamalya, Department of English, Chiangmai University in editing the final version of this paper.
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Chen, C., Wang, R., Sun, G., Fang, H., Ma, D., Yi, S., 2010. Effects of high pressure level and holding time on properties of duck muscle gels containing 1% curdlan. Innov. Food Sci. Emerg. Technol. 11 (4), 538–542. Dajanta, K., Chukeatirote, E., Apichartsrangkoon, A., Frazier, R.A., 2009. Enhanced aglycone production of fermented soybean products by Bacillus species. Acta Biol. Szeged. 53 (2), 93–98. Dajanta, K., Apichartsrangkoon, A., Chukeatirote, E., Frazier, R.A., 2011. Free-amino acid profiles of thua nao, a Thai fermented soybean. Food Chem. 125 (2), 342–347. Funami, T., Yada, H., Nakao, Y., 1998. Thermal and rheological properties of curdlan gel in minced pork gel. Food Hydrocoll. 12 (1), 55–64. Galazka, V.B., Dickinson, E., Ledward, D.A., 2000. Influence of high pressure processing on protein solutions and emulsions. Curr. Opin. Colloid Interface Sci. 5 (3–4), 182–187. Gao, Y.-L., Ju, X.-R., Jiang, H.-H., 2006. Studies on inactivation of Bacillus subtilis spores by high hydrostatic pressure and heat using design of experiments. J. Food Eng. 77 (3), 672–679. Ma, F., Chen, C., Sun, G., Wang, W., Fang, H., Han, Z., 2012. Effects of high pressure and CaCl2 on properties of salt-soluble meat protein gels containing locust bean gum. Innov. Food Sci. Emerg. Technol. 14, 31–37. Matos, E., Silva, T.S., Tiago, T., Aureliano, M., Dinis, M.T., Dias, T., 2011. Effect of harvesting stress and storage conditions on protein degradation in fillets of farmed gilthead-sea bream (Sparus aurata): a differential scanning calorimetry study. Food Chem. 126 (1), 270–276. Na-Nakorn, U., Moeikum, T., 2009. Genetic diversity of domesticated stocks of striped catfish, Pangasianodon
hypophthalmus (Sauvage 1878), in Thailand: relevance to broodstock management regimes. Aquaculture 297 (1–4), 70–77. Orban, E., Nevigato, T., Lena, G.D., Masci, M., Casini, I., Gambelli, L., Caproni, R., 2008. New trends in the seafood market. Sutchi catfish (Pangasius hypophthalmus) fillets from Vietnam: nutritional quality and safety aspects. Food Chem. 110 (2), 383–389. Sánchez-Alonso, I., Haji-Maleki, R., Borderias, A.J., 2007. Wheat fiber as a functional ingredient in restructured fish products. Food Chem. 100 (3), 1037–1043. Saowapark, S., Apichartsrangkoon, A., Bell, A.E., 2008. Viscoelastic properties of high pressure and heat induced tofu gels. Food Chem. 107 (3), 984–989. Stewart, C.M., Dunne, C.P., Sikes, A., Hoover, D.G., 2000. Sensitivity of spores of Bacillus subtilis and Clostridium sporogenes PA 3679 to combinations of high hydrostatic pressure and other processing parameters. Innov. Food Sci. Emerg. Technol. 1 (1), 49–56. Supavititpatana, T., Apichartsrangkoon, A., 2007. Combination effects of ultra-high pressure and temperature on the physical and thermal properties of ostrich meat sausage (yor). Meat Sci. 76 (3), 555–560. Takeshi, Kurata, M., Nakamura, T., Ito, T., Fujiki, K., Nakao, M., Yano, T., 1999. Properties of myofibrillar protein from Japanese stingfish (Sebastes inermis) dorsal muscle. Food Res. Int. 32 (6), 401–405. Yamabe, S., Kobayashi-Hattori, K., Kaneko, K., Endo, H., Takita, T., 2007. Effect of soybean varieties on the content and composition of isoflavone in rice-koji miso. Food Chem. 100 (1), 369–374.
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Contents lists available at ScienceDirect
Food and Bioproducts Processing journal homepage: www.elsevier.com/locate/fbp
Physical, chemical and rheological parameters of pressurized swai-fish (Pangasius hypophthalmus) emulsion incorporating fermented soybeans Jiranat Techarang a , Arunee Apichartsrangkoon b,∗ a
Division of Food Science and Technology, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai 50100, Thailand b Postharvest Technology Research Institute, Chiang Mai University, Chiang Mai 50200, Thailand
a b s t r a c t The objective of this study was to investigate the effect of ultra-high pressure on the degree of denaturation of swai-fish emulsion. Accordingly, 90% swai-fish muscle was emulsified with 10% fermented soybeans (as a treatment) and was subjected to high pressure at 400, 500, and 600 MPa for 20 and 40 min at 25 ◦ C. After that, their viscoelastic, physical, chemical and microbiological properties were examined in comparison with those of pressurized 100% swai-fish emulsion (as a control). The results showed that increase of pressure significantly augmented water holding capacity, gel strength and whiteness index of both controls and treatments, while DSC thermograms demonstrated that swai-fish protein was denatured at pressure 400 MPa. However, to eliminate most undesirable microorganism needed higher pressure level. The viscoelastic characterization showed that all storage and loss moduli increased according to the pressure severity which suggested that more cross-link densities were formed at higher levels of pressurization. Furthermore, the electrophoregrams had specified such linking as hydrophobic and disulphide interactions. Some evidences of interaction between fish and soy proteins also indicated by the electrophoregrams. © 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
Keywords: Viscoelastic characterization; Ultra-high pressure; Fermented soybeans; Swai-fish emulsion; SDS-PAGE electrophoregrams
1.
Introduction
Iridescent shark (Pangasius hypophthalmus) or “swai” as its Thai name is classified under the species of shark catfish (family Pangasiidae) (NaNakorn and Moeikum, 2009). Because of its tender flesh and little small bones, swai gains popularity among consumers who appreciate fish filets. Since swai provides various desirable qualities such as low cholesterol levels (21–39 mg/100 g), absence of fishy odor, good flavor after being cooked (Orban et al., 2008) and reasonable price, this fresh water fish has gained not only high economical values but also an excellent source of protein. To develop a high-quality swai flesh emulsion, plant protein such as fermented soybeans thua nao or natto and miso could be simply fortified. Generally, thua nao contains significant amounts of essential amino acids and some phytoestrogen like isoflavones (Dajanta et al., 2009, 2011). Upon fermentation
by Bacillus subtilis TN51, cooked soybeans increased in total phenolic contents, total free amino acids and essential amino acids by 21%, 644% and 583%, respectively, while total isoflavones including glucosides (daidzin, glycin, and genistin) and aglycones (daidzein, glycitein, and genistein) increased roughly 43% and 300%, respectively. To improve flavor and strong odor of thua nao, miso was also incorporated in the swai-fish emulsion. Yamabe et al. (2007) found that the fermentation of rice-koji miso after 6 months, glycosides decreased from 86.4% to 44.9% but aglycones increased from 9.6% to 53.3%. Protein of fish emulsion is commonly coagulated by heat. Ultrahigh pressure is an alternative technique to enable denaturation of fish protein, which is different from heat. Apichartsrangkoon (2003) reported that pressurized soy-protein gels appeared more shining, more elastic, smoother and softer structure than those heated samples, because pressure enhanced differently conformational structure
∗
Corresponding author. Tel.: +66 53944031; fax: +66 53944031. E-mail addresses: [email protected], [email protected] (A. Apichartsrangkoon). Received 1 April 2014; Received in revised form 28 July 2014; Accepted 2 September 2014 http://dx.doi.org/10.1016/j.fbp.2014.09.002 0960-3085/© 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
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of protein. In addition, Supavititpatana and Apichartsrangkoon (2007) found that a pressure of 700 MPa brought about a complete denaturation of ostrich-meat protein with good water holding capacity. Chattong and Apichartsrangkoon (2009) applied pressure 200, 400 and 600 MPa with 40 and 50 ◦ C for 40 and 60 min for setting ostrich-meat sausage and found that the products responded as weak viscoelastic gel with the predominance of an elastic component. One of the principle concepts for characterizing the viscoelastic behavior of food emulsion is the application of oscillatory stress sweep. Apichartsrangkoon and Ledward (2002) investigated the viscoelastic behavior of pressurized gluten–soy mixtures, and found that the loss tangents of the gels were less than one indicating a solid-like behavior, probably due to the increase of cross-linking with increasing severity of the treatments. Saowapark et al. (2008) examined the viscoelastic properties of pressurized tofu gels and found that these gels showed stronger or had a greater elasticity than those of the equivalent heated gels. In this study, the effects of high pressure on dynamic viscoelastic, physical, chemical and microbiological properties of swai-fish emulsion incorporating fermented soybeans (90% swai muscle, 7% thua nao, and 3% miso) compared with 100% swai-fish emulsion as a control were investigated.
2.
Materials and methods
2.1.
Materials
pressurized fish emulsions. Accordingly, the instrument was calibrated using indium. A weight of 10 ± 3 mg emulsion was heated from 30 to 100 ◦ C at a scanning rate of 10 ◦ C/min using an empty sealed pan as a reference. All samples were rescanned to assess reversibility and at least 10 runs per sample were determined.
2.4.
Determination of water holding capacity
2.4.1.
Released water
A proportion of released water of the product was the weight of sample left after blotting water from the surface following the method described by Funami et al. (1998). Sample with casing was weighed (A) and after removing the casing, the surface water of both product (B) and casing (C) was wiped with filter paper (Whatman No. 2, 90 mm in diameter) then reweighed (B, C). The proportion of released water was calculated according to Eq. (1): Released water =
2.4.2.
(A − B) − C A−C
(1)
Expressible water
The swai-fish muscle with proximate compositions of 75.50% moisture, 15.88% protein and 9.69% fat (AOAC, 2000) was purchased from a market in Chiangmai, Thailand. An alkaline fermented soybean (thua nao) was produced following a modified method of Dajanta et al. (2011) using Bacillus subtilis TISTR001 as a pure starter culture. The proximate compositions of the prepared thua nao were 36.63% moisture, 39.75% protein, and 16.68% fat (AOAC, 2000). Rice-koji miso (Ken Co. Ltd., Japan) with proximate compositions of 35.21% moisture, 27.04% protein and 11.07% fat (AOAC, 2000) was purchased from a market in Chiangmai, Thailand.
A proportion of expressible water of the product was measured as described by Funami et al. (1998). The samples were cut into 15 mm thickness then placed between double layers of filter paper (Whatman No. 2, 90 mm in diameter) and subsequently subjected to compression using a Texture Analyzer TA-XT Plus (Stable Micro Systems Ltd., Surrey, UK) with a cylindrical aluminum probe (50 mm in diameter). The measurement was performed with a 50 kg load cell at a cross-head speed of 3 mm/s to 70% strain for 60 s. The proportion of expressible water was calculated as the ratio of apparent expressible water to the total moisture content measured by the method of AOAC (2000). The proportion of expressible water was calculated according to Eq. (2):
2.2.
Expressible water =
Preparation of pressurized fish emulsions
The preparation of swai batters was divided for the control using 100% (w/w) chopped swai-muscle and for the treatment using 90% (w/w) chopped swai-muscle plus 7% (w/w) thua nao and 3% (w/w) miso of which this formula was selected earlier. The control or treatment was blended for 6 min with 1% (w/w) sodium chloride and 0.3% (w/w) sodium tripolyphosphate using a blender (Model HR1372, Philips Electronics Thailand Limited). Subsequently, all fish emulsions were stuffed in collagen casing (2.3 cm in diameter, Food EQ Ltd., Thailand) prior to pressurization. The stuffed emulsions were then subjected to ultra-high pressure at 400, 500, and 600 MPa for 20 and 40 min at 25 ◦ C. The high pressure rig was a model ‘Food Lab’ 900 (Stansted Fluid Power, Essex, UK) with increasing pressure rate of about 330 MPa/min. The pressure transmitting medium was a mixture of castor oil and 98% ethanol (Chemical & Lab Supplies, Thailand) at the ratio of 20:80 (v/v). After pressurization, the products were kept at 4 ◦ C until use.
2.3. Analysis of thermal denaturation of swai-fish emulsion Differential scanning calorimeter (Perkin-Elmer Diamond DSC, USA) was used to examine thermal denaturation of swai proteins (myosin and actin) in the unpressurized and
App TM
(2)
where App = apparent expressible water, TM = total moisture content of fish emulsion. Apparent expressible water =
W1 − W2 W1
(3)
where W1 = weight before compression, W2 = weight after compression. Water holding capacity was calculated by combining values of released water plus expressible water subtracted from 1 (×100).
2.5.
Determination of gel strength
Gel strength of the fish emulsions was determined using a Texture Analyzer TA-XT Plus (Stable Micro Systems Ltd., Surrey, UK) with a 50 kg load cell. The samples without casing were cut into pieces of 23 mm in diameter × 20 mm length. For this purpose, shear force was determined using a Warner Blatzler blade moved perpendicularly toward the samples with a distance of 40 mm using 10 mm/s cross-head speed. The maximum peak of shear force was recorded. Gel strength was then calculated by multiplying shear force (N) with the distance of shearing (m) (Supavititpatana and Apichartsrangkoon, 2007).
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G' and G" (Pa)
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3
1000
G' of treatment (600 MPa/ 40 min) G" of treatment (600 MPa/ 40 min) G' of control (400 MPa/ 20 min) G" of control (400 MPa/ 20 min)
100
0.2
2
20
200
Oscillatory stress (Pa) Fig. 1 – Stress amplitude sweep (0.1–500 Pa) at a frequency of 1 Hz of the controls (400 MPa/20 min) and the treatments (600 MPa/40 min).
2.6.
Color measurement
A colorimeter (Minolta Chroma Meter, CR-300, Japan) was used with a white plate standard to measure the color of the pressurized emulsions. Color parameters [L (lightness), a* (redness), and b* (yellowness)] were then measured and used to calculate whiteness index (WI = 100 − [(100 − L*)2 + a*2 + b*2 ]1/2 (Sánchez-Alonso et al., 2007).
2-mercaptoethanol/100 mL water and boiled for 10 min for reducing protein (Apichartsrangkoon and Ledward, 2002).
2.9.
Standard plate count of pressurized fish emulsions was performed following the AOAC method no. 966.23 (AOAC, 2000).
2.10. 2.7.
Dynamic viscoelastic characterization
Dynamic viscoelastic behavior of the fish emulsions was measured following a method of Chattong and Apichartsrangkoon (2009) with some modification. Intentionally, a controlled stress rheometer (Advance Rheometer AR2000, TA Instruments-Waters LLC, New Castle, USA) equipped with a 25 mm parallel plate measuring system with 1 mm gap width was used. Stress sweeps were initially performed at a frequency of 1 Hz for all samples in order to ensure that all measurements were carried out within a linear viscoelastic region. Samples of the typical plots are shown in Fig. 1. Based on the finding, stress amplitude of 10 Pa was chosen for further frequency-sweep measurement. The edges of samples were covered with light mineral oil (Sigma–Aldrich Co., Ltd., Gillingham, UK) to prevent from drying out. Oscillatory frequency-sweeps were carried out at 25 ◦ C with a frequency range of 0.1–10 Hz. Consequently, storage (G ) and loss (G ) moduli as well as loss tangent were obtained.
2.8.
Electrophoretic analysis
Sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed following a method of Chattong and Apichartsrangkoon (2009) using an electrotransfer unit (Bio-Rad Laboratories Ltd., Waukegan, Illinois, USA). A weight of 0.05 g sample was dissolved in 1 mL of 2 M Tris–HCl (pH 6.8) buffer containing 40 g SDS/100 mL water and 2 g bromophenol blue/100 mL water. Subsequently, 10 L of extracted sample was loaded to each well of 7.5% gel. High range standards (BioRad Laboratories Ltd., Hercules, California, USA) were used to compare the molecular weights. The gels with protein bands were stained with 1 g Coomassie Blue R-250 (USB Corporation, UK)/100 mL water. The samples were dissolved in 2 mL
Microbiological examination
Statistical analysis
Statistical design was 3 × 2 factorial experiment in CRD consisted of pressure 400, 500, 600 MPa and holding times 20 and 40 min. Means of the controls and treatments of each examination were subjected to analysis of variance (ANOVA) and Duncan’s multiple range test (DMRT) was applied to analyze the differences among variables (P ≤ 0.05). All data were analyzed using SPSS 17 software (SPSS Inc., Chicago, USA).
3.
Results and discussions
3.1.
Examination of thermal denaturation
Fig. 2 shows DSC thermograms of 100% swai-fish emulsion (control), emulsion of 90% swai-fish plus 7% thua nao and 3% miso (treatment), pressurized 100% swai-fish emulsion at 400 MPa for 20 min and rescanned thermogram of the control. Two discernible peaks of the control was found at the transition temperatures 56.09 (H = 0.25 J/g) and 68.09 ◦ C (H = 0.18 J/g), respectively, which corresponded to the transition temperatures of myosin and actin, while the transition temperatures of the endothermic peaks for the treatment were 55.51 ◦ C (H = 0.17 J/g) and 66.96 ◦ C (H = 0.13 J/g), respectively (Fig. 2). These suggested that incorporating fermented soy proteins in the fish emulsion induced slight shift of the transition temperatures, presumably due to the effect of interaction between fish and soy proteins which was better clarified by the gel electrophoregrams in Fig. 4. After rescanning, all endothermic peaks had disappeared, presumably due to a complete denaturation of both proteins from earlier scanning. Other findings for example, Matos et al. (2011) reported that myosin and actin denaturation peaks of gilthead-sea bream (Sparus aurata) muscle were in the range of 47–50 ◦ C and 73–75 ◦ C; in addition, the endothermic peaks of Japanese-sting fish (Sebastes inermis) dorsal muscle were 40.9 ◦ C for myosin and 61.1 ◦ C for actin (Takeshi et al., 1999).
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Fig. 2 – DSC thermograms of unpressurized and pressurized samples at 400 MPa for 20 min obtained at a scanning rate of 10 ◦ C/min. Nonetheless, the endothermic peaks of myosin and actin for pressurized (400 MPa/20 min) control and treatment were not found, which might be due to the impacts of pressure triggering a complete denaturation. This result was matched with the work of Angsupanich and Ledward (1998) showing that myosin, actin and most of the sarcoplasmic proteins of cod muscle were denatured by low levels of pressure 100–300 MPa.
3.2.
Determination of water holding capacity
Table 1 shows that water holding capacities (WHC) of the controls and treatments were significantly increased (P ≤ 0.05) with increasing pressure levels regardless of the holding time. Moreover, the effect of pressure levels was more prominent beyond 500 MPa indicating by significantly higher (P ≤ 0.05) WHC of both controls and treatments compared to those of lower pressurized samples. This suggested that the firm structure had been set at 500 MPa. However, WHC of the controls and treatments were not different, in other words, composition of the emulsion had no effect on WHC. Angsupanich et al. (1998) stated that during pressurization, high pressure induced a partial unfolding of myosin and promoted non-covalent interactions between the subunits such as disulphide, hydrophobic, hydrogen, and ionic bonds. On releasing pressure, a flexible network which could entrap water was formed. Similarly, Chattong and Apichartsrangkoon (2009) and Supavititpatana and Apichartsrangkoon (2007) pressurized
ostrich-meat sausages and found that the levels of pressure significantly influenced WHC of the products. Apart from pressure, the salt content also had some effects on WHC of the pork emulsion (Ma et al., 2012).
3.3.
Determination of gel strength
Similar to WHC, the levels of pressure significantly took an influence (P ≤ 0.05) on the gel strength of the controls and treatments, whereas pressurized samples at 600 MPa received the highest gel strength. On the other hand, the holding time showed less effect on gel strength (Table 1). However, on a precise clarification of each pressure level, pressure 600 MPa/20 min onward had settled texture of the controls and treatments. When comparing between the controls and treatments, gel strength of the treatments was slightly higher than those of the controls, presumably due to the impacts of the interactions among fish, thua nao and miso proteins which could be more specified by electrophoregrams in Fig. 4. Previous study elucidated that pressurized ostrich-meat sausage at 300–700 MPa with temperature 40 and 60 ◦ C for 40 and 60 min exhibited rise of gel strength according to the treatment severities (Supavititpatana and Apichartsrangkoon, 2007). Chen et al. (2010) studied the effects of pressure levels (0–500 MPa) and holding time (10–40 min) on textural properties of duck muscle gels containing 1% curdlan and noticed that the hardness, springiness, cohesiveness and chewiness of the gels
Table 1 – Physical properties of pressurized fish emulsions for 100% fish muscle (controls) and 90% fish muscle plus 7% thua nao and 3% miso (treatments). High pressure conditions
Water holding capacity (%) Controls
400 MPa 20 min 400 MPa 40 min 500 MPa 20 min 500 MPa 40 min 600 MPa 20 min 600 MPa 40 min
87 88 91 91 92 92
± ± ± ± ± ±
1.1b 2.7b 1.7a 1.7a 0.9a 1.4a
Treatments 86 87 89 90 91 91
± ± ± ± ± ±
1.3c 1.7c 1.8b 0.8ab 0.8a 1.4a
Gel strength (N m) Controls 0.6 0.7 0.7 0.7 0.8 0.8
± ± ± ± ± ±
0.1b 0.1b 0.1b 0.1b 0.0a 0.0a
Treatments 0.7 0.7 0.7 0.8 0.9 0.9
± ± ± ± ± ±
0.1b 0.1b 0.1b 0.1ab 0.1a 0.1a
Whiteness index Controls 80 81 82 83 84 84
± ± ± ± ± ±
0.7c 0.6bc 0.7b 1.4ab 0.5a 1.0a
Treatments 67 68 71 71 72 72
± ± ± ± ± ±
1.0b 1.0b 1.0a 1.0a 0.4a 1.0a
Means within each column followed by the same letters have no significant difference (P > 0.05). Each parameter is an average of three replications.
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a) Controls G'/ 400 MPa/ 20 min (k=0.16)
G' and G" (Pa)
G"/ 400 MPa/ 20 min (k=0.20) G'/ 400 MPa/ 40 min (k=0.16) G"/ 400 MPa/ 40 min (k=0.19)
1000
G'/ 500 MPa/ 20 min (k=0.15) G"/ 500 MPa/ 20 min (k=0.17) G'/ 500 MPa/ 40 min (k=0.15) G"/ 500 MPa/ 40 min (k=0.16) G'/ 600 MPa/ 20 min (k=0.14) G"/ 600 MPa/ 20 min (k=0.15) G'/ 600 MPa/ 40 min (k=0.13) G"/ 600MPa/ 40 min (k=0.15)
100
1
0.1
10
Frequency (Hz)
b) Treatments G' 400 MPa/ 20 min (k=0.15)
G' and G" (Hz)
G" 400 MPa/ 20 min (k=0.16) G' 400 MPa/ 40 min (k=0.15) G" 400 MPa/ 40 min (k=0.15)
1000
G' 500 MPa/ 20 min (k=0.15) G" 500 MPa/ 20 min (k=0.14) G' 500 MPa/ 40 min (k=0.15) G" 500 MPa/ 40 min (k=0.14) G' 600 MPa/ 20 min (k=0.14) G" 600 MPa/ 20 min (k=0.13) 100
G' 600 MPa/ 40 min (k=0.14) 1
0.1
10
G" 600 MPa/ 40 min (k=0.13)
Frequency (Hz) Fig. 3 – Typical plots of storage (filled symbols) and loss (unfilled symbols) moduli of pressurized samples at 400, 500 and 600 MPa for 20 and 40 min; k = slopes of each actual plot of the controls (a) and the treatments (b). were increased (P ≤ 0.05) above 300 MPa while the holding time had no significant influence.
3.4.
pressure, while the holding time had also no influence on this attribute. It was noteworthy that the WI of pressurized controls and treatments lay in the range of 80–84 and 67–72, respectively, whereas the WI for unpressurized (raw muscle) control and treatment were 69 and 62, respectively. The sharp increase of WI after pressurization triggered off the loss of translucency from unpressurized state to more opaquewhite-cooked color, indicating that the fish protein had been undergone denaturation by pressure 400 MPa onward.
Color measurement
Different colors of the swai-fish emulsions had been identified by whiteness index (WI) calculated from L, a* and b* parameters. Table 1 shows that WI of the controls and treatments significantly increased (P ≤ 0.05) with the increase of
Table 2 – The loss tangent measured at a frequency of 1 Hz and standard plate count of pressurized fish emulsions for 100% fish muscle (controls) and 90% fish muscle plus 7% thua nao and 3% miso (treatments). Loss tangent (G /G )
High pressure conditions
Controls 400 MPa 20 min 400 MPa 40 min 500 MPa 20 min 500 MPa 40 min 600 MPa 20 min 600 MPa 40 min
0.25 0.25 0.24 0.22 0.22 0.22
± ± ± ± ± ±
0.01a 0.01a 0.00a 0.01b 0.01b 0.01b
Standard plate count (log CFU/g)
Treatments 0.28 0.25 0.24 0.24 0.23 0.23
± ± ± ± ± ±
0.01a 0.01b 0.00c 0.01cd 0.01d 0.00d
Controls 3.0 ± 2.4 ± 2.0 ± 1.4 ± <1e <1e
0.1a 0.1b 0.1c 0.1d
Treatments 3.9 3.9 3.8 3.7 3.6 3.6
± ± ± ± ± ±
0.0a 0.2a 0.0b 0.0c 0.0d 0.0d
Means within each column followed by the same letters have no significant difference (P > 0.05). Each parameter is an average of three replications.
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However, this finding agreed with the DSC thermograms in 3.1. Previously, Angsupanich and Ledward (1998) stated that pressure of 200 MPa enabled the denaturation of cod protein by changing L values with an opaque appearance compared to that of unpressurized cod muscle. To sum up, as comparison between the control and treatment batches, the treatments displayed markedly lower WI than those of the controls, presumably due to the addition of fermented soybeans (thua nao and miso) in the treatments which were visualized darker than the fish muscle.
3.5.
Dynamic viscoelastic characterization
Fig. 3 shows the plots of elastic or storage (G ) and viscous or loss (G ) moduli against frequency 0.1–10 Hz. The plots of G were greater than G over most of the measured frequency range and the ratio of G to G of each plot was nearly 10, indicating a rigid-gel-like structure for which all gels gave the frequency profiles expected for weak viscoelastic systems (Saowapark et al., 2008). Such profiles were consistent with the
behavior of a material formed by the specified cross linking of the denatured proteins such as hydrophobic and disulphide interaction as described in Section 3.6. All plots of storage and loss moduli for the controls and treatments were frequency dependent, but the slopes were lower for those treated at higher levels indicated by the reduction of slopes or k values (Fig. 3). Moreover, higher G and G of fish emulsions treated at a higher pressure and a holding time suggested that the higher pressure and the holding time gave rise to stronger gel system with more cross-link densities (Apichartsrangkoon and Ledward, 2002), consequently, these agreed with the gel strength in Table 1. Similarly, loss tangent (G /G ) at 1 Hz in Table 2 of the controls and treatments significantly reduced (P ≤ 0.05) with the increase of pressure and holding time, which were complied with the slopes. In addition, the pressure of 500 MPa/40 min was a critical level of these alleviations, meaning that this pressure enabled setting of firm viscoelastic structure. Apichartsrangkoon (2002) also studied the viscoelastic properties of heated gluten/soy protein gels and found that lower loss tangent or less
Fig. 4 – SDS-PAGE electrophoregrams of the controls (a) and reduced with 2-mercaptoethanol (c); the treatments (b) and reduced with 2-mercaptoethanol (d). Please cite this article in press as: Techarang, J., Apichartsrangkoon, A., Physical, chemical and rheological parameters of pressurized swai-fish (Pangasius hypophthalmus) emulsion incorporating fermented soybeans. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.002
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frequency dependence of the shear moduli indicated a stronger gel structure with more cross-link densities. It was noticed that the treatments with the addition of thua nao and miso (Fig. 3b) brought about a higher overall moduli than those of the controls (100% swai muscle, Fig. 3a), which could be less moisture in the treatments (∼80%) than the controls (∼83%). Nonetheless, this finding also agreed with the gel strength (Table 1).
inactivate most of the undesirable microbes with the presence of less than 10 CFU/g left. Stewart et al. (2000) revealed that pressure 404 MPa/70 ◦ C/15 min could reduce B. subtilis 168 by 5 log cycles at pH 6.0–7.0, while the pressurization with the same condition at 25 ◦ C could inactivate this bacteria by less than 0.5 log cycle. In addition, Gao et al. (2006) stated that pressure 576.0 MPa/87 ◦ C/13 min gave rise to inactivate spores of B. subtilis As.1.1731 in milk buffers by 6 log cycles.
3.6.
4.
Electrophoretic analysis
Pressure took an effect on protein denaturation, i.e., the disruption of hydrophobic and electrostatic interactions, enhancing hydrogen bonding, and promoting formation of disulphide bonds, consequently, the proteins were denatured and aggregated leading to the improved gelation or protein structure modification (Galazka et al., 2000). In this context, the extent of swai-fish protein denaturation had been shown by SDS-PAGE electrophoregrams in Fig. 4. The electrophoregrams in Fig. 4 displayed that hydrophobic interactions of myosin (200 kDa) or actin (45 kDa) for the pressurized controls (Fig. 4a) and treatments (Fig. 4b) (lanes 2–7) were dissociated in the presence of SDS by dissolving band densities of the myosin but increasing band densities of the lower molecular weight protein (between 66 and 97 kDa and lower than 45 kDa) as compared to the unpressurized samples (lane 1). It was noticed in particular that all myosins at 200 kDa were present as thick bands (Fig. 4a and b) presumably not being dissolved by SDS. After the addition of a reducing agent (2mercaptoethanol), these bands were thinner (Fig. 4c and d) which could be due to the dissociation of disulphide bond. All these phenomena indicated that the pressure denaturation of swai-fish proteins might be dealt with hydrophobic and disulphide interaction. In addition, the distinct proteinbands occurred especially between the molecular weight of 66 kDa and 97 kDa of the treatment (Fig. 4b and d), while the electrophoregrams of the controls (Fig. 4a and c) had bands with much less density. These suggested that upon pressurization, soy proteins present in the treatments had shown some degree of interaction with fish protein in the specified bands. A previous finding by Chattong and Apichartsrangkoon (2009) illustrated that the structural changes of pressurized ostrichmeat emulsion might be associated with the hydrophobic and disulphide interactions.
3.7.
Microbiological examination
Table 2 shows standard plate counts of the pressurized controls and treatments. Treatments with the addition of thua nao and miso exhibited very much higher microbial counts than those of the controls. It was obvious that thua nao was fermented by spore-forming bacteria (Bacillus subtilis TISTR001) which could resist pressure up to 600 MPa, hence the existence of these high viable counts after pressurization could be some survival B. subtilis. However, the residuals of survival B. subtilis in the pressurized emulsion were roughly 10–1000 CFU/g, which could enhance the emulsion with slimy appearance during storage. The effects of pressure levels and two holding time on the standard plate counts of the controls showed significant difference (P ≤ 0.05), but the holding time was less influent on the treatments. These meant that higher pressure level with longer holding time would efficiently reduce the undesirable microbes except B. subtilis. To sum up, the pressure beyond 600 MPa would satisfactorily
Conclusion
The DSC thermograms revealed that pressure 400 MPa/20 min was able to denature fish protein, while the levels of pressure significantly affected other parameters including water holding capacity, gel strength and whiteness index of both controls (100% swai-fish emulsion) and treatments (emulsion of 90% swai fish plus 7% thua nao and 3% miso) regardless the holding time. The increase of these parameters was in accordance with raising pressure. The viscoelastic characterization illustrated that the storage modulus was greater than the loss modulus over most of the measured frequency range. All storage and loss moduli were frequency dependent, whereas the loss tangent at 1 Hz significantly decreased with the increase of pressure and time. These indicated that more cross-link densities or solid-like structure occurred with the products treated at higher levels. electrophoregrams elucidated that the pressure induced denaturation of swai-fish proteins with the association of hydrophobic and disulphide interactions. Some evidences of interaction between fish and soy proteins also indicated by the electrophoregrams. Results of microbiological examination showed that pressure beyond 600 MPa was efficiently eliminated undesirable microbes, however, this pressure could not completely inactivate spore-forming bacteria such as B. subtilis in fermented thua nao.
Acknowledgements The authors wish to thank National Research Council of Thailand and Chiang Mai University, Chiang Mai, Thailand for their financial support. We also would like to express our appreciation to Associate Professor Dr. Saranya Savetamalya, Department of English, Chiangmai University in editing the final version of this paper.
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Please cite this article in press as: Techarang, J., Apichartsrangkoon, A., Physical, chemical and rheological parameters of pressurized swai-fish (Pangasius hypophthalmus) emulsion incorporating fermented soybeans. Food Bioprod Process (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.002