2016 Volume 22 Issue 6 Pages 787-792
Meat homogenates and intestinal extracts were prepared from walleye pollack (Theragra chalcogramma) and white croaker (Pennahia argentata). Then, the proteolytic profiles of meat homogenate in the presence of the intestinal extracts were determined. The proteolytic activity in the intestinal extracts from walleye pollack was the maximum at 50°C and pH 8.53. The maximum activity was recognized also at 50°C when the intestinal extracts from walleye pollack were added to the meat homogenate prepared from white croaker, while it was at 60°C when the intestinal extracts from white croaker were added to the meat homogenate prepared from walleye pollack. These results suggest that the proteolytic profile is predominantly attributable to proteolytic enzymes and not to the substrate species of myosin heavy chain. These findings are useful for managing heating conditions in the industry in order to avoid modori and thus improve the quality of surimi-based products.
Quality levels and accompanying commercial values of surimi-based products such as kamaboko, chikuwa, and imitation crab meat largely depend on their thermal gel properties, which provide a characteristic elasticity called “ashi”, a technical Japanese word representing good texture. This elasticity is especially important for kamaboko, one of the most traditional surimi-based products made in Japan. Various fish species are currently used for kamaboko production: white croaker (Pennahia argentata) is used as a high-quality material producing thermal gels with a high value of thermal gel properties, including tensile strength and breaking extension, whereas walleye pollack (Theragra chalcogramma) is used as a conventional material for thermal gels with standard thermal gel properties (Satoh et al., 2006; Matsuoka et al., 2013; Ueki et al., 2014).
Such thermal gel properties are mostly attributed to the abundance of covalently bound polymers of the myosin heavy chain, a large subunit of the major muscle protein, myosin (Nishioka et al., 1983; Akahane et al., 1984; Iwata et al., 1977; Matsuoka et al., 2013). It is well known that myosin properties change depending on fish species and water temperature in the environment where fish inhabit (Fukushima et al., 2003; Fukushima et al., 2005), resulting in different rheological profiles of their thermal gels (Shimizu et al., 1981; Satoh et al., 2006; Fukushima et al., 2007; Matsuoka et al., 2013).
Surimi-based products are generally prepared by a two-step heating procedure, where the first heating (or pre-heating) is conducted at temperatures less than 40°C and the second one at around 90°C. At first, surimi paste is produced by adding salt to fish meat and subsequently grinding at low temperatures, usually less than 10°C. This salted, ground surimi paste is subjected to pre-heating treatment (suwari), where myosin heavy chains are covalently bound and thus soft gels are formed. The second heat treatment (honkanetsu) at high temperatures facilitates the suwari gels to form elastic gels characteristic of surimi-based products. However, there is a problem called “modori” (return), which often occurs in the procedure between the two heating steps. The suwari gels are degraded by modori during this procedure between 50 and 70°C, thus decreasing the quality of surimi-based products prepared by finally treating at a high temperature around 90°C (Shimizu et al., 1981; Matsuoka et al., 2013). At the industry level, the contamination of fish meat with internal organs has been believed to induce modori. Meanwhile, it has been reported that the gel network is weakened even in the absence of protein degradation, probably due to the detachment of myosin from denatured actin (Sano et al., 1989; Ni et al., 1999, 2001). Under this background, we reported previously that the addition of intestinal extract from white croaker into its own meat degraded myosin heavy chains during heat treatment around 60°C, mainly due to the activity of proteases contaminating the extracts, accompanying the decrease in the elasticity of thermal gels, while the addition of kidney extract had no apparent effect (Ueki et al., 2016). It was also revealed that modori occurring in the thermal gels could not be completely prevented by washing the meat after contamination with intestinal substances. Thus, it is important to avoid contamination with organ tissues during meat separation. Once the meat is contaminated with organ tissues, we have to pass temperatures showing modori as quickly as possible between the temperature regions for suwari and honkanetsu.
Surimi-based products are frequently made from not just one fish species but several fish species together. It seems possible that organ tissues from different fish species have their respective proteolytic properties. Thus, it is important to examine proteolytic activities in the meat in the presence of intestinal extracts from different fish species and to determine the protein degradation profiles for myosin heavy chain from the viewpoint of quality control of surimi-based products.
Under this background, the present study was undertaken to elucidate the effect of the intestinal extracts from white croaker or walleye pollack added to its own or different meat on the protein degradation profiles at different incubation temperatures and pHs.
Materials Dorsal ordinary meats were dissected from white croaker and walleye pollack which had been captured in the East China Sea and Pacific Ocean off Miyagi Prefecture, respectively, and subsequently stored on ice for 2 days. The reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) unless otherwise specified.
Preparation of washed meat Dorsal ordinary meats from white croaker and walleye pollack were minced and washed at 15°C with 4 volumes (v/w) of water, and this procedure was repeated 4 times. After centrifugation at 10,000 × g and 4°C for 15 min, the precipitate was stored at −80°C until used for experiments as washed meat.
Preparation of intestinal extracts Intestinal extracts were prepared according to the method of Ueki et al. (2016). Briefly, the intestine was dissected from the region just beneath the stomach and that immediate to the anus each from white croaker and walleye pollack, whereas the inner materials (possibly containing baits) were removed using running tap water. The washed intestine was homogenized with 3 volumes of ice-cold MilliQ water (v/w) for 2 min at 10,000 rpm (ULTRA-TURRAX T25, IKA®-Labortechnik, Staufen, Germany) and filtered through gauze. After centrifugation at 20,000 × g and 4°C for 15 min, the supernatant was lyophilized and stored at −20°C until use.
Proteolytic activity in the intestinal extracts at different temperatures Proteolytic activities of the intestinal extracts were examined at different temperatures according to the method of Ueki et al. (2016). Briefly, washed meat of white croaker or walleye pollack was homogenized for 2 min at 10,000 rpm (ULTRA-TURRAX T25) in 9 volumes of 100 mM Na-phosphate (pH 7.0) containing 0.6 M NaCl in the presence of intestinal extracts from their own or different species. The intestinal extracts were used at 100 µg/mL from walleye pollack or 50 µg/mL from white croaker. These concentrations were nearly equal to those in the body for both fish. Then, the mixture was incubated at a temperature ranging from 30 to 80°C using a temperature-controlled water bath. After incubation for 30 min, an equal volume of 15% trichloroacetic acid (TCA) was added and the mixture was centrifuged at 20,000 × g and room temperature for 15 min. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed with the precipitate as described below, while an optical density at 280 nm (OD280) for TCA-soluble fraction was measured as an index of the proteolytic activity.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis The protein precipitate prepared as described above was washed with acetone and then an appropriate volume of 20 mM Tris-HCl (pH 8.0) containing 8 M urea, 2% SDS, and 2% 2-mercaptethanol was added in a microtube and the mixture was stirred overnight at room temperature using a rotator. After the mixture was centrifuged at 20,000 × g and room temperature for 15 min, an aliquot of the supernatant was mixed with an equal volume of 15% TCA. The mixture was subjected to centrifugation at 20,000 × g and room temperature for 15 min. Then, the precipitate was dissolved in 1 N NaOH and its protein concentration was determined by the biuret method (Gornall et al., 1949) using bovine serum albumin as the standard. After determining protein concentrations as above, another aliquot of the supernatant containing 15 µg of protein was subjected to SDS-PAGE using 10% gels (Laemmli, 1970), and Precision Plus Protein Standards (Bio-Rad Laboratories, Hercules, CA, USA) was used as the standard molecular weight marker.
Proteolytic activity in the intestinal extracts of walleye pollack at different pHs Washed meat from walleye pollack was homogenized with a few different types of buffers, as described in our previous study (Ueki et al., 2016), to set up pHs from 5.0 to 10.0. To the homogenate was added 100 µg/mL of the intestinal extract from walleye pollack, and the mixture was incubated at 50°C for 30 min. A 20-µg aliquot of the precipitate after centrifugation at 20,000 × g and room temperature for 15 min was subjected to SDS-PAGE analysis, and the TCA-soluble fraction in the supernatant was measured at OD280, as described above.
Proteolytic activity in the intestinal extracts from walleye pollack at different temperatures After the meat homogenate of walleye pollack with its own intestinal extract was incubated at pH 7.0 for 30 min at various temperatures, the proteolytic activity was determined by SDS-PAGE. The degradation of myosin heavy chains was hardly recognized at all temperatures in washed dorsal meat without the intestinal extract as a reference (Fig. 1a). On the addition of the intestinal extract, the myosin heavy chain monomer band almost disappeared in the temperature range 30 to 65°C, while the band remained in the temperature range 70 to 80°C (Fig. 2b). It was noted that the actin band was also markedly degraded in the temperatures range 45 to 55°C.
Proteolytic activities at different temperatures in the meat homogenate of walleye pollack meat added with its own intestinal extracts.
The meat homogenate of walleye pollack without (a) and with (b) its own intestinal extracts was heated at different temperatures and pH 7.0 for 30 min. Numerals on the top of lanes in 10% SDS-PAGE gels indicate incubation temperatures. Relative values of OD280 (c) in the TCA-soluble fraction from the homogenate are shown with the maximum at 50°C as 100%. Values are indicated with mean ± SD (n = 3). M, molecular weight markers; C, control without incubation of the homogenate; MHC, myosin heavy chain; Ac, actin.
Proteolytic activities at different pHs in the meat homogenate of walleye pollack added with its own intestinal extracts.
The meat homogenate of walleye pollack without (a) and with (b) its own intestinal extracts was heated at different pHs and 50°C for 30 min. Numerals on the top of lanes in 10% SDS-PAGE gels indicate incubation pH. Relative values of OD280 (c) in the TCA-soluble fraction from the homogenate are shown with the maximum at pH 8.53 as 100%. Values are indicated with mean ± SD (n = 3). M, molecular weight markers; C, control without incubation of the homogenate; MHC, myosin heavy chain; Ac, actin.
The relative OD280 level of the TCA-soluble fraction was increased from 11.6% at 30°C to 100% at 50°C as taken for the maximum and then rapidly decreased to 12.6% at 60°C (Fig. 1c).
Proteolytic activity in the intestinal extracts from walleye pollack at different pHs Proteolytic activities at different pHs following incubation of the walleye pollack meat homogenate added with its own intestinal extract at 50°C for 30 min were examined using SDS-PAGE analysis and OD280 measurement, as described above. No degradation of myosin heavy chain was observed at any pH in the meat homogenate without intestinal extract (Fig. 2a). The myosin heavy chain band disappeared in the presence of the intestinal extracts in the pH range 5.65 to 9.94, while the band remained at pH 5.12 (Fig. 2b).
The relative OD280 levels of the TCA-soluble fraction from the homogenate gradually increased from 22.6% at pH 5.12 to 100% at pH 8.53 as the maximum value and then decreased to 64.1% at pH 9.94 (Fig. 2c).
Experiment using the meat homogenate containing intestinal extracts from different fish species The proteolytic activity was examined at various temperatures in meat homogenate prepared from washed meat and intestinal extracts from different fish species. When the meat homogenate from white croaker was added with the intestinal extract from walleye pollack, a marked degradation of myosin heavy chain and actin was recognized in the temperature range 45 to 55°C, as revealed by SDS-PAGE analysis (Fig. 3a). The relative OD280 value of the TCA-soluble fraction rapidly increased from 21.2% at 30°C to 100% at 50°C as the maximum and then rapidly decreased to 20.0% at 60°C (Fig. 3b).
Proteolytic activities at different temperatures in the meat homogenate of white croaker added with the intestinal extracts of walleye pollack.
The meat homogenate with the intestinal extracts was heated at different temperatures and pH 7.0 for 30 min. Numerals on the top of lanes in 10% SDS-PAGE gels (a) indicate incubation temperatures. Relative values of OD280 (b) in the TCA-soluble fraction from the homogenate are shown with the maximum at 50°C as 100%. Values are indicated with mean ± SD (n = 3). M, molecular weight markers; C, control without incubation of the homogenate; MHC, myosin heavy chain; Ac, actin.
When walleye pollack meat homogenate was added with the intestinal extract of white croaker, walleye pollack myosin heavy chain and actin were markedly degraded in the temperature range 50 to 65°C, as revealed by SDS-PAGE (Fig. 4a), while the OD280 level of the TCA-soluble fraction rapidly increased from 16.9% at 40°C to 100% at 60°C as the maximum and then rapidly decreased to 23.3% at 70°C (Fig. 4b).
Proteolytic activities at different temperatures in the meat homogenate of walleye pollack added with the intestinal extracts of white croaker.
The meat homogenate with the intestinal extracts was heated at different temperatures and pH 7.0 for 30 min. Numerals on the top of lanes in 10% SDS-PAGE gels (a) indicate incubation temperatures. Relative values of OD280 (b) in the TCA-soluble fraction from the homogenate are shown with the maximum at 60°C as 100%. Values are indicated with mean ± SD (n = 3). M, molecular weight markers; C, control without incubation of the homogenate; MHC, myosin heavy chain; Ac, actin.
The proteolytic activity of meat homogenate of walleye pollack with or without intestinal extract at different temperatures and pHs was examined. Proteolytic activity in washed dorsal meat without the intestinal extract was at a very low level (Fig. 1a), which is consistent with our previous study on white croaker (Ueki et al., 2016). On the addition of the intestinal extract, marked protein degradation was observed in the temperature range 45 to 55°C (Fig. 1b, c). These results suggest that the proteolytic activity in the intestinal extract from walleye pollack was strong at moderately high temperatures, around 50°C. Thus, proteolytic enzymes which are contained in the extracts could be closely related to modori, in which disintegrated gels are observed between 50 and 60°C, although some fish exhibit modori even at 70°C (Shimizu et al., 1981). Meanwhile, walleye pollack surimi shows no apparent reduction in breaking strength and breaking strain rate at around 50°C without contamination of organ tissues (Matsuoka et al., 2013). This temperature-dependent profile of the proteolytic activity is different from that of white croaker, where a marked degradation of myosin heavy chain and actin was observed at around 60°C (Ueki et al., 2016). It is thus suggested that the proteolytic activity in the intestinal extracts is altered depending on various factors, such as fish species, habitat, and water temperature, probably due to the different thermostabilities of proteins. In addition, differences in the proteolytic activity could be associated with the feeding habitat of fish and the species of their baits, although white croaker and walleye pollack are both carnivorous.
Marked protein degradation was observed around pH 8.53 when meat homogenate was added with the intestinal extract (Fig. 2b, c), while no degradation of myosin heavy chain was observed at any pH without the intestinal extract (Fig. 2a). These results are consistent with our previous data for white croaker in which the maximum activity was observed at pH 8.9 (Ueki et al., 2016), suggesting that the intestinal extract from walleye pollack also contains alkaline proteases. It is reasonable that these proteases would be contained in the intestine, since this organ is under alkaline conditions; for instance, the intestinal pH of rainbow trout (Oncorhynchus mykiss) is 7.8 (Kitamikado and Tachino, 1960).
Some proteases have been purified from the intestine of other fish species and identified and characterized. Grass carp (Ctenopharyngodon idellus) intestine has two trypsin isoforms with optimal pHs of 8.0 and 8.5 and optimal temperatures of 38.5 and 44°C, respectively (Liu et al., 2007), together with an acidic protease with an optimal pH of 2.5 and optimal temperature of 37°C (Liu et al., 2008). The optimal temperatures of these proteases are different from that of the intestinal extract from walleye pollack in this study (see Fig. 1). Nile tilapia (Oreochromis niloticus) contains a trypsin-like serine protease, cysteine protease (Yamada et al., 1991), and carboxypeptidase (Taniguchi-Yamada and Takano, 2001) in the intestine. These proteases are activated at around pH 9 and 55°C, and this profile is similar to that of the intestinal extract from walleye pollack in the present study, although the optimal temperature of carboxypeptidase from Nile tilapia is 30°C. However, the proteolytic activities of these proteases from grass carp and Nile tilapia intestines in the presence of NaCl are unclear. Identification and characterization of proteolytic enzymes contained in the intestinal extract of walleye pollack will be necessary in order to better understand the molecular mechanisms involved in modori.
The proteolytic profiles at different temperatures in the meat homogenate prepared from the washed meat and intestinal extracts from other fish species were examined. When the meat homogenate from white croaker was added with the intestinal extract from walleye pollack, the most marked protein degradation was observed at around 50°C (Fig. 3). These results were similar to those for the combination of the meat homogenate of walleye pollack and its own intestinal extract (Fig. 1). When walleye pollack meat homogenate was added with the intestinal extract of white croaker, proteins were markedly degraded at around 60°C (Fig. 4). These results were similar to those for the meat homogenate of white croaker meat and its own intestinal extract (Ueki et al., 2016). Taken together, these results suggest that the degradation profiles of proteins depend strongly on the proteases rather than the substrate proteins. Thus, it is suggested that the proteolytic profile reflecting modori is largely attributable to proteolytic enzymes. It should be noted that the modori phenomenon is observed even after the washing step in a variety of fish species (Kinoshita et al., 1990; Toyohara et al., 1990; Nomura et al., 1993), suggesting that digestive enzymes bind to muscle proteins strongly.
At the industrial level, heating conditions are important for producing high-quality surimi-based products; thus, it is necessary to understand the mechanisms involved in the deterioration of thermal gels as occurs in modori. In addition, the pH conditions of meat are affected by various factors, such as fish species (Shimizu et al., 1981), freshness (Watabe et al., 1991), season (Osako et al., 2001), and seasoning reagents. Therefore, it is important to determine the temperature- and pH-dependence of the proteolytic enzymes that induce modori. The elucidation of factors that reduce the quality of surimi-based products is difficult, since the products are frequently made from not just one fish species but multiple fish species. Thus, the characterization of the proteolytic profiles when washed meat and intestinal extracts from different fish species were mixed in this study would be useful in managing heating conditions in the industry to avoid modori and improve the quality of surimi-based products.
Contamination of abdominal meat and washing water have been implicated as causing modori, and contamination by the intestine has been demonstrated to induce protein degradation. Thus, whether other proteolytic enzymes are localized in muscle tissues and not in organ tissues must be determined, and the proteolytic profiles of such proteases should be characterized.
Acknowledgements The authors thank Dr. Y. Matsuoka and Mr. D. Suzuki, Fish Protein Laboratory, Suzuhiro Kamaboko Honten Co., Ltd., for useful suggestions and discussions in the present study.
