Mode of IMP and Pyrophosphate Enhancement of Myosin and Actin Extraction from Porcine Meat

Transcription

1 Biosci. Biotechnol. Biochem., 77 (6), , 2013 Mode of IMP and Pyrophosphate Enhancement of Myosin and Actin Extraction from Porcine Meat Yukinobu NAKAMURA, 1;y Koshiro MIGITA, 2 Akihiro OKITANI, 2 and Masanori MATSUISHI 2 1 Japan Meat Science and Technology Institute, Ebisu, Shibuya-ku, Tokyo , Japan 2 Department of Food Science and Technology, Nippon Veterinary and Life Science University, Kyonan-cho, Musashino, Tokyo , Japan Received December 18, 2012; Accepted March 15, 2013; Online Publication, June 7, 2013 [doi: /bbb ] We examined the mode of IMP and pyrophosphate enhancement of myosin and actin extraction from porcine meat. Extractabilities were determined after homogenates, prepared by adding 9 volumes of 0.3, 0.4, or 0.5 M NaCl solutions containing 0 to 36 mm IMP and 0 to 9 mm tetrapotassium pyrophosphate (KPP) to minced pork, were incubated at 4 C for 0 or 12 h. Irrespective of the NaCl concentrations, IMP-induced extraction of both proteins increased with increasing extraction time. In contrast, that of KPP did not. When 0.3 M NaCl solutions containing both IMP and KPP were used, the solutions with 1.5 mm KPP showed marked enhancement of IMPinduced myosin and actin extraction. Incorporating these results with our previously published data (Nakamura et al., Biosci. Biotechnol. Biochem., 76, (2012)), we hypothesized that IMP and KPP have the ability to release thick and thin filaments from restraints in myofibrils, in addition to the ability to dissociate actomyosin into myosin and actin, and that the restraintreleasing ability of IMP is dependent on reaction time and NaCl concentration while that of KPP is not. Key words: IMP; pyrophosphate; myosin extraction; actin extraction Heat-induced gel produced from the salted emulsion of comminuted meats is used in the production of sausage. It derives from the binding properties of muscle proteins in the meats. Fukazawa et al. 1 3) and Samejima et al. 4) have reported that the extraction of myosin from myofibrils was necessary for the high binding quality. It is known that pyrophosphate is highly effective in enhancing the extraction of myosin from myofibrils. Yasui et al. 5) found that its effectiveness derives from its ability to dissociate actomyosin into myosin and actin. In addition, Morita et al. 6) found that pyrophosphate at 9mM was sufficient to obtain optimal extraction in the presence of 0.3 M NaCl. IMP, a popular umami seasoning, has also been found to possess the ability to dissociate actomyosin, 7) and was found to enhance the extraction of myosin and actin from porcine meat. 8) This IMP-induced extraction of both proteins was enhanced by increasing the NaCl concentration from 0.3 to 0.5 M. 8) Thus IMP may have the potential to improve the gel-forming ability of meats in the production of sausage. In a previous paper, 8) we reported the extractabilities of myosin and actin measured after incubating saltedmeat homogenates with IMP at 4 C for 12 h to achieve optimal extraction. In the current industrial production of sausage, a heating process is employed immediately following the cutting process. In the present study, to investigate the effects of extraction time on the extractabilities of myosin and actin from meat homogenates, we determined and compared the extractabilities of the proteins immediately (0 h) and at 12 h after the addition of IMP and/or tetrapotassium pyrophosphate (KPP) at various concentrations of NaCl. Materials and Methods Materials. Cuts of fresh meat (mainly M. semimembranosus, M. adductor, and M. gracilis) of Large White Landrace Duroc crossbred pigs (n ¼ 3) were obtained from retail stores and immediately minced in a meat chopper after fat and connective tissues were removed. The minced meat was vacuum packed and stored at 20 C until use. Tetrapotassium pyrophosphate (KPP) was purchased from Wako Pure Chemical Industries (Osaka, Japan), and IMP was from Tokyo Chemical Industries (Tokyo). All other chemicals used were of analytical grade. Extraction of myofibrillar proteins. Minced meat (3 g) was mixed with 9 volumes (27 ml) of each of the extraction solutions and homogenized twice at 12,000 rpm for 30 s with a homogenizer (Excel Auto, Nippon Seiki, Nagaoka, Japan). The extraction solutions were 0.3, 0.4, and 0.5 M NaCl solutions containing 0, 9, 18, or 36 mm IMP and 0, 3, 6, or 9 mm KPP. The effects of KPP on IMP-induced myosin and actin extraction was examined in experiments I and II. In experiment I, meat homogenates were prepared by adding 9 volumes of 0.3 M NaCl solutions containing 9 to 36 mm IMP and 1.5 mm KPP to minced pork. In experiment II, meat homogenates were prepared by adding 9 volumes of 0.3 M NaCl solutions containing 9 to 36 mm IMP and 3.0 mm KPP. After the addition of the 0.3, 0.4, and 0.5 M NaCl solutions, the total KCl and NaCl concentrations of the homogenates were 0.29, 0.38, and 0.47 M respectively, assuming that the KCl concentration of the minced meat was 0.2 M. 9) The addition of IMP and KPP diluted the concentration of the homogenate to 0.9-fold that of the initial concentration. One portion of the homogenate was immediately centrifuged at 12;000 g for 20 min, and centrifugation was finished within 30 min after homogenization. The other portion of the homogenate was incubated at 4 C for 12 h before centrifugation. The dissolved proteins in the supernatant were defined as the extracted proteins. The supernatant and the homogenate were diluted with 2 volumes of 10 mm Tris HCl buffer (ph 7.2) containing 0.1 M NaCl and 1mM NaN 3 and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE). Each of the solution loaded into the well contained protein derived from 74 mg of minced meat. y To whom correspondence should be addressed. Tel: ; Fax: ; nakamura@niku-kakou.or.jp Abbreviations: KPP, tetrapotassium pyrophosphate; MHC, myosin heavy chain

2 SDS-polyacrylamide gel electrophoresis (SDS PAGE). SDS PAGE was carried out by the method of Laemmli 10) with a precast 10% gel (e-pagel E-T 10 L, ATTO, Tokyo) in electrophoresis buffer (AE-1410 EzRun, ATTO). The proteins were stained with Coomassie Brilliant Blue R250 (AE-1340 EzStain Aqua, ATTO). Determination of the extractability of myosin and actin by densitograph. The density of the bands on each of the SDS PAGE profiles was measured with a densitograph (AE-6933FXCF, ATTO). Extractability was calculated by the following formula: Extractability (%) ¼ D s =D h 100 where D s is the density of the myosin heavy chain (MHC) or the actin protein band from the supernatant, and D h is that from the homogenate. Statistics. Extractability was expressed as means of triplicates standard errors. Results Time dependence of IMP- and KPP-induced myosin extraction Figure 1 shows a typical SDS PAGE pattern of proteins extracted with 0.5 M NaCl solutions containing 0, 9, 18, and 36 mm IMP. The extractability of myosin obtained under various IMP conditions is shown in Fig. 2A, B, and C, while that obtained under various KPP conditions is shown in Fig. 2D, E, and F. In the extraction solutions containing 0.3 M NaCl, with IMP at 9 and 18 mm, the 0- and 12-h extractions exhibited similar extractabilities. However, with IMP at 36 mm, 12-h extraction exhibited higher extractabilities than 0-h extraction (Fig. 2A). Thus increasing the extraction time from 0 to 12 h enhanced myosin extraction. On the other hand, extraction with KPP at 3, 6, and 9 mm exhibited high extractability even at 0 h, and the values obtained at 0 h were almost the same as those at 12 h (Fig. 2D). This indicates that KPP-induced myosin extraction from porcine meat was almost complete by 0 h. Myosin and Actin Extraction from Porcine Meat 1215 In the extraction solutions containing 0.4 M NaCl with IMP at 9 mm, 0- and 12-h extraction exhibited similar extractabilities, but with IMP at 18 and 36 mm, 12-h extraction (Fig. 2B), indicating that increasing the extraction time from 0 to 12 h enhanced myosin extraction. On the other hand, extraction with KPP at 3, 6, and 9 mm exhibited high extractability even at 0 h. This indicates that the increased extraction time had no effect on KPP-induced myosin extraction from the porcine meat (Fig. 2E). In the extraction solutions containing 0.5 M NaCl, at all concentrations of IMP, 12-h extraction exhibited higher extractabilities than 0-h extraction (Fig. 2C). On the other hand, extraction with KPP at 3, 6, and 9 mm exhibited high extractability even at 0 h. This indicates that increasing the extraction time had no effect on KPPinduced myosin extraction from porcine meat (Fig. 2F). Time dependence of IMP- and KPP-induced actin extraction The extractability of actin obtained under various IMP conditions is shown in Fig. 3A, B, and C, while that obtained under various KPP conditions is shown in Fig. 3D, E, and F. In the extraction solutions containing 0.3 M NaCl with IMP at 9 and 18 mm, 0- and 12-h extraction exhibited similar extractabilities, but with IMP at 36 mm, 12-h extraction (Fig. 3A). Thus increasing the extraction time from 0 to 12 h enhanced actin extraction. On the other hand, with KPP at 3, 6, and 9 mm, 0- and 12-h Fig. 1. Typical SDS PAGE Pattern Showing Proteins Extracted with 0.5 M NaCl Solutions Containing 0, 9, 18, and 36 mm IMP. Minced pork meat was mixed with 9 volumes of extraction solution and homogenized. The homogenate was centrifuged after incubation at 4 C for 0 and 12 h. The dissolved protein in the supernatant was defined as extracted protein. The supernatant obtained was subjected to SDS PAGE. a, molecular weight marker; b, homogenized whole muscle obtained with 0.5 M NaCl solution containing 0 mm IMP at 0 h of extraction; c j, supernatants from meat extracted with 0.5 M NaCl solutions containing the specified concentrations of IMP after 0 and 12 h, shown under the lanes. MHC, myosin heavy chain. Fig. 2. Effects of Extraction Time on IMP- and KPP-Induced Myosin Extraction. The extractability of myosin was determined after porcine meats homogenized with 9 volumes of 0.3 (A and D), 0.4 (B and E), and 0.5 M (C and F) NaCl solutions containing 0 to 36 mm IMP or 0 to 9mM KPP were incubated for 0 ( ) and 12 h ( ). Results are presented as mean standard error for three independent measurements. Other conditions are described in Materials and Methods.

3 1216 Y. NAKAMURA et al. (Fig. 4A). On the other hand, in the presence of IMP and 1.5 mm KPP, extractabilities with 9, 18, and 36 mm IMP were 55%, 69%, and 87% respectively, representing 1.8-, 2.1-, and 2.0-fold increases respectively as compared with the corresponding sums (31, 34, and 44%) of myosin extractability with 1.5 mm KPP alone (28%) and those with the various concentrations of IMP alone (3, 6, and 16%) (Fig. 4A). Similar results to those obtained at 0 h were obtained at 12 h (Fig. 4B). Thus the presence of 1.5 mm KPP enhanced IMP-induced myosin extraction. In experiment II, as shown in Fig. 4C and D, in the presence of IMP and 3.0 mm KPP, at 0 h the extractabilities with 9, 18, and 36 mm IMP (61, 68, and 80%) exhibited almost the same values as the corresponding sums (65, 68, and 78%) for myosin extractability with 3.0 mm KPP alone (62%) and those for the various concentrations of IMP alone (3, 6, and 16%). Similar results to those obtained at 0 h were obtained at 12 h (Fig. 4D). Thus the presence of 3.0 mm KPP had no influence on IMP-induced myosin extraction. Fig. 3. Effects of Extraction Time on IMP- and KPP-Induced Actin Extraction. The extractability of actin was determined after porcine meats homogenized with 9 volumes of 0.3 (A and D), 0.4 (B and E), and 0.5 M (C and F) NaCl solutions containing 0 to 36 mm IMP or 0 to 9mM KPP were incubated for 0 ( ) and 12 h ( ). Results are presented as mean standard error for three independent measurements. Other conditions are described in Materials and Methods. extraction exhibited similar extractabilities (Fig. 2D). This indicates that KPP-induced actin extraction from the meat was already almost complete at an early stage of extraction. In the extraction solutions containing 0.4 M NaCl with IMP at 9 mm, 0- and 12-h extraction exhibited similar extractabilities, but with IMP at 18 and 36 mm, 12-h extraction (Fig. 3B). On the other hand, with KPP at 3, 6, and 9 mm, 0- and 12-h extraction exhibited similar extractabilities, indicating that the increased extraction time had no effect on KPP-induced actin extraction (Fig. 3E). In the extraction solutions containing 0.5 M NaCl, at all concentrations of IMP, 12-h extraction exhibited higher extractabilities than 0-h extraction (Fig. 3C). On the other hand, with KPP at 3, 6, and 9 mm, no significant effect from the prolonged extraction time was seen, similarly to the results obtained for the 0.3 and 0.4 M NaCl solutions (Fig. 3F). As the mode of myosin and actin extraction enhancement appeared to differ between IMP and KPP, next we examined the effects of KPP on IMP-induced extraction of myosin and actin. Effects of KPP on IMP-induced myosin extraction We determined the effects of KPP on IMP-induced myosin extractability in the presence and the absence of 1.5 and of 3.0 mm KPP, at 0 and 12 h. The results are shown in Fig. 4. In experiment I, at 0 h extractability with 1.5 mm KPP alone was 28% and the extractabilities with 9, 18, and 36 mm IMP alone were 3%, 6%, and 16% respectively Effects of KPP on IMP-induced actin extraction We determined the effects of KPP on IMP-induced actin extractability under the same conditions as those for determining the effect on IMP-induced myosin extraction. The results obtained are shown in Fig. 5. In experiment I, at 0 h extractability with 1.5 mm KPP alone was 10% and extractability with 9, 18, and 36 mm IMP alone were 7%, 9%, and 17% respectively (Fig. 5A). On the other hand, in the presence of IMP and 1.5 mm KPP, the extractabilities with 9, 18, and 36 mm IMP were 13%, 19%, and 40% respectively, representing 0.8-, 1.0-, and 1.5-fold increases respectively as compared with the corresponding sums (17, 19, and 27%) for actin extractability with 1.5 mm KPP alone (10%) and those with the various concentrations of IMP alone (7, 9, and 17%) (Fig. 5A). Thus the presence of 1.5 mm KPP enhanced 36 mm IMP-induced actin extraction. At 12 h, the presence of 1.5 mm KPP enhanced actin extraction induced by 18 and 36 mm IMP (Fig. 5B). In experiment II, as shown in Fig. 5C and D, in the presence of IMP and 3.0 mm KPP, at 0 h the extractabilities with 9, 18, and 36 mm IMP (15, 23, or 38%) exhibited almost the same values as the corresponding sums (22, 24, and 32%) for actin extractability with 3.0 mm KPP alone (15%) and those with various concentrations of IMP alone (7, 9, and 17%). Similar results to those obtained at 0 h were obtained at 12 h (Fig. 5D). Thus the presence of 3.0 mm KPP had no influence on IMP-induced actin extraction. Discussion We found that IMP-induced extraction of myosin and actin from meat homogenates was enhanced by increasing the extraction time in the present study and by increasing the NaCl concentration in our previous study. 8) In contrast, KPP-induced extraction of both proteins was not influenced by these factors. We considered several possible reasons for these differences between IMP and KPP, as follows: First we considered whether there was time dependence in the IMP- and KPP-induced dissociation reaction

4 Myosin and Actin Extraction from Porcine Meat 1217 Fig. 4. Effects of KPP on IMP-Induced Myosin Extraction for 0 and 12 h. The extractability of myosin was determined at 0 (A and C) and 12 h (B and D) after the homogenization of porcine meat with 9 volumes of 0.3 M NaCl solutions containing 0, 9, 18, or 36 mm IMP alone ( on the abscissa), 1.5 mm KPP and 0, 9, 18, or 36 mm IMP (A and B, þ on the abscissa), or 3.0 mm KPP and 0, 9, 18, or 36 mm IMP (C and D, þ on the abscissa). Horizontal dotted lines show the values obtained for 0.3 M NaCl solutions containing 1.5 or 3.0 mm KPP alone. Other conditions are described in Materials and Methods. Fig. 5. Effects of KPP on IMP-Induced Actin Extraction for 0 and 12 h. The extractability of actin was determined at 0 (A and C) and 12 h (B and D) after the homogenization of porcine meat with 9 volumes of 0.3 M NaCl solutions containing 0, 9, 18, or 36 mm IMP alone ( on the abscissa), 1.5 mm KPP and 0, 9, 18, or 36 mm IMP (A and B, þ on the abscissa), or 3.0 mm KPP and 0, 9, 18, or 36 mm IMP (C and D, þ on the abscissa). The horizontal dotted lines show the values obtained with 0.3 M NaCl solutions containing 1.5 or 3.0 mm KPP alone. Other conditions are described in Materials and Methods.

5 1218 Y. NAKAMURA et al. of actomyosin into myosin and actin, as dissociation was assumed to be a prerequisite for the IMP and KPPinduced extraction of myosin and actin from meat homogenates, as found in a previous study. 8) The present study revealed that there was no difference in the KPPinduced extractability of myosin and actin between 0- and 12-h extraction. Hence the KPP-induced dissociation reaction of actomyosin was found to be timeindependent. As for IMP, in a previous paper, 7) we reported that the dissociation reaction of isolated actomyosin was complete within 2 h, and more recently we found that this reaction was complete within 10 min (unpublished data). Hence the IMP-induced dissociation reaction of actomyosin was also assumed to be timeindependent. As we indicated in a previous paper, 7) with 8 mm IMP, complete dissociation of isolated actomyosin occurred in 0.2 M KCl at 0 C. On the other hand, the present study indicates that the addition of a 0.3 M NaCl solution containing 9 or 18 mm IMP to meat homogenate induced very little extraction of myosin (Fig. 2A) or actin (Fig. 3A). This suggests that even if actomyosin is dissociated into myosin and actin by IMP, it does not become solubilized in the 0.3 M NaCl solution, resulting in lower extractability values. This may be due to restraints that prevent the thick filaments containing myosin and the thin filaments containing actin from dissolving. Our previous study 11) suggests that these restraints in myofibrils were released by high concentrations of an alkaline salt solution (Weber-Edsall solution), because the actins that were liberated irreversibly from the thick filaments when the meats heated at 65 C were dissolved into 0.2 M KCl and NaCl only after the scaffolds of the myofibrils were destroyed by treatment of the heated meats with Weber-Edsall solution. These restraints might be bonds between thick and thin filaments and the scaffolds of myofibrils. We presumed that IMP and KPP have activities to release these restraints, which function at certain concentrations of NaCl. Thus the results obtained in the present study might be explained by the fact that the restraintreleasing activities of IMP are time-dependent and are enhanced by increased concentrations of NaCl, while those of KPP are time-independent and function optimally in 0.3 to 0.5 M NaCl. It is noteworthy that the 0.3 M NaCl solution appeared to reduce the amounts of IMP and KPP necessary for optimal extraction of myosin from the meat when IMP and KPP were simultaneously present, as the presence of 9 to 36mM IMP and 1.5 mm KPP markedly enhanced myosin extraction (Fig. 4A and B). We are now studying to determine whether this phenomenon occurs in the production of sausage. We presumed that 1.5 mm KPP has a low activity to dissociate actomyosin into myosin and actin at 0.3 M NaCl (as an extractability of only 28% was seen) even though it exhibits a high activity to release thick filaments from the restraints in myofibril at this salt concentration. On the other hand, we presumed that 9 to 36 mm IMP exhibits a high activity to dissociate actomyosin, but a low activity to release thick filaments from the restraints in the myofibrils at 0.3 M NaCl. Thus when thick filaments are released from the restraints in myofibrils by 1.5 mm KPP, high extractability of myosin appears to be brought about by the optimal actomyosindissociating activity of IMP under these conditions. Based on the results described above, we conclude that there are differences between IMP and KPP in mode of enhancement of myosin and actin extraction from porcine meat. These differences might be due to differences in their reaction mechanisms toward myofibrils. Further studies are needed to clarify these mechanisms in detail. References 1) Fukazawa T, Hashimoto Y, and Yasui T, J. Food Sci., 26, (1961a). 2) Fukazawa T, Hashimoto Y, and Yasui T, J. Food Sci., 26, (1961b). 3) Fukazawa T, Hashimoto Y, and Yasui T, J. Food Sci., 26, (1961c). 4) Samejima K, Hashimoto Y, Yasui T, and Fukazawa T, J. Food Sci., 34, (1969). 5) Yasui T, Fukazawa T, Takahashi K, Sakanishi M, and Hashimoto Y, J. Agric. Food Chem., 12, (1964). 6) Morita J, Kume H, Nagahashi T, and Yasui T, J. Fac. Agric. Hokkaido Univ., 61, (1983). 7) Okitani A, Ichinose N, Koza M, Yamanaka K, Migita K, and Matsuishi M, Biosci. Biotechnol. Biochem., 72, (2008). 8) Nakamura Y, Migita K, Okitani A, and Matsuishi M, Biosci. Biotechnol. Biochem., 76, (2012). 9) Offer G and Knight P, Developments in Meat Science Vol. 4, ed. Lawrie R, Elsevier Science Publishers, Barking, Essex, pp (1988). 10) Laemmli UK, Nature, 227, (1970). 11) Okitani A, Ichinose N, Itoh J, Tsuji Y, Oneda Y, Hatae K, Migita K, and Matsuishi M, Meat Sci., 81, (2009).