Original papers
Effect of salmon protamine on the physicochemical properties of porcine myofibril
2021 Volume 27 Issue 6 Pages 915-921
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2021 Volume 27 Issue 6 Pages 915-921
Though salmon protamine (PRO) is known to exhibit strong antibacterial action, the effect of PRO on the physicochemical properties of porcine myofibril (Mf) are not yet fully understood. In this study, we investigated the effect of PRO on the rheological properties of Mf proteins. The viscosity of the Mf homogenate decreased with increasing PRO in a concentration-dependent manner. The breaking stress of the thermal gel was reduced in the presence of PRO. Scanning electron microscopy examination showed that PRO had a 3-dimensional network structure with thread-like protein filaments. SDS-PAGE analysis demonstrated that myosin binding protein-C and myosin light chain 1 in Mf were solubilized by the addition of PRO. However, PRO inhibited the solubilization of α-actinin. These results showed that PRO had an adverse effect on the gelling properties of porcine Mf through the protein-protein interaction between PRO and Mf proteins.
Various additives are used in the process of manufacturing, processing or preserving foods. As of January 2021, there are 472 kinds of designated additives and 365 kinds of existing food additives such as preservatives, sweeteners, coloring agents, and flavoring agents in Japan (Ministry of Health, Labour and Welfare)i). Fish milt is listed as one of the existing food additives, and the main protein component is protamine (PRO), with a molecular mass of about 4 000 Da (Callanan, 1957; Sorgi et al., 1997). Generally, it is known that PRO has some physiological functions, such as the protection of DNA and the anticoagulant action of heparin (Gill et al., 2006). In the food-processing industry, PRO has been widely used as an antibacterial agent against bacteria, yeast, molds, and so on. (Islam et al., 1984; Potter et al., 2005). There are also some reports on the effect of PRO on the rheological properties of other proteins. For example, PRO has been reported to enhance the physicochemical properties of serum albumin, such as increase in foaming, viscosity, and emulsification (Glaser et al., 2007; Xu et al., 2020).
In meat processing, NaCl is an essential additive because the physicochemical properties of meat depend on the characteristics of salt-soluble proteins (myofibril, Mf). Myosin is particularly important for gelation, while actin plays a role as a promoter of gelation (Stanley et al., 1994). However, various non-meat proteins are added to meat to improve its water-holding capacity and gelation of the products as consumers become increasingly concerned about the effect of salt intake on health. To date, there are very few reports on the effect of PRO on the gelation of Mf. In this study, we investigated the effect of PRO on the rheological properties of Mf, such as viscosity and thermal gelation.
Samples and reagents Commercial pork loin purchased at a meat shop (Shimadaya-Shoji, Ibaraki, Japan) was vacuum packaged in a nylon bag and stored at −80 °C until use. Protamine sulfate from salmon (Oncorhynchus keta) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Other reagents used were of analytical grade.
Preparation of porcine Mf Porcine Mf was prepared from pork loin by partially modifying the method of Perry and Grey (1956). Briefly, sarcoplasmic protein was removed from the minced meat by washing with 25 mM KCl-50 mM imidazole-HCl buffer (pH 6.5). The obtained insoluble fraction (slurry form) was recovered as Mf. The protein concentration of the Mf was determined by the Kjeldahl method.
Determination of the physicochemical properties of Mf Porcine Mf homogenate was prepared by dispersing PRO and Mf slurry in 50 mM imidazole buffer with NaCl (final concentration, 0.2 M) (pH 6.5). The PRO-added Mf homogenate was then poured into a weighing bottle (2.0 cm × ;⌀1.5 cm) and incubated at 4 °C for 1 h, whereafter the viscosity was measured with a vibration viscometer (VM-KA, SEKONIC, Tokyo, Japan). Then, the homogenate was heated in a water bath at 70 °C for 30 min. The breaking stress of the Mf gel was measured by compressing with a plunger (⌀8 mm) using a rheometer (Creep-rheoner II RE-3305, Yamaden, Tokyo, Japan) after cooling on ice for 3 h. The data were analyzed on a computer and expressed as breaking stress (N/m2). The microstructure of the gel was observed with a scanning electron microscope (SEM; JSM-6360A, JEOL, Tokyo, Japan). Briefly, a piece of the gel was cut into a 5 mm square and dehydrated in a freeze-drier (LFD-100NPS1, Laytant Life Science, Kanagawa, Japan) after freezing by immersion in liquid nitrogen. After coating of the sample with platinum using a magnetron sputtering device (JUC-5000, JEOL,), the gel network structure of the sample was observed using the SEM at an acceleration voltage of 10 kV and a magnification of 2 000 or 4 000 times.
The molecular behavior of Mf proteins in the presence of PRO was analyzed by SDS-PAGE based on the method of Laemmli (1970). Briefly, the sample was centrifuged at 10 000 g for 5 min and the supernatant was used for SDS-PAGE analysis. The thermal gel described above was treated with SDS and β-mercaptoethanol, and the resulting soluble fraction was also used as a sample. They were subjected to SDS-PAGE using a slab-type electrophoresis device (ATTO, AE-6500, Tokyo, Japan) at a constant current (30 mA). Protein bands were detected by staining with Coomassie Brilliant Blue G-250. Several protein bands were cut from the gel and treated with trypsin followed by peptide sequencing by MALDI-TOF-mass spectrometry. Protein identification was carried out using the Mascot search engine. This analysis was performed by Cosmo-Bio (Tokyo, Japan). The outline of these experiments is summarized in the flow diagram shown in Fig. 1.
Flow chart for determination of the physicochemical properties of porcine myofibril with salmon protamine.
The amino acid composition of PRO was analyzed using a Hitachi amino acid analyzer (L-8800, Hitachi hi-tech, Tokyo, Japan) after hydrolysis with 6 mol/L hydrochloric acid at 110 °C for 22 h under vacuum conditions. As expected, commercial PRO consisted of Arg, which accounted for about 70 % of the total amino acids (Fig. 2). Other constituent amino acids, such as Pro, Ser, Gly, Val, and Met, were also detected. The results showed almost perfect agreement with the theoretical data from the protein sequence database of UniProtii).
Amino acid composition and sequence of salmon protamine.
Arg, arginine (21 mol/mol PRO); Ser, serine (4 mol/mol PRO); Pro, proline (3 mol/mol PRO); Gly, glycine (2 mol/mol PRO); Val, valine (2 mol/mol PRO); Met, methionine (1 mol/mol PRO).
Theoretical data were obtained from UniProtii).
Fig. 3 shows the effect of PRO on the viscosity of the Mf homogenate. The viscosity decreased drastically as the concentration of PRO increased. The viscosity with 1 % PRO was about 1/18 of that without PRO. Fig. 4 shows the appearance of the Mf homogenate placed on a petri dish. The homogenate of Mf alone remained on the petri dish when it was tilted. On the other hand, PRO decreased the adhesiveness of Mf, as the homogenate flowed on the dish.
Viscosity of porcine myofibril homogenate with or without protamine.
Homogenate was prepared with 5 % porcine myofibril and protamine.
The statistical significance of viscosity was evaluated by a one-way ANOVA. Each bar represents mean ±SE (n = 3). Data bearing same superscripts are not significantly different (p < 0.05).
Appearance of porcine myofibril homogenate with or without protamine.
Cont., 5 % porcine myofibril alone +PRO, 1 %-protamine added 5 % porcine myofibril.
Fig. 5 shows the breaking stress of the thermal Mf gels with and without PRO. The addition of PRO reduced the gel hardness by half. Fig. 6 shows an SEM image of the Mf gel with PRO. In the gel containing Mf alone, floc-like protein filaments were observed to form a network structure at 2000-fold magnification. On the other hand, the gel with PRO also had a network structure with thin thread-like protein filaments, compared to that without PRO. Further, the SEM images at 4000-fold magnification indicated numerous tiny particles on the surface of the protein filaments in the presence of PRO.
Breaking stress of thermal gel prepared from porcine myofibril with or without protamine.
Cont., 5 % Porcine myofibril alone at 0.2 M of NaCl.
+PRO, 1 % PRO-added 5 % porcine myofibril at 0.2 M of NaCl.
The statistical significance of gel hardness was evaluated by a one-way ANOVA. Each bar represents mean ±SE (n = 3). Data bearing same superscripts are not significantly different (p < 0.05).
Scanning electric microscopic images of porcine myofibril gels in the presence of protamine (magnification 2000- or 4000-fold).
Cont. and +PRO, Refer to Fig. 5
Fig. 7 shows the SDS-PAGE band pattern for Mf in the presence of PRO. Without heat treatment, some soluble proteins in Mf were detected as shown in Fig. 7A. The addition of PRO reduced the protein band intensity of 100 kDa proteins. However, the protein band intensities of the 140 and 25 kDa proteins increased after the addition of PRO. MALDI-TOF-MS analysis showed that the 140, 100 and 25 kDa bands were due to myosin binding protein-C (MBP-C), α-actinin, and myosin light chain 1 (MLC1), respectively.
SDS-PAGE patterns of PRO-added myofibril with or without thermal treatment.
A, Soluble fraction of porcine myofibril without heat treatment
B, SDS-soluble fraction of porcine myofibril with heating
12.5 % acrylamide of separation gel; MW, molecular weight marker
Cont., Porcine myofibril alone.
+PRO, PRO-added porcine myofibril.
MBP-C, myosin binding protein-C; α-Act, α-actinin; MLC1, myosin light chain 1; MHC, myosin heavy chain; Ac, actin.
Fig. 7B shows the SDS-PAGE band pattern for proteins extracted from the heated Mf with or without PRO. Some protein bands corresponding to myosin heavy chain (MHC), α-actinin, actin, and MLC1 were detected with or without PRO. The intensities of all protein bands with PRO were almost equivalent to those without PRO.
PRO is a highly cationic peptide that is rich in basic amino acids such as Arg and Lys (Pugsley et al., 2002). A theoretical amino acid analysis showed that PRO mainly consisted of Arg (Fig. 2). The practical composition ratio was slightly different from the theoretical composition, likely because some foreign amino acids, such as Ile and Ala, may have contaminated the PRO, or because amino acids like Met degrade easily by hydrolysis in 6 M HCl (Darragh et al., 1996).
The viscosity of the Mf homogenate decreased with an increase in PRO. After heating, the gel hardness of Mf decreased by the addition of PRO. Solubilization of myosin is required to increase the viscosity of Mf, followed by it's gelation. It is known that solubilized myosin plays an important role in the formation of a three-dimensional gel network structure after heating (Ishioroshi et al., 1981). Sano et al. (1989) reported that actin is responsible for the viscous element of Mf. In the control group, Mf formed weak gels because myosin and actin could not be sufficiently solubilized by 0.2 M NaCl. The gelation properties of Mf decrease with increase PRO content, suggesting that the addition of PRO would not improve the solubility of myosin and actin or that it may inhibit the solubilization of PRO.
In addition, SEM observations showed that the change in the gel network structure of Mf occurred after the addition of PRO. In the presence of PRO, tiny particles on the protein filaments were observed in SEM images. It was previously reported that the antibacterial activity of PRO was mainly attributed to binding to the positively charged Arg-rich domains in the bacterial cell membrane (White et al., 1995). PRO was also reported to show anticoagulant activity by formation of stable bonds with heparin (Carr and Silverman, 1999). The findings of these reports imply that PRO would decrease the viscosity and gelation properties of Mf due to the binding of positively charged PRO to negatively charged Mf proteins.
To clarify the protein-protein interactions between PRO and Mf, an SDS-PAGE analysis was conducted. It was shown that PRO selectively changed the solubility of Mf proteins. PRO specifically insolubilized α-actinin that is located in the Z disks of Mf. α-Actinin has been reported to bind to negatively charged phospholipids which are also a component of Z disks (Bullard et al., 1990). These results suggest that positively charged PRO may interact with the phospholipids in the Z disks, followed by the insolubilization of α-actinin.
Conversely, MBP-C and MLC1 were solubilized in the presence of PRO. As the name aptly implies, MBP-C and MLC1 are major binding partners for MHC. It has been reported that MBP-C plays an important role in the assembly of filaments through strong interactions with myosin rods (Moos et al., 1975). The present results suggested that the solubilization of MBP-C and MLC1 may occur through a protein-protein interaction between MHC and PRO. Although there have been few reports on the effect of MBP-C on the rheological properties of meat, we must investigate the relationship between the state of MBP-C and the texture of meat.
Further, PRO decreased the breaking stress of thermal Mf gels which were mainly formed by MHC and actin, as shown in the SDS-PAGE analysis, suggesting that PRO may alter the electrical charge of Mf without proteolysis, even though the solubilities of myosin-binding proteins (MBP-C and MLC1) and actin-binding protein (α-actinin) were changed.
In conclusion, this study demonstrated that PRO decreased the viscosity of porcine Mf, and that the inhibition of thermal gelation was unpredictable. Generally, Mf protein contributes to the achievement of a desirable texture in processed meat products. PRO had a negative effect the gelation of Mf. However, it is an interesting point that PRO changes the rheological properties Mf proteins from the viewpoint of food engineering. Based on these findings, we are further investigating the effect of PRO on the physicochemical properties of Mf proteins.
Acknowledgements We thank Dr. Naomi Asagi at Ibaraki University for providing technical assistance with SEM analysis. This work was supported by JSPS KAKENHI Grant Number JP26660102.
Conflict of interest There are no conflicts of interest to declare.
