Jun-ichi Wakamatsu

A Zn-porphyrin complex contributes to bright red color in Parma ham

The Italian traditional dry-cured ham (Parma ham) shows a stable bright red color that is achieved without the use of nitrite and/or nitrate. In this study we examined the pigment spectroscopically, fluoroscopically and by using HPLC and ESI-HR-MASS analysis. Porphyrin derivative other than acid hematin were contained in the HCl-containing acetone extract from Parma ham. A strong fluorescence peak at 588 nm and a weak fluorescence peak at 641 nm were observed. By HPLC analysis the acetone extract of Parma ham was observed at the single peak, which eluted at the same time as Zn-protoporphyrin IX and emitted fluorescence. The results of ESI-HR-MS analysis showed both agreement with the molecular weight of Zn-protoporphyrin IX and the characteristic isotope pattern caused by Zn isotopes. These results suggest that the bright red color in Parma ham is caused by Zn-protoporphyrin IX.

Decorin enhances the proliferation and differentiation of myogenic cells through suppressing myostatin activity

Decorin, a small leucine‐rich proteoglycan, plays an important role in the regulation of cell growth. Our recent study has shown that immobilized decorin in the collagen matrix sequesters myostatin into the extracellular matrix and prevents its inhibitory action to myoblast proliferation in vitro. However, it still remains unclear whether free decorin could affect the proliferation and differentiation of myogenic cells by regulating myostatin activity. In the present study, we generated stable clonal C2C12 myoblasts that were over‐expressing decorin, and showed that decorin over‐expressing cells had an increased rate of proliferation as compared to control cells. Decorin over‐expressing cells formed multi‐giant hypertrophic myotubes with an elongated morphology and larger size as compared to control cells, although the initiation of differentiation in decorin over‐expressing cells was somewhat delayed as compared to control cells. Western blot analysis demonstrated that MyoD expression in decorin over‐expressing cells was lower than that in control cells until 12 h after induction to differentiate. At 48‐h differentiation, the expressions of MyoD, p21 and myogenin were dramatically increased in cells that over‐expressed decorin. Furthermore, we revealed that over‐expression of decorin suppressed the activity of myostatin endogenously synthesized in C2C12 myoblasts and attenuated the signaling of exogenous myostatin. Consistent with these results, knock‐down of decorin impairs C2C12 myoblast growth by increasing the sensitivity to exogenous myostatin. These results clearly show that decorin enhances the proliferation and differentiation of C2C12 myoblasts through suppressing myostatin activity. J. Cell. Physiol. 215: 856–867, 2008.

Establishment of a model experiment system to elucidate the mechanism by which Zn–protoporphyrin IX is formed in nitrite-free dry-cured ham

The aim of this study was to establish a model experiment system to elucidate the mechanism by which Zn-protoporphyrin IX (ZPP) is formed in Parma ham. The established model consisted of myoglobin, meat and antibiotics, and incubation under anaerobic conditions resulted in a greater yield of ZPP. Formation of ZPP was observed even in the presence of various antiseptics. The amount of ZPP formed increased as the period of incubation increased. ZPP formation was inhibited by heating meat homogenate depending on the heating temperature. Our results show that anaerobic conditions are suitable for the formation of ZPP in meat products without nitrate or nitrite and that endogenous enzymes as well as microorganisms may be involved in ZPP formation.

Species and muscle differences in L-carnitine levels in skeletal muscles based on a new simple assay

We have adapted the enzymatic method [Biochemical and Biophysical Research Communications 176 (3) (1991) 1617] for the safe and rapid assay of L-carnitine (L-CA) in skeletal muscle using a microplate reader. The concentration of L-CA in fresh semitendinosus muscle from broiler chicken, pig, beef cattle, deer, horse and goat muscle were 0.69, 1.09, 1.86-3.57, 4.57, 4.95 and 11.36 μmol/g wet weight, respectively. The animals which had higher concentration of L-CA, also had the highest amounts of myoglobin as an index to the redness of the muscle. Furthermore, we investigated this relationship between white muscle, M. pectoralis profundus, and red muscle, M. soleus, in laying hens. The L-CA and myoglobin concentration in red muscle were significantly higher than those in white muscle (p<0.01). These findings suggest that L-CA concentration in muscle is related to oxygen metabolism and to myofiber types.

Myosin is solubilized in a neutral and low ionic strength solution containing L-histidine.

Myosin, one of the major myofibrillar proteins, is insoluble at low and physiological ionic strength and soluble at high ionic strength. In this study, the behavior and morphology of myosin solubilized in a low ionic strength solution containing l-histidine (l-His) was investigated. More than 80% of myosin was solubilized in a low ionic strength solution with dialysis against a solution containing 1mM KCl and 5mM l-His. Transmission electron microscopy with rotary shadowing demonstrated that the rod of myosin in a low ionic strength solution containing l-His is longer than that of myosin in a high ionic strength solution. The elongation of the myosin rod in a low ionic strength solution containing l-His would inhibit the formation of a filament, resulting in the solubilization of myosin.

Heat-induced gelation of myosin in a low ionic strength solution containing L-histidine

Binding properties are important for meat products and are substantially derived from the heat-induced gelation of myosin. We have shown that myosin is solubilized in a low ionic strength solution containing L-histidine. To clarify its processing characteristics, we investigated properties and structures of heat-induced gels of myosin solubilized in a low ionic strength solution containing L-histidine. Myosin in a low ionic strength solution formed transparent gels at 40-50°C, while myosin in a high ionic strength solution formed opaque gels at 60-70°C. The gel of myosin in a low ionic strength solution with L-histidine showed a fine network consisting of thin strands and its viscosity was lower than that of myosin in a high ionic strength solution at 40-50°C. The rheological properties of heat-induced gels of myosin at low ionic strength are different from those at high ionic strength. This difference might be caused by structural changes in the rod region of myosin in a low ionic strength solution containing L-histidine.

Myosin filament depolymerizes in a low ionic strength solution containing l-histidine

Myosin, one of the major myofibrillar proteins, forms a filamentous polymer and is insoluble in physiological and low ionic strength solutions. We have shown that myosin is soluble in a low ionic strength solution containing L-histidine. In this study, to clarify the role of L-histidine in the solubilization of myosin, we investigated effects of L-histidine on the filament formation and the morphology of myosin at a low ionic strength. In the presence of L-histidine, myosin formed a filamentous polymer in a physiological ionic strength solution and dispersed in a low ionic strength solution. Transmission electron microscopy showed that light meromyosin (LMM), the rod region of myosin, in a low ionic strength solution containing L-histidine was longer than that in a high ionic strength solution without L-histidine. L-histidine causes the elongation of LMM region of myosin contributing to the weakening of the myosin filament and the dissociation of myosin in a low ionic strength solution.

Interaction between myostatin and extracellular matrix components

Myostatin, a member of the TGF-beta superfamily, is a negative regulator of skeletal muscle mass. We have recently demonstrated that decorin binds to myostatin in vitro, and that immobilized decorin within the collagen matrix prevents myostatin-mediated inhibition of myoblast proliferation. However, little is known about other ECM molecules that bind to myostatin and modulate its activity. Thus, in the present study, we investigated the interaction of several other ECM molecules with myostatin. We here show that fibromodulin, fibronectin and laminin bind to myostatin in the presence of Zn(2+) with a dissociation constant (K(D)) of 10(-10) approximately 10(-8) mol/L. Fibromodulin shows the highest affinity for myostatin among them. These results suggest that these ECM molecules may modulate myostatin activity like decorin does.

Zn protoporphyrin IX is formed not from heme but from protoporphyrin IX

We examined the effects of exogenous myoglobin, a bivalent chelator, and nitrite on Zn protoporphyrin IX (ZPP) formation by using model systems. ZPP was formed in a model solution without addition of exogenous myoglobin. After incubation, the amount of ZPP in a model solution was increased but that of heme was not decreased compared with the amounts before incubation. Protoporphyrin IX (PPIX) instead of ZPP also accumulated in a model solution with addition of EDTA, but the amount of heme was not reduced. These results suggested that ZPP was not formed by the Fe-Zn substitution in heme but was formed by the insertion of Zn into PPIX, which was formed independently. The fact that the effects of various factors in model systems with/without addition of a bivalent chelator were similar suggested that ZPP formation was strongly affected by PPIX formation. Inhibition of PPIX formation by nitrite might be the reason for the low levels of ZPP in cured meats.

Effect of liquid whey feeding on fecal microbiota of mature and growing pigs

The effect of liquid whey feeding on fecal bacteria and their metabolites was assessed in five pregnant sows and 66 growing pigs. Sows were fed a control diet for 4 weeks (control period) followed by the same diet but with whey feeding (5 L/day/pig) for 4 weeks (whey period). One group of growing pigs was given 267 L of whey per pig (whey group), while the other group was not (control group). In both cases, liquid whey was given separately from control diet. Sows in the whey period had feces showing lower pH, lower ammonia concentration, and larger population sizes of total bacteria, lactobacilli and bifidobacteria. The bacterial gene library analysis indicated that Mitsuokella and Megasphaera were more frequently detected, while Clostridium disporicum were detected less frequently in the whey period. Feces from whey-fed growing pigs showed lower pH than that from control pigs in the early stage of growing. Also, larger populations of total bacteria, lactobacilli and bifidobacteria were recorded in the whey group. From the bacterial gene library analysis, the detection frequency of Lactobacillus reuteri tended to be higher in the whey group. These results indicate that whey feeding influences the hindgut microbiota of pigs, possibly leading to a fermentation shift that is favorable for animal health.