Science and technology of meat and meat products in Japan-Pursuit of their palatability under the influence of Washoku, traditional Japanese cuisine.

This review aimed to provide an overview of meat science and technology in Japan influenced by Washoku (traditional Japanese cuisine) from the aspect of meat palatability. The tradition of umami (savory taste) research would contribute to a better understanding of the mechanism of taste improvement by aging, processing, and feeding. Paying close attention to delicate food aroma in Washoku would facilitate finding preferable Wagyu beef aroma, as well as odor substances affecting human physiological conditions. Sticking to various food textures could potentially facilitate the research of heating gel of meat and enzymatic and nonenzymatic theories for meat tenderization. High-pressure studies of meat propose a novel approach to producing low-salt products. Raw ham, Lachs-type ham, might fascinate people due to its moist texture similar to that of raw fish. Methods to season traditional fake meat using plant materials in Washoku could give a hint for improving substituted meat flavor.

Characterization of myofibrillar adenosine triphosphatase activity and liberation of actin from myofibrils upon heating chicken breast meat.

Denaturation of actin and myosin in myofibrils induced by heating at 50°C was investigated to reveal the mechanism of irreversible liberation of actin from myofibrils on heating at lower temperatures than conventional cooking. Denaturation of these proteins was determined by Mg2+ -ATPase (adenosine triphosphatase) and Ca2+ -ATPase activities. When minced meat was heated for 20 min, actin was liberated accompanying denaturation of 80% of actin and 50% of myosin. Heating of the myofibrillar fraction (MFF) isolated from meat homogenate induced much slower denaturation of actin than myosin. When MFF was heated with sarcoplasmic fractions, denaturation of actin was facilitated, suggesting that sarcoplasmic fractions contain factors to facilitate actin denaturation. Inosine-5'-monophosphate, a component of sarcoplasmic fractions, was shown to have no effect on actin and myosin denaturation. These results suggest that heating meat at 50°C dissociates binding ('Bond A') between actin and myosin participating in ATPase activities, resulting in denaturation of both proteins under influence of sarcoplasmic components. Although denaturation of actin and myosin disrupted Bond A, actin was not liberated simultaneously, suggesting the presence of another bond ('Bond B', more heat-stable than Bond A) between both proteins and necessity of disruption of Bond B for actin release from myofibrils.

Retort beef aroma that gives preferable properties to canned beef products and its aroma components.

The objective of this study is to identify the properties and responsible compounds for the aromatic roast odor (retort beef aroma) that commonly occurs in canned beef products and could contribute to their palatability. The optimal temperature for generating retort beef aroma was 121°C. An untrained panel evaluated both uncured corned beef and canned yamato-ni beef and found that they had an aroma that was significantly (P < 0.01) similar to the odor of 121°C-heated beef than 100°C-heated beef. The panel also noted that the aroma of 121°C-heated beef tended to be (P < 0.1) preferable than that of 100°C-heated beef. These results suggest that retort beef aroma is one constituent of palatability in canned beef. GC-MS (gas chromatography-mass spectrometry) analysis of the volatile fraction obtained from 100°C- and 121°C-heated beef showed that the amounts of pyrazine, 2-methylpyrazine and diacetyl were higher in the 121°C-heated beef than in the 100°C-heated beef. GC-sniffing revealed that the odor quality of pyrazines was similar to that of retort beef aroma. Therefore, pyrazines were suggested to be a candidate responsible for the retort beef aroma. Analysis of commercial uncured corned beef and cured corned beef confirmed the presence of pyrazine, 2-methylpyrazine and 2,6-dimethylpyrazine.

Inosine-5'-monophosphate is a candidate agent to resolve rigor mortis of skeletal muscle.

The object of the present study was to reveal the action of inosine-5'-monophosphate (IMP) toward myofibrils in postmortem muscles. IMP solubilized isolated actomyosin within a narrow range of KCl concentration, 0.19-0.20 mol/L, because of the dissociation of actomyosin into actin and myosin, but it did not solubilize the proteins in myofibrils with 0.2 mol/L KCl. However, IMP could solubilize both proteins in myofibrils with 0.2 mol/L KCl in the presence of 1 m mol/L pyrophosphate or 1.0-3.3 m mol/L adenosine-5'-diphosphate (ADP). Thus, we presumed that pyrophosphate and ADP released thin filaments composed of actin, and thick filaments composed of myosin from restraints of myofibrils, and then both filaments were solubilized through the IMP-induced dissociation of actomyosin. Thus, we concluded that IMP is a candidate agent to resolve rigor mortis because of its ability to break the association between thick and thin filaments.

IMP improves water-holding capacity, physical and sensory properties of heat-induced gels from porcine meat.

Water-holding capacity (WHC) of heat-induced pork gels was examined. The heat-induced gels were obtained from meat homogenates prepared by adding nine volumes of 0.3-0.5 mol/L NaCl solutions containing 9-36 mmol/L disodium inosine-5'-monophosphate (IMP) or 9 mmol/L tetrapotassium pyrophosphate (KPP) to minced pork. IMP at 36 mmol/L enhanced the WHC to the same level as attained by KPP. Physical and sensory properties of heat-induced gels were also examined. The heat-induced gels were prepared from porcine meat homogenates containing 0.3 mol/L NaCl and 9-36 mmol/L IMP or 9 mmol/L KPP. IMP at 36 mmol/L enhanced the values of hardness, cohesiveness, gumminess and springiness, measured with a Tensipresser, and several organoleptic scores to the same level as the score attained by KPP. Thus, it is concluded that IMP is expected to be a practical substitute for pyrophosphates to improve the quality of sausages.

Mode of IMP and pyrophosphate enhancement of myosin and actin extraction from porcine meat.

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 IMP-induced myosin and actin extraction. Incorporating these results with our previously published data (Nakamura et al., Biosci. Biotechnol. Biochem., 76, 1611-1615 (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 restraint-releasing ability of IMP is dependent on reaction time and NaCl concentration while that of KPP is not.

Enhancing effect of IMP on myosin and actin extraction from porcine meat.

We examined the effects of nucleoside monophosphates on the dissociation of actomyosin into myosin and actin. GMP was effective only among GMP, CMP, dTMP, and UMP. Hence we concluded that purine-based nucleoside monophosphates such as GMP, AMP, and IMP are effective, incorporating this with our previous results (Okitani A et al., Biosci. Biotechnol. Biochem., 72, 2005-2011 (2008)). Then we examined whether IMP enhances the extraction of myosin and actin as well as pyrophosphate (KPP), using homogenates of pork with 9 volumes of 0.3, 0.4, and 0.5 M NaCl solutions containing 0-36 mM IMP or 0-9 mM KPP. Maximum extractability, about 70% for both proteins, was attained by means of NaCl solutions containing 36 mM IMP. These values were comparable to the maximum values, about 90% for myosin and 50% for actin, attained by means of solutions containing 9 mM KPP. Hence we concluded that IMP enhances the extraction of myosin and actin from porcine meat.

Liberation of actin from actomyosin in meats heated to 65°C.

This study investigated whether actin liberation from myofibrils occurs during the heating of various muscles, as well as squid mantle muscle at temperatures, such as 60°C, employed for vacuum cooking of meats. Actin liberation was demonstrated in scallop striated adductor muscle, but not in beef, pork, or chicken, using the detection method previously employed with squid muscle, in which liberated actin was detected with SDS-PAGE, in the supernatant obtained by centrifugation of the homogenate of heated muscle in 0.2M KCl at a neutral pH. However, actin liberation was demonstrated in beef, pork and chicken by a new detection method, in which heated muscle was homogenized in 0.6M KCl or NaCl at a slightly alkaline pH and maintained at 4°C for 16h with stirring, after which the homogenate was diluted three times with water and centrifuged to obtain the supernatant containing the liberated actin. This new method indicated that actin liberation in beef, pork, and chicken was marked by heating at 65°C, but scarcely induced at 80°C. Thus, the liberation of actin from myofibrils may contribute to the greater tenderness of vacuum-cooked meat (meat heated at a low temperature for long time), as compared with meat prepared by cooking at a higher temperature.

AMP and IMP dissociate actomyosin into actin and myosin.

We investigated to determine why heating of squid muscle at 60 degrees C induced the liberation of actin from myofibrils. When a mixture of a myofibrillar fraction and a low-molecular sarcoplasmic fraction prepared from squid muscle was heated at 60 degrees C, actin liberation occurred. When a myofibrillar fraction was heated with ATP, AMP, or IMP, actin liberation occurred. Hence, AMP is perhaps one of the factors causing actin liberation in postmortem squid muscle. It was found that AMP and IMP reversibly dissociated actomyosin of chicken, bovine, and porcine skeletal muscles into actin and myosin on incubation at 0 degrees C at pH 7.2 in 0.2 M KCl. These results led us to conclude that AMP and IMP were the most responsible factors causing actin liberation from myofibrils in the heated muscle and causing reversible dissociation of actomyosin on storage of skeletal muscle at a low temperature. Hence, AMP and IMP are possible factors causing the resolution of rigor mortis in muscles.

Purification and properties of cysteine proteinase inhibitors from rabbit skeletal muscle.

Two cysteine proteinase inhibitors, CPI-L and CPI-H, were purified from rabbit skeletal muscle by means of successive extraction with a neutral buffer solution, precipitation at pH 3.7, acetone fractionation and gel permeation on Sephadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. The molecular mass of CPI-L was 13 kDa on gel permeation chromatography and SDS-PAGE under reducing conditions and was 15 kDa on SDS-PAGE under non-reducing conditions. The molecular mass of CPI-H was 23 kDa on gel permeation chromatography and it was converted to 13 kDa by SH-reducing agent. Although CPI-H showed single protein band with 13 kDa on SDS-PAGE under reducing conditions, it showed four protein bands with 21, 20, 15 and 13 kDa on SDS-PAGE under non-reducing conditions. Therefore, CPI-H was suggested to have a complicated subunit structure for which S-S bonds and some non-covalent bonds would be responsible. CPI-L and CPI-H were stable in the range of pH 3.0-9.5 and up to 80 degrees C. CPI-L and CPI-H were suggested to inhibit cathepsins B, H and L by a non-competitive mechanism. The inhibition constants (Ki) of CPI-L and CPI-H showed that both CPIs have much higher affinity against cathepsins H and L than against cathepsin B.

Purification and some properties of cathepsin H from rabbit skeletal muscle.

Rabbit muscle cathepsin H classified as an aminoendopeptidase was purified and its properties were investigated to clarify its contribution to the proteolysis of postmortem muscle. The purification was performed by ammonium sulfate fractionation and successive chromatographies on Sephadex G-75, phosphocelluose, DEAE-Sephadex A-50 and Sephadex G-100. The purified enzyme gave a single protein band on SDS-PAGE. Its molecular mass was found to be 28 kDa by gel permeation and 30 kDa by SDS-PAGE. The optimum pHs for alpha-N-benzoyl-DL-arginine-beta-naphthylamide (BANA)- and L-leucine-beta-naphthylamide (Leu-NA)-hydrolyzing activities were 6.6 and 7.0, respectively. This enzyme was almost stable in the range of pH 4-5 and up to 50 degrees C at pH 5.0. The Km values of BANA- and Leu-NA-hydrolyzing activities were 0.367 and 0.203 mM, respectively. The enzyme was inhibited by monoiodoacetic acid, antipain, leupeptin, TLCK and TPCK, but was not affected by pepstatin, bestatin, puromycin, PMSF or trypsin inhibitor. This enzyme strongly acted on Arg-, Lys-, Met-, Ala-, Ser- and Leu-NAs, weakly acted on Val- and Glu-NAs, and hardly acted on Pro- and Gly-NAs. The amount of cathepsin H in muscle was estimated to be less than one-fourth of the sum of the amount of aminopeptidases C and H by the Leu-NA-hydrolyzing activity on the chromatography. This enzyme degraded myosin heavy chain, actin, tropomyosin and troponin I clearly at pH 4.0, while it slightly degraded troponin I at pH 5.0-5.6. Therefore, the contribution of cathepsin H to the proteolysis of postmortem muscle is presumed to be relatively small.