松石 昌典

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.

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 degrees 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 degrees C-heated beef than 100 degrees C-heated beef. The panel also noted that the aroma of 121 degrees C-heated beef tended to be (P<0.1) preferable than that of 100 degrees 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 degrees C- and 121 degrees C-heated beef showed that the amounts of pyrazine, 2-methylpyrazine and diacetyl were higher in the 121 degrees C-heated beef than in the 100 degrees 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.

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 0.3-0.5mol/L NaCl solutions containing 9-36mmol/L disodium inosine-5-monophosphate (IMP) or 9mmol/L tetrapotassium pyrophosphate (KPP) to minced pork. IMP at 36mmol/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 0.3mol/L NaCl and 9-36mmol/L IMP or 9mmol/L KPP. IMP at 36mmol/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.

We examined the mode of IMP and pyrophosphate enhancement ofmyosin 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 restraintreleasing ability of IMP is dependent on reaction time and NaCl concentration while that of KPP is not.

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.

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.

The aim of the present study was to clarify by means of sensory tests whether taste, aroma, or texture is the dominant contributor to people's ability to identify the animal species of meat. The meat samples used were loins of beef, pork, and lamb; chicken thigh; and aigamo (crossbreed of domestic duck and wild duck) breast. Panelists (32 to 35 persons) wearing eye masks ate cooked pieces, patties, and heated soups prepared from the meats of these 5 species with and without pinching their noses to regulate the aroma sensation, attempted to identify the species, and gave reasons for their identifications. In the test involving meat pieces and pinched noses, the percentage of panelists identifying chicken correctly was the highest, at 72%, whereas the percentages of correct identification for all other animal species were less than 50%; however, values were higher for each species in tests without nose pinching. In the tests of meat patties involving pinched noses, the percentage of correct identification for each animal species was slightly lower in comparison with the tests of meat pieces; however, the values were higher in tests without pinched noses. In. the tests of soups using pinched noses, the percentage of correct identification for each animal species was lower than 30%; however, the values were higher in tests without pinched noses. These results led us to the conclusion that aroma is the most important contributor to the identification of the animal species of meat, and texture is the 2nd most important contributor. The contribution of taste appears much smaller than that of aroma and texture.

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 degreesC. CPI-L and CPI-H were suggested to inhibit cathepsins B, H and L by a non-competitive mechanism. The inhibition constants (K-i) of CPI-L and CPI-H showed that both CPIs have much higher affinity against cathepsins H and L than against cathepsin B. (C) 2003 Elsevier Inc. All rights reserved.

Rabbit muscle cathepsin H classified as an amincendopeptidase 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-p-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 degreesC 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. (C) 2003 Elsevier Science Ltd. All rights reserved.

We investigated the factor which increased the maximum value of the Mg-ATPase activity of myofibrils existing at low KCI concentrations during meat conditioning. On the treatment of myofibrils with the solution extracted with the buffer of pH 7.2 from muscle, the Mg-ATPase activity in the presence of 0-0.15 M KCI increased time-dependently. This change was most remarkable in the range of pH 5.6-7.0. Trypsin treatment of the extract abolished such effect, suggesting that the responsible factors were proteins. The fractionation of the extract with isoelectric focusing demonstrated that the factors were basic proteins (pI 8.3-9.6). The treatment of myofibrils with those basic proteins under various conditions suggested that the time-dependent adhesion of those basic proteins, through a denaturation at around pH 5.5, to myofibrils was assumed to raise the Mg-ATPase activity. Analysis of myofibrils prepared from rabbit muscles stored at 0 degreesC for 12 days postmortem showed the appearance of the 35,000 Da protein, accompanied the increase in the Mg-ATPase activity. Therefore, the adhesion of this protein to myofibrils in situ probably caused the increase in the Mg-ATPase activity. Successive treatment with the basic protein and the crude cathepsin increased the dependency of the Mg-ATPase activity on KCI concentrations and the maximum value of the Mg-ATPase activity. Therefore, the coordinate action of a basic 35,000 Da protein and cathepsins was presumed to induce the changes in the Mg-ATPase activity of myofibrils during meat conditioning. The basic protein was concluded to be glyceraldehyde-3-phosphate dehydrogenase (its subunit molecular mass: 35,000 Dal, since the incubation of myofibrils with its commercial preparation raised the Mg-ATPase activity of myofibrils. (C) 2000 Elsevier Science Ltd. All rights reserved.

In our previous paper, a desirable odor generated in raw beef by conditioning, i. e., the conditioned raw beef aroma, was suggested to be produced by some kinds of bacteria at the site containing both leans and fats in the presence of oxygen. Thus, we isolated such a bacterium and examined some of its features. Bacterial isolates from beef loins were screened on sterile lean extract with floating sterile adipose tissue or sterile fat to obtain the bacterium producing the conditioned raw beef aroma. Of 89 isolates, one isolate (H 25W) producing this odor apparently was obtained. H 25W was gram-positive and catalase-positive, and occurred as unbranched rods in short chains. It was nonmotile and grew on the streptomycin-thallous acetate-actidione medium. These features implied that H 25W was Brochothrix thermosphacta, a very common bacterium in market meats. Some other features (oxygen-requirement, etc.) of H 25W coincided with those of Brochothrix thermosphacta. Furthermore, the type culture strain of Brochothrix thermosphacta ATCC 11509 demonstrated to have produced the conditioned raw beef aroma. Thus, H 25W which produced the conditioned raw beef aroma was strongly suggested to be Brochothrix thermosphacta.

In order to clarify factors inducing the changes in the Mg-ATPase activity of myofibrils during meat conditioning, we treated myofibrils in vitro with various muscle proteinases and Ca2²⁺. Ca²⁺ or proteasome did not change the Mg-ATPase activity at 25°C and pH5.5 or 7.0. m-Calpain enhanced the dependency of the Mg-ATPase activity of myofibrils on KCl concentrations (DAAK) in the range of 0 to O.05M KCl at 25°C and pH7.2 as similarly as observed during meat conditioning, but did not at pH5.5. Therefore, m-calpain was assumed to contribute to the increase in DAAK in the early stage of meat conditioning, i. e., before the pH of muscle declined to the ultimate pH. Crude cathepsins raised DAAK in the range of 0 to 0.1M KCl at 25°C and both pH5.5 and 7.0. As a result of the chromatographic separation, cathepsins D and L were shown to enhance DAAK remarkably. Although cathepsins B and H alone raised DAAK only a little, their action was enhanced by the coexistence of cathepsin L. During meat conditioning cathepsins D and L are thus considered to contribute to the increase in DAAK involving a synergistic action of cathepsins B and H. However, all these proteinases investigated here did not cause so much remarkable increase in the maximum value of the Mg-ATPase activity of myofibrils occurring at low KCl concentrations as observed during meat conditioning, suggesting the existence of other unknown factors inducing such changes.

Rabbit proteasome, likely to be a 20S proteasome, was purified and ifs properties were investigated to clarify its contribution to proteolysis during meat conditioning. The purified enzyme migrated as a single band on non-denaturing polyacrylamide gel and dissociated to a number of subunits (20 000-29 000 Da) under denaturing conditions. The molecular mass of this enzyme was found to be 580 000-800 000 Da by Sephacryl S-300 column chromatography. The isoelectric point of this enzyme was 5.5. The optimum pH for hydrolysis of succinyl-Leu-Leu-Val-Tyr-(4-methylcoumaryl-7-amide) (Suc-LLVY-MCA) was 8. This enzyme was almost stable in the range of pH 5-9 and up to 60 degrees C at pH 7.2. The enzyme activity was inhibited by diisopropylfluorophosphate (DFP) and chymostatin, but was not affected by EDTA, leupeptin, E-64, bestatin, monoiodoacetic acid or pepstatin. The enzyme was activated about 8-fold by 0.01% sodium dodecyl sulfate (SDS), but was not by ATP or CaCl2. Remarkably, SDS increased the V-max value of the enzyme. Rabbit proteasome was shown to degrade myosin heavy chain, alpha-actinin, actin, tropomyosin, troponins and myosin light chains in the presence of SDS. In the absence of SDS, no change in myofibrillar proteins was observed. This enzyme did not degrade any sarcoplasmic proteins regardless of the presence of SDS. (C) 1997 Elsevier Science Ltd.

牛の生乳特有の芳香の1つである生乳乳様香に対する加熱の影響を調べた.ホルスタイン種の市販殺菌乳の生乳乳様香の強度は,低温殺菌乳>高温短時間殺菌乳>超高温殺菌乳の順であった.鼻孔を閉じて感知する味の濃厚感(コク味)は3者間で差がなかったが,鼻孔を開けて感知する濃厚感強度の順は生乳乳様香のそれと同じであった.したがって,本香は濃厚感を感じさせる性質をもつことが示唆された.生乳を湯浴で各種条件下で加熱したときの結果および牛乳を分画したときの結果から,生乳乳様香はおもに脂肪球皮膜に存在すると推定され,それは63°Cで30分間の低温加熱殺菌では完全に保持されるが,75°Cで15秒間の高温短時間加熱殺菌ではある程度保持され,120°Cで3秒間の超高温加熱殺菌では完全に消失することがわかった.さらに本香は生乳の63°Cで30分間の予備加熱によってより高い温度での加熱でも保持される存在形態に変わることがわかった.

一般に輸入牛肉は国産の牛肉よりも不味であると言われている.本研究では,その原因解明のために輸入牛肉の軟らかさと香味を調べた.真空包装された豪州産のチルドビーフ,フローズンビーフ,エージドビーフを加熱して軟らかさを比較した結果,3者間で差がなく,いずれも充分軟らかかった.また,いずれにおいても筋原線維のZ線の脆弱化とMg-ATPase活性の変化が充分進行していた.したがって,このような輸入牛肉では不味さの原因を軟らかさの不足に求められないことが判明した.これらの牛肉の加熱後の香味にも3者間で差がなかったが,いずれにも前報で示した牛肉熟成香が著しく不足しており,これが輸入牛肉の不味さの一因であると考えられた.米国産のフローズンビーフを解凍後に含気貯蔵した結果,熟成香が生成した.これにより輸入牛肉に熟成香が不足している原因の1つは真空包装による酸素の遮断にあることが強く示唆された.また,枝肉の状態で冷蔵輸入された米国産のアンガス種の牛肉を含気貯蔵した場合にも熟成香が生成し,熟成香が国内産の牛肉に特有なものでないことが示された.一方,豪州産のチルドビーフを含気貯蔵した場合には熟成香は生じなかったが,これは細菌叢の違いや交雑脂肪の少なさに原因があると考えられた.フローズンビーフを解凍後熟成した場合熟成香のみならずドリップに由来する不快な汗臭も生成したが,これはドリップの除去により抑制することが可能であった.

1. The mode of degradation of myofibrillar proteins and the structural changes in myofibrils due to the action of cathepsin B highly purified from rabbit skeletal muscle were studied. 2. Cathepsin B degraded myosin heavy chain, actin and troponin T, but not alpha-actinin, tropomyosin, troponin I or troponin C among myofibrillar proteins. 3. Cathepsin B optimally degraded myosin heavy chain, actin and troponin T at around pH 5. Degradation of myosin heavy chain produced 6 fragments, 180,000, 150,000, 87,000, 81,000, 75,000 and 69,000 Da, respectively. Actin was hydrolyzed into fragments of 41,000, 38,000 and 30,000 Da. Troponin T was degraded into fragments of 21,000, 12,000 and 10,000 Da. 4. Cathepsin B caused the fragmentation of myofibrils and disturbance of the lateral arrangement of myofibrils. 5. Cathepsin B partly disintegrated the Z-line and the M-line, and induced disordering of the arrangement of filaments in the I-band.

Activities of intracellular proteinases of a strain of Lactococcus lactis were surveyed using a fluorometric method. The cell extract obtained upon disruption of harvested L. lactis cells, using a porcelain mortar and ultrasonic oscillation, was subjected to chromatography on a DEAE-cellulose column and then a Sephadex G-100 column. The eluates were assayed by a fluorometric method using benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-(4-methyl) coumarylamide and casein as substrates. Four different caseinolytic fractions were obtained that were ascribable to two metalloproteinases of molecular weights 62,000 and 98,000; a serine proteinase of 160,000; and a cysteine proteinase of 12,000, respectively. Eight different benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-(4-methyl) coumarylamide-hydrolyzing fractions were also obtained, three of which were attributable to cysteine proteinases with the molecular weights of 125,000, 125,000, and 300,000. None of the eight hydrolyzing fractions seemed to be able to hydrolyze casein.

凍結輸入牛肉が国産牛肉に比べて不味であることの原因の一部を,凍結貯蔵か,あるいは熟成の不充分さに求めうるかを知るためにいくつかの実験を行ない以下の結果を得た.1) 最大硬直期後の乳牛のロイン部について熟成させずに-20°Cで数週間凍結貯蔵した後に解凍したもの(A) と0°Cで18日間貯蔵したもの(B) を官能評価した結果,BがAより有意に軟らかく香味でも優れていた.2) Aを更に0°Cで14日間貯蔵したもの(C) とAを官能評価した結果,CがAより有意に軟らかかった.香味はCが優れている傾向であった.しかし口ざわりは粗く,Bより劣っているように感じられるというコメントがいくつかあった.3) 牛肉の筋原線維の脆弱化は凍結貯蔵中には進行しなかったが,解凍後の0°Cでの貯蔵中には進行した.4) 筋原線維の低イオン強度でのMg-ATPase活性は牛肉の0°Cでの貯蔵中には著しく上昇したが,凍結貯蔵中にはほとんど変化せず,更に解凍後の0°C貯蔵においても余り変化しなかった.これは筋原線維タンパク質が凍結変性したことを示唆している.5) 牛肉の凍結貯蔵によってカテプシンHとBの活性は減少しなかったが,カテプシンL活性はかなり減少した.6) 以上の結果から,凍結輸入牛肉の不味さの原因が熟成不足にある場合は解凍後の貯蔵により軟らかさと香味を向上させうることが判明した.更に凍結牛肉に時に付随する口ざわりの悪さは一部,筋原線維タンパク質の凍結変性に起因する可能性が示唆された.

ニワトリ胸筋中に, カテプシンBを阻害する分子量の異なる4種類のタンパク性のシステインプロテイナーゼインヒビターの存在することを明らかにした。すべてのインヒビターは胸筋をpH 7.2の中性緩衝液で抽出後, DEAE-セルロースカラムに吸着し, NaClの0.07～0.15M濃度で溶出した。溶出した活性区分の33～55%アセトン沈澱画分をセファデックスG-75にかけゲル濾過を行った結果, 分子量58,000のインヒビターが得られた。55～77%アセトン沈澱画分をセファデックスG-75でゲル濾過した結果, 分子量10,000 (I-S), 22,000および32,000のインヒビターが得られた。I-SはさらにセファデックスG-50のゲル濾過を経て, その比活性が粗抽出物のそれの400倍にまで精製された。分子量22,000と32,000のインヒビターは0.1%になるようにメルカプトエタノールを添加し, セファデックスG-75にかけゲル濾過を行ったところ低分子化し, 分子量11, 500のモノマーCPI (I-M) に転換することがわかった。I-MはさらにセファデックスG-50のゲル濾過を経てその比活性が粗抽出物のそれの600倍にまで精製された。I-SとI-MはともにpH 4から7で安定であり, またpH7.2, 100℃で10分間の加熱でもそれぞれ初期値の70%と64%の活性を保持していた。