EFFECTS OF FOUR SPECIES OF BACTERIA ON PORCINE MUSCLE 2. Electrophoretic Patterns of Extracts of Salt-Soluble Protein

SUMMARY– Electrophoresis of 0.6 M KCI extracts of porcine longissimus dorsi muscle revealed little change in the type or number of protein bands found either by starch-urea or disc-urea gel electrophoresis. The 0.6 M KCI extracts of muscle samples inoculated with Pediococcus cerevisiae, Leuconostoc mesenteroides and Micrococcus luteus and stored at 2 and 10°C for 20 days did not differ electrophoretically from control samples. Extracts of samples inoculated with Pseudomonas fragi showed a loss in the number of protein bands on starch-urea gel and disc-urea gel electrophoresis, indicating this organism exhibited some proteolytic effect upon the myofibrillar proteins .     

EFFECTS OF FOUR SPECIES OF BACTERIA ON PORCINE MUSCLE. 1. Protein Solubility and Emulsifying Capacity

SUMMARY– A technique was used to obtain aseptic porcine muscle, portions of which were inoculated with cultures of Pediococcus cerevisiae, Micrococcus luteus, Leuconostoc mesenteroides and Pseudomonas fragi. The inoculated samples were compared with aseptic controls throughout a 20-day storage period at temperatures of 2 and 10°C. All 4 organisms grew at 10°C, but only P. fragi and L. mesenteroides grew at 2°C. The solubilities of the various protein fractions were affected by inoculation treatment. This was exemplified by correlation coefficients ranging from —.37—0.50. The coefficients indicated the interrelationships affected by storage conditions and bacterial growth. Protein solubility studies revealed a loss in the water-soluble fraction during storage of the controls and the M. luteus- and L. mesenteroides- treated samples. Samples inoculated with P. fragi evidenced an initial loss, followed by an increase. The solubility of meat proteins in a salt solution increased during the first 8 days of storage, then decreased or remained relatively constant for all samples. In comparison with controls, samples inoculated with P. fragi increased in salt-soluble protein solubility during the first 8 days, whereas those inoculated with L. mesenteroides decreased during the latter part of storage. Insoluble protein generally increased except for P. fragi- inoculated samples, which decreased. Nonprotein nitrogen (NPN) increased for all treatments and controls during the 20-day storage period. NPN extracted from the samples inoculated with P. fragi increased greatly. The pH increased with growth of M. luteus and P. fragi and decreased with growth of P. cerevisiae and L. mesenteroides. The emulsifying capacity was not influenced by the growth of M. luteus or P. cerevisiae. However, the emulsifying capacity of samples inoculated with L. mesenteroides decreased, whereas that of samples inoculated with P. fragi increased.     

EFFECT OF MICROBIAL GROWTH UPON MYOFIBRILLAR PROTEINS

SUMMARY— Aseptic samples from pig and rabbit muscles were inoculated with Achromobacter liquefaciens, Micrococcus luteus, Pediococcus cerevisiae, Pseudomonas fluorescens, Streptococcus faecal is end a mixed culture obtained from commercial hamburger. Some difficulty was encountered in getting the organisms to grow, and good growth was achieved only with A. liquefaciens end mixed culture from commercial meat. Both inoculated and uninoculated control samples were incubated at 3 and 10°C for 0, 8 and 20 days. The salt soluble proteins were extracted with Weber-Edsall solution and subjected to sucrose density gradient centrifugation, gel filtration and disc gel electrophoresis. The microorganisms utilized in this study had no measurable effect upon the myofibrillar proteins from either pig or rabbit muscle. However, bacterial growth decreased the amount of certain non-protein ultra-violet absorbing components in the ultracentrifugal pattern of Weber-Edsall extract. These components did not appear to be of myofibrillar origin. Disc gel patterns of Weber-Edsall extracts from pig muscle produced a more intensely staining band than those from rabbit muscle at R_m, = 0.59.     

Effect of a proteolytic enzyme produced by Clostridium perfringens upon porcine muscle

Purified proteolytic enzyme preparations isolated from actively growing cultures of Clostridium perfringens (ATCC 13124—Type A) were added to aseptic porcine muscle and incubated at 37°C. Samples were removed after one, two and four days incubation. The sarcoplasmic and myofibrillar proteins were extracted and subjected to both conventional and SDS-polyacrylamide disc gel electrophoresis to assess the changes due to the enzyme treatment. The enzyme preparation caused little change in the sarcoplasmic protein fraction but caused some proteolysis in the myofibrillar extract. Comparison of the protein profile from muscle incubated with the purified enzyme preparation and that incubated with the actively growing organisms per se showed that the organisms caused more degradation, suggesting that they produced additional enzymes causing proteolysis of porcine muscle.     

Degradation of myofibrils and formation of premer omyosin by a neutral protease produced by Pseudomonas fragi

Rabbit muscle myofibrils were treated with a neutral protease (200: 1-protein: enzyme) isolated from Pseudomonas fragi (P. fragi) at 0° C in 100 mm KCl, 5·0 mm CaCl₂, 0·2 mm DTT (dithiothreitol) and 20 mm TRIS. HCl (pH 7·50) for 0–60 min. SDS-polyacrylamide disc gel electrophoresis showed extensive fragmentation of the myofibrillar proteins. Three of the major products correspond in molecular weight to heavy meromyosin (HMM), light meromyosin (LMM) and the recently discovered premeromyosin (PMM). Results demonstrated that the neutral protease produced by P. fragi is capable of hydrolysing myosin and probably plays an important role in microbial breakdown of the muscle proteins during meat spoilage.     

Protein Degradation by Lactobacillus plantarum and Lactobacillus casei in a Sausage Model System

ABSTRACT: :
The proteolytic activity of a starter culture involving Lactobacillus plantarum and Lactobacillus casei towards meat sarcoplasmic and myofibrillar proteins during the fermentation of a sausage-like system was studied. After 96 h of incubation the proteolytic system of L. plantarum CRL681 caused a degradation of both sarcoplasmic and myofibrillar proteins, whereas L. casei CRL705 showed a different affinity to meat proteins. The inoculation of both strains showed a higher activity toward sarcoplasmic fraction. These results correlated with a high rate of sarcoplasmic protein degradation observed in SDS-PAGE analysis. The generation of free amino acids as well as the pH drop at the end of the incubation period was maximal in presence of the mixed starter culture, thereby demonstrating the suitability of these strains to be used in the fermentation of meat products.     

Proteolytic activity of Lactobacillus strains isolated from dryfermented sausages on muscle sarcoplasmic proteins

The proteolytic activity of seven strains of Lactobacillus from two species isolated from dry cured sausages was assayed using a soluble muscle extract as a source of proteins, at a temperature of 30 °C. The results indicated that the strains of Lactobacillus plantarum tested had the more active proteolytic system, showing the highest amino acid release in the medium after 72 hr of incubation (L. plantarum CRL 681) when the microorganism was in the stationary phase of growth. The strains of L. casei showed a continued hydrolytic activity with a lower amino acids concentration along the studied period. The SDS-PAGE profiles showed that the major changes in sarcoplasmic proteins were produced in the 13 kDa and 36-40 kDa molecular weights region.     

Some Biochemical Changes in Sarcoplasmic Depleted, Intact Beef Muscle Inoculated with Pseudomonas frari

Twenty-four hour postmortm intact bovine Longissimus dorsi muscle strips were (1) washed to remove sarcoplasmic fluid, or (2) left intact, and were either uninoculated or inoculated with Pseudomonas fragi ATCC 4973 to evaluate the effect of decreased concentration of sarcoplasm on bacterial growth and subsequent spoilage during storage at 4°C for 12 days. Washing removed the majority of the water-soluble components of the muscle. Significantly (P < 0.01) higher growth rates were observed on intact muscle than on washed muscle. Increased extractability of water-soluble and salt-soluble proteins was observed in intact inoculated (II) muscle as growth of P. fragi progressed. Alterations in SDS-gel electrophoretic patterns of water-soluble, salt-soluble, urea-soluble and urea-insoluble proteins were evident in the II muscle. Relatively little change in nonprotein nitrogen, water-soluble and salt-soluble protein content occurred in washed inoculated (WI) muscle with increased bacterial growth. Only minor changes in the SDS-gel electrophoretograms of the salt-soluble proteins from the WI tissue were apparent.     

Thermal Denaturation of Hake (Merluccius hubbsi) Myofibrillar Proteins. A Differential Scanning Calorimetric and Electrophoretic Study

Denaturation of hake (Merluccius hubbsi) proteins was studied with DSC by monitoring T_max of transitions and denaturation enthalpies. Whole muscle free of connective tissue showed two transitions (T_max 46.5°C and 75.3°C), and ΔH of 4.27 cal/g. The exudative sarcoplasmic fraction showed three transitions (T_max 45.2°C, 59.0°C and 75.5°C) and ΔH of 3.92 cal/g. The sarcoplasmic proteins from whole hake muscle contributed to both denaturation peaks. Muscle depleted of sarcoplasmic proteins by chloride extraction showed a higher thermal sensitivity and a diminished denaturation enthalpy on the second transition. This suggested an additional effect of chloride upon actin in addition to sarcoplasmic protein extraction. pH had an effect upon the native conformation of thick filament proteins, specifically myosin.     

二甲基二碳酸盐联合动态超高压对模拟果汁的杀菌效果研究

本文研究了二甲基二碳酸盐（DMDC）联合动态超高压对模拟果汁中三种挑战污染菌（大肠杆菌、酿酒酵母、肠膜状明串珠菌）的杀菌效果。结果表明：单独动态超高压对三种挑战污染菌的杀菌效果随着压力增大而显著增强（p〈0.05）,其中在相同处理压力下,对大肠杆菌的杀菌最好,但即使在100 MPa的压力处理下,大肠杆菌也仅下降1.2个对数;向含挑战污染菌的模拟果汁（25 ℃）中添加250 mg/L的DMDC 2 h后,大肠杆菌和酿酒酵母均被完全杀灭,而肠膜状明串珠菌对DMDC具有很强的耐受性,仅下降了1.7个对数,且继续延长DMDC的处理时间,其数量下降也不再明显;当DMDC（250 mg/L）与动态超高压联合处理时,它们对模拟果汁中的肠膜状明串珠菌的杀菌作用具有明显的协同增效效果（p〈0.05）。另外,DMDC与动态超高压先后处理次序对肠膜状明串珠菌的杀菌效果也有影响。     

Postmortem breakdown of ATP and glycogen in ground muscle: A review

The postmorte metabolism of ATP and glycogen in ground bovine muscle (in some cases also in rabbit muscle) was studied. At pH 7 protons released during postmortem glycolysis are bound by the phosphorylation of ADP to ATP and are liberated during enzymic hydrolysis of ATP. At lower pH values the protons are immediately released during glycolysis. Not more than 10% of the total drop in pH postmortem is due to protons liberated by the hydrolysis of ATP present in the tissue at death. Half of the buffering capacity of bovine muscle is caused by the myofibrillar proteins. The myofibrillar ATPases, rather than the membrane ATPases, seem to predominate in hydrolysing ATP postmortem in the intact as well as in the ground muscle. The rate of the breakdown of ATP determines the rate of postmortem glycolysis. Phosphofructokinase and, to a lesser extent, phosphorylase play the major role in the control of glycolytic metabolite levels in ground muscle. The mean values of glycogen recovery, obtained by measuring all glycolytic metabolites and lactate, indicate a general stoichiometric relationship in ground tissue, although several muscles did not fit in the scheme. Grinding of the prerigor muscle causes an accelerated hydrolysis of ATP and ADP, resulting in a faster increase in the IMP concentration and in an accelerated glycolysis. The increase in the turnover of ATP by grinding might be due to a faster release of Ca(++) ions from the damage sarcoplasmic reticulum. Addition of sodium chloride (2-4%) to the ground pregidor muscle causes an increase in the rate of the breakdown of ATP to IMP. NaCl changes the steady-state of glycolysis without a major change in the rate of glycogen breakdown. The stimulation of phosphofructokinase observed is probably due to the faster disapearance of ATP in the presence of NaCl. The faster turnover of ATP could be due to an enhanced release of Ca(++) ions from the sarcoplasmic reticulum by exchange against Na(+) After several hours postmortem an inhibition of glycolysis occurs in the salted tissue which is probably due to a denaturation of glycolytic enzymes by the combined effect of low pH (<6) and high ionic strength. The high water-holding capacity of prerigor salted ground beef does not decrease postmortem in spite of the high rate of ATP breakdown. This effect can be explained by an inhibition of rigor mortis in the fibre fragments caused by the combined effect of ATP, high pH and salt ions. Addition of diphosphate to the prerigor ground tissue in the absence or presence of added NaCl results in an acceleration of ATP and glycogen breakdown. A hypothesis for the high ATP turnover is discussed. The higher rate of glycogen breakdown in the presence of diphosphate is caused mainly by an acceleration of the phosphofructokinase step. A rate limitation, by diphosphate, in the glyceraldehyde-3-phosphate dehydrogenase step and/or the following glycolytic steps was also observed.     

Contribution of cathepsins B,L and D to muscle protein profiles correlated with texture in rainbow trout (Oncorhynchus mykiss)

Post-mortem softening of fish tissue often results in low yield and decreased product quality. In this study, proteolytic profiles of trout stored 5 days on ice were obtained by SDS–PAGE. The link between protein band intensities and firmness of trout fillets was examined through a correlation study. In parallel, trout extracts were incubated with cathepsin B, cathepsin L and cathepsin D, alone or in combination, in order to evaluate the effect of each cathepsin on the texture-related proteins. Proteins from both myofibrillar (α-actinin, actin, MLC1, MLC2, and N-terminal 70 kDa MHC fragment) and sarcoplasmic (glycogen phosphorylase, creatine kinase, and TPI) fractions correlated closely with firmness. Cathepsins D, B and L affected, respectively, 10, 9 and 4 out of the 17 protein bands correlating with firmness, and most changes induced by cathepsin D were unfavourable to firmness. This implies that cathepsin D is likely to be involved in textural change of trout, due to breakdown of the muscle structure.     

Gel Electrophoretic Analysis of the Protein Changes in Ground Beef Stored at 2°C

Purified myofibrils and sarcoplasmic proteins were prepared from ground (GR) and intact (CON) beef semitendinosus muscle samples after 0, 1, 3, 6, and 10 days of storage at 2°C. SDS-polyacrylamide gel electrophoresis analysis revealed the following major postmortem changes in GR samples: the gradual disappearance of nebulin and desmin, appearance of 110,000-, 95,000- and 30,000-dalton polypeptides, and an increased content of myosin light chain-3 and 55,000-dalton component in myofibrils. Also noted was emergence of 100,000- and ?500,000-dalton polypeptides and diminution of 300,000-dalton protein in the sarcoplasmic fraction. Since GR samples showed proteolytic changes similar to those of CON samples, it was concluded that grinding had little effect on postmortem muscle protein degradation.     

Proteolytic activity of lactic acid bacteria strains and fungal biota for potential use as starter cultures in dry-cured ham

During the processing of dry-cured meat products, sarcoplasmic and myofibrillar proteins undergo proteolysis, which has a marked effect on product flavor. Microbial proteolytic activity is due to the action of mostly lactic acid bacteria (LAB) and to a lesser extent micrococci. The proteolytic capacity of molds in various meat products is of interest to meat processors in the Mediterranean area. Eleven LAB and mold strains from different commercial origins were tested for proteolytic activity against pork myosin, with a view to possible use of these strains as starter cultures for Iberian dry-cured ham. Proteolytic activity was tested by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The LAB strains with the highest proteolytic activity were Lactobacillus plantarum (L115), Pediococcus pentosaceus (Saga P TM), and Lactobacillus acidophilus (FARGO 606 TM). The best fungal candidate was Penicillium nalgiovense LEM 50I followed by Penicillium digitatum, Debaryomyces hansenii, and Penicillium chrysogenum.     

真空滚揉腌制对伊拉兔肉品质特性的影响

本实验研究了真空滚揉腌制前后伊拉兔肉食用品质特性、质构特性、肌浆蛋白、肌原纤维蛋白、总蛋白溶解度和游离氨基酸含量的变化。结果表明：真空滚揉腌制可以显著改善兔肉的食用品质,提高兔肉的pH值,改善兔肉的色泽,降低兔肉的蒸煮损失和压榨损失,提高保水性,改善兔肉嫩度和质构特性;真空滚揉腌制会导致肌原纤维蛋白分解、肌浆蛋白溶出、游离氨基酸含量下降,引起兔肉品质特性的变化。     

Texture Changes Associated with Insolubilization of Sarcoplasmic Proteins During Salt-vinegar Curing of Fish

The effect of salt-vinegar curing on mackerel meat was investigated. In salt curing, the firmness of the meat increased concomitantly with the decrease in water content and the increase in insolubilization of sarcoplasmic protein of phosphorylase. In subsequent vinegar curing, the firmness and cohesiveness of the meat increased concomitantly with the increase in insolubilization of the sarcoplasmic proteins of enolase, creatine kinase, aldolase, and glyceraldehydephosphate dehydrogenase. No apparent proteolytic breakdown was detected in the salt-vinegar curing. The findings suggested that the precipitation of substantial amounts of sarcoplasmic proteins might contribute to the texture change caused by salt-vinegar curing.     

Proteolytic and lipolytic modifications during the manufacture of dry-cured lacón,a Spanish traditional meat product: Effect of some additives

The extractability of sarcoplasmic and myofibrillar proteins, the myofibrillar proteins and their degradation products, classical nitrogen fractions, free amino acids, acidity of the fat, and free fatty acids were determined throughout the manufacturing process of dry-cured lacón, a traditional dry-salted and ripened meat product made in the northwest of Spain from the foreleg of the pig, following a similar technological process to that of dry-cured ham. The effect of the use of additives (glucose, sodium nitrite, sodium nitrate, sodium ascorbate and sodium citrate) on the proteolytic and lipolytic changes was also studied.     

乳酸菌发酵对草鱼肉理化特性和蛋白质降解变化的影响

研究了乳酸片球菌、乳杆菌-Lo3、乳杆菌-B5407和清酒乳杆菌4种不同乳酸菌在发酵草鱼肉时鱼肉理化特性及蛋白质降解变化的规律和特征。结果表明:添加乳酸菌后,鱼肉pH值迅速由最初的6.25降至2d时的5.06～5.40,对照组则为5.98;接种发酵剂的草鱼肉的TBA和TVB-N值的上升较缓慢,整个发酵过程中显著低于对照组(P<0.05);腌制草鱼肉的蛋白质在发酵期发生了轻微的降解,游离氨基酸略有增加;贮藏期间游离氨基酸量显著增加,肌浆蛋白和肌原纤维蛋白均发生了强烈的水解,接菌组的蛋白降解显著多于对照组,其中,接种乳酸片球菌和乳杆菌-B5407组降解程度最大。     

Relationship Between Thermal Denaturation of Porcine Muscle Proteins and Water-holding Capacity

ABSTRACT: Differential scanning calorimetry was used to investigate denaturation characteristics of pork muscle proteins from carriers and noncarriers of the RN-gene. Pork from RN-carriers deviated from noncarriers in maximum denaturation temperatures and denaturation enthalpy, with proteins of RN-carriers being the most heat-labile. Correlation studies on the results showed that water-holding capacity was significantly correlated to changes in enthalpy of the population mainly representing myosin tails and sarcoplasmic proteins (p < 0.001). Finally, the influence of ultimate pH and preheating on thermal characteristics of porcine muscle proteins was studied. Myosin tails and sarcoplasmic proteins were most sensitive to pH changes, while myosin heads were most sensitive to preheating simulating stress-induced temperature increases.     

Ultramicroscopical Structures and Liquid Loss in Heated Cod ( Gadus morhuaL) and Salmon ( Salmo salar) Muscle

This study was performed in order to assess the effect of heating in pre- and post-rigor muscle of fed cod, wild cod and farmed salmon harvested at different times of the year. The structural changes in muscle samples pre-heated from 5 to 60°C were qualitatively evaluated using both light and transmission electron microscopy techniques. The microstructural changes are discussed in relation to the liquid loss measured by a low-speed centrifugation test. The heat-induced structural changes varied between the fish tested, reflecting different degrees of post mortem degradation prior to heating, the muscle-pH and species-specific structural properties. The fed fish, both cod and salmon, underwent the most severe structural degradation. This reflected both the low muscle pH and the more severe post mortem degradation observed in these fish prior to heating, compared with the wild cod. Heating caused extensive shrinkage of the myofibrils and hence, widened intermyofibrillar and extracellular spaces in both the fed cod and the salmon muscle. In the sample of wild cod muscle, the extracellular spaces were narrow and the myofibrils were closely packed. The difference in heat-induced liquid loss of the fed compared with the wild cod muscle coincides with their different structural features, as observed both by LM and TEM. The better liquid-holding properties of the salmon muscle than the cod muscle are attributed to the species-specific ultrastructural features as observed with TEM. In addition to the denser appearance of the salmon myofibres, it is suggested that both fat droplets and aggregated sarcoplasmic proteins filling the intermyofibrillar and extracellular spaces are important in preventing release of liquid upon heating.