Lactic acid bacteria of meat and meat products

When the growth of aerobic spoilage bacteria is inhibited, lactic acid bacteria may become the dominant component of the microbial flora of meats. This occurs with cured meats and with meats packaged in films of low gas permeability. The presence of a flora of psychrotrophic lactic acid bacteria on vacuum-packaged fresh chilled meats usually ensures that shelf-life is maximal. When these organisms spoil meats it is generally by causing souring, however other specific types of spoilage do occur. Some strains cause slime formation and greening of cured meats, and others may produce hydrogen sulphide during growth on vacuum-packaged beef. The safety and stability of fermented sausages depends upon fermentation caused by lactic acid bacteria. Overall the presence on meats of lactic acid bacteria is more desirable than that of the types of bacteria they have replaced.     

A Newer Method for Isolation of the 7S Globulin in Soybean Seeds

The effects of freezing on the proteolysis of beef during storage at 4°C after being thawed was investigated.

A sarcoplasmic 32-kDa protein in frozen as well as unfrozen beef decreased rapidly during storage at 4°C, and a more than 100-kDa protein appeared in both beef samples. And the increment of peptides in the frozen beef during the storage at 4°C was larger than that in the unfrozen beef, suggesting that the proteolysis was faster during the storage of the former than the latter. However, its increment in the frozen beef for 10 weeks during the storage at 4°C became smaller than that of the one frozen for less than 5 weeks.

To discover an indicator for evaluation of the conditioning of frozen and unfrozen beef, peptides produced during the storage of beef at 4°C were surveyed. A peptide, APPPPAEVPEVHEEV, was detected and seemed to be available as an indicator in the conditioning of beef.     

Microbiology of Lebanon Bologna

Various aspects of the microbiology of the Lebanon bologna process were studied. Manufacture of Lebanon bologna appeared to be similar to that of summer sausage and other fermented sausages and consisted of a lactic acid fermentation by lactobacilli accompanied by the production of cured meat color from the reduction of nitrate by micrococci. The traditional process consists of aging coarse ground beef at 5 C for several days. Aging the beef for about 10 days was necessary to allow development of lactic acid bacteria; for successful fermentation, the concentration of lactic acid producers must be 10(4)/g or more. At least 3% NaCl was necessary to suppress the development of pseudomonads during the aging period; higher concentrations of salt suppress the development of the lactic acid-producing flora. During aging, in the presence of salt, the predominant flora developing on the meat consisted of catalase-positive, gram-positive rods and cocci; during fermentation at 35 C, the predominant flora became catalase-negative, gram-positive rods with characteristics of lactobacilli. Lebanon bologna could be made from frozen beef if the meat was thawed, salted, and aged. However, bolognas could not be made from unaged beef unless a lactic acid starter culture was used. The microflora of several commercial bolognas is reported also.     

The proteolytic release of genotoxins from cooked beef

Dietary factors are important in the aetiology of human cancer and carcinogens, mostly heterocyclic aromatic amines, have been isolated from cooked proteinaceous foodstuffs. Whilst such carcinogens have induced tumours in rodent bioassays, the dosages required were much higher than estimates of human exposure levels. We have examined the possibility that genotoxins, which were not extractable prior to enzymic digestion, may be released from cooked beef by proteolysis. Dichloromethane and/or a solid-phase tandem extraction procedure were used with aqueous homogenates of pan-fried or uncooked beef, both before and after proteolysis (proteinase K). Genotoxicity was measured using the alkaline single cell-gel electrophoresis ('Comet') assay in MCL-5 cells and mutagenicity in Salmonella typhimurium strains TA1538 or YG1019. Proteolysis released significant amounts of DNA-damaging material that was not extractible prior to enzymic digestion, suggesting that human exposures to diet-derived genotoxins may have been underestimated.     

Chlorine resistance of strains of Staphylococcus aureus isolated from poultry processing plants

Strains of Staphylococcus aureus which colonize defeathering machinery and become endemic within poultry processing plants appear to be resistant to the normal cleaning and disinfection processes. The resistance of endemic strains to chlorine was compared with that of isolates from the natural skin flora of poultry. Endemic strains were almost eight times more resistant and this was due primarily to their ability to grow in macroclumps but also to the production of an extracellular slime layer.     

Comparison of microbial counts on beef carcasses by using the moist-swab contact method and secondary tissue removal technique.

When a tissue removal rinse technique was compared to the moist-swab contact method, significantly greater numbers of bacteria were recovered from beef carcasses, especially when the flora exceeded log10 4.5/6.45 cm2. Secondary treatment of the removed surface tissue by blending resulted in a significantly greater number of bacteria being recovered than when the same sample was swabbed and/or rinsed. Data indicate that blending of the carcass surface tissue provides a more representative value of the true microbial flora.     

Comparison of microbial counts on beef carcasses by using the moist-swab contact method and secondary tissue removal technique.

When a tissue removal rinse technique was compared to the moist-swab contact method, significantly greater numbers of bacteria were recovered from beef carcasses, especially when the flora exceeded log10 4.5/6.45 cm2. Secondary treatment of the removed surface tissue by blending resulted in a significantly greater number of bacteria being recovered than when the same sample was swabbed and/or rinsed. Data indicate that blending of the carcass surface tissue provides a more representative value of the true microbial flora.     

Mutual conformational changes of tryptophanyl-tRNA synthetase and tRNATrp in the course of their specific interaction

tRNATrp (beef, yeast) is capable of accelerating limited tryptic hydrolysis of the N-terminal part in the polypeptide chains of dimeric beef pancreas tryptophanyl-tRNA synthetase; it can also eliminate the protective effect of tryptophanyl adenylate on the enzyme proteolysis. The effect of tRNA on the proteolysis is manifested even when the 3'-CCA terminus is removed. It has been concluded that the conformation of the synthetase changes when it forms a complex with tRNATrp. Yeast tRNATrp lacking the 3'-half of the acceptor stem can still interact with the synthetase and, to certain extent, induces changes in the conformation of the latter. The susceptibility of single-stranded and double-stranded regions of tRNATrp to cleavage with endonucleases has been studied, and the results are indicative of the fact that, regardless of considerable differences in the nucleotide sequence of yeast and beef tRNATrp, their three-dimensional structures are similar. This fact is consistent with the finding that parameters for the interaction of these tRNAsTrp with beef tryptophanyl-tRNA synthetase are rather close. The three-dimensional structure of tRNATrp is altered when the enzyme forms a complex with it, as seen from (a) a change in the circular dichroic spectrum and (b) an elevated susceptibility of the anticodon and, apparently, acceptor stems to cleavage with nuclease. The conversion of exposed cytidine residues in tRNATrp into uridine residues results in a loss of the acceptor activity; the capability to accelerate limited tryptic hydrolysis of tryptophanyl-tRNA synthetase is also lost although the enzyme-substrate complex, as seen from circular dichroic spectra, can still be formed. The conversion of cytosine in the anticodon stem into uracil modifies the conformation of the anticodon stem. The anticodon arm (including the anticodon) and the acceptor stem play an essential role in the interaction between tRNATrp and tryptophanyl-tRNA synthetase.     

Fate of Staphylococci and Enteric Microorganisms Introduced into Slurry of Frozen Pot Pies

A slurry was prepared from six frozen pot pies diluted 1:5 with distilled water, two chicken, two turkey, and two beef pies of different brands. This slurry formed a reference sample and was placed in sterilized jars, frozen, and used as needed throughout the experiments. A second slurry was prepared in a similar manner from a frozen beef pot pie and a chicken pot pie, and was used as a control in only one experiment. The total count of microorganisms and the number of coliforms, Escherichia coli, salmonellae, and coagulase-positive staphylococci per gram were determined. Samples of slurry were inoculated in decimal dilutions with one or more of the following: Salmonella typhimurium, E. coli, and a strain of staphylococcus that causes food poisoning. The natural flora was found to exert an inhibitory effect upon the growth of the added microorganisms after incubation at 35 C for 18 hr. The inhibitory effect on growth was in part due to pH. The predominating organism isolated from the natural flora after incubation was a lactobacillus, which, when added in mixture with the test organisms in sterilized slurry, did not exert the profound inhibitory effect observed in the case of the natural flora. Some factors which may be concerned with the inhibition were investigated.     

Use of a Titrimetric Method to Assess the Bacterial Spoilage of Fresh Beef

A new method of determining bacterial spoilage in fresh beef is presented. The technique is based upon the fact that as beef undergoes refrigerator spoilage, there is a gradual increase in the production of alkaline substances by the spoilage flora. The level of these substances was measured by titrating meat homogenates to a pH 5.00 end point, employing 0.02 n HCl and an autotitrator. When 23 samples of ground beef from retail stores were tested, an average of 1.32 ml of acid was required for titration of 1 g of fresh beef to pH 5.00, whereas 2.58 ml was required for the same meat at the onset of spoilage. Preliminary data indicate that beef which requires more than 2 ml of 0.02 n HCl/g to lower its pH to 5.00 under the conditions of the test is in some state of incipient spoilage. The statistical correlation between titration values, log bacterial numbers, and extract-release volume was high (P < 0.001). The technique is simple to execute and is highly reproducible, and duplicate samples can be run within 15 min.     

Response of the Extract-Release Volume and Water-Holding Capacity Phenomena to Microbiologically Spoiled Beef and Aged Beef

The aging of ground beef was effected by storing in gas-impermeable, sterile plastic bags with incubation at 7 and 15 C. Control meat from the same preparations was wrapped in aluminum foil and stored at the same temperature. In three experiments where control meat was tested, aged meat did not attain a log bacterial number of ca. 8.4 per gram until an average of 6 days after this level was reached in control meats. This degree of difference was shown in values for both extract-release volume (ERV) and water-holding capacity. The previously reported ERV value of around 25, which was found to correspond to an average log bacterial number of ca. 8.5 per gram for ground beef allowed to spoil in aluminum foil and freezer paper, was approximated for aged meats, which required an average of 9.7 days to attain this number compared with 4.1 days for unaged meats. Plate count methods indicated the predominant flora of aged beef to be gram-negative, facultatively psychrophilic rods.     

Phosphofructokinase from Dictyostelium discoideum is a potent inhibitor of tubulin polymerization

We identified the nonallosteric phosphofructokinase from the slime mold Dictyostelium discoideum as a potent protein factor that inhibits the rate of polymerization of tubulin at a molar ratio of 1 molecule to about 300 tubulin dimers for half-maximal action (IC50 = 32 nM). This effect was (i) assessed by turbidity measurements, pelleting of microtubules, and electron microscopy, (ii) observed when tubulin assembly was induced by taxol as well as by GTP in the presence of microtubule-associated proteins or glutamate, and (iii) specific as it was not produced by the phosphofructokinase from rabbit muscle. Also in contrast to the latter, neither tubulin nor microtubules modified the catalytic activity of the slime mold isozyme. Immunoelectron microscopy provided further evidence that D. discoideumphosphofructokinase physically interacts with tubulin, leading to the formation of aggregates. The process seems to be reversible since microtubules eventually formed in the presence of the inhibitor with concomitant reduction of tubulin aggregates. Limited proteolysis by subtilisin showed that the hypervariable C-termini of tubulin is not involved in the interaction with the enzyme. The possible physiological relevance of this novel function of D. discoideum phosphofructokinase different from its glycolytic action is discussed.     

The application of NMR to study changes in polar metabolite concentrations in beef <Emphasis Type="Italic">longissimus dorsi</Emphasis> stored for different periods post mortem

In this study data generated by ¹H NMR spectroscopy were combined with chemometrics to analyse beef samples aged over a 21 day period. In particular, the amino acids, of which 12 were identified were found to increase over the ageing period with samples matured for 3 days having notably lower concentrations than carcasses aged for 21 days. This is believed to be a result of increased proteolysis within the muscle. This novel approach of using high resolution NMR spectrometry to analyse beef samples has not previously been reported and these findings demonstrate the potential of this technique linked with HPLC to be used as a suitable method for profiling meat samples.     

The eye lens has an active ubiquitin-protein conjugation system

Using exogenous 125I-ubiquitin, ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lens epithelial cells and lens cortex tissue. Conjugation was ATP-dependent with the greatest variety and amount of conjugates larger than 150 kDa. In vivo production of ubiquitin-protein conjugates in cultured rabbit and beef lens epithelial cells and rabbit lens tissues of different developmental age was established using immunological detection. There were limited similarities between conjugates found in youngest as opposed to oldest tissue. Cultured rabbit cells contained 27 pmol/mg free ubiquitin and 18 pmol/mg conjugated ubiquitin. Levels of free ubiquitin in lens tissue epithelium, cortex, and core were 36, 5, and 5 pmol/mg, respectively. There were only 2 pmol/mg conjugated ubiquitin in each of these tissues. Hydrolysis of 125I-ubiquitin was catalyzed by supernatants of cultured lens cells, beef and human lens tissues, and reticulocytes. Degradation was greatest in epithelial tissues, and least in core. This corroborates studies which show that proteolytic capabilities are attenuated in older tissue. Decreased initiation of proteolysis by ubiquitination as well as diminished proteolysis in older lens tissue may be related to the accumulation of damaged proteins in aging lens tissue.     

Substrate specificity of cyclic nucleotide phosphodiesterase from beef heart and from Dictyostelium discoideum

The substrate specificity of beef heart phosphodiesterase activity and of the phosphodiesterase activity at the cell surface of the cellular slime mold Dictyostelium discoideum has been investigated by measuring the apparent Km and maximal velocity (V) of 24 derivatives of adenosine 3',5'-monophosphate (cAMP). Several analogs have increased Km values, but unaltered V values if compared to cAMP; also the contrary (unaltered Km and reduced V) has been observed, indicating that binding of the substrate to the enzyme and ring opening are two separate steps in the hydrolysis of cAMP. cAMP is bound to the beef heart phosphodiesterase by dipole-induced dipole interactions between the adenine moiety and an aromatic amino acid, and possibly by a hydrogen bond between the enzyme and one of the exocyclic oxygen atoms; a cyclic phosphate ring is not required to obtain binding. cAMP is bound to the slime mold enzyme via a hydrogen bond at the 3'-oxygen atom, and probably via a hydrogen bond with one of the exocyclic oxygen atoms. A cyclic phosphate ring is necessary to obtain binding to the enzyme. A specific interaction (polar or hydrophobic) between the base moiety and the enzyme has not been demonstrated. A negative charge on the phosphate moiety is not required for binding of cAMP to either enzyme. The catalytic reaction in both enzymes is restricted to the phosphorus atom and to the exocyclic oxygen atoms. Substitution of the negatively charged oxygen atom by an uncharged dimethylamino group in axial or equatorial position renders the compound non-hydrolyzable. Substitution of an exocyclic oxygen by a sulphur atom reduces the rate of the catalytic reaction about 100-fold if sulphur is placed in axial position and more than 10000-fold if sulphur is placed in equatorial position. A reaction mechanism for the enzymatic hydrolysis of cAMP is proposed.     

Toxicity and proteolysis in gnotobiotic rats with thermal burns]

Experiments were conducted on germ-free (gnotobiotic) rats. A study was made of the toxic properties of the organs and the activity of proteolytic enzymes in them after thermal burns. Despite the absence of pathogenic microbial flora the germ-free rats developed the state of toxemia with an increase of the level of proteolytic enzymes. The extent of the toxic and catabolic reaction depended on the severity of the burn. Intoxication and an increase in proteolysis in the gnotobiotic rats were similar, and, in a number of cases greater than in usual animals. A conclusion was drawn on the important role of the tissue sources of intoxication in burns. A correlation between the toxemia and proteolysis was established.     

Tyrosyl-tRNA synthetase from the bovine liver. Isolation and physico-chemical properties

Tyrosyl-tRNA synthetase of beef liver has been isolated and its properties have been studied. Tyrosyl-tRNA synthetase is a structural dimer of alpha 2 type. Mr of the enzyme subunit is about 59 kDa. Km values for substrates have been determined and compared with kinetic properties of tyrosyl-tRNA synthetases from different sources. The polymorphism of tyrosyl-tRNA synthetase was studied. The enzyme was separated into two different forms by chromatography on phosphocellulose P 11. P1-form is active only in the amino acid activation reaction. This form is not due to the phosphorylation of the enzyme. The low molecular weight form (38 kDa) was also isolated. This form appeared due to the limited endogenic proteolysis of the main form and retained full activity in the aminoacylation reaction. Tyrosyl-tRNA synthetase from beef liver has non-specific affinity to rRNA-sepharose.     

Characterization of cadmium-responsive MicroRNAs and their target genes in maize (Zea mays) roots

MicroRNAs (miRNAs) are small noncoding RNAs of approximately 22 nucleotides, highly conserved among species, which modulate gene expression by cleaving messenger RNA target or inhibiting translation. MiRNAs are involved in the regulation of many processes including cell proliferation, differentiation, neurogenesis, angiogenesis, and apoptosis. Beef tenderness is an organoleptic characteristic of great influence in the acceptance of meat by consumers. Previous studies have shown that collagen level, marbling, apoptosis and proteolysis are among the many factors that affect beef tenderness. Considering that miRNAs can modulate gene expression, this study was designed to identify differentially expressed miRNAs that could be modulating biological processes involved with beef tenderness. Deep sequence analysis of miRNA libraries from longissimus thoracis muscle allowed the identification of 42 novel and 308 known miRNAs. Among the known miRNAs, seven were specifically expressed in skeletal muscle. Differential expression analysis between animals with high (H) and low (L) estimated breeding values for shear force (EBVSF) revealed bta-mir-182 and bta-mir-183 are up-regulated (q value < 0.05) in animals with L EBVSF, and bta-mir-338 is up-regulated in animals with H EBVSF. The number of bovine predicted targets for bta-mir-182, bta-mir-183 and bta-mir-338 were 811, 281 and 222, respectively, which correspond to 1204 unique target genes. Among these, four of them, MEF2C, MAP3K2, MTDH and TNRC6B were common targets of the three differentially expressed miRNAs. The functional analysis identified important pathways related to tenderness such as apoptosis and the calpain–calpastatin system. The results obtained indicate the importance of miRNAs in the regulatory mechanisms that influence muscle proteolysis and meat tenderness and contribute to our better understanding of the role of miRNAs in biological processes associated with beef tenderness.     

Probing stability and dynamics of proteins by protease digestion. I: Comparison of protease susceptibility and thermal stability of cytochromes c

Protease susceptibility of homologous proteins in their native conformations was studied. This work aims to establish a broad and quantitative basis for the utilization of protease digestion to analyze the local stability of native proteins. Using high-performance liquid chromatography (HPLC) the time course of the proteolytic degradation of intact proteins was quantitatively traced. Rapid separation of peptide fragments with HPLC made possible the elucidation of sequential digestion originating from the cleavage at a very few sites which are locally unstable in the protein structure. Using four serine proteases, chymotrypsin, trypsin, elastase and subtilisin BPN', we found some common trends in proteolysis for a group of proteins of the cytochrome c family. By comparing of the proteolysis and thermal denaturation with ten homologous cytochromes c extracted from horse, beef, Candida krusei, Saccharomyces cerevisiae, chicken, tuna, pigeon, rabbit, dog and rat, protease susceptibility was related to locally unfolding states intrinsic to the native conformation.