Decomposition of extremely hard-to-degrade animal proteins by thermophilic bacteria

Hard-to-degrade animal proteins are ubiquitously present throughout animal bodies. Enormous numbers of these proteins generated in the meat industry are converted to industrial wastes, the disposal of which is tremendously difficult. Most hard-to-degrade animal proteins are currently disposed of by incineration; however, this method has ecological disadvantages in terms of an apparent energy loss and the production of a large amount of carbon dioxide. As a result, an innovative solution to these problems has been sought. In this review, we focus on the degradation of three hard-to-degrade animal proteins (extracellular matrix proteins, collagen in particular, keratin, and prion proteins) and discuss the decomposing capability of thermophilic bacteria. These proteins are strongly resistant to proteinases because of their structural features; therefore, new approaches employing bacterial proteases with strong activity and broad specificity are required for practical application.     

Entrapment of Rhodobacter sphaeroides RV in cationic polymer/agar gels for hydrogen production in the presence of NH4+

Cationic polyelectrolytes (chitosan, poly-L-lysine (PLL), polyethyleneimine (PEI) and trimethylammonium glycol chitosan iodide (TGCI)) were used to entrap anoxygenic phototrophic bacteria in order to prevent the inhibitory effect of NH4+ on hydrogen production. When combined with agar gel, chitosan and PLL demonstrated no obvious repressive effect on hydrogen production by Rhodobacter sphaeroides under light-anaerobic conditions with lactate and glutamate as the carbon and nitrogen sources, respectively. On the other hand, both PEI and TGCI exerted a detrimental effect on hydrogen production under these conditions. Hydrogen production in the presence of NH4+ by the bacteria entrapped in the complex gel containing chitosan and agar improved considerably compared to that in the control containing only agar. Evidence shows that chitosan improves the hydrogen production via various effects. Diffusion tests demonstrated that the addition of chitosan increased to some extent the resistance to the diffusion of positively charged NH4+, but had no effect on negatively charged lactate. A buffer effect in the complex gels was also revealed.     

Pressure-induced conversion of α-connectin to β-connectin

The mechanism of the pressure-induced tenderization of meat has not been fully established in spite of its beneficial effect. To detect the changes in the large structural proteins of the myofibrils induced by pressurization without heat treatment, high hydrostatic pressure (100-300 MPa) was applied to rabbit at-death skeletal muscle for 10 min at low temperature (0-2°C). Significant differences in the electrophoretic pattern of connectin (also called titin) in isolated myofibrils were observed between the control and pressurized muscle samples. The conversion of α-connectin (2800 kDa) to β-connectin (2100 kDa) was accelerated with increasing pressure applied to the muscle; also nebulin (800 kDa) was degraded by pressure treatment. From the results it is clear that the degradation of connectin is induced by pressurization alone without heat treatment. If the conversion of α-connectin to β-connectin during conditioning has some influence on meat tenderization, the pressure-induced conversion of α- to β-connectin is possibly one of the causes of pressure-induced tenderization of meat.     

Purification and characterization of antioxidative peptides derived from rice bran protein hydrolysates

Rice bran protein fraction (RBPF)—albumin, globulin, glutelin and prolamin were hydrolyzed with proteases M, N, P, S and pepsin under their optimal conditions for 24 h. Hydrolysates of various hydrolysis periods were collected and subjected to peptide mapping and the antioxidative activity measured by the 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) method. Protease M hydrolysates showed high degree of hydrolysis (DH), but low antioxidative activity. On the contrary, pepsin hydrolysates showed low DH with high activity. Albumin and globulin hydrolysates had higher DH values, but lower values for glutelin and prolamin. The globulin hydrolysate (Opep2) from 2 h-pepsin hydrolysis was separated by using three consecutive purification steps with RP-HPLC. Nineteen antioxidative peptides were isolated and their amino acid sequences were determined by a gas-phase protein sequencer and MALDI-TOF mass spectrometry. These peptides were composed of 6–30 amino acid residues with molecular masses ranging from 670–3,611 Da. Tyr-Leu-Ala-Gly-Met-Asn had the highest antioxidative activity among them.     

Effects of secretin and cholecystokinin on abdominal blood circulation in unanesthetized rat (microsphere method)]

Mucous barrier was searched by the histochemical method of mucopolysaccharides and by the measurement of precipitated mucus of gastric juice. The neutral polysaccharide stored within the surface epithelial cells must be a source of the mucus of the gastric juice. The storage of the polysaccaride was decreased by a long-term stimulation by histamine, and the precipitated mucus volume was also decreased shortly after the stimulation. Therefore, some kind of the stimulation for the gastric secretion must be avoided for prevention of peptic ulcer. The oral administration of the sulfuric polysaccharide extracted from seaweed prevented the occurrence of experimental ulcer. It may be concluded that the exogeneous polysaccaride can take the place of the endogenous polysaccharide.     

The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venon and characterization of the new active intermediates

Bovine prothrombin was activated, in both the absence and presence of dissopropyphosphofluoridate (DEP) and benzamidine, by an activator which was highly purified from the venom of Echis carinatus (saw-scaled viper, ECV). The process of activation was monitored by sodium dodecysulfate (SDS)-polyacrylamide gel electrophoresis, and the reaction products were isolated and chemically characterized. In the absence of the inhibitors, prothrombin yielded two fragments with molecular weights of 28,000 and 57,000, of which the former was the N-terminal fragment of the zymogen and the latter was intermediate 1, consisting of a single polypeptide chain. Intermediate 1 was subsequently converted to an active intermediate, named intermediate ECV, without decrease of molecular weight. This new intermediate ECV, which showed little clotting activity but a strong alpha-N-tosyl-L-arginine methyl ester (TAME)-esterolytic activity and which bound with hirudin or antithrombin III, consisted of two polypeptide chains with molecular weights of 35,000 of 27,000 daltons. The former was indentified as the thrombin B chain with the N-terminal sequence Ile-Val-Glu-Gly and C-terminal serine, and the latter was a fragment with N-terminal Ser-Gly-Gly, linked to the thrombin A chain. On prolonged incubation, intermediate ECV autocaralytically yielded a fragment (inner fragment) of 14,000 daltons with N-terminal serine and the clotting enzyme alpha-thrombin [EC 3.4.21.5], which consists of A and B chains. In the presence of the inhibitors, intermediate ECV and the N-terminal fragment were accumulated in the activation mixture. On the other hand, when prothrombin was activated by the venom activator in the presence of hirudin, antithrombin III, or p-nitrophenyl p'-guanidinobenzoate, it did not yield any fragments but was converted to a derivative with two polypeptide chains having molecular weights of 51,000 and 34,000 daltons, of which the former consisted of N-terminal fragment, the inner fragment, and thrombin A chain, and the latter was thrombin B chain. This new prothrombin derivative, named prothrombin ECV, formed a high-molecular-weight complex, associating with antithrombin III. The complex was not dissociable even in the presence of SDS. Moreover, prothrombin ECV reacted with p-nitrophenyl p'-guanidinobenzoate. On the basis of the results described above, the mechanism of activaton of prothrombin by Echis carinatus venom activator can be summarized as follows: The venom activator first cleaves an Arg-Ile bond liniking thrombin A and B chains in the zymogen molecule, forming an active derivative, prothrombin ECV. This active derivative converts autocatalytically to intermediate ECV, liberating the N-terminal fragment, and active intermediate ECV generates alpha-thrombin, releasing the inner fragment. Thus, only a single peptide bond cleavage along the polypeptide chain of prothrombin is associated with activation by the venom activator...