Glycogen debranching enzyme activity in the muscles of meat producing animals

Muscle glycogen exists in two forms: low molecular weight pro-glycogen and high molecular weight macro-glycogen. The degradation of glycogen to glucose 1 phosphate and free glucose is catalysed by glycogen phosphorylase together with glycogen debranching enzyme (GDE). The process in which glycogen is broken down via anaerobic pathways to lactate, results in the acidification of the muscles and has a great influence on meat quality. Thus, the overall aim of this thesis was to characterise the post mortem action of GDE in muscles of meat production animals (pigs, cattle and chickens). Interest was focused on the differences in GDE activity between fast twitch glycolytic muscles and slow twitch oxidative muscles. The effects of pH, temperature, RN genotype (PRKAG3 gene), and of time post mortem on GDE activity were also investigated. This thesis showed that there are differences in GDE activity between animal species and between different muscles of an animal. It was shown that in pigs and cattle, higher GDE activity and phosphorylase activity exists in the fast twitch glycolytic muscles than in slow twitch oxidative muscles of the same animal. Thus, the high activity of these enzymes enables a faster rate of glycogenolysis in glycolytic M. longissimus dorsi compared to oxidative M. masseter. In chicken muscles, the GDE activity was low compared to pig or cattle muscles. Furthermore, the GDE activity in the glycolytic M. pectoralis superficialis was lower than in more oxidative M. quadriceps femoris despite the high phosphorylase activity in the former. The relative ratios between phosphorylase and GDE activity were higher in fast twitch glycolytic muscles than in slow twitch oxidative muscles of all studied animals. This suggests that the relatively low GDE activity compared to the phosphorylase activity in fast twitch glycolytic muscles may be a protection mechanism in living muscle against a very fast pH decrease. Chilling significantly decreased GDE activity and below 15 C porcine GDE was almost inactive. The effect of pH on GDE activity was only minor at the range normally found in post mortem muscles (pH 7.4 to 5.0). The GDE activity remained level for several hours after slaughter. During the first hours post mortem, GDE activity was similar in RN- carrier pigs and in wild type pigs. However, the GDE activity declined faster in M. longissimus dorsi from wild type pigs than in the RN carrier pigs, the difference between genotypes was significant after 24 h post mortem. Pro-glycogen and macro-glycogen contents were higher, pH decrease was faster and ultimate pH was lower in RN- carrier pigs than in wild type pigs. In the RN- carriers, the prolonged high GDE activity level may enable an extended pH decrease and lower ultimate pH in their muscles. In conclusion, GDE is not the main factor determining the rate or the extent of post mortem glycogenolysis, but under certain conditions, such as in very fast chilling, the inhibition of GDE activity in meat may reduce the rate of pH decrease and result in higher ultimate pH. The rate and extent of pH decrease affects several meat quality traits.

Relationship between differential retention of Escherichia coli and Enterococcus faecalis and variations in enzyme activity in the scallop Patinopecten yessoensis

Uptake of Escherichia coli and Enterococcus faecalis and variations of trypsin amylase activity acid phosphatase and superoxide dismutase in tissue of the scallop Patinopecten yessoensis were detected. The results showed that P. yessoensis accumulated E. faecalis in larger numbers and more rapidly than E. coli, both with the highest concentration in the digestive tract and lowest in hemolymph. Compared to E. coil, all scallops exposed to E. faecalis showed significantly higher trypsin and AMS activity. SOD activity in hemocytes and ACP activity in hemolymph was significantly higher in the treatments with 5 log(10)CFU/ml E. colt than with E. faecalis. But no significant differences in ACP activity of P. yessoensis exposed to a 3 log(10)CFU/ml inoculum of both bacteria were recorded. In conclusion, the mass retention of gut microflora in P. yessoensis is positively correlated with digestive enzymes activity and negatively correlated with ACP activity in the hemocyte. (C) 2010 Published by Elsevier Ltd.

Enhanced cAMP generation and insulin-releasing potency of two novel Tyr(1)-modified enzyme-resistant forms of glucose-dependent insulinotropic polypeptide is associated with significant antihyperglycaemic activity in spontaneous obesity-diabetes

Glucose-dependent insulinotropic polypeptide (GIP) is an important incretin hormone, which potentiates glucose-induced insulin secretion. Antihyperglycaemic actions of GIP provide significant potential in Type 11 diabetes therapy. However, inactivation of GIP by the enzyme dipeptidyl peptidase IV (DPP IV) and its consequent short circulating half-life limit its therapeutic use. Therefore two novel Tyr(1)-Modified analogues of GIP, N-Fmoc-GIP (where Fmoc is 9-fluorenylmethoxycarbonyl) and N-palmitate-GIP, were synthesized and tested for metabolic stability and biological activity. Both GIP analogues were resistant to degradation by DPP IV and human plasma. In Chinese hamster lung (CHL) cells expressing the cloned human GIP receptor, both analogues exhibited a 2-fold increase in cAMP-generating potency compared with native GIP (EC50 values of 9.4, 10.0 and 18.2 nM respectively). Using clonal BRIN-BD11 cells, both analogues demonstrated strong insulinotropic activity compared with native GIP (P <0.01 to P <0.001). In obese diabetic (ob/ob) mice, administration of N-Fmoc-GIP or N-palmitate-GIP (25 nmol/kg) together with glucose (18 mmol/kg) significantly reduced the peak 15 min glucose excursion (1.4- and 1.5-fold respectively; P <0.05 to P <0.01) compared with glucose alone. The area under the curve (AUC) for glucose was significantly lower after administration of either analogue compared with glucose administered alone or in combination with native GIP (1.5-fold; P <0.05). This was associated with a significantly greater AUC for insulin (2.1-fold; P <0.001) for both analogues compared with native GIP. A similar pattern of in vivo responsiveness was evident in lean control mice. These data indicate that novel N-terminal Tyr(1) modification of GIP with an Fmoc or palmitate group confers resistance to degradation by DPP IV in plasma, which is reflected by increased in vitro potency and greater insulinotropic and antihyperglycaemic activities in an animal model of Type 11 diabetes mellitus.

Production of angiotensin-I-converting enzyme (ACE) inhibitory activity in milk fermented with probiotic strains: Effects of calcium, pH and peptides on the ACE-inhibitory activity

Recently, probiotic fermented milk products have raised interest regarding their potential anti-hypertensive activity mainly due to the production of angiotensin-I-converting enzyme (ACE) inhibitory peptides. Ionic calcium released upon milk acidification during fermentation is also known to exert hypotensive activity. Thus, the main aim of this study was to screen probiotic strains for their ability to induce ACE-inhibitory activity upon fermentation of milk. The relationship of ACE-inhibitory activity percentage (ACEi%) with cell growth, pH, degree of hydrolysis and the concentration of ionic calcium released during the fermentation was also investigated. Compared with other lactic acid bacteria, Lactobacillus casei YIT 9029 and Bifidobacterium bifidum MF 20/5 were able to induce strong ACE-inhibitory activity. Furthermore, it was found that the ionic calcium released during milk fermentation could contribute to the ACE-inhibitory activity. These findings will contribute to the development of new probiotic dairy products with anti-hypertensive activity.

Chemical properties and enzyme activity in a sewage sludge-treated soil

A soil sample was taken from the top 0-20cm at Jaboticabal county, São Paulo State, Brazil, air dried, sieved to 5mm, and placed into pots (2700g per pot). Sewage sludge was air-dried, ground to 2mm, and thoroughly mixed to the top 0-10cm soil of each pot, which were irrigated with distilled water in a total volume equivalent to the last 30years average rainfall in the region. Sorghum was sowed 120days after sewage sludge incorporation and then the irrigation was made according to the plants' requirement. When the plants were about 10 cm high, they were thinned to two per pot. Soil samples (0-10, 10-20, and 20-30 cm depth) were obtained immediately after the incorporation of sewage sludge and at 30, 60, 120, and 170 days after, air dried, sieved to 2 mm and analyzed for organic matter (OM), pH (0,01 mol L-1 CaCl2), extractable P (resin), potassium (K), calcium (Ca), and magnesium (Mg), amylase and cellulase activity. Sewage sludge increased soil OM, pH, extractable phosphorus (P), K. Ca. amylase and cellulase activity, especially at the rate 16 t ha(-1). Organic matter, extractable P, K, Ca, Mg. and amylase activity were higher in the top 0-10cm, while pH was higher in the 20-30cm layer. Amylase activity was not affected by sampling depth. Organic matter, pH, extractable P. K, Ca, and Mg decreased during the experimental period. Amylase activity decreased until sorghum was sowed and increased afterwards. Cellulase activity increased until 90 days after sewage sludge application and then decreased. Sewage sludge used in the experiment should already contain some amylase activity or a substance that was a soil enzyme activator and also a substance that was an inhibitor of soil cellulase inhibitor. Sonic of the plant nutrients contained in sewage sludge, mainly P, did not migrate down the soil column. an indication that sewage sludge should be incorporated into the soil to improve nutrient bioavailability. Sorghum roots increased amylase activity but did not affect cellulase activity.

The long-term administration of calcineurin inhibitors decreases antioxidant enzyme activity in the rat parotid and submandibular salivary glands

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