Development of a stable continuous flow immobilized enzyme reactor for the hydrolysis of inulin

A 23.5-fold purified exoinulinase with a specific activity of 413 IU/mg and covalently immobilized on Duolite A568 has been used for the development of a continuous flow immobilized enzyme reactor for the hydrolysis of inulin. In a packed bed reactor containing 72 IU of exoinulinase from Kluyveromyces marxianus YS-1, inulin solution (5%, pH 5.5) with a flow rate of 4 mL/h was completely hydrolyzed at 55 °C. The reactor was run continuously for 75 days and its experimental half-life was 72 days under the optimized operational conditions. The volumetric productivity and fructose yield of the reactor were 44.5 g reducing sugars/L/h and 53.3 g/L, respectively. The hydrolyzed product was a mixture of fructose (95.8%) and glucose (4.2%) having an average fructose/glucose ratio of 24. An attempt has also been made to substitute pure inulin with raw Asparagus racemosus inulin to determine the operational stability of the developed reactor. The system remained operational only for 11 days, where 85.9% hydrolysis of raw inulin was achieved.

Gut health immunomodulatory and anti-inflammatory functions of gut enzyme digested high protein micro-nutrient dietary supplement-Enprocal

Background: Enprocal is a high-protein micro-nutrient rich formulated supplementary food designed to meet the nutritional needs of the frail elderly and be delivered to them in every day foods. We studied the potential of Enprocal to improve gut and immune health using simple and robust bioassays for gut cell proliferation, intestinal integrity/permeability, immunomodulatory, anti-inflammatory and anti-oxidative activities. Effects of Enprocal were compared with whey protein concentrate 80 (WPC), heat treated skim milk powder, and other commercially available milk derived products.

Results: Enprocal (undigested) and digested (Enprocal D) selectively enhanced cell proliferation in normal human intestinal epithelial cells (FHs74-Int) and showed no cytotoxicity. In a dose dependent manner Enprocal induced cell death in Caco-2 cells (human colon adencarcinoma epithelial cells). Digested Enprocal (Enprocal D: gut enzyme cocktail treated) maintained the intestinal integrity in transepithelial resistance (TEER) assay, increased the permeability of horseradish peroxidase (HRP) and did not induce oxidative stress to the gut epithelial cells. Enprocal D upregulated the surface expression of co-stimulatory (CD40, CD86, CD80), MHC I and MHC II molecules on PMA differentiated THP-1 macrophages in coculture transwell model, and inhibited the monocyte/lymphocyte (THP-1/Jurkat E6-1 cells)-epithelial cell adhesion. In cytokine secretion analyses, Enprocal D down-regulated the secretion of proinflammatory cytokines (IL-1β and TNF-α) and up-regulated IFN-γ, IL-2 and IL-10.

Conclusion: Our results indicate that Enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies. Furthermore, through downregulation of proinflammatory cytokines, Enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease (IBD). Stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies.

Amylase and glucosidase enzyme inhibitory activity of ginger (Zingiber officinale Roscoe) an in vitro study

The prevalence of type 2 diabetes has reached to an epidemic proportion in Sri Lanka. The need for achieving better control of blood glucose level has been evident in diabetes management. However it is not easy to achieve this goal in a large proportion of patients. This is partly due to limitations of currently available pharmacological agents which stimulate research on novel anti-diabetic agents with different mechanisms. Digestive enzymes have been targeted as potential avenues for modulation of blood glucose concentration through inhibition of the enzymatic breakdown of complex carbohydrates to meal derived glucose absorption. Acarbose is a widely used oral anti-diabetic drug which inhibits the α-glucosidase, enzyme responsible for breaking down of disaccharides and polysaccharides into glucose. Many herbal extracts have been found to posses similar inhibitory effects. Ginger (Zingiber officinale Roscoe) has developed a reputation in treatment of several diseases. In vitro enzymic inhibitory effect of ginger was investigated in this study. Enzymes α -amylase and α -glucosidase treated with either Acarbose or ginger extract were allowed to react with cooked rice and percentages of glucose content were measured. The glucosidase and amylase activities on the rice were inhibited by addition of ginger cause significant reduction in glucose percentages (36.86± 1.05 to 26.87± 2.17, P<0.05 and 49.04±0.65 to 35.35±2.22, P<0.05) which showed comparable results with Acarbose on glucosidase activity (36.86± 1.05 to, 27.8±1.32 P<0.05). Results of the study indicates ginger as a potential plant based amylase and glucosidase inhibitor in carbohydrate digestion but usage in glycaemic control in human has to be investigated further.

Elevated intracardiac angiotensin II leads to cardiac hypertrophy and mechanical dysfunction in normotensive mice

Introduction
Angiotensin II (Ang II) is known to induce cardiac growth and modulate myocardial contractility. It has been reported that elevated levels of endogenous Ang II contribute to the development of cardiac hypertrophy in hypertensives. However, the long-term functional effects of cardiac exposure to Ang II in normotensives is unclear.

A recently developed transgenic mouse (TG1306/1R), in which cardiac-specific overproduction of Ang II produces primary hypertrophy, provides a new experimental model for investigation of this phenotype. The aim of the present study was to use this model to investigate whether there is a functional deficit in primary hypertrophy that may predispose to cardiac failure and sudden death. We hypothesised that primary cardiac hypertrophy is associated with mechanical dysfunction in the basal state.

Methods
Normotensive heterozygous TG1306/1R mice harbouring multiple copies of a cardiac-specific rat angiotensinogen gene were studied at age 30—40 weeks and compared with age-matched wild-type littermates. Left ventricular function was measured ex vivo in bicarbonate buffer-perfused, Langendorffmounted hearts ( at a perfusion pressure of 80 mmHg, 37°C) using a fluid-filled PVC balloon interfaced to a pressure transducer and digital data acquisition system.

Results
There was no difference in the mean (±SEM) intrinsic heart rate of TG1306/1R and wild-type control mice (357.4±11.8 vs. 367.5±20.9 bpm, n=9 & 7). Under standardised end-diastolic pressure conditions, TG1306/1R hearts exhibited a significant reduction in peak developed pressure (132.2±9.4 vs. 161.5±3.1 mmHg, n=9 & 7, p<0.05) and maximum rate of pressure development (3566.7±323.7 vs. 4486.3±109.4 mmHg, n=9 & 7, p<0.05). TG1306/1R mice show a significant correlation between incidence of arrhythmia and increasing heart size (Spearman's correlation coefficient 0.61).

Conclusion
These data demonstrate that chronic in vivo exposure to elevated levels of intra-cardiac Ang II is associated with significant contractile abnormalities evident in the ex vivo intact heart. Our findings suggest that endogenous overproduction of cardiac Ang II, independent of changes in blood pressure, is sufficient to induce ventricular remodelling that culminates in impaired cardiac function which may precede failure.

Angiotensin II receptor imbalance associated with neonatal cardiac growth restriction is a prelude to adult cardiac hypertrophy

The Hypertrophic Heart Rat (HHR) displays spontaneous cardiomyocyte hypertrophy in association with an apparent reduction in myocyte number in adulthood. This suggests the possibility of reduced hyperplasia or increased apoptosis during early cardiac development. The angiotensin AT1 and AT2 receptor subtypes have been implicated in both cellular growth and apoptosis, but the precise mechanisms are unclear. The aim of this study was to determine the relationship between cardiac AngII receptor expression levels and neonatal cardiomyocyte growth and apoptotic responses in the HHR compared with the Normal Heart Rat (NHR) control strain. Cardiac tissues were freshly harvested from male HHR and NHR at several developmental stages (p2 and 4, 6, 8, 12wks). HHR cardiac weight indices were considerably smaller than NHR at day 2 (4.330.19 vs 5.010.08 mg/g), but ‘caught-up’ to NHR by 4 weeks (5.100.15 vs 5.160.11 mg/g). By 12 weeks, HHR hearts were 27% larger than NHR. Tissue AT1A and AT2 mRNA expression levels were quantified by real-time RT-PCR. Relative to NHR, HHR neonatal hearts exhibited a 4.6-fold higher AT2/AT1 mRNA expression ratio. Cultured neonatal cardiomyocytes were infected with AT1A and/or AT2 receptor-expressing adenoviruses to achieve a physiological level of receptor expression (150 fmol receptor protein/mg total cell protein). In addition, to emulate receptor expression in neonatal HHR hearts, cells were co-infected with AT1A and AT2 receptors at a 4:1 ratio. Apoptosis incidence was studied by morphological analysis after 72 hours exposure to 0.1 M AngII. When infected with the AT1A receptor alone, a higher proportion of HHR myocytes appeared apoptotic than NHR (22.7 4.1% vs 1.1 0.6%, P 0.001). This implies that intrinsic differences predispose HHR cells to accentuated AT1-mediated apoptosis. Interestingly, the bax-1/bcl-2 mRNA expression ratio was significantly higher (50%) in HHR neonatal hearts. When cells were co-infected with AT1A and AT2 receptors, evidence of apoptosis in HHR cells virtually disappeared (0.4 0.1%). These findings suggest a novel capacity of AT2 receptors to counteract accentuated AT1A receptor-induced apoptosis in the HHR in early cardiac growth.

Elevated dietary sodium intake exacerbates myocardial hypertrophy associated with cardiac-specific overproduction of angiotensin II

Introduction/hypothesis
Cardiac hypertrophy is an independent risk factor predictive of cardiovascular disease and is significantly associated with morbidity and mortality. The mechanism by which angiotensin II (Ang II) and dietary sodium exert additive effects on the development of cardiac hypertrophy is unclear. The goal of this study was to evaluate the hypothesis that, where there is a genetic predisposition to Ang II-dependent hypertrophy, there is also an increased susceptibility to sodium-induced hypertrophy mediated by AT₁-receptor expression.

Methods
Diets of low sodium (LS, 0.3% w:w) and high sodium (HS, 4.0% w:w) content were fed to adult (age 25 weeks) control wild-type mice (WT) and to weeks) control wild-type mice (WT) and to transgenic mice exhibiting cardiac specific overexpression of angiotensinogen (TG). At the conclusion of a 40-day dietary treatment period, cardiac tissue weights were compared and the relative expression levels of Ang II receptor subtypes (AT_1A and AT₂) were evaluated using RT-PCR.

Results
WT and TG mice fed HS and LS diets maintained comparable weight gains during the treatment period. The normalised heart weights of TG mice were elevated compared to WT, and the extent of the increase was greater for mice maintained on the HS diet treatments (WT 12% vs. TG 41% increase in cardiac weight index). While a similar pattern of growth was observed for ventricular tissues, the atrial weight parameters demonstrated an additional significant effect of dietary sodium intake on tissue weight, independent of animal genetic type. No differences in the relative (GAPDH normalised) expression levels of AT_1A- and AT₂-receptor mRNA were observed between diet or animal genetic groups.

Conclusion
This study demonstrates that, where there is a pre-existing genetic condition of Ang II-dependent cardiac hypertrophy, the pro-growth effect of elevated dietary sodium intake is selectively augmented. In TG and WT mice, this effect was evident with a relatively short dietary treatment intervention (40 days). Evaluation of the levels of Ang II receptor mRNA further demonstrated that this differential growth response was not associated with an altered relative expression of either AT_1A- or AT₂-receptor subtypes. The cellular mechanistic bases for this specific Ang II-dietary sodium interaction remain to be elucidated.

Renal responses to angiotensin II in conscious dogs : effects of aspirin and indomethacin

1. Angiotensin II was infused into the renal artery of unanaesthetized dogs at 0.4 and 2.0 ng/kg per min for 40 min each.

2. Indomethacin (3 mg/kg, and 1 mg/kg per h infusion i.v.) accentuated the angiotensin II-induced falls in glomerular filtration rate, renal blood flow and urine flow rate. Indomethacin did not alter the effects of angiotensin II on Na+ or K+ excretions.

3. Aspirin (35 mg/kg p.o. 2.5 h and 0.5 h prior to experiment) did not significantly change the renal effects of angiotensin II.

4. Both aspirin and indomethacin accentuated renal vasoconstriction during briefer (5 min) angiotensin II infusion.

5. Thus indomethacin and aspirin had markedly different effects on the actions of angiotensin II in the kidney. This suggests that at least one of these drugs has actions which affect angiotensin II-mediated vasoconstriction other than via cyclooxygenase inhibition.

Fermentative production of rhamnosidase from Staphylococcus xylosus MAK 2 in a bioreactor for bio-energy generation

Increasing concern about the environment, food and feed shortages and hike in the price of petroleum have stimulated interest in new ways of producing biofuels. The interest is rapidly increasing towards converting agricultural wastes to commercially valuable products. Biofuels made from waste biomass can offer immediate and sustained greenhouse gas advantages. In this direction, we are focusing on Citrus processing waste, a byproduct of juice manufacture, which contains high amount of flavonoids and polysaccharides. There is a considerable industrial interest in the enzymatic transformation of flavonoids to hydrolysis products; that offers a pathway to bio-energy generation. Rhamnosidase of bacterial origin are very few and thus are potentially subject for research.

Staphylococcus xylosus, Gram positive cocci, a nonpathogenic member of CNS family, isolated from soil was used to produce α-L-rhamnosidase. This new strain, so far unknown for the production of α-L-Rhamnosidase, was identified and characterized as Staphyloccocus sp. through biochemical tests and 16S DNA sequence analysis. Effect of various medium and process parameters like pH, temperature, aeration and agitation rates and inducer concentration were studied. Further, the enzyme activity was enhanced by adding the inducer and divalent metal ion to the optimised fermentation medium. We have recovered important sugars “rhamnose” and “galacturonic acid” from the processed waste which would be utilized for ethanol production. This presentation will summarize current efforts to develop an enzymatic treatment which would facilitate the economical processing of citrus waste for bioenergy generation.

Anti-amnesic effect of enzyme extracted stevioside from Stevia rebaudiana

The increasing consumption of sucrose has resulted in several nutritional and medicinal problems, including obesity. There is an alarming rise in the prevalence of obesity, type 2 diabetes mellitus, and metabolic syndrome in children and adults around the world, partly related to increasing availability of energy-dense, high-calorie foods, and perhaps to increased consumption of sugar and particularly fructose sweetened beverages. Therefore, low calorie sweeteners are urgently required to substitute table sugar.

Stevioside, a diterpene glycoside, is well known for its intense sweetness and is used as a non-caloric sweetener. Its potential widespread use requires an easy and effective extraction method. Enzymatic extraction of stevioside from Stevia rebaudiana leaves with cellulase, pectinase and hemicellulase using various parameters such as concentration of enzyme, incubation time and temperature was optimized. The extraction conditions were further optimized using response surface methodology (RSM). Under the optimized conditions, the experimental values were in close agreement with predicted model and resulted in a three times yield enhancement of stevioside.

Various studies have revealed that in addition to sweetening nature of stevisoide, it exerts beneficial effects including antihypertensive, anti-hyperglycemic, anti-human rotavirus, antioxidant, anti-inflammatory and antitumor actions. Its anti-amnesic potential remains to be explored, therefore the present study has been undertaken to investigate the beneficial effect of stevioside in memory deficit of rats employing scopolamine induced amnesia as an animal model.

Significance: Stevia is gaining significance in different parts of the world and is expected to develop into a major source of high potency sweetener for the growing natural food market. There is a strong possibility that Stevia sweeteners could replace aspartame in some diet variants. In addition, Stevia is expected to be used as a part substitute for sugar and also used in combination with other artificial sweeteners in the emerging phase of life cycle.

Parameters important in fabricating enzyme electrodes using self-assembled monolayers of alkanethiols

The fabrication of enzyme electrodes using self-assembled monolayers (SAMs) has attracted considerable interest because of the spatial control over the enzyme immobilization. A model system of glucose oxidase covalently bound to a gold electrode modified with a SAM of 3-mercaptopropionic acid was investigated with regard to the effect of fabrication variables such as the surface topography of the underlying gold electrode, the conditions during covalent attachment of the enzyme and the buffer used. The resultant monolayer enzyme electrodes have excellent sensitivity and dynamic range which can easily be adjusted by controlling the amount of enzyme immobilized. The major drawback of such electrodes is the response which is limited by the kinetics of the enzyme rather than mass transport of substrates. Approaches to bringing such enzyme electrodes into the mass transport limiting regime by exploiting direct electron transfer between the enzyme and the electrode are outlined.

Converting potential organ donors : a situational analysis of Australian physicians

This theory-building research suggests that the key to unlocking Australia's potential organ donor pool is to reduce the ethical ambiguity and legal uncertainty plaguing physicians' interepretation of the patients's best interests standard. This may generate a greater acceptance of organ donation as part of end-of-life patient care amongst our physicians.