Application of Response Surface Methodology to study the effect of different calcium sources in fish muscle-alginate restructured products

Sodium alginate needs the presence of calcium ions to gelify. For this reason, the contribution of the calcium source in a fish muscle mince added by sodium alginate, makes gelification possible, resulting a restructured fish product. The three different calcium sources considered were: Calcium Chloride (CC); Calcium Caseinate (CCa); and Calcium lactate (CLa). Several physical properties were analyzed, including mechanical properties, colour and cooking loss. Response Surface Methodology (RSM) was used to determine the contribution of different calcium sources to a restructured fish muscle. The calcium source that modifies the system the most is CC. A combination of CC and sodium alginate weakened mechanical properties as reflected in the negative linear contribution of sodium alginate. Moreover, CC by itself increased lightness and cooking loss. The mechanical properties of restructured fish muscle elaborated were enhanced by using CCa and sodium alginate, as reflected in the negative linear contribution of sodium alginate. Also, CCa increased cooking loss. The role of CLa combined with sodium alginate was not so pronounced in the system discussed here.

Production of isomaltulose obtained by Erwinia sp. cells submitted to different treatments and immobilized in calcium alginate

In recent decades, there has been an increase in the studies of isomaltulose obtainment, due to its physicochemical properties and physiological health benefits. These properties, which include low cariogenicity, low glycemic index and greater stability, allow the use of this sweetener as a substitute for sucrose in foods; besides the fact that it can be converted to isomalt, a dietary non-cariogenic sugar alcohol used in pharmaceuticals as well as in the food industry. Isomaltulose (6-O-α-D-glucopyronosyl-1-6-D-fructofuranose) is a disaccharide reducer obtained by the enzymatic conversion of sucrose - the α-glucosyltransferase enzyme. Different treatments were performed for the preparation of whole cells; lysed cells; and crude enzyme extract of Erwinia sp. D12 strain immobilized in calcium alginate. The packed bed column of granules, containing Erwinia sp. cells sonicated and immobilized in calcium alginate (CSI), reached a maximum conversion of 53-59% sucrose into isomaltulose and it presented activity for 480 hours. The converted syrup was purified and the isomaltulose crystallization was performed through the lowering of temperature. The isomaltulose crystals presented purity of 96.5%.

Elaboration of biscuits with oatmeal and fat palm with added L-leucine and calcium for sarcopenia

The objective of this study was to evaluate the effect of the addition of oatmeal and palm fat in the elaboration of biscuits with added L-leucine and calcium in order to develop a product for sarcopenia in the elderly. The biscuits, or cookies, were elaborated applying a central composite rotational design with surface response methodology, and the significant linear, quadratic and interaction terms were used in the second order mathematical model. Physical, physicochemical and sensory analyses were performed by a trained panel. Based on the best results obtained, three cookie formulations were selected for sensory evaluation by the target group and physicochemical determinations. The formulations with the highest sensory scores for appearance and texture and medium scores for color and expansion index were selected. The addition of calcium and leucine increased significantly the concentration of these components in the biscuits elaborated resulting in a cookie with more than 30% of DRI (Dietary Reference Intake) for calcium and leucine. The formulations selected showed high acceptance by the target group; therefore, they can be included in the diet of elderly with sarcopenia as a functional food.

Effect of different calcium sources on the antioxidant stability of tortilla chips from extruded and nixtamalized blue corn (Zea mays L.) flours

This research aimed to develop tortilla chips (TC) high in antioxidants from extruded and nixtamalized blue corn flours prepared with calcium hydroxide Ca(OH)2 and calcium lactate C6H10O6Ca. Tortilla chips were made with extruded flours [0.1% Ca(OH)2; 0.9% C6H10O6Ca; without calcium] and nixtamalized flours [1% Ca(OH)2; 2.95% C6H10O6Ca] using the frying process. Total anthocyanin, total phenolics content, antioxidant activity, color, texture, and oil content were determined. The color of tortilla chips from extruded flours (TCEF) showed high values of the parameters a* and b* indicating a reduction in the blue color. These color parameters were significantly different from those observed in tortilla chips from nixtamalized flours (TCNF), which tended to be more blue. The TCEF retained 15% anthocyanins, 34% phenolics, and 54% antioxidant activity. Pearson's correlation analysis indicated that anthocyanins and phenolics correlated significantly with antioxidant activity and color. TCEF with both calcium sources showed higher fracturability compared with that of TCNF. Oil absorption showed an opposite effect, with lower oil content in TCEF. Nixtamalization and extrusion with C6H10O6Ca resulted in flours and TC high in anthocyanins and antioxidant activity, representing an alternative production process for corn snack high in antioxidants.

Effect of preparation practices and the cowpea cultivar Vigna unguiculata L.Walp on the quality and content of myo-inositol phosphate in akara (fried bean paste)

Akara is one of Brazil's national treasures prepared from cowpea (Vigna unguiculata L.Walp), grated onions and salt and deep-fried in crude palm oil. The results of this study on akara preparation methods showed that, in general, cowpeas were soaked for up 3 hours at room temperature, and the seed coats were then removed. The akara makers preferred the olho de pombo cultivar, because of its cream hue, or the macassar cultivar because it produces a crispier paste. The seeds purchased from street markets had lower ranges of InsP6, InsP5, and InsP4 (1.03-7.62 ∝mol.g- 1; 0.14-1.31 ∝mol.g- 1; and 0.0-0.10 ∝mol.g- 1, respectively) than both the paste and akara (6.72-19.24 ∝mol.g- 1; 1.29-4.57 ∝mol.g- 1; 0.0-0.76 ∝mol.g- 1; 3.31-13.71 ∝mol.g- 1; 0.0-4.48 ∝mol.g- 1; and 0.0-1.32 ∝mol.g- 1). These results suggest that other beans or cowpea varieties have been used in the preparation of akara and that the phytate levels do not affect its nutritional quality.

Biochemical changes and color properties of fresh-cut green bean (Phaseolus vulgaris L. cv.gina) treated with calcium chloride during storage

Calcium chloride is widely used in industries as a firming agent, and also to extend shelf-life of vegetables. The aim of this study was to determine, the effect of different doses of calcium chloride on biochemical and color properties of fresh-cut green bean. Fresh-cut green beans were dipped for 90 seconds in 0.5%, 1%, 2% and 3% solution of calcium chloride at 25°C. The fresh-cut green bean samples were packaged in polystyrene foam dishes, wrapped with stretch film and stored in a cold room at 5±1°C temperature and 85-90% RH. Calcium chloride treatments did not retain the green color of samples. Whiteness index, browning index and total color difference (ΔE) values of CaCl2 treated samples were high. Saturation index and hue angle were low compared to the control, especially at higher doses of CaCl2. Polyphenol oxidase (PPO) enzyme activity in samples treated with CaCl2 at 3% doses, was low at the 7th days of storage than with other treatments. Fructose and sucrose content of samples increased in all treatment groups whereas glucose level decreased during the first 4th days of storage.

Calcium citrate improves the epithelial-to-mesenchymal transition induced by acidosis in proximal tubular cells

INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) is a key event in renal fibrosis. The aims of the study were to evaluate acidosis induced EMT, transforming-growth-factor (TGF) β1 role and citrate effect on it. METHODS: HK2 cells (ATCC 2290) were cultured in DMEM/HAM F12 medium, pH 7.4. At 80% confluence, after 24 hr under serum free conditions, cells were distributed in three groups (24 hours): A) Control: pH 7.4, B) Acidosis: pH 7.0 and C) Calcium citrate (0.2 mmol/L) + pH 7.0. Change (Δ) of intracellular calcium concentration, basal and after Angiotensin II (10-6M) exposition, were measured to evaluate cellular performance. EMT was evaluated by the expression of α-smooth muscle actin (α-SMA) and E-cadherin by immunocytochemistry and/or Western blot. TGF-β1 secretion was determined by ELISA in cell supernatant. RESULTS: At pH 7.0 HK2 cells significantly reduced E-cadherin and increased α-SMA expression (EMT). Supernatant TGF-β1 levels were higher than in control group. Calcium citrate decreased acidosis induced EMT and improved cells performance, without reduction of TGF-β production. CONCLUSIONS: Acidosis induces EMT and secretion of TGF-β1 in tubular proximal cells in culture and citrate improves cellular performance and ameliorates acidosis induced EMT.

Novel cancer-related regulatory targets for the signalling sphingolipids sphingosine-1-phosphate and sphingosylphosphorylcholine

The signalling sphingolipid sphingosine-1-phosphate (S1P) is necessary for development of the immune system and vasculature and on a cellular level regulates migration, proliferation and survival. Due to these traits S1P has an important role in cancer biology. It is considered a primarily cancer-promoting factor and the enzyme which produces it, sphingosine kinase (SphK), is often over-expressed in tumours. S1P is naturally present in the blood, lymph, tissue fluids and cell cytoplasm and functions through its cell surface receptors (S1P1-5) and as an intracellular second messenger. Sphingosylphosphorylcholine (SPC) is closely related to S1P and has similar regulatory functions but has not been extensively studied. Both S1P and SPC are able to evoke either stimulatory or inhibitory effects on cancer cells depending on the context. The aim of this thesis work was to study novel regulatory targets of S1P and SPC, which mediate the effects of S1P/SPC signalling on cancer cell behaviour. The investigated targets are the transcription factor hypoxia-inducible factor 1 (HIF-1), the intermediate filament protein vimentin and components of the Hippo signalling pathway. HIF-1 has a central role in cancer biology, as it regulates a multitude of cancer-related genes and is potently activated by intratumoural hypoxia through stabilization of the regulatory subunit HIF-1α. Tumours typically harbour high HIF-1α levels and HIF-1, in turn, facilitates tumour angiogenesis and metastasis and regulates cancer cell metabolism. We found S1P to induce follicular thyroid cancer cell migration in normal oxygen conditions by increasing HIF-1α synthesis and stability and subsequently HIF-1 activity. Vimentin is a central regulator of cell motility and is also commonly over-expressed in cancers. Vimentin filaments form a cytoskeletal network in mesenchymal cells as well as epithelial cancer cells which have gone through epithelial-mesenchymal transition (EMT). Vimentin is heavily involved in cancer cell invasion and gives tumours metastatic potential. We saw both S1P and SPC induce phosphorylation of vimentin monomers and reorganization of the vimentin filament network in breast and anaplastic thyroid cancer cells. We also found vimentin to mediate the anti-migratory effect of S1P/SPC on these cells. The Hippo pathway is a novel signalling cascade which controls cancer-related processes such as cellular proliferation and survival in response to various extracellular signals. The core of the pathway consists of the transcriptional regulators YAP and TAZ, which activate predominantly cancer-promoting genes, and the tumour suppressive kinases Lats1 and Lats2 which inhibit YAP/TAZ. Increased YAP expression and activity has been reported for a wide variety of cancers. We found SPC to regulate Hippo signalling in breast cancer cells in a two-fold manner through effects on phosphorylation status, activity and/or expression of YAP and Lats2. In conclusion, this thesis reveals new details of the signalling function of S1P and SPC and regulation of the central oncogenic factors HIF-1 and vimentin as well as the novel cancer-related pathway Hippo.

Timing of di-ammonium phosphate addition to fermenting chardonnay must : effect on nitrogen utilization, ethyl-carbamate formation, and CAR1 expression by yeast /

The maximum amount of ethyl carbamate (EC), a known animal carcinogen produced by the reaction of urea and ethanol, allowed in alcoholic beverages is regulated by legislation in many countries. Wine yeast produce urea by the metabolism of arginine, the predominant assimilable amino acid in must. This action is due to arginase (encoded by CARl). Regulation of CARl, and other genes in this pathway, is often attributed to a well-documented phenomenon known as nitrogen catabolite repression. The effect of the timing of di-ammonium phosphate (DAP) additions on the nitrogen utilization, regulation of CARl, and EC production was investigated. A correlation was found between the timing of DAP addition and the utilization of nitrogen. When DAP was added earlier in the fermentations, less amino nitrogen and more ammonia nitrogen was sequestered from the media by the cells. It was also seen that early DAP addition led to more total nitrogen being used, with a maximal difference of ~25% between fermentations where no DAP was added versus addition at the start of the fermentation. The effect of the timing ofDAP addition on the expression of CARJ during fermentation was analyzed via northern transfer and the relative levels of CARl expression were determined. The trends in expression can be correlated to the nitrogen data and be used to partially explain differences in EC formation between the treatments. EC was quantified at the end of fermentation by GC/MS. In Montrachet yeast, a significant positive correlation was found between the timing of DAP addition, from early to late, and the final EC concentration m the wine (r = 0.9226). In one of the fermentations, EC levels of 30.5 ppb was foimd when DAP was added at the onset of fermentation. A twofold increase (69.5 ppb) was observed when DAP was added after 75% of the sugars were metabolized. When no DAP was added, the ethyl carbamate levels are comparable at a value of 38 ppb. In contrast, the timing of DAP additions do not affect the level EC produced by the yeast ECU 18 in this manner. The study of additional yeast strains shows that the effect of DAP addition to fermentations is strain dependent. Our results reveal the potential importance of the timing of DAP addition to grape must with respect to EC production, and the regulatory effect of DAP additions on the expression of genes in the pathway for arginine metabolism in certain wine yeast strains.

Relationship between photoperiod, plasma concentration of ionic calcium and the histology of the prolactin-secreting cells of the rostral pars distalis of the pituitary gland, the corpuscles of stannius and the ultimobranchial gland in the goldfish, Carassius auratus L. /

The relationship between photoperiod, plasma concentration of ionic calcium and the histology of the prolactin-secreting cells of the rostral pars distalis of the pituitary gland, the Corpuscles of Stannius and the Ultimobranchial gland were investigated. Neither the plasma concentration of ionic calcium nor histologically apparent prolactin cell activity could be correlated with photoperiod. Some evidence of a photoperiodic effect on both the Corpuscles of Stannius and the Ultimobranchial gland was obtained. The expected reciprocal relationship between the activity of these glands was not obvious at the histological level . Quantitative and qualitative analysis at the light microscope level revealed, however, that the hormone prolactin-secreting eta cells of the rostral pars distalis and the hypocalcin-secreting cells of the Corpuscles of Stannius may be arranged in a lamellar pattern comprized of synchronous bands of cells in like-phase of a secretory cycle consisting of four stages - synthesis, storage, release and reorganization. Such synchronized cell cycles in these glands have not heretofore been described in literature. It is suggested that the maintenance of at least 255? of the cells in any one phase of the cycle ensures a supply of the required hormone at all times.

A possible role for intrinsic calcium buffers in the long- term adaptation of crayfish neurons

Increasing the impulse activity of neurons in vivo over 3 or more days causes a reduction in transmitter release that persists for days or weeks (eg. Mercier and Atwood, 1989). This effect is usually accompanied by decreased synaptic fatigue. These two changes involve presynaptic mechanisms and indicate "long-term adaptation" (LTA) of nerve terminals. Previous experiments have shown that LTA requires extracellular calcium and protein synthesis (eg. Hong and Lnenicka, Soc. Neurosci. Abstr. 17:1322) and appears to involve communication between the cell body and the nerve terminals. The present study examines the possibility that the reduction in transmitter release is caused by an -increase in the calcium buffering ability within the nerve terminals. It examines the responses of adapted and control nerve terminals to exogenously applied calcium buffer, BAPTA-AM, which decreases transmitter release (Robitialle and Charlton, 1992). If LTA increases intrinsic Ca2+-buffering, the membrane permeant form of BAPTA should have less effect on adapted nerve terminals than on controls. Experiments are performed on the phasic abdominal extensor motor neurons of the crayfish, Procambarns clarkii. BAPTA-AM decreases excitatory postsynaptic potentials (EPSP's) of the phasic extensor muscles in a dosedependent manner between 5 and 50 JLM. LTA is elicited by in vivo stimulation at 2.5 Hz for 2-4 h per day over 3 days, which reduces EPSP's by over 50%. Experiments indicate that BAPTA-AM produces no significant change in EPSP reduction in adapted neurons when compared to controls. These results do not support the hypothesis that increased daily activity alters rapid intrinsic calcium buffers, that are able to reduce transmitter output in the same manner as BAPTA.

Cytosolic calcium levels and stress induced y-amino butyrate synthesis in asparagus mesophyll cells

Numerous investigations have demonstrated large increases in y-amino butyrate (GABA) levels in response to a variety of stresses such as touch or cold shock (Wallace et ale 1984) Circumstantial evidence indicating a role of Ca2 + in these increases includes elevated Ca2+ levels in response to touch and cold shock (Knight et ale 1991), and the demonstration of a calmodulin binding domain on glutamate decarboxylase (GAD), the enzyme responsible for GABA synthesis (Baum et al 1993) In the present study the possible role of Ca2+ and calmodulin in stimulation of GAD and subsequent GABA accumulation was examined using asparagus mesophyll cells. Images of cells loaded with the Ca2+ indicator Fluo-3 revealed a rapid and transient increase in cytosolic Ca2+ in response to cold shock. GABA levels increased by 106% within 15 min. of cold shock. This increase was inhibited 70% by the calmodulin antagonist W7, and 42% by the Ca2+ channel blocker La3+.. Artificial elevation of intracellular Ca2+ by the Ca2+ionophore A23187 resulted in an 61% increase in GABA levels. Stimulation of GABA synthesis by ABA resulted in an 83% increase in GABA levels which was inhibited 55% by W7. These results support the hypothesis that cold shock stimulates Ca2+ entry into the cytosol of the cells which results in Ca2+/calmodulin mediated activation of GAD and consequent GABA synthesis.