Exchangeability of Colloidal Calcium in Milk with Soluble Calcium

Approximately 40% of the calcium existing in colloidal phase of skimmilk was estimated to be hardly exchanged with the calcium psesent in soluble phase by applying a radioisotopic technique. This type of calcium was designated hard-to-exchange calcium. Hard-to-exchange calcium was absent or nearly zero in calcium caseinate dispersion or colloidal phosphate-free milk, but was present in composite calcium caseinate phosphate dispersion. It is suggested that hard-to-exchange calcium is present in a part of colloidal phosphate portion of casein micelles.     

Change of Salt Distribution in Milk during Frozen Storage and Its Partial Reversion after Thawing

The change of salt distribution of skimmilk during frozen storage at ?7°C and its reversion after thawing were investigated. Losses of ultrafiltrable calcium, inorganic phosphate and citrate were indicated. A part of insolubilized calcium and citrate reversed to a soluble form after thawing, but insolubilized phosphate hardly reversed when the storage period was prolonged. Comparison on the changes in skimmilk and in its dialyzate suggested that the insolubilization of salt constituents in milk due to frozen storage involved the interaction of these constituents with calcium caseinate phosphate complex.     

Changes of Casein Complex during Storage of Sterilized Skim Milk

Two types of sterilized skim milk were prepared; one was HTS–1 milk which was heated at 130°C flashly and the other was HTS–2 milk which was heated at 130~135°C for 75 sec. The changes of casein complex during storage of HTS–1 and HTS–2 milks were examined and compared with those of AUT milk which was heated at 120°C for 15 min. The results obtained are summarized as follows.

(1) Visible sediment was formed in HTS–1 and HTS–2 milks after 8 and 14 months of storage, respectively, while no sediment was observed in AUT miik throughout 15 months of storage. (2) The amount of calcium in the ultracentrifugal wheys of HTS–1 and HTS–2 milks decreased gradually with prolonged storage, while that in the ultracentrifugal whey of AUT milk was kept constant after 1 month of storage. (3) Almost no differences among the three samples were observed in the increments of Ca/N ratio of ultracentrifuged casein complex during storage. (4) The amount of soluble casein increased in AUT milk during storage, but decreased in HTS–1 and HTS–2 milks.

On the basis of the above results, the destabilization of casein complex during storage was discussed.     

Studies on Destabilization of Caseinate Complex during Frozen Storage of Milk

Unpasteurized skim milk was storaged in a frozen state at ?7°C or ?20°C for up to several months. There was no increase of non casein and non protein nitrogens, but a slight increase of free tyrosine and a slight decrease of alkaline phosphatase activity were detected when storage period was prolonged. Destabilization occurred solely in caseinate complex, but non micellar casein appeared to be stable.

The contents of calcium and inorganic phosphate in the caseinate complex separated by ultracentrifugation were increased appreciably after frozen storage. The viscosity characteristics of frozen storaged skim milk was also investigated.

Caseinate complex was ultracentrifugally separated from skim milk before and after frozen storage, and then lyophilized. Skim milk itself was also lyophilized before and after frozen storage. Dispersibility was examined on the reconstituted suspension of the lyophilized samples.

The lyophilized sample from frozen storaged milk was much less dispersible than the lyophilized control sample prepared before frozen storage. However, when lyophilized samples were once resolved with reagents such as urea and potassium oxalate and then dialyzed against fresh milk, stable micelle resulted in both samples prepared before and after frozen storage.

Some reduction of dispersibility occurred during lyophilization and subsequent storage in a dried state in the caseinate complex prepared before frozen storage. This reduction was small when skim milk was lyophilized and stored.     

Properties of Acid Casein and Casein Micelles from Milk Destabilized by Frozen Storage

No difference was found in calcium sensitivity, electrophoretic and optical properties between acid caseins prepared from skimmilk before and after frozen storage (up to 180 days at ?7°C).

Destabilization of casein micelles can not be explained either by the reduction of solvation of the micelles or by the liberation of κ-casein from the micelles. However, when storage period was extended (about six months), splitting of a part of κ- and β-casein from the micelles to soluble form was observed, suggesting a drastic change of structure of the destabilized casein micelles.     

Comparison of conformations of κ-casein,para-κ-casein and glycomacropeptide

The conformation of κ-casein was compared with those of para-κ-casein and glycomacropeptide formed by the cleavage of κ-casein with chymosin. Para-κ-casein is insoluble in water at room temperature, but is slightly soluble in 0.07 M NaCl (pH 7.0) at 3°C. The secondary structure of κ-casein, para-κ-casein and glycomacropeptide was estimated from CD spectra measured at 3°C by the method of Yada and that of Provencher and Glockner. The surface hydrophobicity of these molecules was estimated by the fluorometric method. It was concluded that the secondary structures of para-κ-casein and glycomacropeptide segments were changed slightly by cleavage with chymosin. Para-κ-casein was estimated to have more β-sheet structure than glycomacropeptide. Para-κ-casein had larger hydrophobic regions on the molecular surface compared with the corresponding part in κ-casein.     

Calcium enhanced inactivation of calmodulin dependent protein kinase from synaptosomes

Calcium-calmodulin dependent protein kinase from synaptosomal cytosol rapidly loses activity upon storage at 4°C. In the presence of calcium, the loss of activity is greatly enhanced with only trace levels remaining after two hours. Calcium-calmodulin dependent protein kinase, purified by affinity chromatography on calmodulin-Sepharose, is also quite labile and the loss of enzyme activity in the partially purified preparation is similarly accelerated in the presence of calcium. Removal of calcium improves stability somewhat, whereas calmodulin itself apparently has no protective effect on the enzyme.     

Effects of storage time on the motility,mortality and calcium levels of Atlantic salmon Salmo salar spermatozoa

This study estimates spermatozoa mortality, morphology, motility and intracellular calcium levels in Atlantic salmon Salmo salar milt after prolonged storage. Milt samples were preserved at 4° C for 25 days and then evaluated for mortality. Motility remained high for the first 3 days and the mortality was low during the first 5 days of storage. A decrease of >50% in calcium content was observed after 5 days of storage. When spermatozoa were activated, calcium levels increased >200% in relative fluorescence units (RFU); this rate of increase was lost when the samples were stored for extended periods of time and was only partially manifested in a zero calcium solution. The results suggest that in vitro storage of S. salar spermatozoa at 4° C for a period of 3 days preserves motility and limits mortality to levels similar to those of fresh spermatozoa. This method also maintains intracellular calcium storage critical for spermatozoa performance.     

Effects of hot water treatments and storage temperatures on the ripening and the use of electrical impedance as an index for assessing post-harvest changes in mango fruits

The effects of hot water treatment and storage temperature (4°C, 13°C or 22°C) on the quality and impedance of outer and inner mesocarp of mango were assessed in two experiments during storage, impedance being a potential non‐destructive measure of tissue damage following heat treatment. Fruits were subjected to equivalent heat units at 36.5°C for 60 min plus 46.5°C for 43 min or 46.5°C for 90 min by hot water treatments (hwt) on the assumption of cumulative heat effects and a base temperature of 12–13°C. Fruit reflectance decreased whereas chroma and hue angle increased over storage time and also with increase in storage temperature. The yellow colour increased with a rise in storage temperature in hot water treated mangoes. Soluble solids content of mangoes held at 22°C was highest at 5 days of storage but decreased subsequently over storage time. Impedance of all fruits decreased with increase in frequency, storage temperature and time in store. The impedance of hwt mangoes was lower than that of non‐hwt fruits 8 h after immersion, but recovered almost to control levels on day 5 at 4°C or 13°C, but decreased gradually after 5 days at 13°C. Impedance of all mangoes stored at 22°C decreased continuously during storage. Impedance was higher in the inner mesocarp than outer pulp. Impedance of hwt fruits was poorly correlated with soluble solid content and chroma but well correlated with reflectance of fruit pulp at 22°C. Changes in impedance of mangoes are discussed in relation to physiological and biochemical changes that occur during heat treatment and storage.     

A New Criterion by Which to Distinguish Aspergillus sojae,a Koji-mold,from Related Taxa Producing Echinulate Conidia

Milk powders (commercial skim milk and modified milk powders) were stored at 30°C and 40°C for one month under various water activities (Aw), 0.23~0.82. After storage under Aw 0.57 and 0.80, they turned a dark brown color, and the contents of methionine and lysine had significantly decreased: methionine had been oxidized to methionine sulfoxide and lysine had changed to an unavailable form. The contents of arginine, tyrosine and tryptophan had also decreased, as had the contents of leucine and histidine in the skim milk powder. The tryptic digestibility of the milk powders had markedly decreased, and both the chymotryptic and peptic digestibilities of the skim milk powder had also decreased. The samples lost the property of being clotted by chymosin treatment.

No significant change was observed with casein under these conditions, nor with milk powders under dry conditions and Aw 0.23.     

Calcium dependency of the binding and mitogenicity of phytohemagglutinin: Differentiation between calcium-dependent and independent binding events

The same amount of phytohemagglutinin binds to lymphocytes at 37 °C and at 0 °C. The binding is inhibited by calcium chelators at 37 °C, but not at 0 °C, during the very first minutes of contact between lectin and cells. N-Acetylgalactosamine in high concentrations inhibits binding at both temperatures. The binding at 0 °C is abortive in the sense that it does not result in subsequent DNA synthesis. These findings indicate that the binding of phytohemagglutin (PHA) to the glycoprotein receptor on the cell surface does not in itself activate the immediate transducer of the mitogenic signal. The binding has to be accompanied by some change which involves calcium and occurs very rapidly, but cannot take place at 0 °C, to give a proper mitogenic anchorage of PHA to the cells.     

Factors affecting the measurement of chemiluminescence in stimulated human polymorphonuclear leucocytes

Optimum conditions were established for the generation and measurement of luminoldependent chemiluminescence (CL) in human polymorphonuclear leucocytes (PMNL) stimulated with a variety of particulate and soluble agents. Several factors had a particular influence on the kinetics of CL stimulated by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). Two peaks, both azide-sensitive, were observed at 21°C and 25°C. but these increased in magnitude and merged t o give a single, early peak when the temperature was increased t o 37°C. Pre-exposure of PMNL to a buffer containing calcium was essential for the expression of both phases of fMLP-stimulated CL, while the second peak decreased dramatically if the cells were stored at 4°C for 4 hours before assay. In contrast, storage of PMNL at 4°C for up t o 8 hours in a buffer without divalent cations did not alter the kinetics or magnitude of CL induced by other stimuli, and had the benefit of minimizing the rate of cell aggregation. This study confirms that measurement of luminol-dependent CL in stimulated PMNL is a useful analytical tool, but shows that careful attention t o experimental design is required t o ensure that the observed CL provides a true measure of the parameter under investigation.     

Calcium Transport by Frankia sp. Strain EAN1pec

When incubated at 25°C, N₂-grown cells of Frankia strain EAN1pec actively accumulated calcium, while NH₄Cl-grown cells did not accumulate calcium. When incubated at 0°C, both N₂-grown and NH₄Cl-grown cells did not actively accumulate calcium. Inhibitors of respiration inhibited calcium accumulation by N₂-grown cells at 25°C. Isolated vesicles also accumulated calcium in an energy- and temperature-dependent manner. Two lines of evidence show that Frankia strain EAN1pec has an active calcium extrusion mechanism. First, NH₄Cl-grown cells incubated under deenergizing conditions accumulated calcium. Second, calcium efflux from calcium-loaded cells required an energy source and was blocked by inhibitors. The results of this study indicate that Frankia strain EAN1pec has two systems for calcium transport: a calcium extrusion system and a developmentally regulated calcium uptake system. Received: 1 December 1997 / Accepted: 9 January 1998     

Properties of Chitosanase from Bacillus cereus S1

Chitosanase from Bacillus cereus S1 was purified, and the enzymatic properties were investigated. The molecular weight was estimated to 45,000 on SDS-PAGE. Optimum pH was about 6, and stable pH in the incubation at 40°C for 60 min was 6–11. This chitosanase was stable in alkaline side. Optimum temperature was around 60°C, and enzyme activity was relatively stable below 60°C. The degradations of colloidal chitosan and carboxymethyl cellulose (CMC) were about 30 and 20% relative to the value of soluble chitosan, respectively, but colloidal chitin and crystalline cellulose were not almost hydrolyzed. On the other hand, S1 chitosanase adsorbed on colloidal chitin completely and by about 50% also on crystalline cellulose, in contrast to colloidal chitosan, which it did not adsorb. S1 chitosanase finally hydrolyzed 100% N-deacetylated chitosan (soluble state) to chitobiose (27.2%), chitotriose (40.6%), and chitotetraose (32.2%). In the hydrolysis of various chitooligosaccharides, chitobiose and chitotriose were not hydrolyzed, and chitotetraose was hydrolyzed to chitobiose. Chitobiose and chitotriose were released from chitopentaose and chitohexaose. From this specificity, it was hypothesized that the active site of S1 chitosanase recognized more than two glucosamine residues posited in both sides against splitting point for glucosamine polymer. Received: 8 June 1999 / Accepted: 20 July 1999     

Effect of Water Content on Glass Transition and Protein Aggregation of Whey Protein Powders During Short-Term Storage

The objectives of this study were to investigate the moisture-induced protein aggregation of whey protein powders and to elucidate the relationship of protein stability with respect to water content and glass transition. Three whey protein powder types were studied: whey protein isolate (WPI), whey protein hydrolysates (WPH), and beta-lactoglobulin (BLG). The water sorption isotherms were determined at 23 and 45°C, and they fit the Guggenheim–Andersson–DeBoer (GAB) model well. Glass transition was determined by differential scanning calorimeter (DSC). The heat capacity changes of WPI and BLG during glass transition were small (0.1 to 0.2 Jg⁻¹ °C⁻¹), and the glass transition temperature (T _g) could not be detected for all samples. An increase in water content in the range of 7 to 16% caused a decrease in T _g from 119 down to 75°C for WPI, and a decrease from 93 to 47°C for WPH. Protein aggregation after 2 weeks’ storage was measured by the increase in insoluble aggregates and change in soluble protein fractions. For WPI and BLG, no protein aggregation was observed over the range of 0 to 85% RH, whereas for WPH, ∼50% of proteins became insoluble after storage at 23°C and 85% RH or at 45°C and ≥73% RH, caused mainly by the formation of intermolecular disulfide bonds. This suggests that, at increased water content, a decrease in the T _g of whey protein powders results in a dramatic increase in the mobility of protein molecules, leading to protein aggregation in short-term storage.     

Effect of Heat Treatment on Changes in Acid Phosphatase Activity during Tomato Ripening

Acid phosphatase (APase) activity of mature green tomatoes decreased during storage at 22°C. Heat-treated tomatoes stored at 33°C showed a rapid decrease in APase activity, and failed to ripen. When heat-treated tomatoes were transferred to 22°C storage, APase activity increased. For most of the storage period (Day 10 excepted), activity of APase II was higher than that of APase I.

Changes in the level of APase II during the storage were estimated using rocket immunoelectrophoresis. Decrease in the specific activity of APase II was observed by the rocket height during storage at 22 and 33°C it declined more rapidly at 33°C. The specific activity of this APase increased when the heat-treated tomatoes were transferred to 22°C storage.     

Calcium and the release from dormancy of freshwater sponge gemmules.

Divalent cations (Zn, Mn, Ba, Sr) inhibit the development of dormant gemmules of the freshwater sponge Spongilla lacustris. This inhibition is overcome by calcium which can be interpreted to mean that this divalent ion is essential for germination (cell division) in this system. Inhibitory divalent cations have different effective concentrations which indicate differing binding affinities for sites which may normally bind calcium. Ethylene glycol bis(β-aminoethyl ether)N,N-tetraacetic acid does not effect gemmule development at 15°C but stimulates it at 4°C, indicating that a dislocation of endogenous calcium stimulates release from dormancy. Magnesium will only partially substitute for calcium in overcoming divalent cation inhibition implying a different specificity for this ion in gemule development. Calcium is also indicated as being essential for hatching (cell motility) in this system.     

Calcium uptake by isolated rat intestinal cells

Intestinal cells were isolated by a combination of mechanical and enzymatic means, and their calcium uptake was assayed by a rapid filtration procedure. Calcium uptake was a time- and concentration-dependent process that was markedly elevated at 25 and 37°C, as compared to 0°C. Cells isolated from rat duodenum exhibited higher uptakes than cells from jejunum, which in turn took up more calcium than cells from the ileurn. Duodenal cells from vitamin D-deficient animals took up less calcium than cells from vitamin D-replete cells. In vivo vitamin D repletion with 1,25-dihydroxyvitamin D₃ raised calcium uptake by duodenal cells from treated animals toward that of cells from replete rats. Furthermore, calcium uptake by duodenal cells from vitamin D-deficient animals approximated that of ileal cells from replete rats. These findings with isolated cells parallel prior findings of tissue calcium transport and suggest that cellular calcium uptake may be related to the saturable component of intestinal calcium absorption. Isolated intestinal cells may therefore constitute one experimental model for the study of transcellular calcium transport.     

The effect of soluble alginate and calcium on β-galactosidase activity produced by the thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus imb3

Since it has previously been demonstrated that ethanol production by the thermotolerant yeast strain, Kluyveromyces marxianus IMB3 is more efficient in calcium alginate-based immobilization systems during growth on lactose-containing media, it was decided to examine the separate effects of soluble alginate and free calcium on the β-galactosidase activity produced by that organism. It was found that the presence of Ca²⁺ significantly increased the thermal stability of the activity at 45?°C, although the pH?and temperature optima remained the same in the presence and absence of that cation. It was also found that the presence of 2% (w/v) sodium alginate (soluble) had a very limited positive effect on the thermal stability of the enzyme at 45?°C, although it was found that activity was very significantly stimulated at that temperature. The activity was found to have an enhanced thermal stability at 30?°C in the presence of sodium alginate. The presence of sodium alginate in assay mixtures had no significant effect on the Km of the activity for the substrate o-nitrophenyl-β-D-galactoside. The results observed in the presence of either free calcium or soluble alginate may at least partially explain enhanced ethanol production by this microorganism in alginate-based immobilization systems.     

Immobilization and characterization of cellulase on concanavalin A (Con A)-layered calcium alginate beads

Cellulase has been immobilized on hybrid concanavalin A (Con A)-layered calcium alginate–starch beads. Immobilized cellulase retained about 82% of its activity. Con A was extracted from jack bean and the obtained crude protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The immobilized beads showed high mechanical and storage stability; immobilized cellulase retained 100% and 85% activity at 4°C and 30°C, respectively, over one month. The immobilized cellulase retained about 70% of its activity after five cycles of use. The immobilized cellulase retained 70% activity after 120-min exposure to 60°C, whereas the soluble form only retained about 20%, showing that immobilization improved thermal stability. Surface morphology and elemental analysis of immobilized cellulase were examined using scanning electron microscope equipped with energy-dispersive X-ray. Based on the enzyme stability and reuse, this method of immobilization is both convenient and cheap.