Synthesis of α-Adenosine 5′-Monophosphate

The distributions of protein, calcium and inorganic phosphate among casein micelles of skimmilk before and after frozen storage were investigated by gel filtration through Sephadex G-200 with 6.6 m urea solution as eluant.

The results showed that the native casein micelles were fractionated into two fractions. Fast eluting fraction contained large amount of calcium and inorganic phosphorus. Slow eluting fraction contained calcium but was essentially free of inorganic phosphorus. Frozen storage caused the increase of proportion of the fast eluting fraction accompanying the increase of calcium and inorganic phosphorus contents in it. It is suggested that the salt linkage newly formed during frozen storage causes the increase of proportion of the fast eluting fraction.     

Sedimentation Property of Casein Micelles from Milk Destabilized by Frozen Storage

Casein micelles destabilized in milk under frozen storage was compared with stable casein micelles before frozen storage by means of analytical ultracentrifugation.

The stable micelles disaggregated with urea-containing buffer showed a single homogeneous peak of ~0.9S, while a fast sedimenting subpeak, in addition to the major peak of ~0.9 S, appeared in the destabilized micelles. However, any difference was not found between the stable and destabilized caseins when they were analyzed after removal of calcium. It is suggested that a new type of association is formed, possibly through salt linkages, in the casein system destabilized under frozen storage. Sedimentation pattern of calcium paracaseinate phosphate complex in urea-containing buffer suggests that the destabilization of the micelles by rennin does not involve the change of salt linkages.     

Denaturation of Soybean Protein by Freezing

When a solution of soybean acid-precipitated or 11S protein was frozen and stored at ?1 to ?5°C, the protein became partially insoluble after thawing. Ultracentrifugation and disc-electrophoresis of freeze-stored 11S protein solution after removing insoluble components revealed that new components which may be aggregates or associates of the 11S component were formed. When concentrated and stored at 5°C, disc-electrophoresis of 11S component showed that associates were formed. Mercaptoethanol could dissolve the insoluble protein and also convert the associates to the original 11S component. NEM–11S was not insolubilized by frozen storage at ?5°C or storage at 5°C after being concentrated. From these facts it can be concluded that denaturation of soybean protein by freezing may be caused by intermolecular reactions through S-S bonds as a result of concentration by freezing. This may suggest a mechanism of the formation of sponge-like texture in kori-tofu which is made by frozen storage of soybean curd for 15 to 20 days at ?1 to ?3°C.     

Separation of the epithelial and mesenchymal components of the newt limb regenerate with salt.

After incubation of isolated forelimb regenerates of Notophthalmus (Triturus) viridescens at all developmental stages for 60 minutes at 37 degrees C in a salt medium containing 111 mM sodium chloride, 5.6 mM potassium chloride and 100 mM sodium phosphate buffer at pH 7.5, the wound epithelium of each regenerate was removed intact from its underlying mesenchymal component. The suggestion is made that the salt medium is an effective epithelial-mesenchymal separating agent due to a combination of its hypertonicity, high ionic strength and the fact that the medium precipitates calcium as calcium phosphate. Attempts to dissect away the epithelium from the mesenchyme after incubation of isolated regenerates in sodium phosphate containing 1% or 3% Difco 1:250 trypsin, 10 mM EDTA or 150 units collagenase/ml medium were unsuccessful. Epidermis of adult newt forelimb skin was removed only after extended incubation of the forelimbs in the salt medium for three hours at 37 degrees C or after freezing isolated forelimbs in buffer and subsequent thawing.     

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.     

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.     

Effect of salts on the activity of carrot phosphofructokinase

Phosphofructokinase (EC 2.7.1.11) from carrot roots was activated by a number of salts. Increase in salt concentration beyond the optimum generally led to a decrease in enzyme activity. Salts of the multivalent anions sulfate and phosphate were very effective activators and inhibitors. Potassium acetate and potassium succinate were also activators. Potassium tartrate and potassium citrate produced a small stimulation at low concentration but with further increase they became inhibitory. The results suggested that the salt effect was largely due to anions rather than cations. Salts such as NaCl, KCl, and in particular potassium phosphate, relieved the inhibition of carrot phosphofructokinase by phosphoenolpyruvate. KCl and potassium phosphate also reversed the inhibition of carrot phosphofructokinase by citrate. The possible significance of these observations in the regulation of glycolysis and carbohydrate metabolism, and in salt respiration is discussed.     

Effects of low-molecular-weight fractions (LMWF) from milk, egg yolk, and seminal plasma on freezability of bovine spermatozoa

The effects of the dialyzable fractions from bovine seminal plasma, egg yolk, and milk and of two buffer systems (TEST and sodium citrate) on post-thaw sperm motility were studied. Each basic salt solution was used in the experimental design. These solutions were used as extender systems in combination with egg yolk and glycerol. After collection, semen samples were extended (1:20), cooled to 5 degrees C in 1.5 hr, and frozen in 0.5-cc French straws after 3 hr of equilibration. Post-thaw samples were assayed for percentage of motile cells immediately after thawing and after 4 hr of incubation at room temperature (22 degrees C). Egg yolk (25%) provided the same protection as did the combination of colloidal material present in the skim milk-yolk extenders. The use of TEST as a buffer provided significantly higher (P less than 0.01) sperm post-thaw motility than milk salts or Na citrate. Sperm survival in extenders containing high concentrations of seminal plasma and/or egg yolk salts was significantly lower (P less than 0.01). Spermatozoa frozen in the presence of 6% glycerol resulted in sperm motility significantly (P less than 0.05) higher than that of spermatozoa frozen with 3% glycerol. However, no difference was observed between these two concentrations when TEST solution was used.     

Effect of cooling and rewarming rates on glycerolated human erythrocytes.

Human erythrocytes suspended in a sodium-free buffered salt solution containing glycerol in 1 m concentration (1 part of packed cells to 4 parts buffered salt solution) were frozen by slow, moderately rapid, or very rapid cooling to various subzero C temperatures. The frozen specimens, after a 5-min storage period at a given temperature, were thawed at low, moderately high, or very high rates. The hemolysis in the frozen and thawed samples was measured by a colorimetric determination of the hemoglobin released from the damaged cells. At ?10 °C, the highest freezing temperature employed, nearly 100% recovery of intact erythrocytes was obtained irrespective of the cooling and rewarming conditions. The extent of the hemolysis after exposure to lower freezing temperatures depended upon the cooling and rewarming conditions. Moderately rapid and very rapid freezing to, and thawing from temperatures below ?40 °C permitted significantly higher recoveries of intact cells than the other freezing/ thawing combinations. In the temperature range ?15 to ?30 °C the combination slow cooling and slow rewarming afforded maximum protection. Very rapid freezing/ slow thawing was the most damaging combination throughout the entire freezing range. The results were interpreted in part by a conventional two-factor analysis, lower cooling rates allowing concentrated salts to determine hemolysis, higher cooling rates destroying the cells by intracellular freezing. Apparent anomalies were explained in terms of a generalized “thermal/osmotic” shock according to which the erythrocytes were subject to greater hemolysis the higher the rates of cooling and/or warming.     

A comparative study of the effects of freezing and frozen storage on intact and demembranated bull spermatozoa

The damage caused to bull sperm by freezing and thawing them without cryoprotectants was assessed in both intact and membrane-extracted cells. Preparations of membrane-extracted cells were produced by treating the sperm with 0.1% Triton X-100 and motility was restored with exogenously applied ATP and Mg²⁺. Motile demembranated sperm showed no detectable reduction in motility after freezing and thawing. In contrast, when intact cells where subjected to freezing and thawing they lost all motility. These damaged cells were also restored to motility when exogenous ATP and Mg²⁺ were added to the sperm mixture. Apparently freezing and thawing sperm cells causes damage to the plasma membrane which permits ATP and Mg²⁺ to freely enter or leave the cells, but does not damage the components of the sperm cell which generate motility.The effects of storage temperature on frozen demembranated sperm were also explored. Sperm held at ?20 °C showed marked structural changes and progressively decreased motility after prolonged storage. When sperm were frozen at ?20 °C the mitochondrial structures were completely lost after 48 to 72 hr and ATP caused the disintegration of the flagellum rather than initiating motility. Sperm which were frozen at ?76 °C retained motility after short periods of storage, but showed a significant decline in motility when thawed after 8 days. Demembranated sperm which were kept frozen at ?196 °C showed no significant loss of motility when thawed after 1 year of storage.     

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.     

An equilibrium thermodynamic model of the sequestration of calcium phosphate by casein micelles and its application to the calculation of the partition of salts in milk

An equilibrium thermodynamic model of the interaction of calcium, phosphate and casein in milk is described in which the micellar calcium phosphate is assumed to be in the form of calcium phosphate nanoclusters. A generalized empirical formula for the nanocluster is used to define the molar ratios of small ions (Ca, Mg, P_i and citrate) to a casein phosphorylated sequence (phosphate centre, PC). From this model, a method of calculating the partition of milk salts into diffusible and non-diffusible fractions is obtained. No arbitrary assumptions are made, no fitting of adjustable parameters is done and the PCs in the caseins are defined by inspection of their primary structures. In addition to the salt partition, the mole fractions of the individual caseins not complexed to the calcium phosphate through one or more of their PCs are computed and a generic stability rule for milks is derived. The use of the model is illustrated by calculations of the partition of salts in a standard milk and by comparison with experimental data on the partition of salts in the milk of individual cows. The generic stability rule is applied to the individual milks to determine whether the micellar calcium phosphate is thermodynamically stable. According to the calculations, compositions that might lead to pathological calcification in the lumen of the mammary gland were seldom found in primiparous healthy cows in early or mid lactation but occurred more often in multiparous animals, in late lactation and during mastitic infection.Abbreviations ACP amorphous calcium phosphate - Cit citrate - CN casein - CPN calcium phosphate nanocluster - DCPD dicalcium phosphate dihydrate - HA hydroxyapatite - IAP ion activity product - MCP micellar calcium phosphate - MWCO molecular weight cut-off - OCP octacalcium phosphate - PC phosphate centre - TCC tricalcium citrate     

Caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate

Artificial casein micelles were prepared by adding 30 mM calcium, 22 mM phosphate and 10 mM citrate to sodium caseinate solutions, and the content of the casein aggregates cross-linked by colloidal calcium phosphate was determined by high-performance gel chromatography on a TSK-GEL G4000SW column in the presence of 6 M urea. The content of the casein aggregates cross-linked by colloidal calcium phosphate in artificial whole casein micelles was 48% of total casein, and their relative casein composition determined by high-performance ion-exchange chromatography was 53.1% for alpha s1-casein, 15.8% for alpha s2-casein, 31.1% for beta-casein and 0% for kappa-casein. The order of cross-linking by colloidal calcium phosphate agreed with that of the ester phosphate content of casein constituents. The content of the casein aggregates cross-linked by colloidal calcium phosphate was higher in alpha s1-kappa-casein micelles than in beta-kappa-casein micelles. kappa- and gamma-caseins and dephosphorylated alpha s1-casein were not cross-linked by colloidal calcium phosphate. Although kappa-casein was not cross-linked, chemically phosphorylated kappa-casein, of which the average phosphate content was 8.5 per molecule, was cross-linked. It is concluded that caseins are cross-linked through their ester phosphate groups by colloidal calcium phosphate.     

Growth inhibition ofChlorella pyrenoidosa produced by sodium dihydrogen phosphate and its reversal by calcium

Summary The growth ofChlorella pyrenoidosa in a special medium based on the critical concentrations of nutrients for autotrophic growth has been shown to be stimulated more by chloride with the sodium salt than with the potassium salt, more by sulphate in the presence of sodium than in the presence of potassium, and to be inhibited by sodium dihydrogen phosphate and not by potassium dihydrogen phosphate. Furthermore, it has been found that calcium reversed the growth inhibition produced by sodium dihydrogen phosphate, and that strontium only partially substituted for calcium.     

The minerals of milk

The salt of milk constitutes a small part of milk (8-9 g.L(-1)); this fraction contains calcium, magnesium, sodium and potassium for the main cations and inorganic phosphate, citrate and chloride for the main anions. In milk, these ions are more or less associated between themselves and with proteins. Depending on the type of ion, they are diffusible (cases of sodium, potassium and chloride) or partially associated with casein molecules (cases of calcium, magnesium, phosphate and citrate), to form large colloidal particles called casein micelles. Today, our knowledge and understanding concerning this fraction is relatively complete. In this review, the different models explaining (i) the nature and distribution of these minerals (especially calcium phosphate) in both fractions of milk and (ii) their behaviour in different physico-chemical conditions, are discussed.     

Fertility of stallion semen processed, frozen and thawed by a new procedure

The fertility of frozen-thawed and fresh semen from three stallions was compared in a trial using a randomized block design and 90 mares for 108 cycles. Semen was collected every third day, diluted to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium, and centrifuged. The cells were resuspended at 700 x 10(6) progressively motile sperm/1.0 ml of added lactose-EDTA-egg yolk extender containing 4% glycerol, packaged by placing 0.55 ml into polypropylene straws, and frozen. Semen was thawed by immersion in 75 degrees C water for 10 sec. All of the 43 ejaculates collected were frozen, but 21 were discarded because progressive sperm motility was <35% immediately after thawing or <40% after 30 min of incubation at 37 degrees C. semen from the same stallions was collected daily for inseminations with fresh semen. Semen containing 200 x 10(6) progressively motile sperm was added to 10 ml of heated skimmilk extender. Mares were inseminated daily starting on the third day of estrus or when a >/=4-cm follicle was detected, whichever came later, and continuing through the end of estrus or for nine days. Based on palpation per rectum on day 50 postovulation, the pregnancy rates from inseminations during one estrus were 50, 56 and 61% with frozen semen and 67, 67 and 61% with fresh semen (P>0.05) from the three stallions, respectively. Thus, mean pregnancy rate with frozen semen was 86% of the rate attained with fresh semen.     

Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage

The ability of two strains of Lactobacillus acidophilus, CRL 640 and CRL 800, to survive and retain their biological activities under frozen storage was determined. Freezing and thawing, as well as frozen storage, damaged the cell membrane, rendering the microorganisms sensitive to sodium chloride and bile salts. Both lactic acid production and proteolytic activity were depressed after 21 days at -20 degreesC, whereas beta-galactosidase activity per cell unit was increased. Cell injury was partially overcome after repair in a salt-rich medium. Copyright 1998 Academic Press.     

Calcium-Induced Alteration of Cellular Morphology Affecting the Resistance of Lactobacillus acidophilus to Freezing

Examination of factors affecting the resistance of Lactobacillus acidophilus NCFM culture concentrates to freeze injury induced during frozen storage at -20°C revealed that calcium supplementation of the growth medium contributed to the storage stability of cells prepared in static culture. Culture concentrates of L. acidophilus NCFM were prepared from cells propagated in MRS broth or MRS broth supplemented with 0.1% calcium carbonate, calcium chloride, or calcium phosphate. After 28 days of frozen storage at -20°C, concentrated cells (3.2 × 10⁹ colony-forming units per ml) prepared from MRS broth cultures showed an 84% reduction in viable cells. Of the remaining viable cells, 88% were sublethally injured and unable to form colonies on MRS agar supplemented with 0.15% bile. Cells prepared in calcium-supplemented MRS broths demonstrated more resistance to frozen storage. Viability and injury losses in the frozen concentrates were limited to 10 to 39% and 3 to 23%, respectively. It was observed that calcium supplementation of MRS medium resulted in a morphological transition of L. acidophilus NCFM from filamentous to bacilloid rods, and the bacilloid cells were more resistant to freezing and storage at conventional freezer temperatures. The results suggest that the morphology of the L. acidophilus cell may be an important consideration in the preparation of freeze-stable culture concentrates.     

Injury and Death of Streptococcus lactis due to Freezing and Frozen Storage

Cells of Streptococcus lactis were harvested in the early stationary phase, washed, and resuspended in either skim milk (10% nonfat milk) or buffered distilled water (0.0003 m dipotassium phosphate, pH 7.2). Samples of each suspension were frozen and stored at -20 C for intervals up to 28 days. Colony counts of the frozen culture were made using lactic agar and a “restricted” lactic agar medium (Tryptone reduced to 0.5%) to determine injury and death. Death was determined by the difference in plate counts on lactic agar before and after freezing. Injured cells were determined by the difference in plate counts on the two plating media. Greatest injury of the cells occurred during early stages of frozen storage and decreased with time, and death continuously increased. Injury and death were more pronounced when cells were frozen in water than when frozen in 10% nonfat milk solids. Certain cultures survived better when frozen rapidly, whereas with others survival was greater when freezing was slow. Successive freezing, thawing, and propagation of the culture gradually eliminated cells which showed injury by freezing.