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.     

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.     

Changes of the Casein Complex in Sterilized Concentrated Skim Milk during Storage

Visible and chemical changes of sterilized concentrated skim milk prepared laboratorially were studied over a long period of storage, and on the basis of the results the changes of the calcium-caseinate-phosphate complex were discussed.

The results obtained are summarized as follows. (1) The formation of visible sediment and “whey off” were observed on the 50th day of storage. The increment of the viscosity was not so large during storage. (2) The remarkable increase of the soluble casein was observed, and its proportion to total casein amounted to about 2/3 on the 120th day of storage. Only small amount of calcium bound to the soluble casein. (3) Each amount of calcium and inorganic phosphorus in the ultracentrifugal whey decreased gradually, and that of magnesium increased during storage. (4) The ratios of Ca/N and P/N in the casein complex increased extremely after storage.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

Streptococcus cremoris was cultivated for 7 days at 30°C in sterilized skim milk or in the sterilized 10% solution of dry skim milk. This skim milk culture was divided into precipitate and supernatant by centrifugation. The absorbancy at 280 mμ of the supernatant prepared from the skim milk culture of S. cremoris was higher than that of the control supernatant.

Casein prepared from the skim milk culture of S. cremoris was less hydrolyzed by rennet than control casein at pH 7.0.

According to the free boundary electrophoretic analysis of the treated casein in m/10 veronal buffer of pH 8.5 containing urea, α-casein seemed to be hydrolyzed by S. cremoris but β-casein did with more difficulty.     

Studies on the Changes of the Milk Casein by Various Treatments

A study has been made with the changes occurred in the milk casein during frozen storage. The flocculated protein formed during storage of frozen milk was resolved in 4 ~ 5 peaks in free-boundary electrophoretic pattern, using veronal buffer containing 7 m urea.

The flocculated protein was mainly casein. It may be considered that portions of casein have undergone some modification or denaturation during the destabilization process.

Significant increase has occurred in the relative proportion of β-casein in the casein remaining in supernatant after removal of flocculate, indicating that the partial fractionation of casein component occurred as a result of flocculation.     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

In sterilized skim milk or sterilized 10% solution of dry skim milk at 120°C for 15 min, Lactobacillus bulgaricus, Lactobacillus helveticus and Streptococcus lactis were cultivated for 7 days at given temperature.

Both NCN (non casein type nitrogen) content and pH in each culture of lactic acid bacteria were rapidly decreased until 2 days after cultivation, But NCN content increased and the pH change got small after 3 days cultivation.

Caseins prepared from the cultures of these three kinds of lactic acid bacteria were examined electrophoretically. From the results of electrophoresis of these caseins, we have concluded that α-casein could be hydrolyzed by these lactic acid bacteria. And, it seemed that β-casein could not be hydrolyzed by these lactic acid bacteria.

Rennet easily hydrolyzed casein treated with L. bulgaricus and L. helveticus but hardly hydrolyzed that treated with S. lactis compared with control-casein. Caseins treated with L. bulgaricus and L. helveticus were hydrolyzed easier than control-casein.

Particle weights of caseins prepared from fermented milk by lactic acid bacteria, Streptococcus cremoris, Streptococcus lactis, Lactobacillus bulgaricus and Lactobacillus helveticus, and of hydrolyzed casein by rennet, trypsin or pepsin were measured according to the light scattering experiment.

Particle weights of various treated caseins were larger than that of raw native casein at both pH 7.0 and 12.0. And the heating caused the polymerization of casein to large particle.     

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.     

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.     

Hydrolysis by Indigenous Plasmin: Consequences for Enzymatic Cross-Linking and Acid-Induced Gel Formation of Non-Micellar Casein

Casein is a group of milk proteins with high nutritional value, and the exploitation of its techno-functional potentials has been investigated for decades. In this study, acid casein powder was dissolved in 0.1 mol/L phosphate buffers with different pH, resulting in casein solutions with pH 5.9, 6.6 and 7.3. During preparation and storage (40 °C) of the samples, casein hydrolysis was observed in size exclusion chromatography and gel electrophoresis. The degree of hydrolysis increased with increasing pH, and treatment of casein with commercial plasmin resulted in similar polypeptides, suggesting that the hydrolysis was caused by residual indigenous plasmin present in the acid casein powder. Most polypeptides could be cross-linked by microbial transglutaminase, except for one particular fraction which appeared at constant intensity in the chromatograms. The stiffness of acid-induced gels as determined in small amplitude oscillatory shear rheology decreased with increasing degree of hydrolysis, and was also lower for cross-linked samples when the preceding casein hydrolysis was more pronounced. Enzymatic cross-linking increased the resistance of casein against plasmin-related hydrolysis, presumably because of the resulting lysine modification. However, one particular fraction of polypeptides was released by hydrolysis in spite of cross-linking, suggesting that they did not contain lysine residues that are susceptible for mTGase. The results indicate that plasmin-related hydrolysis should be taken into account for the application of acid casein or sodium caseinate as additive in food design.

     

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.     

Level and distribution of progesterone in bovine milk in relation to storage in the mammary gland.

The study investigated the effect of the place of storage of milk in the mammary gland on progesterone concentrations in whole milk, skim milk and milk fat. Skim milk, milk fat and whole milk progesterone concentrations were lower (P < 0.05) in milk fractions obtained from the cisternal part of the mammary gland compared to those in the milk fractions from the alveoli. Mean milk fat concentrations did not mirror the changes in the mean skim milk, milk fat and whole milk progesterone concentrations. After administration of oxytocin, milk fat concentrations rose significantly (P < 0.01). At the same time, skim milk and milk fat progesterone concentrations remained unchanged (P > 0.05), compared to those in the milk fractions of alveolar origin, obtained before oxytocin administration. Skim milk and whole milk progesterone concentrations were higher (P < 0.01) in composite milk and in milk samples collected 1 h after milking, compared to concentrations in the milk samples collected before morning milking and at 3, 5, 7 and 9 h after milking. The results suggest that defatted milk, milk fat and whole milk progesterone concentrations were affected by the place of storage of the milk in the mammary gland, and that this effect is independent of milk fat content. Time of milk sampling, not the milk fat concentration, in relation to time of milking, was a critical factor in determining skim milk, milk fat and whole milk progesterone. The study also revealed that the concentrations of the other milk components, somatic cell count, lactose and protein were affected by the place of storage of milk in the mammary gland.     

Effects of Additives on the Survival of Lactic Streptococci in Frozen Storage

Three single-strain cultures, Streptococcus lactis C₂, S. cremoris R₁, and S. diacetilactis DRC₂, were frozen and stored in skim milk, in skim milk containing apple juice, and in skim milk containing one of the following additives: glycerol (10%, v/v), dimethyl sulfoxide (10%, v/v), l-malic acid (0.5 and 2.0%, w/v), acetamide (0.5 and 2.0%, w/v), or succinimide (0.5 and 2.0%, w/v). Cultures were frozen and stored at -23.3 C, frozen and stored at -196 C in liquid nitrogen, or frozen at -196 C and stored at -23.3 C. Cultures frozen and stored at -196 C in liquid nitrogen gave the greatest recovery of viable cells. The number of cells surviving after storage at -23.3 C was greater when the cells had been frozen in liquid N₂ than when they had been frozen at -23.3 C. All strains stored at -23.3 C showed a decrease in numbers of surviving cells; additives, particularly l-malic acid and apple juice, were advantageous in preserving the viability of the S. lactis C₂ and S. cremoris R₁ strains, but had little or no effect on the survival of S. diacetilactis DRC₂. l-Malic acid and apple juice stimulated acid production for all cultures in activity tests following incubation after thawing, whereas glycerol and dimethyl sulfoxide retarded its development.     

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.     

Enrichment of n-Alkane Assimilation-Deficient Mutants of Candida Yeast by Synergistic Effect of Nystatin and Pyrrolnitrin

In order to clarify further the relationship between the heat stability of casein micelles and the formation of soluble casein upon heating concentrated milk, the effect of formaldehyde was examined. The addition of formaldehyde up to 20 mM markedly increased the heat stability of both concentrated skim milk and concentrated whey protein-free (WPF) milk. The stabilizing effect of formaldehyde was greater for concentrated skim milk than for concentrated WPF milk. The addition of formaldehyde depressed the formation of soluble casein upon heating concentrated milk. No soluble casein was formed on the addition of 20 mM formaldehyde. It was confirmed by Sephadex G-200 gel filtration in the presence of 6.6 M urea that cross-links among the casein components were formed in heated concentrated WPF milk containing formaldehyde. These facts suggest that formaldehyde may introduce cross-links among the casein components and prevent the formation of soluble casein accompanying the release of K-casein from micelles, thus stabilizing the casein micelles.     

Effect of cooling of equine spermatozoa before freezing on post-thaw motility: preliminary results

The ability to ship cooled stallion semen to a facility that specializes in cryopreservation of spermatozoa would permit stallions to remain at home while their semen is cryopreserved at facilities having the equipment and expertise to freeze the semen properly. To accomplish this goal, methods must be developed to freeze cooled shipped semen. Three experiments were conducted to determine the most appropriate spermatozoal extender, package, time of centrifugation, spermatozoal concentration and length of time after collection that spermatozoa can be cooled before cryopreservation. In the first experiment, spermatozoa were centrifuged to remove seminal plasma, resuspended in either a skim milk extender, a skim milk-egg yolk-sugar extender or a skim milk-egg yolk-salt extender, cooled to 5 degreesC and frozen in 0.5- or 2.5-mL straws either 2.5 or 24 h after cooling. Samples frozen 2.5 h after cooling had higher percentages of progressively motile (PM) spermatozoa (27%) than samples frozen 24 h after cooling (10%; P < 0.05). Samples frozen 2.5 h after cooling in skim milk extenders containing egg yolk had higher percentages of PM spermatozoa (average 32%) than did spermatozoa frozen in extender containing skim milk alone (average 16%; P < 0.05). The percentages of PM spermatozoa frozen in 0.5- or 2.5-mL straws were similar (21 and 28%, respectively; P > 0.05). In the second experiment, spermatozoa were centrifuged to remove seminal plasma either before (25 degreesC) or after cooling (5 degreesC), and spermatozoa were frozen after being cooled to 5 degreesC for 2, 6, or 12 h. The percentages of PM spermatozoa were higher (P < 0.05) for spermatozoa centrifuged before cooling (30%) than for spermatozoa centrifuged after cooling (19%). Spermatozoa centrifuged at 25 degreesC then cooled for 12 h to 5 degreesC had higher (P < 0.05) post-thaw progressive motility (23%) compared to spermatozoa cooled for 12 h and centrifuged at 5 degreesC (13%). In the third experiment, spermatozoa were centrifuged for seminal plasma removal, resuspended at spermatozoal concentrations of 50,250 or 500 x 10(6)/mL, cooled to 5 degreesC for 12 h and then frozen. Samples with spermatozoa packaged at 50 or 250 x 10(6)/mL had higher (P < 0.05 percentages of PM spermatozoa (25 and 23%) after freezing than did samples packaged at 500 x 10(6) spermatozoa/mL (17%). We recommend that semen be centrifuged at 25 degreesC to remove seminal plasma, suspended to 250 x 10(6) spermatozoa/ml and held at 5 degreesC for 12 h prior to freezing.     

Physical and Textural Properties of Mayonnaise Prepared Using Virgin Coconut Oil/Fish Oil Blend

Physical and textural properties of mayonnaise prepared using virgin coconut oil (VCO)/fish oil (FO) blends at different ratios were examined in comparison with that prepared using soybean oil (SO) as affected by storage time (30 days). At day 0, sample prepared with SO showed the highest L*, a*, and b* values among all the samples, whereas the lowest values were noticeable for VCO containing sample. At day 30 of storage, decreases in L*, and b* values of all mayonnaise samples were observed (p < 0.05). However, a* values were increased at day 30 of storage (p < 0.05). For texture analysis, highest firmness, consistency and cohesiveness were obtained for the sample containing SO. Increasing levels of FO in VCO/FO samples increased the firmness, consistency and cohesiveness. For all the samples, loss modulus (G″) values were lower than G′. After 30 days of storage, all samples demonstrated slight decreases in G′ and viscosity than freshly prepared mayonnaise (day 0). When the sample containing VCO/FO (90:10) blend was further characterized, slight difference was observed in microscopic structure and droplet size distribution before and after storage of 30 days. Increase in droplet size was noticeable because of coalescence after the storage. Overall, type of oil used for preparation of mayonnaise as well as storage time affected the physical properties including textural and rheological properties of mayonnaise.

     

Storage of lyophilized cultures of Lactobacillus bulgaricus under different relative humidities and atmospheres

The viability of lyophilized cultures of Lactobacillus bulgaricus in skim milk, during storage at different temperatures, relative humidities, and atmospheres was investigated. Survival was greatest at 11% relative humidity and at 5°C. Indirect and direct evidence is presented supporting the hypothesis that membrane damage occurs during storage. Experiments on the lipid composition of the cell membrane demonstrate that changes occur with time that are probably the result of oxidation. A study on the lipid composition of the cell membrane by gas chromatography showed that the unsaturated/saturated fatty acid index changes with time during storage.     

Long-term preservation of anammox bacteria

Deposit of useful microorganisms in culture collections requires long-term preservation and successful reactivation techniques. The goal of this study was to develop a simple preservation protocol for the long-term storage and reactivation of the anammox biomass. To achieve this, anammox biomass was frozen or lyophilized at two different freezing temperatures (−60°C and in liquid nitrogen (−200°C)) in skim milk media (with and without glycerol), and the reactivation of anammox activity was monitored after a 4-month storage period. Of the different preservation treatments tested, only anammox biomass preserved via freezing in liquid nitrogen followed by lyophilization in skim milk media without glycerol achieved stoichiometric ratios for the anammox reaction similar to the biomass in both the parent bioreactor and in the freshly harvested control treatment. A freezing temperature of −60°C alone, or in conjunction with lyophilization, resulted in the partial recovery of the anammox bacteria, with an equal mixture of anammox and nitrifying bacteria in the reactivated biomass. To our knowledge, this is the first report of the successful reactivation of anammox biomass preserved via sub-zero freezing and/or lyophilization. The simple preservation protocol developed from this study could be beneficial to accelerate the integration of anammox-based processes into current treatment systems through a highly efficient starting anammox biomass.     

Dietary leucine improves whole-body insulin sensitivity independent of body fat in diet-induced obese Sprague–Dawley rats

Dairy foods and dietary calcium (Ca) are potential regulators of body weight and insulin sensitivity. The specific components of dairy responsible for these actions are not known but may include leucine. Our objective was to determine the effect of dietary protein (casein, skim milk or leucine) and Ca level [low, 0.67% (LC) or high, 2.4% (HC)] on adiposity and insulin sensitivity. Obesity was induced in Sprague–Dawley rats with a 6-week period of high-fat/high-sucrose (HFHS) diet intake. Rats were randomly assigned to one of six HFHS diets for 8 weeks where dietary protein was provided as casein, skim milk or casein enriched with leucine, and contained either LC or HC. Body composition via dual-energy x-ray absorptiometry and insulin sensitivity via euglycemic–hyperinsulinemic clamp were measured. Microarray was used to assess gene expression in liver and skeletal muscle. Rats fed leucine had greater insulin sensitivity than those fed casein or skim milk (P<.05). Dietary protein differentially regulated hepatic and skeletal muscle genes associated with insulin, peroxisome proliferator-activated receptor and mammalian target of rapamycin pathways. Specifically, two key genes responsible for insulin sensitivity, hepatic insulin receptor substrate (IRS) and protein kinase B (Akt), were altered in hepatic tissue in response to leucine. Rats fed skim milk and leucine diets had lower body weight compared to those fed casein (P<.05). HC reduced fat mass compared to LC (P<.05). While skim milk and leucine both reduced fat mass, only leucine improved insulin sensitivity compared to casein. Differential expression of genes such as IRS and Akt may be responsible for changes in insulin sensitivity in obese rats.     

The preservation of infective spores of Octosporea muscaedomesticae in Phormia regina,of Nosema algerae in Anopheles stephensi,and of Nosema whitei in Tribolium castaneum by lyophilization

Infective spores of three species of microsporidia were subjected to the lyophilization process by employing varying media as cryoprotectants. The infectivity of the lyophilized spores was then tested against a standard fresh spore preparation in the appropriate host insect. Spores of Octosporea muscaedomesticae served as an experimental model and were rendered noninfective in host Phormia regina (Calliphoridae: Diptera) after lyophilization with the following cryoprotective agents: skim milk (12%), ascorbic acid (5%) combined with thiourea (5%), glycerol (10%), mesoinositol (5%), and equine serum. Spores of O. muscaedomesticae lyophilized or vacuum-dried in 50% sucrose as well as in the hosts' tissues remained highly infective for as long as 2 years at a dose of 10⁶ spores/fly and a trial length of 12 days. At a dose of 5 × 10⁴ spores/fly there was a slight decrease in infectivity of the spores which had been lyophilized in the host's abdomen after a 2-year storage period compared with that of fresh, nonlyophilized spores. Naked spores of Nosema algerae suspended in 50% sucrose and lyophilized produced infection in 50% of the host population of Anopheles stephensi (Culicidae: Diptera) compared with 70% infection produced by fresh non-lyophilized spores. Spores of Nosema whitei lyophilized within its host larva Tribolium castaneum (Tenebrionidae: Coleoptera) remained 100% infective at a dose of 5 × 10⁵ spores/gram diet. It is concluded that an aqueous solution of 50% sucrose and/or the host's tissues are excellent protectants for the cryogenic or vacuum-drying process of the above-named spores, and their protective function may apply also to other microsporidian species.