Influence of Cross-linked Waxy Maize Starch on the Aggregation Behavior of Casein Micelles During Acid-induced Gelation

The influence of cross-linked waxy maize starch on the aggregation behavior of casein micelles was investigated using a combination of physico-chemical techniques. Milk was homogenized at two different temperatures (55 and 65 °C) and then heated at 95 °C for 5 min in a pilot scale system. The possible interactions between modified starch and milk proteins during lactic acid fermentation were evaluated. While 1% starch did not show differences in the whey protein complexes formed during heating compared to milk with no starch (as measured by size exclusion chromatography), a higher (2.5%) concentration of starch clearly showed an increased amount of heat-induced whey protein aggregates. The gelation pH also increased significantly with 2.5% starch compared to that of the control samples. The storage modulus (G′) increased with increasing levels of starch, and confocal microscopy confirmed that the microstructure of the casein gels was altered by the presence of modified starch. Milk-starch mixtures preheated and homogenized at 55 or 65 °C exhibited similar physico-chemical behavior during acidification. The results suggested a lack of interaction between starch granules and casein micelles during acidification, and scanning electron microscopy images collected with a self-assembled monolayer technique also confirmed that starch granules were not attached to milk caseins but only embedded in the protein gel matrix.     

Enzymatic method to determine biomass in skim milk whey permeate medium

Summary In this study, a new technique for biomass determination was developed. Proteases were used to separate cells from the culture broth in skim milk whey permeate medium and the solubilization of casein was achieved 99% by elastase, 95% by pepsin and 90% by trypsin. Elastase was found to be efficient and reproducible for determining biomass in skim milk whey permeate medium.     

Adsorption of Staphylococcal Bacteriophage by Milk Proteins

Propagation of homologous bacteriophage in a culture of Staphylococcus aureus (1:1 ratio of phage to bacteria) in Trypticase Soy Broth (TSB) and in skim milk indicated more activity of phage in TSB. Early lysis of bacteria in skim milk followed by a pronounced rise in bacterial population suggested that staphylococcal phages were being inactivated by milk. Titration of phages from skim milk, whey, and TSB indicated about 90% adsorption of phages by acid- and heat-precipitable proteins of skim milk, whereas numbers recovered from whey were quite comparable to those recovered from TSB. Reducing the pH from 6.5 to 4.0 increased the percentage of phages recoverable from skim milk from 10 to 56%. Apparently, the changes in electrical charges on the casein micelles at this low pH were responsible for release of many phages from their complex with casein.     

Model process for removal of caseins from milk of transgenic animals

We describe a method for selective removal of caseins from milk. The method was developed as a model for transgenic milk processing. Raw cow milk spiked with nonmilk proteins was chosen as the model to resemble transgenic animal milk containing recombinant proteins. The most important elements of the process are (1) "deconstruction" of casein micelles in milk by destroying their Ca(2+) core using a chelating agent (EDTA), thus freeing any protein that might be entrapped in casein aggregates, and (2) "reconstruction" of micelles by providing them with a new Ca(2+) core, thus precipitating them away from the whey proteins, and the protein of interest. Calcium phosphate particles (CAP) were used to reform the disrupted casein micelles. The crystal clear supernatant fraction generated by this method provided >90% recovery and 6- to 13-fold concentration of the desired protein. Product-rich supernatant contained no detectable casein residues, as silver-stained SDS-PAGE and Western blot analyses demonstrated.     

High pressure-induced changes in bovine milk proteins: a review

High pressure (HP)-induced changes in the proteins of bovine milk have become an area of considerable research interest in recent years; as a result, there is now a detailed understanding of the effects of HP on casein micelles and whey proteins. HP treatment at pressures >400 or >100 MPa denatures the two most abundant whey proteins, alpha-lactalbumin (alpha-la) and beta-lactoglobulin (beta-lg), respectively. The majority of denatured beta-lg in HP-treated milk associates with the casein micelles, although some denatured beta-lg remains in the serum phase or is attached to the milk fat globule membrane; HP-denatured alpha-la is also associated with the milk fat globules. Casein micelles are disrupted on treatment at pressures >200 MPa; the rate and extent of micellar disruption increases with pressure and is probably due to the increased solubility of calcium phosphate with increasing pressure. On prolonged treatment at 250-300 MPa, reassociation of micellar fragments occurs through hydrophobic bonding; this process does not occur at a pressure >300 MPa, leading to considerably smaller micelles in such milk. As a result of HP-induced changes, the size, number, hydration, composition and light-scattering properties of casein micelles in HP-treated milk differ considerably from those in untreated milk.     

The use of starch and skim milk in the regeneration of Bacillus amylotiquefaciens and Bacillus subtilis protoplasts

The addition of starch or skim milk to bovine serum albumin-containing regeneration medium enhances the regeneration of both Bacillus amyloliquefaciens and B. subtilis protoplasts. With B. amyloliquefaciens starch could replace bovine serum albumin; with B. subtilis either starch or skim milk could be used instead of bovine serum albumin. The maximum regeneration frequency achieved was 10% with B. amyloliquefaciens and 290% with B. subtilis .     

Preparation and characterization of milk calcium salts by using casein phosphopeptide

Milk calcium salt solution was prepared by the following procedures using casein phosphopeptides (CPP). To CPP solution, 1 M citric acid, 1 M CaCl2 and 1 M K2HPO4 were added with stirring, while adjusting the pH to 6.7. The prepared solution was left for 12 hr at 25 degrees C and then used for subsequent experiments, or lyophilized. The concentrations of organic phosphate of CPP, calcium, inorganic phosphate, and citrate in the typical milk salt solution were 7, 30, 22, and 10 mM, respectively, which were close to those in bovine milk. The lyophilized sample was easily dissolved in water. No crystal structure of hydroxyapatite was shown in the lyophilized milk calcium salts by X-ray diffraction analysis, although the pattern of KCl crystal was observed. The X-ray diffraction pattern of commercial whey mineral, which was prepared by precipitation at alkaline pH from rennet whey, was similar to that of hydroxyapatite. It was confirmed by high-performance gel chromatographic analysis that the form of calcium phosphate in the milk calcium salts was similar to that of casein micelles.     

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.     

Disulphide bonds in casein micelle from milk

Mammary epithelial cells synthesised and secreted caseins, the major milk proteins in most mammals, as large aggregates called micelles into the alveolar lumen they surround. We investigated the implication of the highly conserved cysteine(s) of kappa-casein in disulphide bond formation in casein micelles from several species. Dimers were found in all milks studied, confirming previous observation in ruminants. More importantly, the study of interchain disulphide bridges in mouse and rat casein micelles revealed that any casein possessing a cysteine is engaged in disulphide bond interchange; these species express four or five cysteine-containing caseins, respectively. We found that the main rodent caseins form both homo- and heterodimers. Additionally, disulphide bond formation among milk proteins was specific since the interaction of the caseins with cysteine-containing whey proteins was not observed in native casein micelles.     

A new innovative process to produce lactose-reduced skim milk

The research field for applications for lactose hydrolysis has been investigated for some decades. Lactose intolerance, improvement for technical processing of solutions containing lactose and utilisation of lactose in whey are main topics in development of biotechnological processes. In this article, the establishment of a hollow fiber membrane reactor process for enzymatic lactose hydrolysis is reported. Mesophilic beta-galactosidases were circulated abluminally during luminal flow of skim milk. The main problem, microorganisms growth in the enzyme solution, was minimised by sterile filtration and UV irradiation. In order to characterise the process parameters, such as skim milk concentration, enzyme activity and flow rates were varied. In comparison to a batch process, enzyme activity could be used longer and enzyme rest into the product should not occur. Furthermore, the three-dimensional separation of the substrate from the enzyme solution minimise blocking and washing out effects, which restrict processes with immobilised enzymes. A conversion rate of 78.11% was achieved at a skim milk flow rate of 9.9l h(-1), enzyme activity of 120 Uml(-1) and a temperature of 23+/-2 degrees C in a hollow fiber reactor with a membrane area of 4.9 m2.     

Influences of milk components on biofilm formation of Cronobacter spp. (Enterobacter sakazakii)

Aim:  To determine the critical component(s) of skim milk for biofilm formation of Cronobacter species.
Methods and Results:  Biofilm forming ability of 72 Cronobacter strains in skim milk preparation was assayed by crystal violet staining. The results revealed that whey protein and casein are more important determinants of skim milk for biofilm formation than lactose, although there was a wide variation in biofilm forming ability. Biofilm structure and capsular material of six strains exhibiting different biofilm forming ability was investigated via electron microscopes. Scanning electron microscopy showed visually that while the strong biofilm formers (E27B, FSM 30 and 2·82) resulted in almost complete coagulation of skim milk, the weak biofilm formers (55, FSM 290 and 2·84) caused less coagulation. No capsule was clearly delineated in transmission electron micrographs of either strong or weak biofilm formers.
Conclusion:  These results indicate that, for biofilm formation of Cronobacter species in skim milk, nitrogen source is probably a more important determinant than carbohydrate, and that strong biofilm formers are responsible for substantial coagulation of skim milk.
Significance and Impact of the Study:  This study provides information for better understanding of the underlying mechanisms by which Cronobacter species form biofilm in infant formula milk.     

Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 degrees C

A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen was diluted with PBS or defined milk protein extender and was either stored directly or 90% of seminal plasma was removed by centrifugation and replaced by defined milk protein extender before storage. In both experiments, eight stallions were available for semen collections. Motility, velocity and membrane integrity of spermatozoa were determined by CASA immediately after semen processing and after 24, 48 and 72 h of storage at 5 degrees C. Total motility after 24 h of storage was lowest in semen diluted with PBS (p<0.05 versus all extenders). At 48 and 72 h, motility of spermatozoa in defined milk protein extender was significantly (p<0.05) higher than in PBS or skim milk extender. Velocity of spermatozoa after storage was highest in defined milk protein extender. Membrane integrity after storage was significantly (p<0.05) lower in semen diluted with PBS than in semen diluted with both extenders. Addition of NAC was without effect on the examined parameters. Centrifugation further increased the percentage of motile and membrane-intact spermatozoa in the defined milk protein extender (p<0.05). Velocity of spermatozoa in this extender was not negatively affected by centrifugation.     

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.     

Influence of Molecular Weight of Chitosan on Interaction with Casein

The process of complex formation of casein from skimmed milk and purified casein with chitosan of different molecular weights was studied. It was shown that at pH 6.3 casein micelles and parts of whey proteins coagulated with positively charged chitosan molecules with molecular weights of 45.3, 25.4, 7.7 and 1.5 kDa. As a result of ionic interaction of chitosan with skimmed milk proteins the yield of target product reached 90–92%. It consisted of all forms of casein: α-casein, β-casein, κ-casein and small amount of whey proteins.     

Proteome mapping of human skim milk proteins in term and preterm milk

The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. However, relatively little is known about the expression of the low abundance proteins that are present in human milk because of the technical difficulties associated with their detection. We used a combination of electrophoretic techniques, ProteoMiner treatment, and two-dimensional liquid chromatography to examine the proteome of human skim milk expressed between 7 and 28 days postpartum by healthy term mothers and identified 415 in a pooled milk sample. Of these, 261 were found in human skim milk for the first time, greatly expanding our understanding of the human skim milk proteome. The majority of the proteins identified were involved in either the immune response (24%) or in cellular (28%) or protein (16%) metabolism. We also used iTRAQ analysis to examine the effects of premature delivery on milk protein composition. Differences in protein expression between pooled milk from mothers delivering at term (38-41 weeks gestation) and preterm (28-32 weeks gestation) were investigated, with 55 proteins found to be differentially expressed with at least 90% confidence. Twenty-eight proteins were present at higher levels in preterm milk, and 27 were present at higher levels in term milk.     

The replacement of skim milk by dried microbial cells and whey in milk diets for calves

In two experiments, the performance and health of calves fed on diets containing dried microbial cells (DMC) was monitored. In the first experiment 32 seven-day-old British Friesian bull calves, reared on a conventional early weaning system to 8 weeks of age, were given milk replacers in which 5, 10 and 20% spray-dried DMC and whey replaced 14–55% of skim milk protein. All DMC diets contained 36 g/kg of non-nucleic acid nitrogen. In Experiment 2, 38 two-day-old Ayrshire bull calves were fed for 63 days solely on one of two milk diets containing 0 or 10% DMC at 5 levels of intake designed to produce body gains of 0–1 kg/day. While the inclusion of up to 10% DMC in both experiments had little effect on performance or health, 20% DMC was associated with severe diarrhoea, loss of appetite and growth retardation, and impaired clotting of the milk in vitro. In Experiment 2 the response, in gain, by calves given increasing intakes of milk dry matter was linear and was unaffected by the type of diet offered.It was concluded that when levels of DMC in excess of 10% inclusion are used in association with maize starch, deleterious effects on performance are likely.     

A Novel Method for Separation of Caseins from Milk by Phosphates Precipitation

Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze–thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock.     

Isolation of recombinant proteins from milk.

Milk is a complex bio-colloid which presents some unique problems for the protein isolation chemist, but the majority of the processing criteria for purifying recombinant proteins are the same as with any complex biological mixture. The casein micelles and fat globules behave as separate phases; they prevent filtration of the milk and interfere with the usual separation methods. The usual first step is to centrifuge the milk to remove the fat and precipitate the casein micelles with low pH or precipitating agents. Some recombinant proteins may associate to some degree with the micelles which may necessitate solubilizing them with chelating agents. If the majority of the product protein associates with either the fat or micelles, this can be used to advantage. Once the casein micelles have been removed or disrupted, the clarified milk can be processed by the usual separation methods. There also are proteases in milk which can degrade recombinant proteins. The greatest advantage of producing recombinant proteins in milk is the high concentration which can be obtained. The high levels of product protein can alleviate many problems associated with the application of classical purification strategies to transgenic milk proteins.     

Composition and ultrastructure of calcium phosphate-citrate complexes in bovine milk systems

The present studies show that the colloidal calcium phosphate of cow's milk has a (Ca + Mg)/P_i ratio of 1.67 (± 0.10; n = 22) and contains citrate, Mg and Zn at molar ratios to Ca averaging 0.05, 0.03 and 0.003, respectively. The composition of the natural colloidal phosphate of milk is similar to the precipitates formed by neutralization of ultrafiltrates obtained from acidified milks, and to that of the calcium phosphate-enriched fraction produced by extensive enzymic hydrolysis of the casein micelles in milk. Examination by electron microscopy of these artificial preparations of milk calcium phosphate revealed in both a very fine and uniform substructure which consisted of granules having an average, true diameter of approx. 2.5 nm. The size and shape of these tiny granules closely resemble the morphologies reported for the colloidal phosphate particles in native casein micelles, as well as for the subunits of amorphous calcium phosphate observed during calcification in other biological systems such as mitochondria and bone.     

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.