Characterization of ethanol-induced casein micelle dissociation using a continuous protein monitoring unit.

The effect of ethanol on milk has been shown to be temperature-dependent, with higher ethanol concentrations and temperatures reversibly dissociating casein micelles. This work looked to expand on this knowledge, while also demonstrating the efficiency and precision of a custom-made continuous monitoring unit that combines solutions at defined concentrations and temperatures while measuring various parameters (i.e., absorbance, fluorescence, pressure). Caseins were found to self-associate at moderate ethanol concentrations (i.e., 12-36% vol/vol ethanol); however, they dissociated and remained in the serum at higher ethanol concentrations (≥48% vol/vol) and temperatures (24 and 34°C). Although serum casein content was found to be positively correlated with protein hydrophobicity, the addition of ethanol only increased protein hydrophobicity when the sample was held at high temperatures (34-64°C). Overall, the greatest dissociation of casein micelles was found between 40 and 60% (vol/vol) ethanol concentration at elevated temperatures (≥34°C). At these ethanol concentrations and temperatures, skim milk absorbance was minimized, serum casein content (including ß-casein content) was maximized, and protein hydrophobicity reached a relative maximum.

Prevention of low-temperature gelation in milk protein concentrates by calcium-binding salts.

The objective of this study was to determine the effect of adding low concentrations of calcium-binding salts on the prevention of low-temperature gelation in milk protein concentrates (MPC). The MPC were created by a combination of ultrafiltration and diafiltration, standardized from 14 to 17% (wt/vol) protein content and mixed with one of 5 calcium-binding salts (sodium citrate, sodium hexametaphosphate, sodium polyphosphate, sodium pyrophosphate, and sodium monophosphate) adjusted to a pH of 6.75. The flow properties, apparent viscosity, and gel strength were determined for MPC containing a wide range of calcium-binding salt concentrations. Low-temperature gelation occurred in MPC with 16.0% and higher protein content. Low-temperature gelation at 16.0% protein content was prevented by the addition of any of the 5 salts tested at low concentrations (0.30 mM or less; sodium citrate, sodium hexametaphosphate, sodium polyphosphate, sodium pyrophosphate or sodium monophosphate), with sodium polyphosphate and sodium monophosphate being the most consistent in preventing low-temperature gels. All MPC samples exhibited shear-thinning behavior (n = 0.52-0.72), which increased (lower n values) as the protein content increased and decreased by addition of salt. At concentrations of salt above 1.00 mM, thermally irreversible gels were observed with relative strength dependent on the salt and protein content.

Characterization of high pressure jet-induced fat-protein complexation.

High-pressure-jet (HPJ) processing of various dairy systems has been shown to disrupt fat droplets and casein micelles and cause a strong association between fat and casein proteins. The present work seeks to better describe this association between fat and casein using a model milk formulated from confectionary coating fat (3.6% wt/wt), micellar casein (3.4% wt/wt), and water (93% wt/wt), which was then pasteurized, homogenized, and then either HPJ-treated (400 MPa) or not (non-HPJ-treated, control). Upon ultracentrifugation, fat in the non-HPJ-treated model milk creamed due to its low density. In the HPJ-treated model milk, fat precipitated with protein into a thick bottom layer upon ultracentrifugation, reflecting a strong association between protein and fat. Differential scanning calorimetry (DSC) and time-domain nuclear magnetic resonance of the non-HPJ-treated model milk revealed fat in 2 physical states: (1) fat that is physically similar to the bulk fat and (2) fat that was in smaller droplets (i.e., homogenized) and crystallized at a lower temperature than the bulk fat. In contrast, DSC of HPJ-treated model milks supported the presence of fat in 3 states: (1) fat that is physically similar to the bulk fat, (2) fat in small droplets that required substantial supercooling beyond the non-HPJ-treated model milk to crystallize, and (3) fat in such small domains that it crystallizes in a less stable polymorphic form than the non-HPJ-treated model milk (or does not crystallize at all). The state of fat within the HPJ-treated model milk changed minimally with acidification, indicating that the association is not dependent on the charge on the casein. Cryogenic transmission electron microscopy (Cryo-TEM) of the non-HPJ-treated model milk revealed uniform casein micelles, which likely adsorbed to the surface of fat globules post-homogenization. In contrast, Cryo-TEM of the HPJ-treated model milk revealed a porous protein aggregate that likely had dispersed fat throughout. Together, these results suggest that HPJ treatment causes fat to be entrapped by casein proteins in very small domains.

The effect of high-pressure jet processing on cocoa stability in chocolate milk.

Fat-free chocolate milk formulations containing skim milk, cocoa powder, and sugar were thermally treated and then processed using high-pressure jet (HPJ) technology from 125 to 500 MPa. The rheological properties and stability of HPJ-treated chocolate milks were compared with controls (no HPJ processing) prepared both with and without added κ-carrageenan. As expected, carrageenan-free chocolate milk exhibited immediate phase separation of the cocoa powder, whereas formulations containing κ-carrageenan were stable for 14 d. An increased stability was observed with increasing HPJ processing pressure, with a maximum observed when chocolate milk was processed at 500 MPa. The apparent viscosity at 50 s-1 of HPJ-processed samples increased from ~3 mPa·s to ~9 mPa·s with increasing pressure, and shear-thinning behavior (n < 0.9) was observed for samples processed at HPJ pressures ≥250 MPa. We suggest that HPJ-induced structural changes in casein micelles and new casein-cocoa interactions increased cocoa stability in the chocolate milk. Because casein seemed to be the major component enhancing cocoa stability in HPJ-treated samples, a second study was conducted to determine the effect of additional micellar casein (1, 2, or 4%) and HPJ processing (0-500 MPa) on the stability of fat-free chocolate milk. Formulations with 4% micellar casein processed at 375 and 500 MPa showed no phase separation over a 14-d storage period at 4°C. The addition of micellar casein together with HPJ processing at 500 MPa resulted in a higher apparent viscosity (~17 mPa·s at 50s-1) and more pronounced shear-thinning behavior (n ≤ 0.81) compared with that without added micellar casein. The use of HPJ technology to improve the dispersion stability of cocoa provides the industry with a processing alternative to produce clean-label, yet stable, chocolate milk.

Freezing kinetics and microstructure of ice cream from high-pressure-jet processing of ice cream mix.

The effect of high-pressure-jet (HPJ) processing (0-500 MPa) on low-fat (6% fat) ice cream was studied by evaluating physiochemical properties before freezing, during dynamic freezing, and after hardening. An HPJ treatment ≥400 MPa decreased the density, increased the apparent size of colloidal particles, and altered rheological behavior (increased non-Newtonian behavior and consistency coefficients) of low-fat ice cream mix before freezing. During dynamic freezing, the particle size and consistency coefficient decreased but remained higher in 400 MPa-treated samples vs. non-HPJ-treated controls at the conclusion of freezing. The resulting ice creams (400 and 500 MPa-treated) had similar hardness values (3,372 ± 25 and 3,825 ± 14 g) and increased melting rates (2.91 ± 0.13 and 2.61 ± 0.31 g/min) compared with a control sample containing polysorbate 80 (3,887 ± 2 and 1.62 ± 0.25 g/min). Visualization of ice cream samples using transmission electron microscopy provided evidence of casein micelle and fat droplet disruption by HPJ treatment ≥400 MPa. In the 400 MPa-treated samples, a unique microstructure consisting of dispersed protein congregated around coalesced fat globules likely contributed to the altered physiochemical properties of this ice cream. High-pressure-jet processing can alter the microstructure, rheological properties, and hardness of a low-fat ice cream, and further modification of the formulation and processing parameters may allow the development of products with enhanced properties.

Critical phosphate salt concentrations leading to altered micellar casein structures and functional intermediates.

We investigated the effect of different phosphate salts on the structural integrity of micellar casein (MC) at pH 7.0. With the increase of salt concentration, a reduction in turbidity was observed for the MC solutions, and it was modeled using an exponential decay function. The inflection point of the model was defined as the first critical salt concentration (C*), and it is suggested that the salt concentration initiates the disintegration of MC. For linear polyphosphates, C* decreased with the number of phosphate groups. Apparent viscosity (ηapp) of MC solutions increased with the increase of salt concentration, and they recorded a peak while the turbidity decreased to a minimum. The salt concentration that resulted in the highest ηapp was identified as the second critical salt concentration (C**). It is hypothesized that the interactions among protein species present in the mixtures are at an optimum state at C**. Both C* and C** were found to be dependent on the MC concentration. The work presented herein supports an understanding of the concentration effect of phosphate salts on MC for structuring dairy products.

The association of lipophilic phospholipids with native bovine casein micelles in skim milk: Effect of lactation stage and casein micelle size.

A known biological role of casein micelles is to transport calcium from mother to young and provide amino acids for growth and development. Previous reports demonstrated that modified casein micelles can be used to transport and deliver hydrophobic probes. In this study, the distribution of lipid-soluble phospholipids, including sphingomyelins (SM) and phosphatidylcholines (PC), was quantified in whole raw milk, skim raw milk, and casein micelles of various sizes during early, mid, and late lactation stages. Low-pressure size exclusion chromatography was used to separate casein micelles by size, followed by hydrophobic extraction and liquid chromatography-mass spectrometry for the quantification of PC and SM. Results showed that the SM d18:1/23:0, d18:1/22:0, d18:1/16:0, d16:1/22:0, d16:1/23:0, and d18:1/24:0 and the PC 16:0/18:1, 18:0/18:2, and 16:0/16:0 were dominating candidates appearing in maximum concentration in whole raw milk obtained from late lactation, with 21 to 50% of total SM and 16 to 35% of total PC appearing in skim milk. Of the total SM and PC found in skim milk, 35 to 46% of SM and 22 to 29% of PC were associated with the casein micelle fraction. The highest concentrations of SM d18:1/22:0 (341 ± 17 µg/g of casein protein) and PC 16:0/18:1 (180 ± 20 µg/g of casein protein) were found to be associated with the largest casein micelles (diameter = 149 nm) isolated in milk from late lactation, followed by a decrease in concentration as the casein micelle size decreased.

Effect of high-pressure-jet processing on the viscosity and foaming properties of pasteurized whole milk.

The processing of milk using high-pressure technologies has been shown to dissociate casein micelles, denature whey proteins, and change the appearance and rheological properties of milk. A novel high-pressure processing technology called high-pressure-jet (HPJ) processing is currently being investigated for use in the food industry. Few studies have evaluated the effects of HPJ technology on dairy foods. The present study investigated the physicochemical and foaming properties of homogenized pasteurized whole milk processed at pressures from 0 to 500 MPa using HPJ processing. The apparent particle size exhibited a monomodal distribution in whole milk samples processed up to 125 MPa and a bimodal distribution for samples processed at 250, 375, and 500 MPa. The viscosity increased from approximately 2 to 5 mPa·s when whole milk was processed using HPJ at 375 MPa, and foam expansion increased from approximately 80 to 140% after processing at >125 MPa. Foam stability was limited to pressures in the 375 to 500 MPa range. We hypothesized that the increase in apparent particle size was due to the dissociation of casein micelles into surface-active casein protein monomers, and the formation of casein-casein and casein-fat particles. Ultracentrifugation of samples into 3 milk fractions (supernatant, serum, and precipitate), and subsequent fat and protein analysis on the 3 fractions, showed that a strong interaction between casein proteins and fat triglycerides occurred, evidenced by the increase in fat content associated with the precipitate fraction with increasing pressure. This suggests that stable casein-fat aggregates are formed when whole milk is processed using HPJ at pressure >125 MPa.

The binding of orally dosed hydrophobic active pharmaceutical ingredients to casein micelles in milk.

Casein proteins (αS1-, αS2-, ß- and κ-casein) account for 80% of the total protein content in bovine milk and form casein micelles (average diameter = 130 nm, approximately 1015 micelles/mL). The affinity of native casein micelles with the 3 hydrophobic active pharmaceutical ingredients (API), meloxicam [351.4 g/mol; log P = 3.43; acid dissociation constant (pKa) = 4.08], flunixin (296.2 g/mol; log P = 4.1; pKa = 5.82), and thiabendazole (201.2 g/mol; log P = 2.92; pKa = 4.64), was evaluated in bovine milk collected from dosed Holstein cows. Native casein micelles were separated from raw bovine milk by mild techniques such as ultracentrifugation, diafiltration, isoelectric point precipitation (pH 4.6), and size exclusion chromatography. Acetonitrile extraction of hydrophobic API was then done, followed by quantification using HPLC-UV. For the API or metabolites meloxicam, 5-hyroxy flunixin and 5-hydroxy thiabendazole, 31 ± 3.90, 31 ± 1.3, and 28 ± 0.5% of the content in milk was associated with casein micelles, respectively. Less than ∼5.0% of the recovered hydrophobic API were found in the milk fat fraction, and the remaining ∼65% were associated with the whey/serum fraction. A separate in vitro study showed that 66 ± 6.4% of meloxicam, 29 ± 0.58% of flunixin, 34 ± 0.21% of the metabolite 5-hyroxy flunixin, 50 ± 4.5% of thiabendazole, and 33 ± 3.8% of metabolite 5-hydroxy thiabendazole was found partitioned into casein micelles. Our study supports the hypothesis that casein micelles are native carriers for hydrophobic compounds in bovine milk.

The effect of emulsifying salts on the turbidity of a diluted milk system with varying pH and protein concentration.

Solutions of 10 commonly used emulsifying salts (ES) listed in the Code of Federal Regulations (21CFR133.179) for pasteurized process cheese were tested for their effect on the turbidity of a diluted milk system at different pH and protein concentrations to characterize the conditions that affect micellar structure. Emulsifying salt solutions were made by mixing the ES in a 1-in-20 dilution of water in skim milk ultrafiltrate (3 kDa molecular weight cut-off) to obtain ES concentrations from 0 to 248 mM. Skim milk was added to solutions containing nanopure water, skim milk ultrafiltrate, and a specific ES ranging in concentration from 0 to 248 mM and pH 5, 5.8, 6.8, 7.8, and 8.8. The turbidity of the samples was measured as the optical density at 400 nm immediately after mixing (time, t = 0), after 30 s (t = 30s), and after 30 min (t = 30min). Emulsifying salts were found to cause a decrease in the turbidity of the system, which was modeled using an exponential decay model, where C* represents a threshold salt concentration at which rapid dissociation occurs. At pH values 5.8 and 6.8, the ES caused the greatest decrease in turbidity of the diluted milk system. At pH 5, the ES had the least effect on the turbidity of the system. Sodium hexametaphosphate was found to have the strongest dissociative effect, with a C* value of 0.33 mM for t = 0 at pH 6.8. In contrast, the largest C* value calculated at pH 6.8 was monosodium phosphate at 278.22 mM. Increased time resulted in lower C* values. The model established for this study can be used to predict the dissociation of casein micelles in the presence of various types of ES.

The association of low-molecular-weight hydrophobic compounds with native casein micelles in bovine milk.

The agreed biological function of the casein micelles in milk is to carry minerals (calcium, magnesium, and phosphorus) from mother to young along with amino acids for growth and development. Recently, native and modified casein micelles were used as encapsulating and delivery agents for various hydrophobic low-molecular-weight probes. The ability of modified casein micelles to bind certain probes may derive from the binding affinity of native casein micelles. Hence, a study with milk from single cows was conducted to further elucidate the association of hydrophobic molecules into native casein micelles and further understand their biological function. Hydrophobic and hydrophilic extraction followed by ultraperformance liquid chromatography-high resolution mass spectrometry analysis were performed over protein fractions obtained from size exclusion fractionation of raw skim milk. Hydrophobic compounds, including phosphatidylcholine, lyso-phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, showed strong association exclusively to casein micelles as compared with whey proteins, whereas hydrophilic compounds did not display any preference for their association among milk proteins. Further analysis using liquid chromatography-tandem mass spectrometry detected 42 compounds associated solely with the casein-micelles fraction. Mass fragments in tandem mass spectrometry identified 4 of these compounds as phosphatidylcholine with fatty acid composition of 16:0/18:1, 14:0/16:0, 16:0/16:0, and 18:1/18:0. These results support that transporting low-molecular-weight hydrophobic molecules is also a biological function of the casein micelles in milk.

Aqueous extracts of yerba mate (Ilex paraguariensis) as a natural antimicrobial against Escherichia coli O157:H7 in a microbiological medium and pH 6.0 apple juice.

Ilex paraguariensis is popularly used in the preparation of a tea infusion (yerba mate), most commonly produced and consumed in the South American countries of Uruguay, Paraguay, Argentina, and Brazil. In this study, aqueous extracts of commercial tea, derived from the holly plant species I. paraguariensis were evaluated for their ability to inhibit or inactivate Escherichia coli O157:H7 in a microbiological medium and modified apple juice. Dialyzed, lyophilized aqueous extracts were screened for antimicrobial activity against E. coli O157:H7 strains ATCC 43894 and 'Cider' in tryptic soy broth (TSB) and apple juice (adjusted to pH 6.0 to allow for growth of the bacterium). A mixture of the two strains was used as the inoculum when apple juice was used as the medium. MBCs were determined to be ca. 5 and 10 mg/ml for ATCC 43894 and 'Cider', respectively, in TSB. Higher concentrations of the extract were required to inactivate E. coli O157:H7 in pH-adjusted apple juice. An approximate 4.5-log reduction was observed for E. coli O157:H7 treated with 40 mg/ml extract. It was concluded that aqueous extracts from commercial yerba mate have potential to be used as antimicrobials in foods and beverages against pathogenic E. coli O157:H7.

Effect of glycation on sodium caseinate-stabilized emulsions obtained by ultrasound.

This work explores the potential of high-intensity ultrasound to produce fine-dispersion, long-time-stable, oil-in-water emulsions prepared with native and glycated bovine sodium caseinate (SC). Regardless the ultrasound amplitude and time assayed, the sonicated emulsions of native SC at 0.5 mg/mL had much higher emulsifying activity indexes compared with those emulsions formed by Ultra-Turrax (IKA Werke GmbH & Co., Staufen, Germany) homogenization. Nevertheless, the native SC emulsions were very unstable despite the optimization of parameters such as protein concentration, amplitude of ultrasound wave, and sonication time by using a Box-Behnken design. Early glycation of SC with either galactose, lactose, or 10 kDa dextran substantially improved both emulsifying activity and the stability, whereas at advanced stages of glycation, SC emulsions showed notably reduced emulsifying properties, likely because extensive glycation of SC promoted its polymerization mainly through covalent cross-linking, as was demonstrated by particle size measurements. The increase in particle diameter of glycoconjugates likely affected the diffusion of SC from bulk to the oil-water interface and slowed the reorientation process of the protein at the interface. These findings show that the combined effect of early-stage glycation of SC and high-intensity ultrasound as an emergent technique to form emulsions has the potential to provide improved emulsions that could be used in several food applications.

Isolation of caseins from whey proteins by microfiltration modifying the mineral balance in skim milk.

The objective of this work was to study the effect of different salts and salt concentration on the isolation of casein micelles from bovine raw skim milk by tangential flow microfiltration. Tangential flow microfiltration (0.22 microm) was conducted in a continuous process adding a modified buffer to maintain a constant initial sample volume. This buffer contained calcium chloride (CaCl2), sodium phosphate (Na2HPO4), or potassium citrate (K3C6H5O7) in concentrations ranging from 0 to 100 mM. The concentrations of caseins and whey proteins retained were determined by sodium dodecyl sulfate-PAGE and analyzed using the Scion Image software (Scion Corporation, Frederick, MD). A complete isolation of caseins from whey proteins was achieved using sodium phosphate in the range of 10 to 50 mM and 20 times the initial volume of buffer added. No whey proteins were detected at 50 mM but this was at the expense of low caseins being retained. When lower sodium phosphate concentrations were used, the amount of caseins retained was higher but a small amount of whey proteins were still detected by sodium dodecyl sulfate-PAGE. Among the salts tested, calcium chloride at 50 mM and all volumes of buffer showed the higher retention of casein proteins. The highest casein:whey protein ratio was found at 30 mM CaCl2, but no complete casein micelle isolation was achieved. Potassium citrate was the most ineffective salt because a rapid loss of caseins and whey proteins was observed at all concentrations and with low quantities of buffer added during the filtration process. Our results show the potential of altering the mineral balance in milk for isolation of casein micelles from whey proteins in a continuous tangential flow microfiltration system.

Manufacture of acid gels from skim milk using high-pressure homogenization.

The effect of high-pressure homogenization (HPH) alone or in combination with a thermal treatment (TT) was investigated for the manufacture of acid gels from skim milk. Raw skim milk was subjected to HPH (0 to 350 MPa) or a TT (90 degrees C, 5 min), or both, in the following processing combinations: 1) HPH, 2) HPH followed by TT, 3) TT followed by HPH, 4) TT, and 5) raw milk (control). After treatments, L* (lightness) values were measured, and then skim milk was acidified with 3% glucono-delta-lactone and rheological properties (G' and gelation time), and whey holding capacity was evaluated. Treatments in which HPH and TT were combined showed greater L* values than those in which just HPH was applied. In all treatments, the L* values decreased as the pressure was increased up to 300 MPa with little change afterward. Gelation times were lower when HPH was combined with TT compared with the acid skim milk gels that were just pressure treated. The final G' in gels obtained from skim milk subjected to the combined process (HPH and TT) was greater and pressure-dependent compared with all other gels. A maximum G' (~320 Pa) was observed with skim milk subjected to a combination of thermal processing before or after HPH at 350 MPa. Acid gels obtained from HPH milk at 350 MPa showed a linear decrease in whey holding capacity over time, retaining 20% more whey after centrifugation for 25 min compared with samples treated at lower pressures and all other treatments. Our results suggest that HPH in combination with TT can be used to improve the rheological properties and stability of yogurt, thus decreasing the need for additives.

Solubilized micellar calcium induced low methoxyl-pectin aggregation during milk acidification.

Low methoxyl (LM) pectin was combined with 3-kDa molecular weight cut-off permeates from milk subjected to pH 6.7 to 5 and 7 degrees C or 40 degrees C with the objective of studying the effect of solubilized micellar calcium on viscoelastic properties of LM-pectin-milk mixes. Lowering the pH of skim milk with hydrochloric acid during ultrafiltration gradually promoted permeates to exhibit gel-like behavior when combined with LM-pectin. The onset of the gel-like behavior (G' > 1) occurred at a higher pH when permeates were obtained from milk filtered at 7 degrees C compared with 40 degrees C. As pH value during ultrafiltration approached 5 and regardless of temperature, G' for permeate-pectin mixes approached the same values (approximately 70 Pa) as G' for skim milk-pectin mixes. In all cases G' was highly correlated with free calcium concentration (r > 0.95). The gradual acidification of skim milk-LM-pectin using glucono-delta-lactone, promoted a sharp increase in storage modulus as pH approached 5.2 and a maximum G' increment (DeltaG') at pH approximately 4.9. From pH 4.9 to 4, G' continued to increase but at smaller increments. It was concluded that LM-pectin-casein micelle interaction in milk is a 2-step process: 1) solubilized micellar calcium dependent pectin-pectin interaction as pH approaches 5.0 to 4.9, and 2) pectin-casein micelle interaction in the 5.0-4.9 to 4.0 pH range.