Available lysine,protein digestibility and lactulose in commercial infant formulas

Available lysine, in vitro protein digestibility and lactulose values were determined in 23 commercial infant formulas. The mean available lysine content of the formulas based on dairy proteins was 66.7±9.5 mg g⁻¹ protein, similar to that of human milk, while that of soy based formulas was considerably lower (45.0±8.3 mg g⁻¹ protein). In vitro protein digestibility values ranged 85.5–88.9% for soy-based formulas and 90.5–98.3% for formulas based on dairy proteins. Formulas based on milk enriched with whey had higher lactulose content than those based on cow's milk. However, all values were below the limit of 600 mg L⁻¹ recommended for UHT milk.     

Effect of composition and antioxidants on the oxidative stability of fluid milk supplemented with an algae oil emulsion

The oxidative stability of an algal oil emulsion dispersed in water, or fluid milk of varying fat contents, was assessed from measurements of lipid hydroperoxide and propanal concentration. All of the milk samples, independent of their milk fat content, were stable compared to the aqueous samples. The extent of oxidation was unaffected when sodium azide (200 ppm) was added to inhibit microbial growth. Added iron (100 ppm) accelerated the oxidation rate in the aqueous samples, but had no effect on the milk samples. The antioxidant properties of milk were ascribed to the iron binding of casein. Added protein antioxidants (0.8 wt%) [i.e. sodium caseinate, whey protein isolate (WPI) and thermally denatured WPI] had minimal effects whereas EDTA and ascorbic acid (160 ppm) were effective antioxidants.     

Economic aspects of cheese making as influenced by whey processing options

Economic consequences of the cheese making process are illustrated through several sample calculations concerning processing of whey in relation to cheese making throughput and several whey processing alternatives. Small cheese plants with daily milk throughput of approximately 100 000 kg cannot economically justify the capital for water removal equipment. For small plants that have to convert whey to a dry product, alternatives include pre-concentrating with a reverse osmosis unit or a small plate evaporator and drying on a double roller dryer. The economics are evaluated at several price levels. At the upper scale of cheese plant size (2–3 million kg d⁻¹ of milk), the investment for whey processing is about half the total investment. Cash flows are calculated for electricity, natural gas and whey powder prices. Increased investment for further processing into whey protein concentrate and dried whey solubles or lactose is evaluated at several price levels.     

Immunochromatographic lateral-flow test strip for the rapid detection of added bovine rennet whey in milk and milk powder

An immunochromatographic lateral-flow test dipstick test was developed for the fast detection of bovine rennet whey in liquid milk and milk powder. The test is based on the binding of casein glycomacropeptide (cGMP) by two specific anti-bovine κ-casein monoclonal antibodies and has a visual detection limit of around 15 ng mL^?1 for cGMP and 1% (v/v) for rennet whey in milk using standards and spiked samples. The dipstick performance was evaluated in a collaborative trial using skim milk powder and raw, pasteurized and UHT milk. The suitability of the dipstick was demonstrated in comparison with gel permeation-high performance liquid chromotography and a colorimetric method, by analysing 60 raw milk samples collected from farms in Brazil. The dipstick results correlated well with the HPLC results and were more reliable than those obtained with the colorimetric method. The dipstick test correctly identified all raw milk samples with a rennet whey content above 4%.     

Pepsin treatment of whey proteins under high pressure produces hypoallergenic hydrolysates

BALB/c mice were used to assess the ability of a whey protein hydrolysate obtained by pepsin treatment under high pressure (400 MPa, 37 °C, 30 min, HWP), to induce anaphylaxis, antibody production and cytokine responses in comparison with the whey protein isolate (WP) from which it is derived. HWP did not contain intact allergens and 50% of its peptides ranged between 10 and 3 kDa. Challenge with HWP did not induce clinical signs, body temperature changes or release of mast cell proteinase-1 in mice sensitized to WP. Immunization of mice with HWP did not produce WP-specific antibodies or allergic reactions upon HWP or WP challenge and thus, it can be considered hypoallergenic. However, HWP stimulated Th2 responses in splenocytes from sensitized mice. These characteristics make HWP a good candidate to be used in the management of milk allergy in diagnosed patients or to induce tolerance to whey proteins.Industrial relevanceHydrolysis with pepsin under high pressure produces in minutes a whey protein hydrolysate that complies with the health claims of the European guidelines on infant and follow-on formulas related to the reduction of risk to allergy to milk proteins. This process constitutes an alternative to the exhaustive enzymatic hydrolysis treatments used in the processing of hypoallergenic formulas that release short peptides and free amino acids to adversely affect organoleptic properties and technological value.     

Purification of angiotensin I-converting enzyme inhibitory peptides and antihypertensive effect of milk produced by protease-facilitated lactic fermentation

Fresh low-fat milk was fermented with five mixed lactic acid bacteria for up to 30 h at 42 °C. A protease, prozyme 6, was added 5 h after the beginning of fermentation. The whey was separated from the fermented milk and freeze-dried. As the fermentation time extended to 30 h, soluble protein content increased from 30.9 to 195.9 mg g⁻¹, free amino acid content increased from 2.8 to 192.8 mg g⁻¹, peptide content increased from 6.4 to 402.8 mg g⁻¹ and γ-aminobutyric acid (GABA) increased from 0 to 80.6 mg 100 g⁻¹, while inhibition of angiotensin I-converting enzyme (ACE) increased as indicated by a decrease of IC₅₀ from 1.18 to 0.24 mg mL⁻¹, respectively. The amino acid sequences of two ACE inhibitory peptides were Gly–Thr–Trp and Gly–Val–Trp, of which the IC₅₀ values were 464.4 and 240.0 μm, respectively. The systolic blood pressure and diastolic blood pressure of spontaneously hypertensive rat (SHR) were reduced 22 and 21.5 mm Hg, respectively, after 8 weeks of oral administration of diluted whey (peptide concentration 5 mg mL⁻¹) from the 30 h fermentation.     

Influence of processing conditions on reducing γ-aminobutyric acid content during fortified milk production

The work studied the effects of processing conditions on the γ-aminobutyric acid (GABA) loss during fortified milk production. Bovine milk or their proteins/lactose fractions (0.66% whey protein and 2.6% casein or 4.9% lactose, w/v) containing 0.05–1.0% added γ-aminobutyric acid (w/w, based on bulk milk or these fractions) were subjected to a simulated milk technological process as following the sequential preheating (25–60 °C), homogenization (0–20 MPa), and pasteurization (62 °C/30 min, 72 °C/15 s, 95 °C/5 min, and 138 °C/2 s) or their unit processes to treat GABA. The resulting samples were characterized through GABA and lactose concentrations under various processing conditions. The amine and carboxyl groups and the structural characteristics of the resulting protein (lactose) were also examined through their concentrations (for lactose) and mass/spectral analyses, respectively. The results showed that the increase in temperature significantly promoted a reduction in GABA content. Whey protein fractions than caseins were primarily responsible for inducing GABA, whereas lactose had no remarkable effect on it. The rationale for GABA reduction is potential reactions with milk proteins/lactose, which preliminarily confirmed by the measurement of protein modification and lactose mass spectrometry.     

Antihypertensive activity of milk fermented by Enterococcus faecalis strains isolated from raw milk

A total of 231 microorganisms were isolated from raw cow milk samples and the angiotensin-converting enzyme-inhibitory (ACEI) activity of the resultant fermented milk produced with the isolated microorganisms was assayed. Forty-six of these microorganisms were selected on the basis of high ACEI activity. Four Enterococcus faecalis strains stood out as producers of fermented milk with potent ACEI activity (IC₅₀ (the protein concentration that inhibits 50% of ACE activity): 34–59 μg mL⁻¹). Single doses (5 mL kg⁻¹) of the whey fraction obtained from these fermented milk samples were administered to spontaneously hypertensive rats (SHR) and to normotensive Wistar-Kyoto (WKY) rats in order to investigate their possible antihypertensive activity. Highly significant decreases in the systolic blood pressure (SBP) and in the diastolic blood pressure (DBP) were observed when the fermented milk was administered to SHR. Nevertheless, the fermented milk did not modify the SBP and the DBP of the WKY rats. Raw cow milk is an excellent source of wild lactic acid bacteria able to produce fermented milk with antihypertensive activity and antihypertensive activity of milk fermented by Enterococcus faecalis strains was associated with peptides different from Ile-Pro-Pro and Val-Pro-Pro.     

Formation of soluble protein complexes and yoghurt properties influenced by the addition of whey protein concentrate

The effects of whey protein concentrate (WPC) on the formation of soluble protein complexes and yoghurt texture were evaluated. Skim milk (SM) and skim milk enriched with 1% WPC (SM + 1%WPC) or 2% WPC (SM + 2%WPC) were left unheated or heated and then made into yoghurt gels. Yoghurt prepared from heated SM + 2%WPC had significantly higher storage modulus, water holding capacity and firmness values and a denser microstructure than those prepared only from skim milk. Electrophoretic analysis of the milk showed that the level of β-lactoglobulin and κ-casein in the serum phase increased with increasing WPC concentration, indicating that the content of disulfide-linked β-lactoglobulin and κ-casein was higher in SM + 2%WPC than in SM, suggesting that more soluble protein complexes had been formed. Consequently, yoghurt prepared from heated SM enriched with WPC may have more bonds and more protein complexes in the protein network than yoghurt prepared only from SM, thus resulting in firmer gels.Practical applicationsYoghurt, one of the most popular fermented milk products, is of high economic importance to the dairy industry worldwide. In particular, high-protein yoghurt, such as Greek-style or set-type yoghurt, has been driving its ongoing popularity over recent years. In current industrial production of high-protein yoghurt, protein fortification and heat treatment of milk are two of the most important processing parameters affecting yoghurt texture. Whey protein concentrate has been added to milk to reduce whey separation and to increase the firmness of the yoghurt. From a technological point of view, the interaction of the denatured whey proteins with casein micelles or with κ-casein in the serum phases is regarded as responsible for obtaining a good yoghurt structure. The present research has shown that it is possible to produce yoghurt with a range of textural properties by precisely controlling the rate of whey protein fortification during its manufacture. Therefore, this study provides a better understanding of the effect of WPC fortification and aims to extend this insight for the production of good-quality yoghurt.     

Effect of chemical phosphorylation on solubility of buffalo milk proteins

Buffalo milk proteins (casein, co-precipitate or whey protein concentrate) were phosphorylated with phosphorus oxychloride (POCl₃) at three different pH values (5.0, 7.0 and 9.0). The solubilities of phosphorylated milk proteins were examined over the pH range 3.0–9.0 in water and ionic (0.1 m NaCl or 10–70 mm Ca²⁺) systems. The solubilities of buffalo milk proteins decreased at pH 3.0, while there was an increase in the solubilities of casein and co-precipitate near their isoelectric points upon phosphorylation. Solubilities of these phosphorylated milk proteins were pH dependent in 0.1 m NaCl but there was a decrease in their solubilities with increase in calcium ion concentration. This alteration could be due to the shifting of isoionic points of phosphorylated buffalo milk proteins towards acidic pH.     

A sensitive HPLC method to detect hen's egg white lysozyme in milk and dairy products

Because of the lytic activity on the cell wall of bacteria like Clostridium tyrobutyricum, hen's egg white lysozyme is used in cheese manufacturing to prevent late blowing. A HPLC method capable to quantify as low as 0.8 ppm lysozyme in milk and cheese is proposed. Lysozyme was extracted with 1 m NaCl at pH 6.0 and the extract was deproteinized at low pH values, reaching a recovery up to 90%. Reversed-phase HPLC was performed on a polymeric column and monitoring lysozyme under fluorescence detection (excitation at 280 nm and emission at 340 nm). The repeatability of this determination tested on a 15-month-aged hard cheese and expressed as relative standard deviation was 1.47 (n=6) and no interference of peptides formed during ripening was observed. Four commercial preparations of egg white lysozyme gave a similar fluorescence response and the partitioning over cheese and whey was studied with one of them. About 80% of the lysozyme added to the cheesemilk at concentrations up to 80 ppm was retained in the cheese and the concentration factor of lysozyme from the cheesemilk to the cheese proved to be 8.2 on average. This HPLC method and the microbiological assay using Micrococcus luteus were compared in cheese analysis, proving the former to be more accurate and reliable than the latter. Eighteen commercial samples including both generic cheeses and cheeses having protected designation of origin, all of them not declared to contain lysozyme, showed concentrations of this enzyme ranging from 0 to 111 ppm.     

Fractionation of pasteurized skim milk proteins by dynamic filtration

This paper describes a two-stage process for separating milk proteins from pasteurized skim milk in three fractions: casein micelles, β-Lactoglobulin (β-Lg) and other large whey proteins, and α-Lactalbumin (α-La). Casein micelles were extracted in the retentate of a microfiltration using rotating ceramic disk membranes. α-La and β-Lg transmissions remained between 0.8 and 0.98. Their yields in permeate reached 81% for α-La and 76.6% for β-Lg at a VRR of 5.4. The separation between β-Lg and α-La was carried out by UF using a rotating disk module equipped with a 50 kDa PES circular membrane. Permeate fluxes were very high, remaining above 340 L h^?1 m^?2 at VRR = 5 and 40 °C. α-La transmission remained generally between 0.2 and 0.13 giving yields from 28% to 34%. β-Lg rejection was above 0.94, giving a maximum selectivity of 4.2. These data confirm the potential of dynamic membrane filtration for separating α-La and β-Lg proteins from skim milk.     

Small and large deformation rheology and microstructure of κ-carrageenan gels containing commercial milk protein products

The rheological behaviour of commercial milk protein/κ-carrageenan mixtures in aqueous solutions was studied at neutral pH. Four milk protein ingredients; skim milk powder, milk protein concentrate, sodium caseinate, and whey protein isolate were considered. As seen by confocal laser microscopy, mixtures of κ-carrageenan with skim milk powder, milk protein concentrate, and sodium caseinate showed phase separation, but no phase separation was observed in mixtures containing whey protein isolate. For κ-carrageenan concentrations up to 0.5 wt%, the viscosity of the mixtures at low shear rates increased markedly in the case of skim milk powder and milk protein concentrate addition, but did not change by the addition of sodium caseinate or whey protein isolate. For κ-carrageenan concentrations from 1 to 2.5 wt%, small and large deformation rheological measurements, performed on the milk protein/κ-carrageenan gels, showed that skim milk powder, milk protein concentrate or sodium caseinate markedly improved the strength of the resulting gels, but whey protein isolate had no effect on the gel stength.     

Effect of feeding whey peptide fractions on the immune response in healthy and Escherichia coli infected mice

The antimicrobial and immunomodulatory activities of whey proteins and peptides make them promising candidates for protection against pathogens. To test this hypothesis, a whey protein isolate and three peptide fractions obtained from its trypsin/chymotrypsin digestion were given orally to mice for 7 days. Half the mice were then infected with Escherichia coli O157:H7 and serum cytokines and total immunoglobulin A (IgA) were measured over the next 7 days. All whey products strongly stimulated total IgA production in non-infected mice, suggesting a potential adjuvant role. The peptide fractions produced contrasting immunomodulatory effects: the neutral fraction (4.5 < pH < 7) stimulated serum interferon-gamma (IFN-γ), whereas the acidic fraction (pH < 4.5) inhibited it. In the infected model, only the basic fraction (pH > 7) induced a sustained serum IgA secretion, which coincided with increased transforming growth factor-beta 1 (TGF-β1) levels. These results indicate that some whey peptides modulate immune parameters in healthy mice, whereas the basic peptide fraction increased immune vigilance during the infection.     

Skim milk protein distribution as a result of very high hydrostatic pressure

This work studies the micellar size and the distribution of caseins, major and minor whey proteins in different fractions of skim milk treated up to 900 MPa for 5 min. Transmission electron microscopy showed that the smallest casein micelles were formed around 450 MPa with no variations at higher pressures. The changes found in micellar size correlated with the concentration of soluble casein, because treatments at 250 MPa significantly enhanced the level of non-sedimentable casein while, between 700 and 900 MPa, there were no further increases with respect to lower pressures. There was a severe β-lactoglobulin (β-Lg) denaturation at pressures ≥ 700 MPa, which reached 77–87%. α-Lactalbumin (α-La) was stable up to 550 MPa, but it denatured at higher pressures. The content of soluble lactoferrin (Lf) decreased with pressure, particularly from 550 to 800 MPa, while that of secretory IgA (sIgA) progressively decreased from 250 up to 700 MPa. Our results indicated that treatment of milk at very high pressures, from 700 to 900 MPa, did not reduce micellar size nor released more soluble casein with respect to treatments at lower pressures (250–550 MPa). However, these treatments led to a severe denaturation of the whey proteins, in particular of β-Lg and the minor proteins Lf and sIgA. The possibility of using high hydrostatic pressure to obtain a soluble milk fraction with a casein and whey protein composition similar to that of human milk is discussed.     

Identification of bioactive peptides after digestion of human milk and infant formula with pepsin and pancreatin

Seven human milks were subjected to an in vitro digestion with pepsin and pancreatin to identify the peptides released from human proteins. On the basis of their sequences, 11 of the 23 peptides were synthesised and their angiotensin converting enzyme (ACE)-inhibitory and antioxidant activities were measured. The β-casein peptides HLPLP and WSVPQPK showed potent ACE-inhibitory and antioxidant activity, with a protein concentration needed to inhibit 50% ACE activity (IC₅₀) of 21 μm and a Trolox Equivalent Antioxidant Capacity (TEAC) of 1.297 μmol 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivs μmol⁻¹ of peptide, respectively. These activities were determined after digestion of eight infant formulas and compared with those found in digested human milk. One of the infant formulas exhibited a low IC₅₀ value (60.11 μg protein mL⁻¹ of reconstituted formula) and a high TEAC value (1.7056 μmol Trolox equivs mg⁻¹ of protein) and was therefore selected to identify the peptides responsible of these activities.     

Development and application of an optical biosensor immunoassay for α-lactalbumin in bovine milk

An automated, label-free biosensor-based immunoassay for α-lactalbumin in bovine milk utilising surface plasmon resonance (SPR) detection is described. α-Lactalbumin content was estimated from the specific interaction with an anti-bovine α-lactalbumin antibody immobilised on the sensor surface in a direct-binding assay format, although an alternative inhibition assay format is also described. Samples were prepared for analysis by direct dilution into buffer. Ligand selection and analysis conditions are defined, and non-specific binding considerations evaluated. Performance parameters include a working range of 10–1000 ng mL^?1, a method detection limit of 0.12 mg mL^?1 in milk, an overall instrumental reproducibility relative standard deviation (RSD_R) of 5.71%, a mean inter-assay RSD_R of 7.61% for an infant formula control sample and a surface stability of approximately 500 cycles. Accuracy was confirmed by comparison against an independent liquid chromatographic method. The technique was applied to the measurement of the α-lactalbumin content of consumer milk, colostrum, whey protein concentrates and infant formulae, the temporal change during early bovine lactation and a preliminary study of thermal denaturation.     

Effect of milk fat concentration and gel firmness on syneresis during curd stirring in cheese-making

An experiment was undertaken to investigate the effect of milk fat level (0%, 2.5% and 5.0% w/w) and gel firmness level at cutting (5, 35 and 65 Pa) on indices of syneresis, while curd was undergoing stirring. The curd moisture content, yield of whey, fat in whey and casein fines in whey were measured at fixed intervals between 5 and 75 min after cutting the gel. The casein level in milk and clotting conditions was kept constant in all trials. The trials were carried out using recombined whole milk in an 11 L cheese vat. The fat level in milk had a large negative effect on the yield of whey. A clear effect of gel firmness on casein fines was observed. The best overall prediction, in terms of coefficient of determination, was for curd moisture content using milk fat concentration, time after gel cutting and set-to-cut time (R² = 0.95).     

Functional properties and Angiotensin-I converting enzyme inhibitory activity of soy–whey proteins and fractions

Soy proteins when prepared to high purity can confer good functional properties and the whey by-product is a potential source for bioactivity. In this study, we determined the protein, moisture, fiber, solubility, foaming, emulsion properties, as well as Angiotensin-I converting enzyme (ACE-I) inhibitory activity of prepared soy–whey proteins and its fractions. The soy–whey proteins were fractionated into < 5, > 5, > 10, and > 50 kDa using ultrafiltration. The expanded AACC methods were used to determine protein, moisture, and fiber analyses of the whey and its fractions. Solubility method was conducted to determine the protein solubility profile of the soy–whey and its fractions at varying pHs. Turbidimetric method was used to evaluate emulsifying activity (EA) and emulsion stability (ES). There were significant differences observed in moisture, protein and salt contents between unfractionated, > 50 kDa and smaller sized fractions. No significant differences were observed with phytic acid and total dietary fiber contents among all samples. The unfractionated whey protein and > 50 kDa fraction showed better solubility than other fractions. Unfractionated whey protein had the highest foam capacity (42.7 mL) while the fraction > 5 kDa showed the greatest foaming stability (46 min). The highest emulsion activity (0.33 ± 0.1) and stability (825.1 ± 45.1) was obtained with the > 50 kDa fraction while the unfractionated whey protein had the highest ACE-I inhibition activity. The findings indicate that soy–whey protein fraction (> 50 kDa) had good solubility, emulsion activity and stability, while the unfractionated whey protein exhibited the strongest ACE-I inhibition percentage (19%).     

Angiotensin-converting enzyme inhibitory activity of milk protein hydrolysates: Effect of substrate,enzyme and time of hydrolysis

Nine milk protein substrates were hydrolysed in vitro with five proteases for various times (0, 3, 6, and 24 h), and the angiotensin-converting enzyme (ACE)-inhibitory activity of hydrolysates was assessed. Overall, the casein substrates gave rise to hydrolysates with significantly higher ACE-inhibitory activity than the whey protein (WP) substrates (85% vs. 79%). No significant difference between 3 and 24 h of hydrolysis was found. A reasonable correlation was found between the ACE inhibition of the 6 h hydrolysates determined in vitro and estimated by in silico modelling. The highest ACE-inhibitory activity was found in hydrolysates made with thermolysin followed by proteinase K, trypsin, pepsin and Bacillus licheniformis protease. The IC₅₀ values for thermolysin hydrolysates of caseins and WPs were 45–83 and 90–400 μg mL⁻¹, respectively, with α-lactalbumin giving the highest inhibitory activity. Thermolysin, proteinase K and trypsin were useful for the release of highly potent ACE-inhibitory peptides from both WPs and caseins.