Influence of enzymatic cross-linking on milk fat globules and emulsifying properties of milk proteins

The enzyme transglutaminase (TGase) can modify dairy protein functionality through cross-linking of proteins. This study examined the effects of TGase treatment on milk fat globules and the emulsifying properties of milk proteins. The extent of TGase-induced cross-linking of caseins increased with incubation time, with no differences between whole and skim milk. Extensive clustering of fat globules in extensively cross-linked raw whole milk occurred on homogenisation at 400 or 800 bar. Considerably less clustering of fat globules was observed when recombined milk (90 g fat L^–1) was prepared from TGase-treated skim milk and homogenised at 400 or 800 bar. TGase treatment did not affect fat globule size in cream, but prevented coalescence of fat globules therein, possibly through cross-linking of milk fat globule membrane components. TGase-induced cross-linking of milk proteins affected their emulsifying properties and may increase the stability of natural milk fat globules against coalescence.     

Effect of hydrolysis of casein by plasmin on the heat stability of milk

This study examined the effect of hydrolysis of casein by added plasmin (6 mg L⁻¹) on the heat stability of raw, pre-heated, serum protein-free or concentrated skim milk. Plasmin activity markedly affected the heat stability–pH profile of skim milk and serum protein-free milk, apparently by altering the properties of the casein micelles. It is probable that changes in the surface charge of the micelles, as a result of the hydrolysis of caseins, contributed to this effect. Hydrolysis by plasmin reduced the zeta-potential of the casein micelles from ∼−19 to ∼−16 mV. The effect of hydrolysis of casein by plasmin on the heat stability of pre-heated milk was less pronounced, shifting the heat stability–pH profile to more alkaline values; the heat stability of concentrated milk was unaffected by plasmin. A very high (50 mg L⁻¹) level of added plasmin resulted in clearing of the skim milk; the L^* value decreased from ∼75 to ∼35 after 24 h incubation at 37 °C. Clearing was correlated with a change in casein micelle diameter from an initial value of ∼175 to ∼250 nm. It is suggested that plasmin-induced changes in zeta-potential may promote micellar aggregation or changes in micelle stucture.     

Properties of casein micelles cross-linked by transglutaminase

Factors affecting the cross-linking of milk proteins by transglutaminase (TGase) were studied. Cross-linking of caseins in bovine skim milk was optimal over a very wide pH range. The role of micellar calcium phosphate (MCP) in maintaining the integrity of TGase-treated casein micelles was studied by incubating skim milk with 0.01% (w/v) TGase at 30°C for 1–24 h, followed by removal of MCP from untreated or TGase-treated milk by acidification and dialysis. The protein content and profile of the samples were determined by Kjeldahl and SDS-PAGE, respectively. Whey proteins in unheated milk were not susceptible to TGase-induced cross-linking. The higher level of sedimentable protein in MCP-free TGase-treated milk than in MCP-free control milk indicated that TGase treatment partially prevented disintegration of the micelle on removal of MCP, probably due to extensive intramicellar TGase-induced cross-linking of casein molecules which led to the formation of sedimentable covalently bonded casein aggregates.     

Effects of varying time/temperature-conditions of pre-heating and enzymatic cross-linking on techno-functional properties of reconstituted dairy ingredients

The effects of varying time/temperature-conditions of pre-heating and cross-linking with transglutaminase (TG) on the functional properties of reconstituted products from skim milk, WPC and sodium caseinate was analyzed. The degree of cross-linking (DC) of skim milk proteins could be increased from 54.4% to 70.5% by varying process conditions. Thereby the water-holding capacity (WHC) increased from 10% to 20%, while the heat stability decreased. The burning-on was lower than that of the non-treated products at optimum pre-heating conditions (90 °C/30 s). Using sodium caseinate as substrate for TG the DC increased from 39.2% to 100% due to the improvement of the process. As a result the WHC increased by 30% and the heat stability up to 380%. However, the burning-on of casein increased as well. TG-treated sodium caseinate started to gel at 10% protein, whereas untreated sodium caseinate gelled not before 15% protein. The WHC of enzyme-treated whey proteins was lowered. The heat stability of WPC could be doubled by TG-treatment, and the burning-on of the products was, especially at optimum pre-heating conditions, less pronounced. The degree of denaturation of TG-treated whey proteins was 2–5% higher than that of untreated samples.     

Inactivation of an indigenous transglutaminase inhibitor in milk serum by means of UHT-treatment and membrane separation techniques

An indigenous inhibitor in raw milk inhibits cross-linking by transglutaminase (TG). The enzymatic cross-linking of micellar casein, compared with sodium caseinate, taking thermal inactivation of the TG inhibitor in the milk serum into consideration, was investigated. Inhibitor-free micellar casein was prepared by membrane separation combined with heat treatment of the UF permeate. The inhibitor permeated through MF (nominal pore size 0.1 μm) and UF (cutoff 25 kDa) membranes. TG-catalyzed cross-linking of casein micelles was clearly enhanced by UHT-treatment of UF permeate. Variation of the enzyme concentration showed that the inhibitory effect could not be compensated by higher enzyme concentrations when the casein micelles were suspended in unheated milk serum. Sodium caseinate, however, underwent high degrees of cross-linking even in unheated milk serum. By mixing an unheated milk serum and a UHT-treated milk serum at different ratios, the relative TG inhibitor activity was analysed. High inactivation (>80%) of the TG inhibitor is necessary to achieve high degrees of protein cross-linking.     

Molecular and structural characteristics of cod gelatin films modified with EDC and TGase

Cod gelatin films before and after cross-linking of gelatin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or transglutaminase (TGase) have been characterized by Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry (DSC) analysis. For comparison, a film prepared from unmodified pig gelatin has been also analysed. The difference spectra showed that cod gelatin during the film formation involved first of all water-to-amide hydrogen bonds, and the film from pig gelatin contained water-to-amide, amide-to-amide and water-to-water hydrogen bonds. A higher number of hydrogen bonds in the structure of the film from pig gelatin contributed to much better recovering of the helical structure in this film than in the film from cod gelatin, as the peaks at about 1663 cm^?1 in the amide I band and at about 1537 cm^?1 in the amide II band in the second-derivative spectra revealed. The recovered helical structure, in turn, resulted in a significantly higher melting enthalpy value in the case of the film from the pig gelatin. After modification of cod gelatin with EDC or TGase, the inter-chain cross-linkages formed in the films led to the conformation of gelatin with no indications of helical ordering. An increase of melting temperature of gelatin films by 7 °C on EDC and by 10 °C on the TGase modifications was related to the formation of covalent cross-links, and a decrease of glass temperature by 28 °C and 7 °C on EDC and TGase cross-linking, respectively, demonstrated the plasticizing effect of water.     

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.     

Baroprotection of vegetative bacteria by milk constituents: A study of Listeria innocua

The baroprotective effect of milk constituents on Listeria innocua 4202 treated at 350 or 500 MPa for 5 min was examined. High-pressure (HP) treatment of L. innocua 4202 (1×10⁹ cfu mL⁻¹) resulted in complete inactivation in simulated milk ultra-filtrate (SMUF), a ∼5.0 log reduction in phosphate-buffered saline and a ∼2.9 log reduction in milk. The addition of micellar casein to SMUF increased survival of the bacterium by 3 logs, compared with SMUF alone, but the protective effect was negated if the minerals associated with the casein micelles were removed. The colloidal minerals calcium (30 mm), magnesium (5 mm), citrate (10 mm) and phosphate (20 mm), suspended in SMUF increased survival by ∼3.3, ∼1.7, ∼3.3 and ∼3.5 logs, respectively. The buffering capacity of the suspending medium was found to be a key factor in microbial baroresistance. Buffering by phosphate and citrate in milk may protect microorganisms against changes in pH during HP treatment, whereas the divalent cations calcium and magnesium may protect cell membranes against HP.     

Effect of NaCl on some physico-chemical properties of concentrated bovine milk

The influence of added NaCl (75–850 mmol L⁻¹) on some physicochemical properties of 2× or 3× concentrated milk (18 or 27%, w/v, total solids, respectively) was investigated. Adding NaCl did not influence average casein micelle size, but reduced the net-negative charge on the casein micelles and milk pH. The level of soluble and ionic calcium was increased by addition of NaCl, but the level of soluble inorganic phosphorus was not influenced. Addition of NaCl shifted the maximum in the pH–heat coagulation time (HCT) profile of 2× or 3× concentrated milk to a higher pH value and certain concentrations increased the maximum HCT, probably due to the fact that NaCl reduced the extent of heat-induced dissociation of κ-casein. Added NaCl reduced the ethanol stability, with the extent of this effect increasing with the concentration of NaCl. The key-mechanism though which added NaCl induces changes in the physico-chemical stability of casein micelles appears to be through changes in the charge on the casein micelles.     

Determination of lactoferrin in bovine milk,colostrum and infant formulas by optical biosensor analysis

An automated, rapid, sensitive and label-free biosensor-based immunoassay for lactoferrin in bovine milk utilising surface plasmon resonance (SPR) optical detection is described. Lactoferrin content is estimated from its specific interaction with an anti-bovine lactoferrin antibody immobilised on the sensor surface in a direct- binding assay format. Samples are prepared for analysis by direct dilution into buffer. Analysis conditions, including ligand immobilisation, flow-rate, contact time and regeneration have been defined and non-specific binding considerations evaluated. Performance parameters include a working range of 0–1000 ng mL⁻¹, a method-detection limit of 19.9 μg mL⁻¹ in undiluted milk, overall instrument response RSD_R of 3.50%, a mean inter-assay RSD_R of 10.8% for milk and surface stability over ca 500 samples. The technique was applied to the measurement of lactoferrin content of consumer milks, colostrum and infant formulas, and temporal change during early bovine lactation.     

Improvement of enzymatic cross-linking of casein micelles with transglutaminase by glutathione addition

The impact of glutathione (GSH) on cross-linking of micellar casein and sodium caseinate by microbial transglutaminase (TG) was investigated. Micellar casein was obtained by removing whey proteins from skim milk by means of membrane separation techniques. The addition of GSH (0.05–0.1 mm) was found to enhance the cross-linking of micellar casein suspended in milk serum. Cross-linking of sodium caseinate remained unaffected by GSH addition. Mixing TG and milk serum before addition of substrate proteins, however, resulted in an almost complete inhibition of the enzyme. When GSH and TG were present in the system before substrate addition, the reactivity of TG can be maintained. Hence, it was concluded that the addition of GSH mainly affected interactions between TG and an indigenous TG inhibitor present in milk serum. The addition of GSH allows cross-linking in milk products by TG without requiring a prior heat treatment of the milk beyond pasteurisation conditions.     

Entrapment in food-grade transglutaminase cross-linked gelatin–maltodextrin microspheres protects Lactobacillus spp. during exposure to simulated gastro-intestinal juices

Dietary intake of probiotic bacteria has been shown to impart health effects, however, maintaining viable cells in foods and during passage of the adverse conditions in the upper gastro-intestinal tract is often a problem. The objective of this research was to develop and characterize novel food-grade phase-separated gelatin–maltodextrin (G-MD) microspheres, where the gelatin was cross-linked with transglutaminase (TGase), to determine if encapsulated probiotic lactic acid bacteria were protected during exposure to simulated upper gastro-intestinal tract conditions. The stability, size, structure and protective ability of G-MD microspheres as a function of different TGase concentrations and gelatin bloom strengths were tested. The G-MD microspheres made with gelatin A 300 bloom and a TGase concentration of 10 U/g prevented pepsin-induced degradation of the microspheres in simulated gastric juice (pH 2.0, 2 h, 37 °C), resulting in significantly (p < 0.05) higher numbers of survivors due to the buffering effect of intact microspheres (average diameter 46 μm). After sequential incubation in simulated gastric (1 h) and intestinal juices (pH 7.4, 4 h, 37 °C), survivor levels of each of the three encapsulated Lactobacillus sp. (3C2–10, 21C2–10 and 21C2–12) were reduced by 0.2–1 log(CFU/g) as compared to 3–4 log(CFU/g) for the free non-encapsulated cells. This study presents a new protein based microencapsulation method, which using all food-grade ingredients protects probiotic lactic acid bacteria during exposure to adverse environmental conditions.     

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.     

The use of sedimentation field-flow fractionation in the size characterization of bovine milk fat globules as affected by heat treatment

Size distribution of fat globules affects the appearance, taste and stability of milk and milk-based products. Full-fat, semi-fat and chocolate bovine milk were subjected to heat treatment within a temperature range of 50–125 °C for 1 h. Sedimentation field-flow fractionation was employed to determine the changes in mean particle diameter of milk fat globules as affected by heat treatment. The mean particle diameter of fat droplets increased with increasing heating temperature for most samples. The particle size of fat globules increased on average 40 nm (4.65%) for full-fat and 72 nm (8.52%) for semi-fat milk following the heat treatment (50–125 °C). Chocolate milk exhibited considerable increase in particle size (104 nm, 12.53%) within a certain temperature range (50–110 °C), followed by a decrease in particle size when heated at 125 °C for 1 h. Heat-induced flocculation due to attractive interactions between hydrophobic sites on denatured protein molecules on different droplets was assumed to be mainly responsible for the increases in particle size observed in this study. Extensive heat-induced denaturation of milk proteins was also indicated by Native PAGE. Sedimentation field-flow fractionation proved to be a useful technique for adequately monitoring heat-induced changes in particle size distributions in milk.     

Growth-inhibition of hiochi bacteria in namazake (raw sake) by bacteriocins from lactic acid bacteria

The bacteriocins produced by Lactococcus lactis subsp. lactis C101910 (C101910) and NBRC 12007 (NBRC 12007) were used to prevent the growth of sake spoiling hiochi bacteria (Lactobacillus hilgardii, Lactobacillus fructivorans, and Lactobacillus paracasei) in namazake, which is raw (unpasteurized) sake. The bacteriocin concentrations required for decreasing the viable cell concentrations of L. hilgardii and L. fructivorans below the detection limit (1.0 × 10² cells/ml) in 24 h from the initial concentration of 4.0–9.5 × 10⁵ cells/ml in the namazake at pH 4.5 and at 4°C, were 18–35 U/ml and 5.6 U/ml for the bacteriocin from C101910 and NBRC 12007, respectively. To decrease the viable cell concentration of L. paracasei from the initial concentration of 7.5 × 10⁵ cells/ml to below the detection limit (1.0 × 10² cells/ml) in 24 h, 350 U/ml bacteriocin from C101910 and 140 U/ml bacteriocin from NBRC 12007 were required. In experiments using McIlvaine buffer (pH 4.5) with 15% ethanol instead of namazake as the medium, the viable cell concentrations of L. hilgardii and L. paracasei decreased to less than 1.0 × 10² cells/ml, whereas those of L. fructivorans decreased to less than 1.0 × 10³ cells/ml, when bacteriocins were added at the concentrations that had proven effective in namazake. The membrane depolarization assay using a fluorescent probe showed that the presence of ethanol stimulated the collapse of the membrane potential induced by bacteriocins. The ethanol induced collapse of the membrane potential suggests that the application of bacteriocins at the storage stage of namazake is more beneficial than when used in other stages of the sake brewing process.     

Impact of process conditions on the rheological detectable structure of UHT treated milk protein–carrageenan systems

UHT treated dairy based drinks containing carrageenan to produce a weak gel type of structure are often found to vary considerably in textural properties. Target of this study was to investigate the effect of the heating temperature in the range of 120–139 °C and the filling temperature after cooling down to 4–14 °C prior to storage for 24 and 96 h at 4 °C on a system comprising milk and 400 ppm kappa-2 type of carrageenan. The textural properties and the stability of such systems mainly depend on the interaction of the carrageenan and the casein micelle surface which was assessed by means of the hysteresis loop area between the upward and downward flow curves upon variation of the shear stress. The loop area was used as a dimension for the energy required breaking down the systems’ textural structure. The hysteresis loop area was found to be significantly increased the higher the heating temperature and the lower the cooling temperature was. During storage, a further influence of the process parameters on structural development between casein and carrageenan was observed. Structure point analysis, small angle oscillatory rheology and particle size measurements were used for further explanation of the results of the hysteresis loop area. Particle size measurements indicated an aggregation phenomenon in the micellar casein dominated microdomains at increasing UHT heating temperatures. Variation of the carrageenan concentration enlightened the participation of casein dominated microdomains in the mixed system’s texture.     

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.     

Hepatoprotective effect of peptic hydrolysate from salmon pectoral fin protein byproducts on ethanol-induced oxidative stress in Sprague–Dawley rats

Bioactive peptides were generated by pepsin hydrolysis from salmon pectoral fin protein byproduct, and the hepatoprotective effect of the peptic hydrolysate (PH) on ethanol-induced oxidative stress was investigated in Sprague–Dawley rats. Administration of ethanol for 4 weeks significantly (p < 0.05) increased serum markers of liver damage, such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase; however, these activities decreased significantly (p < 0.05) after PH administration. Elevated thiobarbituric acid reactive substance levels in liver tissue and serum decreased significantly (p < 0.05) following administration of PH. Ethanol exposure significantly (p < 0.05) decreased glutathione contents in liver and serum, and hepatic antioxidant enzyme activities such as superoxide dismutase and glutathione peroxidase, while the PH treatment significantly (p < 0.05) increased these altered parameters. These results indicate that the PH had a protective effect against ethanol-induced hepatotoxicity that was comparable to that of silymarin, which was supported by evaluating liver histopathology in the rats.     

Pulsed-electric-field-assisted extraction of anthocyanins from purple-fleshed potato

The influence of pulsed electric field (PEF) treatment on the anthocyanin extraction yield (AEY) from purple-fleshed potato (PFP) at different extraction times (60–480 min) and temperatures (10–40 °C) using water and ethanol (48% and 96%) as solvents has been investigated. Response surface methodology was used to determine optimal PEF treatment and optimise anthocyanin extraction. A PEF treatment of 3.4 kV/cm and 105 μs (35 pulses of 3 μs) resulted in the highest cell disintegration index (Z_p = 1) at the lowest specific energy requirements (8.92 kJ/kg). This PEF treatment increased the AEY, the effect being higher at lower extraction temperature with water as solvent. After 480 min at 40 °C, the AEY obtained for the untreated sample using 96% ethanol as the solvent (63.9 mg/100 g fw) was similar to that obtained in the PEF-treated sample using water (65.8 mg/100 g fw). Therefore, PEF was possible with water, a more environmental-friendly solvent than ethanol, without decreasing the AEY from PFP.     

Sensory stability and oxidation of fish oil enriched milk is affected by milk storage temperature and oil quality

The experiments evaluated the influence of fish oil quality and cold storage temperature on the oxidative stability of milk emulsions containing 1.0% w/w milk fat and 0.5% w/w of either a pure fish oil or a fish oil:rapeseed oil mixture. The results showed that it was possible to produce a pasteurised milk product enriched with the important n-3 PUFA from fish oil with acceptable sensory characteristics if (1) the emulsions were based on a mixture of fish oil and rapeseed oil and (2) the initial peroxide value (PV) of the added oil blend was below 0.5 meq kg⁻¹. The sensory analysis showed a clear distinction between emulsions based on oil with PV 0.1 and 0.5 meq kg⁻¹, whereas the PV and the gas chromatographic (GC) analysis of volatile oxidation products were not sensitive enough to reveal these differences clearly. The GC analyses showed that the onset of formation of the volatiles was earlier with increased storage temperature in the range of 2–9 °C.