High-pressure-induced changes in the rennet coagulation properties of bovine milk

The mechanism of high-pressure (HP)-induced changes in rennet coagulation properties of milk, particularly the role of whey protein-casein micelle associations, was studied. Treatment at 100 or 250 MPa reduced the rennet coagulation time (RCT) of raw skimmed bovine milk, compared with untreated milk. Treatment at 400 MPa had little effect, but at 600 MPa, RCT increased considerably. HP-induced increases in RCT did not occur in serum protein-free milk or milk treated with the sulphydryl-oxidising agent KIO₃, which prevents association of denatured β-lactoglobulin with casein micelles. Treatment at 5 or 10 °C at 250–600 MPa resulted in shorter RCT than treatment at 20 °C. In milk without KIO₃, coagulum strength was highest after treatment at 250 or 400 MPa, whereas in milk with KIO₃ it was highest after treatment at 400 MPa. These results indicate the significance of HP-induced association of whey proteins with casein micelles for rennet coagulation properties of milk.     

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.     

An investigation of the plasmin–plasminogen system in caprine milk and cheese

The variability in activities of plasmin (PL) and plasminogen (PG) in bulk milk samples from different breeds of goats was investigated. The mean PL activity was higher (19.56±4.72 U g⁻¹) than in milk from other ruminant species, while PG-derived activity was, surprisingly, lower (12.84±5.31 U g⁻¹). No significant differences in PL and PG levels were observed among goat breeds, but PL activity and PL/PG ratio increased in late lactation compared with early lactation. PL activities in pilot-scale curds (acid or rennet) and in some Italian cheeses made from caprine milk were also measured. Generally, acid curds and cheeses had lower residual PL and PG-derived activities than rennet curds and semi-hard cheeses. In addition, the PL/PG ratio was greatly reduced in rennet curds, due to a higher extent of PG-derived activity. Measurement of proteolysis of β-casein in curds confirmed that more extensive proteolysis occurred in rennet curds, which had higher residual PL activities.     

A rapid,two-hour method for the enumeration of total viable bacteria in samples from commercial milk powder and whey protein concentrate powder manufacturing plants

A new protocol specifically designed to enumerate total viable bacteria in milk powder using flow cytometry was validated against aerobic plate counts using 178 samples of whole milk powder. Viable counts, as measured using flow cytometry, correlated well ($r^2>0.80$) with plate counts across the range of powders with counts of 10²–10⁸ cfu g⁻¹. The protocol was then used to enumerate total viable bacteria in samples of milk and whey collected at different stages during the manufacture of milk powder and whey protein concentrate. Flow cytometer results for samples taken during both processes were similar to those from the aerobic plate counts. However, the flow cytometer results were obtained within 2 h compared with 3 days for the plate counts. The strategic advantage of the close-to-real-time data obtained using flow cytometry is that results can be relayed back quickly to the management of the manufacturing plant for use in decision-making with respect to controlling the manufacturing process.     

Cheese made from instant infusion pasteurized milk: Rennet coagulation,cheese composition,texture and ripening

Milk subjected to instant infusion pasteurization (IIP) at 72 °C, 100 °C and 120 °C (holding time 0.2 s) exhibited increased rennet coagulation time and decreased curd firming rate for increasing heat treatment temperature, when compared with raw or high temperature short time pasteurized (HTST) milk. However, addition of 4.5 mm or 9.0 mm of calcium restored the impaired rennet coagulation ability. Open texture cheeses produced from IIP milk (100 °C and 120 °C) contained significantly more moisture, had lower pH and shorter texture than similar cheese from IIP at 72 °C and HTST pasteurized milk. Cheese ripening was also affected by heat treatment, and different patterns of casein breakdown and peptide formation resulted from cheeses made from milk treated to IIP at 100 °C and 120 °C compared with cheeses made using IIP at 72 °C or HTST.     

Optimisation of a coupled enzymatic assay of glutathione peroxidase activity in bovine milk and whey

Glutathione peroxidase (GSHPx) may have important functions in milk but studies of its activity during dairy processing are hampered by the lack of suitable assays. Thus, a coupled enzymatic assay of GSHPx activity was developed for bovine milk and whey. The reaction mixture contained reduced glutathione, tert-butylhydroperoxide, glutathione reductase and NADPH in phosphate buffer. The effect of reaction conditions on enzymatic and non-enzymatic NADPH consumption was studied, and blanks without added GSHPx sample or complete incubations containing 4 mmol L⁻¹ mercaptosuccinate as an inhibitor of GSHPx were used. With increasing pH and glutathione concentration the reaction rates increased both in the complete incubation and the blank but optimal GSHPx activity was attained at a glutathione concentration of 0.63 mmol L⁻¹. A pH of 7.6 was chosen to attain near-optimal activity without reaching high blanks. Application of the new procedure showed that bulk whey samples had rather constant GSHPx activity with an average (SD) of 36.1 (2.9) U mL⁻¹, but whey samples prepared from individual cows showed a two-fold variation (range 24.5–50.6 U mL⁻¹). The procedure described was more reproducible and precise than previous methods and will be employed in further studies of dairy products.     

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).     

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.     

Development of a fermented goat's milk containing probiotic bacteria

A set-type fermented milk manufactured from goat's milk was developed. Optimal curd tension was achieved by supplementation of milk with skim milk powder and whey protein concentrate (WPC). Milk was fermented employing a commercial probiotic starter culture (ABT-2), which contained Streptococcus thermophilus ST-20Y, Lactobacillus acidophilus LA-5, and Bifidobacterium BB-12. Supplementation of milk with 3% WPC reduced fermentation time by 2 h due to the increase in viable counts of S. thermophilus and Bifidobacterium by 0.3 and 0.7 log units, respectively. Addition of WPC increased the protein content (1%) as well as potassium and magnesium content (0.3 and 0.02 g kg⁻¹, respectively). Increase of the protein content led to an increase in the apparent viscosity and gel firmness of the product, and at the same time whey syneresis was reduced. As a consequence, the product received a high score for appearance, taste, aroma, texture and overall acceptance.     

Manufacture of Cheddar cheese from high-pressure-treated whole milk

The cheese-making characteristics of high-pressure (HP)-treated milk were examined. The rennet coagulation time of pasteurised milk decreased after HP treatment at 400 MPa but increased after treatment at 600 MPa. The L-value (whiteness) of milk decreased directly after HP treatment but, over the course of coagulation, whiteness of HP-treated milk increased to the same level as in the control. Cheddar cheese was then manufactured from raw whole milk or whole milk treated by high-pressure (HP) at 400 MPa (HP400) or 600 MPa (HP600) for 10 min at 20 °C. HP treatment of raw milk at 600 MPa resulted in a 3.66 log reduction in the initial counts of non-starter lactic acid bacteria (NSLAB), decreased protein and fat content, as well as a lower pH compared to the control. Furthermore, higher treatment pressures resulted in increased incorporation of β-lactoglobulin into the cheese curd, with parallel increases in yield by 1.23% and 7.78% for HP400 and HP600 cheeses, respectively. Overall, this study showed that the effects of HP treatment on milk proteins increased rennet coagulation times and changes in cheese composition at day 1.Industrial relevanceHigh-pressure treatment is a novel technology which has been applied to a number of commercial food products. In this study, HP-induced changes in milk proteins resulted in increased cheese yields and increased cheese whiteness. In addition, HP treatment significantly reduced the microflora of raw milk cheese. Those attributes could be of interest for both industry and consumer.     

Proteolysis and storage stability of UHT milk produced in Turkey

The effect of raw milk quality (total and psychrotrophic bacterial and somatic cell counts, proteinase and plasmin activity) and UHT temperature (145 or 150 °C for 4 s) on proteolysis in UHT milk processed by a direct (steam-injection) system was investigated during storage at 25 °C for 180 d. High proteinase activity was measured in low-quality raw milk, which had high somatic cell count, bacterial count and plasmin activity. The levels of 12% trichloroacetic acid–soluble and pH 4.6-soluble nitrogen in all milk samples increased during storage, and samples produced from low-quality milk at the lower UHT temperature (145 °C) showed the highest values. Bitterness in UHT milk processed from low-quality milk at 145 °C increased during storage; gelation occurred in that milk after 150 d. The RP-HPLC profiles of pH 4.6-soluble fraction of the UHT milk samples produced at 150 °C showed quite small number of peaks after 180 d of storage. Sterilization at 150 °C extended the shelf-life of the UHT milk by reducing proteolysis, gelation and bitterness.     

HPLC-MS/MS-Detection of Caseins and Whey Proteins in Meat Products

Screening methods for the mass spectrometric detection of caseins and whey proteins in meat products have been developed. After tryptic digestion, two α-S1-casein and two β-lactoglobulin marker peptides were measured by HPLC-MS/MS. For matrix calibrations, emulsion-type sausages with different concentrations of milk and whey protein (ppm level) were produced. The limits of detection (LODs) were below 1 ppm for milk protein and about 3 ppm for whey protein. The determination coefficients for the correlation between peak area of the marker peptides and the concentrations of milk and whey proteins were R²≥0.9899.     

Partial renneting of pasteurised bovine milk: Casein micelle size,heat and storage stability

The effects of partial renneting at low temperature on the casein micelle (CM) size and the storage stability of milk were investigated. Low chymosin concentrations (≤ 0.03 IMCU mL^− 1) was applied to pasteurised skim milk at 4 °C and enzyme activity was terminated by thermal application at 60 °C/3 min and 85 °C/30 min, referred to as low heat (LHT) and high heat (HHT) treatment milk, respectively. The addition of rennet with concentrations of 0.01, 0.02 and 0.03 IMCU mL^− 1 for 15 min resulted in κ-casein hydrolysis of 10, 20 and 25%, respectively. Moreover, mean CM size of milk was reduced by up to 10 nm. For LHT milk, the renneted micelles appeared to be stable for up to 17 days, especially in response to the application of 0.01 IMCU mL^− 1 and at a storage temperature of 4 °C. Severe heating at 85 °C/30 min to inactivate the enzyme caused an increase in CM size.     

Microbiological and physicochemical stability of raw,pasteurised or pulsed electric field-treated milk

Pulsed electric field (PEF) processing was investigated as an alternative dairy preservation technology that would not compromise quality yet maintain safety. PEF processing of raw whole milk (4% fat) was conducted at two processing conditions (30 kV/cm, 22 μs, at either 53 or 63 °C outlet temperature) and compared with two thermal treatments (15 s, at either 63 or 72 °C) and a raw milk control and replicated twice. Milk bottles (2 L) from each treatment were incubated for two weeks, at 4 and 8 °C, and assessed for total plate count, pH, colour, rennetability, plasmin activity and lipid oxidation. The microbial quality of the thermal (72 °C/15 s) and PEF (63 °C) were similar. A drop in pH occurred after a change in counts was observed. Rennetability was not different between the treatments. Short chain acids dominated the volatile profile of the milk samples. Concentration of volatiles derived from microbial activity, namely 2,3-butanedione, acetic acid and other milk lipid derived short chain free fatty acids (e.g. butanoic and hexanoic acids), followed the trend of microbial activity in milk samples.Industrial relevanceResearch on the application of PEF to control spoilage and pathogenic microorganisms and enzyme systems in milk spans a wide array of processing equipment and reaction conditions. While industrial scale PEF processing of liquid milk for preservation and improved quality seems generally possible, substantiation of lower thermal damage under safe and scalable PEF conditions is required to enable economic feasibility.     

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.     

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.     

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.     

Analysis of cyclopiazonic acid in milk by capillary electrophoresis

A Micellar Electrokinetic Capillary Chromatography (MEKC) method to detect cyclopiazonic acid (CPA) in milk and compare its quantifying efficiency to the Reverse Phase Liquid Chromatography (RPLC) method was evaluated. Alkaline milk samples were defatted, then acidified before being twice liquid–liquid extracted with chloroform. Bare fused-silica capillary-extended light path with 50 μm i.d. and Nova-pak C18-column, were used for the CPA separation in MEKC and RPLC respectively. The analytical response was linear from 40 ppb to 100 ppm CPA in MEKC (correlation coefficient, r=0.99995). Recoveries of spiked CPA in milk were 78–81% over the range of 20 ppb to 500 ppb in MEKC and 71–80% in the range of 50 ppb to 500 ppb in RPLC. The detectable limit of CPA by MEKC was 0.27×10⁻⁰⁷ pg ml⁻¹. Capillary electrophoresis (MEKC) is a better and rapid method for CPA detection in milk.     

Determination of major metal cations in milk by capillary zone electrophoresis

A reliable and rapid capillary zone electrophoresis method for the determination of the major of cations in milk samples was developed. Sample preparation consisted of dilution, acidification to pH 4.0 with 1 mol L⁻¹ acetic acid and filtration. The complete separation of K⁺, Ca²⁺, Na⁺ and Mg²⁺ could be achieved in 4 min with a simple electrolyte composed of 10 mmol L⁻¹ imidazole and 1 mol L⁻¹ acetic acid (pH 3.6). The running voltage was 25 kV at 25 °C. Indirect UV detection was achieved at 185 nm. Detection limits ranged from 0.06 to 0.57 mg L⁻¹ and quantification limits ranged from 0.16 to 0.62 mg L⁻¹. Precision data showed relative standard deviations (RSD%) lower than 4.1% for relative migration time and 2.4% for milk concentrations, respectively. Recoveries of cations in samples analysed ranged from 97.7% to 101.1%. Thirty samples of milk were analysed, obtaining mean values of 1.46, 1.19, 0.505 and 0.126 g L⁻¹ for K⁺, Ca²⁺, Na⁺ and Mg²⁺, respectively.