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.     

Heat-Induced Protein Changes in Milk Processed by Vat and Continuous Heating Systems

This study investigated and compared the effects of heating system and residence times on the physicochemical properties and interactions of casein and whey proteins in heated milk. Milk was processed by vat heating (85°C for 10 to 40 min), HTST heating (98°C for .5 to 1.87 min), UHT heating (140°C for 2 to 8 s), cooled, and fractionated into casein and whey by isoelectric precipitation.β-Lactoglobulin A and B variants were partially denatured by HTST and UHT heating and totally denatured by vat heating. Increasing residence time caused significant (P<.01) increases in denaturation of both β-lactoglobulin variants in UHT and HTST heating systems and of α-lactalbumin in the vat heating system. Surface hydrophobicity and sulfhydryl content were negatively correlated with whey protein denaturation. Sodium dodecyl sulfate electrophoresis of the casein fraction of heated milk indicated the presence of a high molecular weight component that would not enter the gel. Addition of 2-mercaptoethanol to heated casein samples dissociated this component, with the concurrent appearance of β-lactoglobulin and α-lactalbumin bands. In HTST and UHT heating systems, ratio of β-lactoglobulin to κ-casein increased linearly from the complex with increasing residence time.     

Influence of lupin-based milk alternative heat treatment and exopolysaccharide-producing lactic acid bacteria on the physical characteristics of lupin-based yogurt alternatives

In the present study we investigated the influence of heat treatment of lupin-based (LB) milk alternatives and different exopolysaccharide (EPS)-producing lactic acid bacteria on the physical characteristics of set-type LB yogurt alternatives. LB milk alternatives, obtained from protein isolate of Lupinus angustifolius cv. Boregine, were either pasteurized at 80 °C for 60 s or ultra-high temperature (UHT) heated at 140 °C for 10 s and was fermented with Lactobacillus plantarum TMW 1.460 and 1.1468, Pediococcus pentosaceus BGT B34 and Lactobacillus brevis BGT L150. Fermentation duration was strongly affected by heat treatment: different strains needed between 25 to 35 h in UHT LB milk alternative to reach a pH of 4.5 compared to 14 to 24 h in pasteurized LB milk alternative. EPS extraction revealed slightly higher amounts of EPS for UHT LB yogurt alternatives (~ 0.5–0.9 g/l; pasteurized: ~ 0.4–0.7 g/l). The more intensive heat treatment (UHT) resulted also in better rheological (apparent viscosity, hysteresis loop area, flow point, elastic, viscous and complex modulus) and textural properties (firmness, consistency, cohesiveness and index of viscosity) of the investigated LB yogurt alternatives. Furthermore, LB yogurt alternatives out of UHT milk alternative revealed a lower tendency to syneresis, measured with siphon and centrifugation method. This work contributes to the fundamental knowledge of the textural properties of LB yogurt alternatives.     

Effect of incubation temperature and caseinates on the rheological behaviour of Kefir

The effect of incubation temperature and the addition of caseinates on the rheological behaviour of kefir was studied by using a pneumatic tube viscometer of novel design. The results indicated that the incubation time increased as the incubation temperature was reduced and the casein concentration was increased. Kefir samples incubated at 25 °C showed the highest values of viscosity, while the samples incubated at 30 °C exhibited the lowest viscosity. The addition of caseinates caused the viscosity of the samples to increase and their flow behaviour index values to decrease. Kefir samples incubated at 30 °C exhibited the highest flow behaviour index values.     

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.     

Plasmin activity in direct-steam-injection UHT-processed reconstituted milk: Effects of preheat treatment

Ultra-high-temperature (UHT)-processed reconstituted milk that is subjected to a minimal preheat treatment during the direct-steam-injection heating process may have a shortened shelf-life as a result of plasmin-mediated proteolysis. Some manufacturers apply a preheat treatment before UHT treatment (140 °C for 4 s) with the aim of prolonging the shelf-life. Preheat treatments are, however, often arbitrary in terms of temperature and holding time. The aim of the current work was to determine guidelines for the minimum preheat treatment that will effectively inhibit or prevent plasmin-type enzyme activity in UHT milk. A selected range of preheat treatments was applied to milk preparations reconstituted from several batches of low-heat skim milk powder. Increased plasmin-type proteolysis was observed after intermediate preheat treatments at ⩾80 and <90 °C. Effective inhibition of plasmin-type proteolysis was obtained by preheating at 90 °C for 30 or 60 s.     

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.     

A Naturally Buffered Bulk Retentate Starter from Ultrafiltered Milk

Whole milk was ultrafiltered to approximately 4:1 protein concentration, heated to 85°C for 30 min, and cooled to 22°C. It was inoculated with a commercial frozen concentrated lactic starter to give approximately 10⁷ cfu/ml and incubated at 22°C for 12 h. A commercial phage inhibitory medium and 11% nonfat dry milk were used as controls. After 12 h, retentate had significantly higher colony forming units per milliliter (3.2 × 10⁹) and pH (5.21) than phage inhibitory medium (2.5 × 10⁹ and pH 5.02) and nonfat dry milk (2.4 × 10⁹ and pH 4.58). Retentate starter and phage inhibitory medium starter had equal activity in skim milk (.3% developed acidity in 4 h at 32°C) whereas nonfat dry milk starter had significantly lower activity (.26% developed acidity). After a further 8 h incubation at 22°C, retentate starter had the highest pH (4.95) compared with phage inhibitory medium (4.76) and nonfat dry milk (4.51). At this time retentate starter activity was higher (.3%) than phage inhibitory medium (.27%) and nonfat dry milk (.19%). In highly concentrated retentates (3.5:1 and 5:1), retentate starter lowered pH considerably quicker than nonfat dry milk starter.     

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.     

Graininess and roughness of stirred yoghurt as influenced by processing

The aim of this work was to study how heat treatment, the type of starter culture, incubation temperature, and storage time can affect the physicochemical characteristics of stirred yoghurt. A four-factor experimental design was used for data analysis. Yoghurt milk was heated at 95 °C for 5 min or 130 °C for 80 s. Yoghurts were produced with three different starter cultures that had been incubated at 37, 42 or 45 °C and stored at 4 °C for 15 days. Visual roughness, number of grains, perimeter of grains, storage modulus, and yield stress all decreased when heating temperature was increased, when an exopolysaccharide-producing starter culture was used, or when incubation temperature was decreased. Storage time did not affect any of the physicochemical properties of yoghurt, except for the pH.     

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.     

Effect of Serine Proteolytic Enzymes (Trypsin and Plasmin), Trypsin Inhibitor,and Plasminogen Activator Addition to Ultra-High Temperature Processed Milk

Proteolysis and gelation were investigated in single strength, 2% fat, UHT-processed milk following aseptic addition of combinations of plasmin, plasminogen, trypsin, trypsin inhibitor (Kunitz), and urokinase (plasminogen activator). Individual 250-ml milk containers processed by direct or indirect methods were examined for the following attributes over 10 mo: growth on slants, appearance, pH, apparent viscosity, gel formation, enzymatic activity, and casein breakdown. Control milk samples in the study did not gel. Addition of trypsin at 1.5 or 7.5 mg protein/L of milk or addition of plasmin at .3 or 1.5 mg protein/L did not result in gelation. However, containers with plasminogen at .3 mg protein/ L began forming a gel at 5.5 mo. Enzyme activity in plasminogen-treated samples was not detected spectrophotometrically using an L-lysine-p-nitroanilide substrate, but extensive casein breakdown was apparent by SDS-PAGE. The evidence suggests plasminogen-derived activity promotes UHT milk gelation.     

Effect of exopolysaccharides and proteolytic activity of Lactococcus lactis subsp. cremoris strains on the viscosity and structure of fermented milks

Two exopolysaccharide (EPS)-producing Lactococcus lactis subsp. cremoris strains (B35 and B891) were used to study the effect of the kinetics of EPS production and bacterial proteolytic activity on the structure of milk gels and the viscosity of stirred milk gels. Strains were grown at 20 °C in milk containing either yeast extract or casitone and at 30 °C in either milk alone or milk containing casitone. Lactococcal counts, pH decrease and production and molecular characteristics (molar mass and radius of gyration) of both EPSs were followed during milk fermentation. The level of proteolysis in the fermented milks was determined after 24 h of incubation. The results obtained showed that the yield of EPS and the timing of EPS production during milk-gel formation were the most important factors that influenced the structure of the milk gels and the viscosity of the stirred product. The proteolytic activity of the strains did not seem to play any significant role.     

Ethanol stability of casein micelles cross-linked with transglutaminase

The influence of transglutaminase (TGase)-induced cross-linking on the ethanol stability of skimmed milk was investigated. The stability of milk against ethanol-induced coagulation increased in sigmoidal fashion with milk pH (5.0–7.5) for all samples; ethanol stability also increased upon incubation (0–24 h) with 0.05 g L⁻¹ TGase at 30 °C. In untreated milk, addition of ethanol induced a collapse of the polyelectrolyte brush of κ-casein on the micelle surface, thereby facilitating micellar aggregation. Dynamic and static light scattering measurements indicated that in TGase-treated milk, the ethanol-induced collapse of the polyelectrolyte brush was far less than in untreated milk, suggesting that the increased ethanol stability of TGase-treated casein micelles is caused by the cross-linking of the polyelectrolyte brush on the micellar surface.     

Heat resistance of Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk

Nine strains of non-starter Lactobacillus paracasei isolated from semi-hard cheese made of pasteurised milk were selected for their anticlostridial activity. Resistance to thermisation (60 °C, 5 min) and pasteurisation (73 °C, 15 s) was investigated using a submerged-coil apparatus. MRS broth-grown cultures of all nine strains survived thermisation in buffer. The level of resistance to thermisation was strain dependent and lower for freshly grown cells (stationary phase cells) than for resting cells (freshly grown cells kept diluted 10-fold in MRS broth at 17 °C for 6 days). None of the nine Lb. paracasei strains survived or recovered after pasteurisation in buffer when grown in MRS broth, while seven of the nine strains survived pasteurisation in UHT whole milk when grown in milk. Identity of the strains was successfully confirmed during the experiments using repetitive-PCR analysis. The potential of Lb. paracasei strains to survive pasteurisation of cheese milk was demonstrated.     

Rennet-induced coagulation of enzymatically cross-linked casein micelles

The enzymatic cross-linking of casein micelles with transglutaminase had an adverse influence on rennet-induced coagulation. Incubation with transglutaminase at 30 °C progressively reduced the levels of monomeric caseins and increased rennet flocculation time (RFT) in a Berridge test. For incubation up to 3 h at 30 °C, the reciprocal of the RFT was linearly correlated with the level of residual monomeric κ-casein, indicating that at complete cross-linking flocculation is absent. After treatment for 4–24 h at 30 °C, no residual monomeric κ-casein was detected and no rennet-induced flocculation of the casein micelle suspension was observed. Monitoring rennet-induced coagulation by diffusing wave spectroscopy revealed that transglutaminase-induced inhibition of rennet-induced coagulation of casein micelles is primarily due to an inhibition of the secondary phase of rennet coagulation, i.e., the gelation and gel-firming phase of the casein micelle coagulation. The gelation and fusion of κ-casein-depleted para-casein micelles as in normal milk appears to be absent if the casein macropeptide remains attached to the casein micelle.     

Transglutaminase-induced caseinate gelation for the microencapsulation of probiotic cells

A novel method for the encapsulation of probiotic cells in foodgrade casein microcapsules was developed. The process is based on a transglutaminase-catalysed gelation of casein suspensions containing probiotic cells. Water insoluble, spherical capsules with a volume-based median diameter of 165 ± 23 μm resulted from the process. Encapsulation yields of 70 ± 15% and 93 ± 22% were achieved for Lactobacillus paracasei ssp. paracasei F19 and Bifidobacterium lactis Bb12, respectively. Analysis of living cell numbers after incubation of free and encapsulated probiotics in simulated gastric juice without pepsin at pH 2.5 and pH 3.6 (37 °C, 90 min) showed a protective effect due to microencapsulation under all conditions tested. The study indicates that transglutaminase-induced caseinate gelation can be applied to the microencapsulation of probiotics. Furthermore, it could be shown that an entrapment in a dense casein matrix can protect these microorganisms from damage due to pH-levels similar to those in the human stomach.     

Biocontrol of Staphylococcus aureus in curd manufacturing processes using bacteriophages

The ability of specific bacteriophages to inhibit Staphylococcus aureus growth in curd manufacturing processes was determined. Two lytic bacteriophages specific against S. aureus were obtained by DNA random deletion from the milk-isolated temperate phages, ΦH5 and ΦA72. A cocktail of these lytic phages, Φ88 and Φ35, at multiplicity of infection (MOI) of 100, produced a complete elimination of 3×10⁶ cfu mL⁻¹ of the pathogen in ultra-high-temperature (UHT) whole milk at 37 °C. Furthermore, the frequency of emergence of bacteriophage-insensitive mutants was reduced up to 200-fold in the presence of the two lytic phages compared with that detected with the combination of the temperate counterparts. The lytic phage derivatives, added to milk, were able to decrease rapidly the viable counts of S. aureus during curd manufacture. In acid curd, the pathogen was not detected after 4 h of incubation at 25 °C, whereas pathogen clearance was achieved within 1 h of incubation at 30 °C in renneted curd. These results indicate that lytic bacteriophages could be used as biopreservatives in the manufacture of particular dairy products.     

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.     

Physical Properties of Yogurt: A Comparison of Vat Versus Continuous Heating Systems of Milk

Milk was processed by vat (85°C for 10 to 40 min), high temperature, short time (98°C for .5 to 1.87 min), and ultra-high temperature (140°C for 2 to 8 s) heating systems and made into yogurt. Yogurt firmness ranged from 90 to 104 g force for vat treatments, 74 to 96 g for high temperature, short time treatments, and 47 to 65 g for ultra-high temperature treatments. Planned contrasts between heating systems indicated significantly higher yogurt firmness and viscosity for vat versus high temperature, short time and ultra-high temperature systems. Yogurt from high temperature, short time milk showed the highest water-holding capacity, followed by ultra-high temperature and vat treatments. Correlation coefficient between yogurt firmness and whey protein denaturation was .83 and between apparent viscosity and whey protein denaturation was .89. Sensory evaluation indicated an overall preference for yogurt made from high temperature, short time (1.87 min) milk.