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.     

Contribution of proteolytic activity associated with somatic cells in milk to cheese ripening

The effect of proteolytic enzymes from somatic cells on cheese quality was studied. In preliminary experiments, milk and two sodium caseinate systems (pH 6.5 and pH 5.2, the latter in the presence of 5% NaCl) were used as substrates to investigate the proteolytic activity of somatic cells recovered from mastitic milk. Urea-polyacrylamide gel electrophoretograms of hydrolysates suggested that somatic cell extracts contributed directly to proteolysis both in buffer and in milk, but that such activity was reduced by batch pasteurisation (63 °C for 30 min). Sodium caseinate was readily hydrolysed by somatic cell extracts; hydrolysis of α_s1-casein was greater at pH 5.2 and increased with level of somatic cells, suggesting that somatic cells contain proteolytic enzymes which are more active at acidic pH values. Subsequently, miniature Cheddar-type cheeses were made from batches of milk to which somatic cells were added (at levels of levels of 3×10⁵ or 6×10⁵ cells mL⁻¹), either before or after pasteurisation. Proteolysis during ripening of cheese (as measured by levels of pH 4.6-soluble nitrogen) increased with somatic cell addition, although this effect was reduced by pasteurisation after cell addition. Somatic cells may also have directly influenced cheese moisture content, which has been established as a principal indicator of quality of Cheddar-type cheese. Proteolytic enzymes of somatic cells from milk were shown to contribute directly to proteolysis in milk and cheese.     

Proteolysis in Milk Associated with Increasing Somatic Cell Counts

Individual cow samples were collected and preserved with potassium dichromate. Somatic cells counts were determined. Tyrosine value was used as an index of proteolysis. Sixty-six samples ranged in somatic cell count from < 50,000 to > 2,000,000/ml. Initial milk tyrosine values and tyrosine values for milks incubated for 24 h at 37°C showed proteolytic activity increased with increasing somatic cell count. The increase in proteolysis in preserved milk refrigerated for 72 h at 6.7°C was over 1.5 times greater in milks with > 1,000,000 cells/ml than in milks with < 60,000 cells/ml. When preserved milks were laboratory pasteurized, cooled, and stored at 6.7°C for 14 d, some proteolytic activity was detected in milks at all concentrations of somatic cells, and proteolysis increased as somatic cell counts increased. Laboratory-pasteurized samples of milk with various somatic cell counts were also incubated at 30°C for 3 and 6 h to duplicate the proteolysis that could occur during the ripening, coagulation, cutting, and cooking steps of cheese making. Again, the greatest increases in tyrosine were in milks with high somatic cell count. Protease(s) associated with elevated somatic cell counts will damage raw milk quality upon storage, pasteurized fluid milk over shelf-life, and milk during cheese making.     

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.     

HPLC of proteose peptones for evaluating ageing of packaged pasteurized milk

The proteolysis of casein (CN) occurring in packaged pasteurized milk (PM) during refrigerated storage was studied with relation to hygienic and microbiological characteristics of starting raw milk. Six batches of raw milk having standard plate count (SPC) from 1.5×10⁴ to 2.5×10⁵ cfu mL⁻¹ and somatic cell count (SCC) from 1.6×10⁵ to 4.4×10⁵ units mL⁻¹ were pasteurized (73 °C for 15 s), packaged and stored at 4 °C for 12 days. Capillary zone electrophoresis of CN showed breakdown of β-CN in all PM samples during storage. An HPLC method for monitoring proteose peptones (PP) formation was developed. Level of PP in PM samples increased, with keeping time from 667–789 to 947–1383 mg L⁻¹ and PP formation was significantly (P<0.05) related to SCC of starting raw milk. Electrospray ionization–mass spectrometry showed that PP were mainly represented by PP-5 from either A¹ or A² variants of β-CN. Five commercial samples of PM were analysed for PP formation during 14-day storage at 4 °C. Commercial samples prepared by microfiltration process or bactofugation combined with pasteurization showed the slowest formation of PP. The effect of storage temperature on PP formation was evaluated by keeping a conventional PM sample at either 8 or 12 °C for 12 days. Proteolysis of all major CNs upon action of plasmin and bacterial proteinases was observed under these conditions. PP level thus proves to be a reliable analytical index for evaluating the ageing of packaged PM during refrigerated storage.     

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.     

Thermal stability of plasminogen activators and plasminogen activation in heated milk

Thermal stabilities of bovine plasminogen (PG) activators, tissue and urokinase type (t-PA and u-PA), and their impact on activation of PG, were studied after application of various heating conditions (65, 75, 85, or 90 °C for 15, 20, or 30 s), in both milk and buffer systems. Both t-PA and u-PA were thermally stable in milk heated at ⩽75 °C. However, almost half of the t-PA activity and 30% of u-PA activity was lost after heating milk at 85 °C for 30 s. A lower heat stability of both t-PA and u-PA was observed in buffer than in milk. The decrease in level of PG was more pronounced than that of active plasmin in milk heated at ⩾85 °C for 30 s; however, the residual level of PG was considerably higher than the residual level of active PL. Overall, results indicated that u-PA plays a more significant role than t-PA in the activation of PG during storage of heated milk.     

Determination of Whey Protein Denaturation in Heat-Processed Milks: Comparison of Three Methods

Fast protein liquid chromatography was used to determine the extent of whey protein denaturation in various heat-treated milk samples: Sordi-indirect UHT (145°C/3 s), Dasi-direct UHT (142°C/3 s), HTST (80°C/30 s), and batch (63°C/30 min). Results were compared with other published methods (differential scanning calorimetry, whey protein nitrogen index, and Kjeldahl nitrogen on salt fractions). Results of the differential scanning calorimetry method were too erratic to be used to quantify whey protein denaturation. The remaining methods (fast protein liquid chromatography, Kjeldahl nitrogen, and whey protein nitrogen index) gave reproducible results and the extent of denaturation (highest to lowest) was consistently predicted as Sordi > Dasi > HTST > batch. There was no difference between fast protein liquid chromatography and Kjeldahl nitrogen, but there was a significant difference between fast protein liquid chromatography and whey protein nitrogen index and between Kjeldahl nitrogen and whey protein nitrogen index. Fast protein liquid chromatography appears to be an effective method to determine whey protein denaturation in heat-treated milks.     

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.     

Effect of addition of CO₂ to raw milk on quality of UHT-treated milk

The objective of this study was to evaluate the effect of addition of CO(2) to raw milk on UHT milk quality during storage. Control milk (without CO(2) addition) and treated milk (with CO(2) addition up to pH 6.2) were stored in bulk tanks at 4°C for 6d. After storage, both samples were UHT processed using indirect heating (140°C for 5s). Samples were aseptically packed in low-density polyethylene pouches and stored in the dark at room temperature. Raw milk was evaluated upon receipt for physicochemical composition, proteolysis, lipolysis, standard plate count, psychrotrophic bacteria, and Pseudomonas spp. counts, and after 6d of storage for proteolysis, lipolysis, and microbial counts. After processing, UHT milk samples were evaluated for physicochemical composition, proteolysis, and lipolysis. Samples were evaluated for proteolysis and lipolysis twice a month until 120d. Peptides from pH 4.6-soluble N filtrates were performed by reversed-phase HPLC after 1 and 120d of storage. A split-plot design was used and the complete experiment was carried out in triplicate. The results were evaluated by ANOVA and Tukey's test. After 6d of storage, CO(2)-treated raw milk kept its physicochemical and microbiological quality, whereas the untreated milk showed significant quality losses. A significant increase in proteolysis occurred during 120d of storage in both treatments, but the increase occurred 1.4 times faster in untreated UHT milk than in CO(2)-treated UHT milk. In both UHT milks, the proteolysis was a consequence of the action of plasmin and microbial proteases. However, the untreated UHT milk showed higher microbial protease activity than the treated UHT milk. The addition of CO(2) to the raw milk maintained the quality during storage, resulting in UHT milk with less proteolysis and possibly longer shelf life, which is usually limited by age gelation of UHT milk.     

Gelation Profiles of Yogurt as Affected by Heat Treatment of Milk

Milk was heated at 140°C for 6 s (UHT), 98°C for 1.87 min (HTST) and 85°C for 10 min (vat-pasteurized), homogenized, inoculated with yogurt culture, and incubated at 45°C. During incubation, pH, ionic Ca, and viscosity changes were continuously monitored by interfacing to a microcomputer. Samples for transmission electron microscopy were taken at selected pH intervals.Acid fermentation of milk was a multistage process comprising 1) an initial lag period of low viscosity; 2) a period of rapid viscosity change, and 3) a stage of high constant viscosity. The shapes of the viscosity versus incubation time curves were the same for all heat treatments differing only in the extent of viscosity change in stage 2. A microstructural study of UHT milk indicated that at pH 5.1, casein micelles appeared to dissociate into subparticles of approximately 30 to 40 nm diameter. By pH 4.8, subparticles reassociated into large casein aggregates of nonspecific shape and dimensions. Micellar dissociation was thought to be influenced by conversion of colloidal Ca to ionic Ca.     

Effect of ultra high temperature (UHT) treatment on coffee brew stability

In this work, the influence of an Ultra High Temperature (UHT) treatment on chemical and sensory composition of Arabica coffee brews for a longer shelf-life has been studied. A temperature of 120 °C for 2 s allows to obtain a microbiologically safe coffee brew, good valued from the sensory point of view. The behavior of the UHT vs non UHT treated coffee brew was followed throughout 120 days of storage at 4 °C. The UHT treatment keeps the typical acidity of the brews longer, delaying and softening the pH decrease and the development of sourness, which is one of the main causes for the rejection of stored coffee brews. The UHT treatment hardly affects the concentrations of caffeine and trigonelline, and of some phenolic compounds such as 5-caffeoylquinic (5-CQA), caffeic or ferulic acids. Sixteen key odorants and staling volatiles were analyzed by HS–GC–MS and lower changes were observed in the UHT treated coffee brew throughout storage. Higher DPPH scavenging activity was observed in the UHT treated coffee brew from days 60 to 120. In conclusion, the application of an UHT treatment is proposed to extend the shelf-life (up to 60 days) of stored coffee brews.     

Bovine milk procathepsin D: Presence and activity in heated milk and in extracts of rennet-free UF-Feta cheese

Milk samples were heat-treated at 72, 85 and 99°C for 15 or 60 s, and the effect on the stability of the milk acid proteinase zymogen procathepsin D was studied by combining immunoblotting using antibodies directed against bovine cathepsin D and its propeptide and by measuring residual procathepsin D-derived activity. Approximately half of the procathepsin D-derived activity detected in milk serum remained after heat treatment at 72°C/15 s or 72°C/60 s, while heat treatment at increased temperature further reduced the detectable activity. In accordance, immunoreactive procathepsin D was detected in serum from milk heated at 72°C/15 s and 72°C/60 s, while very low amounts of immunoreactivity were observed after treatment at higher temperatures. Contrary to the decrease in milk serum, the amount of procathepsin D antigen associated with casein micelles slightly increased with the temperature of the heat treatment, but still the measurable proteolytic activity derived from procathepsin D in the casein micelle samples decreased with temperature treatment. Moreover, the presence of procathepsin D and derived proteolytic activity was demonstrated in rennet free UF-Feta cheese. These results correlated with the finding of α_s1-I and para-κ-caseins in rennet free cheese. This is the first demonstration of procathepsin D in cheese, and of activity derived from indigenous procathepsin D in milk contributing to the proteolysis process in UF-products.     

Flavour profiles and survival of starter cultures of yoghurt produced from high-pressure homogenized milk

Volatile carbonyl compounds, organic acids and yoghurt bacteria counts were investigated in yoghurts made from ultra-high pressure homogenized milk. Yoghurts were manufactured from milk treated using ultra-high pressure homogenization at 200 or 300 MPa and at 30 °C or 40 °C, and compared with those produced from heat-treated milk with 3% added skim milk powder. To study the evolution of these parameters, samples were analysed after days 1, 14 and 28 of storage. Yoghurts from milk heat-treated or treated at 300 MPa had very similar profiles of organic acids and volatile compounds, as well as similar bacterial counts of both starter cultures. In comparison, yoghurts from milk treated at 200 MPa at either 30 °C or 40 °C gave different profiles, together with a sharp decrease in counts of lactobacilli. During storage, only slight differences in flavour compounds and yoghurt bacteria counts were detected, except in those samples from milk treated at 200 MPa.     

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.     

Effect of polysaccharides with different ionic charge on the rheological,microstructural and textural properties of acid milk gels

The effects of addition of polysaccharides with different ionic charge on rheology, microstructure, texture and water holding capacity (WHC) of acid milk gels were studied and compared to that of gelatin addition. Similar to gelatin, starch (neutral) and xanthan gum (anionic) did not prevent milk gelation in the first 30 min of the acidification stage, even at high concentrations, and the typical casein network in acid milk gels could still be seen from electron micrographs; gelling and melting of these hydrocolloids were observed during the cooling and heating stages at specific concentrations. On the other hand, two neutral polysaccharides, guar gum (≥ 0.05%) and locust bean gum [LBG] (≥ 0.1%) inhibited milk gelation from the beginning of the acidification stage; the microstructure of the gel was modified greatly and no gelling/melting was observed during the cooling or heating stages. Another anionic polysaccharide, carrageenan, induced earlier milk gelation at low concentration (≤ 0.05%), but inhibited gelation entirely at high concentration (0.2%); inflections at ~ 27 °C and 21 °C were also observed during the cooling and heating stages at 0.05% concentration. The gel microstructure was not changed greatly, but showed smaller particle size at a carrageenan concentration of 0.05% than control sample. None of the polysaccharides showed as much improvement in WHC of the milk gels as gelatin did. Hence, xanthan and starch were found to be closer to gelatin in their effect on acid milk gels compared to guar gum, LBG and carrageenan.     

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.     

High-pressure and temperature effects on the inactivation of Bacillus amyloliquefaciens,alkaline phosphatase and storage stability of conjugated linoleic acid in milk

Milk rich in conjugated linoleic acid (CLA, 42 ± 3 mg g^− 1 fat) was used to evaluate the impact of high-pressure sterilization (HPS). The pressure, temperature and time needed to reduce 7-log of Bacillus amyloliquefaciens endospores were determined in the presence of nisin (4–64 mg L^− 1). In addition, the inactivation of alkaline phosphatase was evaluated. After HPS treatment, the remaining CLA and formation of hydroperoxides were monitored during storage up to 60 d at 25 °C. The addition of nisin (≥ 16 mg L^− 1) to milk significantly enhanced the inactivation of B. amyloliquefaciens (7-log reduction) after treatment at 600 MPa, 120 °C and 5 min of holding time. These conditions were selected to evaluate the impact of HPS on the CLA retention and hydroperoxides formation. Milk with the addition of nisin and treated with HPS delivered higher retention of CLA and a lower concentration of hydroperoxides compared with the UHT equivalent process (125 °C/15 s and 135 °C/10 s).Industrial relevanceHigh-pressure sterilization is a valuable alternative to produce superior quality milk products in cases where traditional thermal treatments have failed. This study evaluated the impact of processing conditions on the conjugated linoleic acid content at conditions where commercial sterilization has been achieved (7-log reduction of B. amyloliquefaciens). The outcomes of this study are considered as a step further for the development of high-pressure sterilized milk.     

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.     

Temperature effects on Fossomatic cell counts in goats milk

In cows milk, analytical temperature has been identified as one of the possible factors affecting the somatic cell count (SCC). To establish the effect of the temperature used for SCC in goats milk, counts were performed on 45 goats milk samples using the Fossomatic counter. Tests were performed at two temperatures (40° and 60 °C) on preservative-free samples 3 h after collection, and on samples preserved with bronopol (BR) at 3 h, 1, 2, 3, and 4 d post-collection. The test temperature did not modify the SCC of the milk samples analyzed. Incubating samples 3 h post-collection at 60 °C failed to improve the SCC results. Similar counts were obtained for BR-preserved samples stored for 1–4 d at refrigeration temperature, suggesting the possibility of performing Fossomatic SCC in goats milk samples stored for this length of time.