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.     

Aflatoxin M1 in Manufactured Dairy Products Produced in the United States in 1979

In a 1979 survey of manufactured dairy products (992 samples of nonfat dry milk, vanilla ice cream, yogurt, Cheddar cheese, and cottage cheese) for aflatoxin M₁ contamination, one sample, a cottage cheese, had detectable aflatoxin equivalent to .08 ng/ml in the milk from which the product was made. Samples were taken by Food and Drug District inspectors from randomly selected establishments at three times throughout the year. The distribution of sample quotas to each District was weighted to double the representation of establishments in the southern tier of states. The conclusion from this survey is that in a “normal” year aflatoxin M₁ should not be in a manufactured dairy product in the United States at a level in excess of that from milk with .1 ng aflatoxin M₁/ml.     

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.     

Incorporation of Ultrafiltration Concentrated Whey Solids into Cheddar Cheese for Increased Yield

A process for incorporating whey solids into Cheddar cheese was evaluated. Whey was concentrated by ultrafiltration to between 9.8 and 20.3% solids (4.3 to 7.1% protein) and then heated at 75°C for 30 min. Return of this concentrate to cheese milk increased average yield 4.0% at constant cheese moisture. Cheese made by this procedure was lower in fat than control cheese and had a higher moisture content. Setting time was shorter, and acid development was faster. The pH was lower than that of the control cheese. Specific body, texture, and flavor characteristics were identified. Acid was the only flavor defect more prominent in experimental than in control cheese. None of the specific body or texture characteristics was significantly different.     

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.     

Ultrafiltration of Milk on French Farms and in the Making of a New Specialty Cheese Industry

Ultrafiltration and thermization of milk on dairy farms in France have been under study since 1979. More recently five dairy farms in Brittany have been routinely producing 2:1 whole milk retentate for Emmental and St. Paulin cheese making. The milk processed is ultrafiltered at 35°C and thermized at 72°C for 15 s in an Ultratherm unit. Cheese quality generally appears satisfactory.In Eastern France a new specialty cheese industry has been started utilizing 4.5:1 whole milk retentate produced by ultrafiltration conducted at 40°C. The specialty cheese attains its characteristic white surface from Penicillium album mold. It has a bland, nutty flavor and very soft, smooth texture. A surface bluing phenomenon occurs after 14 d.     

Cottage Cheese from Ultrafiltered Skim Milk Retentates in Industrial Cheese Making

Ultrafiltered skim milk retentates were transported to a large industrial cottage cheese plant for milk supplementation leading to cottage cheese. The resulting industrial products were observed for composition, yields, whey component losses, and quality.Ten lots of small curd cottage cheese were made in vats containing up to 6593 kg skim milk. Retentate supplemented skim milks, concentrated approximately 10% (1.1:1) and 20% (1.2:1) in total protein, were very similar in initial composition to the controls. Mean cheese yield values from milks supplemented to 1.2:1 total protein were significantly higher than mean unsupplemented control milk values. Cheese yield efficiencies, per kilogram total solids, were also significantly higher in the retentate cheese but not when calculated per kilogram total protein.Total solids, total protein, and ash were higher in cottage cheese wheys from retentate supplemented cheese and were directly related to retentate supplementation concentration. Mean whey component loss per kilogram cheese exhibited significant decreases from milks of higher retentate supplementation. Retentate supplemented skim milk produced industrial cottage cheese of comparable quality to cheese made from unsupplemented control skim milks.     

Use of high-pressure-treated milk for the production of reduced-fat cheese

Pasteurized (65°C, 30 min), pressurized (400 MPa, 22°C, 15 min) and pasteurized–pressurized milks were used for reduced-fat (approximately 32% of total solids) cheese production. Pressurization of milk increased the yield of reduced-fat cheese through an enhanced β-lactoglobulin and moisture retention. In addition, pressurisation of pasteurized skim milk improved its coagulation properties. The cheeses made from pasteurized–pressurized and pressurized milks showed a faster rate of protein breakdown than the cheese made from pasteurized milk, that might be mainly attributed to a higher level of residual rennet. Hardness of the experimental cheeses, as determined by both the sensory panel and instrumental analyses, decreased as the moisture content and proteolytic degradation of the cheese increased (pasteurized>pressurized>pasteurized–pressurized). In general terms, pressurization of reduced-fat milk prior to cheese-making improved cheese texture and thus accounted for a higher overall acceptability, except for the cheeses made from pasteurized–pressurized milk at 60 d of ripening, whose acceptability score was adversely affected by bitterness.     

Prediction and Characterization of Normal and Vat-Failed Cottage Cheese

An acidification-heat-coagulation test has been developed for predicting cottage cheese vat-failure potential of milk. Milk is fist acidified to pH 5.06 at 10°C and then heated at a slow rate (1°C increment per min). Poor quality acidified milk (> 10⁴ CFU/ml) forms small curds at 37°C and below. Good quality acidified milk (< 10⁴ CFU/ml) will form small curds at higher temperatures. By this procedure cottage cheese vat-failure potential of milk containing different levels of psychrotrophs can be predicted. Normal and vat-failed cottage cheese curds are characterized by % of grit in cottage cheese and amount of curd fines in whey.     

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.     

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.     

Growth of Lactobacillus bulgaricus in Milk. 1. Cell Elongation and the Role of Formic Acid in Boiled Milk

An abnormal elongation of cells occurred when Lactobacillus bulgaricus B5b was cultivated in milk heated at 100°C for 15 min. Nuclear staining revealed that the elongated cells were multinucleate, and septum staining indicated that the septum had not yet formed. Similar cell elongation was confirmed using other strains of L. bulgaricus.Extent of cell elongation varied with strains and heating conditions of milk; severe heating shortened the cell length. This cell elongation was not observed in autoclaved milk (121°C, 15 min), milk containing added formic acid and heated to 100°C for 15 min, and in mixed culture with Streptococcus thermophilus. The formate added to the milk was incorporated into purines of ribonucleic acid and deoxyribonucleic acid of the cells. Addition of formic acid or adenine stimulated ribonucleic acid synthesis in the cells cultivated in milk heated to 100°C for 15 min. The cell elongation was always accompanied by a remarkable depression of ribonucleic acid synthesis.Lactobacillus bulgaricus cells grown in milk heated to 100°C for 15 min are not able to produce adequate amounts of formic acid required for synthesis of purines de novo.     

Relationship of Protease Activity to Shelf-Life of Skim and Whole Milk

This study was to investigate causes of a possible difference in shelf-life of pasteurized skim milk and whole milk. Samples of skim and whole milk were obtained the day of processing, in 235 ml containers, from commercial dairies throughout South Carolina. They were stored at 4.5°C for 0,4,8,10,12,14, and 16 days; 7°C for 0,4,6,8,10, and 12 days; and 10°C for 0,1,2,3,4,5, and 6 days. On each sampling day milks were tested for coliform count, psychrotrophic count, flavor score, and relative protease activity. The shelf-life of skim milk was significantly less than that of whole milk when both were stored at 4.5°C and 7°C, but not at 10 C. Bacteria counts were not significantly different; thus, they were of no predictive value as anticipated changes in flavor score. Relationship between flavor score and relative protease activity of skim and whole milk was linear. Also, relative protease activity was significantly higher in skim milk as compared to whole milk stored at 4.5 and 7° C. Therefore, a higher protease activity in skim milk may account partially for its decreased shelf-life.     

Effect of thermal treatment on the activation of bovine plasminogen

The effect of heat on plasminogen (PG) was studied in both buffer and milk. The denaturation temperature for bovine PG was determined. Kinetic analysis of heated PG was conducted using a coupled assay and Spectrozyme^®PL. The effect of heat on PG activation was studied in milk samples with and without addition of PG activator. The temperature range for denaturation of PG was between 50.1 and 61.6 °C. The overall catalytic efficiency increased for PG heated to 60 °C and above, as indicated by the significant increase in k_cat/K_M. In a milk system, a significant increase in activation of PG was measured at 57 °C. A drop in activation of PG was observed at higher temperatures, suggesting counteracting effects of other milk components that were affected by heat. Results indicated that the increased activation of heated bovine PG observed in buffer and milk systems was probably due to the unfolding of PG as a result of thermal denaturation.     

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.     

Age of Ultrafiltered Fresh Farm Milk and Effect on Quality

Nonultrafiltered fresh whole raw and heated milks and their retentates concentrated by ultrafiltration to 2.07:1 volume concentration ratio were stored at 4.5 and −20°C and periodically scored for flavor quality.Nonultrafiltered whole raw milks were unclean, fermented, and rancid after 4 to 5 d. Nonultrafiltered heated milks over a 7 to 8-d period developed strong oxidized flavors. Milk retentates produced at 54°C for 120 min showed little or no flavor deterioration after 7 to 8 d at 4.5°C. In the same period at 4.5°C, raw milks from four trials showed 20 defects, heated milks 11 defects and 2.07:1 retentates 2 defects. Milks and retentates held frozen at −20°C for 2 mo showed greater deterioration than split samples held at 4.5°C, but whole milk retentates ultrafiltered to approximately 2:1 volume concentration ratio and stored frozen for 2 mo still displayed good to very good flavor.Thiobarbituric acid values of milks and retentates of ultrafiltration held 4 d at 4.5°C confirmed the presence of strong oxidized flavor in nonultrafiltered heated milks and its virtual absence in whole milk retentates.     

Activation energy measurements in rheological analysis of cheese

Activation energy of flow (E_a) between 30 and 44 °C was calculated from temperature sweeps of cheeses with contrasting characteristics to determine its usefulness in predicting rheological behavior upon heating. Cheddar, Colby, whole milk Mozzarella, low-moisture part-skim Mozzarella, Parmesan, soft goat, and Queso Fresco cheeses were heated from 22 to 70 °C, and E_a was calculated from the resulting Arrhenius plots. Protein and moisture content were highly correlated with E_a. The E_a values for goat cheese and Queso Fresco, which did not flow when heated, were between 30 and 60 kJ mol^?1. Cheddar, Colby, and the Mozzarellas did flow upon heating, and their E_a values were between 100 and 150 kJ mol^?1. Parmesan, the hardest cheese, flowed rapidly with heat and had an E_a > 180 kJ mol^?1. E_a provides an objective means of quantitating the flow of cheese, and together with elastic modulus and viscous modulus provides a picture of the behavior of cheese as it is heated.     

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.     

Response of High Producing Dairy Cows in Early Lactation to the Feeding of Heat-Treated Whole Soybeans

Fifty-eight multiparous cows were assigned randomly to one of two rations. Control cows received a concentrate mixture that contained 20% soybean meal as the protein supplement, and the experimental cows were fed a concentrate that contained 25% heat-treated whole soybeans. The experimental period started 10 d after calving and continued for 15 wk.Experimental cows peaked later in milk production (5 vs. 3 wk) but at a higher level (39.8 vs. 39.4 kg/d) than control cows. Although milk production was less during the first 4 wk, experimental cows surpassed the controls in wk 5 and increased the advantage to 2.0 kg/cow/d by wk 15. For the total 15-wk period, average milk production was 37.0 kg/d for the experimental cows compared with 36.2 kg/d for the controls.Total dry matter intake, lactation efficiency, body weight, and reproductive performance were similar for both treatments. Cows fed heated soybeans consumed more metabolizable energy, 61.6 vs. 60.4 Mcal/d for controls. Cows on experimental diet also had higher free fatty acids in plasma (5.6 vs. 4.8 mg/100 ml) and triglycerides (25.0 vs. 20.9 mg/100 ml). The acetate-to-propionate ratio of rumen acids was significantly lower in the experimental group (3.36 vs. 3.61).     

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.