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Physical stability and textural properties of eighteen low fat stirred yoghurts, differing in type of added dairy protein ingredients and in exopolysaccharide (EPS)-producing cultures, were studied by instrumental and sensorial measurements. Image analysis was performed to quantify the amount of grains and the amount of EPS. Enhanced textural properties and physical stability was observed in systems containing whey protein concentrate or particulated whey proteins as well as EPS-producing cultures. Irrespective of the culture used, casein and fractionated whey protein did not contribute to the textural properties. Sensory analysis confirmed the results obtained by instrumental measurements. Oral gel firmness was highly correlated to G′, viscosity at low shear rate, and instrumental measured firmness (R ≈ 0.82). Mouth thickness was well described by viscosity at high shear rate (R = 0.81), as well as by the hysteresis loop area and ropiness (R ≈ 0.81). Measured syneresis showed a significant relationship with visually evaluated syneresis (R = 0.76), and the amount of grains also correlated with visual evaluated graininess (R ≈ 0.81). The effect of the cultures on the sensory properties was revealed by multilevel simultaneous component analysis (MLSCA-P).

The effect of addition of low and high acyl gellan (LAG/HAG) at three different concentrations on the microstructure and texture of acidified milk systems with different casein to whey protein ratios was investigated. The systems with added LAG exhibited a continuous gel network whereas micro-phase separation occurred when HAG was added. The continuous gel network was indicative of complexation between the polysaccharides and casein, which in turn led to enhanced textural properties and reduced syneresis. Those properties were reduced by decreasing the casein to whey protein ratio as fewer complexes were formed. HAG appeared to be incompatible with casein micelles, which resulted in a micro-phase separated system. This caused decreased textural properties but a higher physical stability due to HAG present in the serum phase available to bind water. By decreasing the casein to whey protein ratio the textural properties were improved at expense of the physical stability, indicating a preferential binding between HAG and whey proteins.

Lactic acid bacteria with antifungal properties are applied for biopreservation of food. In order to further our understanding of their antifungal mechanism, there is an ongoing search for bioactive molecules. With a focus on the metabolites formed, bioassay-guided fractionation and comprehensive screening have identified compounds as antifungal. Although these are active, the compounds have been found in concentrations that are too low to account for the observed antifungal effect. It has been hypothesized that the formation of metabolites and consumption of nutrients during bacterial fermentations form the basis for the antifungal effect, i.e., the composition of the exometabolome. To build a more comprehensive view of the chemical changes induced by bacterial fermentation and the effects on mold growth, a strategy for correlating the exometabolomic profiles with mold growth was applied. The antifungal properties were assessed by measuring mold growth of two Penicillium strains on cell-free ferments of three strains of Lactobacillus paracasei pre-fermented in a chemically defined medium. Exometabolomic profiling was performed by reversed-phase liquid chromatography in combination with mass spectrometry in electrospray positive and negative modes. By multivariate data analysis, the three strains of Lb. paracasei were readily distinguished by the relative difference of their exometabolomes. The relative differences correlated with the relative growth of the two Penicillium strains. Metabolic footprinting proved to be a supplement to bioassay-guided fractionation for investigation of antifungal properties of bacterial ferments. Additionally, three previously identified and three novel antifungal metabolites from Lb. paracasei and their potential precursors were detected and assigned using the strategy.

Havarti 45+ cheese was manufactured from milk concentrated 1.8–4.6-fold by ultrafiltration (UF) and from normal milk, and the effect of concentration factor on plasmin activity and subsequent proteolysis in cheese during ripening was examined. There was decreased plasmin activity and a reduced rate of proteolysis of α S2 -casein and β -casein in the UF-cheeses, compared with the corresponding controls, independent of concentration factor. The decreased plasmin activity and slower breakdown of α S2 -casein and β -casein in UF-cheeses compared with traditional cheeses can be partly explained by the inclusion of an increased amount of plasmin inhibitors into the UF-cheeses. However, it is suggested that the differences in plasmin activity and proteolysis arise mainly as a result of inactivation of the plasminogen activation system during UF-concentration, due to a combination of time, temperature and the presence of air in the UF-equipment. The effect of milk treatment on the plasminogen activation system should be further investigated.

Lactic acid bacteria with antifungal properties can be used to control spoilage of food and feed. Previously, most of the identified metabolites have been isolated from cell-free fermentate of lactic acid bacteria with methods suboptimal for detecting possible contribution from volatiles to the antifungal activity. The role of volatile compounds in the antifungal activity of Lactobacillus paracasei DGCC 2132 in a chemically defined interaction medium (CDIM) and yogurt was therefore investigated with a sampling technique minimizing volatile loss. Diacetyl was identified as the major volatile produced by L. paracasei DGCC 2132 in CDIM. When the strain was added to a yogurt medium diacetyl as well as other volatiles also increased but the metabolome was more complex. Removal of L. paracasei DGCC 2132 cells from CDIM fermentate resulted in loss of both volatiles, including diacetyl, and the antifungal activity towards two strains of Penicillium spp. When adding diacetyl to CDIM or yogurt without L. paracasei DGCC 2132, marked inhibition was observed. Besides diacetyl, the antifungal properties of acetoin were examined, but no antifungal activity was observed. Overall, the results demonstrate the contribution of diacetyl in the antifungal effect of L. paracasei DGCC 2132 and indicate that the importance of volatiles may have been previously underestimated.

Chromatographic and electrophoretic methods have been established as valuable tools in characterising cheese ripening. In a recent IDF-Bulletin, a number of such methods have been described. Some of the methods have been used in the analyses of Danbo 45+ cheese from 41 vats produced in two different dairies. The purpose of this work was to investigate if the two dairies produced cheese with systematic differences in ripening characteristics and thereby possibly also in quality. Casein breakdown was monitored by capillary electrophoresis, peptides by reversed phase liquid chromatography and flavour compounds by gas chromatography-mass spectrometry. Multivariate analysis of the data was performed by principal component analysis, which revealed systematic differences between the dairies, most clearly in caseins and in peptides. Data from chemical and sensory analyses of a subset of the cheeses showed systematic variations in the chemical variables but only minor differences were found in the sensory attributes.

Two batches of rindless cheese of the type Danbo 45+ (semi-hard cheese with 45% fat in dry matter) were manufactured from milk subjected to heat treatment at 72°C, 80°C or 90°C for 15, 30 or 60 s. The effect of heat treatment on the proteolytic process in the cheeses during a 16-week ripening period was monitored by the use of reversed phase HPLC analysis of pH 4.6 soluble peptides. Peak-areas from peptide profiles were calculated essentially in 5-min intervals, and the resulting areas as well as the total peak-area were examined using principal component analysis (PCA). This resulted in a PCA-model with 5 principal components (PC) which accounted for more than 83% of the total variance in the data. Score plots revealed that PC1 separated the cheese samples mainly according to temperature of cheese milk heat treatment, whereas it was found that PC2 separated the samples mainly according to variations in age and trial number. The results were in agreement with observations on plasmin activity and digestion of casein in the experimental cheeses during the ripening period. To verify these observations it was shown that regions in the peptide profiles displaying large variation between cheeses manufactured from milk heated at low and high temperatures, respectively, included peptides created by plasmin digestion of casein.

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.

Danbo 45+ was manufactured from milk subjected to heat treatment at 72, 80 or 90°C for 15, 30 and 60 s. The effect of heat treatment on the activity of plasmin in the resulting cheeses and the subsequent proteolysis of caseins during ripening were examined. This revealed that the plasmin activity decreased as the temperature and the holding time increased. In addition, the cheeses showed decreased digestion of β - and α s2 -caseins during ripening, which was explained by variations in plasmin activity. The digestion of para- κ casein was slightly affected by heat treatment, which may be explained by steric hindrance due to the formation of thermally induced complexes with whey proteins. The digestion of α s1 -casein, however, was unaffected by the heat treatment and probably results primarily from the action of residual rennet. It is suggested that the altered proteolytic digestion of casein during ripening of cheese manufactured from heated milk results from reduced plasmin activity in the cheese as a consequence of thermal inactivation of the plasminogen activation system and thermally induced interactions between the components of the plasminogen activation system and β -lactoglobulin.

A procedure was developed for the isolation of plasminogen and plasmin from bovine milk. Pure undegraded plasminogen with N-terminal amino acid sequence and mobility in SDS-PAGE similar to plasminogen isolated from bovine blood was obtained. Additionally, the preparation contained the proteolytically modified midi-plasmin, consisting of kringle 4,5 and the light chain with an apparent molecular weight of 50 k Da in unreduced SDS-PAGE, and resulting in two bands of 30 and 26 k Da after reduction. Some 15% of the amino acid sequence of plasminogen/midi-plasmin isolated from milk was determined. The partial amino acid sequence was identical to that previously reported for plasminogen isolated from bovine blood (J. Schaller et al., (1985). Eur. J. Biochem., 149, 267–278), and to the bovine liver plasminogen cDNA sequence (L. Berglund et al., (1995). Int. Dairy J., 5, 593–603). Enzyme-linked immunosorbent assay (ELISA) and Western blotting experiments revealed that immunoreactive plasminogen was associated with acid-precipitated casein, rennet-coagulated casein and casein micelles. Some was found in acid whey and to a lesser extent in rennet whey. The amount of plasminogen associated with the milk fat globule membrane was very low and reflected the presence of casein. By Western blotting, immunoreactive plasminogen was found in zones with apparent molecular weights of 85, 80 and 50 k Da in unreduced gels. These hands may represent two plasminogen forms (85 and 80 k Da) and midi-plasmin/midi-plasminogen (50 k Da). The total concentration of plasminogen in bovine milk was estimated to 1.5 μg/mL.

We have investigated the content of plasminogen activators in bovine milk during mastitic inflammation induced by Staphylococcus aureus. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with fibrin agarose zymography and a coupled peptidyl anilide plasminogen activation assay of samples of whey prepared by acidification, we found that the level of tisue-type plasminogen activator (t-PA) in milk was increased immediately after infection and remained elevated during an experimental period of 42 days. The maximal increase was 10 to 20-fold. By zymography, we also demonstrated a strong increase in urokinase-type plasminogen activator (u-PA) associated with the bovine cells in the milk. By ligand blotting, we demonstrated an increase in the level of the urokinase-receptor (u-PAR) on the milk cells during inflammation. Plasma kallikrein was also detected as a plasminogen dependent proteolytic activity by zymography of whey samples. When analyzed in the presence of the t-PA in the milk, the plasma kallikrein lysis zone was strongly increased in mastitic whey, but when analyzed after separation from t-PA, its level was unaffected by mastitis; this could be ascribed to a t-PA dependent stimulation of plasma prekallikrein. These results suggest an important role for plasminogen activators in the inflammatory response during bovine mastitis. Using an enzyme-linked immunosorbent assay we measured the plasminogen/plasmin level during the inflammation, but found a less than 2-fold increase during the experimental period.

Bovine tissue-type plasminogen activator (tPA) was heterologously expressed in the methylotrophic yeast Pichia pastoris and characterized structurally and kinetically. The bovine single-chain tPA-mediated activation of bovine plasminogen was studied in the presence and absence of fibrinogen fragments. We have proposed a refined new method of kinetic analysis which allows examination of both stationary and prestationary phases of this process. The investigation revealed the presence of two interconvertible forms of the recombinant bovine tPA being in equilibrium at a 1 to 50 ratio. Only the minor form was able to bind and activate plasminogen. Saturation of the whole pool of tPA required high plasminogen concentration (Km >/= 5 microM) in order to reverse the equilibrium between the two forms. Fibrinogen fragments activated the single-chain tPA due to preferential binding and stabilization of the minor "active" form of the enzyme until all the molecules of tPA were converted. The same mechanism could be applied to human tPA as well. The Km values, obtained for recombinant bovine and human tPA in the presence of fibrinogen fragments, were found to be similar (Km = 0.1 microM) while kcat of human tPA was 5-10 times higher.