Classification of Lactococcus lactis cell envelope proteinase based on gene sequencing, peptides formed after hydrolysis of milk, and computer modeling.

Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from αS1-casein and ß-casein. A model structure of Lc. lactis CEP based on the crystal structure of Streptococcus C5a CEP was used to investigate the AA positions in the substrate-binding region. New AA positions were suggested, which could be relevant for the cleavage specificity of CEP; however, these could only explain 2 out of 3 found subgroups. The third subgroup could be explained by 1 to 5 AA positions located opposite the substrate binding region.

Composition and effect of blending of noncoagulating, poorly coagulating, and well-coagulating bovine milk from individual Danish Holstein cows.

The aim of the present investigation was to study the underlying causes of noncoagulating (NC) milk. Based on an initial screening in a herd of 53 Danish Holstein-Friesians, 20 individual Holstein-Friesian cows were selected for good and poor chymosin-induced coagulation properties; that is, the 10 cows producing milk with the poorest and best coagulating properties, respectively. These 20 selected cows were followed and resampled on several occasions to evaluate possible changes in coagulation properties. In the follow-up study, we found that among the 10 cows with the poorest coagulating properties, 4 cows consistently produced poorly coagulating (PC) or NC milk, corresponding to a frequency of 7%. Noncoagulating milk was defined as milk that failed to form a coagulum, defined as increase in the storage modulus (G') in oscillatory rheometry, within 45min after addition of chymosin. Poorly coagulating milk was characterized by forming a weak coagulum of low G'. Milk proteomic profiling and contents of different casein variants, ionic contents of Ca, P and Mg, κ-casein (CN) genotypes, casein micelle size, and coagulation properties of the 4 NC or PC samples were compared with milk samples of 4 cows producing milk with good coagulation properties. The studies included determination of production of caseinomacropeptide to ascertain whether noncoagulation could be ascribed to the first or second phase of chymosin-induced coagulation. Caseinomacropeptide was formed in all 8 milk samples after addition of chymosin, indicating that the first step (cleavage of κ-CN) was not the cause of inability to coagulate. Furthermore, the effect of mixing noncoagulating and well-coagulating milk was studied. By gradually blending NC with well-coagulating milk, the coagulation properties of the well-coagulating samples were compromised in a manner similar to titration. Milk samples from cows that consistently produced NC milk were further studied at the udder quarter level. The coagulation properties of the quarter milk samples were not significantly different from those of the composite milk sample, showing that poor coagulation traits and noncoagulation traits of the composite milk were not caused by the milk quality of a single quarter. The milk samples exhibiting PC or NC properties were all of the κ-CN variant AA genotype, and contained casein micelles with a larger mean diameter and a lower fraction of κ-CN relative to total CN than milk with good coagulation properties. Interestingly, the relative proportions of different phosphorylation forms of α-CN differed between well-coagulating milk and PC or NC milk samples. The PC and NC milk samples contained a lower proportion of the 2 less-phosphorylated variants of α-CN (α(S1)-CN-8P and α(S2)-CN-11P) compared with samples of milk that coagulated well.

Identification of peptides in aggregates formed during hydrolysis of beta-lactoglobulin B with a Glu and Asp specific microbial protease.

The purpose of the present study was to identify the peptides responsible for aggregate formation during hydrolysis of beta-lactoglobulin by BLP at neutral pH. Hydrolysates taken at various stages of aggregate formation were separated into a precipitate and a soluble phase and each was analyzed by CE and mass spectrometry. The aggregates consisted of six to seven major peptides of which four were tentatively identified. The peptides were positively charged at neutral pH and had a high charge-to-mass ratio at low pH. The fragment f135-158 seemed to be the initiator of aggregation, since it was present at high concentration in the aggregates at all stages, and the concentration of this peptide remained low in the supernatant. F135-158 contains several basic and acid amino acids alternating with hydrophobic amino acids, which is in accordance with formation of noncovalently linked aggregates, as previously shown.

Molecular self-assembly of partially hydrolysed alpha-lactalbumin resulting in strong gels with a novel microstructure.

Gelation of alpha-lactalbumin (alpha-la) incubated with a protease from Bacillus licheniformis (BLP) at 50 degrees C for 4 h was monitored using small oscillatory shear and the large deformation properties of final gels were characterized by uniaxial compression. Transmission electron microscopy was used to visualize the microstructure. Gels made from alpha-la (10 g/l) using BLP were almost transparent, although somewhat whitish, and they were more than 20 times stiffer (measured as complex modulus) than equivalent gels made from beta-lactoglobulin (beta-lg) at the same concentration. The microstructure of the gels consisted of non-branching, apparently hollow strands with a uniform diameter close to 20 nm, similar in overall structure to microtubules. Adding Ca2+ in amounts of 50 or 100 mM changed the spatial distribution of the strands and resulted in a reduction in the failure stress recorded in uniaxial compression. Apart from affecting the microstructure, Ca2+ was shown to be essential for the formation of the gels. It is proposed. that the mechanism behind the self-assembly of the partially hydrolysed alpha-la into long tubes is a spatially restricted creation of ionic bonds between Ca2+ and carboxyl acid groups on peptide fragments resulting from the action of BLP on alpha-la. Proteolysis of alpha-la with BLP in the presence of Ca2+ thus results in formation of a strong gel with a microstructure not previously observed in food protein systems.

Standardized reaction times used to describe the mechanism of enzyme-induced gelation in whey protein systems.

Whey protein isolate (WPI), either untreated or pretreated at 80 degrees C for 30 min, was incubated with a proteinase from Bacillus licheniformis until a gel was formed. Standardized reaction times, directly linked to the degree of hydrolysis, were obtained from plots of the relative amount of peptides released v. reaction time obtained under different conditions (enzyme concentration, temperature, pH, NaCl addition). This provided a connection between the gelation profile and the degree of hydrolysis. In the case of untreated WPI, gelation occurred at lower degrees of proteolysis when the enzyme concentration was decreased, demonstrating that a rate-limiting aggregation process occurred at the same time as the proteolysis in a manner similar to the renneting of milk. This was not the case for preheated WPI, when gelation was found to take place at a constant degree of proteolysis, independent of the enzyme concentration. In this case, the mechanism could be described by assuming the thermally induced aggregates present in this substrate had progressively more stabilizing peptide segments shaved off, resulting in increased attraction between individual aggregates that ultimately led to gelation. Results obtained at 40-60 degrees C supported this, as we found no effect of temperature on the degree of proteolysis at gelation for the untreated WPI, whereas the degree of proteolysis decreased with increasing temperature when heated WPI was hydrolysed. The effect of pH and NaCl addition on the process was to reduce repulsion between the aggregating species so that gelation was induced at a decreased degree of proteolysis.

Effect of partial hydrolysis with an immobilized proteinase on thermal gelation properties of beta-lactoglobulin B.

We have investigated the influence of partial hydrolysis with an immobilized proteinase from Bacillus licheniformis on the thermal gelation of isolated beta-lactoglobulin B. Gelation behaviour was determined by dynamic rheological measurements (small deformation) and the gels were characterized with respect to microstructure and water-holding properties. A fine-stranded gel with a complex modulus of approximately 2000 Pa was formed from beta-lactoglobulin (50 g/l in 75 mM-Tris-HCl, pH 7.5). Limited hydrolysis prior to thermal gelation resulted in coarser gels with thicker protein strands and larger pores. Gel structure correlated with its permeability, proton mobility and water-holding capacity. Total stiffness gel increased with low degrees of hydrolysis, but decreased after prolonged hydrolysis. Maximal gel stiffness was 1.5-fold that gels made from of unhydrolysed beta-lactoglobulin. This was much lower than the stiffening effect obtained after partial hydrolysis of whey protein isolate, showing that the gel strengthening effect of partial hydrolysis was depedent on the protein composition and/or the hydrolysis and gelatin conditions. A mechanism to explain the observed effects of hydrolysis on gelation and gel properties is presented.

Effect of high hydrostatic pressure on the enzymic hydrolysis of beta-lactoglobulin B by trypsin, thermolysin and pepsin.

Hydrolysis of beta-lactoglobulin B (beta-lg B) by pepsin, a process slow at ambient conditions, is facilitated at a moderately high hydrostatic pressure such as 300 MPa, corresponding to an apparent volume of activation delta V# = -63 ml mol-1 at pH 2.5, 30 degrees C and gamma/2 = 0.16. Digestion of beta-lg by trypsin and thermolysin is likewise enhanced by pressure, and the pressure effect has been traced to pressure denaturation of beta-lg B, which by high-pressure fluorescence spectroscopy has been shown to have a large negative volume of reaction, delta V(o) = -98 ml mol-1, at pH 6.7, 30 degrees C and gamma/2 = 0.16. Pressure denaturation is only slowly reversed following release of pressure and the enhanced digestibility is maintained at ambient pressure for several hours.

Microstructure and rheology of yogurt made with cultures differing only in their ability to produce exopolysaccharides.

Yogurt was made using an exopolysaccharide-producing strain of Streptococcus thermophilus and its genetic variant that only differed from the mother strain in its inability to produce exopolysaccharides. The microstructure was investigated using confocal scanning laser microscopy, allowing observation of fully hydrated yogurt and the distribution of exopolysaccharide within the protein network. Yogurt made with the exopolysaccharide-producing culture exhibited increased consistency coefficients, but lower flow behavior index, yield stress, viscoelastic moduli and phase angle values than did yogurt made with the culture unable to produce exopolysaccharide. The exopolysaccharides, when present, were found in pores in the gel network separate from the aggregated protein. These effects could be explained by the incompatibility of the exopolysaccharides with the protein aggregates in the milk. Stirring affected the yogurt made with exopolysaccharide differently from yogurt without exopolysaccharide, as it did not exhibit immediate syneresis, although the structural breakdown was increased. The shear-induced microstructure in a yogurt made with exopolysaccharide-producing culture was shown to consist of compartmentalized protein aggregates between channels containing exopolysaccharide, hindering syneresis as well as the buildup of structure after stirring.