High Pressure Effects on Proteolytic and Glycolytic Enzymes Involved in Cheese Manufacturing

The activity of chymosin, plasmin, and Lactococcus lactis enzymes (cell envelope proteinase, intracellular peptidases, and glycolytic enzymes) were determined after 5-min exposures to pressures up to 800 MPa. Plasmin was unaffected by any pressure treatment. Chymosin activity was unaffected up to 400 MPa and decreased at 500 to 800 MPa. Fifty percent of control chymosin activity remained after the 800 MPa treatment. The lactococcal cell envelope proteinase (CEP) and intracellular peptidase activities were monitored in cell extracts of pressure-treated cells. A pressure of 100 MPa increased the CEP activity, whereas 200 MPa had no effect. At 300 MPa, CEP activity was reduced, and 400 to 800 MPa inactivated the enzyme. X-Prolyl-dipeptidyl aminopeptidase was insensitive to 5-min pressure treatments of 100 to 300 MPa, but was inactivated at 400 to 800 MPa. Aminopeptidase N was unaffected by 100 and 200 MPa. However, 300 MPa significantly reduced its activity, and 400 to 800 MPa inactivated it. Aminopeptidase C activity increased with increasing pressures up to 700 MPa. High pressure did not affect aminopeptidase A activity at any level. Hydrolysis of Lys-Ala-ρ-NA doubled after 300-MPa exposure, and was eliminated at 400 to 800 MPa. Glycolytic enzyme activities of pressure-treated cells were evaluated collectively by determining the titratable acidity as lactic acid produced by cell extracts in the presence of glucose. The titratable acidities produced by the 100 and 200 MPa samples were slightly increased compared to the control. At 300 to 800 MPa, no significant acid production was observed. These data demonstrate that high pressure causes no effect, activation, or inactivation of proteolytic and glycolytic enzymes depending on the pressure level and enzyme. Pressure treatment of cheese may alter enzymes involved in ripening, and pressure-treating L. lactis may provide a means to generate attenuated starters with altered enzyme profiles.Keywords: protease, cheese ripening, Lactococcus, peptidaseKeywords: protease, cheese ripening, Lactococcus, peptidas

MAPK feedback encodes a switch and timer for tunable stress adaptation in yeast

Signaling pathways can behave as switches or rheostats, generating binary or graded responses to a given cell stimulus. We evaluated whether a single signaling pathway can simultaneously encode a switch and a rheostat. We found that the kinase Hog1 mediated a bifurcated cellular response: Activation and commitment to adaptation to osmotic stress are switch-like, whereas protein induction and the resolution of this commitment are graded. Through experimentation, bioinformatics analysis, and computational modeling, we determined that graded recovery is encoded through feedback phosphorylation and a gene induction program that is both temporally staggered and variable across the population. This switch-to-rheostat signaling mechanism represents a versatile stress adaptation system, wherein a broad range of inputs generate an “all-in” response that is later tuned to allow graded recovery of individual cells over time

Water vapor barrier and rheological properties of simulated and industrial milkfat fractions

The applicability of anhydrous milkfat fractions in edible films and coatings was determined through moisture barrier, penetrometer, and stress-relaxation testing. Industrial milkfat fractions were compared along with tripalmitin:anhydrous milkfat blends which simulated milkfat fractions having different melting points. The water vapor permeability (WVP) was determined gravimetrically using a modified ASTM E96-92 method, while rheological properties were determined via cone penetration and stress-relaxation tests. The WVP of the lipid films, at 27.5°C and 0 to 100% RH difference, ranged from 0.0093 g·mm/kPa·h·m² for pure tripalmitin to 0.5993 g·mm/kPa·h·m² for anhydrous milkfat. Industrial milkfat fraction WVPs were intermediate to these extremes, with the higher-melting-point fraction having the lower WVP.This is the publisher’s final pdf. The published article is copyrighted by American Society of Agricultural Engineers and can be found at: https://elibrary.asabe.org/Keywords: Milkfat fractions, Edible films, Rheology, Water vapor permeabilit

Effects of High Pressure on the Viability, Morphology, Lysis, and Cell Wall Hydrolase Activity of Lactococcus lactis subsp. cremoris

Viability, morphology, lysis, and cell wall hydrolase activity of Lactococcus lactis subsp. cremoris MG1363 and SK11 were determined after exposure to pressure. Both strains were completely inactivated at pressures of 400 to 800 MPa but unaffected at 100 and 200 MPa. At 300 MPa, the MG1363 and SK11 populations decreased by 7.3 and 2.5 log cycles, respectively. Transmission electron microscopy indicated that pressure caused intracellular and cell envelope damage. Pressure-treated MG1363 cell suspensions lysed more rapidly over time than did non-pressure-treated controls. Twenty-four hours after pressure treatment, the percent lysis ranged from 13.0 (0.1 MPa) to 43.3 (300 MPa). Analysis of the MG1363 supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed pressure-induced lysis. Pressure did not induce lysis or membrane permeability of SK11. Renaturing SDS-PAGE (zymogram analysis) revealed two hydrolytic bands from MG1363 cell extracts treated at all pressures (0.1 to 800 MPa). Measuring the reducing sugars released during enzymatic cell wall breakdown provided a quantitative, nondenaturing assay of cell wall hydrolase activity. Cells treated at 100 MPa released significantly more reducing sugar than other samples, including the non-pressure-treated control, indicating that pressure can activate cell wall hydrolase activity or increase cell wall accessibility to the enzyme. The cell suspensions treated at 200 and 300 MPa did not differ significantly from the control, whereas cells treated at pressures greater than 400 MPa displayed reduced cell wall hydrolase activity. These data suggest that high pressure can cause inactivation, physical damage, and lysis in L. lactis. Pressure-induced lysis is strain dependent and not solely dependent upon cell wall hydrolase activity

Changes in the mechanical properties of corn tortillas due to the addition of glycerol and salt and selective high pressure treatments

The effects of high pressure processing (HPP) at 500 and 800 MPa for 1 and 10 min, and the addition of glycerol (4% db) and salt (1% db) on the thermal and physical properties of corn tortillas were investigated. Instron analysis illustrated an increase in stiffness following processing at 800 MPa for 10 min as compared to the control. Glycerol and salt decreased the temperature range of the major peak transition characterized by Dynamic Mechanical Analysis and decreased the stress and strain of the tortillas (Instron), thereby suggesting a more homogeneous transition and an increase in softness of the product due to additive addition. Therefore, addition of glycerol and salt along with HPP at short hold times does not adversely effect the mechanical properties of the corn tortillas

Effect of high pressure processing and addition of glycerol and salt on the properties of water in corn tortillas

The effects of high pressure processing (HPP) at 500 and 800 MPa for 1 and 10 min, and the addition of glycerol (4% w/w) and salt (1% w/w) on the properties of water in corn tortillas were investigated. Moisture content and water activity were not affected by either HPP or addition of glycerol/salt. Thermal analysis techniques (such as differential scanning calorimetry and thermogravimetric analysis) and nuclear magnetic resonance spectroscopy (1H cross-relaxation and 1H T1 and T2 relaxation times) were used to characterize these samples at a structural and molecular level, respectively. Addition of glycerol and salt significantly decreased the ‘freezable’ water content of the tortillas while HPP had little effect on the measured structural properties. The proton mobility was significantly decreased by HPP but not affected by addition of glycerol and salt. These results show that both composition and HPP may alter the structural and molecular properties of water in corn tortillas