Leszek Stepaniak

Enzymes in cheese technology

Abstract The significance and developments in the application of the principal exogenous enzymes, rennets, lipases, lysozyme, catalase, glucose oxidase and β-galactosidase, used in cheese manufacture and ripening are discussed. Some indigenous milk enzymes, notably plasmin, lipoprotein lipase, acid phosphatase and xanthine oxidase, may also be significant in cheese ripening.

Isolation and some properties of extracellular heat-stable lipases from Pseudomonas fluorescens strain AFT 36.

Aeration increased the growth and lipase production in milk by Pseudomonas fluorescens strain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented approximately 71% of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8.0 and 35 degrees C; it had a Km on tributyrin of 3.65 mM and was inhibited by concentrations of substrate greater than approximately 17 mM. The enzyme was very stable over the pH range 6-9; it was relatively heat-labile in phosphate buffer in the temperature range 60-80 degrees C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100-150 degrees C: the D values at 150 degrees C were approximately 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the corresponding Z values in the temperature range 100-150 degrees C were approximately 40 and approximately 42 degrees C and the Ea for inactivation were 7.65 X 10(4) J mol-1 and 6.97 X 10(4) J mol-1 respectively.

Characterization of the principal intracellular endopeptidase from Lactococcus lactis subsp. lactis MG1363

Abstract The major 70 kDa endopeptidase from the cytoplasm of plasmid-free Lactococcus lactis subsp. lactis MG1363 was purified to homogeneity. The enzyme was immunologically identical with the 70 kDa endopeptidase isolated by others from L. lactis subsp. cremoris Wg2. The enzyme did not hydrolyse α s1 -, β- or k-casein (CN) but it readily hydrolysed α s1 -CN f1–23, α s1 -CN f165–199 and three of five minor peptides released from α s1 -CN by chymosin. The enzyme also hydrolysed a number of peptide hormones. It cleaved preferentially the peptide bonds of α s1 -CN f1–23, α s1 -CN f165–199, oxidized insulin chain B and synthetic peptides which contained hydrophobic amino acids at position P 1′ , including ProLeu bonds. The enzyme also hydrolysed the GlyPhe bond of methionine enkephalin. More than 95% of enzyme activity was inhibited by 1 mM of o-phenanthroline or phosphor amidon. The enzyme was also inhibited strongly by a peptide fraction isolated from Cheddar cheese.

Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36

A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0.1 M-phosphate buffer, pH 6.6, being completely denatured above 70 degrees C in 1 min. It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 degrees C (minimum at 55 degrees C), but stability was very high above 80 degrees C in this buffer. D-values were determined at 10 degrees C intervals in the range 70-150 degrees C in SMS from which a Z value of 31.9 degrees C and an Ea of 8.82 X 10(4) J mol-1 were calculated; the half-life at 150 degrees C was 9 s. Instability at 55 degrees C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups. The extent of inactivation experienced at 80 degrees C was inversely related to the rate of heating to 80 degrees C, i.e. length of time spent in the neighbourhood of 55 degrees C. Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 degrees C, presumably due to substrate binding. Attempts to stabilize the enzyme at 55 degrees C by addition of EDTA or by adjusting the reaction pH to 4.2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented. Unlike the proteinase from Ps. fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 degrees C from 80, 100 or 150 degrees C, but did regain autolytic activity on cooling to below 50 degrees C to an extent dependent on the duration of holding at the lower temperature. It is suggested that on heating to approximately 55 degrees C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 degrees C.

Chemometrical analysis of proteolytic profiles during cheese ripening.

This research note gives a presentation of chemometrical analysis of proteolytic profiles obtained by electrophoresis and chromatography. An explanation of how the proteolytic profiles can be transformed to a multivariate data set and some basic information about multivariate statistical techniques as principal component analysis and discriminant analysis are provided. Some of the recent and most relevant research is presented to illustrate how this technique can be used in research on proteolysis during cheese ripening.

Isolation and characterization of heat stable proteinases from Pseudomonas isolate AFT 21.

Pseudomonas strain AFT 21 produced three heat stable extracellular proteinases in milk and nutrient broth at 7 or 21 degrees C, but the proportions depended on medium and cultivation temperature. The three proteinases were EDTA- and o-phenanthroline-sensitive metalloenzymes and were not inhibited by N-ethylmaleimide or phosphoramidon. Proteinases I and II showed maximum activity at pH 7-7.5 and proteinase III at pH 8.5. All three enzymes showed maximum activity at 45-47.5 degrees C, but had relatively high (19-27% of maximum) activity at 4 degrees C. They were unstable at 55 degrees C in phosphate buffer, pH 6.6, or synthetic milk ultrafiltrate (SMUF) containing 12 mmol Ca2+, but were stabilized by short preheating at 100 degrees C. They were extremely heat stable in both phosphate buffer and SMUF, pH 6.6, at 70-150 degrees C. Their D-values at 140 degrees C were 69, 54 and 80 s respectively. The Z-values for Pseudomonas AFT 21 proteinase III in phosphate buffer and SMUF were 29.7 and 30.3 degrees C respectively; the corresponding activation energies for inactivation were 8.7 x 10(4) J mol-1 and 9.2 X 10(4) J mol-1.

Peptides inhibitory to endopeptidase and aminopeptidase fromLactococcus lactis ssp.lactis MG1363, released from bovineβ-casein by chymosin, trypsin or chymotrypsin

Peptides inhibitory to the 70-kDa endopeptidase (PepO) from the cytoplasm ofLactococcus lactis ssp.lactis MG1363 were isolated from the supernatant (pH 4.6) of chymosin, tryptic and α-chymotryptic hydrolysates ofβ-casein (β-CN) by reversed-phase HPLC and identified by sequencing and mass spectrometry. Chymosin releasedβ-CN f193–209, kinetic constant (Ki) of which for inhibition of PepO was 60 μM. This peptide also inhibited (Ki=1700 μM) the 95-kDa aminopeptidase (PepN) fromL. lactis ssp.lactis MG 1363. Trypsin released two PepO-inhibitory peptides: one,β-CN f69–97, was not degradable by PepO (Ki=4.7 μM), while the other,β-CN f141–163, was degradable by PepO but competitively inhibited hydrolysis of methionine enkephalin by PepO. A peptide,β-CN f69–84, which inhibited PepO with aKi of 8.1 μM, was isolated from the α-chymotryptic hydrolysate. Peptides released fromβ-CN by trypsin or chymotrypsin had very little inhibitory activity against PepN. PepO degradedβ-CN f193–209 very slowly compared with the hydrolysis of methionine enkephalin. All four inhibitory peptides (β-CN f193–209, f69–97, f69–84, f141–163) were readily degraded by thermolysin.