Emanuele Smacchi

Peptides from several italian cheeses inhibitory to proteolytic enzymes of lactic acid bacteria, Pseudomonas fluorescens ATCC 948 and to the angiotensin I-converting enzyme

Abstract Water-soluble peptides from Mozzarella, Italico, Crescenza, and Gorgonzola cheeses were fractionated by reverse-phase fast protein liquid chromatography. Peptide fractions with inhibitory activity to amino- and endo-peptidases from Lactobacillus delbrueckii ssp. bulgaricus B397, Streptococcus thermophilus 305, and Lactococcus lactis ssp. cremoris Wg2 were found. Enzymes from Lactobacillus casei ssp. casei 2752 were less sensitive. Endopeptidase from Lactobacillus casei ssp. casei 2752 also had a different response to the effect of some inhibitors. It probably showed limited differences in catalysis and substrate positioning. Most of these inhibitory peptides were also effective in reducing the activity of the Pseudomonas fluorescens ATCC 948 endopeptidase and the angiotensin I-converting enzyme. Inhibitory peptide fractions from Mozzarella, Italico, and Crescenza cheeses had a certain degree of hydrophobicity while the peptide fraction from Gorgonzola cheese eluted in the initial part of the acetonitrile gradient. One of the inhibitory peptides contained in the water-soluble extract of Crescenza cheese was further purified and sequenced. It corresponded to the β-casein fragment 58-72.

Microbiology and biochemistry of gorgonzola cheese during ripening

Abstract A microbiological and biochemical characterization of Gorgonzola cheese during ripening is reported. Analyses were conducted on the core and under the rind of the cheese. In 86 day-old cheese there were high numbers of Streptococcus thermophilus, Penicillium roqueforti , lactococci and surface growth of micrococci, yeasts and moulds. A salt-in-moisture gradient from the surface to the core persisted until about 63 days. The increase in pH was initially higher in the cheese surface; the final value was ca. 6.80 in both cheese layers. After 86 days of ripening, the concentration of the water-soluble N in the core was more than 50% of the total N but was markedly less in the external region. The 12% TCA-soluble N was the only N fraction which increased markedly during late ripening. Proteolytic (peptidases and proteinase) activity strongly depended on the growth and enzyme activity of P. roqueforti . The proteolytic activity decreased from the centre to the surface of the cheese. Enzymes from the mould veins showed a very limited diffusion in the outer layer, giving zonal variations in the cheese. Electrophoresis showed extensive hydrolysis of α s 1 - and β-caseins, with the fastest degradation in the core. Electrophoresis of the pH 4.6-soluble fraction and RP-HPLC of pH 4.6-soluble, ethanol-soluble and -insoluble fractions showed a complex production of various sized peptides which were, in part, degraded subsequently. Limited differences were observed in the level of lipolysis and the two cheese layers had about the same final value of free fatty acids. The surface microflora contributed markedly to lipolysis, which depended less on the activity of P. roqueforti .

Purification and characterization of a cell surface-associated esterase from Lactobacillus fermentum DT41

Abstract A cell surface-associated esterase from Lactobacillus fermentum DT41, a starter used to produce Parmesan cheese, was purified to homogeneity by chromatography on Q-Sepharose, Sephacryl 200, Phenyl-Superose and Mono Q. The enzyme, together with a distinct cytoplasmic esterase, expressed the highest activity during the exponential phase of growth. The esterase was a monomer with a Mr of ca 67 kDa and was most active at pH 7.0 and 30–35 °C, retaining considerable activity at pH 5.0 and 15 °C. The enzyme was stable at the cooking temperature (54–56 °C for a few minutes) of Parmesan cheese, its D55 °C value was 35 min. The highest activity was determined on β-naphthyl butyrate, but marked hydrolysis also occurred with β-naphthyl esters of C2 to C10 fatty acids. β-Naphthyl esters of C14 to C18:1 fatty acids were not hydrolyzed and only tributyrin was degraded among the triglycerides. The Km on β-naphthyl butyrate was 0.31 mM with a Vmax of 140 μmol min−1mg−1. The esterase was strongly inhibited by 5 mM phenylmethylsulfonyl fluoride and by 1 mM Hg2+ and Ag+, and moderately stimulated by Ca2+ and Mg2+.

Microbiology and biochemistry of taleggio cheese during ripening

Abstract A microbiological and biochemical characterization of Taleggio cheese during ripening is reported. Analyses were conducted on the surface, middle and core layers of the cheese from three production batches. In the 42 day-old cheese there were high numbers of starter lactic acid bacteria and surface growth of yeasts and moulds. Coliforms were present at low number and a moderate increase of micrococci and contaminating bacteria also occurred at the cheese surface. A gradient of salt-in-moisture from the surface to the core persisted until about 21 days. A marked increase in pH was observed only on the cheese surface. Initially, the concentration of pH 4.6-soluble N was similar in the three layers, but from 21 days onwards, hydrolysis was more pronounced in the external region. No pronounced differences were observed in the proteolytic (peptidases and proteinase), esterolytic and lipolytic activities of the three cheese layers during the first 21 days of ripening but thereafter a marked gradient of the enzymatic activity was noted from high at the cheese surface to low in the core. Electrophoresis showed hydrolysis of α s 1 - rather than β-casein and primary proteolysis was very similar to that in Cheddar cheese. Electrophoresis of pH 4.6-soluble, ethanol-insoluble fractions showed that little production of various sized peptides occurred before 21 days and greater hydrolysis was detected in the cheese surface. The ratio of volatile to non-volatile free fatty acids did not change during ripening; butyric, palmitic, myristic and oleic acids were the principal fatty acids.

Accelerated ripening of Pecorino Umbro cheese

We have investigated accelerating the ripening of Pecorino Umbro cheese by adding crude cytoplasmic extract from Pseudomonas fluorescens, non-starter lactic acid bacteria (NSLAB) or cheese slurry. Microbiological and biochemical analyses and sensory evaluation were carried out on control and experimental cheeses over 28 d ripening. In the cheeses containing NSLAB or slurry, counts of mesophilic lactobacilli ranged from log 7.6 at day 1 to ∼ log 8.6 cfu/g after 28 d ripening. ∼ 2 log cycles higher than in the control cheese. All the experimental cheeses contained higher levels than the control of total free amino acids and N soluble at pH 4.6 and in 120 g trichloroacetic acid/l. Compared with the control, higher aminopeptidase and dipeptidase activities were found in the cheeses containing NSLAB and slurry, and especially in those containing the Pseudomonas enzyme. The cheeses containing NSLAB or slurry were characterized by an accumulation of short peptides (M r < 2000) detected by FPLC. Although the cheese containing enzyme had an atypical flavour, the addition of mesophilic lactobacilli reduced from 60 to 28 d the ripening period of Pecorino Umbro cheese, without the appearance of off flavour.

Purification and characterization of an extracellular proline iminopeptidase from Corynebacterium variabilis NCDO 2101

Aims: To screen the extracellular proteolytic and lipolytic activities of Corynebacterium variabilis NCDO 2101 and to purify and characterize a proline iminopeptidase enzyme in order to investigate the role of the major component of the smear of bacterial surface‐ripened cheeses.