scholarly journals Altered Specificity of Lactococcal Proteinase PI(Lactocepin I) in Humectant Systems Reflecting the Water Activity and Salt Content of Cheddar Cheese

1998 ◽  
Vol 64 (2) ◽  
pp. 588-593 ◽  
Author(s):  
Julian R. Reid ◽  
Tim Coolbear
Keyword(s):  
Enzyme Activity ◽  
Polyethylene Glycol ◽  
Lactococcus Lactis ◽  
Water Activity ◽  
Cell Envelope ◽  
Salt Content ◽  
Cheddar Cheese ◽  
Major Product ◽  
Cheese Ripening ◽  
Hydrolysis Of

ABSTRACT By using various humectant systems, the specificity of hydrolysis of αs1-, β-, and κ-caseins by the cell envelope-associated proteinase (lactocepin; EC 3.4.21.96 ) with type P1 specificity (i.e., lactocepin I) from Lactococcus lactis subsp. lactis BN1 was investigated at water activities (aw) and salt concentrations reflecting those in cheddar type cheese. In the presence of polyethylene glycol 20000 (PEG 20000)-NaCl (aw = 0.95), hydrolysis of β-casein resulted in production of the peptides comprising residues 1 to 6 and 47 to 52, which are characteristic of type PIII enzyme activity (lactocepin III) in buffer. The fragment comprising residues 1 through 166, inclusive (fragment 1-166), which is typical of lactocepin I activity in buffer systems, was not produced. Similarly, peptide 152-160 from κ-casein, which is usually produced in aqueous buffers exclusively by lactocepin III, was a major product of lactocepin I. Most of the specificity differences obtained in the presence of PEG 20000-NaCl were also obtained in the presence of PEG 20000 alone (aw = 0.99). In addition, αs1-casein, which normally is resistant to lactocepin I activity, was rapidly hydrolyzed in the presence of PEG 20000 alone. Hydrolysis of casein in the presence of PEG 300-NaCl or glycerol-NaCl (both having an aw of 0.95) was generally as expected for lactocepin I activity except that β-casein peptide 47-52 and κ-casein fragment 1-160 were produced; both of these are normally formed by lactocepin III in buffer. The differences in lactocepin specificity obtained in the humectant systems can be attributed to a combination of aw and humectant hydrophobicity, both of which are parameters that are potentially relevant to the cheese-ripening environment.

scholarly journals Specificity of Lactococcus lactis subsp.cremoris SK11 Proteinase, Lactocepin III, in Low-Water-Activity, High-Salt-Concentration Humectant Systems and Its Stability Compared with That of Lactocepin I

1999 ◽  
Vol 65 (7) ◽  
pp. 2947-2953 ◽  
Author(s):  
Julian R. Reid ◽  
Tim Coolbear
Keyword(s):  
Lactococcus Lactis ◽  
Water Activity ◽  
Salt Concentration ◽  
Cheddar Cheese ◽  
High Salt Concentration ◽  
Nacl Concentration ◽  
Peptide Composition ◽  
The Stability ◽  
The Relationship ◽  
Hydrolysis Of

ABSTRACT Marked changes in the specificity of hydrolysis of αs1-, β-, and κ-caseins by lactocepin III fromLactococcus lactis subsp. cremoris SK11 were found in humectant systems giving the equivalent water activity (aw) and salt concentration of cheddar cheese. Correlations were noted between certain peptides produced by the activity of lactocepin III in the humectant systems and peptides found in cheddar cheese. The stability of lactocepin III was compared with that of lactocepin I from L. lactis subsp. cremoris HP in the humectant systems at different pHs. Significant differences between the stability of each of the lactocepin types were evident. The relationship between stability and humectant type, aw, pH, and NaCl concentration was complex. Nevertheless, in those systems where aw, pH, and NaCl concentration were equivalent to those in cheddar cheese, lactocepin I was generally more stable than lactocepin III. It was concluded that differences in the specificity and/or stability of various lactocepin types are likely to persist in cheese itself and therefore potentially contribute to differences in the peptide composition of ripened cheese.

scholarly journals Contribution of Lactococcus lactis Cell Envelope Proteinase Specificity to Peptide Accumulation and Bitterness in Reduced-Fat Cheddar Cheese

2002 ◽  
Vol 68 (4) ◽  
pp. 1778-1785 ◽  
Author(s):  
Jeffery R. Broadbent ◽  
Mary Barnes ◽  
Charlotte Brennand ◽  
Marie Strickland ◽  
Kristen Houck ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Cell Envelope ◽  
Cheddar Cheese ◽  
Gene Replacement ◽  
Reversed Phase ◽  
Reduced Fat ◽  
Key Factor ◽  
Industrial Strains ◽  
Group A ◽  
Or Gene

ABSTRACT Bitterness is a flavor defect in Cheddar cheese that limits consumer acceptance, and specificity of the Lactococcus lactis extracellular proteinase (lactocepin) is widely believed to be a key factor in the development of bitter cheese. To better define the contribution of this enzyme to bitterness, we investigated peptide accumulation and bitterness in 50% reduced-fat Cheddar cheese manufactured with single isogenic strains of Lactococcus lactis as the only starter. Four isogens were developed for the study; one was lactocepin negative, and the others produced a lactocepin with group a, e, or h specificity. Analysis of cheese aqueous extracts by reversed-phase high-pressure liquid chromatography confirmed that accumulation of αS1-casein (f 1-23)-derived peptides f 1-9, f 1-13, f 1-16, and f 1-17 in cheese was directly influenced by lactocepin specificity. Trained sensory panelists demonstrated that Cheddar cheese made with isogenic starters that produced group a, e, or h lactocepin was significantly more bitter than cheese made with a proteinase-negative isogen and that propensity for bitterness was highest in cells that produced group h lactocepin. These results confirm the role of starter proteinase in bitterness and suggest that the propensity of some industrial strains for production of the bitter flavor defect in cheese could be altered by proteinase gene exchange or gene replacement.

The influence of phage-assisted lysis of Lactococcus lactis subsp. lactis ML8 on cheddar cheese ripening

1995 ◽  
Vol 5 (5) ◽  
pp. 451-472 ◽  
Author(s):  
Vaughan L. Crow ◽  
Frank G. Martley ◽  
Tim Coolbear ◽  
Sally J. Roundhill
Keyword(s):  
Lactococcus Lactis ◽  
Cheddar Cheese ◽  
Lactococcus Lactis Subsp ◽  
Cheese Ripening

Accelerated ripening of Cheddar cheese with the aminopeptidase of Brevibacterium linens and a commercial neutral proteinase

1990 ◽  
Vol 57 (4) ◽  
pp. 571-577 ◽  
Author(s):  
Kiyoshi Hayashi ◽  
Dean F. Revell ◽  
Barry A. Law
Keyword(s):  
Enzyme Activity ◽  
Sensory Analysis ◽  
Cheddar Cheese ◽  
Brevibacterium Linens ◽  
Neutral Proteinase ◽  
Cheese Ripening ◽  
Accelerated Ripening

SummaryPartly purified extracellular aminopeptidase from Brevibacterium linens was used to accelerate Cheddar cheese ripening. It was found that although the aminopeptidase was unstable in acidic buffer, it was highly stable in Cheddar cheese; negligible amounts of the enzyme activity were lost during 3 months' maturation. A better score for flavour in sensory analysis of enzyme-treated cheese was obtained by the combination of the aminopeptidase and a commercially available metalloproteinase (Neutrase) than by the metalloproteinase alone.

Isolation and identification of further peptides in the diafiltration retentate of the water-soluble fraction of Cheddar cheese

1997 ◽  
Vol 64 (3) ◽  
pp. 433-443 ◽  
Author(s):  
TANOJ K. SINGH ◽  
PATRICK F. FOX ◽  
ÁINE HEALY
Keyword(s):  
Soluble Fraction ◽  
Cell Envelope ◽  
Cheddar Cheese ◽  
Water Soluble ◽  
Soluble Extract ◽  
Amino Acid Sequencing ◽  
Isolation And Identification ◽  
Water Soluble Fraction ◽  
Water Soluble Extract ◽  
Hydrolysis Of

Several peptides were isolated from the diafiltration retentate, prepared using 10 kDa membranes, of the water-soluble extract from a commercial mature Cheddar cheese and identified by amino acid sequencing and mass spectrometry. Most of the peptides were from the N-terminal half of β-casein, but peptides from αs1- and αs2-caseins were also identified; the extract also contained α-lactalbumin. Identified peptides showed the important role played by lactococcal cell envelope proteinases in the degradation of primary proteolytic products from αs1- and β- caseins, produced by chymosin and plasmin respectively. Plasmin seemed to be involved in the hydrolysis of αs2-casein. Several phosphopeptides were identified and the action of phosphatase on these peptides was evident.

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

2015 ◽  
Vol 98 (1) ◽  
pp. 68-77 ◽  
Author(s):  
M.W. Børsting ◽  
K.B. Qvist ◽  
E. Brockmann ◽  
J. Vindeløv ◽  
T.L. Pedersen ◽  
...  
Keyword(s):  
Computer Modeling ◽  
Lactococcus Lactis ◽  
Cell Envelope ◽  
Gene Sequencing ◽  
Hydrolysis Of

Hydrolysis of β-casein by the cell-envelope-located PI-type protease of Lactococcus lactis: A modelling approach

2011 ◽  
Vol 21 (10) ◽  
pp. 755-762 ◽  
Author(s):  
Rafael Muñoz-Tamayo ◽  
Jolan de Groot ◽  
Edwin Bakx ◽  
Peter A. Wierenga ◽  
Harry Gruppen ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Cell Envelope ◽  
Hydrolysis Of ◽  
Modelling Approach

scholarly journals Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides

2005 ◽  
Vol 71 (6) ◽  
pp. 3025-3032 ◽  
Author(s):  
Vidya R. Sridhar ◽  
Joanne E. Hughes ◽  
Dennis L. Welker ◽  
Jeffery R. Broadbent ◽  
James L. Steele
Keyword(s):  
Genomic Sequence ◽  
Cheddar Cheese ◽  
Lactobacillus Helveticus ◽  
Model Systems ◽  
Cheese Ripening ◽  
Bitter Peptides ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Hydrolysis Of

ABSTRACT Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5α derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, β-casein (β-CN) (f193-209) and αS1-casein (αS1-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards αS1-CN (f1-9) and β-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze β-CN (f193-209) and αS1-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides αS1-CN (f1-9), αS1-CN (f1-13), and αS1-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with β-CN (f193-209) and αS1-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.

scholarly journals Starter strain related effects on the biochemical and sensory properties of Cheddar cheese

2006 ◽  
Vol 74 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Dara K Hickey ◽  
Kieran N Kilcawley ◽  
Tom P Beresford ◽  
Elizabeth M Sheehan ◽  
Martin G Wilkinson
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Lactococcus Lactis ◽  
Milk Fat ◽  
Cheddar Cheese ◽  
Lactobacillus Helveticus ◽  
Sensory Characteristics ◽  
Milk Supply ◽  
Cheese Ripening ◽  
The Impact

A detailed investigation was undertaken to determine the effects of four single starter strains, Lactococcus lactis subsp. lactis 303, Lc. lactis subsp. cremoris HP, Lc. lactis subsp. cremoris AM2, and Lactobacillus helveticus DPC4571 on the proteolytic, lipolytic and sensory characteristics of Cheddar cheese. Cheeses produced using the highly autolytic starters 4571 and AM2 positively impacted on flavour development, whereas cheeses produced from the poorly autolytic starters 303 and HP developed off-flavours. Starter selection impacted significantly on the proteolytic and sensory characteristics of the resulting Cheddar cheeses. It appeared that the autolytic and/or lipolytic properties of starter strains also influenced lipolysis, however lipolysis appeared to be limited due to a possible lack of availability or access to suitable milk fat substrates over ripening. The impact of lipolysis on the sensory characteristics of Cheddar cheese was unclear, possibly due to minimal differences in the extent of lipolysis between the cheeses at the end of ripening. As anticipated seasonal milk supply influenced both proteolysis and lipolysis in Cheddar cheese. The contribution of non-starter lactic acid bacteria towards proteolysis and lipolysis over the first 8 months of Cheddar cheese ripening was negligible.

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