Use of high pressure treatment to attenuate starter bacteria for use as adjuncts for Cheddar cheese manufacture

Attenuated starter bacteria cannot produce acid during cheese manufacture, but contain enzymes that contribute to cheese ripening. The aim of this study was to investigate attenuation of starter bacteria using high pressure treatment, for use in combination with a primary starter for Cheddar cheese manufacture, and to determine the effect of such adjunct cultures on secondary proteolysis during ripening. Lactococcus lactis ssp. cremoris HP and L. lactis ssp. cremoris 303 were attenuated by pressure treatment at 200 MPa for 20 min at 20 °C. Cheddar cheese was manufactured using untreated cultures of both these starter strains, either alone or in combination with their high pressure-treated equivalents. High pressure-treated starters did not produce acid during cheese manufacture and starter counts in cheeses manufactured using high pressure-treated starter did not differ from those of the controls. Higher levels of cell lysis were apparent in cheese manufactured using high pressure-treated strains than in the controls after 26 d of ripening. Small differences were observed in the peptide profiles of cheeses, analysed by reversed-phase HPLC; cheeses manufactured using high pressure-treated starters also had slightly higher levels of amino acids than the relevant controls. Overall, addition of high pressure-treated starter bacteria as a secondary starter culture accelerated secondary proteolysis in Cheddar cheese.

Industrial relevance

Attenuated starters provide extra pool of enzymes, which can influence cheese ripening, without affecting the cheese making schedule. This paper presents an alternative method for attenuation of starter bacteria using high pressure treatment and their subsequent use to accelerate secondary proteolysis in Cheddar cheese during ripening.     

Influence of proteolytic Lactococcus lactis subsp. cremoris on ripening of reduced-fat Cheddar cheese made with camel chymosin

This study investigated proteolysis in reduced-fat Cheddar cheese produced with camel chymosin and Lactococcus lactis subsp. cremoris with the ability to cleave the N-terminus of α_S1-casein. The aim was to match the activity of bovine chymosin, which leads to softer cheese structure than camel chymosin. Cheeses were analysed for gross composition, casein and peptide breakdown, release of free amino acids, structure parameters and sensory characteristics. Selected Lc. lactis subsp. cremoris increased the amount of peptides and, to a limited extent, the total amount of free amino acids in the cheeses. One group of experimental cheeses was found to have a significantly firmer structure, higher stress at fracture and modulus of deformability than the reference cheeses. The addition of the selected proteolytic dairy strains of Lc. lactis subsp. cremoris to the cheeses did not result in extended breakdown of α_S1-casein or a softer cheese structure.     

Influence of starters on chemical, biochemical, and sensory changes in Turkish White-brined cheese during ripening

Turkish White-brined cheese was manufactured using Lactococcus strains (Lactococcus lactis ssp. lactis NCDO763 plus L. lactis ssp. cremoris SK11 and L. lactis ssp. lactis UC317 plus L. lactis ssp. cremoris HP) or without a starter culture, and ripened for 90 d. It was found that the use of starters significantly influenced the physical, chemical, biochemical, and sensory properties of the cheeses. Chemical composition, pH, and sensory properties of cheeses made with starter were not affected by the different starter bacteria. The levels of soluble nitrogen fractions and urea-PAGE of the pH 4.6-insoluble fractions were found to be significantly different at various stages of ripening. Urea-PAGE patterns of the pH 4.6-insoluble fractions of the cheeses showed that considerable degradation of α_s1-casein occurred and that β-casein was more resistant to hydrolysis. The use of a starter culture significantly influenced the levels of 12% trichloroacetic acid-soluble nitrogen, 5% phosphotungstic acid-soluble nitrogen, free amino acids, total free fatty acids, and the peptide profiles (reverse phase-HPLC) of 70% (vol/vol) ethanol-soluble and insoluble fractions of the pH 4.6-soluble fraction of the cheeses. The levels of peptides in the cheeses increased during the ripening period. Principal component and hierarchical cluster analyses of electrophoretic and chromatographic results indicated that the cheeses were significantly different in terms of their peptide profiles and they were grouped based on the use and type of starter and stage of ripening. Levels of free amino acid in the cheeses differed; Leu, Glu, Phe, Lys, and Val were the most abundant amino acids. Nitrogen fractions, total free amino acids, total free fatty acids, and the levels of peptides resolved by reverse phase-HPLC increased during ripening. No significant differences were found between the sensory properties of cheeses made using a starter, but the cheese made without starter received lower scores than the cheeses made using a starter. It was found that the cheese made with strains NCDO763 plus SK11 had the best quality during ripening. It was concluded that the use of different starter bacteria caused significant differences in the quality of the cheese, and that each starter culture contributed to proteolysis to a different degree.     

Application of exopolysaccharide-producing cultures in reduced-fat Cheddar cheese: composition and proteolysis

Proteolysis during ripening of reduced fat Cheddar cheeses made with different exopolysaccharide (EPS)-producing and nonproducing cultures was studied. A ropy strain of Lactococcus lactis ssp. cremoris (JFR1) and capsule-forming nonropy and moderately ropy strains of Streptococcus thermophilus were used in making reduced-fat Cheddar cheese. Commercial Cheddar starter was used in making full-fat cheese. Results showed that the actual yield of cheese made with JFR1 was higher than that of all other reduced-fat cheeses. Cheese made with JFR1 contained higher moisture, moisture in the nonfat substance, and residual coagulant activity than all other reduced-fat cheeses. Proteolysis, as determined by PAGE and the level of water-soluble nitrogen, was also higher in cheese made with JFR1 than in all other cheeses. The HPLC analysis showed a significant increase in hydrophobic peptides (causing bitterness) during storage of cheese made with JFR1. Cheese made with the capsule-forming nonropy adjunct of S. thermophilus, which contained lower moisture and moisture in the nonfat substance levels and lower chymosin activity than did cheese made with JFR1, accumulated less hydrophobic peptides. In conclusion, some EPS-producing cultures produced reduced-fat Cheddar cheese with moisture in the nonfat substance similar to that in its full-fat counterpart without the need for modifying the standard cheese-making protocol. Such cultures might accumulate hydrophobic (bitter) peptides if they do not contain the system able to hydrolyze them. For making high quality reduced-fat Cheddar cheese, EPS-producing cultures should be used in conjunction with debittering strains.     

Isolation and characterisation of exopolysaccharide-producing Weissella and Lactobacillus and their application as adjunct cultures in Cheddar cheese

This study characterised exopolysaccharide-producing lactic acid bacteria and examined their potential for use in Cheddar cheese manufacture. Two strains were chosen for incorporation as adjunct cultures in Cheddar cheese manufacture: namely, the homopolysaccharide-producers Weissella cibaria MG1 and Lactobacillus reuteri cc2. These strains both produce dextrans with molecular masses ranging from 10⁵ to 10⁷ Da. Both strains were used in the production of miniature Cheddar cheeses that employed a conventional commercial cheese starter culture Lactococcus lactis R604. A cheese was also included that used purified dextran as an ingredient. The W. cibaria strain survived in cheese with levels increasing by 1.5 log cycles over the ripening period. All experimental cheeses (adjunct or exopolysaccharide ingredient) had higher moisture levels compared with the control cheese made using starter alone. Inclusion of the adjunct strains had no detectable negative effects on cheeses in terms of proteolysis.     

Application of ruthenium red and colloidal gold-labeled lectin for the visualization of bacterial exopolysaccharides in Cheddar cheese matrix using transmission electron microscopy

The objective of the present study was to develop a methodology for direct observation of capsular and ropy strains and their exopolysaccharides (EPS) in a Cheddar cheese matrix. Cheddar cheeses with 50% reduced fat were made from milk containing 1.7% fat using mixed starter culture containing either capsule-forming Lactococcus lactis subsp. cremoris (SMQ-461) or ropy L. lactis subsp. cremoris (JRF-1) strains. Control cheese was made using the EPS-negative L. lactis subsp. cremoris (RBL132) strain. Following cheese pressing, samples were taken from each cheese treatment and examined by transmission electron microscopy (TEM). Samples were divided into two series: the first was prepared following the conventional methods (involving fixation, post fixation, dehydration and embedding in resin) and the second with added ruthenium red at 0.15% (w/v) during the fixation, post fixation and washing procedures. Gold-labeled lectin was also used for the visualization and localization of EPS in cheese matrix. Electron micrographs showed that ruthenium red makes it possible to visualize and enhance the resolution of the EPS in a Cheddar matrix compared with the conventional method. The EPS layer of the capsular strain appeared regular and evenly distributed around the cell, whereas the cell-associated EPS layer produced by the ropy strain was longer, more irregular (having a filamentous structure) and unevenly surrounded the cell. EPS released from the ropy strain appeared to form a network-like structure located principally in whey pockets and appeared to interact with the casein matrix and fat globule membrane. Labeling EPS by lectin conjugated to colloidal gold could only be performed with conventional preparation of cheese samples and appeared to react only with the cell surface rather than with liberated EPS. Besides their ability to bind water and increase cheese yield, capsular and ropy strains used in this study appear to have potential autolytic characteristics, which may have an impact on cheese proteolysis, texture and flavor quality.     

Impact of ropy and capsular exopolysaccharide-producing strains of Lactococcus lactis subsp. cremoris on reduced-fat Cheddar cheese production and whey composition

Cheddar cheese mixed starter cultures containing exopolysaccharide (EPS)-producing strains of Lactococcus lactis subsp. cremoris (Lac. cremoris) were characterized and used for the production of reduced-fat Cheddar cheese (15% fat). The effects of ropy and capsular strains and their combination on cheese production and physical characteristics as well as composition of the resultant whey samples were investigated and compared with the impact of adding 0.2% (w/v) of lecithin, as a thickening agent, to cheese milk. Control cheese was made using EPS-non-producing Lac. cremoris. Cheeses made with capsular or ropy strains or their combination retained 3.6–4.8% more moisture and resulted in 0.29–1.19 kg/100 kg higher yield than control cheese. Lecithin also increased the moisture retention and cheese yield by 1.4% and 0.37%, respectively, over the control cheese. Lecithin addition also substantially increased viscosity, total solid content and concentrating time by ultra-filtration (UF) of the whey produced. Compared with lecithin addition, the application of EPS-producing strains increased the viscosity of the resultant whey slightly, while decreasing whey total solids, and prolonging the time required to concentrate whey samples by UF. The amount of EPS expelled in whey ranged from 31 to 53 mg L⁻¹. Retention of EPS-producing strains in cheese curd was remarkably higher than that of non-producing strains. These results indicate the capacity of EPS-producing Lac. cremoris for enhanced moisture retention in reduced-fat Cheddar cheese; these strains would be a promising alternative to commercial stabilizers.     

Enhanced nutty flavor formation in cheddar cheese made with a malty Lactococcus lactis adjunct culture

Nutty flavor in Cheddar cheese is desirable, and recent research demonstrated that 2- and 3-methyl butanal and 2-methyl propanal were primary sources of nutty flavors in Cheddar. Because malty strains of Lac-tococcus lactis (formerly Streptococcus lactis var. malti-genes) are characterized by the efficient production of these and other Strecker aldehydes during growth, this study investigated the influence of a malty L. lactis adjunct culture on nutty flavor development in Cheddar cheese. Cheeses made with different adjunct levels (0, 10⁴ cfu/mL, and 10⁵ cfu/mL) were ripened at 5 or 13°C and analyzed after 1 wk, 4 mo, and 8 mo by a combination of instrumental and sensory methods to characterize nutty flavor development. Cheeses ripened at 13°C developed aged flavors (brothy, sulfur, and nutty fla-vors) more rapidly than cheeses held at 5°C. Additionally, cheeses made with the adjunct culture showed more rapid and more intense nutty flavor development than control cheeses. Cheeses that had higher intensities of nutty flavors also had a higher concentration of 2/3-methyl butanal and 2-methyl propanal compared with control cheeses, which again confirmed that these compounds are a source of nutty flavor in Cheddar cheese. Results from this study provide a simple methodology for cheese manufacturers to obtain consistent nutty flavor in Cheddar cheese.     

Effect of exopolysaccharide produced by isogenic strains of Lactococcus lactis on half-fat Cheddar cheese

Fat-reduced cheeses often suffer from undesirable texture, flavor, and cooking properties. Exopolysaccharides (EPS) produced by starter strains have been proposed as a mechanism to increase yield and to improve the texture and cooking properties of reduced-fat cheeses. The objective of this work was to assess the influence of an exopolysaccharide on the yield, texture, cooking properties, and quality of half-fat Cheddar cheese. Two pilot-scale half-fat Cheddar cheeses were manufactured using single starters of an isogenic strain of Lactococcus lactis ssp. cremoris (DPC6532 and DPC6533) that differed in their ability to produce exopolysaccharide. Consequently, any differences detected between the cheeses were attributed to the presence of the exopolysaccharide. The results indicated that cheeses made with the exopolysaccharide-producing starter had an 8.17% increase in actual cheese yield (per 100 kg of milk), a 9.49% increase in moisture content, increase in water activity and water desorption rate at relative humidities ≤90%, significant differences in the cheeses microstructure, and a significant improvement in both textural and cooking properties, without negatively affecting the flavor profiles of the cheeses.     

Genotypic identification of some lactic acid bacteria by amplified fragment length polymorphism analysis and investigation of their potential usage as starter culture combinations in Beyaz cheese manufacture

In this study, 2 different starter culture combinations were prepared for cheesemaking. Starter culture combinations were formed from 8 strains of lactic acid bacteria. They were identified as Lactococcus lactis ssp. lactis (2 strains), Lactobacillus plantarum (5 strains), and Lactobacillus paraplantarum (1 strain) by amplified fragment length polymorphism analysis. The effects of these combinations on the physicochemical and microbiological properties of Beyaz cheeses were investigated. These cheeses were compared with Beyaz cheeses that were produced with a commercial starter culture containing Lc. lactis ssp. lactis and Lc. lactis ssp. cremoris as control. All cheeses were ripened in brine at 4°C for 90 d. Dry matter, fat in dry matter, titratable acidity, pH, salt in dry matter, total N, water-soluble N, and ripening index were determined. Sodium dodecyl sulfate-PAGE patterns of cheeses showed that α_S-casein and β-casein degraded slightly during the ripening period. Lactic acid bacteria, total mesophilic aerobic bacteria, yeast, molds, and coliforms were also counted. All analyses were repeated twice during d 7, 30, 60, and 90. The starter culture combinations were found to be significantly different from the control group in pH, salt content, and lactobacilli, lactococci, and total mesophilic aerobic bacteria counts, whereas the cheeses were similar in fat, dry matter content, and coliform, yeast, and mold counts. The sensory analysis of cheeses indicated that textural properties of control cheeses presented somewhat lower scores than those of the test groups. The panelists preferred the tastes of treatment cheeses, whereas cheeses with starter culture combinations and control cheeses had similar scores for appearance and flavor. These results indicated that both starter culture combinations are suitable for Beyaz cheese production.     

Comparative study by multivariate statistical analysis of proteolysis in a sodium caseinate solution under cheese-like conditions caused by strains of Lactococcus

Sonicated cells of Lactcoccus lactis subsp. cremoris 223, 227, SK11, AM1, Wg2 or L. lactis subsp. lactis UC317 were added to a sodium caseinate solution under conditions found in Cheddar or Gouda cheese during ripening (pH 5.25 and 5.0% (w/v) NaCl). Proteolysis was assessed by urea-polyacrylamide gel electrophoresis of the pH 4.6- and ethanol (70%)-insoluble fractions and reverse-phase-high-performance liquid chromatography of the ethanol (70%)-soluble fraction, and the resulting peptide profiles were analysed by principal component analysis and hierarchical cluster analysis. Statistical analysis of peptide profiles of the ethanol (70%)-soluble fraction from 2, 9, 17 or 23 d-old samples grouped the strains into three clusters which were similar to those found earlier for the same strains in an experiment with miniature Cheddar-type model cheeses by Shakeel-Ur-Rehman, Pripp, McSweeney and Fox (1999, Lait, 79, 361–383).     

Efficacy of four conjugal lactococcal phage resistance plasmids against phage in commercial Lactococcus lactis subsp. cremoris cheese starter strains

The efficacy of four lactococcal phage resistance plasmids (pNP40, pMU1311, pDI60 and pKP100) against phage was assessed after their conjugal transfer to four commercial Lactococcus lactis subsp. cremoris cheese starter strains and to the plasmid-free strain L. lactis subsp. cremoris MG1363. In MG1363, only pNP40 conferred resistance to prolate phages c2 and 643. Highest levels of resistance to small isometric phages in MG1363 occurred when pNP40 was stacked together with pMU1311 or pDI60. In the four starter strains, the plasmids conferred varying levels of resistance to small isometric phages. Growth and acidification rates in milk of most transconjugants derived from the starter strains decreased, but this was not always due to loss of plasmid-encoded cell wall proteinase (lactocepin) activity. Only one transconjugant grew during repeated subculture in milk with addition of factory wheys containing phages. This and the presence of bacteriocins encoded on pMU1311 and pDI60 limited application of the plasmids to protect L. lactis subsp. cremoris starters against phages in industry. However, some of the plasmids could be useful in extending the industry life of starters where fast acid production is not required or where bacteriocin production is acceptable.     

Culture Effect on Ripening Characteristics and Rheological Behavior of Cheddar Cheese

Six defined strains of bacteriophage-insensitive Streptococcus cremoris, grown in whey-based starter media, were used over a period of 10 months to produce more than 2 million kg of Cheddar cheese on continuous cheesemaking equipment. Flavor development in this cheese was less than that in cheese made with conventional bulk starter. Proteolysis in the trichloracetic acid (TCA) soluble cheese fraction was less in cheese manufactured using S. cremoris compared to conventional bulk starter. Rheological properties were studied and the force-compression curves related to the age, composition and pH of the cheese. Pattern recognition techniques were used to analyze the multivariate data. The texture was affected by the mechanical process, moisture content and yield point of the cheese. Casein proteolysis, age, culture type and firmness were the most discriminating variables affecting maturity.     

Improvement of texture and structure of reduced-fat Cheddar cheese by exopolysaccharide-producing lactococci

The objective of this study was to evaluate the effect of capsular and ropy exopolysaccharide (EPS)-producing strains of Lactococcus lactis ssp. cremoris on textural and microstructural attributes during ripening of 50%-reduced-fat Cheddar cheese. Cheeses were manufactured with added capsule- or ropy-forming strains individually or in combination. For comparison, reduced-fat cheese with or without lecithin added at 0.2% (wt/vol) to cheese milk and full-fat cheeses were made using EPS-nonproducing starter, and all cheeses were ripened at 7°C for 6 mo. Exopolysaccharide-producing strains increased cheese moisture retention by 3.6 to 4.8% and cheese yield by 0.28 to 1.19 kg/100 kg compared with control cheese, whereas lecithin-containing cheese retained 1.4% higher moisture and had 0.37 kg/100 kg higher yield over the control cheese. Texture profile analyses for 0-d-old cheeses revealed that cheeses with EPS-producing strains had less firm, springy, and cohesive texture but were more brittle than control cheeses. However, these effects became less pronounced after 6 mo of ripening. Using transmission electron microscopy, fresh and aged cheeses with added EPS-producing strains showed a less compact protein matrix through which larger whey pockets were dispersed compared with control cheese. The numerical analysis of transmission electron microscopy images showed that the area in the cheese matrix occupied by protein was smaller in cheeses with added EPS-producing strains than in control cheese. On the other hand, lecithin had little impact on both cheese texture and microstructure; after 6 mo, cheese containing lecithin showed a texture profile very close to that of control reduced-fat cheese. The protein-occupied area in the cheese matrix did not appear to be significantly affected by lecithin addition. Exopolysaccharide-producing strains could contribute to the modification of cheese texture and microstructure and thus modify the functional properties of reduced-fat Cheddar cheese.     

Autolysis of Lactococcus lactis

During cheese making, autolysis of Lactococcus lactis starter bacteria affects cheese flavour development through release of intracellular enzymes. The gene for the major autolysin in L. lactis, N-acetyl muramidase (AcmA), has been cloned and sequenced. The activity of AcmA alone, however, does not explain the huge variation in the extent of autolysis found in commercial L. lactis starter strains. Many such strains have multiple cell wall hydrolases that can be seen as different sized clearance bands in zymograms. In addition, the recently completed L. lactis subsp. lactis IL1403 genome sequence shows the presence of several open reading frames that putatively encode cell wall hydrolases having up to 42% predicted amino acid identity to AcmA. These enzymes could have roles in the autolysis of L. lactis. In this paper, we review the literature on autolysis of L. lactis and provide experimental evidence, based on Western blot and zymogram analysis, that commercial L. lactis starter strains express varying levels of AcmA and contain other cell wall hydrolases.     

Production of fresh Cheddar cheese curds with controlled postacidification and enhanced flavor

Cheddar cheese in curd form is very popular in eastern Canada. It is retailed immediately after cheese manufacturing and can be maintained at room temperature for 24 h to provide better texture and mouthfeel. Subsequently, the cheese curds must be stored at 4°C. The shelf life is generally 3 d. In this study, Cheddar cheese curds were produced by adding a high diacetyl flavor-producing strain (Lactococcus diacetylactis) to a thermophilic-based starter. The objective was to achieve both postacidification stability to increase the shelf life and enhanced flavor. The addition of L. diacetylactis increased processing time but did not affect cheese composition or the evolution of proteolysis and texture. During cheese manufacturing, streptococci became the dominant microflora in all cheeses, whereas populations of Lactococcus cremoris and L. diacetylactis decreased. During cheese storage, viable counts of L. diacetylactis and Streptococcus thermophilus increased but the counts of L. cremoris decreased. During cheese manufacturing and storage, the concentrations of lactic acid and diacetyl increased rapidly in cheeses produced with L. diacetylactis. Citric acid and galactose contents remained high in cheese made without L. diacetylactis. Sensory evaluation indicated that cheeses containing the L. diacetylactis strain were more flavorful and also had less sourness and could be stored at 4°C for up to 7 d.     

Application of exopolysaccharide-producing cultures in reduced-fat Cheddar cheese: cryo-scanning electron microscopy observations

The microstructure of reduced- and full-fat Cheddar cheeses made with exopolysaccharide (EPS)-producing and nonproducing cultures was observed using cryo-scanning electron microscopy. Fully hydrated cheese samples were rapidly frozen in liquid nitrogen slush (−207°C) and observed in their frozen hydrated state without the need for fat extraction. Different EPS-producing cultures were used in making reduced-fat Cheddar cheese. Full-fat cheese was made with a commercial EPS-nonproducing starter culture. The cryo-scanning electron micrographs showed that fat globules in the fully hydrated cheese were surrounded by cavities. Serum channels and pores in the protein network were clearly observed. Young (1-wk-old) full-fat cheese contained wide and long fat serum channels, which were formed because of fat coalescence. Such channels were not observed in the reduced-fat cheese. Young reduced-fat cheese made with EPS-nonproducing cultures contained fewer and larger pores than did reduced-fat cheese made with a ropy strain of Lactococcus lactis ssp. cremoris (JFR1), which had higher moisture levels. A 3-dimensional network of EPS was observed in large pores in cheese made with JFR1. Major changes in the size and distribution of pores within the structure of the protein network were observed in all reduced-fat cheeses, except that made with JFR1, as they aged. Changes in porosity were less pronounced in both the full-fat and the reduced-fat cheeses made with JFR1.     

Dynamics of starter,adjunct non-starter lactic acid bacteria and propionic acid bacteria in low-fat and full-fat Dutch-type cheese

The microbial dynamics of Dutch-type cheeses differing in starter (commercial DL starter or single strain of Lactococcus lactis ssp. cremoris), adjunct (Lactobacillus or Propionibacterium) and fat contents (10% or 28% fat) were investigated by culture-dependent and culture-independent analysis. The cheese microbiota was dominated by the adjunct Lactobacillus after 4 weeks of ripening and the fat content did not influence the microbial diversity. The Leuconostoc sp., presumably from the DL starter, was detected in cheeses made with added Lactobacillus plantarum and Lactobacillus rhamnosus and was not detected in cheese made with added Lactobacillus paracasei after 4 and 7 weeks. No Lactobacillus spp. were detected in cheese with added Propionibacterium, while Leuconostoc was the only species detected. In cheeses made with Lc. lactis ssp. cremoris as starter, the Lactobacillus microbiota was similar to the cheese milk microbiota after 24 h while after 4 weeks different species of Lactobacillus and Leuconostoc were detected.     

Differentiation of intracellular peptidases of starter and adjunct cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Selection of starter and adjunct cultures is important to minimize bitterness of Cheddar and Gouda cheeses. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry may be useful for rapid screening of cheese cultures for propensity to produce bitter cheese. The objective of this study was to demonstrate the application of MALDI-TOF for differentiating intracellular peptidase activities of starter and adjunct cultures on β-CN f193-209 under simulated cheese condition. Bovine β-casein was incubated with chymosin in 9.55 g/l citrate buffer (pH 5.4, 40 g/l sodium chloride) at 30°C for 24 h, followed by incubation with cell-free extract (CFE) of starter or adjunct culture. Mixed strains of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris designated as 56 and 105 were the sources of nonbitter and bitter starter cultures, respectively. Lactobacillus helveticus WSU-19 and W900R represented adjunct cultures having high and low debittering activities, respectively. The degradation pattern of β-CN f193-209 by CFE of WSU-19 indicates general aminopeptidase and endopeptidase activities, while degradation of the peptide by CFE of W900R, 56, and 105 are mainly from endopeptidase activity. The rates of β-CN f193-209 hydrolysis by CFE of WSU-19, W900R, 56, and 105 are 6.90, 0.38, 0.39, and 0.23 mg/l per h, respectively.