Preliminary observations on proteolysis in Manchego cheese made with a defined-strain starter culture and adjunct starter (Lactobacillus plantarum) or a commercial starter

Different authors have demonstrated the potential of adding lactobacilli as adjunct cultures to pasteurized milk used in cheese manufacture. The aim of this work was to observe the effect of the use of a defined-strain starter culture and the addition of an adjunct culture (Lactobacillus plantarum) to cheesemilk in order to determine their effect on the ripening of Manchego cheese. Manchego cheeses were manufactured using one of the following starter culture systems: a defined starter consisting of Lactococcus lactis ssp. lactis and Leuconostoc mesenteroides ssp. dextranicum; a defined starter, as described above, and Lb. plantarum, which were isolated from a good quality Manchego cheese made from raw milk, or a commercial starter comprised of two strains of Lc. lactis. The cheeses were sampled at 15, 45, 90 and 150 d of ripening. Principal component analysis of peak heights of reversed-phase HPLC chromatograms of 70% (v/v) ethanol-insoluble and -soluble fractions distributed the samples according to the starter used in their manufacture. Quantitative differences in several peptides were evident between the three cheeses. Cheeses made with the defined-strain starters received higher scores for the flavour quality and intensity and for overall impression than the cheeses made with the commercial starter.     

Biogenic amine content and proteolysis in Manchego cheese manufactured with Lactobacillus paracasei subsp. paracasei as adjunct and other autochthonous strains as starters

Two different autochthonous strain starter cultures, in which the acidifying starter was composed of strains of Lactococcus lactis, were used for the manufacture of pasteurised milk Manchego cheese. Proteolysis parameters, biogenic amines and sensory characteristics were evaluated and compared with those of commercial starter Manchego cheese and raw milk Manchego cheese manufactured without starter. Autochthonous starter cheeses, and especially those including Lactobacillus paracasei subsp. paracasei as adjunct, presented higher levels of proteolysis than in commercial starter cheese. The concentrations of total biogenic amines in autochthonous starter cheeses were much lower than in raw milk cheese and even lower than in commercial starter cheese. Cheese manufactured with the adjunct strain gave the best results for both flavour intensity and flavour quality, and was the most preferred by panellists. The results suggest that the culture containing Lb. paracasei subsp. paracasei as adjunct could be used for the manufacture of industrial Manchego cheese.     

Aroma development in semi-hard reduced-fat cheese inoculated with Lactobacillus paracasei strains with different aminotransferase profiles

Reduced-fat, semi-hard round-eyed cheese was made from bovine milk with a mesophilic DL-starter and three Lactobacillus paracasei subsp. paracasei single-strain adjuncts with different aminotransferase (AT) activity profiles. The pilot-plant procedure was not influenced by the adjuncts, and similar cheese was made from all vats. The growth of each adjunct to dominate the cheese was confirmed by using DNA fingerprinting. Flavour profiles were different for cheeses made with the different Lactobacillus strains. Use of the adjunct CHCC 4256 significantly increased the content of flavour compounds that were produced from the branched-chain amino acids (BcAAs: Leu, Ile and Val) and Asp. These cheeses also had superior sensory characteristics as they tasted aromatic and sweet without bitterness. The adjunct CHCC 4256 did not have glutamate dehydrogenase activity, or the highest activity on BcAA, but showed typical AT activity, with a similar activity on Leu, Phe and Asp substrates.     

Influence of pasteurization,brining conditions and production environment on the microbiota of artisan Gouda-type cheeses

To monitor the effect of the indigenous milk microbiota and of technological and environmental parameters on the microbiota established in ripened cheese, the diversity and dynamics of the predominant microbial communities in artisan Gouda-type cheeses produced under different conditions was studied. A total of 22 cheese types differing in milk source, milk treatment, production environment and brining conditions were analyzed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) using total DNA extracts as well as DNA extracted from culturable fractions. Through band position analysis and band sequencing, the majority of DGGE bands could be attributed to lactic acid bacteria (LAB), although a few bands also belonged to staphylococci and gamma-Proteobacteria. Aided by principal component analysis (PCA) and multivariate analysis of variance (MANOVA), cheeses produced at different locations could clearly be differentiated. The same approach also allowed to distinguish raw and pasteurized milk cheeses, the former showing a more diverse microbiota in terms of a higher species richness and number of DGGE bands. No substantial differences were found between cheeses brined at two different locations. In conclusion, the combined PCR-DGGE approach relying on both total DNA extracts and culturable fractions proved its value for analyzing the effect of technological and environmental parameters on the diversity and dynamics of the microbiota in Gouda-type cheeses.     

Volatile composition and improvement of the aroma of industrial Manchego cheese by using Lactobacillus paracasei subsp. paracasei as adjunct and other autochthonous strains as starters

The use of several autochthonous strains of lactic acid bacteria, including Lactobacillus paracasei subsp. paracasei as adjunct of the starter in the manufacture of Manchego cheese, was evaluated in an attempt to improve the aroma of the industrial Manchego cheese. Volatile composition and odour characteristics were evaluated and compared to those in Manchego cheese manufactured with a commercial starter (CS) culture and with raw milk cheese manufactured without starter. Manchego cheeses manufactured with two autochthonous strains of Lactococcus lactis subsp. lactis displayed a similar volatile profile and odour characteristics to the cheese made with the CS. The use of the strain Lactobacillus paracasei subsp. paracasei CECT 7882 as adjunct of the Lactococcus strains produced cheeses with higher amounts of some free fatty acids and alcohols, acetoin, lactones, phenylacetaldehyde, 2-phenylethanol and linalool, and higher scores of the odour intensity, odour quality, and ewe’s milk odour than the CS cheeses. It resulted in an intensification and improvement of industrial Manchego cheese aroma.     

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.     

Short Communication: Culture-Independent Detection of Lactic Acid Bacteria Bacteriocin Genes in Two Traditional Slovenian Raw Milk Cheeses and Their Microbial Consortia

Two Slovenian traditional raw milk cheeses, Tolminc (from cows’ milk) and Kraški (from ewes’ milk), were examined for the presence of 19 lactic acid bacteria bacteriocin genes by PCR analysis of total DNA extracts from 9 cheeses and from consortia of strains isolated from these cheeses. Eleven bacteriocin genes were detected in at least one cheese or consortium, or from both. Different cheeses or consortia contained 3 to 9 bacteriocin determinants. Plantaricin A gene determinants were found in all cheese and consortia DNA extracts. Genes for enterocins A, B, P, L50A, and L50B, and the bacteriocin cytolysin were commonly detected, as were genes for nisin. These results indicate that bacteriocinogenic strains of Lactobacillus, Enterococcus, and Lactococcus genera with protective potential are common members of indigenous microbiota of raw milk cheeses, which can be a good source of new protective strains.     

Starter culture development for improving safety and quality of Domiati cheese

Eleven lactococci strains (sp. lactis and cremoris) were collected according to specific or selected characteristics for development of defined strain starter (DSS) to improve safety and nutritional quality of traditional and low salt Domiati cheese. Thirteen DSS; nisin-producing system or/and folate-producing strains were prepared. The behaviour of the strains in DSS was studied in milk and in two series of Domiati cheese; the first one made with 5% NaCl and salt tolerant strains, the second made with 3% NaCl and the control cheeses were made without starters. The population dynamics of strains and sensory evaluation of cheese corroborated the results in milk. All strains can grow well together and appeared to produce pleasant flavours, normal (typical) body and texture Domiati cheese. There was no apparent difference in cheese composition between cheeses in each series; the levels were within margins for composition of Domiati cheese. The levels of nisin (IU g⁻¹) ranged from 204 to 324 IU g⁻¹ in 3-months' cheeses. Folate concentration increased in cheeses made with DSS cultures than control and the level ranged from 5.5 to 11.1 μg 100 g⁻¹ in cheeses after 3 months. All results revealed that selected DSS can be used for improving Domiati cheese.     

Autolysis of selected Lactobacillus helveticus adjunct strains during Cheddar cheese ripening

Cheddar cheeses were manufactured on a pilot scale (500 L vats) with three different Lactobacillus helveticus strains, which showed varying degrees of autolysis, added as adjuncts to the starter. Autolysis of adjunct strains was monitored by reduction in cell numbers, level of intracellular enzymes released into the cheese, and by the consequent changes in the degree of proteolysis and concentration of free amino acids in the cheese. The flavour profiles of the cheeses at 6 months were also determined. Significant variation in viability of the Lb. helveticus strains, which showed a positive correlation with the indicators of autolysis, was observed. However, cheese manufactured with the most autolytic strain did not receive the highest flavour scores. The results indicate that whereas autolysis of adjunct strains is an important factor in Cheddar cheese flavour development, other factors also contribute to the overall flavour improvement observed.     

Influence of adjunct use and cheese microenvironment on nonstarter bacteria in reduced-fat cheddar-type cheese

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7 degrees C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 10(4) cfu/ml did not completely suppress "wild" NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety.     

Influence of microfiltration and adjunct culture on quality of Domiati cheese

The effects of microfiltration and pasteurization processes on proteolysis, lipolysis, and flavor development in Domiati cheese during 2 mo of pickling were studied. Cultures of starter lactic acid bacteria isolated from Egyptian dairy products were evaluated in experimental Domiati cheese for flavor development capabilities. In the first trial, raw skim milk was microfiltered and then the protein:fat ratio was standardized using pasteurized cream. Pasteurized milk with same protein:fat ratio was also used in the second trial. The chemical composition of cheeses seemed to be affected by milk treatment—microfiltration or pasteurization—rather than by the culture types. The moisture content was higher and the pH was lower in pasteurized milk cheeses than in microfiltered milk cheeses at d 1 of manufacture. Chemical composition of experimental cheeses was within the legal limits for Domiati cheese in Egypt. Proteolysis and lipolysis during cheese pickling were lower in microfiltered milk cheeses compared with pasteurized milk cheeses. Highly significant variations in free amino acids, free fatty acids, and sensory evaluation were found among the cultures used in Domiati cheesemaking. The cheese made using adjunct culture containing Lactobacillus delbrueckii ssp. lactis, Lactobacillus paracasei ssp. paracasei, Lactobacillus casei, Lactobacillus plantarum, and Enterococcus faecium received high scores in flavor acceptability. Cheeses made from microfiltered milk received a higher score in body and texture compared with cheeses made from pasteurized milk.     

Production of hard cheese from caprine milk by the use of two types of probiotic cultures as adjuncts

Hard cheeses (Kefalotyri-like) were manufactured from caprine milk with yoghurt as a starter (A), and with its partial replacement with the probiotic adjuncts Lactobacillus rhamnosus LC 705 (B) and/or Lactobacillus paracasei ssp. paracasei DC 412 (C). Both adjuncts retarded the growth of enterococci, and the environment in cheese B did not favour the recovery of lactic acid bacteria (LAB) on Rogosa agar. However, better recovery of the LAB population on M17 agar from cheeses B and C made with adjuncts was recorded early in ripening, and this was accompanied by a greater decrease in pH. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell protein demonstrated that cheese C, made with Lb. paracasei ssp. paracasei as adjunct, is a better vehicle for delivery of live probiotic cells (10 ⁷   cfu/g) to the gastrointestinal tract than cheese B, made with Lb. rhamnosus ; the latter did not belong to the predominant microflora of one out of the two B cheeses. Urea-PAGE electrophoresis results indicated that adjunct lactobacilli enhanced the degradation of both α _S -casein (α _S -CN) and β-casein (β-CN). In the fresh cheese, hydrolysis of α _S -CN was more rapid than β-CN, and the free amino acid content of B and C was higher than in A. Lipolysis products were also higher in B and C than in A as ripening progressed, and the organoleptic characteristics of these cheeses resulted in higher scores, in the order C > B > A. Thus, making Kefalotyri-like cheese from caprine milk with probiotic lactobacilli, particularly Lb. paracasei ssp. paracasei, as adjunct can be considered an effective way of producing a cheese with a large number of probiotic cells.     

Utilisation of microfluidisation to enhance enzymatic and metabolic potential of lactococcal strains as adjuncts in Gouda type cheese

Lactococcal strains were selected based on the release of key intracellular enzyme activities post-attenuation by microfluidisation. Attenuated strains were evaluated as adjuncts with commercial lactococcal starter and compared with a control without adjunct, and to a commercial Lactobacillus helveticus (FLAV54) adjunct in a mini vat Gouda type cheese system. No significant differences were noted in compositional parameters apart from a lower pH in one cheese, but differences were evident for proteolysis, volatile and sensory attributes after 12 weeks ripening. The effectiveness of adjuncts to augment cheese flavour was strain specific, and the influence of the starter in terms of release of intracellular enzymes was affected by the choice of adjunct. The sensory character of one attenuated lactococcal strain was similar to FLAV54. This study has highlighted the potential of microfluidisation to enhance enzymatic and metabolic performance of adjuncts in the development of cheese flavour.     

Functionality of enterococci in dairy products

Enterococci have important implications in the dairy industry. They occur as nonstarter lactic acid bacteria (NSLAB) in a variety of cheeses, especially artisan cheeses produced in southern Europe from raw or pasteurised milk, and in natural milk or whey starter cultures. They play an acknowledged role in the development of sensory characteristics during ripening of many cheeses and have been also used as components of cheese starter cultures. The positive influence of enterococci on cheese seems due to specific biochemical traits such as lipolytic activity, citrate utilisation, and production of aromatic volatile compounds. Some enterococci of dairy origin have also been reported to produce bacteriocins (enterocins) inhibitory against food spoilage or pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Vibrio cholerae, Clostridium spp., and Bacillus spp. The technological application of enterocins, shown to be produced during cheese manufacture, led to propose enterococci as adjunct starter or protective cultures in cheeses. There is evidence that enterococci, either added as adjunct starters or present as nonstarter NSLAB, could find potential application in the processing of some fermented dairy products. Literature suggest that the complex biochemical and ecological phenomena explaining the technological functionality of the enterococci in dairy products, are still to be fully understood. Clearly, the clinical research on enterococci underlines also that the safety of dairy products containing enterococci is an issue that the industry must carefully address before proceeding to their application.     

Employment of autochthonous microflora in Pecorino Sardo cheese manufacturing and evolution of physicochemical parameters during ripening

The isolation and identification of lactic acid bacteria (LAB) from raw ewes’ milk and traditional Pecorino Sardo cheese made from this milk without the addition of starter culture was carried out to define the autochthonous lactic microflora present in milk and the evolution of LAB during cheese ripening. Isolation of 275 strains belonging to different Lactococcus, Lactobacillus, Streptococcus and Enterococcus species was achieved. Coccal-shaped LAB were found to predominate during cheese fermentation, while lactobacilli were preponderate during the latter phase of ripening. The technological selection of a total of 174 LAB strains belonging to the species Lactococcus lactis, Streptococcus thermophilus, Lactobacillus helveticus and Lb. casei allowed an experimental starter to be prepared, in which a potentially probiotic species, Lb. casei was used. The suitability of the autochthonous starter culture was tested in cheese-making trials, using thermised ewes’ milk, by comparing experimental Pecorino Sardo cheese with a control cheese produced at industrial scale using a whey starter culture from previous batches of manufacture. In particular, microbiological and physicochemical parameters were determined over 210 days of cheese ripening. Although sensory evaluation did not show any significant difference between experimental and control Pecorino Sardo cheeses, the use of the selected autochthonous starter allowed the production of experimental cheese with a significantly higher level of free amino acids, in particular essential amino acids, in comparison with the Pecorino Sardo control cheeses.     

Nonstarter Lactobacillus strains as adjunct cultures for cheese making: in vitro characterization and performance in two model cheeses

Nonstarter lactic acid bacteria are the main uncontrolled factor in today's industrial cheese making and may be the cause of quality inconsistencies and defects in cheeses. In this context, adjunct cultures of selected lactobacilli from nonstarter lactic acid bacteria origin appear as the best alternative to indirectly control cheese biota. The objective of the present work was to study the technological properties of Lactobacillus strains isolated from cheese by in vitro and in situ assays. Milk acidification kinetics and proteolytic and acidifying activities were assessed, and peptide mapping of trichloroacetic acid 8% soluble fraction of milk cultures was performed by liquid chromatography. In addition, the tolerance to salts (NaCl and KCl) and the phage-resistance were investigated. Four strains were selected for testing as adjunct cultures in cheese making experiments at pilot plant scale. In in vitro assays, most strains acidified milk slowly and showed weak to moderate proteolytic activity. Fast strains decreased milk pH to 4.5 in 8 h, and continued acidification to 3.5 in 12 h or more. This group consisted mostly of Lactobacillus plantarum and Lactobacillus rhamnosus strains. Approximately one-third of the slow strains, which comprised mainly Lactobacillus casei, Lactobacillus fermentum, and Lactobacillus curvatus, were capable to grow when milk was supplemented with glucose and casein hydrolysate. Peptide maps were similar to those of lactic acid bacteria considered to have a moderate proteolytic activity. Most strains showed salt tolerance and resistance to specific phages. The Lactobacillus strains selected as adjunct cultures for cheese making experiments reached 10⁸ cfu/g in soft cheeses at 7 d of ripening, whereas they reached 10⁹ cfu/g in semihard cheeses after 15 d of ripening. In both cheese varieties, the adjunct culture population remained at high counts during all ripening, in some cases overcoming or equaling primary starter. Overall, proximate composition of cheeses with and without added lactobacilli did not differ; however, some of the tested strains continued acidifying during ripening, which was mainly noticed in soft cheeses and affected overall quality of the products. The lactobacilli strains with low acidifying activity showed appropriate technological characteristics for their use as adjunct cultures in soft and semihard cheeses.     

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.     

Screening and selection of Lactobacillus strains for use as adjunct cultures in production of semi-hard cheese

Thirty-three Lactobacillus strains were tested as adjuncts in a cheese model system. Eighteen strains originated from cheese (nine Lactobacillus spp. and nine Lb. paracasei/casei) and 15 from human intestinal mucosa (11 Lb. rhamnosus; three Lb. paracasei; one Lb. plantarum). Model cheeses weighing 120 g were made of cheese grains from full-scale production of washed curd semi-hard cheese (Herrg?rd). The model system was reproducible and similar to full-scale production with respect to moisture, salt content, pH and microbial flora. The model cheeses were sampled for aerobic and anaerobic plate count and viable counts of Lactobacillus and Lactococcus. The presence of adjuncts in the model cheeses was confirmed by typing isolates with Randomly Amplified Polymorphic DNA (RAPD). The sensory properties of model cheeses were described. In a first trial 23 of the 33 adjuncts were re-isolated from the corresponding model cheeses after 9 or 13 weeks. Adjuncts of Lb. paracasei were re-isolated more frequently than adjuncts of Lb. rhamnosus. Nine strains were selected, on the basis of their ability to grow and be a dominating part of the microflora of model cheese with interesting sensory properties. These strains were further studied together with two commercial cultures. The sensory influences on model cheeses of six of the adjuncts were confirmed, and flavour scores were in the range of 2.9-7.1 for model cheeses with different adjuncts while the control had a flavour score of 5.6 (0-10 scale). Survival and growth of seven out of the nine strains correlated with the results of the first trial. Growth and influence on flavour of four adjunct cultures were confirmed in experimental cheese manufactured in a 400-1 open vat.     

Production of peptides and free amino acids in a sterile extract describes peptidolysis in hard-cooked cheeses

Hard cooked cheeses are mostly manufactured with lactic starters of Lactobacillus helveticus, which constitute a major proteolytic agent in the food. In this work, we assessed the proteolysis produced by enzymes of two strains of L. helveticus in a new cheese model, which consisted of a sterile substrate prepared with hard-cooked cheeses, and identified the time of ripening when main changes in proteolysis are produced. The extract, a representative model of the aqueous phase of the cheeses, was obtained from Reggianito cheeses of different ripening times (3, 90, and 180 days) made with starters composed of the strains tested, either SF138 or SF209. To obtain the substrate, the cheese was extracted with water, then centrifuged and the aqueous phase was sterilized by filtration through membrane (0.45 ??m). The substrates were incubated at 34 °C during 21 days; samples were taken at 0, 3, 7, 14, and 21 days. Sterility was verified by plating samples on skim milk agar and incubating at 37 °C for 48 h. Proteolysis was determined by liquid chromatography of soluble peptides and free amino acids. Great variation in peptide profiles was found as incubation progressed in cheese extracts, which evidenced that proteases and peptidases from the starter were active and able to degrade the proteinaceous material available in the extracts. The extracts derived from cheeses with L. helveticus SF138 showed low production of peptides and a notable increase in free amino acids content during incubation. L. helveticus SF209, on the contrary, caused an increase on soluble peptides, but the free amino acids accumulation was lower than in the first case, which suggested that L. helveticus SF209 had either a low peptydolitic activity or produced an intense amino acids breakdown. This trend was more evident for extracts prepared with 90-day-old cheeses. It was concluded that the strains of L. helveticus assayed showed potentially complementary proteolytic abilities, as SF209 was able to provide a continuous replenishment of peptides during incubation, while SF138 increased their hydrolysis to free amino acids. The extract was an appropriate medium to model hard cooked cheese ripening in short periods of time.