Production of pyroglutamic acid by thermophilic lactic acid bacteria in hard-cooked mini-cheeses

Pyroglutamic acid is present in high amounts (0.5g/ 100g) in many cheese varieties-and particularly in extensively ripened Italian cheeses such as Grana Padano and Parmigiano Reggiano. An in vivo model system for cooked mini-cheese production and ripening acceleration was set up to demonstrate the ability of thermophilic lactic acid bacteria, used as a starter, to produce pyroglutamic acid (pGlu). In mini-cheeses stored at 38 and 30 degrees C for up to 45 d, all starters tested produced different amounts of pGlu. In descending order of pGlu production, the bacteria analyzed were: Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactobacillus delbrueckii subsp. lactis. Evidence for the presence of glutamine to pGlu cyclase activity in lactic acid bacteria was provided. Cell lysates obtained from cultures of L. helveticus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, and S. thermophilus showed the ability to cyclize glutamine to pGlu, resulting in processing yields from 1.4 to 30.3%, depending on the subspecies. Formation of pGlu from free glutamine appeared to be similar to that observed using a glutamine-glutamine dipeptide substrate. Under the experimental conditions applied, pGlu aminopeptidase activity was only detected in L. helveticus. Thus, pGlu formation in long-ripened cooked cheese may depend on the activity of thermophilic lactic acid bacteria.     

Streptococcus thermophilus 580 produces a bacteriocin potentially suitable for inhibition of Clostridium tyrobutyricum in hard cheese

A strain of Streptococcus thermophilus that inhibits Clostridium tyrobutyricum has been isolated from raw milk. The active compound produced disappears after a treatment with protease. However, unlike most bacteriocins, it is not thermoresistant, and the activity is completely lost after 1 h at 60 degrees C. Its inhibitory spectrum is limited to other thermophilic streptococci, Brochothrix, and sporulated gram-positive rods. So this bacteriocin could be different from those already described. This bacteriocin-producing strain could be used in thermophilic starter for hard cheese making because the bacteriocin is not active against thermophilic lactobacilli. It is produced in M17 medium during the decreasing temperature phase of the hard cheese-making process temperature cycle and is also produced in milk. Moreover, when Streptococcus thermophilus was cocultured with a Lactobacillus delbrueckii subsp. lactis starter strain, it seems to enhance the bacteriocin production. However the level of activity always decreases drastically during the stationary phase. But inhibition of Clostridium tyrobutyricum spores can be obtained in small-scale curds.     

Characterization of natural isolates of Lactobacillus strains to be used as starter cultures in dairy fermentation

The technological relevant characteristics of five homofermentative lactobacilli strains, isolated from natural fermented hard cheeses, were studied. Isolates CRL 581 and CRL 654, from Argentinian artesanal hard cheeses, and isolates CRL 1177, CRL 1178, and CRL 1179, from Italian Grana cheeses, were identified as Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus, respectively, by physiological and biochemical tests, SDS-PAGE of whole-cell proteins and sequencing of the variable (V1) region of the 16S ribosomal DNA. All strains showed high levels of beta-galactosidase activity. However, proteolytic activity varied widely among isolates. Strains CRL 581, CRL 654, and CRL 1177 hydrolyzed alpha- and beta-caseins and were able to coagulate reconstituted skim milk in less than 16 h at 42 degrees C. According to the substrate specificity, these proteinases have a caseinolytic activity comparable to that of the P(III)-type of lactococcal proteinases. No strains produced inhibitor substances (bacteriocin) and all were insensitive to attack by 14 L. helveticus- and L. delbrueckii subsp. lactis-specific bacteriophages.     

Indigenous raw milk microbiota influences the bacterial development in traditional cheese from an alpine natural park

Nostrano di Primiero is a 6-month ripened cheese produced from raw milk collected in the Paneveggio-Pale di San Martino Natural Park area in the Italian Dolomites. In summer, this cheese is made using milk collected from two different areas, Passo Rolle and Vanoi, in the Paneveggio Natural Park. During the experiment, the milk from the two areas was separately processed, and cheeses were made in the same cheese factory using the same technological process. The microbiota of raw milk and cheeses of the two areas was isolated and the dominant population was monitored by RAPD analysis and identified by 16S rRNA sequence. The milk of the Passo Rolle area was mainly composed of mesophilic strains, thermophilic Streptococcus thermophilus, and low amounts of enterococci were also found; the milk of the Vanoi area was dominated by mesophilic microbiota mostly Lactococcus lactis ssp. cremoris and ssp. lactis and Lactobacillus paracasei ssp. paracasei. The plating of the natural starter culture revealed the presence of a relevant community of thermophilic cocci and lower amounts of enterococci. The dynamic population analysis showed the importance of the natural starter culture in the first 2 days of cheese ripening in both cheeses. Moreover, the large biodiversity observed in the raw milks was also detected in the cheeses during ripening. The Vanoi cheese was dominated by Enterococcus faecium and Streptococcus macedonicus in the first two days and mesophilic 21 Lb. paracasei ssp. paracasei became the most represented population after 15 days of ripening. In the first few days, the Rolle cheese was characterized by being mainly composed of thermophilic S. macedonicus and S. thermophilus and secondarily by mesophilic cocci. During ripening, the microbiota composition changed, and at 15 days, mesophilic lactobacilli were the dominant population, but later, this was mainly composed of mesophilic cocci and lactobacilli. The taxonomical identification by 16S rRNA sequence confirmed a large biodiversity related to raw milk microbiota and only five strains of S. macedonicus, Lactobacillus plantarum, 21 Lb. paracasei ssp. paracasei, Lactobacillus fermentum and E. faecium were detected in both cheeses.     

Manufacture of Fior di Latte cheese by incorporation of probiotic lactobacilli

This work aimed to select heat-resistant probiotic lactobacilli to be added to Fior di Latte (high-moisture cow milk Mozzarella) cheese. First, 18 probiotic strains belonging to Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus reuteri were screened. Resistance to heating (65 or 55°C for 10 min) varied markedly between strains. Adaptation at 42°C for 10 min increased the heat resistance at 55°C for 10 min of all probiotic lactobacilli. Heat-adapted L. delbrueckii ssp. bulgaricus SP5 (decimal reduction time at 55°C of 227.4 min) and L. paracasei BGP1 (decimal reduction time at 55°C of 40.8 min) showed the highest survival under heat conditions that mimicked the stretching of the curd and were used for the manufacture of Fior di Latte cheese. Two technology options were chosen: chemical (addition of lactic acid to milk) or biological (Streptococcus thermophilus as starter culture) acidification with or without addition of probiotics. As determined by random amplified polymorphic DNA-PCR and 16S rRNA gene analyses, the cell density of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 in chemically or biologically acidified Fior di Latte cheese was approximately 8.0 log(10)cfu/g. Microbiological, compositional, biochemical, and sensory analyses (panel test by 30 untrained judges) showed that the use of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 enhanced flavor formation and shelf-life of Fior di Latte cheeses.     

Combined effects of concentrated thermophilic and mesophilic cultures and conditions of curd acidifications on the manufacture and quality of kasseri cheese

The effect offour process variables on the biological acidification of curd to be usedfor the manufacture of kasseri cheese was examined. The variables were (i) concentrated starter cultures: a thermophilic mixture of Streptococcus thermophilus and Lactobacillus delbrueckii subsp bulgar-icus compared with a blend of Lactococcus lactis subsp lactis, Lactococcus lactis subsp cremoris and Lactobacillus casei subsp casei, and (ii) acidification temperatures: 30 and 40° C for the thermophilic cultures and 20 and 30°C for the mesophiles. The use of concentrated starter cultures enabled the cheese to be made in one day as compared with three in the traditional system usually using raw milk and no culture. The mesophilic blend combined with a lower scalding temperature (35°C) gave a higher yield than the alternative culture, and the cheeses were higher in total solids, acidity andsalt; the same cheeses were also harder and less elastic. The use of mesophilic cultures and a low acidification temperature (20° C) is recommended.     

Molecular methods for identification of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus using methionine biosynthesis and 16S rRNA genes

Yoghurt and starter culture producers are still searching strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus to produce healthier yogurt with longer shelf life, better texture, taste and quality. However, selective identification of Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus from a mixed population using microbiological and biochemical methods is difficult, time consuming and may not be accurate. In this study, a quick, sensitive and accurate method is proposed to identify both Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus using PCR. The method is comprised of two parts. In the first part, methionine biosynthesis genes, known to be present in both species were partially amplified by designed primers (cysmet2F and cysmet2R). Partial amplification of the methionine biosynthesis gene which gives 700 bp fragment resulted in selective identification of Lb. bulgaricus and Strep. thermophilus. All 16 Lb. bulgaricus and 6 Strep. thermophilus isolates assessed by this method gave the expected amplification. On the other hand, further analysis of other closely related species with the same primers have indicated that the same product was also amplified in two more lactobacilli namely, Lb. delbrueckii subsp. lactis and Lb. helveticus species. Thus, in the second part of the method, further differentiation of Lb. delbrueckii subsp. bulgaricus and Strep. thermophilus from each other and these species was achieved using restriction analysis of 16S rRNA gene with EcoRI.     

Effect of thermophilic lactic acid bacteria on the viability of Salmonella serovar Typhimurium PT8 during milk fermentation and preparation of buffalo's yogurt

Viability of dairy-borne Salmonella enterica ssp. enterica serovar Typhimurium PT8 was studied during the fermentation of skim milk by thermophilic lactic acid bacteria (LAB). Longer generation times of Salmonella were found in mixed cultures of skim milk containing Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus or a mixture of them (1:1), as compared with single cultures of the pathogen. Salmonella was less able to survive in mixed cultures with these LAB during prolonged incubation at 41°C and also during cold storage of the fermented milk. L actobacillus ssp. bulgaricus and its mixture with S. thermophilus were more inhibitory to the growth and survival of Salmonella than was S. thermophilus . This was associated with higher ability of L . ssp. bulgaricus and the mixture to develop acidity in milk than S. thermophilus . Examining the antibacterial activity of these LAB towards Salmonella showed that other factors including heat-resistant and heat-labile compounds were involved in inhibiting the pathogen by these cultures. The viability of the same Salmonella strain during the preparation and cold storage of buffalo's yogurt was also examined. Salmonella was found to survive longer in yogurt made with starter containing probiotic bacteria than in that prepared with the traditional starter. This was ascribed to the development of lower pH by the traditional starter.     

Biopreservation by Lactobacillus paracasei in coculture with Streptococcus thermophilus in potentially probiotic and synbiotic fresh cream cheeses

The viability of Lactobacillus paracasei and its effect on growth of the microbiota in potentially probiotic and synbiotic fresh cheeses during storage at 4 +/- 1 degree C was investigated. Three cheese-making trials (T1, T2, and T3) were prepared in quadruplicate, all supplemented with a Streptococcus thermophilus culture. L. paracasei subsp. paracasei was added to cheeses in T1 and T2, and inulin was added to cheeses in T2. Counts of L. paracasei, S. thermophilus, coliforms, Escherichia coli, Staphylococcus spp., DNase-positive Staphylococcus, and yeasts and molds were monitored during storage for up to 21 days. Viable counts of L. paracasei in probiotic (T1) and synbiotic (T2) cheeses remained above 7 log CFU/g during the entire storage period, whereas counts of S. thermophilus remained above 9.5 log CFU/g for cheeses from TI, T2, and T3. Populations of coliforms, Staphylococcus spp., and DNase-positive Staphylococcus were higher in T3 cheese and differed significantly from those in cheeses from T1 and T2 (P < 0.05). Inhibition of contaminants prevailed when both L. paracasei and S. thermophilus were present in fresh cream cheese and probably was due to acid production by both strains; bacteriocin production was not found. Addition of inulin in T2 did not impact microbial viability (P > 0.05). L. paracasei subsp. paracasei in coculture with S. thermophilus was inhibitory against microbial contaminants in fresh cream cheese with or without the addition of inulin, indicating the potential use of this combination in a probiotic and synbiotic product.     

Influence of capsular and ropy exopolysaccharide-producing Streptococcus thermophilus on Mozzarella cheese and cheese whey

We investigated the effect of capsular and ropy exopolysaccharide-producing Streptococcus thermophilus starter bacteria on Mozzarella cheese functionality and whey viscosity. Mozzarella cheeses were manufactured with Lactobacillus helveticus LH100 paired with one of four S. thermophilus strains: MR-1C, a bacterium that produces a capsular exopolysaccharide; MTC360, a strain that secretes a ropy exopolysaccharide; TAO61, a nonexopolysaccharide-producing commercial cheese starter; and DM10, a nonencapsulated, exopolysaccharide-negative mutant of strain MR-1C. As expected, cheese moisture levels were significantly higher in Mozzarella cheeses made with exopolysaccharide-positive versus exopolysaccharide-negative streptococci, and melt properties were better in the higher moisture cheeses. Whey viscosity measurements showed that unconcentrated and ultrafiltered, fivefold concentrated whey from cheeses made with S. thermophilus MTC360 were significantly more viscous than whey from cheeses made with MR-1C, TAO61, or DM10. No significant differences were noted between the viscosity of unconcentrated or concentrated whey from cheeses made with S. thermophilus MR-1C versus the industrial cheese starter TAO61. These data indicate that encapsulated, but not ropy, exopolysaccharide-producing S. thermophilus strains can be utilized to increase the moisture level of cheese and to improve the melt properties of Mozzarella cheese without adversely affecting whey viscosity.     

Interactions among lactic acid starter and probiotic bacteria used for fermented dairy products

Interactions among lactic acid starter and probiotic bacteria were investigated to establish adequate combinations of strains to manufacture probiotic dairy products. For this aim, a total of 48 strains of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis, Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium spp. (eight of each) were used. The detection of bacterial interactions was carried out using the well-diffusion agar assay, and the interactions found were further characterized by growth kinetics. A variety of interactions was demonstrated. Lb. delbrueckii subsp. bulgaricus was found to be able to inhibit S. thermophilus strains. Among probiotic cultures, Lb. acidophilus was the sole species that was inhibited by the others (Lb. casei and Bifidobacterium). In general, probiotic bacteria proved to be more inhibitory towards lactic acid bacteria than vice versa since the latter did not exert any effect on the growth of the former, with some exceptions. The study of interactions by growth kinetics allowed the setting of four different kinds of behaviors between species of lactic acid starter and probiotic bacteria (stimulation, delay, complete inhibition of growth, and no effects among them). The possible interactions among the strains selected to manufacture a probiotic fermented dairy product should be taken into account when choosing the best combination/s to optimize their performance in the process and their survival in the products during cold storage.     

Proteolytic specificity of Lactobacillus delbrueckli subsp. bulgaricus influences functional properties of mozzarella cheese

Low-moisture part-skim Mozzarella cheeses were manufactured from 2% fat milk and aged for 21 d. Treatments included cheeses made with one of three different strains of Lactobacillus delbrueckii ssp. bulgaricus in combination with a single strain of Streptococcus thermophilus. A fourth, control treatment consisted of cheeses made with only S. thermophilus. Although total proteolytic ability of these strains, as indicated by the o-phthaldialdehyde analysis, was similar in each of the three strains of L. bulgaricus, these strains exhibited different proteolytic specificities toward the peptide, alpha(s1)-CN (f 1-23). On the basis of their alpha(s1)-CN (f 1-23) cleavage patterns and a previously described classification, these strains were assigned to the groups I, III, and V. The objective of this study was to investigate the influence of lactobacilli proteolytic systems, based on specificity toward alpha(s1)-CN (f 1-23), on functionality of part-skim Mozzarella cheese. Moisture, fat, protein, salt-in-moisture, and moisture in nonfat substances content of cheeses made with groups I, III, and V strain were similar. Control cheese had a lower moisture content than did other treatments. Significant differences were observed in functional properties between cheeses manufactured using groups III and V strains. Cheeses made with groups I and III strains were similar in their meltability, hardness, cohesiveness, melt strength, and stretch quality. Meltability and cohesiveness increased with age, while melt strength and stretch quality decreased with age for all cheeses. Additionally, HPLC showed that total peak areas of water-soluble peptides derived from cleavage of alpha(s1)-CN (f 1-23) by different strains of lactobacilli could be highly correlated to meltability and stretch characteristics of cheeses made with those strains.     

Novel Streptococcus infantarius subsp. infantarius variants harboring lactose metabolism genes homologous to Streptococcus thermophilus

The biodiversity and growth dynamics of Lactic Acid Bacteria (LAB) in farm-house Ossau-Iraty cheeses were investigated from vat milk to 180 days of ripening in six independent batches made from six raw ewe’s milks using five typical cheese-making methods. Commercial starter S1 was used for three batches, starter S1 combined with S2 for one batch and no starter for two batches.Up to ten LAB species from five genera and up to two strains per species were identified per milk; up to eleven species from five genera and up to three strains per species were identified per cheese. Lactococcus lactis, Lactobacillus paracasei, Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, and Leuconostoc mesenteroides were detected in all cheeses. Lactococci reached the highest counts irrespective of the milk and starter used. Lactococci and enterococci increased during manufacture, and mesophilic lactobacilli increased during ripening. Strain and species numbers, the percentage of isolates originating from the raw milk, maximum counts of each genus/species and time for reaching them, all varied according to whether or not a starter was used and the composition of the starter. The genotypes of strains within species varied according to the raw milk used. This generated distinct LAB microbiotas throughout manufacture and ripening that will certainly impact on the characteristics of the ripened cheeses.     

Changes of composition and free fatty acid contents of Urfa cheeses (a white-brined Turkish cheese) during ripening: Effects of heat treatments and starter cultures

The influences of heat treatments (at 65 °C for 20 min or 72 °C for 5 min) applied to the milk and addition of mesophilic or thermophilic starter cultures, prior to cheese-making, on the composition and free fatty acid contents of Urfa cheeses were evaluated throughout the ripening period. Sensory evaluation of cheese samples was also performed on 90th day. The basic composition of ripened cheese samples was not significantly affected by the heat treatments and starter cultures. Heat treatments adversely affected the lipolysis and sensory properties of Urfa cheeses, particularly at 72 °C. The FFA contents of cheeses made from mesophilic and thermophilic cultures were similar. Cheese made from raw milk had a higher level of lipolysis than the cheeses made from milk inoculated with mesophilic or thermophilic lactic starters (p < 0.05).     

Evaluation of the possible use of potentially probiotic Lactobacillus strains in dairy products

In the study, the ability of two potentially probiotic strains Lactobacillus plantarum 14 and Lactobacillus fermentum 4a to milk fermentation and the possibility to use them in yogurt production were investigated. The strains did not acidify milk during 24 h and 72 h fermentation at 37C, but grew well and remained at the level of 10⁸ colony-forming units (CFU)/mL during 21 days of cold storage. Their application to yogurt production along with commercial starter culture consisted on L. delbrueckii ssp. bulgaricus and S. thermophilus allowed to obtain products with typical sensory properties, pH values and numbers of potentially probiotic bacteria at desired level 10⁷ CFU/mL.     

Selection and use of autochthonous multiple strain cultures for the manufacture of high-moisture traditional Mozzarella cheese

Lactobacillus plantarum 18A, Lactobacillus helveticus 2B, Lactobacillus delbrueckii subsp. lactis 20F, Streptococcus thermophilus 22C, Enterococcus faecalis 32C and Enterococcus durans 16E were the most acidifying strains within 146 isolates for natural whey starters. The effect of media and temperature on 2 autochthonous multiple strain cultures (AMSI: 18A, 2B, 20F and 22C, 32C and 16E and AMSII: 18A, 2B, 20F and 22C) was studied. Genomic analysis showed a constant cell numbers for AMSII during 16 days of propagation in whey milk. Mozzarella cheese was made by using AMSII, commercial starter (CS) or citric acid (DA). Compared to other cheeses, the DA had a lower level of protein, ash, Ca, free amino acids and a higher level of moisture. Based on confocal laser scanning microscopy analysis, AMSII cheese showed the lowest microstructural variations during the period of storage compared to other cheeses. All the sensory attributes were scored highest for AMSII cheese. ASMII extend the shelf-life to ca. 12-15 days instead of the 5-7 days of traditional high-moisture Mozzarella cheese.     

Proteolysis on Reggianito Argentino cheeses manufactured with natural whey cultures and selected strains of Lactobacillus helveticus

Reggianito Argentino cheese is traditionally manufactured with whey starter cultures that provide typical and intense flavor but can cause poor quality standardization. In this study, the influence of natural and selected starters on Reggianito Argentino cheese proteolysis was investigated. Cheeses were manufactured with three strains of Lactobacillus helveticus (SF133, SF138 and SF209) cultured individually in sterile whey and used as single or mixed starters. Control cheeses were made with natural whey starter culture. Cheeses were analyzed to determine gross composition, as well as total thermophilic lactic flora. Proteolysis was assessed by N fractions, electrophoresis and liquid chromatography. Gross composition of the cheeses did not significantly differ, while viable starter cell counts were lower for cheeses made with strain SF209 alone or combined with other strains. Soluble N at pH 4.6 was the same for cheeses made with natural or selected starters, but soluble N in 12% trichloroacetic acid and 2.5% phosphotungstic acid was significantly higher in cheeses made with starters containing strain SF209. Nitrogen fractions results indicated that natural whey starter cultures could be replaced by several starters composed of the selected strains without significant changes to proteolysis patterns. Starter cultures prepared only with SF209 or with the three selected L. helveticus strains produced cheese products with significantly more proteolysis than control cheeses. Chromatographic profiles analyzed by principal components showed that three main peaks on chromatograms, presumptively identified as Tyr, Phe, and Trp, explained most of variability. Principal component scores indicated that cheese samples were grouped by ripening time, which was confirmed by linear discriminant analysis. On the contrary, samples did not cluster by Lactobacillus strain or type of starter.     

Modelling the survival of starter lactic acid bacteria and Bifidobacterium bifidum in single and simultaneous cultures

The inactivation kinetics at 4 degrees C of Bifidobacterium bifidum, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, cultured alone or consociated in a laboratory medium (modified MRS broth), were modelled through the Weibull model and a second-order polynomial equation. The initial cell number of S. thermophilus and L. delbrueckii subsp. bulgaricus was approximately 6-7log (CFU/ml); the viability loss after 30 days of storage was 2.87 and 1.99log (CFU/l) for L. delbruckii subsp. bulgaricus and S. thermophilus, cultured alone, respectively; whereas the consociation of lactobacilli and streptococci with bifidobacteria reduced viability loss during storage (0.28 and 0.54log CFU/l for lactobacilli and streptococci, respectively). Finally, the consociation of lactobacilli and streptococci with B. bifidum improved their oxygen uptake.     

Effect of carbon dioxide under high pressure on the survival of cheese starter cultures

A new processing method that rapidly forms curds and whey from milk has the potential to improve cheesemaking procedures if cheese starter cultures can tolerate the processing conditions. The survival of Lactobacillus delbrueckii ssp. bulgaricus, Lactococcus lactis ssp. lactis, or Streptococcus thermophilus through this new process was evaluated. Inoculated milk containing 0, 1, or 3.25% fat or Lactobacillus MRS broth or tryptone yeast lactose broth (depending on microorganism used) was sparged with CO2 to a pressure of 5.52 MPa and held for 5 min at 38 degrees C. Broth contained 7.93 to 8.78 log CFU/ ml before processing and 7.84 to 8.66 log CFU/ml afterward. Before processing, milk inoculated with L bulgaricus, L. lactis, or S. thermophilus contained 6.81, 7.35, or 6.75 log CFU/ml, respectively. After processing, the curds contained 5.68, 7.32, or 6.50 log CFU/g, and the whey had 5.05, 6.43, or 6.14 log CFU/ml, respectively. After processing, the pHs of control samples were lower by 0.41 units in broth, 0.53 units in whey, and 0.89 units in curd. The pH of the processed inoculated samples decreased by 0.3 to 0.53 units in broth, 0.32 to 0.37 units in whey, and 0.93 to 0.98 units in the curd. Storing curds containing L. lactis at 30 degrees C or control curds and curds with L. bulgaricus or S. thermophilus at 37 degrees C for an additional 48 h resulted in pHs of 5.22, 5.41, 4.53, or 4.99, respectively. This study showed that milk inoculated with cheese starter cultures and treated with CO2 under high pressure to precipitate casein-produced curds that contained sufficient numbers of viable starter culture to produce lactic acid, thereby decreasing the pH.