Gas production by Paucilactobacillus wasatchensis WDCO4 is increased in Cheddar cheese containing sodium gluconate.

Paucilactobacillus wasatchensis can use gluconate (GLCN) as well as galactose as an energy source and because sodium GLCN can be added during salting of Cheddar cheese to reduce calcium lactate crystal formation, our primary objective was to determine if the presence of GLCN in cheese is another risk factor for unwanted gas production leading to slits in cheese. A secondary objective was to calculate the amount of CO2 produced during storage and to relate this to the amount of gas-forming substrate that was utilized. Ribose was added to promote growth of Pa. wasatchensis WDC04 (P.waWDC04) to high numbers during storage. Cheddar cheese was made with lactococcal starter culture with addition of P.waWDC04 on 3 separate occasions. After milling, the curd was divided into six 10-kg portions. To the curd was added (A) salt, or salt plus (B) 0.5% galactose + 0.5% ribose (similar to previous studies), (C) 1% sodium GLCN, (D) 1% sodium GLCN + 0.5% ribose, (E) 2% sodium GLCN, (F) 2% sodium GLCN + 0.5% ribose. A vat of cheese without added P.waWDC04 was made using the same milk and a block of cheese used as an additional control. Cheeses were cut into 900-g pieces, vacuum packaged and stored at 12°C for 16 wk. Each month the bags were examined for gas production and cheese sampled and tested for lactose, galactose and GLCN content, and microbial numbers. In the control cheese, P.waWDC04 remained undetected (i.e., <104 cfu/g), whereas in cheeses A, C, and E it increased to 107 cfu/g, and when ribose was included with salting (cheeses B, D, and F) increased to 108 cfu/g. The amount of gas (measured as headspace height or calculated as mmoles of CO2) during 16 wk storage was increased by adding P.waWDC04 into the milk, and by adding galactose or GLCN to the curd. Galactose levels in cheese B were depleted by 12 wk while no other cheeses had residual galactose. Except for cheese D, the other cheeses with GLCN added (C, E and F) showed little decline in GLCN levels until wk 12, even though gas was being produced starting at wk 4. Based on calculations of CO2 in headspace plus CO2 dissolved in cheese, galactose and GLCN added to cheese curd only accounted for about half of total gas production. It is proposed that CO2 was also produced by decarboxylation of amino acids. Although P.waWDC04 does not have all the genes for complete conversion and decarboxylation of the amino acids in cheese, this can be achieved in conjunction with starter culture lactococcal. Adding GLCN to curd can now be considered another confirmed risk factor for unwanted gas production during storage of Cheddar cheese that can lead to slits and cracks in cheese. Putative risk factors now include having a community of bacteria in cheese leading to decarboxylation of amino acids and release of CO2 as well autolysis of the starter culture that would provide a supply of ribose that can promote growth of Pa. wasatchensis.

Increasing stringiness of low-fat mozzarella string cheese using polysaccharides.

When fat content of pasta filata cheese is lowered, a loss of fibrous texture occurs and low-fat (LF) mozzarella cheese loses stringiness, making it unsuitable for the manufacture of string cheese. We investigated the use of various polysaccharides that could act as fat mimetics during the stretching and extruding process to aid in protein strand formation and increase stringiness. Low-fat mozzarella cheese curd was made, salted, and then 3.6-kg batches were heated in hot (80°) 5% brine, stretched, and formed into a homogeneous mass. Hot (80°C) slurries of various polysaccharides were then mixed with the hot cheese and formed into LF string cheese using a small piston-driven extruder. Polysaccharides used included waxy corn starch, waxy rice starch, instant tapioca starch, polydextrose, xanthan gum, and guar gum. Adding starch slurries increased cheese moisture content by up to 1.6% but was not effective at increasing stringiness. Xanthan gum functioned best as a fat mimetic and produced LF string cheese that most closely visually resembled commercial string cheese made using low-moisture part skim (LMPS) mozzarella cheese without any increase in moisture content. Extent of stringiness was determined by pulling apart the cheese longitudinally and observing size, length, and appearance of individual cheese strings. Hardness was determined using a modified Warner-Bratzler shear test. When LF string cheese was made using a 10% xanthan gum slurry added at ~1%, increased consumer flavor liking was observed, with scores after 2wk of storage of 6.44 and 6.24 compared with 5.89 for the LF control cheese; although this was lower than an LMPS string cheese that scored 7.27. The 2-wk-old LF string cheeses containing xanthan gum were considered still slightly too firm using a just-about-right (JAR) test, whereas the LMPS string cheese was considered as JAR for texture. With further storage up to 8wk, all of the LF string cheeses softened (JAR score was closer to 3.0); however, much of the stringiness of the LF string cheeses was also lost during storage. We have demonstrated the potential feasibility of increasing stringiness in LF string cheese using polysaccharides with xanthan gum, although further research is needed to develop quantitative methodology for measuring stringiness and to maintain stringiness through the extended refrigerated shelf life needed for string cheese.

Effect of sodium, potassium, magnesium, and calcium salt cations on pH, proteolysis, organic acids, and microbial populations during storage of full-fat Cheddar cheese.

Sodium reduction in cheese can assist in reducing overall dietary Na intake, yet saltiness is an important aspect of cheese flavor. Our objective was to evaluate the effect of partial substitution of Na with K on survival of lactic acid bacteria (LAB) and nonstarter LAB (NSLAB), pH, organic acid production, and extent of proteolysis as water-soluble nitrogen (WSN) and protein profiles using urea-PAGE, in Cheddar cheese during 9mo of storage. Seven Cheddar cheeses with molar salt contents equivalent to 1.7% salt but with different ratios of Na, K, Ca, and Mg cations were manufactured as well as a low-salt cheese with 0.7% salt. The 1.7% salt cheeses had a mean composition of 352g of moisture/kg, 259g of protein/kg and 50% fat-on-dry-basis, and 17.5g of salt/kg (measured as Cl(-)). After salting, a faster initial decrease in cheese pH occurred with low salt or K substitution and it remained lower throughout storage. No difference in intact casein levels or percentage WSN levels between the various cheeses was observed, with the percentage WSN increasing from 5% at d 1 to 25% at 9mo. A greater decrease in intact αs1-casein than ß-casein was detected, and the ratio of αs1-casein (f121-199) to αs1-casein could be used as an index of ripening. Typical changes in bacteria microflora occurred during storage, with lactococci decreasing gradually and NSLAB increasing. Lowering the Na content, even with K replacement, extended the crossover time when NSLAB became dominant. The crossover time was 4.5mo for the control cheese and was delayed to 5.2, 6.0, 6.1, and 6.2mo for cheeses with 10, 25, 50, and 75% K substitution. Including 10% Mg or Ca, along with 40% K, further increased crossover time, whereas the longest crossover time (7.3mo) was for low-salt cheese. By 9mo, NSLAB levels in all cheeses had increased from initial levels of ≤10(2) to approximately 10(6)cfu/g. Lactococci remained at 10(6) cfu/g in the low-salt cheese even after 9mo of storage. The propionic acid concentration in the cheese increased when NSLAB numbers were high. Few other trends in organic acid concentration were observed as a function of Na content.

Application of ARISA to assess the influence of salt content and cation type on microbiological diversity of Cheddar cheese.

UNLABELLED: The structure and dynamics of microbial populations play a significant role during cheese manufacture and ripening. Therefore, fast and accurate methods for identification and characterization of the microbial populations are of fundamental importance to the cheese industry. In this study, we investigate the application of the automated ribosomal intergenic spacer analysis (ARISA) for the assessment of the microbial dynamics in cheeses differing in salt cation level and type. We developed a database of the observed and theoretical length of the 16S-23S intergenic spacer of common lactic acid bacteria (LAB) found in cheese and used the database to describe the structure and dynamics of microbial populations during ripening. Salt content and cation concentration did not significantly influence the overall bacteria structure, except that lower salt levels resulted in enhanced starter survival. Presence of nonstarter LAB was detected by ARISA and denaturing gradient gel electrophoresis (DGGE) after 3 months for all the cheeses analysed. ARISA used as fingerprinting method, proved to be a rapid and inexpensive technique for the discrimination of LAB in cheese and demonstrated higher resolution and performance in comparison with DGGE. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial communities play important roles during cheese making and ripening, hence rapid inexpensive methods to characterize this microbiota are of great interest to both academic and industrial scientists. The application of automated ribosomal intergenic spacer analysis (ARISA) was used to examine the microbial ecology of Cheddar cheese differing in salt level and type. ARISA is well suited to the analysis of the microbial ecology of cheese during ripening. Additionally, the results confirm that salt concentration influences starter culture survival in the cheese matrix, while significant differences were not observed in the nonstarter lactic acid bacteria.

Survival of probiotic adjunct cultures in cheese and challenges in their enumeration using selective media.

Various selective media for enumerating probiotic and cheese cultures were screened, with 6 media then used to study survival of probiotic bacteria in full-fat and low-fat Cheddar cheese. Commercial strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, or Bifidobacterium lactis were added as probiotic adjuncts. The selective media, designed to promote growth of certain lactic acid bacteria (LAB) over others or to differentiate between LAB, were used to detect individual LAB types during cheese storage. Commercial strains of Lactococcus, Lactobacillus, and Bifidobacterium spp. were initially screened on the 6 selective media along with nonstarter LAB (NSLAB) isolates. The microbial flora of the cheeses was analyzed during 9 mo of storage at 6°C. Many NSLAB were able to grow on media presumed selective for Lactococcus, Bifidobacterium spp., or Lb. acidophilus, which became apparent after 90 d of cheese storage, Between 90 and 120 d of storage, bacterial counts changed on media selective for Bifidobacterium spp., suggesting growth of NSLAB. Appearance of NSLAB on Lb. casei selective media [de man, Rogosa, and Sharpe (MRS)+vancomycin] occurred sooner (30 d) in low-fat cheese than in full-fat control cheeses. Differentiation between NSLAB and Lactococcus was achieved by counting after 18 to 24h when the NSLAB colonies were only pinpoint in size. Growth of NSLAB on the various selective media during aging means that probiotic adjunct cultures added during cheesemaking can only be enumerated with confidence on selective media for up to 3 or 4 mo. After this time, growth of NSLAB obfuscates enumeration of probiotic adjuncts. When adjunct Lb. casei or Lb. paracasei cultures are added during cheesemaking, they appear to remain at high numbers for a long time (9 mo) when counted on MRS+vancomycin medium, but a reasonable probability exists that they have been overtaken by NSLAB, which also grow readily on this medium. Enumeration using multiple selective media can provide insight into whether it is the actual adjunct culture or a NSLAB strain that is being enumerated.

Influence of calcium, pH, and moisture on protein matrix structure and functionality in direct-acidified nonfat Mozzarella cheese.

Influence of calcium, moisture, and pH on structure and functionality of direct-acid, nonfat Mozzarella cheese was studied. Acetic acid and citric acid were used to acidify milk to pH 5.8 and 5.3 with the aim of producing cheeses with 70 and 66% moisture, and 0.6 and 0.3% calcium levels. Cheeses containing 0.3% calcium were softer and more adhesive than cheeses containing 0.6% calcium, and flowed further when heated. Cheeses with the same calcium content (0.6%), the same moisture content, but set at different pH values (pH 5.3 and 5.8), exhibited no significant differences in melting or firmness. Increasing cheese moisture content from 66 to 70% produced a softer cheese but did not increase meltability. Such differences in functionality corresponded with differences in structure and arrangement of proteins in the cheese protein matrix. Microstructure of cheese with 0.6% calcium had an increase in protein folds and serum pockets compared with the 0.3% calcium cheeses that had a more homogeneous structure. Protein matrix in the low-calcium cheese appeared less dense indicating the proteins were more hydrated. In the 0.6% calcium cheeses, the proteins appeared more aggregated and had larger spaces between protein aggregates. Thus, between pH 5.3 and 5.8, calcium controls cheese functionality, and pH has only an indirect affect related to its influence on the calcium in cheese.

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.

Effect of Lactobacillus helveticus and Propionibacterium freudenrichii ssp. shermanii combinations on propensity for split defect in Swiss cheese.

One of the least controlled defects in Swiss cheese is development of splits that appear during refrigerated storage after cheese is removed from the warm room. Such fissures, or cracks, in the body of the cheese can be as short as 1 cm, or long enough to span a 90-kg block. A 2 x 2 x 2 factorial experiment was used to determine the effect of different Lactobacillus helveticus/Propionibacterium freudenreichii ssp. shermanii starter culture combinations on the occurrence of split defect in Swiss cheese. Eights vats of cheese were made in summer and eight in winter. Each 90-kg block of cheese was cut into twenty-four 4-kg blocks and graded based on the presence of splits. Only small variations were found in the composition of cheeses made during the same season. There were no correlations between moisture, pH, fat, protein, calcium, lactose contents, D/L lactate ratio, or protein degradation that could be used to predict splits after 90 d of storage. However, cheese made in the summer had 2% higher moisture content and a greater prevalence of splits. There was a sixfold increase in amount of downgraded cheese between the best and worst culture combinations used during cheese manufacture. After 90-d storage, 14 to 90% of cheese had splits in the summer, and 1 to 6% in the winter. Split formation increased with time from 60 to 120 d of storage and extent of split formation was influenced by both the lactobacilli and propionibacteria cultures used.

Biochemistry, genetics, and applications of exopolysaccharide production in Streptococcus thermophilus: a review.

Many strains of Streptococcus thermophilus synthesize extracellular polysaccharides. These molecules may be produced as capsules that are tightly associated with the cell, or they may be liberated into the medium as a loose slime (i.e., "ropy" polysaccharide). Although the presence of exopolysaccharide does not confer any obvious advantage to growth or survival of S. thermophilus in milk, in situ production by this species or other dairy lactic acid bacteria typically imparts a desirable "ropy" or viscous texture to fermented milk products. Recent work has also shown that exopolysaccharide-producing S. thermophilus can enhance the functional properties of Mozzarella cheese, but they are not phage-proof. As our understanding of the genetics, physiology, and functionality of bacterial exopolysaccharides continues to improve, novel applications for polysaccharides and polysaccharide-producing cultures are likely to emerge inside and outside the dairy industry. This article provides an overview of biochemistry, genetics, and applications of exopolysaccharide production in S. thermophilus.

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.

Temperature effect on structure-opacity relationships of nonfat mozzarella cheese.

Our objective was to determine the effect of heating on the structure of nonfat Mozzarella cheese and then to relate changes in structure to changes in cheese opacity. Cheese was made according to a direct-acid, stirred-curd procedure. Cheese samples, at 4 degrees C, were taken on d 1 and placed into glass bottles, which were sealed and heated. Once the cheese reached 10 degrees C or 50 degrees C, the bottles were placed on a scanner and color values measured. Samples were also taken on d 1 for chemical, micro, and ultrastructural analyses. Applying heat increased cheese opacity. At 50 degrees C the cheese was more opaque than at 10 degrees C. The increase in temperature induced changes in cheese structure. Larger high-density protein aggregates and increased protein concentration in the protein matrix were observed in cheese at 50 degrees C. Applied heat would favor hydrophobic interactions, and possibly, re-association of beta-casein and calcium with the protein matrix, promoting protein-to-protein interactions. Thus, the protein matrix contracts, occupying less cheese matrix area, and microphase separation occurs, causing serum pockets to grow in size, and microstructural heterogeneity to increase. It is proposed that the increased size of aggregates and heterogeneity of the cheese at 50 degrees C promote light reflection, thus increasing cheese opacity. We concluded that applying heat alters protein interactions in the cheese matrix, manifested as changes in cheese structure. Such changes in structure help provide an understanding of changes in cheese opacity.

Diversity in specificity of the extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus.

AIMS: To investigate the diversity in specificity of cell-bound extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus. METHODS AND RESULTS: HPLC analysis of whole-cell preparations of 14 Lact. delbrueckii subsp. bulgaricus and eight Lact. helveticus strains incubated with alpha (s1)-casein (f 1-23) detected at least six distinct proteolytic patterns. Differences between groups were found in both the primary and secondary specificity toward alpha(s1)-casein (f 1-23) and its breakdown products. No correlation was found between the o-phthaldialdehyde (OPA) general proteolysis analysis and alpha(s1)-casein (f 1-23) cleavage profiles. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: Using the alpha(s1)-CN (f 1-23) method, six patterns of proteolysis were found in the dairy lactobacilli tested. Understanding the influence of Lactobacillus proteinase specificity on casein degradation should facilitate efforts to develop starter cultures that predictably improve the functional properties of Mozzarella cheese.

Test for measuring the stretchability of melted cheese.

A test for measuring the stretchability of cheese was developed by adapting a texture-profile analyzer to pull strands of cheese upwards from a reservoir of melted cheese. Seven different cheeses were analyzed using the Utah State University stretch test. The cheeses were also analyzed for apparent viscosity with a helical viscometer, for meltability using a tube melt test, and for stretch using the pizza-fork test. Cheese was placed into a stainless steel cup and tempered in a water bath at 60, 70, 80, or 90 degrees C for 30 min before analysis. The cup was then placed in a water-jacketed holder mounted on the base of the instrument. A three-pronged hook-shaped probe was lowered into the melted cheese and then pulled vertically until all cheese strands broke or 30 cm was reached. This produced a stretch profile as the probe was lifted through the reservoir of melted cheese and then pulled strands of cheese upwards. Three parameters were defined to characterize the stretchability of the cheese. The maximum load, obtained as the probe was lifted through the cheese, was defined as melt strength (F(M)). The distance to which cheese strands were lifted was defined as stretch length (SL). The load exerted on the probe as the strands of cheese were being stretched was defined as stretch quality (SQ). There was a correlation between F(M) and apparent viscosity. There was also some correlation between SL measured by the fork test and SL when the cheese was tested at 90 degrees C, but no correlation occurred at lower temperatures.

Reversibility of the temperature-dependent opacity of nonfat mozzarella cheese.

Salted and unsalted nonfat mozzarella cheese was made by direct acidification and stored at 4 degrees C over 60 d. Changes in cheese opacity were measured by using reflectance L* values while the cheese was heated from 10 to 90 degrees C, then cooled to 10 degrees C, and reheated to 90 degrees C. A characteristic opacity transition temperature (T(OP)) was obtained for each cheese. Both salt content and storage time influenced T(OP). Opacity during heating, cooling, and reheating formed a hysteresis. At d 1, the unsalted cheese became opaque when heated to 20 degrees C, but the salted cheese required heating to 40 degrees C. As the salted cheese was aged, its T(OP) increased so that by 60 d the cheese did not become opaque until it was heated to 70 degrees C.

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.

Characterization of the Lactobacillus helveticus groESL operon.

This study utilized inverse polymerase chain reactions to characterize a 2.7-kb region of the Lactobacillus helveticus LH212 chromosome that included two complete and one truncated open reading frames (ORFs). Protein homology searches showed that the first two ORFs encoded homologs to the universally conserved heat shock proteins GroES and GroEL. Amino acids encoded by the 5' end of the truncated ORF that was downstream of groEL showed good homology to the amino terminal end of the Streptococcus pneumoniae DNA mismatch repair enzyme HexA. Nucleotide sequence analysis identified a putative transcriptional promoter upstream of groES that was comprised of -35 and -10 hexamers flanked upstream and downstream by copies of the conserved Gram-positive heat shock gene regulatory sequence, CIRCE. A large inverted repeat that may function as a rho-independent transcriptional terminator was located between groEL and the third ORF. Northern hybridization of an LH212 groEL gene fragment to RNA isolated from cells that had been heat shocked at 52 degrees C for 0, 5, 10 or 15 min detected a 2.2-kb transcript in each of the cell preparations. Densitometry showed the concentration of this mRNA species was approximately 4-fold higher after heat shock for 5 or 10 min and 3-fold higher after 15 min of heat shock.

Role of Streptococcus thermophilus MR-1C capsular exopolysaccharide in cheese moisture retention.

Recent work by our group has shown that an exopolysaccharide (EPS)-producing starter pair, Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii subsp. bulgaricus MR-1R, can significantly increase moisture retention in low-fat mozzarella (D. B. Perry, D. J. McMahon, and C. J. Oberg, J. Dairy Sci. 80:799-805, 1997). The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. Analysis of low-fat mozzarella made with different combinations of MR-1C, MR-1R, and the non-EPS-producing starter culture strains S. thermophilus TA061 and Lactobacillus helveticus LH100 showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level. To investigate the role of the S. thermophilus MR-1C EPS in cheese moisture retention, the epsE gene in this bacterium was inactivated by gene replacement. Low-fat mozzarella made with L. helveticus LH100 plus the non-EPS-producing mutant S. thermophilus DM10 had a significantly lower moisture content than did cheese made with strains LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-1C EPS indicated that the polymer has a novel basic repeating unit composed of D-galactose, L-rhamnose, and L-fucose in a ratio of 5:2:1.

Development and characterization of lactose-positive pediococcus species for milk fermentation.

Bacteriophages against Streptococcus thermophilus are a growing problem in the Italian cheese industry. One possible control method involves replacing S. thermophilus in mozzarella starter blends with lactic acid bacteria from a different genus or species. In this study, we evaluated lactose-positive pediococci for this application. Because we could not identify any commercially available pediococci with fast acid-producing ability in milk, we transformed Pediococcus pentosaceus ATCC 25744, P. pentosaceus ATCC 25745, and Pediococcus acidilactici ATCC 12697 by electroporation with pPN-1, a 35-kb Lactococcus lactis lactose plasmid. Transformants of P. pentosaceus ATCC 25745 and P. acidilactici ATCC 12697 were then used to examine lactose-positive pediococci for properties related to milk fermentation. Both transformants rapidly produced acid and efficiently retained pPN-1 in lactose broth, and neither bacterium was attacked by bacteriophages in whey collected from commercial cheese facilities. Paired starter combinations of Pediococcus spp. and Lactobacillus helveticus LH100 exhibited synergistic pH reduction in milk, and small-scale cheese trials showed that these cultures could be used to manufacture part-skim mozzarella cheese. Results demonstrate that lactose-positive pediococci have potential as replacement cocci for S. thermophilus in Italian cheese starter blends and may facilitate development of new strain rotation schemes to combat S. thermophilus bacteriophage problems in mozzarella cheese plants.