C. J. Oberg

Use of Exopolysaccharide-Producing Cultures to Improve the Functionality of Low Fat Cheese

Abstract Lactic acid bacteria may produce exopolysaccharide (EPS) that is tightly associated with the bacterial cell wall (capsular EPS) or liberated into the growth medium (ropy EPS). Because EPS have viscosity enhancing and stabilizing properties, exopolysaccharide-producing (EPS + ) starter cultures are commonly used to enhance water binding and viscosity in yogurt and fermented milks. Previous work has shown that low fat Mozzarella cheese manufactured with an EPS + starter pair, Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii subsp. bulgaricus MR-1R, contained significantly more moisture and had better melt properties than cheese made with a control starter pair. Genetic studies demonstrated that this effect was due to the MR-1C capsular EPS, and industrial cheese trials confirmed that MR-1C can effect a 1.5% moisture increase in part-skim Mozzarella. Because EPS accumulation in cheese whey may retard whey protein concentration and drying efficiency, the effect of capsular and ropy S. thermophilus starter bacteria on Mozzarella cheese and whey was also compared. Moisture and melt properties were improved in cheeses made with either EPS + S. thermophilus , but 5× concentrated whey from the ropy S. thermophilus was significantly more viscous than concentrated wheys from capsule-producing or non-EPS-producing S. thermophilus . These data indicate that encapsulated, but not ropy, EPS + S. thermophilus can be used to increase moisture content and improve melt in Mozzarella cheese, without deleteriously affecting whey viscosity.

Antimicrobial Effects of Essential Oils on Streptococcus pneumoniae

Abstract Of 73 essential oils tested for antibacterial activity against Streptococcus pneumoniae R36A (an unencapsulated strain) with a paper disk diffusion assay, three oils were highly inhibitory, fifteen moderately inhibitory and the remainder weakly or non-inhibitory. Three oils from each group were selected and tested with a broth assay in which each oil was added to growing cultures of S. pneumoniae R36A and optical densities (OD) were measured over time. The oils with high antibacterial activity; oregano, thyme and rosewood, induced rapid lysis of R36A as indicated by a decrease in OD, and appearance of dechaining and considerable cell debris within 30 min of addition. The lytic response of R36A to the three oils with moderate activity was variable but all induced some lysis. Oils that were weak inhibitors generally caused slowing of growth but little or no lysis. Several oils were also tested against an encapsulated isolate, S. pneumoniae IC2. Both disk assay and broth results were similar to those obtained with R36A, except that the oils were slightly less effective. Disk assay results showed some correlation with the broth assay, but were not always predictive of an oils ability to induce bacterial lysis. Essential oils that induce lysis in S. pneumoniae may have potential as an alternative treatment for infections caused by drug resistant pneumococci.

Attributes of the Heat Shock Response in Three Species of Dairy Lactobacillus

Summary Lactobacillus acidophilus, L. casei , and L. helveticus are industrially important bacteria for the manufacture of fermented dairy foods. Despite widespread commercial use, there is limited knowledge of basic physiological responses by these bacteria to dairy processing conditions. This study investigated the heat shock (HS) response in L. acidophilus NCFM, L. casei LC301, and L. helveticus LH212. Thermotolerance experiments showed HS improved the ability of log phase L. acidophilus NCFM, L. casei LC301, and L. helveticus LH212 cells to withstand a 20 min high temperature incubation by approximately 27-, 5- and 11-fold, respectively. Two-dimensional polyacrylamide gel electrophoresis showed HS induced synthesis of several proteins in each Lactobacillus species, and Western blots revealed these molecules included homologs to the universally conversed heat shock proteins DnaK, GroEL, ClpB, and GrpE. DnaJ was also detected, but expression of this protein was not stimulated by HS in any of the Lactobacillus species tested.

Effect of sodium, potassium, magnesium, and calcium salt cations on pH, proteolysis, organic acids, and microbial populations during storage of full-fat Cheddar cheese1

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 β-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.

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.

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.

Sequence and structural characterization of great salt lake bacteriophage CW02, a member of the T7-like supergroup.

ABSTRACT Halophage CW02 infects a Salinivibrio costicola-like bacterium, SA50, isolated from the Great Salt Lake. Following isolation, cultivation, and purification, CW02 was characterized by DNA sequencing, mass spectrometry, and electron microscopy. A conserved module of structural genes places CW02 in the T7 supergroup, members of which are found in diverse aquatic environments, including marine and freshwater ecosystems. CW02 has morphological similarities to viruses of the Podoviridae family. The structure of CW02, solved by cryogenic electron microscopy and three-dimensional reconstruction, enabled the fitting of a portion of the bacteriophage HK97 capsid protein into CW02 capsid density, thereby providing additional evidence that capsid proteins of tailed double-stranded DNA phages have a conserved fold. The CW02 capsid consists of bacteriophage lambda gpD-like densities that likely contribute to particle stability. Turret-like densities were found on icosahedral vertices and may represent a unique adaptation similar to what has been seen in other extremophilic viruses that infect archaea, such as Sulfolobus turreted icosahedral virus and halophage SH1.

Growth and gas production of a novel obligatory heterofermentative Cheddar cheese nonstarter lactobacilli species on ribose and galactose

An obligatory heterofermentative lactic acid bacterium, Lactobacillus wasatchii sp. nov., isolated from gassy Cheddar cheese was studied for growth, gas formation, salt tolerance, and survival against pasteurization treatments at 63°C and 72°C. Initially, Lb. wasatchii was thought to use only ribose as a sugar source and we were interested in whether it could also utilize galactose. We conducted experiments to determine the rate and extent of growth and gas production in carbohydrate-restricted (CR) de Man, Rogosa, and Sharpe (MRS) medium under anaerobic conditions with various combinations of ribose and galactose at 12, 23, and 37°C, with 23°C being the optimum growth temperature of Lb. wasatchii among the 3 temperatures studied. When Lb. wasatchii was grown on ribose (0.1, 0.5, and 1%), maximum specific growth rates (µmax) within each temperature were similar. When galactose was the only sugar, compared with ribose, µmax was 2 to 4 times lower. At all temperatures, the highest final cell densities (optical density at 640 nm) of Lb. wasatchii were achieved in CR-MRS plus 1% ribose, 0.5% ribose and 0.5% galactose, or 1% ribose and 1% galactose. Similar µmax values and final cell densities were achieved when 50% of the ribose in CR-MRS was substituted with galactose. Such enhanced utilization of galactose in the presence of ribose to support bacterial growth has not previously been reported. It appears that Lb. wasatchii co-metabolizes ribose and galactose, utilizing ribose for energy and galactose for other functions such as cell wall biosynthesis. Co-utilization of both sugars could be an adaptation mechanism of Lb. wasatchii to the cheese environment to efficiently ferment available sugars for maximizing metabolism and growth. As expected, gas formation by the heterofermenter was observed only when galactose was present in the medium. Growth experiments with MRS plus 1.5% ribose at pH 5.2 or 6.5 with 0, 1, 2, 3, 4, or 5% NaCl revealed that Lb. wasatchii is able to grow under salt and pH conditions typical of Cheddar cheese (4 to 5% salt-in-moisture, pH ~5.2). Finally, we found that Lb. wasatchii cannot survive low-temperature, long-time pasteurization but survives high-temperature, short-time (HTST) laboratory pasteurization, under which a 4.5 log reduction occurred. The ability of Lb. wasatchii to survive HTST pasteurization and grow under cheese ripening conditions implies that the presence of this nonstarter lactic acid bacterium can be a serious contributor to gas formation and textural defects in Cheddar cheese.

Cheese: Pasta-filata Cheeses: Low-Moisture Part-Skim Mozzarella (Pizza Cheese)

Low-moisture part-skim (LMPS) Mozzarella is a commodity cheese whose composition, production, and functionality are strongly linked to its use as pizza cheese. Its traditional manufacture follows that of most pasta-filata or stretched cheeses in that after curd formation, and sufficient syneresis and acid development, the curd is heated and mechanically stretched, then formed into shape, cooled, brined, and vacuum packaged. Many innovations have been implemented in its large-scale manufacture to lower cost, shorten make time, simplify use in the food service industry, or modify baking performance. LMPS Mozzarella needs only a few weeks of aging to develop the desired properties, or if moisture, fat, and calcium levels are correct it can be shredded for immediate use and frozen for storage and delivery. When LMPS Mozzarella is melted, it forms a homogeneous mass with sufficient flow, so shreds fuse together while enough calcium-mediated interactions occur between proteins to retain some elasticity (stretch). Thermophilic starter cultures, such as Streptococcus thermophilus and Lactobacillus helveticus in combination, or St. thermophilus alone, are used to slow proteolysis and provide a longer time before the cheese becomes too soft to shred. The fibrous structure of LMPS Mozzarella allows it to be sold as an individually wrapped ‘string’ cheese.

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

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.