Classification of Lactococcus lactis cell envelope proteinase based on gene sequencing, peptides formed after hydrolysis of milk, and computer modeling.

Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from αS1-casein and ß-casein. A model structure of Lc. lactis CEP based on the crystal structure of Streptococcus C5a CEP was used to investigate the AA positions in the substrate-binding region. New AA positions were suggested, which could be relevant for the cleavage specificity of CEP; however, these could only explain 2 out of 3 found subgroups. The third subgroup could be explained by 1 to 5 AA positions located opposite the substrate binding region.

Genome Sequences of Two Leuconostoc pseudomesenteroides Strains Isolated from Danish Dairy Starter Cultures.

The lactic acid bacterium Leuconostoc pseudomesenteroides can be found in mesophilic cheese starters, where it produces aromatic compounds from, e.g., citrate. Here, we present the draft genome sequences of two L. pseudomesenteroides strains isolated from traditional Danish cheese starters.

Genome Sequence of Leuconostoc mesenteroides subsp. cremoris Strain T26, Isolated from Mesophilic Undefined Cheese Starter.

Leuconostoc is the main group of heterofermentative bacteria found in mesophilic dairy starters. They grow in close symbiosis with the Lactococcus population and are able to degrade citrate. Here we present a draft genome sequence of Leuconostoc mesenteroides subsp. cremoris strain T26.

Physicochemical and sensory characterization of Cheddar cheese with variable NaCl levels and equal moisture content.

The present study investigated the effect of salt (NaCl) on the flavor and texture of Cheddar cheese with the particular aim to elucidate consequences of, and strategies for, reducing the salt concentration. Descriptive sensory analysis and physicochemical mapping of 9-mo-old Cheddar cheeses containing 0.9, 1.3, 1.7, and 2.3% salt and an equal level of moisture (37.6 ± 0.1%) were undertaken. Moisture regulation during manufacture resulted in slightly higher calcium retention (158 to 169 mmol/kg) with decreasing NaCl concentration. Lactose was depleted only at 0.9 and 1.3% salt, resulting in concomitantly higher levels of lactate. Lower levels of casein components and free amino acids were observed with decreasing NaCl concentration, whereas levels of pH 4.6-soluble peptides were higher. Key taste-active compounds, including small hydrophobic peptides, lactose, lactate, and free amino acids, covaried positively with bitter, sweet, sour, and umami flavor intensities, respectively. An additional direct effect of salt due to taste-taste enhancement and suppression was noted. Sensory flavor profiles spanned a principal component dimension of palatability projecting true flavor compensation of salt into the space between cheeses containing 1.7 and 2.3% salt. This space was characterized by salt, umami, sweet, and a range of sapid flavors, and was contrasted by bitter and other off-flavors. Rheological and sensory measurements of texture were highly correlated. Cheeses made with 2.3% salt had a longer and slightly softer texture than cheeses containing 0.9, 1.3, and 1.7% salt, which all shared similar textural properties. Moisture regulation contributed to restoring the textural properties upon a 50% reduction in salt, but other factors were also important. On the other hand, significant flavor deterioration occurred inevitably. We discuss the potential of engineering a favorable basic taste profile to restore full palatability of Cheddar with a 50% reduction in salt.

Genetic diversity in proteolytic enzymes and amino acid metabolism among Lactobacillus helveticus strains.

Lactobacillus helveticus CNRZ 32 is recognized for its ability to decrease bitterness and accelerate flavor development in cheese, and has also been shown to release bioactive peptides in milk. Similar capabilities have been documented in other strains of Lb. helveticus, but the ability of different strains to affect these characteristics can vary widely. Because these attributes are associated with enzymes involved in proteolysis or AA catabolism, we performed comparative genome hybridizations to a CNRZ 32 microarray to explore the distribution of genes encoding such enzymes across a bank of 38 Lb. helveticus strains, including 2 archival samples of CNRZ 32. Genes for peptidases and AA metabolism were highly conserved across the species, whereas those for cell envelope-associated proteinases varied widely. Some of the genetic differences that were detected may help explain the variability that has been noted among Lb. helveticus strains in regard to their functionality in cheese and fermented milk.

Influence of microflora on texture and contents of amino acids, organic acids, and volatiles in semi-hard cheese made with DL-starter and propionibacteria.

The microflora of semi-hard cheese made with DL-starter and propionic acid bacteria (PAB) is quite complex, and we investigated the influence of its variation on texture and contents of organic acids, free amino acids, and volatile compounds. Variation in the microflora within the normal range for the cheese variety Grevé was obtained by using a PAB culture in combination with different DL-starters and making the cheeses at 2 dairy plants with different time and temperature profiles during ripening. Propionic acid bacteria dominated the microflora during ripening after a warm room period at levels of log 8 to log 9 cfu/g, which was about 1 log unit higher than the total number of starter bacteria and about 2 log units higher than the number of nonstarter lactic acid bacteria. Eye formation was observed during the warm room period and further ripening (at 8 to 10°C). The amounts of acetate, propionate, total content of free amino acids, 2-propanol, and ethyl propionate in the ripened cheeses were related to the number of PAB. A decrease in the relative content of Asp and Lys and increase of Phe over the ripening time were different from what is observed in semi-hard cheese without PAB. The occurrence of cracks was higher in cheeses with more hydrolyzed α(S1)- and ß-casein, higher content of free amino acids, lower strain at fracture (shorter texture), and a greater number of PAB.

Isolation of cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and molecular comparison with dairy-related Lactobacillus helveticus strains.

AIMS: To isolate cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and compare them with dairy-related Lactobacillus helveticus strains using molecular typing methods. METHODS AND RESULTS: The number of thermophilic bacteria in seven commercial cheeses manufactured with mesophilic starters was estimated to be <10 CFU g(-1). Implementation of an enumeration step in the isolation method made it possible to isolate one thermophilic strain from each of five of seven cheeses. Comparing repetitive sequence PCR (rep-PCR) profiles of the isolates with dairy-related Lact. helveticus strains indicated that one isolate was a Lact. helveticus. Partial sequencing of 16S rRNA confirmed this, and the remaining four strains were identified as Lactobacillus delbrueckii, Lactobacillus fermentum and Enterococcus faecium. The rep-PCR profile of the isolated Lact. helveticus was identical to the rep-PCR profile of the Lact. helveticus adjunct culture used in the specific cheese, but their pulsed field gel electrophoresis profiles differed slightly. CONCLUSION: It was possible to isolate cultivable thermophilic bacteria from ripened cheeses manufactured with mesophilic starter and thermophilic adjunct cultures by using an enumeration step. SIGNIFICANCE AND IMPACT OF THE STUDY: Isolation of cultivable thermophilic bacteria from ripened cheeses made with mesophilic starters offers an original source for new dairy-relevant cultures.

Relationship between physical properties of casein micelles and rheology of skim milk concentrate.

The properties of casein micelles in milk concentrates are of interest for the use of ultrafiltered (UF) skim milk concentrates in dairy products, and for the general understanding of colloidal stability and behavior of the casein micelle. The rheological behavior of UF skim milk concentrate with a casein concentration of 19.5% (wt/wt) was investigated at different pH and NaCl concentrations by analyzing flow viscometry and small amplitude oscillatory shear measurements. Viscometric flow curves were fitted to the Carreau-Yasuda model with the aim of determining values for the viscosity at infinite high shear rates and thereby estimate the voluminosity of the casein micelles (nu(casein)) in the UF concentrate. The voluminosity of the casein micelles increased with addition of NaCl and decreased when pH was decreased from 6.5 to 5.5. At pH 5.2, nu(casein) increased because of acid-induced aggregation of the casein micelles. The changes in nu(casein) could be interpreted from transmission electron microscopy of freeze-fractured samples of the UF concentrate and partly from dynamic light scattering measurements. Altered interactions between casein micelles due to different pH and NaCl concentrations are proposed to occur due to collapse of the kappa-casein layer, changed ionic strength, and altered distance between casein micelles.

Purification and characterization of a branched-chain amino acid aminotransferase from Lactobacillus paracasei subsp. paracasei CHCC 2115.

AIM: Purification and characterization of an aminotransferase (AT) specific for the degradation of branched-chain amino acids from Lactobacillus paracasei subsp. paracasei CHCC 2115. METHODS AND RESULTS: The purification protocol consisted of anion exchange chromatography, affinity chromatography and hydrophobic interaction chromatography. The enzyme was found to exist as a monomer with a molecular mass of 40-50 kDa. The AT converted isoleucine, leucine and valine at a similar rate with alpha-ketoglutarate as the amino group acceptor; minor activity was shown for methionine. The enzyme had pH and temperature optima of 7.3 and 43 degrees C, respectively, and activity was detected at the pH and salt conditions found in cheese (pH 5.2, 4% NaCl). Hg2+ completely inhibited the enzyme, and the inhibition pattern was similar to that for pyridoxal-5'-phosphate-dependent enzymes, when studying the effect of other metal ions, thiol- and carbonyl-binding agents. The N-terminal sequence of the enzyme was SVNIDWNNLGFDYMQLPYRYVAHXKDGVXD, and had at the amino acid level, 60 and 53% identity to a branched-chain amino acid AT of Lact. plantarum and Lactococcus lactis, respectively. CONCLUSIONS: The results suggest that Lact. paracasei subsp. paracasei CHCC 2115 may contribute to development of flavour in cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of transamination performed by cheese-related bacteria, and in the understanding and control of amino acid catabolism and the production of aroma compounds.