Modelling the biphasic growth of non-starter lactic acid bacteria on starter-lysate as a substrate.

Since cheese is poor in energy for bacterial growth, it is believed that non-starter lactic acid bacteria growth and flavour development are supported by the nutrients from lysis of the starter culture. This study was performed to investigate the dynamics of interaction between starter and non-starter strains from cheese. A starter culture lysate was prepared by enzymatic digestion and tested as a growth substrate for Lactobacillus sp. strains. The two starter culture strains of Lactococcus lactis were also tested on the starter-lysate. All seventeen strains were individually inoculated at the level of 5.0 log10 cfu mL-1 in M17 broth, with or without 10% starter-lysate, and incubated at 30 °C for 140 h. The optical density600 nm was modelled with the primary log-transformed Logistic model with delay and lag phase duration, maximum specific growth rate as well as maximum population density obtained. Biphasic growth was mainly observed when the strains were able to utilize the starter-lysate as an energy source. To deal with the lack-of-fit related to the biphasic growth, the observed data points of the curve were divided after graphic evaluation and according to deviation of the residuals from the range ±0.05. Modelling was then performed in two phases by applying the same primary Logistic model in each of the two parts of the growth curve. Values of root-mean-square error and graphic evaluation indicated the good fitting of the data with the suggested approach. The growth of the two Lactococcus lactis strains was not affected by the starter-lysate. However, thirteen of the non-starter strains had their growth rates increased. The increase was greatest for Lactobacillus rhamnosus KU-LbR1, which reached maximum optical densities of 0.23 and 0.58 in the absence and the presence of starter-lysate, respectively. No effect of the starter-lysate was shown for the growth of Lactobacillus curvatus strains. The extend of the growth of non-starter strains on the starter-lysate was shown to be species and strain dependent.

Histamine forming behaviour of bacterial isolates from aged cheese.

This study was performed to investigate the presence of histamine forming bacteria in commercially available cheeses as well as to evaluate the histamine forming potential using in vitro models. Five long-time-ripened cheeses made from different milk types were analysed for histamine producing bacterial isolates. The ability of the isolates to produce histamine was tested by incubation at 37 °C for five days in a restricted media with a pH indicator. Changes in the amino-compound profile were investigated using ultra performance liquid chromatography. Only eight of 106 isolates were able to produce compounds raising the pH and seven of those were confirmed to be histamine producers. Despite the fact that all isolates were obtained from the same vintage Danish Gouda cheese, made from raw cow milk, the amino-compound profile as well as the response to different environmental conditions diverged between the isolates. Rep-PCR and 16S rRNA gene sequencing was used to further characterize the isolates. Pediococcus pentosaceus was for the first time reported to be a histamine producer in cheese. The presence of the histidine decarboxylase gene (hdcA) was confirmed by PCR amplification of the histidine decarboxylase gene in four of the isolates. The results indicate that evaluating the presence and concentration of histamine is not only a relevant parameter to evaluate quality and safety, but is also an important tool to classify histamine producers in cheese.

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.

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.

Aspects of enzymology and biochemical properties of Brevibacterium linens relevant to cheese ripening: a review.

Brevibacterium linens is a major surface microorganism that is present in the smear of surface-ripened cheeses. The enzymology and biochemical characteristics of B. linens influence the ripening and final characteristics of smear surface-ripened cheeses. Proteolytic, peptidolytic, esterolytic, and lipolytic activities, which are of particular importance in the ripening process, are discussed in detail. This review also describes the production of volatile compounds, especially sulfur-containing ones, by B. linens, which are thought to be important in respect to the flavor of smear surface-ripened cheeses. The unique orange-colored carotenoids and the factors effecting their production by B. linens are also presented. The catabolism of aromatic amino acids, bacteriocin production, plasmids, and miscellaneous biochemical and physiological properties (peptidoglycan type, antibiotic resistance, insecticide degradation, and biotechnological applications) of B. linens are discussed. The problem associated with the current taxonomical classification of B. linens strains caused by strain variation is evaluated. Finally, the application of B. linens cell extracts or its proteolytic enzymes as cheese ripening accelerants for semi-hard or hard cheese varieties is considered.

Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine beta-casein.

The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine beta-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The major sites of hydrolysis were Ser-18-Ser-19, Glu-20-Glu-21, Gln-56-Ser-57, Gln-72-Asn-73, Leu-77-Thr-78, Ala-101-Met-102, Phe-119-Thr-120, Leu-139-Leu-140, Ser-142-Trp-143, His-145-Gln-146, Gln-167-Ser-168, Gln-175-Lys-176, Tyr-180-Pro-181, and Phe-190-Leu-191. The proteinase had a broad specificity for the amino acid residues present at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.

Specificity of an extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein.

The specificity of the extracellular proteinase from Brevibacterium linens ATCC 9174 on bovine alpha s1-casein was studied. Hydrolysis was monitored over time by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and urea-PAGE. The major pH 4.6-soluble peptides were isolated by high-performance liquid chromatography and identified by N-terminal amino acid sequencing and mass spectrometry. The time course of peptide formation indicated that His-8-Gln-9, Ser-161-Gly-162, and either Gln-172-Tyr-173 or Phe-23-Phe-24 were the first, second, and third bonds cleaved, respectively. Other cleavage sites included Asn-19-Leu-20, Phe-32-Gly-33, Tyr-104-Lys-105, Leu-142-Ala-143, Phe-150-Arg-151, Gln-152-Phe-153, Leu-169-Gly-170, and Thr-171-Gln-172. The proteinase had a broad specificity for the amino acid residues at the P1 and P'1 positions but showed a preference for hydrophobic residues at the P2, P3, P4, P'2, P'3, and P'4 positions.

Purification and Characterization of an Extracellular Proteinase from Brevibacterium linens ATCC 9174.

An extracellular serine proteinase from Brevibacterium linens ATCC 9174 was purified to homogeneity. pH and temperature optima were 8.5 and 50(deg)C, respectively. The results for the molecular mass of the proteinase were 56 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 126 kDa by gel filtration, indicating that the native enzyme exists as a dimer. Mg(sup2+) and Ca(sup2+) activated the proteinase, as did NaCl; however, Hg(sup2+), Fe(sup2+), and Zn(sup2+) caused strong inhibition. The sequence of the first 20 N-terminal amino acids was NH(inf2)-Ala-Lys-Asn-Asp-Ala-Val-Gly-Gly-Met-Gly-Tyr-Leu-Ser-Met-Ile-Pro-Se r-Gln-Pro-Gly.