ABSTRACT
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.
Subject(s)
Cheese/analysis , Sensation , Sodium Chloride/analysis , Water/analysis , Adult , Amino Acid Sequence , Amino Acids/analysis , Carbohydrates/analysis , Carboxylic Acids/analysis , Caseins/analysis , Chemical Phenomena , Female , Food Technology , Humans , Hydrogen-Ion Concentration , Male , Minerals/analysis , Molecular Sequence Data , Peptides/analysis , Peptides/chemistry , Rheology , TasteABSTRACT
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 140â¯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.
Subject(s)
Cheese/microbiology , Food Microbiology , Lactobacillales/growth & development , Models, Biological , Animals , Hydrogen-Ion Concentration , Milk/microbiology , TasteABSTRACT
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.
Subject(s)
Bacteria/metabolism , Cheese/microbiology , Food Microbiology , Histamine/metabolism , Bacteria/classificationABSTRACT
Previous studies have suggested the presence of quantitative trait loci (QTLs) influencing blood pressure on rat chromosomes 2 and 13. In this study, we mapped the QTLs in F2 rats derived from a cross of the spontaneously hypertensive rat and the Wistar-Kyoto rat and analyzed the effect of the QTLs on blood pressures measured longitudinally between 12 and 25 weeks of age. We analyzed 16 polymorphic markers spanning 147.3 cM on chromosome 2 and 13 markers spanning 91.6 cM on chromosome 13. Both chromosomes contained QTLs with highly significant effects on blood pressure (peak logarithm of the odds [LOD] scores, 5.64 and 5.75, respectively). On chromosome 2, the peak was localized to a position at anonymous marker D2Wox7, 2.9 cM away from the gene for the sodium-potassium ATPase alpha 1-subunit. On chromosome 13, the major peak coincided with the marker D13Mit2, 21.7 cM away from the renin gene, but there was a suggestion of multiple peaks. The effect of the QTL on chromosome 2 was seen throughout from 12 to 25 weeks of age, whereas interestingly, the effect for the QTL on chromosome 13 was maximal at 20 weeks of age but disappeared at 25 weeks of age, presumably because of the effect of either epistatic factors or environmental influences. The findings provide important information on QTLs influencing blood pressure on rat chromosomes 2 and 13 that will be useful in localizing and identifying the causative genes and emphasize the importance of age being taken into account when the effects of individual QTLs on a trait that shows significant age-related changes are being analyzed.
Subject(s)
Aging/genetics , Blood Pressure/genetics , Animals , Genotype , Rats , Sodium-Potassium-Exchanging ATPase/geneticsABSTRACT
Food-fermenting lactic acid bacteria (LAB) are generally considered to be non-toxic and non-pathogenic. Some species of LAB, however, can produce biogenic amines (BAs). BAs are organic, basic, nitrogenous compounds, mainly formed through decarboxylation of amino acids. BAs are present in a wide range of foods, including dairy products, and can occasionally accumulate in high concentrations. The consumption of food containing large amounts of these amines can have toxicological consequences. Although there is no specific legislation regarding BA content in many fermented products, it is generally assumed that they should not be allowed to accumulate. The ability of microorganisms to decarboxylate amino acids is highly variable, often being strain specific, and therefore the detection of bacteria possessing amino acid decarboxylase activity is important to estimate the likelihood that foods contain BA and to prevent their accumulation in food products. Moreover, improved knowledge of the factors involved in the synthesis and accumulation of BA should lead to a reduction in their incidence in foods.
Subject(s)
Biogenic Amines/toxicity , Fermentation , Food Microbiology , Lactobacillaceae/metabolism , Dairy Products/analysis , Dairy Products/microbiology , Decarboxylation , Food Contamination , Risk Assessment , Wine/analysis , Wine/microbiologyABSTRACT
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.
Subject(s)
Brevibacterium/enzymology , Brevibacterium/metabolism , Cheese/microbiology , Amino Acids/metabolism , Aminopeptidases/metabolism , Brevibacterium/classification , Endopeptidases/metabolism , Esterases/metabolism , Lipolysis , Pigmentation , VolatilizationABSTRACT
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.
Subject(s)
Brevibacterium/enzymology , Caseins/metabolism , Endopeptidases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Caseins/chemistry , Cattle , Cheese/microbiology , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Substrate SpecificityABSTRACT
The p55 gene, which codes for a 55-kDa erythrocyte membrane protein, has been cloned and sequenced from the genome of the Japanese puffer fish Fugu rubripes (Fugu). This organism has the smallest recorded vertebrate genome and therefore provides an efficient way to sequence genes at the genomic level. The gene encoding p55 covers 5.5 kb from the beginning to the end of the coding sequence, four to six times smaller than the estimated size of the human gene, and is encoded by 12 exons. The structure of this gene has not been previously elucidated, but from this and other data we would predict a similar or identical structure in mammals. The predicted amino acid sequence of this gene in Fugu, coding for a polypeptide of 467 amino acids, is very similar to that of the human gene with the exception of the first two exons, which differ considerably. The predicted Fugu protein has a molecular weight (52.6 kDa compared with 52.3 kDa) and an isoelectric point very similar to those of human p55. In human, the p55 gene lies in the gene-dense Xq28 region, just 30 kb 3' to the Factor VIII gene, and is estimated to cover 20-30 kb. Its 5' end is associated with a CpG island, although there is no evidence that this is the case in Fugu. The small size of genes in Fugu and the high coding homology that they share with their mammalian equivalents, both in structure and sequence, make this compact vertebrate genome an ideal model for genomic studies.
Subject(s)
Blood Proteins/genetics , Erythrocyte Membrane/metabolism , Fishes, Poisonous/genetics , Genome , Membrane Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , Cosmids , CpG Islands , DNA, Complementary/genetics , Factor VIII/genetics , Fishes, Poisonous/blood , Humans , Models, Genetic , Molecular Sequence Data , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
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.
Subject(s)
Brevibacterium/enzymology , Caseins/metabolism , Endopeptidases/metabolism , Amino Acid Sequence , Animals , Binding Sites , Caseins/chemistry , Caseins/genetics , Cattle , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Substrate SpecificityABSTRACT
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.
ABSTRACT
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.
Subject(s)
Amino Acids, Branched-Chain/metabolism , Cheese , Food Microbiology , Lactobacillus/enzymology , Transaminases/isolation & purification , Amino Acid Sequence , Amino Acids, Branched-Chain/analysis , Chromatography, High Pressure Liquid/methods , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Hydrogen-Ion Concentration , Molecular Sequence Data , Temperature , Transaminases/metabolismABSTRACT
Intravital capillary video-microscopy is a dynamic method for studying skin capillaries. The technique of direct intravital microscopy (without dyes) depends on the presence of red blood cells inside capillaries for their identification. The aim of the present study was to compare different techniques to try to establish the best method for maximizing the number of visible perfused capillaries during intravital capillary microscopy. We compared the effects of venous congestion with those of post-occlusive reactive hyperaemia (Study 1). We also investigated venous congestion followed first by post-occlusive reactive hyperaemia and then by a core heat load test (Study 2). Finally we investigated venous congestion followed by post-occlusive reactive hyperaemia combined with venous congestion (Study 3). In Study 1, capillary density increased with venous congestion from a baseline value of 74+/-2 (mean+/-S.E.M.) per field to 82+/-3 per field (P<0.0001; analysis of variance). With reactive hyperaemia, there was an apparent decrease in visible capillary density to 69+/-2 per field. In Study 2, baseline capillary density was 69+/-4 per field, and this increased significantly with venous congestion to 74+/-4 per field (P=0.01). With both reactive hyperaemia and core heat load, the apparent density was 62+/-4 per field. In Study 3 the baseline density was 70+/-2 per field, and this increased significantly with venous congestion to 80+/-3 per field (P<0.0001). With reactive hyperaemia combined with venous congestion, the density was 81+/-3 per field (P=0.328 compared with venous congestion alone). The results show that venous congestion at 60 mmHg for 2 min is the most effective method for visualization of the maximal number of perfused skin capillaries during intravital video-microscopy.
Subject(s)
Hypertension/pathology , Microscopic Angioscopy/methods , Skin/blood supply , Adult , Capillaries/pathology , Constriction , Female , Hot Temperature , Humans , Hyperemia/pathology , Male , Microscopy, Video , Middle AgedABSTRACT
BACKGROUND: Rarefaction of skin capillaries in people with intermittent borderline essential hypertension suggests a primary or an early abnormality that may antedate the onset of sustained hypertension. OBJECTIVE: To compare skin capillary density in subjects with and without a family history of essential hypertension. SUBJECTS: 21 normotensive individuals, one or both of whose parents had essential hypertension (mean age 39.3 years; blood pressure 124/79 mm Hg); 21 normotensive controls with no family history of hypertension (age 46.3 years; blood pressure 124/78 mm Hg). METHODS: The skin of the dorsum of the fingers was examined by intravital capillary microscopy before and after venous congestion at 60 mm Hg for two minutes. RESULTS: By analysis of variance, both baseline and maximum skin capillary density were lower in subjects with a family history of essential hypertension than in those with no family history (baseline: 67 v 79 capillaries per field, p = 0.008; maximum: 74 v 93 capillaries per field, p < 0.0005). CONCLUSIONS: Capillary rarefaction in essential hypertension may occur before the increase in blood pressure and could, at least in part, reflect a primary rather than a secondary abnormality.