Ylva Ardö

Lactobacillus strains isolated from Danbo cheese as adjunct cultures in a cheese model system.

Isolates of Non-Starter Lactic Acid Bacteria (NSLAB) from six ripened Danbo cheeses of different ages and of different brands were examined. Special emphasis was on the genus Lactobacillus with the aim of investigating their role in cheese maturation. Thirty-three isolates were typed by the PCR-based method, Randomly Amplified Polymorphic DNA (RAPD). Ten RAPD types were found and 70% of the isolates were of RAPD types found in more than one cheese. The different RAPD types were identified to species level by Temporal Temperature Gradient Gel Electrophoresis (TTGE). Most of the isolates were identified as Lactobacillus paracasei (76%), but also Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus rhamnosus and some taxa originating from the starter culture were detected. In one cheese, no lactobacilli were found. One strain of the most frequent Lactobacillus RAPD type from each of the five cheeses with a Lactobacillus flora was used as adjunct cultures in a cheese model system. Four of the five adjuncts were re-isolated during ripening. Two adjunct containing model cheeses received higher flavour scores than the control while two other were associated with off-flavours. The two model cheeses with off-flavour had a similar microflora and both were after 13 weeks of ripening dominated by a strain identified as L. plantarum.

Genetic diversity in proteolytic enzymes and amino acid metabolism among Lactobacillus helveticus strains1

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.

Exploratory study of proteolysis, rheology and sensory properties of Danbo cheese with different fat contents

The quality of low-fat cheese may be inferior to the corresponding full-fat varieties due to firm texture, bland taste, and bitter after-taste. This study was carried out to identify the critical properties of low-fat Danbo cheese, which is a semi-hard cheese variety that for centuries has been produced in Denmark with low (13%), reduced (17%) and normal (25%) fat contents. Sensory and rheological analysis confirmed that the low-fat cheeses had a firmer and more elastic texture. Peptide profiles and free amino acid composition were quite similar in all cheeses, reflecting the way they were produced. However, proteolysis may need to be altered in the low-fat cheese in order to achieve a pleasant texture and flavour. About 50% more bitter peptides derived from casein could be added to the normal or reduced fat cheese than to the low-fat cheese before bitterness was noticed, indicating masking of bitterness in cheese with more fat.

Characterizing ripening in low-fat, semi-hard round-eyed cheese made with undefined mesophilic dl-starter

Abstract Attractive body in low-fat cheese was achieved by altering cheesemaking schemes. Similar moisture content in non-fat substance and pH after 24 h were obtained and the ripening processes in cheeses with various fat contents were compared. Four semi-hard, round-eyed cheese varieties were made from the same batch of cheesemilk, i.e. Herrgards cheese with fat contents of 28 and 17% and Drabant cheese with fat contents of 17 and 10%. The experiment was repeated five times. It was shown that the bacterial balance in the mesophilic undefined dl -starter used was influenced by manufacturing conditions such as cooking temperature. Only small differences were found in proteolysis, concentrations of carboxylic acids and pH, and they were mainly explained by the manufacturing conditions rather than fat contents. Characteristic mild flavours developed in the cheeses containing 17% fat.

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.

Influence of nitrogen sources on growth and fermentation performance of different wine yeast species during alcoholic fermentation

In this study, the influence of twenty different single (i.e. 19 amino acids and ammonium sulphate) and two multiple nitrogen sources (N-sources) on growth and fermentation (i.e. glucose consumption and ethanol production) performance of Saccharomyces cerevisiae and of four wine-related non-Saccharomyces yeast species (Lachancea thermotolerans, Metschnikowia pulcherrima, Hanseniaspora uvarum and Torulaspora delbrueckii) was investigated during alcoholic fermentation. Briefly, the N-sources with beneficial effects on all performance parameters (or for the majority of them) for each yeast species were alanine, arginine, asparagine, aspartic acid, glutamine, isoleucine, ammonium sulphate, serine, valine and mixtures of 19 amino acids and of 19 amino acids plus ammonium sulphate (for S. cerevisiae), serine (for L. thermotolerans), alanine (for H. uvarum), alanine and asparagine (for M. pulcherrima), arginine, asparagine, glutamine, isoleucine and mixture of 19 amino acids (for T. delbrueckii). Furthermore, our results showed a clear positive effect of complex mixtures of N-sources on S. cerevisiae and on T. delbrueckii (although to a lesser extent) as to all performance parameters studied, whereas for L. thermotolerans, H. uvarum and M. pulcherrima, single amino acids affected growth and fermentation performance to the same extent as the mixtures. Moreover, we found groups of N-sources with similar effects on the growth and/or fermentation performance of two or more yeast species. Finally, the influences of N-sources observed for T. delbrueckii and H. uvarum resembled those of S. cerevisiae the most and the least, respectively. Overall, this work contributes to an improved understanding of how different N-sources affect growth, glucose consumption and ethanol production of wine-related yeast species under oxygen-limited conditions, which, in turn, may be used to, e.g. optimize growth and fermentation performance of the given yeast upon N-source supplementation during wine fermentations.

Comparison of the Hydrolysis of Bovine κ-Casein by Camel and Bovine Chymosin: A Kinetic and Specificity Study

Bovine chymosin constitutes a traditional ingredient for enzymatic milk coagulation in cheese making, providing a strong clotting capacity and low general proteolytic activity. Recently, these properties were surpassed by camel chymosin, but the mechanistic difference behind their action is not yet clear. We used capillary electrophoresis and reversed-phase liquid chromatography-mass spectrometry to compare the first site of hydrolysis of camel and bovine chymosin on bovine κ-casein (CN) and to determine the kinetic parameters of this reaction (pH 6.5; 32 °C). The enzymes showed identical specificities, cleaving the Phe105-Met106 bond of κ-CN to produce para-κ-CN and caseinomacropeptide. Initial formation rates of both products validated Michaelis-Menten modeling of the kinetic properties of both enzymes. Camel chymosin bound κ-CN with ∼30% lower affinity (K(M)) and exhibited a 60% higher turnover rate (k(cat)), resulting in ∼15% higher catalytic efficiency (k(cat)/K(M)) as compared to bovine chymosin. A local, less dense negatively charged cluster on the surface of camel chymosin may weaken electrostatic binding to the His-Pro cluster of κ-CN to simultaneously impart reduced substrate affinity and accelerated enzyme-substrate dissociation as compared to bovine chymosin.

Screening and selection of Lactobacillus strains for use as adjunct cultures in production of semi-hard cheese

Thirty-three Lactobacillus strains were tested as adjuncts in a cheese model system. Eighteen strains originated from cheese (nine Lactobacillus spp. and nine Lb. paracasei/casei) and 15 from human intestinal mucosa (11 Lb. rhamnosus; three Lb. paracasei; one Lb. plantarum). Model cheeses weighing 120 g were made of cheese grains from full-scale production of washed curd semi-hard cheese (Herrgård). The model system was reproducible and similar to full-scale production with respect to moisture, salt content, pH and microbial flora. The model cheeses were sampled for aerobic and anaerobic plate count and viable counts of Lactobacillus and Lactococcus. The presence of adjuncts in the model cheeses was confirmed by typing isolates with Randomly Amplified Polymorphic DNA (RAPD). The sensory properties of model cheeses were described. In a first trial 23 of the 33 adjuncts were re-isolated from the corresponding model cheeses after 9 or 13 weeks. Adjuncts of Lb. paracasei were re-isolated more frequently than adjuncts of Lb. rhamnosus. Nine strains were selected, on the basis of their ability to grow and be a dominating part of the microflora of model cheese with interesting sensory properties. These strains were further studied together with two commercial cultures. The sensory influences on model cheeses of six of the adjuncts were confirmed, and flavour scores were in the range of 2.9-7.1 for model cheeses with different adjuncts while the control had a flavour score of 5.6 (0-10 scale). Survival and growth of seven out of the nine strains correlated with the results of the first trial. Growth and influence on flavour of four adjunct cultures were confirmed in experimental cheese manufactured in a 400-1 open vat.

A comparison between the microflora of Herrgård cheese from three different dairies

The microflora of Herrgard cheese produced at three dairies was grouped by phenotypical key tests and typed by randomly amplified polymorphic DNA (RAPD). Ten cheeses from the same vat at each dairy were analysed, five after 3 and another five after 6 months of ripening. All cheeses were organoleptically graded as Herrgard after uniform national standards, and only small but characteristic differences were found between cheeses from different dairies. Cheese from two dairies were dominated by a spontaneous secondary microflora of Lactobacillus from 3 months of ripening, while strains from the starter culture still dominated in cheese from the third dairy after 6 months. The Lactobacillus flora from the different dairies were mainly of diverse RAPD types, but of the 24 different RAPD types found, three were isolated from cheese made at two different dairies and two other RAPD types were isolated from cheese from all three dairies. A succession of different RAPD types was shown at one dairy.

Camel and Bovine Chymosin Hydrolysis of Bovine αS1- and β-Caseins Studied by Comparative Peptide Mapping

In many cheese varieties, the general proteolytic activity of the coagulant is of great importance to the development of flavor and texture during ripening. This study used capillary electrophoresis and LC-MS/MS to compare the in vitro proteolytic behavior of camel and bovine chymosin (CC/BC) on bovine α(S1)- and β-casein (CN) at pH 6.5 and 30 °C. β-CN hydrolysis was also studied at pH 5.2 and in the presence of 0, 2, and 5% (w/v) NaCl. A total of 25 α(S1)- and 80 β-CN peptides were identified, and initial rates of early peptide formation were determined. The modes of proteolytic action of CC and BC shared a high degree of similarity generally. However, except for a few peptide bonds, CC was markedly less active, the magnitude of which varied widely with cleavage site. Preferential α(S1)-CN (Phe23-Phe24) and β-CN (Leu192-Tyr193) hydrolysis by CC proceeded at an estimated 36 and 7% of the initial rate of BC, respectively. The latter rate difference was largely pH and NaCl independent. Several cleavage sites appeared to be unique to CC and especially BC action, but qualitative differences were often predetermined by quantitative effects. In particular, negligible CC affinity to α(S1)-CN₁₆₄/₁₆₅ and β-CN₁₈₉/₁₉₀ prevented further exposure of the N-terminal products. β-CN hydrolysis by either enzyme was always stimulated at the lower pH, yet either inhibited or stimulated by the presence of NaCl, depending mainly on the predominating type of molecular substrate interactions involved at the specific site of cleavage. The potential impact of this proteolytic behavior on cheese quality is discussed.