Proteolysis as a function of distance from surface to centre in a smear-ripened Irish farmhouse cheese

This study focused on proteolysis in an Irish farmhouse smear-ripened cheese by serial slicing (0.41 mm/slice) the first 2 cm from surface towards the centre of the cheese. Ureapolyacrylamide gel electrophoretograms confirmed higher proteolysis in the outer layers than at the centre. Free amino acid (FAA) analysis confirmed decrease in proteolytic activity from surface to centre. Peptides produced at depths 0.41 mm and 20.5 mm were 720 and 427 from αs1-casein; 691 and 337 from αs2-casein; 807 and 453 from β-casein; 180 and 109 from κ-casein. The study confirms higher proteolytic activity at surface due to action of enzymes of the smear microbiota, than at the centre of cheese and identified the agents responsible for production of many peptides

Nucleic acid-based approaches to investigate microbial-related cheese quality defects

peer-reviewedThe microbial profile of cheese is a primary determinant of cheese quality. Microorganisms can contribute to aroma and taste defects, form biogenic amines, cause gas and secondary fermentation defects, and can contribute to cheese pinking and mineral deposition issues. These defects may be as a result of seasonality and the variability in the composition of the milk supplied, variations in cheese processing parameters, as well as the nature and number of the non-starter microorganisms which come from the milk or other environmental sources. Such defects can be responsible for production and product recall costs and thus represent a significant economic burden for the dairy industry worldwide. Traditional non-molecular approaches are often considered biased and have inherently slow turnaround times. Molecular techniques can provide early and rapid detection of defects that result from the presence of specific spoilage microbes and, ultimately, assist in enhancing cheese quality and reducing costs. Here we review the DNA-based methods that are available to detect/quantify spoilage bacteria, and relevant metabolic pathways in cheeses and, in the process, highlight how these strategies can be employed to improve cheese quality and reduce the associated economic burden on cheese processors.This work was funded by the Department of Agriculture, Food and the Marine under the Food Institutional Research Measure. Daniel J. O’Sullivan is in receipt of a Teagasc Walsh Fellowship, Grant Number:2012205

Temporal and spatial differences in microbial composition during the manufacture of a Continental-type cheese

peer-reviewedWe sought to determine if the time, within a production day, that a cheese is manufactured has an influence on the microbial community present within that cheese. To facilitate this, 16S rRNA amplicon sequencing was used to elucidate the microbial community dynamics of brine salted Continental-type cheese in cheeses produced early and late in the production day. Differences in microbial composition of the core and rind of the cheese were also investigated. Throughout ripening, it was apparent that late production day cheeses had a more diverse microbial population than their early day equivalents. Spatial variation between the cheese core and rind was also noted in that cheese rinds were found to initially have a more diverse microbial population but thereafter the opposite was the case. Interestingly, the genera Thermus, Pseudoalteromonas and Bifidobacterium, not routinely associated with a Continental-type cheese produced from pasteurised milk were detected. The significance, if any, of the presence of these genera will require further attention. Ultimately, the use of high throughput sequencing has facilitated a novel and detailed analysis of the temporal and spatial distribution of microbes in this complex cheese system and established that the period during a production cycle at which a cheese is manufactured can influence its microbial composition.This work was funded by the Department of Agriculture, Food and the Marine under the Food Institutional Research Measure through the ‘Cheeseboard 2015’ project. Daniel J. O’Sullivan is in receipt of a Teagasc Walsh Fellowship, Grant Number: 201220

Primary proteolysis of caseins in cheddar cheese

Studies were undertaken to investigate proteolysis of the caseins during the initial stages of maturation of Cheddar cheese. Isolated caseins were hydrolyzed by enzymes thought to be of importance during cheese ripening and the resulting peptides isolated and identified. Large peptides were also isolated from Cheddar cheese and identified, thus enabling the extent to which casein degradation studies could be extrapolated to cheese to be established. The proteolytic specificity of chymosin on bovine αs1- and αs2-caseins and of plasmin on bovine αs1-casein were determined. The action of cathepsin D, the principal indigenous acid milk proteinase, on caseins was studied and its pH optimum and sensitivity to NaCI determined. The action of cathepsin D on αs1-, αs2-, β- and κ-caseins was compared with that of chymosin and was found to be generally similar for the hydrolysis of αs1- and κ-caseins but to differ for αs2-and β- caseins. β-Casein in solution was hydrolyzed by cell wall-associated proteinases from three strains of Lactococcus lactis; comparison of electrophoretograms of the hydrolyzates with those of Cheddar cheese indicated that no peptides produced by cell wall-associated proteinases were detectable in the cheeses. All the major peptides in the water-insoluble fraction of Cheddar cheese were isolated and identified. It was found that β-casein was degraded primarily by plasmin and αs1 -casein by chymosin. Initial chymosin and plasmin cleavage sites in αs1-, and β-casein, respectively, identified in these and other studies corresponded to the peptides isolated from cheese. The importance of non-starter lactic acid bacteria (NSLAB) to the maturation of Cheddar was studied in cheeses manufactured from raw, pasteurized or microfiltered milks. NSLAB were found to strongly influence the quality and patterns of proteolysis. Results presented in this thesis are consistent with the hypothesis that primary proteolysis in Cheddar is catalysed primarily by the action of chymosin and plasmin on intact αs1- and β-caseins, respectively. The resulting large peptides so produced are subsequently degraded by these enzymes and by proteinases and peptidases from the starter and NSLAB

High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

peer-reviewedBackground The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. Results Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. Conclusions In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.This work was funded by the Department of Agriculture, Food and the Marine under the Food Institutional Research Measure through the ‘Cheeseboard 2015’ project. Daniel J. O’Sullivan is in receipt of a Teagasc Walsh Fellowship, Grant Number: 2012205

Effect of pectin on the composition, microbiology, texture, and functionality of reduced-fat Cheddar cheese

International audienceAbstractHydrocolloids have been extensively studied in low-fat cheeses as a way to improve defects associated with fat reduction, which are often related to texture and functionality (meltability). Pectin is a polysaccharide obtained from plant cells and is commonly used as a stabilizer for acidified dairy beverages. This work aimed to evaluate the effect of three types of commercial pectins on the characteristics of reduced-fat Cheddar cheese during a ripening period of 180 days. Five Cheddar cheeses were made: full-fat control (FF), reduced-fat control (RF), and reduced-fat cheeses with amidated (RA), high-methoxy (RH), or low-methoxy (RL) pectin added to milk prior processing at concentrations of 0.175%, 0.100%, and 0.075% (w/w), respectively; levels were chosen to avoid phase separation of the casein micelles, due to depletion flocculation. Addition of amidated pectin markedly increased the moisture content of the experimental cheese (~49%), compared to RF (~45%; P 100 N in RF at 180 days; P 85 versus <70% at 180 days; P < 0.05). These results suggest that pectin addition can be used to modify the moisture content, texture, and melting properties of reduced-fat Cheddar cheese

Proteolysis in Irish farmhouse Camembert cheese during ripening

Proteolysis in an Irish farmhouse Camembert cheese was studied during 10 weeks of ripening. Urea‐polyacrylamide gel electrophoresis of pH 4.6‐insoluble fractions of cheese showed the degradation of caseins, initially due to the action of chymosin and plasmin and later due to Penicillium camemberti proteinases. Proteolytic specificities of Penicillium camemberti proteinases on the caseins in milk hydrolysates were determined and 64, 6, 28, and 2 cleavage sites were identified in αs1‐, αs2‐, β‐, and κ‐casein, respectively. Proteolysis in cheese was studied and peptides produced were determined and compared to the cleavage specificities of Penicillium camemberti proteinases. Regions most susceptible to proteolysis were 1–40, 79–114, and 168–199 in αs1‐casein; 42–79 and 97–116 in αs2‐casein; 40–57, 101–125, 143–189, and 165–209 in β‐casein; and 31–81 and 124–137 in κ‐casein. The present study describes in detail the proteolytic action of proteinases from Penicillium camemberti in Camembert cheese during ripening. Practical applications: Camembert cheese is a major international cheese variety, made in many countries around the world. The ripening of the cheese involves many biochemical changes and this study provides new information on peptides produced during ripening. Penicillium camemberti is an important mold used in the production of this type of cheese and detailed information is provided on the action of its enzymes on the caseins. Data reported in this study furthers the understanding of the ripening of Camembert cheese

Sequencing of the Cheese Microbiome and Its Relevance to Industry

peer-reviewedThe microbiota of cheese plays a key role in determining its organoleptic and other physico-chemical properties. It is essential to understand the various contributions, positive or negative, of these microbial components in order to promote the growth of desirable taxa and, thus, characteristics. The recent application of high throughput DNA sequencing (HTS) facilitates an even more accurate identification of these microbes, and their functional properties, and has the potential to reveal those microbes, and associated pathways, responsible for favorable or unfavorable characteristics. This technology also facilitates a detailed analysis of the composition and functional potential of the microbiota of milk, curd, whey, mixed starters, processing environments, and how these contribute to the final cheese microbiota, and associated characteristics. Ultimately, this information can be harnessed by producers to optimize the quality, safety, and commercial value of their products. In this review we highlight a number of key studies in which HTS was employed to study the cheese microbiota, and pay particular attention to those of greatest relevance to industry

Composition and properties of bovine colostrum: a review

International audienceAbstractColostrum is the initial milk secreted by mammals following parturition, the composition and physicochemical properties of which are highly dynamic and variable. The composition and physicochemical properties of colostrum during the initial post-partum period has not been systematically reviewed for many years, although the topic remains of interest both to milk producers and processors. In this article, the current understanding of the composition of colostrum, i.e. carbohydrates, proteins, growth factors, enzymes, enzyme inhibitors, nucleotides and nucleosides, cytokines, fats, vitamins and minerals, is reviewed. In addition, the physicochemical properties, i.e. pH and buffering capacity, colour, density and specific gravity, osmotic pressure, somatic cell count, properties of casein micelles, ethanol stability and rennet coagulation properties are discussed, as well as the effects of heat-treating colostrum

Genome Sequence of Staphylococcus saprophyticus DPC5671, a Strain Isolated from Cheddar Cheese

peer-reviewedThe draft genome sequence of Staphylococcus saprophyticus DPC5671, isolated from cheddar cheese, was determined. S. saprophyticus is a common Gram-positive bacterium detected on the surface of smear-ripened cheese and other fermented foods