• Effect of milk centrifugation and incorporation of high heat-treated centrifugate on the microbial composition and levels of volatile organic compounds of Maasdam cheese

      Lamichhane, Prabin; Pietrzyk, Anna; Feehily, Conor; Cotter, Paul D.; Mannion, David T.; Kilcawley, Kieran; Kelly, Alan L.; Sheehan, Diarmuid (JJ); Dairy Levy Trust; Teagasc Walsh Fellowship Programme; et al. (Elsevier, 2018-03-15)
      Centrifugation is a common milk pretreatment method for removal of Clostridium spores which, on germination, can produce high levels of butyric acid and gas, resulting in rancid, gassy cheese. The aim of this study was to determine the effect of centrifugation of milk, as well as incorporation of high heat-treated centrifugate into cheese milk, on the microbial and volatile profile of Maasdam cheese. To facilitate this, 16S rRNA amplicon sequencing in combination with a selective media-based approach were used to study the microbial composition of cheese during maturation, and volatile organic compounds within the cheese matrix were analyzed by HPLC and solid-phase microextraction coupled with gas chromatography–mass spectrometry. Both culture-based and molecular approaches revealed major differences in microbial populations within the cheese matrix before and after warm room ripening. During warm room ripening, an increase in counts of propionic acid bacteria (by ∼101.5 cfu) and nonstarter lactic acid bacteria (by ∼108 cfu) and a decrease in the counts of Lactobacillus helveticus (by ∼102.5 cfu) were observed. Lactococcus species dominated the curd population throughout ripening, followed by Lactobacillus, Propionibacterium, and Leuconostoc, and the relative abundance of these accounted for more than 99% of the total genera, as revealed by high-throughput sequencing. Among subdominant microflora, the overall relative abundance of Clostridium sensu stricto was lower in cheeses made from centrifuged milk than control cheeses, which coincided with lower levels of butyric acid. Centrifugation as well as incorporation of high heat-treated centrifugate into cheese milk seemed to have little effect on the volatile profile of Maasdam cheese, except for butyric acid levels. Overall, this study suggests that centrifugation of milk before cheesemaking is a suitable method for controlling undesirable butyric acid fermentation without significantly altering the levels of other volatile organic compounds of Maasdam cheese.
    • Evaluation of the Potential of Lactobacillus paracasei Adjuncts for Flavor Compounds Development and Diversification in Short-Aged Cheddar Cheese

      Stefanovic, Ewelina; Kilcawley, Kieran; Roces, Clara; Rea, Mary; O'Sullivan, Maurice G.; Sheehan, Diarmuid (JJ); McAuliffe, Olivia; Teagasc Walsh Fellowship Programme; 2012040 (Frontiers, 05/07/2018)
      The non-starter microbiota of Cheddar cheese mostly comprises mesophilic lactobacilli, such as Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and Lactobacillus plantarum. These bacteria are recognized for their potential to improve Cheddar cheese flavor when used as adjunct cultures. In this study, three strains of L. paracasei (DPC2071, DPC4206, and DPC4536) were evaluated for their contribution to the enhancement and diversification of flavor in short-aged Cheddar cheese. The strains were selected based on their previously determined genomic diversity, variability in proteolytic enzyme activities and metabolic capability in cheese model systems. The addition of adjunct cultures did not affect the gross composition or levels of lipolysis of the cheeses. The levels of free amino acids (FAA) in cheeses showed a significant increase after 28 days of ripening. However, the concentrations of individual amino acids in the cheeses did not significantly differ except for some amino acids (aspartic acid, threonine, serine, and tryptophan) at Day 14. Volatile profile analysis revealed that the main compounds that differentiated the cheeses were of lipid origin, such as long chain aldehydes, acids, ketones, and lactones. This study demonstrated that the adjunct L. paracasei strains contributed to the development and diversification of compounds related to flavor in short-aged Cheddar cheeses.
    • Evaluation of the Potential of Lactobacillus paracasei Adjuncts for Flavor Compounds Development and Diversification in Short-Aged Cheddar Cheese

      Stefanovic, Ewelina; Kilcawley, Kieran; Roces, Clara; Rea, Mary; O'Sullivan, Maurice G.; Sheehan, Diarmuid (JJ); McAuliffe, Olivia; Teagasc Walsh Fellowship Programme; 2012040 (Frontiers, 2018-07-05)
      The non-starter microbiota of Cheddar cheese mostly comprises mesophilic lactobacilli, such as Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, and Lactobacillus plantarum. These bacteria are recognized for their potential to improve Cheddar cheese flavor when used as adjunct cultures. In this study, three strains of L. paracasei (DPC2071, DPC4206, and DPC4536) were evaluated for their contribution to the enhancement and diversification of flavor in short-aged Cheddar cheese. The strains were selected based on their previously determined genomic diversity, variability in proteolytic enzyme activities and metabolic capability in cheese model systems. The addition of adjunct cultures did not affect the gross composition or levels of lipolysis of the cheeses. The levels of free amino acids (FAA) in cheeses showed a significant increase after 28 days of ripening. However, the concentrations of individual amino acids in the cheeses did not significantly differ except for some amino acids (aspartic acid, threonine, serine, and tryptophan) at Day 14. Volatile profile analysis revealed that the main compounds that differentiated the cheeses were of lipid origin, such as long chain aldehydes, acids, ketones, and lactones. This study demonstrated that the adjunct L. paracasei strains contributed to the development and diversification of compounds related to flavor in short-aged Cheddar cheeses.
    • High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

      O'Sullivan, Daniel; Fallico, Vincenzo; O'Sullivan, Orla; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D.; Giblin, Linda; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 2012205 (Biomed Central, 17/11/2015)
      Background 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.
    • High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

      O'Sullivan, Daniel; Fallico, Vincenzo; O'Sullivan, Orla; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D.; Giblin, Linda; Teagasc Walsh Fellowship Programme; Department of Agriculture, Food and the Marine; 2012205 (Biomed Central, 17/11/2015)
      Background: 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.
    • Nucleic acid-based approaches to investigate microbial-related cheese quality defects

      O'Sullivan, Daniel; Giblin, Linda; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D.; Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 2012205 (Frontiers Media SA, 21/01/2013)
      The 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.
    • Sequencing of the Cheese Microbiome and Its Relevance to Industry

      Yeluri Jonnala, Bhagya R.; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D.; Teagasc Walsh Fellowship Programme (Frontiers, 2018-05-23)
      The 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.
    • Sequencing of the Cheese Microbiome and Its Relevance to Industry

      Yeluri Jonnala, Bhagya R.; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Cotter, Paul D.; Teagasc Walsh Fellowship Programme (Frontiers, 2018-05-23)
      The 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.
    • Symposium review: Structure-function relationships in cheese

      Lamichhane, Prabin; Kelly, Alan L.; Sheehan, Diarmuid (JJ); Dairy Levy Trust; Teagasc Walsh Fellowship Programme; Ornua; RMIS6259 (Elsevier, 2017-10-18)
      The quality and commercial value of cheese are primarily determined by its physico-chemical properties (e.g., melt, stretch, flow, and color), specific sensory attributes (e.g., flavor, texture, and mouthfeel), usage characteristics (e.g., convenience), and nutritional properties (e.g., nutrient profile, bioavailability, and digestibility). Many of these functionalities are determined by cheese structure, requiring an appropriate understanding of the relationships between structure and functionality to design bespoke functionalities. This review provides an overview of a broad range of functional properties of cheese and how they are influenced by the structural organization of cheese components and their interactions, as well as how they are influenced by environmental factors (e.g., pH and temperature).
    • Temporal and spatial differences in microbial composition during the manufacture of a Continental-type cheese

      O'Sullivan, Daniel; Cotter, Paul D.; O'Sullivan, Orla; Giblin, Linda; McSweeney, Paul L. H.; Sheehan, Diarmuid (JJ); Department of Agriculture, Food and the Marine; Teagasc Walsh Fellowship Programme; 2012205 (American Society for Microbiology, 30/01/2015)
      We 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.