Jeremiah J. Sheehan

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

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.

Relationships between the gross, non-volatile and volatile compositions and the sensory attributes of eight hard-type cheeses

Abstract Relationships between individual flavour attributes of eight hard-type cheeses and their volatile compounds, free amino acids (FAA), free fatty acids (FFA) and gross compositional constituents were determined. Relationships were also determined between individual texture attributes and gross compositional constituents. A trained panel of 15 assessors described the sensory characteristics of the cheeses using eight odour, twenty flavour, four appearance and nine texture attributes. Volatile compounds were isolated using a model-mouth device. FAA, FFA and gross compositional constituents were determined using standard methods. Relationships were determined by using partial least squares regression coupled with a new jack-knife method for identification and elimination of non-contributing variables. Eight flavour attributes were found to be correlated with subsets of volatile compounds, FAA, FFA and gross compositional constituents. For instance, the “nutty” flavour of Emmental was found to be positively correlated with the concentrations of propionic acid, ethyl acetate and 2-pentanone. “Nutty” flavour was negatively correlated with the concentrations of salt in moisture and pH 4.6-soluble nitrogen (pH 4.6-SN). Four texture attributes were correlated with subsets of gross compositional constituents. For example, “firmness” was positively correlated with concentrations of protein, calcium and phosphorous and negatively correlated with pH value and level of pH 4.6-SN.

Relationships between sensory attributes and the volatile compounds, non-volatile and gross compositional constituents of six blue-type cheeses

Abstract Relationships between odour and flavour attributes of six blue-type cheeses and their volatile compounds, free amino acids (FAA), free fatty acids (FFA) and gross compositional constituents were determined. Relationships were also determined between texture attributes and gross compositional constituents. Fifteen assessors described the odour, flavour, appearance and texture profile of cheeses. Volatile compounds were isolated using a model-mouth apparatus. FAA, FFA and gross compositional constituents were determined using standard methods. Using Partial Least Squares Regression two odour and five flavour attributes were found to correlate with subsets of volatile compounds, FAA, FFA and gross compositional constituents. For example, “mouldy” flavour was positively correlated with the concentrations of pH 4.6-soluble nitrogen and 2-pentanone, 2-heptanone, 2-octanone and 2-nonanone. Three texture attributes were found to correlate with subsets of gross compositional constituents. For example, “crumbly” texture was positively correlated with concentration of fat and protein and negatively correlated with levels of moisture in the non-fat substance and moisture.

Temporal and Spatial Differences in Microbial Composition during the Manufacture of a Continental-Type Cheese

ABSTRACT 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 the microbial composition of the core and rind of the cheese were also investigated. Throughout ripening, it was apparent that cheeses produced late in the day had a more diverse microbial population than their early equivalents. Spatial variation between the cheese core and rind was also noted in that cheese rinds were initially found to 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 pasteurized 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.

Growth and location of bacterial colonies within dairy foods using microscopy techniques: a review.

The growth, location, and distribution of bacterial colonies in dairy products are important factors for the ripening and flavor development of cheeses, yogurts, and soured creams. Starter, non-starter, spoilage, and pathogenic bacteria all become entrapped in the developing casein matrix of dairy foods. In order to visualize these bacterial colonies and the environments surrounding them, microscopy techniques are used. The use of various microscopy methods allow for the rapid detection, enumeration, and distribution of starter, non-starter and pathogenic bacteria in dairy foods. Confocal laser scanning microscopy is extensively utilized to identify bacteria location via the use of fluorescent dyes. Further study is needed in relation to the development of micro- gradients and localized ripening parameters in dairy products due to the location of bacteria at the protein–fat interface. Development in the area of bacterial discrimination using microscopy techniques and fluorescent dyes/tags is needed as the benefits of rapidly identifying spoilage/pathogenic bacteria early in product manufacture would be of huge benefit in relation to both safety and financial concerns.

Thermus and the Pink Discoloration Defect in Cheese

Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin. ABSTRACT A DNA sequencing-based strategy was applied to study the microbiology of Continental-type cheeses with a pink discoloration defect. The basis for this phenomenon has remained elusive, despite decades of research. The bacterial composition of cheese containing the defect was compared to that of control cheese using 16S rRNA gene and shotgun metagenomic sequencing as well as quantitative PCR (qPCR). Throughout, it was apparent that Thermus, a carotenoid-producing genus, was present at higher levels in defect-associated cheeses than in control cheeses. Prompted by this finding and data confirming the pink discoloration to be associated with the presence of a carotenoid, a culture-based approach was employed, and Thermus thermophilus was successfully cultured from defect-containing cheeses. The link between Thermus and the pinking phenomenon was then established through the cheese defect equivalent of Koch’s postulates when the defect was recreated by the reintroduction of a T. thermophilus isolate to a test cheese during the manufacturing process. IMPORTANCE Pink discoloration in cheese is a defect affecting many cheeses throughout the world, leading to significant financial loss for the dairy industry. Despite decades of research, the cause of this defect has remained elusive. The advent of high-throughput, next-generation sequencing has revolutionized the field of food microbiology and, with respect to this study, provided a means of testing a possible microbial basis for this defect. In this study, a combined 16S rRNA, whole-genome sequencing, and quantitative PCR approach was taken. This resulted in the identification of Thermus, a carotenoid-producing thermophile, in defect-associated cheeses and the recreation of the problem in cheeses to which Thermus was added. This finding has the potential to lead to new strategies to eliminate this defect, and our method represents an approach that can be employed to investigate the role of microbes in other food defects of unknown origin.

Compromised Lactobacillus helveticus starter activity in the presence of facultative heterofermentative Lactobacillus casei DPC6987 results in atypical eye formation in Swiss-type cheese.

Nonstarter lactic acid bacteria are commonly implicated in undesirable gas formation in several varieties, including Cheddar, Dutch-, and Swiss-type cheeses, primarily due to their ability to ferment a wide variety of substrates. This effect can be magnified due to factors that detrimentally affect the composition or activity of starter bacteria, resulting in the presence of greater than normal amounts of fermentable carbohydrates and citrate. The objective of this study was to determine the potential for a facultatively heterofermentative Lactobacillus (Lactobacillus casei DPC6987) isolated from a cheese plant environment to promote gas defects in the event of compromised starter activity. A Swiss-type cheese was manufactured, at pilot scale and in triplicate, containing a typical starter culture (Streptococcus thermophilus and Lactobacillus helveticus) together with propionic acid bacteria. Lactobacillus helveticus populations were omitted in certain vats to mimic starter failure. Lactobacillus casei DPC6987 was added to each experimental vat at 4 log cfu/g. Cheese compositional analysis and X-ray computed tomography revealed that the failure of starter bacteria, in this case L. helveticus, coupled with the presence of a faculatively heterofermentative Lactobacillus (L. casei) led to excessive eye formation during ripening. The availability of excess amounts of lactose, galactose, and citrate during the initial ripening stages likely provided the heterofermentative L. casei with sufficient substrates for gas formation. The accrual of these fermentable substrates was notable in cheeses lacking the L. helveticus starter population. The results of this study are commercially relevant, as they demonstrate the importance of viability of starter populations and the control of specific nonstarter lactic acid bacteria to ensure appropriate eye formation in Swiss-type cheese.

Measurement of pH micro-heterogeneity in natural cheese matrices by fluorescence lifetime imaging

Cheese, a product of microbial fermentation may be defined as a protein matrix entrapping fat, moisture, minerals and solutes as well as dispersed bacterial colonies. The growth and physiology of bacterial cells in these colonies may be influenced by the microenvironment around the colony, or alternatively the cells within the colony may modify the microenvironment (e.g., pH, redox potential) due to their metabolic activity. While cheese pH may be measured at macro level there remains a significant knowledge gap relating to the degree of micro-heterogeneity of pH within the cheese matrix and its relationship with microbial, enzymatic and physiochemical parameters and ultimately with cheese quality, consistency and ripening patterns. The pH of cheese samples was monitored both at macroscopic scale and at microscopic scale, using a non-destructive microscopic technique employing C-SNARF-4 and Oregon Green 488 fluorescent probes. The objectives of this work were to evaluate the suitability of these dyes for microscale pH measurements in natural cheese matrices and to enhance the sensitivity and extend the useful pH range of these probes using fluorescence lifetime imaging (FLIM). In particular, fluorescence lifetime of Oregon Green 488 proved to be sensitive probe to map pH micro heterogeneity within cheese matrices. Good agreement was observed between macroscopic scale pH measurement by FLIM and by traditional pH methods, but in addition considerable localized microheterogeneity in pH was evident within the curd matrix with pH range between 4.0 and 5.5. This technique provides significant potential to further investigate the relationship between cheese matrix physico-chemistry and bacterial metabolism during cheese manufacture and ripening.

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

BackgroundThe 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.ResultsAmplicons 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.ConclusionsIn 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.

Effect of pasture versus indoor feeding systems on quality characteristics, nutritional composition, and sensory and volatile properties of full-fat Cheddar cheese

The purpose of this study was to investigate the effects of pasture-based versus indoor total mixed ration (TMR) feeding systems on the chemical composition, quality characteristics, and sensory properties of full-fat Cheddar cheeses. Fifty-four multiparous and primiparous Friesian cows were divided into 3 groups (n = 18) for an entire lactation. Group 1 was housed indoors and fed a TMR diet of grass silage, maize silage, and concentrates; group 2 was maintained outdoors on perennial ryegrass only pasture (GRS); and group 3 was maintained outdoors on perennial ryegrass/white clover pasture (CLV). Full-fat Cheddar cheeses were manufactured in triplicate at pilot scale from each feeding system in September 2015 and were examined over a 270-d ripening period at 8°C. Pasture-derived feeding systems were shown to produce Cheddar cheeses yellower in color than that of TMR, which was positively correlated with increased cheese β-carotene content. Feeding system had a significant effect on the fatty acid composition of the cheeses. The nutritional composition of Cheddar cheese was improved through pasture-based feeding systems, with significantly lower thrombogenicity index scores and a greater than 2-fold increase in the concentration of vaccenic acid and the bioactive conjugated linoleic acid C18:2 cis-9,trans-11, whereas TMR-derived cheeses had significantly higher palmitic acid content. Fatty acid profiling of cheeses coupled with multivariate analysis showed clear separation of Cheddar cheeses derived from pasture-based diets (GRS or CLV) from that of a TMR system. Such alterations in the fatty acid profile resulted in pasture-derived cheeses having reduced hardness scores at room temperature. Feeding system and ripening time had a significant effect on the volatile profile of the Cheddar cheeses. Pasture-derived Cheddar cheeses had significantly higher concentrations of the hydrocarbon toluene, whereas TMR-derived cheese had significantly higher concentration of 2,3-butanediol. Ripening period resulted in significant alterations to cheese volatile profiles, with increases in acid-, alcohol-, aldehyde-, ester-, and terpene-based volatile compounds. This study has demonstrated the benefits of pasture-derived feeding systems for production of Cheddar cheeses with enhanced nutritional and rheological quality compared with a TMR feeding system.