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

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

Validation of an Automated Body Condition Scoring System Using 3D Imaging

peer-reviewedBody condition scores (BCS) measure a cow’s fat reserves and is important for management and research. Manual BCS assessment is subjective, time-consuming, and requires trained personnel. The BodyMat F (BMF, Ingenera SA, Cureglia, Switzerland) is an automated body condition scoring system using a 3D sensor to estimate BCS. This study assesses the BMF. One hundred and three Holstein Friesian cows were assessed by the BMF and two assessors throughout a lactation. The BMF output is in the 0–5 scale commonly used in France. We develop and report the first equation to convert these scores to the 1–5 scale used by the assessors in Ireland in this study ((0–5 scale × 0.38) + 1.67 → 1–5 scale). Inter-assessor agreement as measured by Lin’s concordance of correlation was 0.67. BMF agreement with the mean of the two assessors was the same as between assessors (0.67). However, agreement was lower for extreme values, particularly in over-conditioned cows where the BMF underestimated BCS relative to the mean of the two human observers. The BMF outperformed human assessors in terms of reproducibility and thus is likely to be especially useful in research contexts. This is the second independent validation of a commercially marketed body condition scoring system as far as the authors are aware. Comparing the results here with the published evaluation of the other system, we conclude that the BMF performed as well or better

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

Steam and Flame Applications as Novel Methods of Population Control for Invasive Asian Clam (Corbicula fluminea) and Zebra Mussel (Dreissena polymorpha)

Control strategies for established populations of invasive alien species can be costly and complex endeavours, which are frequently unsuccessful. Therefore, rapid-reaction techniques that are capable of maximising efficacy whilst minimising environmental damage are urgently required. The Asian clam (Corbicula fluminea Müller, 1774), and the zebra mussel (Dreissena polymorpha Pallas, 1771), are invaders capable of adversely affecting the functioning and biodiversity of freshwater ecosystems. Despite efforts to implement substantial population-control measures, both species continue to spread and persist within freshwater environments. As bivalve beds often become exposed during low-water conditions, this study examined the efficacy of steam-spray (≥100 °C, 350 kPa) and open-flame burn treatments (~1000 °C) to kill exposed individuals. Direct steam exposure lasting for 5 min caused 100% mortality of C. fluminea buried at a depth of 3 cm. Further, combined rake and thermal shock treatments, whereby the substrate is disturbed between each application of either a steam or open flame, caused 100% mortality of C. fluminea specimens residing within a 4-cm deep substrate patch, following three consecutive treatment applications. However, deeper 8-cm patches and water-saturated substrate reduced maximum bivalve species mortality rates to 77% and 70%, respectively. Finally, 100% of D. polymorpha specimens were killed following exposure to steam and open-flame treatments lasting for 30 s and 5 s, respectively. Overall, our results confirm the efficacy of thermal shock treatments as a potential tool for substantial control of low-water-exposed bivalves. Although promising, our results require validation through upscaling to field application, with consideration of other substrate types, increased substrate depth, greater bivalve densities, non-target and long-term treatment effects

Assessing the ability of nisin A and derivatives thereof to inhibit gram-negative bacteria from the genus Thermus

peer-reviewNisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains

Assessing the ability of nisin A and derivatives thereof to inhibit gram-negative bacteria from the genus Thermus

peer-reviewNisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains

The Use of ICT Tools to Capture Grass Data and Optimise Grazing Management

In temperate regions, where pasture-based milk production systems predominate, the strategic allocation of pasture grazing area to dairy cows is essential for optimal management and increased milk outputs. Rising plate meters (RPM) are frequently used to estimate pasture herbage mass (HM; i.e. dry matter yield per hectare), through the use of simple regression equations that relate compressed sward height (CSH) to HM. Measurement must be accurate and efficient. Despite improved farm management practices aided by a variety of technological advances, the standard design of a RPM has remained relatively unchanged. As part of this thesis, a RPM utilising a micro-sonic sensor and digital data capture capability linked to a smart device application was developed. Further, the ability of the micro-sonic sensor RPM, to accurately and precisely measure fixed heights was examined. As correct allocation of grazing area requires accurate geolocation positioning, the associated GPS technology was assessed. In order to improve the accuracy and precision of these equations, so that inherent variation of grasslands is captured, there is a need to incorporate differences in grass types and seasonal growth As good bassline data are required for the development of effective conversion of CSH to HM, the variation of growth for both perennial ryegrass and hybrid ryegrass was recorded over the seven month growing season, using a total of 308 grass plots. Once the correct HM is established it must be allocated to the herd in an accurate and efficient manner. As intensive pasture-based farming systems rely on precise and frequent allocations of grass to animals, a Virtual Fence (VF) system to enhance automated allocation of correct forage areas to animals was developed and assessed, as was an associated cow training protocol. The micro-sonic sensor RPM was found to be significantly more accurate for height capture than a traditional ratchet counter RPM. The ratchet counter RPM underestimated height by 7.68 ± 0.06 mm (mean ± SE), while the micro-sonic sensor RPM overestimated height by 0.18 ± 0.08 mm. These discrepancies can result in an under- and overestimation of HM by 13.71 % and 0.32 % per Ha-1, respectively. The performance of the on-board GPS did not significantly differ from that of a tertiary device. Subsequently, three dynamic equations were derived for the effective conversion algorithms form CSH to HM incorporating different grass types, time of the year and dry matter percentage, one of algorithms is now in everyday commercial use. Although the operating capacity of the VF system was found to be robust, with dairy cows rapidly associating visual cues with VF boundary lines, and a cue-consequence association with the audio warning and corrective stimulus, the number of boundary challenges made by cows increased upon removal of all visual cues. Overall, although further research will be required, the results presented within this thesis allow for the further development of decision support tools to improve on-farm grassland management

Novel concept to allow automation of grazing management within a dairy farm system

Abstract Precise grass allocation is fundamental to economic return in the Irish and European grassland production systems. There is a strong relationship between the cost of production and the proportion of grass in the cow's diet. It is now widely accepted that grazed grass is the cheapest feed available on most European dairy farms. Grazing management usually involves subjective visual assessment and intuitive decision-making on farms implementing an intensive grazing system. The development of an automated grass measurement tool would add objectivity to the measurement of grass and the use of virtual fence (VF) technology would automate the process of grass allocation. The aim of this project is to develop a decision support tool (DST) with global positioning system (GPS) capability that will precisely measure herbage mass and integrate with an on-cow VF system, which is also GPS based to accurately and automatically allocate the grazing area. One virtual fence approach is to set virtual boundaries via GPS and Global System for Mobile Communications (GSM) integrated in an on-cow device. To stop an animal crossing the virtual boundary, negative reinforcement acting as a warning system will be used. This technology would potentially result in labour savings by removing the requirement to physically setup electric fences for grazing allocation. The measurement of herbage height and mass by the DST will be calibrated and validated along-side conventional measurement techniques on a number of research and pilot farms over an entire year. A variety of sward heights, types and herbage mass from pre-, intermediate and post-grazing swards in Irish paddocks will be measured. Once calibrated and validated the DST will be integrated with virtual fence technology and the communication between these devices will be evaluated on dairy cows. The impact of GPS error on the movement of the virtual fence boundary will be analysed and subsequent alterations implemented to enhance the accuracy of the device. An effective triggering system for negative reinforcement of the virtual fence boundary will be analysed, using acoustic and tactile cues when animals choose to test the boundaries. Improvements to the system will be implemented based on the initial calibration. The goal is to achieve an accurate, labour saving and economical method for precision grazing