The Prevalence and Control of Bacillus and Related Spore-Forming Bacteria in the Dairy Industry

peer-reviewedMilk produced in udder cells is sterile but due to its high nutrient content, it can be a good growth substrate for contaminating bacteria. The quality of milk is monitored via somatic cell counts and total bacterial counts, with prescribed regulatory limits to ensure quality and safety. Bacterial contaminants can cause disease, or spoilage of milk and its secondary products. Aerobic spore-forming bacteria, such as those from the genera Sporosarcina, Paenisporosarcina, Brevibacillus, Paenibacillus, Geobacillus and Bacillus, are a particular concern in this regard as they are able to survive industrial pasteurization and form biofilms within pipes and stainless steel equipment. These single or multiple-species biofilms become a reservoir of spoilage microorganisms and a cycle of contamination can be initiated. Indeed, previous studies have highlighted that these microorganisms are highly prevalent in dead ends, corners, cracks, crevices, gaskets, valves and the joints of stainless steel equipment used in the dairy manufacturing plants. Hence, adequate monitoring and control measures are essential to prevent spoilage and ensure consumer safety. Common controlling approaches include specific cleaning-in-place processes, chemical and biological biocides and other novel methods. In this review, we highlight the problems caused by these microorganisms, and discuss issues relating to their prevalence, monitoring thereof and control with respect to the dairy industry.NG is funded by the Teagasc Walsh Fellowship Scheme and through the Irish Dairy Levy funded project ‘Thermodur-Out.

Model System for the Production of Enzyme Modified Cheese (EMC) Flavours.

End of Project ReportNatural cheese flavour ingredients, in the form of enzyme modified cheeses (EMCs), are widely used in the convenience food industry and can provide high volume added opportunities for the cheese industry. Many EMCs are produced using commercial enzyme preparations and previous studies have indicated that they contain side activities in addition to their stated main activity (see DPRC Report No.10). Therefore, it is critical that the exact enzyme complement of these preparations are known before they can be used to produce EMC of specific requirements on a consistent basis. The scientific basis of rapid enzyme mediated flavour formation in the production of EMCs is not fully understood. Consequently this knowledge gap is a major obstacle in the development of high value cheese flavour ingredients. Hence, a major objective of this project was to deepen the scientific understanding of flavour formation with a view to the production of natural enzyme-mediated dairy flavour ingredients with commercial potential. The ultimate aim was to develop the technology to produce customised high value dairy flavour ingredients in an optimised process.Dairy LevyDepartment of Agriculture, Food and the Marin

Significance of Lactobacilli in Cheddar Cheese

End of Project ReportThe objectives of this project were to isolate and identify the non-starter lactobacilli in mature Cheddar cheese, identify strains which impart mature flavours to cheese and determine their role in developing cheese flavour. The main conclusions were as follows: Based on an analysis of 18 mature Cheddar cheeses, selected from 7 commercial manufacturers, non-starter lactic acid bacteria typically numbered, as expected, 106-108 per gram and were dominated (97 percent) by Lactobacillus paracasei. Although a small number of strains (typically 1 to 4) was found in each cheese there was considerable strain diversity in cheeses within as well as between manufacturing plants. When selected strains were investigated for survival and flavour enhancement when added (as starter adjuncts) with the normal starter cultures in Cheddar cheese manufacture, it was found that they remained dominant for up to 3 months of ripening. Commercial grading of these cheeses at 3 and 6 months confirmed that the added strains did modify flavour development and one (DPC 4103), in particular, had a beneficial effect. It was confirmed that two selected strains of non-starter lactobacilli were capable of metabolising citrate under the conditions of Cheddar cheese ripening and, consequently, if present in sufficient numbers, would influence flavour development. The work was greatly facilitated by the successful and novel adaptation of a modern rapid molecular technique (RAPD) for species and strain classification. In summary these studies found that one species of lactobacilli (Lb. paracasei) was the dominant non-starter lactic acid bacteria in mature Cheddar cheese. Although a wide variety of strains were identified, only a few were found in any particular cheese, suggesting their likely role in cheese flavour diversity even within the same manufacturing plant. This suggests the potential for flavour control or enhancement through the selective and controlled use of non-starter lactic acid bacteria. Preliminary investigations of the metabolism of those organisms supports this view and a follow-up study now in progress should provide greater clarity on this matter.Department of Agriculture, Food and the Marin

Bile salt hydrolase and lipase inhibitory activity in reconstituted skim milk fermented with lactic acid bacteria

peer-reviewedObesity is the main cause of metabolic syndrome, a condition of which includes hypercholesterolemia. Reduced dietary fat absorption through inhibition of pancreatic lipase and/or hydrolysis of bile salts may provoke weight loss and cholesterol reduction. In this study, the potential anti-obesity properties of milk fermented with lactic acid bacteria was assessed by measuring the expression of bile salt hydrolase (BSH) after milk fermentation and the ability of fermentates to inhibit pancreatic lipase in vitro. Thirty BSH positive strains were identified, with 17 strains expressing this enzyme during milk fermentation. Apart from BSH activity, the milks fermented with L. plantarum SC70 and SC80 also displayed the capacity to inhibit pancreatic lipase by >35%. As expression of BSH and inhibition of pancreatic lipase are proposed synergistic activities to reduce fat absorption, fermentates produced with these strains are good candidates for use as functional foods for the treatment of obesity and hypercholesterolemia

Role of Lactobacilli in Flavour Development of Cheddar Cheese.

End of Project ReportCheddar cheese is a complex microbial ecosystem. The internal cheese environment, in particular of hard and semi-hard cheeses, is not conducive to the growth of many microorganisms. At the beginning of ripening the dominant microorganisms are the starter bacteria which are present at high levels (~109/g). However, during ripening, non-starter lactic acid bacteria (NSLAB) grow from relatively low levels (<103/g) at the beginning of ripening, to 108/g within 6 - 8 weeks. Other bacteria, e.g. enterococci and staphylococci, may also be present but in much lower numbers. In a previous study of mature and extra mature Cheddar cheeses from different manufacturers (see End of Project Report No. 1), it was found that the NSLAB population was dominated by strains of Lb. paracasei. However, their contribution to cheese flavour and their source(s) are still unclear, nor is it known if the NSLAB flora is unique to each plant. Hence, understanding the growth of this group of organisms in cheese is a key to defining their role in flavour development. The biochemistry of flavour development in cheese is poorly understood. For most cheese varieties, including Cheddar, proteolysis, which results in the accumulation of free amino acids, is of vital importance for flavour development. Increasing evidence suggests that the main contribution of amino acids is as substrates for the development of more complex flavour and aroma compounds. The manner by which such compounds are generated in cheese is currently the focus of much research. Starter bacteria have been shown to contain a range of enzymes capable of facilitating the conversion of amino acids to potential flavour compounds. However, the potential of lactobacilli (NSLAB) to produce similar enzymes has only recently been investigated. Hence, although, it is generally accepted that the cheese starter flora is the primary defining influence on flavour development, the contribution of NSLAB is also considered significant. The objectives of these studies were: - to develop a greater understanding of the behaviour of NSLAB in cheese, and - to identify suitable strains, and other cheese bacteria, to be used as starter adjuncts for flavour improvement.Department of Agriculture, Food and the Marin

Influence of Enterococci and Thermophilic Starter Bacteria on Cheddar Cheese Flavour

End of Project ReportThis project set out to identify suitable enterococci and thermophilic starter strains which could be added to the cheese during manufacture (as starter adjuncts) with the specific aims of enhancing flavour during ripening as well as facilitating flavour diversity - a trait sought by many commercial Cheddar companies. This project confirmed the potential of thermophilic lactic acid strains to affect flavour when used as starter adjuncts in Cheddar cheese manufacture. Their use can also lead to the development of novel flavours. Many adjunct cultures proposed to-date to enhance Cheddar flavour are composed of strains of lactococcal starter, selected for their flavouring capacity. However, application of such strains in industry would lead to increased probability of phage attack on the primary starter. On the other hand, thermophilic lactic acid strains are phage unrelated to conventional starter and thus would not lead to the introduction of starter specific phage into the cheese plant. A thermophilic strain from the Moorepark collection (DPC 4571) was shown to have major commercial potential as a flavour enhancer.Department of Agriculture, Food and the Marin

The potential of non-starter lactic acid bacteria from Cheddar cheese to colonise the gut

peer-reviewedThis study was undertaken to assess the potential of Non-Starter Lactic Acid Bacteria (NSLAB) from Cheddar cheese to survive gastric transit and display probiotic-related traits including bile salt hydrolase activity, the ability to adhere to the gut epithelium and inhibition of enteropathogen binding. Populations of NSLAB, up to 107 CFU/g per cheese were recovered following exposure of cheese to Simulated Stomach Duodenum Passage (SSDP) conditions. A total of 240 isolates were randomly selected from twelve Cheddar cheeses and assessed for probiotic traits. Two strains Lactobacillus paracasei DPC 7150 and Lactobacillus rhamnosus DPC 7102 showed the most probiotic potential. The Lb. paracasei and Lb. rhamnosus strains displayed adhesion rates of 64% and 79%, respectively and inhibited binding of pathogenic Escherichia coli by >20%. This research demonstrates that Cheddar cheese harbours potentially beneficial bacteria, a large portion of which can survive simulated digestion and potentially exhibit health beneficial effects once ingested.TEAGAS

Health Benefits of Lactic Acid Bacteria (LAB) Fermentates

peer-reviewedConsuming fermented foods has been reported to result in improvements in a range of health parameters. These positive effects can be exerted by a combination of the live microorganisms that the fermented foods contain, as well as the bioactive components released into the foods as by-products of the fermentation process. In many instances, and particularly in dairy fermented foods, the microorganisms involved in the fermentation process belong to the lactic acid group of bacteria (LAB). An alternative approach to making some of the health benefits that have been attributed to fermented foods available is through the production of ‘fermentates’. The term ‘fermentate’ generally relates to a powdered preparation, derived from a fermented product and which can contain the fermenting microorganisms, components of these microorganisms, culture supernatants, fermented substrates, and a range of metabolites and bioactive components with potential health benefits. Here, we provide a brief overview of a selection of in vitro and in vivo studies and patents exclusively reporting the health benefits of LAB ‘fermentates’. Typically, in such studies, the potential health benefits have been attributed to the bioactive metabolites present in the crude fermentates and/or culture supernatants rather than the direct effects of the LAB strain(s) involved

Identification of the key compounds responsible for Cheddar cheese flavour

End of Project ReportThere is a poor understanding of the relationship between organoleptic assessment of cheese and quantitative analysis of flavour compounds. Further, the contribution of particular cheese-making parameters such as ripening temperature and starter culture has not been fully elucidated. During the ripening of most cheese varieties complex chemical conversions occur within the cheese matrix. In most cheese varieties breakdown of protein is the most important flavour development pathway. The primary cheese protein, casein, is degraded enzymatically to short peptides and free amino acids. The agents primarily responsible for these conversions are the residual rennet that is retained in the cheese curd at the end of the manufacturing phase and the proteinases and peptidases that are associated with the starter bacteria. While the rate and degree of proteolysis are of vital significance for desired flavour development, the direct products of proteolysis do not fully define cheese flavour. Much research is now demonstrating that the further biochemical and chemical conversions of the products of proteolysis, in particular the amino acids, are necessary for full flavour development. The products produced by these pathways are volatile at low boiling points and are thus released during mastication of the cheese in the mouth. Many of these volatile compounds contribute to the flavour sensation experienced by the consumer. A very wide spectrum of such compounds have been isolated from cheese, in excess of two hundred in some cheese varieties. It is now generally accepted that there is no individual compound which defines cheese flavour completely and that the flavour sensation is the result of numerous compounds present in the correct proportions. This has become known as the Component Balance Theory . The application of modern analytical techniques as proposed in this project would provide a greater understanding of the significant flavour compounds in Cheddar cheese and help to identify the impact of specific cheese-making parameters such as starter flora and ripening temperature on the production of volatile flavour compounds. This data would assist the general programme on flavour improvement of cheese which should ultimately benefit the cheese manufacturer. Hence this project set out to develop methods to identify the key flavour compounds in Cheddar cheese. These techniques would then be applied to experimental and commercial cheeses during ripening in an effort to identify key compounds and the influence of starter cultures and ripening temperature on their production.Department of Agriculture, Food and the Marin

The potential of non-starter lactic acid bacteria from Cheddar cheese to colonise the gut

peer-reviewedThis study was undertaken to assess the potential of Non-Starter Lactic Acid Bacteria (NSLAB) from Cheddar cheese to survive gastric transit and display probiotic-related traits including bile salt hydrolase activity, the ability to adhere to the gut epithelium and inhibition of enteropathogen binding. Populations of NSLAB, up to 107 CFU/g per cheese were recovered following exposure of cheese to Simulated Stomach Duodenum Passage (SSDP) conditions. A total of 240 isolates were randomly selected from twelve Cheddar cheeses and assessed for probiotic traits. Two strains Lactobacillus paracasei DPC 7150 and Lactobacillus rhamnosus DPC 7102 showed the most probiotic potential. The Lb. paracasei and Lb. rhamnosus strains displayed adhesion rates of 64% and 79%, respectively and inhibited binding of pathogenic Escherichia coli by >20%. This research demonstrates that Cheddar cheese harbours potentially beneficial bacteria, a large portion of which can survive simulated digestion and potentially exhibit health beneficial effects once ingested