• Enterococci in Food Fermentations: Functional and Safety Aspects

      Cogan, Tim; Rea, Mary; Drinan, Finbarr; Gelsomino, R. (Teagasc, 2001-04-01)
      Enterococci are natural residents of the human and animal gastrointestinal tracts; many species are also found in soil, plants and food. These organisms also form an important part of the microflora of many cheeses, especially those made in Southern Europe, where they can reach levels of 107 - 108 cfu/g. There is contradictory information on their role in flavour development in cheese with some studies showing that they have a positive effect and others a negative one. Enterococcus faecalis, Ec. faecium and Ec. durans are the important species found in cheese, though recent results from our laboratory show that Ec. casseliflavus may also be important (see below). Many of these species withstand pasteurisation. Their presence in food has been questioned because they are responsible for many nosocomial infections in hospitals. They are also promiscuous and easily transfer antibiotic resistance to other organisms and acquire resistance to vancomycin themselves. Cheddar cheese has a complex microflora and is conducive to growth of many bacteria, especially lactic acid bacteria. Enterococci are facultative anaerobes, which ferment lactose and can grow in high salt concentrations. Therefore, they should grow in cheese if they are present in the raw milk. Phenotypically they can be confused with starter lactococci. Traditionally, they are separated from lactococci by their ability to grow at 45°C and in 6.5% salt. However, these tests have serious drawbacks since some species of enterococci cannot grow at 45°C and some lactococci can grow at 45°C and in 6.5% salt. The effect of enterococci on flavour development in Cheddar cheese has not been studied to any great extent. The overall objectives of this collaborative project were to investigate the taxonomic relationships between food, veterinary and clinical isolates of enterococci, their virulence, their ability to produce toxins, their antibiotic resistance and their technological performance in cheesemaking. The specific objectives of the Moorepark team were to study the co-metabolism of citrate and sugar by enterococci, develop a DNA probe to distinguish between Enterococcus and Lactococcus and evaluate the contribution of enterococci to flavour development in Cheddar cheese.
    • Establishment of Enabling Technology for Manufacture of Selected Types of Continental and Speciality Cheeses

      Wilkinson, M.G.; Sheehan, Diarmuid (JJ); Guinee, Timothy P.; Cogan, Tim (Teagasc, 1998-09-01)
      The objectives in the project were the development of the science and technology for speciality cheese manufacture, identification and overcoming of the technical constraints to the manufacture of soft speciality cheeses in Ireland and the development of Moorepark Technology Limited (MTL) pilot plant as an integrated, flexible pre-commercial manufacturing platform with which to evaluate the market for speciality cheese.
    • Influence of Enterococci and Thermophilic Starter Bacteria on Cheddar Cheese Flavour

      Beresford, Tom; Cogan, Tim; Wallace, J.; Drinan, D.; Tobin, S.; Piveteau, P.; Carroll, N.; Deasy, B. (Teagasc, 1998-09-01)
      This 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.
    • Role of Lactobacilli in Flavour Development of Cheddar Cheese.

      Beresford, Tom; Cogan, Tim; Rea, Mary; Drinan, Finbarr; Fitzsimons, Nora; Brennan, N.; Kenny, Owen; Fox, P.F. (Teagasc, 2001-05-01)
      Cheddar 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.
    • Significance of Lactobacilli in Cheddar Cheese

      Cogan, Tim; Beresford, Tom; Drinan, Finbarr; Palles, Tony; Fitzsimons, Nora (Teagasc, 1998-09-01)
      The 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.
    • Stimulation of Propionic Acid Bacteria by Lactic Acid Bacteria in Cheese.

      Condon, S.; Cogan, Tim; Piveteau, P.; O'Callaghan, Jim; Lyons, B. (Teagasc, 2001-06-01)
      In the manufacture of Swiss-type cheese two successive fermentations occur. During manufacture, lactic acid bacteria (LAB), particularly Streptococcus thermophilus, Lactobacillus helveticus and Lb. delbrueckii subsp. lactis, convert lactose to lactate while, during ripening, propionic acid bacteria (PAB) convert lactate to propionic acid, acetic acid and carbon dioxide (CO2). CO2 is responsible for eye formation and propionic acid results in the typical nutty flavour of Swiss-type cheese. There have been a few reports of interactions between a small number of LAB and PAB but the compounds involved have not been identified. A better understanding of this phenomenon is necessary to select strains of PAB for cheesemaking and improve the quality of hard cheeses. Cheese cannot be used for such a study because of its complexity and the length of time it is ripened. Hence, a simple whey-based model developed by Piveteau et al (1995) was successfully used to study the interactions between LAB and PAB. In this procedure, the LAB were grown overnight in milk and the whey was collected by centrifugation. After neutralisation and filter-sterilisation, the growth of strains of PAB in this whey and in a control whey produced from the same milk by acidification with lactic acid were compared. The objectives of this study were to refine the model of Piveteau et al (1995) to study the interactions between LAB and PAB and to determine the nature of the stimulant(s) produced by the LAB. * Thirty-two combinations of different commercial strains of PAB and LAB were evaluated in a modified whey model. None showed any inhibition and all showed some degree of stimulation but the extent of the stimulation depended on the particular pair of PAB and LAB used. * An inhibitor of PAB was found in milk, which prevented the growth of PAB from low (105 cfu/ml) but not from high inocula (107 cfu/ml). The inhibitor was heat stable (to autoclaving for 15 min), of low molecular mass and could be removed by pre-growth of some but not all starter LAB in milk. * Growth of P. freudenreichii DPC 3801 in control whey was stimulated by peptone, tryptone, casein hydrolysed by the crude proteinase of Lb. helveticus DPC 4571 and by pre-growth of the lactobacillus in milk, but not by vitamins (riboflavin, thiamine, PABA, Ca panthothenate, biotin and nicotinic acid) or minerals (MgSO4, MnCl2, CoCl2 and CuSO4). * Growth of Lb. helveticus DPC 4571 in milk resulted in significant increases in peptide and amino acid production but the amino acids produced did not stimulate the growth of the PAB. Based on these results it was concluded that the stimulation was due to production of peptides by the LAB from casein. * The whey model developed by Piveteau et al (1995) to study the interactions between PAB and LAB was shown to be reproducible. Adjustment of the pH of the whey to 5.4 rather than 6.0, incubation at 24ºC rather than 30ºC and addition of 1% NaCl, to simulate cheese ripening conditions allowed growth of all the PAB tested. * Several chromatographic procedures, including ion-exchange, gel permeation and reverse-phase, high-pressure liquid chromatography failed to categorically identify the peptide(s) responsible for the stimulation of the PAB. In some of these chromatographic systems,the stimulatory activity was shown to be present in several peaks implying that different peptides were involved.