• Application of Probiotic Bacteria to Functional Foods

      STANTON, CATHERINE; Ross, R Paul; Fitzgerald, Gerald F; Collins, K.; McBrearty, S.; Gardiner, Gillian E.; Desmond, C.; Kelly, J.; Bouchier, Paul J.; Lawless, Fergal; et al. (Teagasc, 2001-05-01)
      Probiotic cultures are described as live microbial feed supplements that improve intestinal microbial balance and are intended for maintenance of health or prevention, rather than the curing of disease. The demand for probiotic foods is increasing in Europe, Japan and the U.S. reflecting the heightened awareness among the public of the relationship between diet and health. Traditionally, the most popular food delivery systems for these cultures have been freshly fermented dairy foods, such as yogurts and fermented milks, as well as unfermented milks with cultures added. However, in the development of functional foods, the technological suitability of probiotic strains poses a serious challenge since their survival and viability may be adversely affected by processing conditions as well as by the product environment and storage conditions. This is a particular concern, given that high levels (at least 107 per gram or ml) of live micro-organisms are recommended for probiotic products. In previous studies (see DPRC No. 29) the successful manufacture of probiotic Cheddar cheese harbouring high levels (>108 cfu/g) of the probiotic Lactobacillus paracasei NFBC 338 strain was reported. Hence, the overall objective of these studies was to continue the development and evaluation of Functional Foods containing high levels of viable probiotic bacteria, with particular emphasis on overcoming the technological barriers and the identification of strains suited to particular applications, such as incorporation into Cheddar cheese and spray-dried powders.
    • Development and Application of Strategies to Generate Bacteriophage Resistant Strains for Use in Milk Fermentation Processes

      Ross, R Paul; Fitzgerald, Gerald F; Coffey, Aidan; Coakley, M.; O'Sullivan, Daniel (Teagasc, 1999-02-01)
      The objectives of this project were firstly, the identification of natural phage resistance systems for exploitation, secondly, the development of methodologies to utilise these systems to improve the bacteriophage resistance of starter strains for use in milk fermentation processes, and thirdly, the actual application of these methodologies to improving starter strains. The main conclusions were as follows: Three new natural plasmid (DNA)-associated bacteriophage resistance systems were identified at Moorepark. The detailed genetic makeup of the phage resistance plasmid (pMRC01) was elucidated. Bacteriophages currently evolving in the industrial cheese-making environment were monitored to facilitate the judicious choice of phage resistance systems for use in commercial starter cultures which can more effectively target the documented problematic phage types. Two highly virulent phages targeting important cheese starters were identified in the industrial cheese-making environment. A reliable food-grade method to facilitate the transfer of phage resistance systems to cheese-making starter strains was developed. This is based on bacteriocin immunity-linked phage resistance. Phage resistant cheese starter cultures were developed through natural selection and by molecular manipulation using phage resistance plasmids. The phage resistance plasmid pMRC01 was introduced to 31 cheese starter strains.
    • Functional Foods in Relation to Health and Disease (New Probiotic Cheddar Cheese).

      STANTON, CATHERINE; Ross, R Paul; Fitzgerald, Gerald F; Collins, K.; Gardiner, Gillian E. (Teagasc, 2000-09-01)
      Growing public awareness of diet-related health benefits has fuelled the demand for probiotic foods. These foods contain probiotic bacteria which are described as live microbial supplements that improve the intestinal microbial balance and are intended for maintenance of health and/or the prevention of disease. Probiotic bacteria for human use must be proven to be safe and beneficial, and should preferably be of human origin as evidence suggests that these bacteria are species specific and perform best in the species from which they were isolated. They must also retain both viability and efficacy in a particular food product throughout its shelf-life, and following consumption. Above all however, probiotic food products must be proved effective in controlled validated clinical trials. Dairy foods, including in particular, fermented milks and yogurt are among the best accepted food carriers for probiotic cultures. The aim of this study was to develop new probiotic foods, particularly, the production of high quality Cheddar cheese containing high levels of probiotic bacteria.
    • Molecular Characterisation of Bacteriophage K Towards Applications for the Biocontrol of Pathogenic Staphylococci.

      O'Flaherty, Sarah; Flynn, James; Coffey, Aidan; Fitzgerald, Gerald F; Meaney, William J; Ross, R Paul (Teagasc, 2006-01-01)
      The aim of this work was to characterise staphylococcal bacteriophage (a bacterial virus) and to assess their potential as therapeutic agents against pathogenic strains of Staphylococcus aureus, particularly mastitis-causing strains. The project included the use of two newly isolated phage CS1 and DW2, and an existing polyvalent phage. The new phage were isolated from the farmyard and characterised by electron microscopy and restriction analysis. Both phage were shown to belong to the Siphoviridae family and were lytic for representatives of all three clonal groups of Irish mastitis-associated staphylococci. A cocktail of three phage (CS1, DW2 and K) at 108 (plaque forming units) PFU/ml was infused into cows teats in animal trials. The lack of an increase in somatic cell counts in milks indicated strongly that the phage did not irritate the animal. In addition, the most potent phage used in this study, phage K, was further studied by genome sequencing, which revealed a linear DNA genome of 127,395 base pairs, which encodes 118 putative ORFs (open reading frames). Interesting features of the genome include; 1) a region exhibiting high homology to the structural module from Listeria phage A511, 2) genes which potentially encodes proteins necessary for its own replisome, 3) an absence of GATC sites and 4) three introns encoding putative endonucleases were located in the genome, (two in the putative DNA polymerase gene and one in the lysin gene). Unlike both CS1 and DW2, the polyvalent phage K, exhibited a broad host range within the genus Staphylococcus. In in vitro inhibitory assays, phage K lysed all staphylococcal strains tested including nine different species. In preliminary application-type studies, anti-staphylococcal activity was also evident in a hand wash Project 4942 2 and phage cream. An unexpected result was the observation that phage K was unable to replicate in raw milk, which could limit its applications in mastitis treatments. This may have been due to clumping of the bacteria caused by immunoglobulins. However, inhibition activity was lost after milk was heat-treated. The overall results in this study provide new insights into the biology of the broad host range phage K and indicate that phage K has potential for treatment and prevention of infections caused by pathogenic staphylococci.