• 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.
    • Assessment and Control of Foodborne Pathogens in Ireland

      Ross, R Paul; Hill, Colin; Murphy, P.; Jordan, Kieran; Arendt, Elke; van Sinderen, Douwe; Morgan, S.M.; Hickey, Rita M.; Maher, M.J.; Kelly, J.; et al. (Teagasc, 2001-05-01)
      Consumers are increasingly demanding food that is free from pathogens, but with less preservatives and additives. As a response to these conflicting demands, current trends in the food industry include minimal processing, and the investigation of alternative inhibitors for use in foods. Additionally, the manufacture of an increasing range of novel foods, and the inclusion of non-dairy ingredients into dairy products, and vice versa, poses additional dangers with respect to safety. Furthermore, the dramatic increase in incidence of food-borne illness internationally, as a result of contamination with food-borne pathogens such as Listeria monocytogenes, is a cause of considerable consumer concern. Bacteriocins are inhibitory peptides produced by a number of Lactic Acid Bacteria which are capable of killing other bacteria. These natural inhibitors have widespread applications in the preservation of foods, since they can kill a number of pathogenic and spoilage bacteria. The broad spectrum bacteriocin Lacticin 3147 (discovered in a previous project and patented - see DPRC No. 3) is produced by Lactococcus lactis subsp. lactis DPC3147, a food-grade strain, similar to strains used for commercial cheese manufacture. Lacticin 3147 is effective in the inhibition of all Gram positive bacteria tested including the food pathogens Listeria monocytogenes and Staphylococcus aureus and food spoilage bacteria such as Clostridia and Bacillus species. As part of this project the bacteriocin Lacticin 3147 was assessed as a food preservative for improving food safety via inhibition of pathogenic organisms. Thus the project plan followed a "twin-track" approach to assessing and controlling the food safety aspects of Irish food. The first of these was designed to investigate the current safety status of Irish dairy products. The second approach involved an attempt to exploit natural antimicrobial substances, including Lacticin 3147, to protect foods from pathogenic bacteria.
    • Control of Cheese Microflora using Bacteriocins.

      Ross, R Paul; Hill, Colin; O'Keeffe, T.; McAuliffe, Olivia; Ryan, Maire; O'Connor, Paula M.; Freyne, T. (Teagasc, 2001-08-01)
      Bacteriocins are proteins, produced by some bacteria which are capable of inhibiting other bacteria. The overall aim of this project was the development and exploitation of bacteriocins such as Lacticin 3147 (produced by a food-grade microorganism), as biological tools to control the microflora of foods. Lacticin 3147-producing strains were evaluated for their ability to improve the microbial quality of a variety of dairy products and in particular, Cheddar cheese. The manipulation of cheese flora using bacteriocins should offer manufacturers greater control in the consistency and quality of the final product, in addition to improving its safety. In concert with these studies, Lacticin 3147 was studied in detail at the molecular level resulting in its biochemical and genetic analysis. These studies have demonstrated the complexity and uniqueness of this potent antimicrobial.
    • 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.
    • High Pressure Processing of Dairy Foods

      Donnelly, W.J.; Beresford, Tom; Lane, C.N.; Walsh-O'Grady, D.; O'Connor, Paula M.; Fitzgerald, Richard J.; Murphy, P.M.; O'Reilly, Conor; Morgan, S.M.; Ross, R Paul; et al. (Teagasc, 2000-09-01)
      The term High Pressure Processing (HPP) is used to describe the technology whereby products are exposed to very high pressures in the region of 50 - 800 MPa (500 - 8000 Atmospheres). The potential application of HPP in the food industry has gained popularity in recent years, due to developments in the construction of HPP equipment which makes the technology more affordable. Applying HPP to food products results in modifications to interactions between individual components, rates of enzymatic reactions and inactivation of micro-organisms. The first commercial HPP products appeared on the market in 1991 in Japan, where HPP is now being used commercially for products such as jams, sauces, fruit juices, rice cakes and desserts. The pioneering research into the application of HPP to milk dates back to the end of the 19th century. Application of HPP to milk has been shown to modify its gel forming characteristics as well as reducing its microbial load. HPP offers the potential to induce similar effects to those generated by heat on milk protein. Recent reports have also indicated that HPP could accelerate the ripening of cheese. Much of the Irish cheese industry is based on the production of Cheddar cheese, the ripening time for which can vary from 4 - 12 months or more, depending on grade. A substantial portion of the cost associated with Cheddar manufacture is therefore attributed to storage under controlled conditions during ripening. Thus, any technology which may accelerate the ripening of Cheddar cheese while maintaining a balanced flavour and texture is of major economic significance. While food safety is a dominant concern, consumers are increasingly demanding foods that maintain their natural appearance and flavour, while free of chemical preservatives. HPP offers the food industry the possibility of achieving these twin goals as this technology can lead to reduced microbial loads without detrimentally effecting the nutritional or sensory qualities of the product. The development of food ingredients with novel functional properties offers the dairy industry an opportunity to revitalise existing markets and develop new ones. HPP can lead to modifications in the structure of milk components, in particular protein, which may provide interesting possibilities for the development of high value nutritional and functional ingredients. Hence these projects set out to investigate the potential of HPP in the dairy industry and to identify products and processes to which it could be applied.
    • 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.
    • Use of Bacteriocins to Improve Cheese Quality and Safety

      Ross, R Paul; Hill, Colin; Ryan, Maire; Cunniffe, Alan; McAuliffe, Olivia; Murray, Deirdre; O'Keefe, Triona; Rea, Mary (Teagasc, 1998-09-01)
      The objectives of this project were to generate, characterise and exploit a range of novel bacteriocin producing starter cultures to improve both the safety and the quality of fermented dairy foods. The main conclusions were as follows: Lacticin 3147 is a broad spectrum bacteriocin which inhibits a wide range of Gram-positive bacteria including lactobacilli, clostridia and Listeria. The bacteriocin has been purified by chromatographic procedures and has been shown to be composed of two peptides, both of which are required for biological activity. The mechanism of action of lacticin 3147 has been elucidated. The entire plasmid encoding lacticin 3147 has been sequenced and the bacteriocin in distinct from any previously characterised lactococcal bacteriocin. The Food Grade introduction of the bacteriocin genes into cheese starters was carried out. Lacticin 3147 producing starters have been used to control the pathogen Listeria monocytogenes on the surface of mould ripened cheese. Lacticin 3147 producing starters have been used to control the non-starter lactic acid bacteria complement in Cheddar cheese during the ripening process. A novel starter system using a bacteriocin (lactococcin)- producing adjunct has been designed which gives increased cell lysis during Cheddar cheese manufacture while ensuring that efficient acid production is not compromised. In summary these studies have found that naturally occurring antimicrobials such as bacteriocins have a wide range of applications in the food industry for improving both the quality and safety of fermented dairy products.