The biotechnology of lactic add bacteria with emphasis on applications in food safety and human health Charles Daly and Ruth Davis Department ofMicrobiology and National Food Biotechnology Centre, University College, Cork, Ireland, e-mail: Dean.food@ucc.ie Fermentalion of various foodsluffs by lactic acid bacteria (LAB) is one of the oldest forms of bio- preservation practised by mankind. In recent years, significant advances have been made in elucidat- ing the genetic and physiological basis of key LAB traits involved in these industrially significant processes. One important attribute of many LAB is their ability to produce antimicrobial compounds called bacteriocins. Interest in these compounds has grown substantially due to their potential useful- ness as natural substitutes for chemical food preservatives in the production of foods with enhanced shelflife and/or safety. There is growing consumer awareness of the link between diet and health. Recent scientific evidence supports the role of probiotic LAB in mediating many positive health effects. In addition, some LAB are currently being assessed for their ability to act as live delivery vectors in the development of new oral vaccines. Key words: biopreservation, functional foods, probiotics, LAB-vaccines ntroduction Mankind throughout the ages has practised fer- mentations by lactic acid bacteria (LAB) as an effective means of improving the shelflife ofoth- erwise perishable foodstuffs and as such they represent a long-standing application of biotech- nology. Many substrates including milk, meats, cereals, vegetables and fruits have been ferment- ed generating a wide range of nutritious end products with desirable flavours and attributes. LAB are a phylogenetically diverse group of bacteria. Members of the genera Lactococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus, in particular, are involved in these fermentations. In addition, some LAB (mainly Lactobacillus spp.) as well as the functionally related, though non-LAB, Bifidobacterium, are known to form part of the normal human intesti- nal microflora and accumulating evidence sug- gests that these bacteria may exert a positive ef- fect on human health. Given the economic value of food fermenta- tions and a growing acceptance thatat least some of these products may contribute to improved © Agricultural and Food Science inFinland Manuscript received May 1998 251 Voi 7 (1998): 251- AGRICULTURAL AND FOOD SCIENCE IN FINLAND health, it is not surprising that LAB are attract- ing major attention at this time. This is reflected in the increased volume of fermented products available world-wide especially in the area of functional foods containing probiotic or health- promoting bacteria. Through the BIOTECHNOLOGY and AGRI- INDUSTRIAL programmes, the European Com- mission (EC) has provided outstanding financial support for research on LAB and has fostered many high-quality transnational collaborations. Two projects within the current European Un- ion (EU) Fourth Framework Programme, in par- ticular, illustrate the integrated approach that has been taken. The STARLAB project within the BIOTECHNOLOGY programme has 56 partici- pating laboratories, 13 of which are industry based (Mercenier et al. 1997). It has the follow- ing research themes: 1. Cell engineering of Lactococcus lactis 2. LAB with modified proteolytic properties in milk fermentation 3. Control of bacteriophage development in LAB: towards a rational solution to a major problem of food fermentation 4. The molecular biology and genetics of ther- mophilic LAB 5. LAB as cell factories for the production and delivery of mucosal immunogens 6. Carbon catabolite control in food grade lacto- bacilli to provide the tools for strain improve- ment. As part of the AGRI-INDUSTRIAL pro- gramme, the PROBDEMO project supports the development of novel probiotic products in the European market by providing a sound assess- ment of their functionality and subsequently dis- seminating the information to relevant authori- ties, consumer organisations and participating industry partners. The PROBDEMO project en- compasses nine groups including four major dairy industries (Mattila-Sandholm 1997). The project’s research tasks include the follow- ing: 1. To establish a scientifically based selection of probiotic bacterial strains currently avail- able for functional foods 2. To demonstrate the beneficial value of pro- biotic products in human pilot testing both in children and in adults, applying molecu- lar tools for identificationof gastrointestinal flora 3. To demonstrate and meet the functional and technological requirements essential for the industrial production of probiotics as func- tional foods 4. To disseminate the knowledge and results to the extended audience consisting of the group of industrial users, authorities and consumer organisations. These and other research efforts world-wide have significantly advanced our understanding ofkey functional processes in LAB. They have already been instrumental in providing well- characterised strains for use in large-scale food fermentationsand they underpin the development of future genetic strategies aimed at construct- ing strains with superior performance character- istics. Many of these developments will, either directly or indirectly, have applications in im- proving the quality and safety of foods. Research on the contribution of various lacto- bacilli and bifidobacteria to the normal healthy functioning of the human gastrointestinal sys- tem and their likely probiotic effects is evolving rapidly, drivenby the eagerness offood compa- nies to satisfy a growing consumer market. Al- ready several products are being marketed with probiotic claims and a number of manufacturers have developed and licensed specific probiotic bacteria - Lactobacillus johnsonii LAI from Nestlé, LA7 from Bauer, Causido culture from MD Foods, the Lacticel strain from Danone and Lactobacillus GG from Valio, Mona and other companies (Young, J. 1996). One of the newer and very exciting areas of LAB research concerns their exploitation as live oral vaccine delivery vehicles. The improved ability to genetically manipulate these bacteria, their ‘generally regarded as safe’ (GRAS) status and the potential ease of production and admin- 252 Seminar in honour of the 100th anniversary ofMTT AGRICULTURAL AND FOOD SCIENCE IN FINLAND istration ofLAB-based vaccines make them very attractive candidates for such applications (Wells etal. 1996). The aim of this paper is to review develop- ments in LAB research that have already impact- ed, or are likely to impact, the production of foods that are safer and of better quality. In ad- dition, it examines the research supporting the potential therapeutic applications of some of these bacteria. Developments in the biotechnology of LAB The past 20 years have seen a major impetus in LAB research. Although initially much of this research focused on dairy lactococci, investiga- tions now encompass many different LAB in- volved in a wide variety of fermentation proc- esses and, more recently, various lactobacilli and bifidobacteria belonging to the human microbi- ota. A very large number of genes have already been cloned, sequenced and subjected to inten- sive analyses regarding their genetic and molec- ular organisations, modes of action and regula- tion. Two of the most important functional prop- erties, lactose utilisation and proteolytic activi- ty, are particularly far advanced. However, sig- nificant developments in other areas such as bac- teriophage biology and resistance mechanisms, pyruvate metabolism and the production of bac- teriocins have also been made (Fitzgerald and Hill 1996, von Wright and Sibakov 1998). Progress in LAB genetics was greatly aided ear- ly on by the fact that many of the industrially significant properties of these bacteria were en- coded by plasmids (Fitzgerald and Hill 1996). However, research on their chromosomal genet- ics is also progressing rapidly. Physical and ge- netic maps have been constructed for a number of strains and there are an increasing number of chromosomally located genetic loci under inves- tigation (Davidson et al. 1996). The development of tools that facilitated the genetic manipulation of LAB has been crucial to the success of these endeavours. In particu- lar, electrotransformation, which mediates high frequency uptake of in vitro DNA, allowed clas- sical recombinant DNA technologies to be ap- plied across a wide range of LAB (Gasson and Fitzgerald 1994, Mercenier et al. 1994). Also conjugation, one of the natural processes ofgene exchange common among lactococci, has played an important role in non-recombinant strategies of strain improvement (Gasson and Fitzgerald 1994). Since the early 1980s, the array of clon- ing vectors available to researchers has expand- ed enormously. In addition to general cloning vectors, there is a wide choice of vectors availa- ble with specialised functions (de Vos and Si- mons 1994).These include genetic signal screen- ing vectors, high expression vectors and induci- ble expression systems. Two further systems are worthy of special note. First is the development of vectors suitable for use in food industry ap- plications. These contain only LAB- derived DNA and use food grade selection markers such as bacteriocin resistance, lactose-fermenting ability, bacteriophage resistance etc (von Wright and Sibakov 1998). The second system of note concerns the development of vectors that facili- tate heterologous gene expression and secretion (de Vos and Simons 1994). These are particular- ly relevant for the exploitation of LAB as vac- cine delivery vehicles, an area of research that will be discussed in more detail in a later sec- tion. The understanding and exploitation of indus- trial traits are not the only aspects of LAB re- search that have benefited from the development of more sophisticated technologies. Reliable methods of strain identification and classifica- tion are vitally important. Newer techniques such as the ability to sequence large tracts of 16S and 23S rRNA genes using polymerase chain reac- tion (RAPD-PCR) and the use ofpulsed field gel electrophoresis (PFGE) to fingerprint genomic restriction patterns have contributed enormous- ly to these efforts (Axelsson 1998). This relates very much to the field of probiotics where the 253 Vol. 7(1998): 251-265. AGRICULTURAL AND FOOD SCIENCE IN FINLAND ability to monitor strains through clinical trials and to evaluate their effects on the gastrointesti- nal tract microflora as well as the protection of their proprietary value depends on exact and re- producible strain identification. The following sections of this review will focus on some of the properties ofLAB that con- tribute to their roles in biopreservation and in modulating the health of their hosts. Bacteriocins of LAB - Roles in biopreservation Despite improved manufacturing facilities and the implementation of effective process control procedures such as HACCP (Hazard Analysis and Critical Control Points) throughout much of the food industry, the number of reported food borne illnesses has continued to rise. Concomi- tantly, there is a strong trend on the part of con- sumers favouring less processed foods contain- ing fewer chemical preservatives (Daeschel 1993). As a result, there is an increased interest in the preservative aspects of LAB particularly in view of their long and safe association with human fermented foods. Several metabolic com- pounds produced by these bacteria have antimi- crobial effects, including organic acids, fatty acids, hydrogen peroxide and diacetyl (Holzap- fel et al. 1995, Ouwehand 1998). However, the majority of attention has focused on the ability of many LAB to produce specific proteinaceous inhibitory substances, bacteriocins, that inhibit the growth of other bacteria and can, therefore, enhance the shelf-life of foods in which they are present. Significantly, some bacteriocins inhibit serious food-borne pathogens such as Listeria, Clostridium, Staphylococcus, and certain Bacil- lus spp. and Enterococcus spp. At present four classes ofLAB bacteriocins have been defined (Table 1). Members of class- es I and II are the most frequently characterised probably reflecting the well-establishedisolation procedures for these bacteriocins and their po- tential for industrial application. Nisin, which is produced by some L. lactis subsp. lactis strains, belongs to the class I lanti- biotics and is by far the most extensively stud- ied bacteriocin of the LAB (Dodd and Gasson 1994, Jack et al. 1995). It was discovered as far back as 1928 and has a broad spectrum of activ- ity against many Gram-positive bacteria includ- ing Listeria spp. It prevents the outgrowth of germinating bacillus and clostridial spores and, through the addition of a calcium chelator, it is possible to broaden its activity to include some Gram negative bacteria (Stevens et al. 1991). The mature nisin molecule is just 34 amino acids long and undergoes extensive post-translational mod- Table 1. Classes of bacteriocins produced by LAB. Class Subclass Description I Lantibiotics - small, heat stable, containing unusual amino acids Small (30-100 amino acids), heat stable, non-lantibioticII Ha Pediocin-like bacteriocins, with anti-listerial effects lib Two peptide bacteriocins lie Sec-dependentsecretion of bacteriocins 111 IV Large (> 30 kDa) heat-labile proteins Complex bacteriocins with glyco- and/or lipid moieties, heat stable Adapted from Nes et al. 1996, Ouwehand 1998 254 Seminar in honour of the 100thanniversary ofMTT AGRICULTURAL AND FOOD SCIENCE IN FINLAND ifications in which serine and threonineresidues are dehydrated and several thio-ether bridges are formed. These modifications result in the for- mation of the five ring structures that are char- acteristic of the molecule. The primary target of nisin’s antimicrobial action is the cell membrane. It is thought that nisin interferes with the energy supply of the cell by creating pores in the mem- brane and dissipating its potential (Sahl et al. 1995). Owing to its extensive genetic and mo- lecular characterisation, nisin has been the tar- get of several protein engineering studiesaimed at broadening its functional attributes. Modified nisins containing specific amino acid substitu- tions have been generated some of which exhib- it enhanced practical features such as increased activity against food pathogens and improved stability and/or solubility under various food- processing conditions (Kuipers et al. 1991, 1995, Rollerna et al. 1995). Other class I lantibiotic type compounds apart from nisin have been isolated from a wide vari- ety ofLAB sources. One, lacticin 3147, was re- cently identified from a lactococcal isolate of Irish kefir grains (used in the manufacture of buttermilk) during a collaborative study between the Teagasc Research Centre, Moorepark, Ireland and the Microbiology Department at University College, Cork, Ireland (Ryan et al. 1996). This bacteriocin is particularly attractive as it inhib- its a wide spectrum of Gram-positive bacteria including potential food-borne pathogens such as Staphylococcus , Clostridium and Listeria spp. as well as several mastitic staphylococci and streptococci (Meaney et al. 1997). Lacticin 3147 requires two peptides for activity. Both peptides are produced in a precursor form and are sub- jected to post-translational modifications involv- ing the dehydration and linkage of a number of amino acids producing typical lanthionine rings and the cleavage ofprepropeptide sequences. The genetic determinants of lacticin 3147 are locat- ed on a large 60 kb conjugative plasmid, pMRCOI, flanked by two iso-1557-like elements (Dougherty et al. in press). The intervening re- gion contains 13 open reading frames (ORFs), eleven of which, arranged in two operon struc- tures, are thought to be associated with the bac- teriocin functions. Six of the ORFs showed sig- nificant sequence homology with genes known to be involved in the production, immunity and transport of other recognised lantibiotics. It has not yet been possible to conclusively identify the structural genes of the bacteriocin. As with ni- sin, lacticin 3147 acts on susceptible cell mem- branes by introducing ion-specific pores that dis- rupt the membrane potential and rapidly cause cell death (McAuliffe et al. 1998). Class II bacteriocins contain a wide variety of bacteriocins and, therefore, are categorised into three further subclasses. In general, howev- er, they are all relatively small cationic peptides (30-100 amino acids) exhibiting a high degree of heat stability. Like the lantibiotics, class II bacteriocins also target the cell membrane as their active site forming oligomeric pores. How- ever, unlike lantibiotics, their bacteriocidal ac- tivity is independent of the membrane’s energi- sation state and appears to require a cell mem- brane receptor molecule. Lactococcin A, whose mode of action has been studied in some detail, is thought to insert itself as an a-helical structure across the cell membrane. Several lactococcin A molecules subsequently combine to form pores in the membrane. These pores cause an efflux of small cytoplasmic molecules and ions resulting in dissipation of the membrane potential (Vene- rna et al. 1995). The genetic determinants of several class II bacteriocins have been cloned and sequenced. Many have been linked to plasmids and, in some cases, an individual host may produce multiple bacteriocins (Dodd and Gasson 1994). In the case of lactococcins A, B and M, all three were located on the same plasmid (van Belkum et al. 1991, 1992). Less is known about the classes 111 and IV bacteriocins. Members of theLactobacillus gen- era produce all of the class 111 bacteriocins iso- lated to date. Helveticin J is the best known com- pound of this class. The legitimacy of the fourth bacteriocin class is somewhatcontroversial. The requirement of the glyco and/or lipid component for the action of these bacteriocins is not well 255 Vol. 7(1998): 251-265. AGRICULTURAL AND FOOD SCIENCE IN FINLAND established and may be a consequence of incom- plete purification procedures. The modes of ac- tion for both these classes are poorly understood (Klaenhammer 1993, Venema et al. 1995). Clearly, many bacteriocins of the LAB, es- pecially those withbroad spectra of activity, have tremendous potential to be exploited as safe and effective ‘natural’ inhibitors of potential patho- genic and food spoilage bacteria in various food systems. Nisin is the classic example with a par- ticularly long and successful history in food ap- plications. Some of its commercial applications include: preventing clostridial spoilage of proc- essed and natural cheeses, inhibiting the growth of some psychrotrophic bacteria in cottage chees- es, extending the shelf-lifeof milk in warm coun- tries, preventing the growth of spoilage lactoba- cilli in beer and wine fermentations and provid- ing additional protection against bacillus and clostridial spores in canned foods. Nisin is a permitted food additive in more than 50 coun- tries including the US and Europe where it is commercially available through Aplin and Bar- rett (UK) under the trade name Nisaplin® (Vanden- berg 1993,Delves-Broughton et al. 1996). The emergence of Listeria, specifically Ls. monocytogenes , as a serious food-borne patho- gen is of major concern in the food industry es- pecially in light of the fact that these bacteria are common contaminants of many raw food materials such as milk, meat and vegetables (Ryser and Marth 1991). Consequently, bacteri- ocins belonging to the subclass Ha, which dem- onstrate antilisterial activity, have received sig- nificant research attention. Pediocin PA-l/AcH produced by Pediococcus acidilactici is regard- ed as the prototype bacteriocin of this subclass and various studies have demonstrated its abili- ty to control Listeria in cheese, vegetable and meat systems. The application ofpediocin in the biopreservation of meats is particularly relevant, as nisin is not very effective in this environment. It is also significant to note that Pediococcus acidilactici is a common starter culture used in the production of most fermented meats (Vandenberg 1993, Stiles 1996). The bacteriocin, lacticin 3147, has also been the subject of food application studies. Ryan et al. (1996) developed a range of lacticin 3147- producing starter strains suitable for use in com- mercial cheese making. When incorporated, these strains effectively controlled the growth of any non-starter LAB in Cheddar cheese and com- pletely eliminated deliberately inoculated Ls. monocytogenes from cottage cheese. Lacticin 3147 has a number of advantages over nisin. It is effective at neutral pH and starter cultures pro- ducing this bacteriocin have good acid produc- ing and bacteriophage resistance properties un- like their counterparts producing nisin. Signifi- cantly, C. Hill (University College, Cork), W.J. Meaney and R Ross (Teagasc, Moorepark) are seeking approval from the European Agency for the Evaluation ofMedicinal Products (Veterinary Medicines Evaluation Unit) for the use of lac- ticin 3147 as a mastitis-controlling therapy in dry cows (C. Hill, pers. comm.). Unfortunately, a major drawback associated with LAB bacteriocins lies in the fact that gram negative bacteria as well as yeasts and moulds are normally refractive to their bactericidal ac- tion. As a result, the usefulness of these com- pounds in commercial practice has been some- what limited as many important food borne path- ogens and food spoilage microorganisms belong to these resistant categories. This has sparked several recent studies aimed at broadening the bactericidal activity ofLAB bacteriocins to en- compass these normally resistant groups. In gen- eral, these studies have focused on the synergis- tic effects of bacteriocins, most notably nisin, with other antibacterial factors such as the lac- toperoxidase system present in milk, hydrolytic enzymes, various chelating agents (including siderophores) and other bacteriocins (Helander et al. 1997). To date, nisinremains the only LAB bacteri- ocin to be legally permitted as a food additive. This has had major implications for the many other bacteriocins that, in recent years, have dem- onstrated commercial potential. Two products, ALTA™243I and Microgard®, have been devel- oped as shelf-lifeextenders based on crude LAB fermentation products and, therefore, do not re- 256 Seminar in honour of the 100th anniversary ofMTT AGRICULTURAL AND FOOD SCIENCE IN FINLAND quire a food additive label. ALTA™234I is pro- duced from a Pediococcus acidilactici fermen- tation and is assumed to rely on the inhibitory effects of Pediocin PA-l/AcH. It is commonly added to Mexican soft cheeses which are partic- ularly susceptible to listerial contamination (Glass et al. 1995). Microgard® is the result of a Propionibacterium fermentation. It is active against Gram negative bacteria such as Pseu- domonas, Salmonella, and Yersinia, as well as yeasts and moulds. Microgard®’s protective ac- tion is probably due to the presence of propion- ic acid as a metabolic end-product. However, a role for a bacteriocin in this product has also been proposed (Al-Zoreky et al. 1991). Microgard® has been approved by the FDA for use in food applications such as cottage cheese and fruit-fla- voured yoghurts. Approximately 30% of the cot- tage cheese produced in the US contains this product as a preservative. In another product, Bioprofit®, a combination of specific Lactoba- cillus and Propionibacterium strains is used as a protective adjunct to normal starter cultures to inhibit the growth of yeasts, moulds. Bacillus spp. Clostridium spp. and heterofermentative lactobacilli during some dairy fermentations (Mäyrä- Mäkinen and Suomalainen 1995). It must be emphasised that the use of bacte- riocins either exogenously or by the adventitious use of bacteriocin-producing cultures should not be regarded as a panacea for poor-quality raw materials or manufacturing practices. Instead, it proposed that bacteriocins be used in combina- tion with other physical, chemical and microbi- al preservation factors as an additional ‘hurdle’ against potential pathogenic or food spoilage bacteria. LAB and health: Probiotic studies As we approach a new millennium, there is a growing appreciation world-wide that a healthy lifestyle, including diet, can play a major role in preventing diseases and promoting human health. Functional foods containing probiotic cultures are a well-established concept in Japan and, in recent years, such products comprise a rapidly expanding, lucrative, internal and export market for the EU. Several factors have fuelled this in- terest in functional foods. Today, consumers are better informed than ever and are keen to take proactive decisions with regard to maintaining their health. Changing population dynamics to- wards older societies and the increased preva- lence of chronic illnesses such as cardiovascu- lar disease and cancer are placing heavy demands on already stretched and expensive healthcare services. In addition, there is serious concern at the dramatic increase in microbial resistance to antibiotics as a result of widespread overpre- scription and misuse. In this context, the World Health Organisation (WHO) has advocated mov- ing towards alternative disease control strategies including the use of probiotic bacteria in the pre- vention and treatment of certain infections (Bengmark 1998). The human gastrointestinal (GI) tract sup- ports a rich and dynamic microbial population of more than 500 bacterial species. Maintaining this delicately balanced ecosystem is important for the normal functioning of the gut, particu- larly withregard to preventing GI infections and stimulating the host’s immune response. Mod- ern antibiotic treatments, radiation therapy, stress and Western dietary preferences can significantly affect the gut microflora predisposing the host to various diseases (Salminen et al. 1995, 1998a, Schaafsma 1995). Probiotic cultures are generally defined as live, non-pathogenic bacteria which when ingest- ed exert a positive influence on the host’s health. Lactobacillus spp. and Bifidobacterium spp. are prominent members of the commensal intesti- nal flora of most healthy individuals and are the most commonly studied probiotic bacteria. Their probable and theoretical benefits have been out- lined in several recent reviews and include re- duced lactose intolerance, alleviation of some diarrhoeas, lowered blood cholesterol, increased immune responses and prevention of cancer (Marteau and Rambaud 1993, 1996, Gilliland 257 Vol. 7 (1998): 251-265. AGRICULTURAL AND FOOD SCIENCE IN FINLAND 1996,Salminen et al. 1996, 1998a). The concept of probiotics is not new, having been proposed originally by Metchnikoff in 1907. However, despite numerous studies in the past, there has been very little convincing scientific evidence to substantiate their health claims until recently. This has been due to difficulties in unequivocal- ly identifying strains, differences in experimen- tal systems and data interpretation and a general lack of coordination between clinicians and microbiologists (Sanders 1994). Considerable efforts have been made recently to redress this situation. Modern taxonomic methods have im- proved the identification of test strains and em- phasis is being placed on performing random double-blind placebo-controlled clinical trials to demonstrate the efficacy of potential probiotic strains and products. Table 2 outlines the agreed criteria that should be fulfilled by such studies. Many different strains of both Lactobacillus and Bifidobacterium have been used in probiot- ic preparations. Few, however, have well docu- mented beneficial properties. Salminen et al. (1998a) presented a comprehensive list of suc- cessful probiotic strains and theirreported clin- ical effects. Lb. acidophilus NCFB 1478, Lb. johnsonii LAI, Lb. casei Shirota strain and Lb. rhamnosus GG are among the best studied and have consistently demonstrated their effective- ness in carefully designed trials that fulfil the requirements in Table 2. Selection criteria for probiotic LAB include: human origin, safety, viability/activity in delivery vehicles, resistance to acid and bile, adherence to gut epithelial tis- sue, ability to colonise the GI tract, production of antimicrobial substances, ability to stimulate a host immune response and the ability to influ- ence metabolic activities such as vitamin pro- duction, cholesterol assimilation and lactase ac- tivity (Huis in’t Veld and Shortt 1996,Salminen et al. 1996). Of course, it is unlikely that any individual strain will be able to present all of these credentials and a blend of strains with com- plementary attributes may be required to deliver optimum probiotic performance. At University College, Cork, Ireland, Collins and co-workers applied stringent in vitro selec- Table 2. Requirements forclinical studies of probiotic foods for functional and clinical use. Each strain documented and tested independently Extrapolation of data from closely related strains not acceptable Well defined probiotic strains, studyproducts, and study populations Double-blind, placebo-controlled, and randomised human studies Result confirmed by several independent research groups Publication in peer-reviewed journals (Salminen et al. 1996, 1998a) tion criteria to a bank of human Lactobacillus isolates in an effort to identify a range of new strains with potential probiotic characteristics. Eight candidates survived the selection process and one. Lb. salivarius UCCIIB producing a broad spectrum anti-microbial protein, was cho- sen for further clinical trials. Lb. salivarius UCCIIB was demonstrated to be efficiently de- livered to the gut following oral administration in milk or yoghurt carriers. In addition, in a pro- portion of volunteers (<10%) significant num- bers were still present in faeces 3 weeks after the cessation of its administration, indicating that this strain was capable of colonising the human GI tract in vivo. Lb. salivarius UCCIIB did not disturb the numbers of other lactobacilli in the gut, but there was a statistically significant re- duction in the numbers of excreted Clostridia. Although it is recognised that further work is required to build up the medical dossier on Lb. salivarius UCCIIB, these preliminary trials strongly support this strain as an effective pro- biotic culture (K. Collins, pers. comm.). Most of the disease states that benefit from LAB therapy are characterised to a greater or lesser extent by a disturbed intestinal microflo- ra, intestinal inflammation and increased gut permeability. For example, there is clear evi- dence that lactose intolerant individuals tolerate fermented dairy products better than their un- fermented counterparts even when they contain significant amounts of lactose. Milks fermented by various LAB, including the common yoghurt 258 Seminar in honour of the I OOth anniversary ofMTT AGRICULTURAL AND FOOD SCIENCE IN FINLAND cultures, Lb. bulgaricus and S. thermophilus, are effective and viable cultures are important to achieve maximum benefits. At least three mech- anisms, or a combination thereof, are thought to contribute to the effect: the reduced lactose con- centration in the fermented product, breakdown of lactose in the gut lumen by residual LAB lactase activity and a slower transit time in the intestines of the fermented products compared to liquid milk (Gilliland 1996, Salminen et al. 1996, 1998a). Probiotic preparations have also been found to be beneficial in the prevention and treatment of certain GI infections including infantile rota- virus diarrhoea and diarrhoeas associated with antibiotic and pelvic radiation treatments. Strongest evidence has been presented forLacto- bacillus GG but positive results were also achieved with Lb. johnsonii LAI and NCFB 1748,Lb. casei Shirota strain and more recently with Lb. reuterii (Lee and Salminen 1995, Salminen et al. 1998a). The mechanisms by which these effects were achieved are not well defined. However, the ability of the lactobacilli to adhere to and potentially modify host mucos- al surfaces is thought to be important. It is like- ly that the lactobacilli suppress the growth of pathogens at the mucosal surface probably by out-competing them for nutrients or by produc- ing antibacterial compounds (Salminen et al. 1998a, Isolauri et al. 1998). Cardiovascular disease is responsible for ap- proximately half of the Western world’s deaths and high serum cholesterol levels are usually indicative of an increased risk of this disease. Consequently, claims regarding the potential cholesterol-lowering properties of probiotic cul- tures have attracted muchresearch attention. The results to date are inconclusive. Several studies demonstrated that various strains could assimi- late cholesterol in vitro. However, reliable data regarding an in vivo function have not yet been reported (Lichtenstein and Goldin 1998). The ability of GI microflora to enzymatical- ly convert precursors naturally present in the diet to carcinogenic forms is well documented and is likely to contribute to the aetiology of colonic cancer. Significantly, LAB and bifidobacteria tend to have low levels of such activities in com- parison to other gut bacteria. Several studies in both animals and humans have demonstrated the ability of these bacteria to reduce the toxicity of intestinal contents by suppressing the levels of bacterial enzymes such as (3-glucoronidase, ni- troreductase, azo-reductase and urease, all of which have been implicated in activating pro- carcinogens (Salminen et al. 1996, 1998a, Iso- lauri et al. 1998). In addition, many LAB pro- duce metabolic end-products (butyrate/butyric acid) that have anti-tumorigenic activities in vitro (Young, G. 1996). There are also a number of in vitro and in vivo animal studies that demonstrate more directly tumour inhibition by LAB. In hu- mans, the evidence for such activities is still largely circumstantial. Recently, however, Aso and co-workers (Aso and Akazan 1992,Aso et al. 1995) reported the first clinical instances in which oral administration of Lb. casei Shirota strain was shown to reduce the recurrence of superficial bladder carcinoma in humans. It has also been documented that variousLAB can modulate the host immuneresponse. Reports have described increased production of immu- noglobulins, interleukins 6 and 10, gamma in- terferon, tumour necrosis factor-a and increased phagocytic activity. Notably, Lactobacillus GG was able to stimulate local and systemic IgA to rotavirus during infection of children with this agent (Kaila et al. 1992).This effect was thought to contribute to protection against reinfection. Lb. salivarius UCCIIB also exhibited a strong mucosal IgA immuneresponse in human volun- teers during clinical trials (Mattila-Sandholm 1997). Both Lactobacillus GG and Lb. johnso- nii LAI have been successfully used as adjuvants to oral vaccines (Isolauri et al. 1998). Another approach to the maintenance of a healthy gut microflora is the provision of sub- strates that preferentially select for the growth ofdesirablebacteria in the host. These substrates, called prebiotics, are based on non- or slowly absorbable complex carbohydrates that can be assimilated by beneficial bacteria such as Bifi- dobacterium and Lactobacillus but in contrast 259 Vol. 7(1998): 251-265. AGRICULTURAL AND FOOD SCIENCE IN FINLAND are hardly ever utilised by potentially pathogen- ic Gram-negativeorganisms. Examples of preb- iotic substrates include inulin, lactulose, various galacto-, fructo-, xylo-oligosaccharides and sug- ar alcohols such as lactitol and xylitol (Salmi- nen et al. 1998b). Many of the functional foods recently launched in Europe contain a combina- tion of a probiotic culture with a prebiotic sub- strate that favours its growth. One such ‘synbi- otic’ product is the fermented drink Fyos (Nu- tricia), which combines the probiotic cultureLb. casei with the prebiotic oligofructose, inulin. LA7 (Bauer), Vifit (Mona) and Actimel (Danone) employ similar strategies (Young, J. 1996). Although in comparison with Japan, the Eu- ropean and US markets for functional foods are still relatively underdeveloped, there are definite indications that this situation is changing. It has been estimated that by the year 2000, the global market for these products will be in the region of $l7 billion (Young, J. 1996). In Europe espe- cially, there is a growing number of dairy-based products available that containprobiotic cultures and/or prebiotic substrates (Table 3). Currently most companies are adopting a prudent approach to marketing their probiotic products relying on general health claims such as ‘helps boost the body’s natural defences’ or ‘restores the body’s natural balance’. In light of the high R+D costs and in order for these products to achieve a max- imum return on investment, it is essential that consumers are presented with clear and substan- tiated health claims. The PROBDEMO project has an important role to play in this respect and underlines the EU’s commitment to supporting this market segment. LAB as live vaccine delivery vehicles In recent years there has been increasing inter- est in exploiting some LAB as live vaccine de- livery vehicles. LAB present a number ofadvan- tages that make them attractive for this function. They have a long history of safe use in foods, there is extensive knowledge already available regarding their production on a large scale and, through fermented products, they are easily ad- ministered orally. Furthermore, it is recognised that the gut is an important site for antigen im- mune education. Different approaches have been adopted in the development of LAB-based vaccines. One relies on colonising lactobacilli that are capable of remaining in the gut or genital tract for a pe- riod of time during which an immune response may be elicited to an expressed antigen. Lacto- bacillus vectors are chosen on the basis of their potential for genetic manipulation and the abili- ty to express foreign antigens as well as for their capacity to stimulate a host immune response. Several lactobacilli including Lb. casei and Lb. plantarum strains have been targeted for re- search. In another approach, the oral commen- sal bacterium S. gordonii has been exploited. This strain is particularly advantageous, as it is easily transformable at high frequencies by nat- ural competence. Also, the strain colonises the oral cavity very efficiently and has been demon- strated to colonise mice vaginal tracts for up to 8 weeks. A third strategy has focused on the use of non-colonising L. lactis strains. In this in- stance, antigens expressed by these bacteria are generally retained intracellularly and, therefore, are not as susceptible to degradation in the gut and, in addition, there is evidence that protein antigens are more immunogenic when they are contained either within or associated with the recombinant bacteria. Results to date with all three approaches are encouraging. Several antigenic epitopes have been expressed in all three host types using var- ious cellular locations (intracellular, cell surface, extracellular) and have demonstrated an ability to elicit local and systemic immune responses (Wells et al. 1996). Concluding remarks Without doubt, advances in biotechnology over the last two to three decades have significantly 260 Seminar in honour of the 100thanniversary ofMTT AGRICULTURAL AND FOOD SCIENCE IN FINLAND Table 3. Examples of fermented milk products containing probiotic bacteria available in food retail outlets in Europe, the UK and Ireland. Product Brand name Company Countries (Organism - 107-108 viable LAB/ml) Yoghurt LCI Nestlé France, Belgium, Spain, (Lb. johnsoniiLAI) Switzerland, Portugal, Italy, Germany, UK. Yoghurt Gefilus Valio (Lb. rhamnosus GG) Finland Yoghurt Vifit Mona Netherlands, Ireland (Lb. rhamnosus GG) Yoghurt Vifit Sudmilch Germany (Lb. rhamnosus GG) Yoghurt drink Yo-Plus Waterford Foods Ireland (Lb. acidophilus) Yoghurt Bio-Pot Onken Europe (Biogarde cultures) Yoghurt LA7 Bauer Germany (Lb. acidophilus) Fermented milk Yakult Yakult Netherlands, UK, drink (Lb. casei Shirota strain) Germany Cultured Gaio MD Foods Denmark yoghurt-style product (E. faecium) Yoghurt SNO Dairygold Ireland (Lb. acidophilus) Yoghurt Actimel Danone Belgium Cholesterol (Lb. acidophilus) Control Fermented milk Actimel Danone Europe drink (Lb. casei) Yoghurt Yoplait Waterford Foods Ireland (Lb. acidophilus) Fermented milk Bra-Mjolk Aria Sweden drink (Bifidus, Lb. reuterii, Lb. acidophilus) Fermented milk Fyos Nutricia Netherlands drink (Lb. casei) Yoghurt Symbalance Tonilait Switzerland (Lb. reuterii, Lb. casei. Lb. acidophilus) Yoghurt Shape St. Ivel Ireland, UK (Lb. acidophilus) (Young, J. 1996 and various sources) expanded our ability to produce high quality, nutritious and tasteful foods that remain fresher for longer, are completely safe and that are less reliant on artificial additives. The potential ap- plications of bacteriocins as ‘consumer friend- ly’ biopreservatives either in the form of protec- tive cultures or as additives are significant. Dis- appointingly, with the exception of nisin and to a much lesser extent Pediocin PA-l/AcH, very little of this potential has been realised in an in- 261 Vol. 7 (1998): 251-265. AGRICULTURAL AND FOOD SCIENCE IN FINLAND dustrial context. Despite strong arguments in favour of their efficacy and safety, the process of obtaining regulatory approval for the more widespread use of these compounds (other than nisin) in various foodstuffs is lengthy and ex- pensive. Progress in this regard will be neces- sary to unblock a major bottleneck facing the practical application of these important but, as yet, underexploited proteins of LAB. In contrast, the development of the function- al foods market, particularly with regard to the use of probiotic cultures, has been exceptional over the last few years and is poised to grow considerably more. In order to support this growth, several fundamental issues need to be addressed. A major challenge for scientists will be unravelling the complex probiotic-host inter- actions and activities that dictate the in vivo func- tionality of these bacteria. Obviously this is quite a daunting task given the complexity of the hu- man microbiota and the multiplicity of interde- pendent reactions that are likely to be involved. Essential to these efforts, however, will be a thor- ough understanding of the genetics and molecu- lar biology of these probiotic strains. Unfortu- nately, many of the strains that show the most probiotic potential are very difficult to manipu- late technically, a factor that is sometimes over- looked in initial selection procedures. While the benefits of probiotic cultures appear to be many and wide-ranging, at present very few have real scientific backing. It is important that in the rush to expand the market for these products, unsub- stantiated claims or adverse publicity do not damage consumer confidence. The public in gen- eral, and especially those involved in consumer affairs and in policy decision-making bodies, must be carefully educated regarding their po- tential benefits. In addition, important consum- er requirements such as taste and convenience should not be compromised in the development of effective probiotic products. Extending the traditional fermentation roles ofLAB is an important goal of scientific research and new product development. In this respect, the use of certain LAB as potential vaccine de- livery vehicles has opened up a completely new avenue for the exploitation of these bacteria. Although this area is in the early stages of de- velopment as yet, the initial successes in elicit- ing immune responses to heterologous antigens bode well for the future development of new oral vaccines. References Al-Zoreky, N., Ayres, J.W. & Sandine, W.E. 1991. Antimi- crobial activity of Microgard® against food spoilage and pathogenic microorganisms. Journal of Dairy Science 74: 758-763, Aso, Y. & Akazan, H, 1992. Prophylactic effect of a Lacto- bacillus casei preparation on the recurrence of su- perficial bladder cancer. Urology International 49: 125-129. - , Akazan, H., Kotake, T., Tsukamoto, T, Imai, K. & Mai- to, S. 1995. Preventative effect of a Lactobacillus casei preparation on the recurrence of superficial bladder cancer in a double blind trial. European Urol- ogy 27: 104-109. Axelsson, L. 1998. Lactic acid bacteria: classification and physiology. In: Salminen, S. & von Wright, A. (eds.). Lactic Acid Bacteria: Microbiology and Functional Aspects 2nd Edition. New York: Marcel Dekker Inc. p. 1-72. Bengmark, S. 1998. Ecological control of the gastroin- testinal tract. The role of probiotic bacteria. Gut 42: 2-7. Daeschel, M.A. 1993. Applications and interactions of bacteriocins from lactic acid bacteria in foods and beverages. In: Hoover, D.B. & Steenson, L.R. (eds.). Bacteriocins of Lactic Acid Bacteria. New York: Aca- demic Press Inc. p. 63-91. Davidson, 8.E., Kordias, N., Dobos, M. & Hillier, A.J. 1996. Genomic organisation of lactic acid bacteria. Antonie van Leeuwenhoek 70: 65-87. de Vos, W.M. & Simons, G.F.M. 1994. Gene cloning and expression systems. In: Gasson, M.J. & de Vos, W.M. (eds.). Genetics and Biotechnology of Lactic acid bacteria. Glasgow: Blackie Academic and Profession- al. p. 52-105, Delves-Broughton, J,, Blackburn, P., Evans, R.J. & Hu- genholtz, J. 1996. Applications of the bacteriocin, 262 Seminar in honour of the 100th anniversary ofMTT AGRICULTURAL AND FOOD SCIENCE IN FINLAND nisin. Antonie van Leeuwenhoek 70: 193-202. Dodd, H.M. & Gasson, M.J. 1994, Bacteriocins of lactic acid bacteria. In: Gasson, M.J. & de Vos, W.M. (eds.). Genetics and Biotechnology of Lactic Acid Bacteria. Glasgow: Blackie Academic and Professional, p. 211- 251. Dougherty, 8., Hill, C. & Ross, R.P. 1998. Complete DNA sequence of the 60 kb lactococcal conjugative plas- mid pMRCOI. Molecular Microbiology (in press). Fitzgerald, G.F. & Hill, C. 1996. Genetics of Starter Cul- tures. In: Cogan, T.M. & Accolas, J.-P. (eds.). Dairy Starter Cultures. New York: VCH Publishers, p. 25- 46. Gasson, M.J. & Fitzgerald, G.F. 1994. Gene transfersys- tems and transposition. In; Gasson, M.J. & de Vos, W.M, (eds.). Genetics and Biotechnology of Lactic Acid Bacteria. Glasgow: Blackie Academic and Pro- fessional. p. 1-51. Gilliland, S.E. 1996. Special additional cultures. In: Cogan, T.M. & Accolas, J.-P. (eds.). Dairy Starter Cultures. New York: VCH Publishers, p. 25-46. Glass, K.A., Bhanu Prasad, 8., Schlyter, J.H., Uljas, H.E., Farkye, N.Y. & Luchansky, J.B. 1995. Effects of acid type and ALTA™243I on Listeria monocytogenes in a Queso Blanco type of cheese. Journal of Food Pro- tection 58: 737-741. Helander, LM., von Wright, A. & Mattila-Sandholm, T.-M. 1997. Potential of lactic acid bacteria and novel anti- microbials against Gram-negative bacteria. Trends in Food Science and Technology 8:146-150. Holzapfel, W.H., Geisen, R. & Schillinger, U. 1995. Bio- logical preservation of foods with reference to pro- tective cultures, bacteriocins and food-grade en- zymes. InternationalJournalofFoodMicrobiology24: 343-362. Huis in’t Veld, J. & Shortt, C. 1996. Selection criteria for probiotic microorganisms. In: Leeds, A.R. & Rowland, I.R. (eds.). Gut Flora and Health - Past, Present and Future. London: The Royal Society of Medicine Press Ltd. p. 19-26. Isolauri, E., Salminen, E. & Salminen, S. 1998. Lactic acid bacteria and immune modulation. In: Salminen, S. & von Wright, A. (eds.). Lactic AcidBacteria: Micro- biology and Functional Aspects 2nd Edition. New York: Marcel Dekker Inc. p. 255-268. Jack, R.W., Tagg, J.R. & Ray, B. 1995. Bacteriocins of gram positive bacteria. Microbiology Reviews 59: 171-200. Kaila, M,, Isolauri, E., Soppi,E., Virtanen, E., Laine, S. & Arvilommi, H. 1992. Enhancement of the circulating antibody secreting cell response in human diarrhoea by a human Lactobacillus strain. PaediatricResearch 32: 141-144. Klaenhammer, T.R. 1993. Genetics of bacteriocins pro- duced by lactic acid bacteria. FEMS Microbiology Reviews 12: 39-86. Kuipers, 0.P., Rollerna, H.S., Beerthuyzen, M.M., Siezen, R.J. & de Vos, W.M. 1995b. Protein engineering and biosynthesis of nisin and regulation of transcription of the structural nisA gene. International Dairy Jour- nal 5: 785-795. - , Yap, W.M.G.J., Rollerna, H.S., Beerthuyzen, M.M., Siezen, R.J. &de Vos, W.M. 1991. Expression of wild- type and mutant nisin genes in Lactococcus lactis. In: Sahl, H.-G. & Jung., G. (eds.). Nisin and Novel Lantibiotics. Leiden: Escom Publishers, p. 250-259. Lee, Y.-K. & Salminen, S. 1995. The coming of age of probiotics. Trends in Food Science and Technology 6: 241-245. Lichtenstein, A.H. & Goldin, B.R. 1998. Lactic acid bac- teria and intestinal drug and cholesterol metabolism. In: Salminen, S. & von Wright, A. (eds ). Lactic Acid Bacteria: Microbiology and Functional Aspects 2nd Edition. New York: Marcel Dekker Inc. p. 269-277. Marteau, P. & Rambaud, J.-C. 1993. Potential of using lactic acid bacteria for therapy and immunomodula- tion in man. FEMS Microbiology Reviews 12: 207- 220. - & Rambaud, J.-C. 1996. Therapeuticapplications of probiotics in humans. In: Leeds, A.R. & Rowland, I.R. (eds.). Gut Flora and Health - Past, Present and Future. London: The Royal Society of Medicine Press Ltd. p. 47-56. Mattila-Sandholm, T. 1997. Demonstration Project FAIR CT96-1028. In: Alander, M. etal. (eds.). Novel Meth- ods for Probiotic Research: 2nd Workshop Demon- stration of the Nutritional Functionality of Probiotic Foods FAIRCT96-1028. Technical Research Centre of Finland (VTT), p, 11-17. Mäyrä-Mäkinen, A. & Suomalainen, T. 1995. Lactobacil- lus casei spp. rhamnosus, bacterial preparations comprising said strain and use of said strain and prep- arations for the controlling of yeast and moulds. Unit- ed States Patent US 5 378 458. McAuliffe, 0., Ryan, M.P., Ross, R.P., Hill, C., Breeuwer, P. &Abee, T. 1998. Lacticin 3147, a broad-spectrum bacteriocin which selectively dissipates the mem- brane potential. Applied and Environmental Microbi- ology 64: 439-445. Meaney, 8., Ryan, M., Flynn, J., Hill, C. & Ross, P. 1997. Mastitis control without antibiotics? In: O’ Rourke, C. (ed) Farm and Food Teagasc Vol 7. Dublin: Teagasc. p. 23-25. Mercenier, A., Pouwels, P.H. & Chassy, B.M. 1994. Ge- netic engineering of lactobacilli, leuconostocs and Streptococcus thermophilus. In: Gasson, M.J. & de Vos, W.M. (eds.). Genetics and Biotechnology of Lactic AcidBacteria. Glasgow: Blackie Academic and Professional, p. 252-293. Mercenier, A. et al. (eds.). 1997. Integrated project STAR- LAB: Strategic and applied research on lactic acid bacteria. STARLAB News. Institute Pasteur de Lille. Issue Nos. 1 & 2. Metchnikoff, E. 1907. The prolongation of life. Optimistic studies. William Heinemann. London. Nes, 1.F., Diep, D.8., Håvarstein, L.S., Brurberg, M 8., Eijsink, V. & Holo, H. 1996. Biosynthesis of bacteri- ocins in lactic acid bacteria. Antonie van Leeuwen- hoek 70: 2-4. Ouwehand, A.C. 1998. Antimicrobial components from lactic acid bacteria. In: Salminen, S. & von Wright, A. (eds.). Lactic Acid Bacteria: Microbiology and Func- tional Aspects 2nd Edition. New York: Marcel Dekker Inc. p. 139-160. 263 Vol. 7(1998): 251-265. AGRICULTURAL AND FOOD SCIENCE IN FINLAND Rollerna, H.S., Kuipers, 0.P., Both, P, de Vos, W.M. & Siezen, R.J. 1995. Improvement ofsolubility and sta- bility of the antimicrobial peptide nisin by protein en- gineering. Applied and Environmental Microbiology 61: 2873-2878. Ryan, M. P„ Rea, M.C., Hill, C. & Ross, R.P. 1996. An application in Cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad- spectrum bacteriocin, laclicin 3147. Applied and En- vironmental Microbiology 62: 612-619. Ryser, E. & Marth, E.H. 1991. Foodborne Listeriosis. In: Ryser, E. & Marth, E.H. (eds.). Listeria, Listeriosis and Food Safety. New York: Marcel Dekker Inc. p. 240-287. Sahl, H.-G., Jack, R.W. & Bierbaum, G. 1995. Biosyn- thesis and biological activities of lantibiotics with unique post-translational modifications. European Journal of Biochemistry 230: 827-853. Salminen, S., Deighton, M.A., Benno, Y. & Gorbach, S.L. 1998a. Lactic acid bacteria in health and disease. In: Salminen, S. & von Wright, A. (eds,). Lactic Acid Bacteria: Microbiology and Functional Aspects 2nd Edition. New York: Marcel Dekker Inc. p. 211-254. - , Isolauri, E. & Onnela, T. 1995. Gut microflora in health and disease. Chemotherapy 41, Suppl. 1: 5-15. - , Isolauri, E. & Salminen, E. 1996. Clinical uses of pro- biotics for stabilising the gut mucosal barrier: suc- cessful strains and future challenges. Antonie van Leeuwenhoek 70: 251-262. - , Roberfroid, M., Ramos, P. & Fonden, R. 1998b. Pre- biotic substrates and lactic acid bacteria. In: Salmi- nen, S. & von Wright, A. (eds.). Lactic Acid Bacteria: Microbiology and Functional Aspects 2nd Edition. New York: Marcel Dekker Inc. p. 343-350. Sanders, M.E. 1994. Lactic acid bacteria as promoters of human health. In: Goldberg, I. (ed.). Functional Foods: Designer Foods, Pharmafoods, Nutraceuti- cals. New York: Chapman and Hall. p. 294-322. Schaatsma, G. 1995. Application of lactic acid bacteria in novel foods from a nutritional perspective. In: Novel, G. & Le Querler, J.-F. (eds.). Lactic Acid Bac- teriä: Actes du Colloque LACTIC 94. Presses Universitaires de Caen. p. 85-93. Stevens, K.A., Sheldon, 8.W., Klapes, N.A. & Klaenham- mer, T.R, 1991. Nisin treatment for the inactivation of Salmonella species and other Gram negative bac- teria. Applied and Environmental Microbiology 57: 3613-3615. Stiles, M.E. 1996. Biopreservation by lactic acid bacte- ria. Antonie van Leeuwenhoek 70: 235-249. van Belkum, MJ., Hayema, 8.J., Jeeninga, R.E., Kok, J. & Venerna, G. 1991. Organisation and nucleotide sequences of two lactococcal bacteriocin operons. Applied and Environmental Microbiology 57: 492- 498. - , Kok, J. &Venerna, G. 1992. Cloning, sequencing and expression in Escherichia coli of Icnß, a third bacte- riocin determinant from the lactococcal bacteriocin plasmid p984-6. Applied and Environmental Micro- biology 58: 572-577. Vandenberg, P.A. 1993. Lactic acid bacteria, their meta- bolic products and interference with microbial growth. FEMS MicrobiologyReviews 12: 221-238. Venerna, K., Venerna, G. & Kok, J. 1995. Lactococcal bacteriocins: mode of action and immunity. Trends in Microbiology 3: 299-304. von Wright, A. & Sibakov, M. 1998. Genetic modification of lactic acid bacteria. In: Salminen, S. & von Wright, A. (eds.). Lactic Acid Bacteria: Microbiology and Functional Aspects 2nd Edition. New York: Marcel Dekker Inc. p. 161-210. Wells, J.M.,Robinson, K., Chamberlain, L.M., Schofield, K.M. & Le Page, R.W. 1996. Lactic acid bacteria as vaccine delivery vehicles. Antonie van Leeuwenhoek 70: 317-330. Young, G. 1996. Prevention of colon cancer: role of short chain fatty acids produced by intestinal flora. Asia Pacific Journal of Clinical Nutrition 5: 44-47. Young, J. 1996. In: Financial Times ManagementReports: Functional Foods - Strategies for Successful Prod- uct Development. London: Pearson Professional Ltd. 264 Seminar in honour of the IOOth anniversary ofMTT AGRICULTURAL AND FOOD SCIENCE IN FINLAND SELOSTUS Terveyttä ja ruoan turvallisuutta edistävät maitohappobakteerien biotekniset sovellukset Charles Daly ja Ruth Davis University College, Cork, Irlanti Ruoka-aineiden käyttäminen maitohappobakteerien avulla on yksi vanhimmista säilöntämenetelmistä. Näiden teollisuustuotannossakin merkittävien maito- happobakteerien geneettisten ja fysiologisten ominai- suuksien tutkimuksessa on viime vuosina edistytty merkittävästi. Yksi maitohappobakteerien tärkeä omi- naisuus on niidenkyky tuottaa mikrobeille vastustus- kykyisiä yhdisteitä. Mikrobeille vastustuskykyiset yhdisteet kiinnostavat aiempaa enemmän, koska nii- tä voidaan käyttää elintarviketeollisuudessa kemial- listen säilöntäaineiden sijaan. Kuluttajat ovat yhä enemmän tietoisia ruoan ja ter- veyden välisestä yhteydestä. Viime aikaiset tutkimus- tulokset tukevat oletuksia, joiden mukaan maitohap- pobakteereilla on probioottisia, terveyttä edistäviä, ominaisuuksia. Lisäksi tällä hetkellä selvitetään eräi- den maitohappobakteerien kykyä toimia suun kautta nautittavien rokotteiden elävinä kuljetusvektoreina. 265 Vol. 7(1998): 251-265. AGRICULTURAL AND FOOD SCIENCE IN FINLAND