View of The biotechnology of lactic acid bacteria with emphasis on applications in food safety and human health

The biotechnology ^{of lactic add bacteria with} emphasis

on applications in food safety and human health

Charles Daly and Ruth Davis

Department ofMicrobiologyand National FoodBiotechnology Centre, University College, Cork, Ireland,e-mail: Dean.food@ucc.ie

Fermentalion of various foodsluffsby lactic acid bacteria (LAB) is oneof the oldest forms of bio- preservation practised bymankind.Inrecent years,significantadvances have been madeinelucidat- ingthe genetic andphysiological basis ofkey LABtraits involved inthese industrially significant processes. Oneimportantattribute of manyLAB is theirabilitytoproduceantimicrobialcompounds calledbacteriocins.^{Interestinthese}compounds has grownsubstantiallydue to theirpotential useful- ness asnatural substitutes for chemical food preservatives intheproduction offoods with enhanced shelflife and/or safety.

There isgrowingconsumerawareness^{of thelink}between diet and health. Recent scientific evidence supports the role ofprobiotic LAB in mediatingmanypositive health effects. In addition,someLAB arecurrently beingassessed for theirability to actaslivedelivery vectorsinthedevelopmentofnew 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_meansof improving the shelflife ofoth- erwise perishable foodstuffs and as such they represent along-standing application of biotech- nology. Many substrates includingmilk, meats, cereals,vegetables and fruits have been ferment- ed generating a wide range of nutritious end products with desirable flavours and attributes.

LAB are aphylogenetically diversegroupof

bacteria. Members of the generaLactococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus, in particular, _areinvolved in these fermentations. In addition, someLAB (mainly Lactobacillus spp.) as wellas the functionally related, though non-LAB, Bifidobacterium, are known ^toformpart of the normal human intesti- nal microflora and accumulating evidence sug- geststhat these bacteria may^exertapositive ef- fectonhuman health.

Given the economic value of food fermenta- tions and agrowingacceptancethat^atleastsome of these products may contribute to improved

©Agriculturaland Food Science inFinland Manuscriptreceived May 1998

Voi 7^(1998):251-

health, it is^not surprising that LAB are^attract- ing major attentionatthis time. This is reflected in the increased volume of fermented products available world-wide especially in the area of functional foods containing probioticor 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 thecurrent 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 (Mercenieretal. 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: towardsarational solutionto amajor problem of food fermentation

4. The molecular biology and genetics of ther- mophilic LAB

5. LAB ascell factories for the production and delivery of mucosal immunogens

6. Carbon catabolite control in food grade lacto- bacilli^toprovidethetoolsfor strainimprove-

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- mentof their functionality and subsequently dis- seminating the informationto relevant authori- ties, consumer organisations and participating industrypartners. The PROBDEMO projecten- 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 identification of gastrointestinal flora

3. To demonstrate and meet the functional and technological requirements essential for the industrial production of probiotics asfunc- tional foods

4. To disseminate the knowledge and results to the extended audience consisting of the group of industrialusers,authorities and_consumer organisations.

These and other research efforts world-wide have significantly advanced our understanding of key functional processes in LAB. They have already been instrumental in providing well- characterised strains forusein large-scale food fermentations and 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.

Researchonthe contribution of various lacto- bacilli and bifidobacteriato the normal healthy functioning of the human gastrointestinal sys- temand their likely probiotic effects is evolving rapidly, driven by the eagerness offood compa- niesto satisfy a growing consumer market. Al- ready several products _arebeing marketed with probiotic claims and_anumber of manufacturers have developed and licensed specific probiotic bacteria ^- Lactobacillus johnsonii LAI from Nestlé, LA7 fromBauer, Causido culture from MDFoods, the Lacticel strain from Danone and Lactobacillus GG from Valio, Mona and other

companies (Young, J. 1996).

One of the newerand very exciting areasof LAB researchconcerns their exploitationaslive oral vaccine delivery vehicles. The improved ability^togenetically manipulate thesebacteria, their ‘generally regarded assafe’(GRAS) status and the potential easeof production and admin- Seminar in honour

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istration ofLAB-based vaccines make them very attractive candidates for such applications (Wells etal. 1996).

The aim of this paper is toreview develop- mentsin LAB research that have already impact- ed, or are likely _to impact, the production of foods that aresafer 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 past20 years have seen amajor impetus in LAB research. Although initially much of this research focused on dairylactococci,investiga- tions _now encompass many differentLAB in- volved in a wide variety of fermentation proc- esses and, morerecently, various lactobacilli and bifidobacteria belonging tothe 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 themostimportant functional prop- erties, lactose utilisation and proteolytic activi- ty, areparticularly far advanced. However, sig- nificant developments in otherareassuchasbac- 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 wasgreatly aidedear- ly onby the fact that many of the industrially significant properties of these bacteriawere en- coded by plasmids (Fitzgerald and Hill ^1996).

However,researchon their chromosomalgenet- ics is also progressing rapidly. Physical and ge- netic maps have been constructed foranumber of strains and thereare anincreasing number of chromosomally located genetic loci under inves- tigation (Davidson etal. 1996).

The development of tools that facilitated the genetic manipulation of LAB has been crucial tothe success of these endeavours. In particu- lar, electrotransformation, which mediates high frequency uptake of in vitroDNA,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,oneof the natural processes of gene exchangecommonamonglactococci,has played animportant role in non-recombinant strategies of strain improvement (Gasson and Fitzgerald 1994).Since the early 1980

s,

the array of clon- ing _vectorsavailabletoresearchers has expand- ed enormously. In addition to general cloning vectors,there isawide choice ofvectorsavaila- ble with specialised functions ^(de Vos and Si- mons ^1994).These include genetic signalscreen- ing ^vectors,high expression ^vectorsand induci- ble expressionsystems.Two furthersystemsare worthy of special_note.First is the development ofvectors suitable for _use in food industry ap- plications. These contain only LAB- derived DNA andusefood grade selection markers such as bacteriocin resistance, lactose-fermenting ability, bacteriophage resistance^etc(vonWright and Sibakov 1998).The secondsystemof^note concerns the development ofvectorsthat facili- tate heterologous gene expression and secretion (de Vos and Simons 1994). Theseareparticular- ly relevant for the exploitation of LAB as vac- cine delivery vehicles, an areaof research that will be discussed in more detail in a latersec- tion.

The understanding and exploitation of indus- trial traitsare ^notthe onlyaspects of LAB re- search that have benefited from the development of more sophisticated technologies. Reliable methods of strain identification and classifica- tionarevitally important.^Newertechniques such asthe abilitytosequencelargetractsof 16S and 23S rRNA genes using polymerase chainreac- tion(RAPD-PCR)and theuseofpulsed field gel electrophoresis (PFGE) tofingerprint genomic restrictionpatterns have contributedenormous- lyto these efforts(Axelsson 1998).This relates

very much to the field of probiotics where the Vol.7(1998):251-265.

ability to monitor strains through clinical trials andtoevaluate their effectson the gastrointesti- nal tract microflora_as well _asthe protection of their proprietary value depends onexactand _re- producible strain identification.

The following sections of this review will focuson someof the properties ofLAB thatcon- 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 continuedtorise. Concomi- tantly, there is a strong trendon thepart ofcon- sumers favouring less processed foods contain- ing fewer chemical preservatives (Daeschel

1993). As a result, there isan increased interest in the preservative aspects of^LABparticularly in view of their long and safe association with human fermented foods. Several metaboliccom- pounds produced by these bacteria have antimi- crobial effects, including organic acids, fatty

acids, hydrogen peroxide and diacetyl (Holzap- fel etal. 1995, Ouwehand 1998).However, the majority of attention has focused_on the ability of many LAB toproduce specific proteinaceous inhibitory substances, bacteriocins, that inhibit the growth of other bacteria and can, therefore, enhance the shelf-life of foods in which theyare present. Significantly,somebacteriocins inhibit serious food-borne pathogens such as Listeria, Clostridium,Staphylococcus, and certain Bacil- lus spp. and Enterococcus spp.

Atpresent four classes of LAB bacteriocins have been defined (Table 1). Members of class- esI and II arethemostfrequently characterised probably reflecting the well-establishedisolation procedures for these bacteriocins and their po- tential for industrial application.

Nisin, which is produced by someL. lactis subsp. lactisstrains,belongs^tothe class I lanti- biotics and is by far the^mostextensively stud- ied bacteriocin of the LAB (Dodd and Gasson

1994, Jacketal. 1995).Itwas discoveredasfar back as 1928 and hasabroadspectrum of activ- ity against many Gram-positive bacteria includ- ing Listeria spp. It prevents the outgrowth of germinating bacillus and clostridial sporesand, through the addition ofa calciumchelator, it is possibletobroaden its activitytoincludesome Gram negative bacteria (Stevens etal. 1991). The maturenisin molecule is just 34 amino acids long and undergoes extensive post-translational mod-

Table 1.^Classesof bacteriocins produced byLAB.

Class Subclass Description

I Lantibiotics^- small,heatstable,containingunusual amino acids Small (30-100amino acids), heatstable,non-lantibiotic II

Ha Pediocin-likebacteriocins,with anti-listerial effects lib Twopeptidebacteriocins

lie Sec-dependentsecretion of bacteriocins

111 IV

Large ^(>30kDa) heat-labileproteins

Complexbacteriocins with glyco-and/orlipid moieties,heat stable Adaptedfrom Nes et al. 1996,Ouwehand 1998

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ifications in which serine and threonineresidues aredehydrated and several thio-ether bridges are formed. These modifications result in the for- mation of the five ring structuresthatarechar- acteristic of the molecule. The primarytargetof 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 themem- brane and dissipating its potential (Sahl et al.

1995).Owing to its extensive genetic and mo- lecularcharacterisation, nisin has been thetar- getof several protein engineering studies aimed atbroadening its functional attributes. Modified nisins containing specific amino acid substitu- tions have been generatedsomeof which exhib- it enhanced practical features suchas increased activity against food pathogens and improved stability and/or solubility under various food- processing conditions (Kuipersetal. 1991, 1995, Rollernaetal. 1995).

Other class I lantibiotictypecompoundsapart from nisin have been isolated fromawide vari- etyof LAB sources. One,lacticin 3147, was re- cently identified from a lactococcal isolate of Irish kefir grains ^(used in the manufacture of buttermilk) during_acollaborative study between the Teagasc ResearchCentre,Moorepark, Ireland and the Microbiology DepartmentatUniversity College,Cork, Ireland (Ryan etal. 1996).This bacteriocin is particularly attractiveas it inhib- its a wide_spectrum of Gram-positive bacteria including potential food-borne pathogens such asStaphylococcus,Clostridium and Listeria spp.

as well as several mastitic staphylococci and streptococci (Meaneyetal. 1997).Lacticin 3147 requires twopeptides for activity. Both peptides are produced in a precursor form and are sub- jectedtopost-translational modifications involv- ing the dehydration and linkage ofanumber of amino acids producing typical lanthionine rings and the cleavage ofprepropeptide sequences. The genetic determinants of lacticin 3147 arelocat- ed on a large 60 kb conjugative plasmid, pMRCOI, flankedbytwoiso-1557-like elements (Dougherty etal. in press). The interveningre- gion contains 13 open reading frames (ORFs), eleven ofwhich, arranged in two operonstruc-

tures, arethoughttobe associated with the bac- teriocin functions. Six of the ORFs showed sig- nificant sequence homology with genes known

tobe involved in the production, immunity and transportof other recognised lantibiotics. It has notyetbeen possibleto conclusively identify the structural genes of the bacteriocin. As with ni- sin, lacticin3147 ^actsonsusceptible cell mem- branes by introducing ion-specific pores that dis- rupt the membrane potential and rapidly cause cell death(McAuliffeetal. 1998).

Class II bacteriocins contain a wide variety of bacteriocins and, therefore, are categorised into three further subclasses. In general, howev- er, theyareall 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 membraneas their active site forming oligomeric pores. How- ever, unlikelantibiotics, their bacteriocidalac- tivity is independent of the membrane’s energi- sationstate and appearstorequire a cell mem- branereceptormolecule. Lactococcin A, whose mode of action has been studied in some detail, is thought^to insert itselfas ana-helicalstructure across the cell membrane. Several lactococcin A molecules subsequently combinetoform pores in the membrane. These porescause anefflux of small cytoplasmic molecules and ions resulting in dissipation of the membrane potential (Vene- rna^etal. 1995).

The genetic determinants of several class II bacteriocins have been cloned and sequenced.

Many have been linkedtoplasmidsand,insome cases, an individual host may produce multiple bacteriocins(Doddand Gasson 1994). Inthecase of lactococcins A, B and M, all three were located on the sameplasmid (van Belkum etal.

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- latedtodate. Helveticin J is the best knowncom- pound of this class. The legitimacy of the fourth bacteriocin class is somewhat controversial. The requirement of the glyco and/or lipidcomponent for the action of these bacteriocins is not well Vol. 7(1998):251-265.

established and may beaconsequence of incom- plete purification procedures. The modes ofac- tion for both these classesarepoorly understood (Klaenhammer 1993, Venemaetal. 1995).

Clearly, many bacteriocins of theLAB, es- pecially those with broadspectraof activity, have tremendous potentialtobe exploitedassafe and effective ‘natural’ inhibitors of potential patho- genic and food spoilage bacteria in various food systems.Nisin is the classic example with ^apar- ticularly long and successful history in food ap- plications. Some of its commercial applications include: preventing clostridial spoilage of proc- essed and naturalcheeses, inhibiting the growth ofsomepsychrotrophic bacteria incottagechees- es,extending the shelf-life of milk inwarm 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 tradenameNisaplin®(Vanden- berg 1993, Delves-Broughtonetal. 1996).

The emergence ofListeria, specifically Ls.

monocytogenes,as a serious food-borne patho- gen is of major^concernin 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 belongingtothe subclassHa,which dem- onstrateantilisterial activity, have received sig- nificant research attention. Pediocin PA-l/AcH produced by Pediococcus acidilactici is regard- edas theprototype bacteriocin of this subclass and various studies have demonstrated its abili- ty to control Listeria in cheese, vegetable and meatsystems.The application ofpediocin in the biopreservation ofmeatsis particularlyrelevant, asnisin is^not very effectivein this environment.

It is also significant to note that Pediococcus acidilactici is a common starterculture used in the production of most fermented meats (Vandenberg ^1993,Stiles 1996).

Thebacteriocin, lacticin 3147, has also been

the subject of food application studies. Ryan et al. (1996) developed a range of lacticin 3147- producing starterstrains suitable foruseincom- mercial cheese making. When incorporated, these strains effectively controlled the growth of anynon-starterLAB in Cheddar cheese andcom- pletely eliminated deliberately inoculated Ls.

monocytogenes fromcottage cheese. Lacticin 3147 has a number of advantages overnisin. It is effectiveatneutral pH and^startercultures pro- ducing this bacteriocin have good acid produc- ing and bacteriophage resistance properties un- like theircounterpartsproducing 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- ticin3147as amastitis-controlling therapy in dry cows(C. Hill,pers. comm.).

Unfortunately,amajor drawback associated with LAB bacteriocins^liesinthefact^that gram negative bacteria as well as yeasts and moulds are normally refractive totheir bactericidal ac- tion. As a result, the usefulness of thesecom- pounds in commercial practice has been some- what limitedasmanyimportant food borne path- ogens and foodspoilage microorganisms belong to these resistant categories. This has sparked severalrecent studies aimed atbroadening the bactericidal activity of LAB bacteriocinstoen-

compass thesenormally resistant groups.Ingen- eral,these studies have focusedonthe synergis- tic effects ofbacteriocins, most notably nisin, with other antibacterial factors such as the lac- toperoxidasesystem presentin milk, hydrolytic enzymes, various chelating agents (including siderophores) and other bacteriocins (Helander etal. 1997).

Todate,nisin remains the only LAB bacteri- ocin tobe legally permitted as afood additive.

This has had major implications for the many other bacteriocinsthat,inrecentyears, have dem- onstrated commercial potential. Two products, ALTA™243I and Microgard®, have been devel- opedasshelf-life extenders basedon crude LAB fermentation products and, therefore, donotre- Seminar in honour

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quire afood additive label. ALTA™234I is pro- duced from aPediococcus acidilactici fermen- tation and is assumedto rely on the inhibitory effects of Pediocin PA-l/AcH. It is commonly added_toMexican soft cheeses whicharepartic- ularly susceptible _to listerial contamination (Glass etal. 1995).Microgard® is the result ofa Propionibacterium fermentation. It is active against Gram negative bacteria such as Pseu- domonas, Salmonella, and Yersinia, as well _as yeastsand moulds. Microgard®’s protective ac- tion is probably due tothe presence of propion- ic acid as ametabolic end-product.However, a role forabacteriocin in this product has also been proposed (Al-Zoreky et al. 1991). Microgard®

has been approved by the FDA foruse in food applications suchas cottagecheese and fruit-fla- voured yoghurts. Approximately 30% of thecot- 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 aprotective adjuncttonormalstartercultures to inhibit the growth of yeasts, moulds.^Bacillus spp. Clostridium spp. and heterofermentative lactobacilli during some dairy fermentations (Mäyrä- Mäkinen and Suomalainen 1995).

It mustbe emphasised that theuseof bacte- riocins either exogenouslyorby the adventitious useof bacteriocin-producing cultures should^not be regarded as a panacea forpoor-quality raw materials ormanufacturing practices. Instead, it proposed that bacteriocins be used in combina- tion with other physical, chemical and microbi- al preservation factors as anadditional ‘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 thatahealthy lifestyle, includingdiet, canplay amajor role in preventing diseases and promoting human health.

Functional foods containing probiotic cultures are a well-establishedconcept in Japan and,in recent years, suchproducts comprise arapidly expanding, lucrative,internal andexportmarket for the EU. Several factors have fuelled this in- terestin functional foods. Today,consumers are better informed than _ever and _arekeen to take proactive decisions with regard^to maintaining their health. Changing population dynamicsto- wards older societies and the increased preva- lence of chronic illnesses such_as cardiovascu- lar disease andcancer areplacing heavy demands on already stretched and expensive healthcare services. Inaddition, there is seriousconcernat 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 advocatedmov- ing towards alternative disease control strategies including theuseof probiotic bacteria in the pre- vention and treatment of certain infections (Bengmark 1998).

The human gastrointestinal (GI)tract sup- ports arich and dynamic microbial population ofmore than500 bacterial species. Maintaining this delicately balanced ecosystem is important for the normal functioning of thegut, particu- larly withregardtopreventing GI infections and

stimulating the host’s immune response. Mod- ernantibiotictreatments,radiation therapy, stress and Western dietary preferencescansignificantly affect thegut microflora predisposing the host to various diseases (Salminenetal. 1995, 1998

a,

Schaafsma 1995).

Probiotic cultures are generally defined as live,non-pathogenic bacteria which when ingest- edexertapositive influenceonthe host’s health.

Lactobacillus spp. and

Bifidobacterium

^{spp. are}

prominent members of the commensal intesti- nal flora ofmosthealthy individuals and _arethe

mostcommonly studied probiotic bacteria. Their probable and theoretical benefits have beenout- lined in severalrecent reviews and includere- duced lactose intolerance, alleviation of some diarrhoeas,lowered bloodcholesterol,increased immune responses and prevention of cancer (Marteau and Rambaud 1993, ^1996, Gilliland Vol.7(1998): 251-265.

1996, Salminenetal. 1996,1998^a).Theconcept of probiotics is not new, having been proposed originally by Metchnikoff in 1907. However, despitenumerous studies in thepast, there has been very little convincing scientific evidence tosubstantiate their health claims until recently.

This has been duetodifficulties in unequivocal- ly identifying strains,differences in experimen- talsystemsand data interpretation and ageneral lack of coordination between clinicians and microbiologists (Sanders 1994). Considerable efforts have been made recentlytoredress this situation. Modern taxonomic methods have im- proved the identification ofteststrains and em- phasis is being placed onperforming random double-blind placebo-controlled clinical trials^to demonstrate the efficacy of potential probiotic strains and products. Table2 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. Salminenet 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 areamong 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 deliveryvehicles,resistance toacid and bile,adherence to gutepithelial tis- sue,ability to colonise the GI tract,production of antimicrobialsubstances, abilitytostimulate ahost immune response and the abilityto influ- ence metabolic activities such as vitamin pro- duction,cholesterol assimilation and lactaseac- tivity (Huisin’t Veld and Shortt 1996, Salminen et al. 1996).Ofcourse, it is unlikely that any individual strain will be able to present all of these credentials andablend of strains withcom- plementary attributes may be required todeliver optimum probiotic performance.

AtUniversity College,Cork, Ireland,Collins and co-workers applied stringent in vitro selec-

Table2. Requirementsfor clinical studies ofprobioticfoods for functional and clinicaluse.

Each strain documented and testedindependently Extrapolation of data from closelyrelated strains not acceptable

Well definedprobiotic strains, studyproducts,andstudy populations

Double-blind,placebo-controlled, and randomised human studies

Result confirmed by several independent research groups

Publication inpeer-reviewedjournals (Salminen etal. 1996, 1998^a)

tion criteria _{to a}bank of human Lactobacillus isolates in an effortto identify arange ofnew strains with potential probiotic characteristics.

Eight candidates survived the selection process and one. Lb. salivarius UCCIIB producing a broadspectrumanti-microbial protein, wascho- sen for further clinical trials. Lb. salivarius UCCIIB wasdemonstratedtobe efficiently de- liveredtothe gutfollowing oral administration in milkoryoghurt carriers. Inaddition,in_apro- portion of volunteers (<10%) significant num- bers were stillpresent in faeces 3 weeks after the cessation of itsadministration,indicating that this strain wascapable of colonising the human GItract in vivo. Lb. salivarius UCCIIB did^not disturb the numbers of other lactobacilli in the

gut, but therewas a statistically significant re- duction in the numbers of excreted Clostridia.

Although it is recognised that further work is required tobuild up the medical dossieronLb.

salivarius UCCIIB, these preliminary trials strongly support this strain as an effective pro- biotic culture(K. Collins, pers. comm.).

Most of the disease ^statesthat benefit from LAB therapy are characterised ^to a greater or lesserextentby adisturbed 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- fermentedcounterparts even when they contain significantamounts of lactose. Milks fermented by variousLAB, including thecommonyoghurt Seminar in honour

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cultures,Lb. bulgaricus and S. thermophilus,are effective and viable cultures are important to achieve maximum benefits. At least three mech- anisms, or acombinationthereof, arethoughtto contributetothe effect: the reduced lactosecon- 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 etal.

1996, 1998^a).

Probiotic preparations have also been found tobe 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 beenpresented forLacto- bacillus GG but positive results were also achieved with Lb. johnsonii LAI and NCFB 1748,Lb. casei Shirota strain andmorerecently with Lb. reuterii (Lee and Salminen 1995, Salminen et al.

1998

a). The mechanisms by which these effects were achieved are notwell defined.However, the ability of the lactobacilli toadheretoand potentially modify hostmucos- al surfaces is thought^tobe important. It is like- ly that the lactobacilli suppress the growth of pathogens at the mucosal surface probably by out-competing them for nutrientsorby produc- ing antibacterial compounds (Salminen et al.

1998

a,

Isolaurietal. 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- tureshave attracted much research attention. The results to dateareinconclusive. Several studies demonstrated that various strains could assimi- late cholesterol in vitro.However, reliable data regarding_an in vivo function have^notyetbeen reported(Lichtenstein and Goldin 1998).

The ability of GI microflorato enzymatical- lyconvertprecursors naturallypresentin the diet to carcinogenic forms is well documented and is likelytocontributetotheaetiologyof colonic

cancer. Significantly, LAB and bifidobacteria tendtohave low levels of such activities incom- parison toothergut bacteria. Several studies in both animals and humans have demonstrated the ability of these bacteriatoreduce the toxicity of intestinal contentsby 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 etal. 1996, 1998

a,

Iso- laurietal. 1998).In ^addition, many LAB pro- duce metabolic end-products (butyrate/butyric acid) that have anti-tumorigenic activities in vitro (Young, G. 1996).Therearealso anumber of in vitro and in vivo animal studies that demonstrate moredirectly tumourinhibition 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 toreduce the recurrence of superficial bladder carcinoma in humans.

It has also been documented that various LAB canmodulate the host immuneresponse. Reports have described increased production of immu- noglobulins, interleukins 6 and 10, gamma in- terferon, tumournecrosis factor-a and increased phagocytic activity. Notably, Lactobacillus GG wasable tostimulate local and systemic IgAto rotavirus during infection of children with this agent^{(Kaila et}al. 1992).This effectwasthought

to contribute ^toprotection against reinfection.

Lb. salivarius UCCIIB also exhibiteda 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_asadjuvants tooral vaccines (Isolauri etal. 1998).

Another approach to the maintenance of a healthy gutmicroflora is the provision of sub- strates that preferentially select for the growth of desirablebacteria in the host. Thesesubstrates, called prebiotics, are based on non- or slowly absorbable complex carbohydrates that canbe assimilated by beneficial bacteria suchas

Bifi-

dobacterium and Lactobacillus but in contrast

Vol. 7(1998):251-265.

arehardlyeverutilisedby potentially pathogen- ic Gram-negative organisms. Examples of preb- iotic substrates includeinulin, lactulose,various galacto-,fructo-,xylo-oligosaccharides and sug- aralcohols such as lactitol and xylitol ^(Salmi- nenetal.

1998

b). Many of the functional foods recently launched in Europe containacombina- tion ofa probiotic culture withaprebiotic sub- stratethat 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, therearedefinite 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^$l7billion (Young, J. ^1996).In Europe espe- cially, there is ^agrowing number of dairy-based products available that contain probiotic cultures and/or prebiotic substrates^{(Table 3).} Currently mostcompaniesareadoptingaprudent approach

tomarketing their probiotic products relying on general health claims suchas ‘helps boost ^the body’s natural defences’or ‘restores the body’s natural balance’. In light of the high R+Dcosts and in order for these products^toachievea max- imum return on investment, it is essential that consumers arepresented with clear and substan- tiated health claims. The PROBDEMO project hasanimportant role toplay in thisrespect and underlines the EU’s commitment tosupporting this market segment.

LAB ^as live vaccine delivery vehicles

In recent years there has beenincreasing inter- estin exploiting someLAB as live vaccine de- livery vehicles. LAB present anumber ofadvan- tagesthat make them attractive for this function.

They havea long history of safe use in foods, there is extensive knowledge already available

regarding ^theirproductionon alarge scaleand, through fermented products, they areeasily ad- ministered orally. Furthermore, it is recognised that the gut is an important site for antigen im-

muneeducation.

Different approaches have been adopted in the development of LAB-based vaccines. One relies oncolonising lactobacilli thatarecapable of remaining in thegut or genital tractforape- riod of time during which animmune response may be elicited toanexpressed antigen. Lacto- bacillusvectors arechosenonthe basis of their potential for genetic manipulation and the abili- tyto expressforeign antigensas wellasfor 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 transformableathigh frequencies bynat- ural competence. Also, the strain colonises the oral cavity very efficiently and has been demon- strated tocolonise mice vaginaltractsfor up ^to 8 weeks. A thirdstrategyhas focusedonthe use of non-colonising L. lactis strains. In this in- stance, antigens expressed by these bacteria are generally retained intracellularlyand, therefore, are not as susceptible to degradation in thegut and, in addition, there is evidence that protein antigens are more immunogenic when they are contained either within orassociated with the recombinant bacteria.

Results todate with all threeapproaches are encouraging. Several antigenic epitopes have been expressed in all three hosttypesusing_var- ious cellular locations(intracellular, cellsurface, extracellular) and have demonstrated_an ability

to elicit local and systemic immune responses (Wellsetal. 1996).

Concluding ^remarks

Withoutdoubt, advances inbiotechnology over the lasttwo to three decades have significantly Seminar in honour

of

the 100thanniversary

ofMTT

Table3. Examplesof fermentedmilkproducts containing probioticbacteria availableinfood retail outlets inEurope,the UK and Ireland.

Product Brandname ^{Company Countries}

(Organism^- 10⁷-10⁸viable LAB/ml)

Yoghurt LCI Nestlé France,Belgium, Spain,

(Lb. johnsonii LAI) 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)

Yoghurtdrink 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 MDFoods 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. 1996and various sources)

expanded our ability ^to produce high quality, nutritious and tasteful foods that remain fresher for longer, arecompletely safe and that_areless reliant on artificial additives. The potential ap- plications of bacteriocins as ‘consumer friend-

ly’ biopreservatives either in the form ofprotec- tive culturesor asadditivesare significant. Dis- appointingly, with the exception of nisin andto amuch lesser extentPediocin PA-l/AcH, very little of this potential has been realised in anin- Vol. 7(1998): 251-265.

dustrial context. Despite strong arguments in favour of their efficacy and safety, the process of obtaining regulatory approval for the more widespread useof 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 importantbut, as yet, underexploited proteins of LAB.

Incontrast,the development of the function- al foods market,particularly with regard tothe 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 _tobe 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 adaunting task given the complexity of the hu- manmicrobiota and the multiplicity of interde- pendent reactions thatarelikelytobe involved.

Essentialtotheseefforts, however,will beathor- 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 arevery difficulttomanipu-

late technically, afactor that is sometimesover- looked in initial selection procedures. While the benefits of probiotic cultures appeartobe many and wide-ranging, ^atpresent 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 damageconsumerconfidence. The public in gen- eral, and especially those involved inconsumer affairs and in policy decision-making bodies, mustbe carefully educated regarding their po- tential benefits. In addition,importantconsum- errequirements such as tasteand convenience shouldnotbe compromised in the development of effective probiotic products.

Extending the traditional fermentation roles ofLAB isanimportant goal of scientific research and new product development. In this respect, theuse of certain LAB aspotential vaccine de- livery vehicles has opened upacompletely new avenue for the exploitation of these bacteria.

Although this ^areais in the early stages of de- velopmentas yet, the initial successes in elicit- ing immune responsesto heterologous antigens bode well for the future development ofneworal vaccines.

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