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Custom Search Bacteriology."The Good, t he Bad , and t he Deadly". (SCIENCE Magazine - June 4,

La ct ic Acid Ba ct e r ia ( LAB )are Gram-positive , non-sporeforming cocci, coccobacilli or rods with a DNA base composition of less than 53mol % G +C. They generally are non respiratory and lack catalase. They ferment glucose primarily to lactic acid, or to lactic acid, CO2and ethanol . All LAB grow anaerobically, but unlike most anaerobes, they grow in the presence of O

2as "aerotolerant anaerobes". Although they lack catalase , they possess superoxide dismutase and have alternative means to detoxify peroxide radicals, generally through peroxidase enzymes.

Although many genera of bacteria produce lactic acid as a primary or secondary end-product of fermentation, the termLa ct ic Acid Ba ct e r iais conventionally reserved for genera in the order Lact obacillales, which includesLact obacillus , Leuconost oc, Pediococcus , Lact ococcusand St rept ococcus ,in addition toCarnobact erium , Ent erococcus, Oenococcus, Tet ragenococcus , Vagococcus,andWeisella.

Because they obtain energy only from the metabolism of sugars, lactic acid bacteria are restricted to environments in which sugars are present. They have limited biosynthetic ability, having evolved in environments that are rich in amino acids, vitamins , purines and pyrimidines, so they must be cultivated in complex media that fulfill all their nutritional requirements . Most are free-living or live in beneficial or harmless associations with animals, although some are opportunistic pathogens . They are found in milk and milk products and in decaying plant materials. They are normal flora of humans in the oral cavity, the intestinal tract and the vagina, where they play a beneficial role.

A few LAB are pathogenic for animals , most notably some members of the genusSt rept ococcus. In humans,St rept ococcus pyogenesis a major cause of disease (strep throat, pneumonia, and other pyogenic infections, scarlet fever and other toxemias),St rept ococcus pneum oniaecauses lobar pneumonia, otitis media and meningitis; some viridans and nonhemolytic oral streptococci play a role in dental caries and may be an insidious cause of endocarditis. The pathogenic streptococci are dealt with elsewhere in the text. This chapter deals primarily with LAB in association with food and dairy microbiology, to a lesser extent with LAB as beneficial components of the human normal flora and probiotics.

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spoilage bacteria by producing growth-inhibiting substances and large amounts of lactic acid. As agents of fermentation LAB are involved in making yogurt , cheese, cultured butter , sour cream, sausage, cucumber pickles, olives and sauerkraut, but some species may spoil beer , wine and processed meats .

D iffe r e n t ia l ch a r a ct e r ist ics of la ct ic a cid ba ct e r ia ba se d on m or ph ology a n d ph ysiology

Ge n u s Lact obacillus Ent erococcus Lact ococcus Leuconost oc Pediococcus St rept ococcus Ch a r a ct e r ist ic

Morphology rods cocci cocci cocci cocci in

tetrads cocci

CO₂from

glucose* ± − − + − −

Growth

at 10°C ± + + + ± −

at 45°C ± + − − ± ±

in 6 .5% NaCl ± + − ± ± −

at pH 4 .4 ± + ± ± + −

at pH 9 .6 − + − − − −

Lactic acid

configuration D, L, DL L L D L , DL L

+ posit ive; - n e ga t ive ; ± va r ie s be t w e e n spe cie s *

t e st for h om o- or h e t e r ofe r m e n t a t ion of glu cose : - h om ofe r m e n t a t ion + h e t e r ofe r m e n t a t ion

chapter continued

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Feat ured Microbe

Web Review of Todar' s Online Text book of Bact eriology."The Good, t he Bad, and t he Deadly". (SCI ENCE Magazine - June 4 , 2004 - Vol 304 : p. 1421 ).

Tag words: lact ic acid bact eria, LAB , hom olact ic, het erolact ic, Lact ococcus, Lact obacillus , acidophilus, St rept ococcus t herm ophilus, Leuconost oc, lact ic acid, cheese, curds , yogurt , but t er , but t erm ilk, sour cream , cheesem aking , bact eriocins, nicin, lant ibiot ics , vaccine delivery , lact ococcus genom e, probiot ics.

The essent ial feat ure of LAB m et abolism is efficient carbohydrat e ferm ent at ion coupled t o subst rat e- level phosphorylat ion . Adenosine t riphosphat e ( ATP) generat ed is subsequent ly used for biosynt hesis. LAB as a group exhibit an enorm ous capacit y t o degrade different carbohydrat es and relat ed com pounds . Generally, t he predom inant end product is lact ic acid ( > 50% of sugar carbon ) . However, LAB adapt t o various condit ions and change t heir m et abolism accordingly . This m ay lead t o significant ly different end- product pat t erns.

Based on sugar ferm ent at ion pat t erns , t wo broad m et abolic cat egories of LAB exist : hom oferm ent at ive and het eroferm ent at ive . The first cat egory ,h om ofe r m e n t a t iveLAB, includes som e lact obacilli and m ost species of ent erococci , lact ococci , pediococci , st rept ococci, t et ragenococci , and vagococci , t hat ferm ent hexoses by t he Em bden -Meyerhof ( E- M ) pat hway. The second cat egory ,h e t e r ofe r m e n t a t iveLAB, includes leuconost ocs, som e lact obacilli, oenococci, and weissella species . The apparent difference on t he enzym e level bet ween t hese t wo cat egories is t he presence or absence of t he key cleavage enzym es of t he E- M pat hway ( fruct ose 1 , 6- diphosphat e ) and t he PK pat hway ( phosphoket olase ) .

H om ola ct ic Fe r m e n t a t ion

Under condit ions of excess glucose and lim it ed oxygen , hom olact ic LAB cat abolize one m ole of glucose in t he Em bden - Meyerhof pat hway t o yield t wo m oles of pyruvat e. I nt racellular redox balance is m aint ained t hrough t he oxidat ion of NADH, concom it ant wit h pyruvat e reduct ion t o lact ic acid. This process yields t wo m oles of ATP per glucose consum ed . Represent at ive hom olact ic LAB genera includeLact obacillus , Lact ococcus, Ent erococcus , St rept ococcusandPediococcusspecies .

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Gram Bacteria Stain

Th e pa t h w a y of h om ola ct ic a cid fe r m e n t a t ion in La ct ic Acid Ba ct e r ia

H e t e r ola ct ic Fe r m e n t a t ion

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Th e pa t h w a y of h e t e r ola ct ic a cid fe r m e n t a t ion in La ct ic Acid Ba ct e r ia

Lact ic acid bact eria have a very lim it ed capacit y t o synt hesize am ino acids using inorganic nit rogen sources. They are t herefore dependent on preform ed am ino acids being present in t he growt h m edium as a source of nit rogen . The requirem ent for am ino acids differs am ong species and st rains wit hin species . Som e st rains are prot ot rophic for m ost am ino acids, whereas ot hers m ay require 13 –15 am ino acids. Since t he quant it ies of free am ino acids present in t heir environm ent are not sufficient t o support t he growt h of bact eria t o a high cell densit y, t hey require a prot eolyt ic syst em capable of hydrolyzing pept ides and prot eins in order t o obt ain essent ial am ino acids. All dairy lact ococci used for acidificat ion of m ilk ( e . g. , in cheese m anufact ure) have prot eolyt ic act ivit y. The lact ococcal prot eolyt ic syst em consist s of enzym es out side t he cyt oplasm ic m em brane , t ransport syst em s, and int racellular pept idases . The prot eolyt ic act ivit y of LAB cont ribut es addit ionally t o t he developm ent of t he flavor , arom a and t ext ure of ferm ent ed product s . For m any variet ies of cheeses, such as Swiss and Cheddar , desirable " flavor t ones" are derived by prot eolysis.

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Feat ured Microbe

Many hum an foods are plant s or anim al product s which have been ferm ent ed by lact ic acid bact eria, since t hese bact eria possess propert ies t hat can benefit food product ion or conversion. The acidic and organolept ic propert ies of ferm ent ed foods result from t he m et abolic act ivit ies of t hese m icroorganism s. Foods such as ripened cheeses, ferm ent ed sausages , sauerkraut and pickles have not only a great ly ext ended shelf life com pared t o t he raw m at erials from which t hey are derived , but also arom a and flavor charact erist ics cont ribut ed direct ly or indirect ly by t he ferm ent ing organism s.

Ferm ent ed dairy product s have been m ade for t housands of years , but only wit hin t he last cent ury have t he m icrobiological bases of t hese ferm ent at ions been elucidat ed . Lact ic acid bact eria are t he principal organism s involved in ferm ent ing dairy product s. Prior t o t he availabilit y of st art er cult ures, m ilk ferm ent at ions relied on t he LAB nat urally present in raw m ilk . The first com m ercial st art er cult ures were unknown m ixes of m icrobes from raw m ilk t hat were prepared in Denm ark around end of t he 19t h cent ury . I n t he 1930s and 40 s, t he idea of pure single- st rain st art er cult ures evolved.

Ferm ent ed dairy product s are enj oying increased popularit y as convenient , nut rit ious, st able, nat ural , and healt hy foods . Lact ic acid bact eria are t he principal organism s involved in t he m anufact ure of cheese, yogurt, but t erm ilk, cot t age cheese, sour cream and cult ured but t er . I n som e ferm ent ed dairy product s , addit ional bact eria, referred t o as secondary m icroflora, are added t o produce carbon dioxide, which influences t he flavor and alt ers t he t ext ure of t he final product .

Sausage is one of t he oldest processed m eat product s . The writ ings of ancient Egypt ians described t he preservat ion of m eat by salt ing and sun drying. The ancient Babylonians, Greeks, and Rom ans used sausage as a food source during t im es of war . Microorganism s were recognized as being im port ant t o t he product ion of sausages about 1921. I n t he 1940s and 1950s, pure m icrobial st art er cult ures consist ing of lact ic acid bact eria becam e available but t heir use was not widespread unt il t he early 1980s.

The ferm ent at ion of veget ables, a pract ice t hat originat ed in t he Orient , has been used as a m eans of preserving food for m ore t han 2 , 000 years . I n t he t hird cent ury B. C . , during t he const ruct ion of t he Great Wall of China, t he Chinese produced ferm ent ed veget ables ( cabbages, radishes, t urnips, cucum bers, et c. ) on a large scale. The m ost com m on ferm ent ed veget ables available in t he Unit ed St at es are pickles, sauerkraut , and olives. Carrot s , cauliflower, celery, okra, onions, and sweet and hot peppers also are sold as ferm ent ed veget able product s.

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Blue

Lact obacillusis very het erogeneous genus, encom passing species wit h a large variet y of phenot ypic, biochem ical , and physiological propert ies.

Most species of lact obacilli are hom oferm ent at ive, but som e are het eroferm ent at ive . The genus has been divided int o t hree m aj or subgroups and over 70 species are recognized . Group I lact obacilli are obligat ely hom oferm ent at ive and produce lact ic acid as a m aj or end product ( > 85 % ) from glucose. They are represent ed byL . delbrueckiiandL.

acidophilus. They grow at 45oC but not at 15oC. Group I I , also hom oferm ent at ive , grow at 15oC and show variable growt h at 45oC . Represent ed byL . caseiandL. plant arum, t hey can produce m ore oxidized ferm ent at ions ( e . g . acet at e) if O2is present . Group I I I lact obacilli are het eroferm ent at ive . They produce lact ic acid from glucose, along wit h CO₂ and et hanol . Aldolase is absent and phosphoket olase is present . Represent at ive species includeL . ferm ent um , L . brevisandL. keferi.

Lact obacilli are oft en found in dairy product s, and som e species are used in t he preparat ion of ferm ent ed m ilk product s. For exam pleL . delbrueckiisubspbulgaricusare used in t he preparat ion of yogurt ;L. acidophilusis used in t he preparat ion of acidophilus m ilk ;L. helvet icus, as well asL. delbrueckiisubspbulgaricus, are used t o m ake Swiss, Mozzarella, provolone, Rom ano , and parm esan cheeses. Ot her species are used in t he product ion of sauerkraut , silage and pickles. The lact obacilli are usually m ore resist ant t o acidic condit ions t han are ot her LAB, being able t o grow at pH values as low as 4 . This enables t hem t o cont inue t o grow during nat ural lact ic ferm ent at ions when t he pH has dropped t oo low for ot her LAB t o grow , so t hey are oft en responsible for t he final st ages of m any lact ic acid ferm ent at ions.

Many sausage ferm ent at ions include aLact obacillusspecies .L . plant arumis used in st art er cult ures for t he m anufact ure of sum m er sausage , pepperoni and salam i.

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plant arumandL . brevisbeing t he m ost im port ant .

St r e pt ococci a n d La ct ococci

Not e on St rept ococcal Classificat ion. Since 1985, m em bers of t he diverse genus St rept ococcushave been reclassified int oLact ococcus, Vagococcus, Ent erococcus, and St rept ococcus, based on biochem ical charact erist ics , as well as ssRNA analysis. Hist orically, st rept ococci were segregat ed int o serological groups based on t he presence of specific carbohydrat e ant igens. Ant igenic groups, or Lancefield groups ( nam ed for Rebecca Lancefield, a pioneer in St rept ococcus t axonom y) , are designat ed by let t ers A t hrough O . Lancefield groups have proven t o correlat e well wit h t he current t axonom ic definit ions. The bet a - hem olyt ic st rept ococci found in hum ans cont ain t he group A ant igen, while " fecal st rept ococci" ( ent erococci ) cont ain t he group D ant igen. Group B st rept ococci, usually found in anim als, are a cause of m ast it is in cows, and have been im plicat ed in hum an infect ions. " Lact ic st rept ococci" ( st rept ococci and lact ococci) cont ain t he group N ant igen and are nonpat hogenic. However ,Lact ococcushas been defined as a genus separat e fromSt rept ococcus. As lact ic acid bact eria,Lact ococcus lact isandSt rept ococcus t herm ophilusare t he cornerst ones of cheese m anufact ure.

St r e pt ococcu s t h e r m oph ilu s

St rept ococcus t herm ophilusis an alpha- hem olyt ic species of t he viridans group. The bact erium is found in m ilk and m ilk product s. I t is not a probiot ic ( it does not survive t he st om ach) and generally is used in t he product ion of yogurt and t he m anufact ure of several t ypes of cheese, especially I t alian and Swiss cheeses. The organism is a m oderat e t herm ophile wit h an opt im al growt h rat e at 45 ° C. Alt houghS. t herm ophilusis closely relat ed t o ot her pat hogenic st rept ococci ( such asS . pneum oniaeandS. pyogenes) ,S. t herm ophilusis classified as a nonpat hogenic, alpha - hem olyt ic species t hat is part of t he viridan' s group. I t is closely relat ed t oS . salivariusin t he oral cavit y .

La ct ococcu s la ct is

Lact ococcusis a genus of of LAB wit h five m aj or species form erly classified as Group N st rept ococci. The t ype species for t he genus isL . lact is ,which has t wo subspecies,lact is andcrem oris. Lact ococci differ from ot her lact ic acid bact eria by t heir pH, salt and t em perat ure t olerances for growt h.

Lact ococcus lact isis crit ical for m anufact uring cheeses such as Cheddar , cot t age , cream , Cam em bert , Roquefort and Brie, as well as ot her dairy product s like cult ured but t er , but t erm ilk , sour cream and kefir . The bact erium can be used in single st rain st art er cult ures, or in m ixed st rain cult ures wit h ot her lact ic acid bact eria such asLact obacillus andSt rept ococcus.

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Class I bact eriocins or lant ibiot ics are sm all pept ides cont aining t he unusual dehydroam ino acids and t hioet her am ino acids lant hionin and 3- m et hyllant hionine, which are synt hesized by Gram - posit ive bact eria during post t ranslat ional m odificat ions. These pept ides are t hought t o at t ach t o t he m em brane of t arget cells and, by an as yet unknown conform at ional rearrangem ent, lead t o increased perm eabilit y and disrupt ion of t he m em brane pot ent ial. There are t wo t ypes of lant ibiot ics, t ypes A and B . The lant ibiot ics produced by LAB all belong t o t ype A, which are elongat ed screw- shaped pept ides, whereas t ype B lant ibiot ics are m ainly globular. Nisin produced byLact ococcus lact isssp. lact is has been st udied ext ensively . I t has a broad spect rum of act ivit y against Gram - posit ive bact eria. The prim ary t arget is believed t o be t he cell m em brane . Unlike som e ot her ant im icrobial pept ides, nisin does not need a recept or for it s int eract ion wit h t he cell m em brane ; however, t he presence of a m em brane pot ent ial is required .

St a r t e r cu lt u r e s

St a r t e r cu lt u r e s con sist in g of la ct ic a cid ba ct e r ia a r e a dde d a t t h e be gin n in g of t h e ch e e se m a k in g pr oce ss. La ct ic a cid ba ct e r ia a r e e sse n t ia l for m a n u fa ct u r e of ch e e se , yogu r t , sou r cr e a m , cu lt u r e d bu t t e r a n d m ost fe r m e n t e d m ilk pr odu ct s.

St art er cult ures play an essent ial part in t he m anufact ure of ferm ent ed dairy product s. They produce t he lact ic acid t hat coagulat es m ilk and t hey cont ribut e t o t ext ure, m oist ure cont ent , freedom from pat hogenic m icroorganism , and t ast e of t he product . The rat e of acid product ion is crit ical in t he m anufact ure of cert ain product s, e . g . Cheddar cheese. Depending on t he product , especially in m echanized cheese product ion unit s, st art ers m ay also be required t o produce acid at a consist ent ly fast rat e t hrough t he m anufact uring period each day and every day . The negat ive redox pot ent ial creat ed by st art er growt h in cheese also aids in preservat ion and t he developm ent of flavor in Cheddar and sim ilar cheeses. Addit ionally ant ibiot ic subst ances, now referred t o as bact eriocins, produced by st art ers, e. g . nisin , m ay also have a role in preservat ion.

Ecology of st art er bact eria

Most st art ers in use t o t oday have t heir origins as lact ic acid bact eria nat urally present as part of t he cont am inat ing m icroflora of m ilk . These bact eria probably cam e from veget at ion in t he case of lact ococci or t he int est inal t ract in t he case of bifidobact eria, ent erococci andLact obacillus acidophilus.

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indust ry his pract ice has been referred t o as " back - slopping" .

Classificat ion of st art er cult ures

While t he m icrobes used in t he m anufact ure of ferm ent ed dairy product s are generally lact ic acid bact eria,Propionibact erium sherm aniiandBifidobact eriumspp . which are not lact ic acid bact eria, are also used . I n addit ion, ot her bact eria includingBrevibact erium linens, responsible for t he flavor of Lim burger cheese; and m olds (Penicilliumspecies) are used in t he m anufact ure of Cam em bert , Roquefort and blue cheeses.

Pr obiot ics a n d La ct ic Acid Ba ct e r ia

Pr obiot icsare product s designed t o deliver pot ent ially beneficial bact erial cells t o t he m icrobiot ic ecosyst em of hum ans and ot her anim als. St rains of lact ic acid bact eria are t he m ost com m on m icrobes em ployed as probiot ics, especiallyLact obacillusand

Bifidobact eriumspecies , but lact ococci, som e ent erococci and som e st rept ococci are also included as probiot ics.

La ct ic a cid- pr odu cin g Ba ct e r ia Use d a s Pr obiot ics

La ct oba cillu s

La ct oba cillu sspe cie s a r e fa cu lt a t ive a n a e r obe s. Th e y gr ow in t h e pr e se n ce of O2, h ow e ve r , a n d m a y con ve r t it t o H2O or H2O2. La ct oba cilli n or m a lly

pr e dom in a t e in t h e sm a ll in t e st in e, a n d t h e y a r e k n ow n for t h e ir be n e ficia l e ffe ct s w h ich m a y a n t a gon iz e pot e n t ia l pa t h oge n s. Of t h e m or e t h a n 1 0 0 La ct oba cillu sspe cie s, t h e follow in g a r e com m on ly u se d pr obiot ics : L . a cidoph ilu s

L . fe r m e n t u m L . pa r a ca se i L . br e vis L . ga sse r i L . pla n t a r u m L . bu lga r icu s L . h e lve t icu s L . r e u t e r i L . ca se i L . j e n se n ii L . r h a m n osu s L . cr ispa t u s L . j oh n son ii L . sa liva r iu s

Bifidoba ct e r iu m

Bifidoba ct e r iu mis n ot in clu de d in t h e t r a dit ion a l La ct ic Acid Ba ct e r ia du e t o it s ge n e t ic u n r e la t e dn e ss, bu t t h e ba ct e r iu m h a s a h a bit a t t h a t ove r la ps w it h LAB , a n d it h a s a m e t a bolism t h a t pr odu ce s la ct ic a cid a s a pr im a r y e n d - pr odu ct of fe r m e n t a t ion . Bifidoba ct e r ia a r e st r ict ly a n a e r obic a n d n or m a lly vie for pr e dom in a n ce in t h e la r ge in t e st in e. Am on g 3 0 spe cie s , t h ose r e cogn iz e d a s pr obiot ics in clu de:

B. a dole sce n t is B. br e ve B. lon gu m B. a n im a lis B. in fa n t is B. t h e r m oph ilu m B. bifidu m B. la ct is

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ba ct e r iu m .

St r e pt ococcu s

St r e pt ococcu sspe cie s a r e n ot t ypica lly a ssocia t e d w it h h e a lt h be n e fit s a n d som e a r e h igh ly pa t h oge n ic. H ow e ve r , on e fa cu lt a t ive a n a e r obic spe cie s ,

St r e pt ococcu s t h e r m oph ilu s, is k n ow n t o pr om ot e h e a lt h. I t is on e of t h e t w o pr im a r y spe cie s fou n d in yogu r t cu lt u r e s, t h e ot h e r be in gL . bu lga r icu s.

En t e r ococcu s

Fou n d in a n u m be r of pr obiot ic pr odu ct s, t h e fa cu lt a t ive a n a e r obeEn t e r ococcu s fa e ciu mis in va r ia bly a com pon e n t of t h e n or m a l in t e st in a l m icr obiot a a n d is con side r e d a be n e ficia l m icr obe. H ow e ve r ,E. fa e ciu mh a s e volve d fr om a r e la t ive ly n on pa t h oge n ic com m e n sa l ba ct e r iu m t o t h e t h ir d m ost com m on ca u se of h ospit a l- a cqu ir e d in fe ct ion s a n d n ow a ccou n t s for ove r 1 0 % of e n t e r ococca l clin ica l isola t e s . Fu r t h e r m or e , it h a s de ve lope d e x t e n sive r e sist a n ce t o a n t ibiot ics, w h ich it is ca pa ble of t r a n sfe r r in g t o ot h e r ba ct e r ia .

The hum an body , prim arily t he gast roint est inal t ract , is hom e t o a large num ber of different species of bact eria, and it is likely we could not survive wit hout t heir presence. Two of t he m ost com m on bact eria t hat com prise t he int est inal m icrobiot a ( "norm al flora" ) areLact obacillus acidophilusandBifidobact erium bifidum. Hence , t hey are a m ain com ponent of probiot ics.

The indigenous bact eria of hum ans serve a wide range of funct ions , which include m anufact ure of som e B vit am ins and vit am in K , synt hesis of som e digest ive enzym es ( e . g. lact ase) , com pet it ion wit h pat hogens for colonizat ion sit es, product ion of ant ibact erial and ant ifungal subst ances t hat prot ect against harm ful organism s , product ion of chem icals t hat have been shown t o be ant i- carcinogenic , and st im ulat ion of t he developm ent and act ivit y of t he im m une syst em .

The nat ural balance of t he body ’s bact eria can be upset by several fact ors such as cert ain m edicines, ant ibiot ics and st eroids, increased acidit y in t he digest ive syst em caused by st ress, lack of sleep and poor diet , const ipat ion or diarrhea , yeast overgrowt h , fat igue, I BS and ot her int est inal condit ions .

I t has been been suggest ed , in a few cases proven , t hat one way t o com bat t hese condit ions is by supplem ent at ion of t he diet wit h probiot ic bact eria in nat ural foods or art ificial supplem ent s.

Probiot ics have been recom m ended or suggest ed for pat ient s receiving radiat ion t reat m ent , individuals who have recurrent t hrush, vaginal yeast infect ions, or urinary t ract infect ions , persons suffering from irrit able bowel syndrom e ( I BS) or ot her bowel problem s , for t ravelers abroad t o prot ect against food poisoning and during any period where ant ibiot ics m ay be t aken .

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Feat ured Microbe

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La ct ic Acid Ba ct e r ia ( pa ge 5 )

( This chapt er has 5 pages)

Th e follow in g se ct ion t h a t focu se s on

La ct ococcu s la ct is

a s t h e

pr ot ot ypica l la ct ic a cid ba ct e r iu m .

La ct ococcu s la ct is

e x h ibit s

vir t u a lly a ll of t h e for e goin g ch a r a ct e r ist ics a n d a pplica t ion s of a

la ct ic a cid ba ct e r iu m . Th is ba ct e r iu m is ch ose n be ca u se of it s

pr e e m in e n ce in ch e e se m a k in g a n d it s st a t u s a s t h e n a t u r a l

n om in e e a s t h e st a t e m icr obe of t h e da ir y st a t e of W iscon sin .

La ct ococcu s la ct is.UW D e pa r t m e n t of Ba ct e r iology st r a in LcL 3 2 5 UW . M a gn ifica t ion 2 0 0 0 0 X . Sca n n in g e le ct r on m icr ogr a ph by Jose ph A. H e in t z , Un ive r sit y of W iscon sin - M a dison .

Lact ococcus lact isis a m icrobe classified inform ally as a Lact ic Acid Bact erium because it ferm ent s m ilk sugar ( lact ose) t o lact ic acid. Lact ococci are t ypically spherical or ovoid cells, about 1 . 2 µm by 1 . 5 µm , occurring in pairs and short chains . They are Gram -posit ive, non m ot ile , and do not form spores . Lact ococci are found associat ed wit h plant m at erial , m ainly grasses , from which t hey are easily inoculat ed int o m ilk . Hence, t hey are found norm ally in m ilk and m ay be a nat ural cause of souring .Lact ococcus lact ishas t wo subspecies,lact isandcrem oris, bot h of which are essent ial in m anufact ure of m any variet ies of cheese and ot her ferm ent ed m ilk product s.

Lact ococcus lact isis relat ed t o ot her lact ic acid bact eria such asLact obacillus acidophilus in our int est inal t ract andSt rept ococcus salivariusin t he m out h . However,Lact ococcus does not norm ally colonize hum an t issues and differs from m any ot her lact ic acid bact eria in it s pH, salt, and t em perat ure t olerances for growt h, which are im port ant charact erist ics relevant t o it s use as a st art er cult ure in t he cheesem aking indust ry .

Lact ococcus lact isis vit al for m anufact uring cheeses such as Cheddar , Colby , cot t age cheese, cream cheese, Cam em bert , Roquefort and Brie, as well as ot her dairy product s like cult ured but t er , but t erm ilk , sour cream and kefir . I t m ay also be used for veget able ferm ent at ions such as cucum ber pickles and sauerkraut . The bact erium can be used in single st rain st art er cult ures, or in m ixed st rain cult ures wit h ot her lact ic acid bact eria such asLact obacillusandSt rept ococcusspecies.

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t he m ilk t hat t hen separat es t o form curds , which are used t o produce cheese and whey. Curdling t he m ilk is not t he bact erium 's only role in cheese product ion . The lact ic acid produced by t he bact erium lowers t he pH of t he product and preserves it from t he growt h by unwant ed bact eria and m olds while ot her m et abolic product s and enzym es produced byLact ococcus lact iscont ribut e t o t he m ore subt le arom as and flavors t hat dist inguish different cheeses.

Fe r m e n t e d da ir y pr odu ct s w h e r e inLa ct ococcu s la ct isis t h e pr im a r y or ga n ism in volve d in m a n u fa ct u r e.

Pr odu ct

Pr in cipa l a cid

pr odu ce r s

Se con da r y m icr oflor a

Cheese

Colby , Cheddar,

cot t age , cream

Lact ococcus lact is

ssp .

crem oris

None

Lact ococcus lact is

ssp .

lact is

Blue

Lact ococcus lact is

ssp .

crem oris

Cit rat e

+

Lact ococcus

lact is

ssp .

lact is

Penicillium roquefort i

Lact ococcus lact is

ssp .

lact is

Ferm ent ed m ilk

But t erm ilk

Lact ococcus lact is

ssp .

crem oris

Leuconost oc

spp .

Cit rat e

+

Lact ococcus

lact is

ssp .

lact is

Lact ococcus lact is

ssp .

lact is

Sour cream

Lact ococcus lact is

ssp .

crem oris

None

Lact ococcus lact is

ssp .

lact is

La ct ococcu s la ct is. M a gn ifica t ion 1 5 0 0 X. Ph a se m icr ogr a ph cou r t e sy of T . D . Br ock , Un ive r sit y of W iscon sin - M a dison.

Ch e e se

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W iscon sin ' s u n iqu e ch e e se cu r ds , Colby , a n d doz e n s of va r ie t ie s of Ch e dda r a r e m a de e x clu sive ly w it h st r a in s ofLa ct ococcu s la ct is. I m a ge s cou r t e sy of W iscon sin Ch e e se M a r t , M ilw a u k e e W iscon sin .

Cu lt u r e d Bu t t e r, Bu t t e r m ilk a n d Sou r Cr e a m

Sour cream is m ade from cream t o which a st art er cult ure ofLact ococcus lact ishas been added t o coagulat e t he cream and t o enhance it s flavor . But t erm ilk is also m ade wit h Lact ococcus lact isin order t o acidify , preserve and flavor t he m ilk . Diacet yl, m ade from cit rat e byLact ococcus, gives but t erm ilk it s dist inct t ast e and enhances it s st orage propert ies .Lact ococcus lact isor m ixed cult ures t hat cont ainLact ococcus lact is, plus a Leuconost ocspecies are used. I n t he m aking of cult ured but t er , fat ( cream ) is separat ed from skim m ilk by cent rifugat ion of m ilk . The cream is past eurized and inoculat ed wit h select ed st art er cult ures. The ripened cream is t hen churned . The cream separat es again int o cream but t er and it s byproduct , sour but t erm ilk.

N isin

Nisin is an ant ibiot ic - like subst ance , called a bact eriocin , produced by t he " food grade" st art er st rain ,Lact ococcus lact isssp .lact is .I t is a nat ural ant im icrobial agent wit h act ivit y against a wide variet y of Gram - posit ive bact eria, including food - borne pat hogens such asList eria , St aphylococcusandClost ridium. The prim ary t arget of nisin is believed t o be t he cell m em brane . Unlike som e ot her ant im icrobial pept ides , nisin does not need a recept or for it s int eract ion wit h t he cell m em brane ; however, t he presence of a m em brane pot ent ial is required . Nisin is a nat ural preservat ive present in cheese m ade wit hLact ococcus lact isssp.lact is, but it is also used as a preservat ive in heat processed and low pH foods . Since nisin cannot be synt hesized chem ically, t he nisin - producing Lact ococcus lact isst rains are used for it s indust rial synt hesis .

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La ct ococcu s la ct isa n d t h e m ole cu le, N isin . M odifie d Sca n n in g EM fr om pr ofoodin t e r n a t ion a l . com w it h pe r m ission .

St a r t e r Cu lt u r e s

St art er cult ures have crucial roles t o play during all phases of t he cheese m aking and m at urat ion process. As t he cult ure grows in t he m ilk , it convert s lact ose t o lact ic acid. This ensures t he correct pH for coagulat ion and influences t he final m oist ure cont ent of t he product . The rat e of acid product ion is crit ical in t he m anufact ure of cert ain product s, e . g. Cheddar cheese. I n m echanized operat ions, st art ers are oft en required t o produce acid at a consist ent ly fast rat e t hrough t he m anufact uring period each and every day . During ripening , cult ure, lipolyt ic and prot eolyt ic enzym es are released from t he bact eria t hat add a balanced arom a, t ast e, t ext ure, and surface appearance t o t he product . The negat ive redox pot ent ial creat ed by st art er growt h in cheese also aids in preservat ion and t he developm ent of flavor in Cheddar and sim ilar cheeses. Addit ionally , ant ibiot ic - like subst ances produced by st art ers ( e. g . nisin ) m ay also have a role in preservat ion.

A com m e r cia lly - a va ila ble st a r t e r cu lt u r e . Th e de scr ipt ion r e a ds " D ir e ct se t M e soph ilic Cu lt u r e - La ct ococcu s la ct is a n d La ct ococcu s cr e m or is . For h a r d a n d fr e sh ch e e se s - Ch e dda r, Colby , Fe t a, Ch e vr e a n d ot h e r s. Use 1 / 4 t sp. pe r 2 - 3 ga llon s of m ilk for h a r d ch e e se s a n d 1 / 4 t sp . pe r 3 - 5 ga llon s of m ilk for soft ch e e se s. Th is pa ck a ge con t a in s e n ou gh D ir e ct se t M e soph ilic cu lt u r e t o se t 1 6 ga llon s of m ilk. St or e r e fr ige r a t e d ( 4 0 - 4 5 D e g F) . Th is cu lt u r e doe s n ot con t a in r e n n e t . "h t t p :/ / w w w . t h e gr a pe. n e t

La ct ococcu sa n d V a ccin e D e live r y

A recent ly discovered applicat ion ofLact ococcus lact isis in t he developm ent of vaccine delivery syst em s . The bact erium can be genet ically engineered t o produce prot eins from pat hogenic species on t heir cell surfaces . I nt ra nasal inoculat ion of an anim al wit h t he m odified st rain will elicit an im m une response t o t he cloned prot ein and provide im m unit y t o t he pat hogen . For exam ple, if one wished t o provide im m unit y t oSt rept ococcus pyogenes, t he causat ive agent of st rep t hroat ,Lact ococcuscould be engineered t o present t he conserved port ion of t he st rept ococcal M prot ein required for st rept ococcal adherence and colonizat ion t o t he nasopharyngeal m ucosa . The result ing local im m une response could prot ect t he individual from st rep t hroat caused by t he st rept ococcus t hat exhibit s t hat form of t he M prot ein . This approach t heoret ically can be adapt ed t o any pat hogen t hat colonizes and or/ ent ers via a m ucosal surface in hum ans or anim als. This includes hum an pat hogens such asSt rept ococcus pyogenes , St rept ococcus pneum oniae , Haem ophilus influenzae , Mycobact erium t uberculosis , Bordet ella pert ussisandNeisseria m eningit idis, am ong ot hers .

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responses t hat defeat of t he pat hogens at t he sit e of infect ion .

Lact ococcus lact ishas been shown t o deliver ant igens t hat st im ulat e m ucosal im m unit y t o nonrespirat ory pat hogens , as well, including HI V, Hum an papillom a virus and t he m alarial parasit e.

Som e of t he research papers t hat have em ployedLact ococcus lact isas a vect or for vaccine delivery are cit ed below.

Lee, M. H . , et al . 2001. Expression ofHelicobact er pyloriurease subunit B gene in Lact ococcus lact isMG 1363 and it s use as a vaccine delivery syst em against H . pylori infect ion in m ice. Vaccine 2001. 19 : 3927- 3931.

Ribeiro, L . A . , et al . 2002. Product ion and Target ing of t he Brucella abort us Ant igen L7 / L 12 inLact ococcus lact is: a First St ep t owards Food- Grade Live Vaccines against Brucellosis. Applied and Environm ent al Microbiology 2002. 68 : 910- 916.

Xin , K. Q. , et al. 2003 I m m unogenicit y and prot ect ive efficacy of orally adm inist ered recom binant Lact ococcus lact isexpressing surface - bound HI V Env . Blood 2003. 10: 223 - 228.

Robinson , K. , et al. 2004. Mucosal and cellular im m une responses elicit ed by recom binant st rains ofLact ococcus lact isexpressing t et anus t oxin fragm ent C. I nfect ion and I m m unit y 2004. 72 : 2753– 2756.

Berm udez - Hum aran, L. G . , et al. 2005. A Novel Mucosal Vaccine Based on Live Lact ococci Expressing E7 Ant igen and I L- 12 I nduces Syst em ic and Mucosal I m m une Responses and Prot ect s Mice against Hum an Papillom avirus Type 16- I nduced Tum ors . The Journal of im m unology 2005. 175: 7297- 7302.

Buccat o, S . , et al. 2006. Use ofLact ococcus lact isExpressing Pili from Group B St rept ococcus as a Broad - Coverage Vaccine against St rept ococcal Disease. The Journal of I nfect ious Diseases 2006. 194: 331- 340 .

Ram asam ay , R. et al. 2006. I m m unogenicit y of a m alaria parasit e ant igen displayed by Lact ococcus lact isin oral im m unisat ions. Vaccine 2006. 24 : 3900- 3908.

Hanniffy , S. B. , et al. 2007. Mucosal Delivery of a Pneum ococcal Vaccine Using Lact ococcus lact isAffords Prot ect ion against Respirat ory I nfect ion . The Journal of I nfect ious Diseases 2007. 195: 185– 93.

Th eLa ct ococcu sGe n om e

Part ly due t o t heir indust rial relevance, bot hLact ococcus lact issubspecies (lact isand crem oris) are widely used as generic LAB m odels for research .L . lact isssp.crem oris, used in t he product ion of hard cheeses, is represent ed by t he laborat ory st rains LM0230 and MG 1363. Sim ilarly ,L . lact isssp.lact isis em ployed in soft cheese ferm ent at ions, wit h t he workhorse st rain I L1403, ubiquit ous in LAB research laborat ories. I n 2001, t he genom e of st rain I L1403 was sequenced leading t o increased underst anding of LAB genom ics and relat ed applicat ions. Current ly, t here are t woL. lact isssp .crem orist hat have been sequenced for public release.

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Ge n om e a t la s of t h e ch r om osom e ofL . la ct isM G 1 3 6 3 .ifr . a c. u k

The sequence also led t o t he ident ificat ion of 29 genes t hat are required t o build t he m esh - like pept idoglycan com ponent of t he bact erium 's cell wall. I nducing som e of t hese enzym es can accelerat e t he slow , expensive process of cheese ripening , during which t he cheese ages and develops it s charact erist ic flavor . Learning how t o select ively act ivat e som e of t hese enzym es could revolut ionize t he cheese m anufact uring process.

TheLact ococcus lact isssp .lact isgenom e has 2 , 365, 589 unit s ( bp) of DNA, w hich cont ain 2 , 310 predict ed genes. About 64 percent of t he genes have assigned roles in t he cell , while 20 percent m at ch ot her hypot het ical genes wit h unknown funct ion . Alm ost 16 percent of t he genes bear no resem blance t o genes from ot her species and are considered t o be unique t o t his bact erium .

TheL . lact isgenom e cont ains six prophages ( carrying nearly 300 genes or about 14 percent of t he t ot al coding capacit y) and 43 insert ion elem ent s. Sequence dat a also revealed new possibilit ies for ferm ent at ion pat hways and confirm ed t he t ot al lack of genes and enzym es involved in t he TCA cycle alt hough, unexpect edly , cert ain genes necessary for aerobic respirat ion were found encoded in t he genom e.

I t is ant icipat ed t hat underst anding t he physiology and genet ic m ake - up of t his bact erium will prove invaluable for food m anufact urers as well as t he pharm aceut ical indust ry , which is exploring t he capacit y ofL. lact ist o serve as a vehicle for delivering drugs and vaccines.

For exam ple, genom ic analysis ofLact ococcus lact issubsplact isrevealed t he presence of several genes t hat encode enzym es involved in t he reduct ion of pyruvat e t o various end-product ot her t han hom olact ic acid, including et hanol, acet ic acid, form ic acid, diacet yl, acet oin and but anediol . Manipulat ion and underst anding of t he regulat ion of t hese genes could defeat or enhance t he synt hesis of t hese end product s leading t o st rain im provem ent in st art er cult ures for cheeses, but t erm ilk , and ot her dairy product s.

Th e pyr u va t e m e t a bolism ofLa ct ococcu s la ct is.LD H: la ct a t e de h ydr oge n a se ; PD H: pyr u va t e de h ydr oge n a se com ple x ;PFL: pyr u va t e for m a t e - lya se;AD H E: a ce t a lde h yde de h ydr oge n a se ;AD H A: a lcoh ol de h ydr oge n a se ;PTA:

ph osph ot r a n sa ce t yla se ;ACKA: a ce t a t e k in a se,ALS/ I LV B: ca t a bolic a n d a n a bolic 2 - a ce t ola ct a t e syn t h a se;ALD B: a ce t ola ct a t e de ca r box yla se ;BUTA: dia ce t yl r e du ct a se;BUTB: a ce t oin r e du ct a se;N OX: N AD H ox ida se . Olive ir a e t a l. BM C M icr obiology . 5 : 3 9 ( 2 0 0 5 ) .

END OF CHAPTER

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La ct ococcu s la ct is

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La ct ococcu s la ct is

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n om in e e a s t h e st a t e m icr obe of t h e da ir y st a t e of W iscon sin .

Pr odu ct

Pr in cipa l a cid

pr odu ce r s

Se con da r y m icr oflor a

Cheese

Colby , Cheddar,

cot t age , cream

Lact ococcus lact is

ssp .

crem oris

None

Lact ococcus lact is

ssp .

lact is

Blue

Lact ococcus lact is

ssp .

crem oris

Cit rat e

Lact ococcus

lact is

ssp .

lact is

Penicillium roquefort i

Lact ococcus lact is

ssp .

lact is

Ferm ent ed m ilk

But t erm ilk

Lact ococcus lact is

ssp .

crem oris

Leuconost oc

spp .

Cit rat e

Lact ococcus

lact is

ssp .

lact is

Lact ococcus lact is

ssp .

lact is

Sour cream

Lact ococcus lact is

ssp .

crem oris

None

Lact ococcus lact is

ssp .

lact is