Metabolism and Biochemical Characteristics of Yogurt Bacteria. a Review a Zourari, Jp Accolas, Mj Desmazeaud

Metabolism and biochemical characteristics of yogurt bacteria. A review A Zourari, Jp Accolas, Mj Desmazeaud

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A Zourari, Jp Accolas, Mj Desmazeaud. Metabolism and biochemical characteristics of yogurt bacteria. A review. Le Lait, INRA Editions, 1992, 72 (1), pp.1-34. hal-00929275

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Metabolism and biochemical characteristics of yogurt bacteria. A review

A Zourari *, JP Accolas, MJ Desmazeaud

Station de Recherches Laitières, INRA, 78352 Jouy-en-Josas Cedex, France

(Received 5 July 1991; accepted 1st October 1991)

Summary - This review reports recent data regarding the metabolism and biochemistry of Streptococcus salivarius subsp thermophilus and Lactobacillus delbrueckii subsp bulgaricus related to yogurt manufacture. The taxonomy of these bacteria is presented. Different proposed pathways for carbohydrate metabolism are then discussed, as weil as recent molecular and genetic studies of the enzymes involved. Acetaldehyde is the major aromatic compound in yogurt, and so the different pathways of its formation are briefly described. Recent studies have concerned threonine aldolase which catalyzes acetaldehyde synthesis by yogurt bacteria. Exocellular polysaccharides produced by lactic acid bacteria improve the texture of stirred and liquid yogurts. Some of the polysaccharides of yogurt bacteria are currently known and particular aspects of their production are discussed. Some other properties, le proteolysis, Iipolysis, urease, oxygen metabolism, are also briefly presented. Interactions between streptococci and lactobacilli are weil established, but more data are required for the complete characterization and control of mixed populations. In particular, Iittle is known about antimicrobial compounds produced by these microorganisms. Bacteriophages of yogurt bacteria are now weIl characterized, but liltle is known about Iysogeny in thermophilic streptococci. Finally, progress in genetics (on both plasmid and chromosomal DNA) is briefly discussed.

Streptoeoeeus sal/varlus subsp thermophllus 1 Laetobaclllus delbrueekli subsp bulgarleus 1 yogurt 1 metabollsm 1 Interaction

Résumé - Métabolisme et caractéristiques biochimiques des bactéries du yaourt. Une revue. Dans le présent article, la position taxonomique de Streptococcus salivarius subsp thermophilus et de Lactobacillus delbrueckii subsp bulgaricus est d'abord décrite. Les différentes voies du métabolisme des glucides sont discutées ainsi que les études récentes des enzymes impliquées, sur le plan moléculaire et génétique. L'acétaldehyde est le composé caractéristique de l'arôme typi- que du yaourt. Les différentes voies métaboliques de sa synthèse sont brièvement décrites, en particulier celle qui met en jeu la thréonine aldolase. Les bactéries lactiques produisent des polysaccharides exocellulaires qui influencent la texture des yaourts brassés et des yaourts liquides. Des polysaccharides produits par les bactéries du yaourt sont maintenant connus et quelques aspects in- téressants de leur production sont actuellement étudiés. D'autres activités (protéolyse, lipolyse, uréese, métabolisme de l'oxygène) sont aussi brièvement présentées, suite à plusieurs études ré- centes. Les interactions entre streptocoques et lactobacilles sont bien établies, mais davantage de données sont nécessaires pour la caractérisation complète et la msttnse des populations mixtes. En particulier, les connaissances des substances antimicrobiennes produites par les bactéries du

• Presentaddress: Boil-GroupeChr Hansen, Le Moulin d'Aulnay, BP 64, 91292 Arpajon Cedex, France 2 A Zourari et al yaourt sont encore limitées. Les bactériophages de ces bactéries sont maintenant relativement bien caractérisés mais les données sur la Iysogénie du streptocoque demeurent encore incomplètes. Enfin, le progrès des études génétiques (ADN plasmidique et chromosomique) est brièvement discuté.

Streptococcus sallvarlus subsp thermophllus / Lactobaclllus delbrueckll subsp bulgarlcus / yaourt / métabolisme / interaction

CONTENTS

Introduction 2

Taxonomy 2 S salivarius subsp thermophilus 2 L delbrueckii subsp bulgaricus 3

Important metabolic activities for yogurt technology 4 Acid production - Carbohydrate metabolism 4 Carbohydrate metabolism of S thermophilus 5 - Lactose and galactose transport 5 - Metabolic pathways of lactose, glucose and galactose 6 - Sucrose utilization 6 Carbohydrate metabolism of L bulgaricus 8 Lactic acid isomers in yogurt 8

Formation of f1avour compounds 9 Flavour compounds of yogurt 9 Metabolic pathways of acetaldehyde synthesis 9 - Formation of acetaldehyde from glucose 9 - Formation of acetaldehyde from threonine 10 - Formation of acetaldehyde from DNA components 10 - Alcohol dehydrogenase 11 Production of polysaccharides 11

Other metabolic activities 12 Proteolytic activity 12 Amino acids in milk and yogurt 12 Utilization of proteins 12 Utilization of peptides 13 Lipolytic activity 13 Vrease activity 14 Oxygen and metabolism 14 Metabolism and biochemistry of yogurt bacteria 3

Particular aspects of yogurt fermentation 15

Interactions between yogurt bacteria 15 Production of antimicrobial compounds by yogurt bacteria 16 Bacteriophages of L bulgaricus and S thermophilus 17 Genetics of S thermophilus and L bulgaricus 21

Conclusion 23

References 24

INTRODUCTION cfu.g-1 of yogurt). It is noteworthy that these 2 conditions are seldom specified by Yogurt and similar fermented milk products existing legislation in most other yogurt- have been very popular for a long time in producing countries (Anonymous, 1989). Mediterranean countries (the Balkans, We review here recent data on some of North Africa), in central and southwest the metabolic and biochemical aspects' of Asia (Mongolia, Turkey, Iraq, Iran, Syria) these starter bacteria in relation to yogurt and in central Europe. In many of these manufacture. Tamime and Deeth (1980) countries, yogurt is still manufactured us- have published an excellent review con- ing traditional procedures. Since the last cerning ail the technological and biochemi- world war, yogurt consumption has been cal aspects of yogurt, followed by a second steadily increasing not only in European article dealing with its nutritional and thera- countries, but also in the United States, en- peutic properties (Deeth and Tamime, hancing its industrial-scale production. At 1981). These reviews include a detailed present, new types of fermented milk are presentation of S salivarius subsp thermo- available, prepared by adding fruits or fla- phi/us and L delbrueckii subsp bulgaricus. vouring, enriched with vitamins or contain- Since 1981, both microorganisms have ing selected intestinal bacteria such as been extensively studied and reviews deal- Lactobacillus acidophilus and several ing with particular aspects of their metabo- Bifidobacterium species (Kurmann, 1984; lism and genetics have been published Puhan, 1988). (Hutkins. and Morris, 1987; Mercenier and ln France, the term "yogurt" can be Lemoine, 1989; De Vos, 1990; Mercenier, used legally only to designate the product 1990). The present review is an update in resulting from milk fermentation brought this field and will give the technological about exclusively with 2 thermophilic lactic and biochemical context of yogurt manu- acid bacteria, Streptococcus ssilvsrius facture. Emphasis is put on bacteria and subsp thermophi/us and Lactobacillus del- not on yogurt and only limited information brueckii subsp bulgaricus, which must be concerning yogurt manufacture will be re- found alive in the final product (== 10 million ported. 4 A Zourariet al

TAXONOMY Of S SALIVARIUS SUBSP 1978). Furthermore, the therrnoresistance THERMOPHILUS AND L DELBRUECKII of their fructose-diphosphate aldolases is SUBSP BULGARICUS different, as is their level of homology with Enteroccoeus faeealis aldolase assessed S salivarius subsp thermophllus by immunodiffusion assays, in spite of very close migration distances (London and S salivarius subsp thermophilus is the new Kline, 1973). name proposed to designate Streptoeoe- ln addition, Garvie and Farrow (1981) eus thermophilus which was originally de- had previously placed these streptococcal scribed by Orla-Jensen (1919) and which strains in the same group, based on ho- stands apart from the other streptococci mology between their DNA and ribosomal and especially lactic streptococci, at RNA of the type-strain of Streptoeoccus present designated as lactococci. It is ex- bovis (this strain now belongs to the Strep- clusively isolated from the dairy environ- toeoccus equinus species; Schleifer and ment, ferments only few carbohydrates, ie Kilpper-Bâlz, 1987), but in different c1us- lactose, sucrose, glucose and sometimes ters. Besides, DNA-DNA molecular hybrid- galactose, and is characterized by its ther- ization between several strains of S ther- moresistance and a rather high growth mophi/us and S salivarius have only temperature which may reach 50-52 "C. yielded a 60% homology in optimum condi- No group-specific antigen has been found tions and 30% under stringent hybridiza- (Hardie, 1986). tion conditions (Schleifer and Klllper-Bâlz, DNA-DNA homology studies might sup- 1987). Based on recent hybridization data, ply more information on this bacterium and phenotypic differences and occurrence in may question its classification. The guano- completely different biotopes (mouth/milk), sine plus cytosine content (mol G + C%) of Schleifer and Kilpper-Bâlz (1987) suggest- DNA ranges from 37.2-40.3% according ed maintaining S thermophi/us and S sali- to Farrow and Collins (1984). These au- varius as 2 separate species. It is obvious thors obtained 61-78% homology between that the taxonomy of S thermophi/us re- DNA of several S thermophi/us strains and quires further discussion and that more a DNA probe of Streptoeoccus salivarius data are required before reaching a defini- (the homology between DNA of S salivari- tive decision. us strains and a DNA probe of S thermo- Colmin et al (1991) developed a DNA phi/us ranged from 67-91%). They thus probe which specifically hybridized to 25 confirmed the high DNA homology (70- strains of subspecies thermophi/us and to 100%) observed in an earlier study by 2 strains of subspecies salivarius of S sali- Kilpper-Bëlz et al (1982) for 2 strains of varius. This cross-hybridization suggests each species. On the basis of these re- that both species share common DNA se- sults, Farrow and Collins (1984) proposed quences, but more data are needed to that S thermophi/us should be reclassified support a possible close relationship be- as a subspecies of S salivarius despite the tween these 2 streptococci. In addition, the large phenotypic differences between probe easily detects RFLP (restriction frag- these 2 bacteria. In fact, lactate dehydro- ment length polymorphism) in S thermophi- genases of the type-strains of both strepto- lus and accordingly can be used for strain cocci have different properties (Garvie, characterization. Metabolism and biochemistry of yogurt bacteria 5

L delbrueckii subsp bulgaricus IMPORTANT METABOLIC ACTIVITIES FOR YOGURT TECHNOLOGY This lactobacillus was first described by Orla-Jensen (1919) and named Thermo- The role of streptococci and lactobacilli in baeterium bulgarieum. It is presently con- yogurt manufacture can be summarized as sidered as one of the subspecies of Lae- follows: milk acidification, synthesis of aro- tobaeillus delbrueekii. Two other sub- matic compounds, development of texture species, subsp delbrueekii and subsp lae- and viscosity. The latter aspect is required tis, also belong to this group. The old ter- mainly for stirred and liquid yogurts. Thus, minology Laetobaeillus leiehmannii is no for industrial yogurt manufacture, starter longer in use. selection takes into account these 3 properties. Evaluation of acidification properties L delbrueekii subsp bulgarieus is homo- is difficult because of the high buffering ca- fermentative, ferments few carbohydrates, pacity of milk and the lack of a standard ie glucose, lactose, fructose, and some- procedure. Efforts have been made to de- times galactose or mannose, and has a velop new methods for an objective evalu- high growth temperature (up to 48 or 50 ation of strains in replacement of former OC).Its DNA mol G + C% ranges from 49 procedures (eg that of Accolas et al, 1977) to 51% (Kandler and Weiss, 1986). based on the measurement of titratable Simonds et al (1971) obtained a DNA acidity in milk. For example, the method homology of 86% between the former patented by Corrieu et al (1989), and de- L bulgarieus and L laetis species, but only scribed by Spinnler and Corrieu (1989), 4.8% with Laetobaeillus helvetieus and no uses continuous automatic recording of pH homology with L helvetieus var jugurti. This during bacterial growth in milk and the was confirmed by Dellaglio et al (1973) strains are compared on the basis of maxi- who obtained < 7% DNA homology be- mum acidification rates and the corre- tween L bulgarieus and L helvetieus or sponding time and pH. In the field of indus- L helvetieus var jugurti. The study of Weiss trial preparation of mixed starter cultures, et al (1983) showed 80 to 100% homology efforts have been made to control the (90-100% for type-strains) between the growth of streptococcus and lactobacillus former L bulgarieus, L leiehmannii, L laetis populations by culture conditions, pH and and L delbrueekii species. DNA homology temperature (Béai et al, 1989). Chamba led to the reclassification of these 4 lactob- and Prost (1989) evaluated the acidifying acilli as subspecies of L delbrueekii in the properties of strains used for cheesemak- most recent classification of Bergey's Man- ing, based on pH measurements during ual (Kandler and Weiss, 1986). growth following the temperature profile of A specifc DNA probe for L delbrueekii a standard cheesemaking operation. has been developed recently, which The evaluation of aroma formation is makes it possible to differentiate this spe- generally based on the production of ace- cies from other lactobacilli, lactococci and taldehyde, a major aromatic compound of propionibacteria (Delley et al, 1990). yogurt, whereas the thickening character is ln the present article, we shall use the based on measurements of milk viscosity former names of the yogurt organisms, ie (Bouillanne and Desmazeaud, 1980, 1981; S thermophilus and L bulgarieus for pur- Zourari et al, 1991; Zourari and Des- poses of convenience. mazeaud,1991). 6 A Zourariet al

ln the first part of this review, we rase system (PTS). Lactose-6-phosphate present the metabolic activities responsi- formed during transport is hydrolyzed by ble for the acidification, f1avouring and an intracellular phospho-B-qalactosidase thickening properties of yogurt bacteria. into glucose and galactose-6-phosphate We then describe some metabolic activi- which are then metabolized into lactic acid ties of lesser importance for yogurt manu- (Thomspon, 1987). S thermophilus does facture, but which determine the growth of not possess lactose PTS or phospho-û- bacteria in milk and their interactions, ie galactosidase (Tinson et al, 1982a; Thom- proteolysis, lipolysis, urease activity, oxy- as and Crow, 1984), but has a lactose per- gen effect. mease system including a proton- dependent membrane-Iocated permease and an intracellular ~-galactosidase (Pool- Acid production. man et al, 1989). Carbohydrate metabolism The lactose permease gene of S thermophilus (lacS) was cloned and expressed The main role of S thermophilus and L bul- in Escherichia coli (Poolman et al, 1989). garicus in yogurt manufacture is to acidify The comparison of amino acid sequences milk by producing a large amount of lactic showed that the streptococcal lactose acid from lactose. Lactic acid reduces the transport protein (LacS) differs from the pH of the milk and leads to a progressive lactose permease of E coli (LacY) and ap- solubilization of micellar calcium phos- pears to be a hybrid protein of 69 kDa. The phate. This causes the demineralization of NH -terminal region is homologous with casein micelles and their destabilization, 2 the melibiose carrier (MeIB) of E coli (23% which generates the complete precipitation overail similarity) whereas the COOH- of casein in a pH range of 4.6-4.7 (Fox, terminal end is partially homologous (34- 1989). In addition, lactic acid provides yog- 41%) with enzyme III (E III) of 3 different urt with its sharp, acid taste and contrib- PEP-PTS systems. utes to flavour. Galactose seems to be directly involved Lactic acid production may also occur in lactose transport. In fact, the presence during yogurt storage at low temperature. of a large amount of galactose in the lt may lead to an excessive acidification growth medium inhibits lactose transport which affects the organoleptic properties (Somkuti and Steinberg, 1979). Biochemi- of the product. This activity depends on cal studies have shown that galactose has the strains used and especially on the lac- a considerable affinity _for lactose per- tobacilli (Accolas et al, 1977; Bouillanne mease and acts as a competitive inhibitor and Desmazeaud, 1980, 1981). of the lactose transport system (Hutkins and Morris, 1987), but more recent data Carbohydrate metabolism supply another explanation. In membrane of S thermophilus vesicles of E coli, lacS permease not only catalyzes a proton motive force-Iinked Lactose and galactose transport transport system, but also exchange of ~- The mechanism of lactose transport in galactosides. This means that the lactose S thermophilus differs from that of lacto- transport reaction proceeds as a lactose- cocci, which possess a specific system for galactose antiport independent of the pro- lactose transport, the phosphoenolpyru- ton motive force (Poolman et al, 1989; Hut- vate (PEP)-dependent phosphotransfe- kins and Ponne, 1991). Metabolism and biochemistry of yogurt bacteria 7

Galactose transport requires an exoqe- The galactose moiety is released into nous energy source and involves the ac- the extracellular medium by most S ther- tion of a galactose permease. A PEP-PTS- mophilus strains which are not able to use like system has not been detected in it, ie Gal- phenotype (Hutkins and Morris, S thermophilus. Glucose and galactose 1987). Galt strains have been isolated transport is slower than that of lactose (Somkuti and Steinberg, 1979; Thomas (Hutkins et al, 1985a). The preferential and Crow, 1984), however, in which galac- utilization of lactose could be due to tose is metabolized via the Leloir pathway limitations in monosaccharide transport involving the enzymes galactokinase, (Somkuti and Steinberg, 1979). In light of galactose-1-phosphate-uridyl transferase the involvement of galactose in lactose and uridine-5-diphospho-glucose-4-epime- transport, however, these data should be rase. The 2 latter enzymes are constitutive re-evaluated. and present in Gal- as weil as in Gal! strains (Thomas and Crown, 1984). The Metabolic pathways of lactose, difference between the 2 phenotypes glucose and galactose utilization seems to be due to a difference in galac- Figure 1 summarizes the proposed path- tokinase and/or galactose permease activi- ways for lactose and galactose transport ty. Both enzymes are induced by galactose and their utilization by S thermophilus. In- and repressed by lactose (Hutkins et al, tracellular ~-galactosidase (~-gal) hydrolyz- 1985a, b). Nucleotide sequences of the es lactose to glucose and galactose. Glu- genes encoding the 2 constitutive en- cose represses synthesis of this enzyme zymes are homologous with the analogous (Tinson et al, 1982a). Streptococcal ~-gal genes of other bacteria and yeasts. Their has bsen purified and characterized (Ra- expression and regulation have been stud- mana Rao and Dutta, 1981; Greenberg ied (Poolman et al, 1990). Until the present and Mahoney, 1982; Smart et al, 1985). time, the use of galactose via the tagatose The ~-gal-encoding gene has been cloned pathway has not been reported for either and expressed in E coli (Herman and S thermophilus or L bulgaricus. McKay, 1986) and in Saccharomyces ce- It is possible to obtain Gal+ derivatives revisiae (Lee et al, 1990). Streptococcal ~- of S thermophilus strains after growth of gal is a protein of about 105 kDa and the Gal- cultures in a chemostat under lactose ~-gal-encoding gene is located in a 3.85-kb limitation (Thomas and Crow, 1984). The region of the chromosomal DNA (Herman use of Gal+ strains in cheesemaking de- and McKay, 1986). This enzyme is useful creases residual galactose in Cheddar, as a potential selective marker in food- Mozzarella, and Emmental cheeses and grade cloninq vectors, and also for its pos- thus avoids sorne serious technological sible industrial production because of its problems. As mentioned by Hutkins et al higher thermostability compared tocurrent- (1986), high residual galactose in cheese Iy produced yeast enzymes. curd: (i) may enable a heterofermentative The glucose moiety of lactose is directly metabolism of galactose during ripening, used via the Embden-Meyerhof-Parnas with concomitant carbon dioxide produc- pathway (fig 1). S thermophilus possesses tion, and may also lead to the development 2 fructose-1,6-diphosphate-independent of a harmful flora generating off-flavours (FDP-independent) lactate dehydrogenas- and texture defects and (ii) has been impli- es (LDH) which reduce pyruvate to lactic cated in browning reactions which occur acid (Garvie, 1978). during processing or cooking at high tem- 8 A Zourari et al

Lactose Galactose Lactose Galactose •1

ADP ATP

2H:.------H+

Galactase-I-P UDP-gIUCaSe A TP Gal-I-P uridyl trans1erasee Hexokinase ~ Glucose-I-phosphote ADP UDP-goloctose ~UDP-glucose -4-eplmeras e

GIUCOse-6-p_G1UCOSe-l-p"7"\ UDP-glucose

~ UTP PPi . Fructose-6-P ATP~ 1 LELOIR PATHWAY 1 ~P~ . Fructose-I.6-DiP ~Aldolase

Oihydroxyacetone C p Glycerqldehyde-3-P 1 1 PhosPholno,pyruvate ADP ----)1 A TP.....- {yruvate kinase

Pyruvate

NADH + H+ ------JLactate dehydrogenase NAD+~

1 LAGTIG AGIO 1

1 EMBDEN-MEYERHOF-PARNAS PATHWAY 1

Fig 1. Proposed pathways for lactose (lac) and galactose (gal) utilization in S thermophilus. From Hut- kins and Morris (1987), Thompson (1987) and De Vos (1990). P: phosphate. PPi: inorganic pyrophos- phate, ATP: adenosine triphosphate, ADP: adenosine diphosphate, UTP: uridine triphosphate, UDP: uridine diphosphate. Voies métaboliques proposées pour l'utilisation du lactose (lac) et du galactose (gal) par S thermophilus. D'après Hutkins et Morris (1987), Thompson (1987) et De Vos (1990). P: phosphate, PPi : pyro- phosphate inorganique, A TP : adénosine triphosphate, ADP : adénosine diphosphate, UTP : uridine triphosphate, UDP : uridine diphosphate. Metabolism and biochemistry of yogurt bacteria 9 peratures of cheeses such as Cheddar or zation are weil conserved in S thermophi- Mozzarella. lus and L bulgaricus. Their lactose per- meases and 13-galactosidasesshare 60 Sucrose utilization and 48% cornmon amino acids, respective- Iy (Mercenier, 1990). Sucrose metabolism has been poorly documented, especially at the molecular and Once inside the cell, lactose is hydro- genetic levels. During the growth of S ther- Iyzed by a 13-gal.Glucose represses lac- mophilus on sucrose, both glucose and tose transport and 13-galsynthesis. Purified fructose are used simultaneously. Fructose 13-galof an L bulgaricus strain has a maxi- accumulates in the growth medium, how- mum activity at 50 "C and pH 7 (Itoh et al, ever, even when the strain can use it 1980). The 13-gal-encodinggene has been (Thomas and Craw, 1983). The higher rate cloned in E coli and expressed using its of sucrose utilization compared to those of own promoter. The molecular mass of the glucose and fructose might be due to a dif- enzyme is about 114 kDa, deduced from ferent uptake rate (Hutkins and Morris, the nucleotide sequence of the corre- 1987). sponding gene (Schmidt et al, 1989). Ga- lactose is metabolized by L bulgaricus via An inhibitory effect of high sucrose con- the Leloir pathway. A galactokinase activity tent in milk (10-12%), on the growth of has been detected in Gal" strains (Hickey yogurt bacteria has often been reported. It et al, 1986). is due to both an adverse osmotic effect of the solutes in milk and a low water activity Lactic acid isomers in yogurt (Tamime and Robinson, 1985). S thermophilus possesses 2 FDP- Carbohydrate metabolism independent L-LDH (Garvie, 1978; Hemme of L bulgarieus et al, 1981a) and produces L(+) lactic acid, while L bulgaricus produces OH lactic L bulgaricus is characterized by the utiliza- acid and possesses an NAD-dependent tion of a small number of carbohydrates, stereospecific LDH (Gasser, 1970). Thus, but their metabolism has not been studied both lactic acid isomers are simultaneously enough. This lactobacillus uses only the produced in yogurt. glucose moiety of lactose and releases ga- The D-LDH of L bulgaricus purified by lactose into the growth medium. Sorne Le Bras and Garel (1991) is a dimer corn- strains can use galactose in a growth me- posed of 2 identical 37-kDa chains. The dium containing Iimiting concentrations of amino-terminal sequence determined on lactose. Glucose transport occurs via a 50 residues is the first D-LDH sequence PTS-like system, while lactose and galac- available and does not show significant ho- tose uptake occurs by means of per- mology with any known LDH. This sug- meases (Hickey et al, 1986). Recent find- gests that the evolutionary process of 0- ings suggest that intracellular galactose is LDH from L bulgaricus is not identical to exchanged with lactose, enabling the that of the NAD-dependent L-LDH family. transport of lactose as in S thermophilus. ln fact, preliminary results indicate that ga- 0(-) lactic acid is metabolized only very lactose efflux from the cells is stimulated slowly in man compared to the L(+) isomer almost 100-fold by the presence of lactose and may cause metabolic disorders if in- in the external medium (Poolman et al, gested in excess. The World Health Or- 1990). The lactose genes and gene organi- ganization therefore recommends a Iimited 10 A Zourariet al daily consumption of D(-) lactic acid to fore yogurt manufacture eg aldehydes, ke- 100 mg.kg-1 bodyweight and some coun- tones, alcohols, lactones, sulfur corn- tries, eg Germany, have decided to mini- pounds (Tamime and Deeth, 1980). mize the D(-) lactic acid content of fer- Acetaldehyde is considered as the ma- mented dairy products (Kondratenko et al, jor flavour component of yogurt (Pette and 1989). In yogurt, this can only be obtained Lolkema, 1950c; Dumont and Adda, 1973; in the absence of L bu/garieus producing Law, 1981) while high concentrations of the D(-) isomer, which accounts for 30- this compound in other dairy products 50% of the total lactic acid content of yo- (cheese or cream) lead to flavour defects gurt (Kunath and Kandler, 1980; Alm, described as "green" or "yogurt-Iike" (Lind- 1982a). Nevertheless, the D(-) lactic acid say et al, 1965). Bottazzi and Vescovo content may reach critical concentrations (1969), however, underlined the impor- that can lead to disturbances especially tance of the acetaldehyde to acetone ratio. when the diet is extremely unbalanced, but Diacetyl contributes to the delicate, full this finding has not yet been reported flavour of yogurt and seems to be impor- (Gurr, 1986). Rare cases of D(-)-Iactic aci- tant when the acetaldehyde content is low dosis have been reported in children and (Groux, 1973a). According to Rasic and adults, in association with altered bowel Kurmann (1978) S thermophilus is the only physiology (short gut syndrome) and pro- microorganism responsible for diacetyl pro- liferation of active D-Iactate bacterial production in yogurt, whereas Dutta et a/ ducers (Szylit and Nugon-Baudon, 1985). (1973) reported that diacetyl is also largely Detection of a weak L-LDH activity in produced by L bu/garieus (12-13 ppm for the type-strain of L bu/garieus grown in a both species in pasteurized milk). Rysstad chemostat is therefore very interesting and Abrahamsen (1987) detected diacetyl (Ragout et al, 1989). The identification of in goat's milk yogurt with a low acetalde- its mechanism of regulation could contrib- hyde content and suggested that diacetyl ute to increase L(+) lactic acid synthesis might be very important for the flavour of by this species and to reduce the D(-) lac- goat's milk yogurt. tic acid content of yogurt. Even though the importance of acetaldehyde, acetone and diacetyl for yogurt Formation of flavour compounds flavour is weil established, it is very difficult to evaluate the contribution of several iden- Flavour compounds of yogurt tified volatile compounds because their sensory perception thresholds vary consid- The typical flavour of yogurt is due to lactic erably (Dumont and Adda, 1973). acid and various carbonyl compounds, ie ln spite of important progress in chro- acetaldehyde, acetone and diacetyl, pro- matographic methods such as head-space duced by S thermophilus and L bu/garieus. techniques which increase the detection ln addition to carbonyl substances, many sensitivity of volatile compounds (Degorce- volatile compounds have also been identi- Dumas et al, 1986), only few recent stud- fied in yogurt, ie volatile fatty acids (Turcic ies are available in this field and none of et al, 1969; Dumont and Adda, 1973) and them has involved a large number of several compounds derived from the ther- strains examined using the recently devel- mal degradation of lipids, lactose and pro- oped techniques. Thus, it ls difficult to teins during the heat treatment of milk be- compare results and conflicting findings Metabolism and biochemistry of yogurt bacteria 11

may be due to the different sensitivity of this enzyme in 2 strains of L bulgaricus, the methods previously used. but not in the 2 strains of S thermophilus investigated. In other studies, however, this enzyme was detected in both species Metabolic pathways (Lees and Jago, 1976b; Wilkins et al, of acetaldehyde synthesis 1986a; Marranzini et al, 1989). Streptococ- cal threonine aldolase activity decreases Formation of acetaldehyde from glucose significantly when growth temperature in- Two possible pathways exist: creases from 30 to 37 oC (Lees and Jago, - The Embden-Meyerhof-Parnas pathway 1976b) or from 30 to 42 -c (Wilkins et al, generates pyruvate. An a-carboxylase cat- 1986b), but that of L bulgaricus remains al- alyzes the formation of acetaldehyde from most identical. Thus, at 37 and 42 oC thre- pyruvate. An aldehyde dehydrogenase onine aldolase activity of the streptococci may also generate acetaldehyde from ace- is significantly lower than that of the lactob- tyl-CoA which is formed from pyruvate by acilli, whereas it is identical at 30 oC.Since the action of a pyruvate dehydrogenase. yogurt is generally manufactured at high Neither significant a-carboxylase nor alde- temperatures (43· OC), acetaldehyde is hyde dehydrogenase activities have been probably.produced mainly by L bulgaricus. detected by Raya et al (1986) in 2 strains The addition of threonine (0.6%) or gly- of S thermophilus and 2 strains of L bulgar- cine (20/0)to the growth medium does not icus. Nevertheless, Lees and Jago (1976a) significantly affect the specific activity of found an aldehyde dehydrogenase activity threonine aldolase in cell extracts (Lees in 4 strains of each species. Therefore it is and Jago, 1976b). High threonine concen- difficult to conclude whether this pathway trations combined with low glycine concen- occurs rarely or frequently in yogurt bacte- trations in the growth medium increase the ria. biosynthesis of the enzyme. When this ra- - The hexose monophosphate (HMP) tio is inverted, a decrease is observed. pathway generates acetylphosphate. A However, the threonine concentration dur- phosphotransacetylase catalyzes the for- ing growth may have a greater effect on in- mation of acetyl-CoA from acetylphos- creasing threonine aldolase activity than phate and an acetate kinase leads to the the glycine concentration may have on information of acetate. Although Raya et al hibiting it. Streptococcal threonine aldolase (1986) detected these 2 activities in both is more affected by hi~ glycine concentra- yogurt bacteria, acetaldehyde cannot be tions than the lactobacillus enzyme (Mar- formed by this pathway since their strains ranzini et al, 1989). The threonine aldolase did not possess aldehyde dehydrogenase of a L bulgaricus strain has been partiaily which catalyzes the biosynthesis of acetal- purified and studied by Manca de Nadra et dehyde from acetyl-CoA or acetate. al (1987). Maximum enzyme activity is observed at 40 oC and pH 6.5 as weil as a Formation of acetaldehyde from threonine strong allosteric pH-dependent inhibition by glycine. Inhibition of threonine aldolase Threonine aldolase catalyzes the c1eavage by glycine could explain the low acetalde- of threonine to acetaldehyde and glycine hyde content of yogurt from goat's rnilk, and appears to be the most important which is richer in glycine than cow's milk pathway for acetaldehyde production in yo- (2.4 versus 0.84 mg.100 ml-1; Rysstad et gurt. In tact, Raya et al (1986) detected al,1990). 12 A Zourariet al

Formation ot acetaldehyde Schellhaass (1983) isolated ropy strains trom DNA components of some thermophilic lactic acid bacteria. Two strains of S thermophilus and one She observed higher exopolysaccharide production in milk at suboptimal growth L bulgaricus strain studied by Raya et al (1986) possess a deoxyriboaldolase which temperatures, as measured by an increase catalyzes the synthesis of acetaldehyde of relative viscosity. The polymer material from 2-deoxyribose-5-phosphate. This en- obtained contained galactose and glucose zyme could be involved in the degradation in the ratio 2:1. The slime secreted by a rather than the synthesis of DNA. The 4 strain of L bulgaricus studied by Groux lactobacilli strains and 3 out of the 4 strep- (1973b) contained mainly galactose and tococcal strains examined by Lees and some glucose, mannose and arabinose. Jago (1977) did not contain this enzyme. Cerning et al (1988) studied an exocellular polysaccharideproduced by S thermophilus, A~oholdehydrogenase which contains galactose (55%), glucose (25%), mannose (15%) and small amounts Alcohol dehydrogenase reduces acetalde- of rhamnose, xylose and arabinose. The L hyde to ethanol. This enzyme has not bulgaricus exopolysaccharide studied by been detected in L bulgaricus, but has Cerning et al (1986) contains galactose, been found in 2 of 4 strains of S thermo- glucose and rhamnose in an approximate philus (Lees and Jago, 1976a; Marshall molar ratio of 4:1:1 with a molecular mass and Cole, 1983; Raya et al, 1986). of about 500 kDa and intrinsic viscosity of 4.7 dl.g-1. Doco et al (1990) studied an exopolysaccharide produced by S thermophi- Production of polysaccharides lus after 3-4 h of incubation and obtained a polymer composed of galactose, glucose Several Gram-negative and Gram-positive and N-acetylgalactosamine in a ratio of bacteria, including lactic acid bacteria, pro- 2:1:1. The molecular mass of the polymer duce exocellular polysaccharides. The is 1 x 103 kDa and intrinsic viscosity 1.54 "ropy" character is often required for the dl.g-1. Rhamnose, present mostly in the manufacture of many fermented milk prod- exopolysaccharides of L bulgaricus, has ucts. Ropy strains of lactococci have been not been identified in the exocellular poly- isolated from Scandinavian fermented mer produced by 3 lactobacillus strains dairy products (Makura and Townsley, isolated from traditional Greek yogurts 1984; Nakajima et al, 1990). In stirred yo- (Zourari, 1991). These molecules are com- gurt, yogurt beverages and low milk solids posed of galactose (5O-70%), glucose yogurts, production of polysaccharides can (2D-40%) and some mannose and arabi- improve viscosity and texture, increase re- nose. sistance to mechanical handling and decrease susceptibility to syneresis. The use The quantities of polymer formed by of ropy strains is particularly important in ropy strains of both species vary consider- France and in the Netherlands where the ably even under identical experimental addition of stabilizers in yogurt is prohibit- conditions (Giraffa and Bergère, 1987; ed. In fact, some strains of S thermophilus Cerning et al, 1990; Zourari, 1991). It is dif- and L bulgaricus produce neutral exopoly- ficult to establish a good correlation be- saccharides. Only few studies are availa- tween the quantity of polysaccharide pro- ble on the characterization and production duced and the corresponding viscosity. of these polymers (Cerning, 1990). This difficulty may be due to changes in Metabolism and biochemistry of yogurt bacteria 13

the 3-dimensional configuration of poly- OTHER METABOLIC ACTIVITIES mers and to their interactions with some milk compounds, mainly caseins that are Proteo/ytie aetivity precipitated at low pH (Olsen, 1989). In addition, viscosity measurements are difficult to interpret for non-Newtonian solutions Amino acids in milk and yogurt such as milk and fermented milk products. Bottazzi and Bianchi (1986) studied by ln yogurt, proteolysis is not determinant for scanning electron microscopy (SEM) the organoleptic properties, but it is an impor- structure of milk fermented with a ropy tant factor for the selection of strains for L bulgaricus strain. It was shown that a cheesemaking. On the other hand, proteo- part of the polysaccharide covers the cells Iytic activity is greatly involved in both nutri- with an uniform layer, and the rest of the tion and interactions of yogurt bacteria, polymer binds cells together and to milk since lactic acid bacteria cannot synthe- casein via a dense network of visible fila- size essential amino acids. Therefore, they ments. Recently, Teggatz and Morris require an exogenous nitrogen source and (1990) used SEM to explain changes oc- utilize peptides and proteins in their growth curing in the microstructure of ropy yogurt medium by more or less complete enzyme when it is subjected to a shear force. Their systems. aim was to understand the interactions of S thermophilus primarily requires glu- exopolysaccharide material with its sur- tamic acid, histidine and methionine, as roundings, and how they may influence weil as cystine, valine, leucine, isoleucine, viscosity. It was observed that an increase tryptophan, arginine and tyrosine for of shear rate first disrupts the attachment growth (Shankar and Davies, 1977; Brac- of polymer to the bacterial surface, but the quart et al, 1978). The uptake of branched- polysaccharide material remains incorpo- chain amine acids has been studied. lt is rated with the casein where it continues to an active transport which requires an exog- influence viscosity. enous energy source, depends on temper- The ropy character of S thermophilus is ature and pH and is inhibited by L-cysteine often unstable (Cerning et al, 1988). This (Akpemado and Bracquart, 1983). may be partially due to the presence of gly- The free amine acid content of cow's co hydrolase enzymes capable of hydrolyz- milk generally does not exceed 10 mg·100 ing the polysaccharide material. There is ml-1 (Rasic and Kurmann, 1978; Miller et no evidence of a possible loss of plasmids al, 1964; Alm; 1982c). In yogurt, the free encoding this character as in Lactococcus amine acid pattern depends on the type of lactis subsp cremoris (Vedamuthu and Ne- milk (animal species, season), its heat ville, 1986) or in Lactobacillus casei subsp treatment, manufacturing techniques, bac- casei (Vescovo et al, 1989). In fact, ropy terial strains used and storage conditions strains of yogurt bacteria studied by Cern- (Rasic et al, 1971 a, b). The free amino ing et al (1986, 1988, 1990) and Zourari acid content generally increases in yogurt (1991) are plasmid-free. compared to that of milk. L bulgaricus ap- 14 A Zourari et al pears to be the main species responsible (1991) who also obtained a proteinase- for these changes (Miller et al, 1964; Alm, negative mutant. Strains which possess 1982c; Feller et al, 1990). this proteolytic activity have a significantly higher acidification rate in milk compared with 13 other randomly chosen strains. Utilization of proteins

Milk proteins (caseins, whey proteins) are Utilization of peptides the main nitrogen source for lactic acid bacteria, which utilize them with the action The low molecular weight peptide fraction of exocellularproteinases, membrane- of milk is an important nitrogen source for bound aminopeptidases and intracellular yogurt bacteria. The importance of pep- exopeptidases and proteinases. tides for their growth stimulation and their Proteinase activity has been detected in acidification is now weil established, espe- several strains of lactobacilli and strepto- cially for S thermophilus (Desmazeaud and cocci by Ezzat et al (1985) and Kalantzo- Devoyod, 1970; Desmazeaud and Her- poulos et al (1990). L bulgaricus possess- mier, 1973; Bracquart and Lorient, 1979; es a firmly cell-bound proteinase with Hernrne etai, 1981b). optimum activity between 45 and 50 "C S thermophilus generally possesses a and pH values ranging from5.2 to 5.8 leucine-aminopeptidase activity (Bouillanne (Argyle et al, 1976). The partiaily purified and Desmazeaud, 1980). Some strains cell-wall-associated proteinase studied by possess an arginine-amino-peptidase ac- Ezzat et al (1987) has maximum activity at tivity which is usually inactive against di- 35 "C and pH 5.5. The proteinase of L bul- peptides. Two intracellular metalloen- garicus is more active on ~-casein than on zymes of this species have been purified whey proteins (Chandan et al, 1982). El and characterized, ie an amino-peptidase Soda and Desrnazeaud (1982) and Laloi with broad specificity and a dipeptidase (1989) revealed a preferential but partial which hydrolyzes dipeptides with a hydro- hydrolysis of ~- and us1-caseins. Laloi phobie and bulky residue at the NH2- (1989) also observed that after growth in terminal end (Rabier and Desma-zeaud, milk, caseinolytic activity is 3 times higher 1973). A non specific prolyl-dipeptidase hy- than that measured after growth in a com- drolyzing severai dipeptides has also been plex medium rich in short peptides. detected '(Desmazeaud and Jugé, 1976). Desmazeaud (1974) studied an intracel- An X-prolyl-dipeptidylamino-peptidase (X- lular metalloproteinase of S· thermophilus pro-DPAP) of a S thermophilus strain has which was more active on the carboxy- been purified, characterized and compared methylated B-chain of insulin or on gluca- with that of a L bulgaricus strain. The gon than on caseins. A weak caseinolytic streptococcal enzyme is composed of 2 activity was detected in both the cell enve- subunits, has a molecular mass of 165 lopes and cytoplasm of S thermophilus. As küa, an isoelectric point of 4.5 and opti- the 2 corresponding enzymes had the mum pH values ranging from 6.5 to 8.2 same electrophoretic mobility they were (Meyer and Jordi, 1987). considered to be identical (Meyer et al, Two intracellular exopeptidases of L bul- 1989). A high cell-wall-associated protei- garicus have been characterized by El nase activity characterizes 2 strains of Soda and Desmazeaud (1982), an amino- S thermophilus studied by Shahbal et al peptidase with specificity limited to argi- Metabolism and biochemistry of yogurt bacteria 15

nine-~-naphthylamide, and a dipeptidase (1990) detected esterase-1 and esterase-2 with broad specificity. Laloi (1989) re- activities in both yogurt bacteria, using 2- vealed the presence of 4 aminopeptidases and 4-nitrophenylbutyrate, respectively. (API, Il, III, IV) and an X-pro-DPAP in the same species. API and APIII (cytoplasmic enzymes) are induced after growth in a Urease activity complex medium, while APII and APIV are constitutive. Mutants partially or totally free ln milk, S thermophilus produces a large of APII or X-pro-DPAP have been isolated amount of carbon dioxide (C02) which is after chemical mutagenesis. Deficiency of not formed from lactose metabolism since these 2 enzymes significantly increases this microorganism is a strictly homofer- the caseinolytic activity of mutants corn- mentative species (Driessen et al, 1982). It pared to the parental strain (Atlan et al, also produces a large quantity of ammonia 1989, 1990). (NH3) which was initially explained as resulting from the deamination of some amino acids (Groux, 1973a). CO2 production Lipolytic activity is due to the activity of urease, which breaks down milk urea (about 250 mg.I-1) Lipolysis is generally low in yogurt and is into CO2 and NH3 (Miller and Kandler, . therefore not significant in terrns of flaveur. 1967b; Tinson et al, 1982b; Juillard et al, The free fatty acid content of yogurt differs 1988). This leads to the alkalinization of only slightly from that of milk (Rasic and the growth medium and directly affects Vucurovic, 1973; Alm, 1982b). Therefore, acidification rate measurements in milk only few studies with these enzyme sys- (Spinnler and Corrieu, 1989; Famelart and tems have been reported in yogurt bacteria. Maubois, 1988; Zourari et al, 1991). Tamime and Deeth (1980) have sum- Urease activity is of technological inter- marized the main lipolytic activities as fol- est for 3 reasons: (i) it enables streptococ- lows: the hydrolytic activity of streptococci cal numbers in mixed cultures with L bul- on tributyrin and triolein is high and, in- garicus (this lactobacillus has no urease versely, is low on milk fat and on Tween 40 activity) to be followed by measuring the or 60. The hydrolytic activity of lactobacilli amount of CO2 produced (Spinnler et al, on tributyrin is weaker, while esterases hy- 1987; Miller and Kandler, 1967a); (ii) NH3 drolyzing several soluble substrates, eg n- production affects the evaluation of strep- naphthylacetate, are active in both bacte- tococcal acidifying properties by pH meas- ria. urements; (iii) this activity may also be involved in the stimulation of lactobacilli by L bulgaricus possesses intracellular es- CO produced by the streptococci (see dis- terases active on ortho- and para- 2 cussion below). nitrophenyl derivatives of short-chain fatty acids and activity levels vary only slightly Besides its technological interest, the between different strains (El Soda et al, presence of urease activity in S thermophi- 1986). Many strains of S thermophilus (30 lus also has taxonomie relevance. AI- out of 32 tested) possess a lipase more ac- though Farrow and Collins (1984) reported tive towards tributyrin than towards natural that S thermophilus does not degrade lipids (De Moraes and Chandan, 1982) urea, the presence of an urease activity and maximum activity was observed at seems to characterize most strains exam- 45 -c and pH 9. Kalantzopoulos et al ined until now. It also characterizes oral 16 A Zourariet al

S salivarius strains (Sissons et al, 1989) coccal strains can degrade H202, eg strain but lactococci do not possess this activity TS2 studied by Smart and Thomas (1987). (Miller and Kandler, 1967b). The absence Nevertheless, no further experiments have of urease activity in the 6 strains of S ther- been performed and the c1eavingactivity of mophilus studied by Bridge and Sneath this strain was attributed by the authors to (1983) could be due to the prolonged incu- a mechanism independent of active metab- bation time (5 days at 37 OC) before olism. urease assays were carried out, since Juil- The differences generally observed be- lard et al (1988) demonstrated that urease tween strains regarding their tolerance to activity dropped during advanced station- and utilization of oxygen are due to varia- ary phase. bilities in their enzymatic systems. Ritchey and Seeley (1976) detected no NADH- oxidase activity in 3 S thermophilus Oxygen and metabolism strains, while many lactococcal strains possess this enzyme. Nevertheless, an- Lactic acid bacteria are facultative anaer- other weil studied strain possesses this en- obes with a preference for anaerobic con- zyme as weil as NADH-peroxidase, super- ditions (Whittenbury, 1978). They cannot oxide dismutase, pyruvate and lactate synthesize porphyrins and consequently dehydrogenases (Smart and Thomas, they do not synthesize cytochromes or cat- 1987). alase. Oxygen is sometimes used for the Teraguchi et al (1987) compared the formation of hydrogen peroxide (H202) S thermophilus STH 450 strain which has which is toxic for lactic acid bacteria which a high oxygen consumption, with the type- do not contain catalase to break it down. strain, ATCC 19258. Strain STH 450 has Some species possess protection mecha- NADH-oxidase, but no pyruvate oxidase, nisms, eg a high content of manganese and very low NADH-peroxidase activity. which is oxidized by O2- radicals in Lac- These enzyme activities are much lower in tobacillus plantarum (Archibald and Frido- the type-strain. Strain STH 450 accumu- vich, 1981). lates a-acetolactate, acetoin and diacetyl L bulgaricus is among the least oxygen- as end-products of aerobic glucose metab- tolerant lactic acid bacteria (Archibald and olism. Fridovich, 1981). lt can produce a very large amount of H202 which activates the LPS system of milk (Iactoperoxidase- PARTICULAR ASPECTS hydrogen peroxide-thiocyanate system) OF YOGURT FERMENTATION and inhibits its own growth as weil as the growth of some other bacteria such as Interactions between yogurt bacterla Lactobacillus acidophilus, associated in the manufacture of some fermented milk products (Gilliland and Speck, 1977). A positive interaction is generally observed S thermophilus does not produce enough between S thermophilus and L bulgaricus H202 to activate the LPS system and in mixed culture, leading to the stimulation therefore inhibition occurs only when exog- of growth and acid production of both bac- enous H202 is added to milk (Premi and teria compared to their single-strain cul- Bottazzi, 1972; Guirguis and Hickey, tures (Pette and Lolkema, 1950a; Bautista 1987). On the other hand, some strepto- et al, 1966; Accolas et al, 1977). In addi- Metabolism and biochemistry of yogurt bacteria 17 tion, total proteolysis in mixed culture (ex- of both cells are almost equal. The com- pressed in Jlg of released tyrosine per ml bined growth of L bulgaricus and S thermo- of culture) exceeds the sum of the values phi/us has the same effect as the addition obtained by each strain alone (Rajagopal of formic acid. A better knowledge of path- and Sandine, 1990). Mixed yogurt cultures ways and kinetics of formic acid production may also stimulate the production of some by S thermophi/us is required to definitively metabolites such as acetaldehyde (Ham- establish the stimulating effect of this acid, dan et al, 1971; Bottazzi et al, 1973) and questioned by Juillard et al (1987). influence carbohydrate utilization. For in- Driessen et al (1982) observed that stance, one L bulgaricus strain studied stimulation of lactobacilli growth could also which cannot use galactose in pure culture result from an increased CO2 content in metabolizes this sugar when it is associat- the growth medium, during continuous cul- ed with one strain of S thermophi/us (Amo- ture at constant pH. S thermophilus pro- roso et al, 1988, 1989). Contrary behavior duces a large quantity of CO2 from milk was observed with one streptococcal strain urea (Tinson et al, 1982b). In this way, it (Oner and Erickson, 1986) indicating that can stimulate lactobaciiii, since the quanti- interactions between yogurt bacteria are ty of CO2 dissolved in milk decreases after very complex and are greatly dependent heat treatment and so remaining CO2 is on the strains involved. too low to meet the requirements of the S thermophi/us does not possess sub- lactobacillus (Driessen et al, 1982). Car- stantial extracellular proteolytic activity and bon dioxide might be involved in the syn- the amino acid and free peptide content of thesis of aspartic acid (Reiter and Oram, milk is not high enough to promote its full 1962). growth. Lactobacillus proteases break ln conclusion, interaction between yo- down caseins and supply the streptococ- gurt bacteria is a good example of integrat- cus with amino acids and peptides (Pette ed metabolism in a mixed culture of lactic and Lolkema, 1950b, Bautista et al, 1966; acid bacteria, but our knowledge on the Shankar and Davies, 1978; Radke-Mitchell stimulation of L bulgaricus by S thermophi- and Sandine, 1984). lus is still incomplete. The growth of L bulgaricus is stimulated by a compound produced by S thermophi- Production of antimicrobial compounds lus, which seems to be formic acid (Gales- by yogurt bacteria loot et al, 1968; Veringa et al, 1968; Acco- las et al, 1971; Shankar and Davies, As mentioned above, there is generally a 1978). Higashio et al (1978) reported a symbiotic relationship between yogurt bac- combined stimulating effect of formic and teria, but growth inhibition is sometimes pyruvic acids. Suzuki et al (1986) observed observed (Moon and Reinbold, 1974; that addition of formic acid to boiled milk Suzuki et al, 1982; Pereira Martins and prevents abnormal cell elongation in L bul- Luchese, 1988). This should be taken into garicus (filamentous forms). Formic acid account when selecting starters. Inhibition synthesis from pyruvate is a limiting step in may be due to competition for one or more purine synthesis and this explains the com- nutrients of the growth medium (Moon and bined action of the 2 acids. When formate Reinbold, 1976) or to inhibitory compounds is lacking, ribonucleic acid (RNA) synthesis produced by the strains, such as bacterio- is depressed. Elongated cells contain less cins and inhibitory peptides (Pereira Mar- RNA than normal cells while DNA contents tins and Luchese, 1988). 18 A Zourariet al

Pulusani et al (1979) extracted at least only little information on the nature of the 3 fractions of '" 700 Da from milk cultured compounds involved, their spectrum and with S thermophilus, which inhibited the mode of action. These antibacterial sub- growth of Pseudomonas, Bacil/us, E coli, stances often seem to be peptides. More Flavobacterium, Shigella, Salmonella and extensive study is required for a better un- Lactococcus strains. These fractions are derstanding of interactions of yogurt bacte- most likely aromatic amines released in ria with other lactic acid bacteria and with the growth medium since cells are free of non-Iactic cheese flora. any antimicrobial activity. Glucose and lactose, but not sucrose, are essential for production of these compounds which are Bacteriophages of L bulgaricus also obtained in milk-free media, eg "soy- and S thermophilus milk" (Rao and Pulusani, 1981). Smaczny and Krârner (1984a) studied Knowledge of the specifie bacteriophages 2 bacteriocins of 10-20 kDa produced by (phages) of thermophilic lactic acid bacte- 2 S thermophilus strains which inhibited ria has been weil documented, primarily on the growth of strains of the same species and after 1980. Specifie phages may at- and to a lesser extent of enterobacteria. tack S thermophilus and L bulgaricus These molecules are sensitive to several strains during yogurt or cheese manufac- proteases and to a lipase, suggesting that ture and seriously affect product quality. a Iipid component participates in their ac- Moreover, even if phage attacks do not de- tive sites. lay acidification during yogurt manufacture, An antimicrobial substance named "bul- they can lead to an important decrease in garican" produced by an L bulgaricus strain the streptococci and to a lower flavour has been partially purified (Reddy and Sha- score of the resulting yogurt (Stadhouders hani, 1971; Reddy et al, 1983). At neutral or et al, 1988). Phage outbreaks are less fre- acid pH, this heat-stable substance was ac- quent in modern yogurt production units tive against several strains of Bacillus, than in cheese factories because: (a) the Streptococcus, Staphylococcus, Sarcina, high heat treatment of milk destroys phag- Pseudomonas, Escherichia and Serratia es present in raw milk; (b) aseptic process- species. Protonated carboxyl groups may ing and packaging may prevent contamina- be important for the activity of this mole- tions by airborne bacteriophages, bacteria, cule. Spillmann et al (1978) could not con- yeasts or moulds; and (c) according to firm, however, bulgarican production. Lawrence and Heap (1986), an increasing viscosity of the yogurt milk below pH 5.2 A newly isolated antibacterial substance and the fact that no whey is drained off from L bulgaricus, active against a Pseu- during yogurt manufacture could efficiently domonas tragi and a Staphylococcus aure- reduce the spreading of phages in yogurt us strains has optimum pH values close to plants. Thus, phage attacks are due either 4. It is a di- or a tripeptide (molecular mass to accidentai contaminations (Stadhouders < 700 Da) which probably contains an aro- et al, 1984) or to the presence of Iysogenic matie group (Abdel-Bar et al, 1987). starter bacteria which might be the major Knowledge of antibacterial substances source of phages by two possible routes: produced by yogurt bacterla is still frag- (i) and temperate phage particles derived mentary and the results are often contra- from a Iysogenic strain may infect a sensi- dictory. Available studies are few and give tive (indicator) strain of the same starter; Metabolism and biochemistry of yogurt bacteria 19

(ii) temperate phages may give rise to virulent mutants able to superinfect and lyse the parental Iysogenic and other related strains. A typical example of the latter incidence has been weil documented for a Iy- sogenic strain of Lactobacillus casei subsp casei used in Japan for the industrial manufacture of a fermented milk beverage, Va- kult (Shimizu-Kadota and Sakurai, 1982; Shimizu-Kadota et al, 1985). Substitution of a prophage-free strain for the Iysogenic strain immediately stopped ail phage infec- tions in the different Vakult production units.

Bacteriophages of L bulgaricus are very 15 _ closely related to those of L delbrueckii subsp lactis (L lactis) but they are corn- pletely different from those of L helveticus, which is usually used in cheesemaking (Séchaud, 1990). Two surveys of L bulgarieus and L lactis phages (Cluzel, 1986; _Iv Mata et al, 1986) led to the description of 4 groups named a, b, c and d. Ali these phages belong to the Siphoviridae family of Matthews (1982) which corresponds to Fig 2. Morphology of bacteriophages which be- Bradley's group B (Bradley, 1967). The long to group a. Their host-ranges include L bul- head of these phages is isometric or pro- garicus and L lactis strains. Phage 15possesses late, collars are frequent, and the tails of- fragile cross-bars on its tail which may be easily ten have diverse terminal structures removed during phage preparation. Coll ars are visible at the head-tail junctions of phage 0448 (pronged or plain plates, fibers) and occa- (triple collar) and phage Iv (single collar). Fibers sionally harbour regularly spaced cross- are present at the tail tip of phages Iv and 0448. bars. Figures 2 and 3 iIIustrate some of Bars = 100 nm; the magnification factor is the these typical morphologies. Group a is the sa me for ail the pictures (micrographs from largest phage group (20 of the 26 phages INRA, Jouy-en-Josas, France). studied by Cluzel, 1986) and includes tem- Morphologie de bactériophages du groupe a. Ces phages attaquent indifféremment un certain perate phages of both subspecies (8 of the nombre de souches de L bulgaricus et L lactis. 20 phages of Cluzel's group a) along with Le phage 15possède des structures transver- Iytic phages isolated in cheese plants or sales fragiles tout le long de la queue, qui peu- yogurt production units (12 of the 20 phag- vent se détacher facilement au cours de la pré- es of Cluzel's group a). Ali phages of group paration du phage. Des colliers sont visibles à la a share common molecular characteristics, jonction de la tête et de la queue des phages 0448 (triple collier) et Iv (collier simple). Des serological properties, Iytic spectra and are fibres sont présentes à l'extrémité distale de la specifie towards both subspecies (typical queue des phages Iv et 0448. Barres = 100 nm; morphology in figure 2). The close relation- l'agrandissement est le même pour toutes les ship between temperate and Iytic phages images (micrographies de /'INRA, Jouy-en- of group a suggests that Iysogeny may be Josas, France). 20 A Zourari et al

a determining factor in the occurrence and 0235 spread of these phages. Each of the , c groups c and d contains a single represen- tative temperate phage of L lactis. These 2 phages have Iytic spectra similar to those of group a phages and consequently can infect some L lactis as weil as L bulgaricus strains. However, no morphological (fig 3), molecular or serological similarities between them or with group a phages have been observed. Their low incidence may limit their importance in technology. Group b is composed of 4 closely related virulent phages. They seem .less widespread than group a phages and attack only L bulgaricus strains. They have different Iytic spectra and do not share molecular and serological relationships with phages of the 3 other groups. Some L bulgaricus strains sensitive to group b phages are Iysogenic and harbour prophages that belong to group a. Prophage- Fig 3. Morphology of bacteriophages which be- free (cured) bacterial clones become sen- long to groups b, c and d. Phages of group b, sitive to the corresponding temperate such as c31 and c5, have a host-range restric- phage and to ail other phages of groups a, ted to L bulgaricus strains whereas phages 0235 (group c) and 0252 (group d) have host- c and d but are unexpectedly resistant to ranges similar to those of group a phages. group b phages. Inversion of phage sensi- Phages c31 and c5 (groupe b) possess a non- tivity occurs along with changes in mor- contractile tail fitted with a pronçed- base plate phology of bacterial colonies (rough form and at least one fiber. Tail tip of prolate-headed for cured clones/smooth form the parental temperate phage 0235 (group c) exhibits a Iysogenic strains; CIuzel et al, 1987). This small base plate and a fiber with 2 swellings. Isometric-headed temperate phage 0252 (group suggests that these strains couId possess d) possasses a fairly long tail with saveral fibers. specifie receptors for phages of ail four Bars = 100 nm; the magnification factor is the groups. In the presence of prophage, only same for ail the pictures (phages c31, 0235 and receptors for group b phages are function- 0252: micrographs from INRA; phage c5: micro- al while those for groups a, c and d phag- graphs from INRNETH-Zürich). es are masked or non-functional. It could Morphologie des phages des groupes b, c et d. Les phages du groupe b, tels que c31 et c5, ont be a Iysogenic conversion. un spectre d'hôtes limité à quelques souches de L bulgaricus, tandis que les phages 0235 (groupe c) et 0252 (groupe d) ont des spectres d'hôtes similaires à ceux des phages du groupe Le phage à tête isométrique 0252 (groupe d) a. Les phages c31 et c5 (groupe b) possèdent possède une queue assez longue, munie de une queue non contractile pourvue d'une plaque plusieurs fibres terminales. Barres = 100 nm; basale dentée et d'au moins une fibre. L'extrémi- l'agrandissement est le même pour toutes les té distale de la queue du phage tempéré à tête images (phages c31, 0235 et 0252 : microgra- allongée 0235 (groupe c) porte une petite pla- phies de l'INRA; phage c5 : micrographies de que basale et une fibre avec deux renflements. /'INRA 1ETH-Zürich). Metabolism and biochemistry of yogurt bacteria 21

The large number of group a phages Recent studies in Germany and France underlines the risk resulting from the pres- have demonstrated the existence of an ap- ence of Iysogenic bacteria in lactic starters. parent homogeneity of the specifie phages Therefore, the use of cured strains ap- of S thermophilus at both the morphologi- pears to be a satisfactory solution, but re- cal and molecular levels (Krush et al, quires strictly aseptic conditions during 1987; Neve et al, 1989; Prévots et al, starter preparation and fermentation in or- 1989; Benbadis et al, 1990; Larbi et al, der to prevent attacks by the widespread 1990). These phages have been isolated group a phages. as Iytic and belong to the Siphoviridae fam- Among phages of L bulgaricus and ily of Matthews (1982) or to Bradley's L lactis, only phage LL-H, isolated in Fin- group B1 (Bradley, 1967), as shown in fig- land and active against L lactis strain LL ure 4. They contain linear double-stranded 23, has been extensively studied (see DNA (34-44 kb) with complementary cohe- Alatossava, 1987). Its morphology is repre- sive ends. Grouping of these phages has sentative of group a, and is closely related been attempted on the basis of pro- to that of phage Iv iIIustrated in figure 2. tein patterns and DNA restriction profiles. It The average burst size reaches 102 phag- is likely that most S thermophilus phages es per infected cell under optimal condi- studied until now were derived from a corn- tions. Phage LL-H contains 2 major struc- mon ancestor. As indicated by Mercenier turai proteins (34 kDa for the head and 19 (1990), their genomes have non-homolo- kDa for the tail) and no less than 5 minor gous sequences flanked by homologous proteins. The DNA molecules are linear regions. It is consistent to apply the hy- (34 kbp) with constant ends. A restriction pothesis of Botstein (1980) who postulated map of LL-H DNA has been established that the observed diversity of phages might and a library of restriction fragments has result from recombination and exchange of been prepared by molecular cloninq into E "modules" ie genome fragments encoding coli. This had led to the localization of 5 for particular biological functions. genes encoding structural proteins and phage Iysin. Phage LL-H DNA contains 2 The existence of Iysogenic strains in polycistronic c1usters of genes ie "early" S thermophilus has been reported several genes which are involved inphaqe replica- times since 1970 (Ciblis, 1970; Kurmann, tion and "late" genes which encode structu- 1979, 1983; Smaczny and Krârner, rai proteins and phage Iysin. Bivalent cat- 1984b), but these studies remained incom- ions (Ca2+ or Mg2+) stabilize the coiled piete. Neve et al (1990) detected prophag- DNA into the capsid, improve adsorption es in 2 strains by probing chromosomal rate and control penetration efficiency of DNA of 17 strains with genomic DNA of vir- phage DNA into the bacterial cells. A mo- ulent phages. Subsequently, the same au- lecular study of the LysA-encoding gene of thors demonstrated that these 2 strains L bulgaricus temperate phage mv1 c1assi- were inducible by mitomycin C (0.2-1 fied in group a (Boizet et al, 1990), re- Ilg.ml-1) and released mostly defective vealed a close relationship between ail Iy- phage particles. However, these phages sins of group a phages (including LL-H and were able to relysogenize cured clones mv1 enzymes). The lysA gene has a nu- and formed turbid plaques on agar medi- c1eotide sequence of 585 bp correspond- um. Presence of prophage led to a modifi- ing to a 21,120 Da protein. Significant ho- cation of sorne phenotypic traits of the mology with the N-terminal domain of strains, ie homogeneous non-c1umpingcul- known muramidases has been observed. tures in broth instead of c1umpingin cured 22 A Zourari et al

strains, and autolysis at 45 oC probably by Mercenier and Lemoine (1989) and due to the thermoinducibility of prophage Mercenier (1990). This is mainly due to im- at elevated temperature. portant methodological problems, but also A survey of S thermophilus phages is to the fact that the major functions are car- currently being carried out at the INRA la- ried by chromosomal genes making stud- boratory at Jouy-en-Josas (8 Fayard and ies more difficult. For this reason, many ef- M Haefliger, unpublished data). Ten of the forts have been made to adapt DNA 120 strains examined were shown to be Iy- transfer methods to this species. The or- sogenic. Induction by mitomycin C gave ganization and expression of genes in- temperate phages active on one or several volved in lactose and galactose metabo- indicator strains for 7 of them. Temperate lism were then studied by Herman and phage DNA exhibited 5 different restriction McKay (1986), Poolman et al (1989, patterns but shared homology. DNA ho- 1990), and Lee et al (1990). mology also existed between temperate The plasmids of lactic acid bacteria phages and 52 phages isolated as Iytic fol- encode important characters for dairy lowing acidification failures. Preliminary re- technology, eg carbohydrate metabolism, sults suggest that prophages might repre- proteolytic activity, citrate utilization, pro- sent a phage source for S thermophilus duction of antimicrobial compounds, bacte- and the impact of Iysogeny in current riophage and antibiotic resistance, poly- phage outbreaks should not be underesti- saccharide production (McKay, 1983; mated. Vedamuthu and Neville, 1986). S thermo- Starter suppliers and yogurt producers philus and L bulgaricus seem to be natural- still Jackbasic information about the origin Iy poor in plasmids (table 1). of disturbing phages and phage-bacteria Ali plasmids mentioned in table 1 are relationships. This explains why they often considered as being cryptic. Most of the adopt a pragmatic approach to the phage repJicons isolated from S thermophilus problem. Thus, an infected strain is re- would be too small to carry a phenotypic placed by an insensitive strain, sometimes trait (Mercenier, 1990). Until the present, by a spontaneously resistant mutant, with the largest extrachromosomal element de- similar technological properties. This is the scribed is the 22.5-kb plasmid harboured case for Australian yogurt manufacture by strain IP6631, along with 2 other small (Hull, 1983). In day-te-day practice, phage replicons (Gavoille, 1989). control requires experience and "know- Only one endogenous plasmid of S ther- how", but progress in the study of yogurt mophilus has been fully sequenced, ie bacteria phages will certainly help to pre- plasmid pA33 (6.9 kb) from strain A033 vent phage attacks. For instance, improve- (Mercenier, 1990). Preliminary results indi- ment of phage resistance by genetic engi- cate that this plasmid integrates and re- neering might be the solution to phage excises from chromosomal DNA. Its pres- problems in the future (Sanders, 1989). ence or absence as an autonomously repli- cating extrachromosomal element provides the host-cella with phenotypes which differ Genet/cs ofS thermophilus by their colony morphology, cell chain and L bulgaricus length, growth and acidification rates in milk, antibiotic resistance and phage sensi- Only few studies have been performed on tivity. The nucleotide sequence of this plas- the genetics of S thermophilus, reviewed mid showed Iimited similarity with coding Metabolism and biochemistry of yogurt bacteria 23

responsible for cell wall changes, but its role remains to be demonstrated. These observations should be considered in relation to specifie chromosomal DNA rear- rangements in S thermophilus, which lead to morphologically different colonies (Pé- bay et al, 1989). These general considerations also apply to L bulgaricus. The rare plasmids are cryptic and genetic studies of different metabolic functions have just started. Mor- elli et al (1983) tried to correlate antlblotlc resistance with the presence of plasmids in three lactobacillus strains. After treatment with curing agents (ethidium bromide, acrif- lavin) these strains lost several plasmids and their resistance to various antibiotics. However, the 2 observations cannot be correlated since the loss of plasmids and of antibiotic resistance was obtained by powerful mutagens which may also act on Fig 4. Morphology of S thermophilus bacteriophages. Ali phages possess an isometric head the chromosome. and a fairly long and flexible tail, generally fitted with a small base plate. Tails of phage c20 exhi- bit a long fiber, whereas those of phages sch CONCLUSION and sc are fitted with a shorter one. The cluster of phage sch particles adsorbed on a streptococcal cell debris is reminiscent of Iysis from with- Since 1980 many studies on S thermophi- out. Bars = 100 nm; the magnification factor is the same for ail the pictures except phage elus- lus and L bulgaricus have led to a better ter sch (micrographs from INRAIETH-Zürich). understanding of sorne metabolic and bio- Morphologie de bactériophages spécifiques de chemical aspects of these bacteria that S thermophilus. Tous les phages possèdent une control their growth, especially in mixed tête isométrique et une queue longue et flexible, généralement pourvue d'une petite plaque ba- cultures. For instance, lactose and galac- sale. La queue du phage c20 se prolonge par tose metabolism have been weil studied at une longue fibre, tandis que celles des phages both the biochemical and molecular levels. sch et sc sont munies d'une fibre plus courte. Le ln addition, sorne enzymes involved in the groupe de particules du phage sch adsorbées production of aroma compounds have sur un débris cellulaire du streptocoque consti- tue une image évocatrice de la lyse externe. been investigated. Production of exocellu- Barres = 100 nm; l'agrandissement est le même lar polysaccharides by yogurt bacteria is pour toutes les images, à l'exception du groupe now weil established and sorne of these de particules du phage sch (micrographies de polymers have been studied. Several en- /'INRAIETH-Zür~ch). zymes such as aminopeptidases, proteases, esterases and lipases have been characterized in both bacteria, especially sequences of sorne proteins of the outer proteases hydrolyzing caseins as weil as membrane or of proteins involved in antibi- urease activity of S thermophilus because otic resistance. This plasmid seems to be of their technological and taxonomical in- 24 A Zourari et al

terest. The taxonomy of S thermophilus is strains better established. In particular, Iy- still debated and its definitive classification sogenic strains seem to be a major source as an S salivarius subspecies requires fur- of phages for L bulgaricus and probably for ther consideration. Many bacteriophages S thermophilus. of yogurt bacteria have been weil charac- Many questions still remain unan- terized and their interactions with host- swered. The molecular aspects of the me-

Table 1. Distribution and characterization of plasmids from S thermophilus and L bulgaricus. Distribution et caractérisation de plasmides de S thermophilus et de L bulgaricus.

Reference No of plasmids Plasmid size Plasmid per strain (kb) characterization

S thermophilus

Pechmann et al (1982) + (1/8)1

2 plasmids in duplicate Herman and McKay (1985) 1 (5/23) 2.1 to 3.3 (restriction profiles) Homology of ail plasmids (DNA-DNA hybridization)

9 distinct types (restriction Somkuti and Steinberg 1 (10/35) 2.20 to 14.75 profiles, obtained (1986a) 2 (3/35) using 19 endonucleases)

8 types (size, restriction Girard et al (1987) 1 (6/50)2 2.9 ± 0.1 to 7.6 ± 0.2 profiles) - 3 groups 2 (3/50)3 (DNA-DNA hybridization)

L bulgaricus

Vescovo et al (1981) + (1/19)

Klaenhammer et al (1979) + (1/1) 52.5, 40.5, 13.5 and 6

Somkuti and Steinberg 4 (1/1) (1986b)

Sosi et al (1990) 1 (1/3) 8.2 to 16.1 2 (213)

+ : No of plasmids not 9iven; - no data available. 1 ln parentheses, number of plasmid-containing strains on tested strains. 2 Four strains isolated from raw milk and 2 industrial starter strains. 3 Two strains isolated from raw milk and strain CNRZ 312.

+ : Le nombre de plasmides n'est pas donné. - Pas de données disponibles. t Entre perentnëses, nombre de souches contenant des plasmides sur le nombre de souches testées. 2 Quatre souches isolées du lait cru et deux souches provenant de levains industriels. 3 Deux souches isolées du lait cru et la souche CNRZ 312. Metabolism and biochemistry of yogurt bacteria 25

tabolism of sucrose and other carbohy- ACKNOWLEDGMENTS drates should be better studied. This is particularly true for L bu/garicus, since The authors are grateful to K Rérat (INRA, Ser- metabolic studies are less advanced than vice de Traduction UCD-CRJ, Jouy-en-Josas, in S thermophi/us. Volatile compounds pro- France) who translated this paper into English duced by S thermophilus and L bu/garicus and to J Cerning for helpful suggestions and dis- remain to be identified because of their im- cussions during preparation of the English version of this manuscript. portance for the organoleptic properties of yogurt. The study of the different biosynthesis pathways of acetaldehyde and other volatile compounds, especially at the mo- REFERENCES lecular level, is also necessary. Further research is needed in the field of exopoly- Abdel-Bar N, Harris ND, Rili RL (1987) Purifica- saccharide production. In particular the tion and properties of an antimicrobial genetics of the metabolic pathways (en- substance produced by Laetobaeillus bulgarieus. J Food Sei 52,411-415 zymes involved, and their regulation) require investigation in order to control and Accolas JP, Veaux M, Auclair J (1971) Étude des interactions entre diverses bactéries lac- stabilize polysaccharide production in milk. tiques thermophiles et mésophiles, en rela- The nutritional and possible therapeutic tion avec la fabrication des fromages à pâte roles of these polymers remain to be con- cuite. Lait 51, 249-272 firmed. Accolas JP, Bloquel R, Didienne R, Régnier J ln mixed cultures, the growth of L bu/- (1977) Propriétés acidifiantes des bactéries garicus and S thermophilus is not yet fully lactiques thermophiles en relation avec la fabrication du yoghourt. Lait 57, 1-23 controllable. Even if the interactions of these bacteria are weil established, many Akpemado KM, Bracquart PA (1983) Uptake of branched-chain amino acids by Streptococ- aspects are still unknown. In particular, the eus thermophilus. Appl Environ Mierobiol 45, real influence of associated growth on sec- 136-140 ondary metabolic activities has not been Alatossava T (1987) Molecular biology of Lae- studied sufficiently. In addition, the charac- tobaeillus laetis bacteriophage LL-H. Acta terization of inhibitory substances which Univ Ouluensis Ser A 191, 1-65 may be produced would be very useful (a) Alm L (1982a) Effect of fermentation on L(+) and for a better understanding of the relation- D(-) lactic acid in milk. J Dairy Sci 65, 515- ship between the yogurt bacteria, and (b) 520 for their action on other micro-organisms Alm L (1982b) Effect of fermentation on milk fat associated with yogurt bacteria for· the of Swedish fermented milk products. J Dairy manufacture of several fermented milk Sei 65, 521-530 products. Alm L (1982c) Effect of fermentation on proteins of Swedish fermented milk products. J Dairy Finally, progress in genetic studies Sei 65, 1696-1704 should contribute to better knowledge on Amoroso MJ, Manca de Nadra MC, Oliver G the yogurt bacteria and their growth and (1988) Glucose, galactose, fructose, lactose activity in milk. and sucrose utilization by Lactobaeillus bul- 26 A Zourari et al

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