Proteolytic enzymes of dairy starter cultures *

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1 - - Genetics, FEMS Microbiology Reviews 46 (1987) Published by Elsevier FER 0069S Proteolytic enzymes of dairy starter cultures * 1. SUMMARY Terence D. Thomas a and Graham G. Pritchard b a New Zealand Dairy Research Institute and b Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand Key words: Proteinase; Peptidase; Starter culture; (Proteolysis in milk and cheese) The synthesis of proteolytic enzymes by starter bacteria is a fundamental requirement for rapid acid production in milk fermentations. These organisms possess a number of proteinases and peptidases which act in concert to hydrolyse milk protein to the free amino acids required for cell growth. The same enzymes have an important secondary role in cheese ripening contributing to rheological and organoleptic changes. A highly complex mixture of both enzymes and substrates is present. The strategic location of these enzymes, in the cell wall and membrane structures and in the cytoplasm, governs enzyme access to the substrates and is central to both roles. An overview of the above topics is presented. 2. INTRODUCTION Starter bacteria are carefully selected organisms that are deliberately added to initiate and carry through the desired fermentation. In milk fermentations the prime function of the starter is lactic acid production (from lactose). For the purpose of this review, starter bacteria include the closely related mesophilic lactic stlcptococci (Streptococcus cremoris, S. lactis and S. diacetylactis) and thermophilic organisms (S. thermophilus, Lactobacillus bulgaricus and L. helveticus). These dairy starter cultures must grow to high cell densities in milk in order to produce acid at the rapid rates required for the manufacture of most products [1]. Numerous amino acids are either stimulatory or essential for starter growth. In milk most of these amino acids are in short supply in free form and as low molecular weight peptides. Therefore nitrogen is growth-limiting, and acid production slow, unless the starter cells have a proteolytic system that produces free amino acids from milk protein. In this review attention will be focussed on milk since the primary justification for research on the proteolytic enzymes of starter bacteria relates to their role for growth in this medium. Other reviews on the proteolytic enzymes of starter bacteria have appeared recently [2-5]. 3. MILK - THE GROWTH MEDIUM FOR STARTER BACTERIA * Presented at the Second Symposium on Lactic Acid Bacteria Metabolism and Applications, September 1987, Wageningen, The Netherlands. Correspondence to: T.D. Thomas, New Zealand Dairy Research Institute, Palmerston North, New Zealand. Growth of typical starter strains of lactic streptococci in milk results in maximum cell densities of -500 /~g (dry weight) bacteria/ml (or about 10 9 colony-forming units/ml). This growth requires the synthesis of -260 /~g bacterial pro /87/$ Federation of European Microbiological Societies

2 246 Table 1 Amino acid requirements of lactic streptococci and the minimum concentrations required for bacterial protein synthesis compared with the levels of amino acids present in milk in low molecular weight form Amino acid Requirement for growth a Minimum concentration required for Concentration in milk (/~g/ml) S. cremoris S. lactis S. crernoris AM2 cell protein free c NPN d synthesis b (~g/ml) Asp Thr Ser _ Glu Pro Gly Ala _ Cys - - ND e nd f nd Val Met 6.5 nd 3.7 Ile Leu Tyr _ ND ND Phe _ 15.8 ND ND Lys _ His Trp ± - ND ND ND Arg _ a = required by all strains tested: = required by some of the strains tested; - = not required [6]. b Based on the amino acid composition of S. cremoris AM2 protein [7], given that the final cell density in coagulated milk is /~g (dry weight) bacteria/ml [8], 52% fo bacterial dry matter is protein [7] and assuming the cell does not synthesize amino acids. Similar data were obtained for S. cremoris E8 [7]. c Values from [7]. Similar levels of free amino acids have been found in aseptically drawn milk [9]. Aston [9] reported Met, Tyr and Phe levels of 0.0, 1.3 and 0.8 #g/ml, respectively. d Amino acids resulting from acid hydrolysis of the non-protein nitrogen (NPN) fraction (soluble in 12% TCA) [7]. e ND = not determined. f nd = not detectable. tein/ml. The concentration of essential amino acids (in free form) in milk is usually well below the minimum required for this cell protein synthesis (Table 1). Even if all the amino acids in the non-protein nitrogen fraction were readily available, the levels of most essential amino acids in low molecular weight form would still limit starter growth in milk. With respect to amino acid requirements, S. thermophilus [10] and L. helveticus [11] appear to be at least as fastidious as the lactic streptococci. Bovine milk contains % (w/w) protein, about 80% of which consists of as1-, as2-, fl- and r-caseins in the approximate proportions 4 : 1 : 3 : 1 [12]. The caseins, which occur as micelles, have an open, largely random structure which makes them readily susceptible to proteolysis. In contrast, the principal whey proteins (fl-lactoglobulin and a- lactalbumin) are globular molecules with a high degree of secondary and tertiary structure. In undenatured form steric factors make whey proteins remarkably resistant to proteolysis. Milk protein may undergo limited hydrolysis during processing resulting in stimulation of starter growth. For example, the proteolysis resulting from high heat treatment of milk [13] is normally stimulatory [14]. Growth of S. thermophilus was stimulated in mastitic milk apparently because of proteolysis caused by elevated levels of indigenous milk proteinase [15]. This serine proteinase, termed plasmin, is derived from blood and occurs in all milk together with its inactive zymogen. However,

3 247 this endogenous activity is low in normal milk where it appears to have little effect on starter growth. The number of specific activity of proteinases synthesized by starter bacteria depend on the composition of the growth medium (see Section 4.1.5). Therefore if proteinase studies are to contribute to an understanding of starter growth in milk, the cells for such studies should be grown in milk (which has had minimal heat treatment). The problems encountered when using milk, which include the difficulties in growth measurement and in isolating cells free of milk protein, have been overcome. Clean cells can be obtained, using low-heat milk which has been subjected to centrifugal pretreatment, by centrifuging cultures prior to precipitation of the casein [16]. To obtain high cell densities the ph may be controlled by automatic alkali addition or by inclusion of sodium glycerophosphate buffer in the milk. The pelleted cells must be washed either at low temperature or in a buffer containing Ca 2 to prevent the spontaneous release of proteinase [17]. Bacterial density may be determined by direct turbidimetric measurement after removal of the opacity of milk with alkaline EDTA [16,18]. The need to use cells that have been grown in milk and then carefully washed has been recognized in several different laboratories [17,19-22]. 4. PROTEOLYTIC ENZYMES The proteolytic system of starter bacteria is usually considered to consist of two functionally distinct classes of enzymes -proteinases, which catalyse the hydrolysis of native or denatured protein molecules, and peptidase, which catalyse the degradation of the smaller peptides produced by proteinases action [2,3] Proteinases Considerable attention has been paid to the study of starter proteinases in recent years but generalizations and comparisons are difficult because of the range of different species and strains studied, the different growth conditions used and the variety of methods employed for release or extraction of proteinases Assay of proteinase activity Definition of an enzyme as a proteinase implies the ability to catalyse hydrolysis of intact protein molecules. Appropriately, most assay methods employed in the study of starter proteinases have used casein or casein derivatives as substrates. Virtually all available methods rely on precipitation of the unhydrolysed casein by trichloroacetic acid (TCA) in samples taken at various times and measurement of TCA-soluble hydrolysis products in the supernatant, preferably using sensitive detection methods. Several studies on starter proteolytic activity have used radiochemical assays employing 14Clabelled casein [17,19,23] or, more recently, a fluorimetric assay based on fluorescamine-labelled casein [24]. The particular fluorometric assay procedure used [25] involved incubation with the derivatized casein at ph 8 which is well above the optimum for many starter proteinases [26,27]. A more satisfactory version of this assay involves detection of hydrolysis products in the TCA supernatant by reaction with fluorescamine at neutral ph when only the free a-amino groups formed as a result of proteolysis will react with this reagent [28]. This enables the assay to be carried out at any desired ph and gives a linear increase in fluorescence with time and a linear dependence of measured activity on enzyme concentration. Another recently developed assay [29] based on fluorescein isothiocyanate-labelled casein has proved very satisfactory for assay of starter proteinase activity (G.G. Pritchard, unpublished data) Cellular location of proteinase activity Unlike many other Gram-positive bacteria, lactic streptococci and other starter bacteria apparently do not secrete significant levels of proteinases into the growth medium. This conclusion rests largely on the absence of evidence to the contrary. A thorough study of the existence of cell-free proteinase activity produced by starter bacteria growing in milk using highly sensitive assay methods, concentrated culture supernatants and satisfying rigorous criteria to eliminate the

4 248 possibility of cell lysis has not apparently been carried out. The determination of low levels of proteinase activity in milk media would present considerable difficulties. An apparent exception is S. cremoris ML1 which grows rapidly in milk but lacks detectable cell wall proteinase activity suggesting the possibility that, in this strain, the proteinase is liberated into the medium [26]. This was verified in a subsequent study of a wide range of starter strains, in which the proteinase activity of strain ML1 was recovered from the growth medium [24]. For all other strains examined, the proteinases primarily responsible for the degradation of extracellular proteins remain bound to the cell wall [2]. The proteinase activity can be partially or wholly released from this site by incubation in CaZ-free buffer [17,19,24,27] or by use of lysozyme [20,30] or phage lysin [31] treatment. In using any of these procedures to obtain preparations of cell-wall-bound proteinase for enzyme characterization it is essential that the extent of release of intracellular enzymes as a result of protoplast lysis or leakage is estimated by assay of suitable intracellular marker enzymes such as lactate dehydrogenase [17,20] or aldolase [20]. A study of the release of cell wall proteinase from Laetobacillus species [21] revealed much more extensive leakage of lactate dehydrogenase from cells treated with lysozyme in the presence of 1 M sucrose than from cells incubated in Ca2-free buffer. The inhibition of release of proteinase from suspensions of whole cells by Ca 2 suggests a role for this ion in binding to the cell wall. Ca ~ is also involved in stabilizing the wall-bound enzyme [32,33]. In a recent study [34] the localization of proteinase in S. cremoris Wg2 has been investigated more directly using immunogold labelling and electron microscopy to detect the proteinase molecules in cell sections after electrophoretic purification of proteinases from this strain and the subsequent isolation of antibodies to the purified enzymes (see below). The published photograph (Fig. 1) shows the presence of gold-labelled antibody in the cell wall region but the rather high intracellular background and the scattered distribution of particles leave open to question the Fig. 1. Immunogold labelling of proteinases in a thin section of S. cremoris Wg2. From Hugenholtz et al. [34]. authors' conclusion [34] that the proteinases are 'clearly seen to be located at the outside of the cell wall'. In view of the possibility that polyclonal antibodies may bind to a variety of bacterial cell wall antigens, the inclusion of proteinase-negative (Prt-; see Section 7) controls treated in the same way would have improved this very useful and direct approach to the study of proteinase localization. The existence and possible functions of intracelhilar proteinases have been thoroughly reviewed [3] and the few recent studies have not clarified the uncertain status and relevance of earlier studies [35-38] on these enzymes. Two distinct types of intracellular proteinase activity, one with an optimal temperature at around C, the other at 5-10 C, have been reported [39,40] from lactic streptococci and L. bulgaricus although the criteria establishing the intracellular location of these enzymes are not clear. Cliffe and Law [20] have attempted to resolve the question as to whether S. lactis contains intracellular proteinases distinct from those in the cell wall by use of polyacrylamide gel electrophoresis of various cell fractions. The proteolytic activity was detected by soaking the gels in casein solution and subsequently in a protein stain revealing proteinases as stain-free zones in the gel. Although the gels from the intracellular fraction contained stain-free zones which could be interpreted as proteinase bands, the same pattern of clear zones was obtained in the presence of proteinase inhibitors in contrast to the cell wall pro-

5 249 teinase gels where the cleared regions were unequivocally indicative of proteinase activity. Whilst the existence of intracellular proteinase activity is of marginal relevance to the use of extracellular protein for the growth of starter organisms the release of such enzymes from lysed bacteria may contribute to ripening and flavour development in the later stages of cheese maturation (see Section 6) Complexity of cell wall proteinase activity A major problem confronting any attempt to review the proteolytic enzyme complex of starter bacteria is the apparent variety of enzymes involved with respect to both the number of enzymes in any one strain and the differences between strains. Some progress has been made towards clarifying the situation for S. cremoris. An early attempt to classify S. cremoris strains with respect to the patterns of cell-wall-associated proteinases [26] involved a survey of the proteolytic activity of whole cells of 14 different strains as a function of ph and temperature. Three types of activity (designated PI, Pit and PIII) were distinguished on the basis of the ph and temperature dependence of proteolysis and the strains were classified according to which combinations of these three activities were present (Table 2). This classification was later used in a study investigating the correlation of bitter peptide production with the type of proteolytic activity [30] (see Section 6). The availability of immunoelectrophoretic techniques opens up the possibility of a more sensitive and specific analysis of relatedness among complex groups of enzymes. This approach has been exploited [24] in a reinvestigation by means of crossed immunoelectrophoresis (CIE) of representatives from the same range of strains as those used in the earlier survey [26]. This procedure allows comparison of enzymes with respect to both electrophoretic mobility and immunological cross-reactivity. In this study, the proteinases were released from the cell wall by incubation in Ca 2free buffer [17], partially purified and used to raise antibodies. On the basis of the precipitation patterns in CIE and the fusion of precipitation lines in tandem Table 2 Compositions of the proteolytic systems of S. cremoris strains (from Hugenholtz et al. [24]) S. crernoris strain Components of the Proteolytic proteolytic system a activities b Wg2, HP, C13 and ML1 A and B PI and PH E8 A and C PI TR, FD27 and US3 A, B and C P1, PII and PIII AM1, SKll A, (B), C and A' PllI a See Fig. 2. b From [26]. CIE, four different enzyme types (designated A, B, C and A') were differentiated (Table 2). Only one of these (component A, common to all strains) was shown to have proteolytic activity in this study. All of the strains studied had at least two different precipitation bands (Fig. 2); some strains had possibly four different components. Indirect evidence that the components other than protein A were also proteolytic was obtained by demonstrating the absence of the components designated B and C in Prt- mutants of strains Wg2 and E8. A fourth component (A') was detected in strains AM1 and SKll but the unavailability of Prt- Fig. 2. Crossed immunoelectrophoresis of the purified proteolytic system from S. crernoris strains Wg2 (a), E8 (b), TR (c) and AM1 (d). From Hugenholtz et al. [24].

6 250 mutants of these strains left the proteolytic identity of this component unconfirmed. All four components were absent when cultures were grown in casein-free medium. In a subsequent study [34], attempts to separate proteins A and B from S. cremoris Wg2 using gel filtration, ion exchange chromatography and isoelectric focusing were unsuccessful indicating that the two proteins had very similar Mrs (140 kda) and isoelectric points (ph 4.5). Separation was ultimately achieved by immunoprecipitation with antibodies raised to the individual proteins excised from the CIE gels. Both proteins had proteolytic activity, each protein contributing about half of the total activity. In view of the difficulties in separating the two proteins, the authors caution against concluding from the demonstration of a single band after electrophoresis on polyacrylamide gels that a single pure proteinase has been isolated [22,27]. The authors considered the possibility that one of the two proteins, A or B, had originated by aut0proteolytic digestion of the other but concluded that this was unlikely since the electrophoretic mobility of the two proteins was very similar. To account for their results a very small change in molecular weight because of autoproteolytic cleavage would have had to confer distinctive antigenic properties on the product. However, in the case of the cell wall proteinase system of S. cremoris HP the presence of several distinct proteolytically active components has been attributed to autoproteolytic modification of a single original proteinase [33]. The two components differed slightly in molecular weight by an amount that may not have been detectable in the CIE system [34]. The lower M r component (126 kda) was believed to be a more stable derivative of the larger protein (130 kda). Further lower Mr, proteolytically active, components originated during diafiltration of solutions of these proteins with a corresponding decline in the amount of the 130 kda form. Amino acid sequence data on the different proteolytic components isolated in the above studies [33,34] are needed to establish whether autoproteolysis is a sufficient explanation for the existence of apparently distinct proteinases produced by a single strain. Another investigation of the number of differ- ent cell wall proteinases present in S. cremoris strains [41] studied the patterns of degradation of the different casein types by partially purified enzymes released by incubation of cells from eight different strains in Ca2-free buffer. Two clearly distinct types of proteolytic activity could be distinguished. One type, designated the AM1 type, showed characteristic degradation patterns with both as1- and fl-casein. The second type, designated the HP type, did not degrade %1- casein but showed a distinctive profile of hydrolysis products with fl-casein differing from that obtained with the AM1 type. The AM1 type, possibly equivalent to the proteinase referred to as PIH in the earlier study [26], was present as the sole type in S. cremoris AM1 and SKll and was present along with the HP type in strains E8 and FD27. The HP type, possibly equivalent to PI in the earlier study [26], was present as the sole type in strains HP, Wg2, C13 and TR. These results, while providing a considerable simplification of earlier classifications of proteolytic enzyme activities in S. cremoris, are difficult to reconcile with other data [34] indicating the presence of a common proteolytic component (component A) in all eight strains of S. cremoris (Table 2). The existence of multiple cell wall proteinases in S. lactis has been less fully investigated. The presence of four proteolytic bands on polyacrylamide gel electrophoresis of cell wall lysates of S. lactis 712 has been demonstrated [20]. At least two of these were encoded on a plasmid since they were not detected in Prt- strains when comparison was made between Prt and Prt- strains grown in M17 broth. Since the other two proteolytic bands were not detected in broth-grown cells their plasmid linkage could not be established. An analysis of the ph and temperature response and the substrate specificity of cell wall proteinase activity in S. lactis suggested only a single type of proteolytic activity in five different strains [42]. The question of the number of different proteinase enzymes in any one strain and the interrelatedness of proteinases in different strains may soon be resolved as a result of rapid progress in mapping the genetic determinants for these enzymes on specific plasmids. The proteinase gene(s) has been mapped on a 33-MDa plasmid of S.

7 251 lactis 712 [43] and on a 17.5-MDa p!asmid in S. cremoris Wg2 [44]. A 4.3-MDa HindlII fragment of the S. cremoris Wg2 plasmid has been cloned in S. lactis and in Bacillus subtilis where it expresses both protein A and protein B [44]. Immunogold detection suggests that the expressed proteinase is cell wall-located in these heterologous systems [34]. The 4.3-MDa restriction fragment from S. cremoris Wg2 is currently being sequenced [34]; this will indicate whether there are two genetically different proteinases in this strain and, if so, the extent to which they are related to each other at the amino acid sequence level. The rapid progress in mapping and cloning of the proteinase genes will make available DNA probes that will provide more precise information on the relationships between the different cell wall proteinases both within and between strains. Considerable clarification of the complexity of the cell wall proteinase system in starter bacteria may be expected in the near future Characterization and substrate specificity of cell wall proteinases In view of the possible multiplicity of cell wall proteinase enzymes and strain differences there is little value in reviewing in any detail the properties of the few enzymes that have been purified to a sufficient level for preliminary characterization to be attempted. Purification or partial purification of cell wall proteinases has recently been reported for S. lactis 763 [45], S. cremoris HP [19,33], S. cremoris AC1 [27] and S. cremoris Wg2 [34]. In general these enzymes appear to be high Mr proteins (- 140 kda for S. cremoris AC1 and Wg2, kda for S. cremoris HP and 80 kda for S. lactis 763), to have ph optima around , to be activated or stabilized by Ca 2 and to be inhibited by the serine protease inhibitors phenylmethanesulphonylfluoride or diisopropylfluorophosphate. Of particular interest is the specificity towards the different caseins present in milk. A survey of the proteolytic activity of a range of S. cremoris strains isolated from mixed starter cultures [46] revealed that all 23 strains that coagulated milk were able to degrade r-casein whilst none was capable of degrading a-casein. These findings are consistent with the results of a number of studies on purified cell wall proteinases. Proteinase from S. cremoris HP preferentially hydrolysed r-casein with only very slow rates of hydrolysis of a~l- and x-casein [19]. Marked preference for r-casein was also found with the proteinases from S. cremoris AC1 [27], from S. cremoris Wg2 [34] and from five strains of S. lactis [42]. However, as discussed above (section 4.1.3), there is a second type of proteolytic activity produced by S. cremoris AM1 and SK11 [41,47] which catalyses the hydrolysis of a~-casein and ~-casein in addition to that of r-casein. It is not yet clear whether this is due to the activity of a single proteinase or a mixture of different enzymes since there are no reported studies on purified enzymes from strains producing this type of activity. Cell-wall-associated proteinases from L. helveticus also catalysed hydrolysis of both asl-casein and r-casein [21]. Similarly, proteinase from L. bulgaricus degraded all types of casein although r-casein was the most susceptible fraction [48]. The question of the bond specificity of the cell wall proteinases has recently been addressed in a study [22] of the peptide products of r-casein hydrolysis by a purified cell wall proteinase from S. lactis 763. Five trifluoroacetic acid (TFA)-sohible peptides were identified after incubation of r-casein with the enzyme for 48 h. These were all ', Ar 9 GI... Glu-Glu-Leu-Asn Va] Pro Giy-Glu-II... I-Glu-Ser-leu-Ser-Ser-Ser-G(l~ - p p p p G,o-~,--I,e-,... ~,,o ~ L.~,.~-I I~-GIu-I..-P.o-G, ~-0,o-0,o G,o GIo-G'L p thr-glu Asp Glu Leu Gln-Asp-lys-lle-Hi~-Pro Phe A1a Gin [hr GInS... Val lyr Pro Phe-Pro-G1y-Pro-[le Pro As Ser leu Prfl-G]n-Asn-]le Pro-Pro-leu-]hr-G1n-lhr-.o v.i.v.,_v,_.._.o_..o,oo ~,n P;o G,...,-~.~ G,. Vo,-~o~ L.~.,,~-~,~ G,o ~or G,.~... t...,....-'/,'-~......,~o:l.:': Set Val-leu-qer-leu-Ser-G]n S~ ~ lys ial Leu Pro-Va~]-Pro-Glu-lys-Ala-Val-Pro-iff~_ rt-, - r s le Gln-AIa-Phc-!eu-leu-lyr-Gln-Gln Pro-Val-Leu-GIy-Pro- Val-Arg-Gly Pro Phe Pro lie [le Vd] L I' Fig. 3. Bonds hydrolysed ($) in B-casein by cell wall proteinase from S. lactis 763. From Monnet et al. [22].

8 252 derived from the C-terminal 43 residue sequence of the 209 residue fl-casein molecule although it cannot be concluded that the remaining 166 residue N-terminal polypeptide remained intact since large hydrophobic peptides derived from this part of the molecule may be insoluble in TFA. The lack of a clear pattern in the bond specificity of the enzyme preparation (Fig. 3) raises the question of its homogeneity. The purity of the enzyme was demonstrated by showing a single protein band on polyacrylamide gel electrophoresis [45] but, as shown for the S. cremoris Wg2 cell wall proteinases [34], this is not a guarantee of homogeneity. On the other hand, if the relatively non-specific cleavage pattern obtained is the result of a single enzyme then further breakdown of the large oligopeptide products between 24 and 48 h would have been expected, yet the 48-h HPLC scan shows no evidence of small peptides. Despite these uncertainties this study is indicative of the type of approach that is now required for a better understanding of proteolysis by starter enzymes Regulation of proteinase synthesis Early studies on starter proteinase activity [26,32] noted that milk-grown cultures had considerably higher levels of cell-wall-bound proteolytic activity than cultures grown in broth media containing high levels of free amino acids and peptides. Two different factors contribute to the difference in proteinase levels between milk-grown and broth-grown cultures. One is a requirement for Ca 2 for the accumulation of proteinase activity in the cell wall. When S. cremoris AM1 was grown in broth media containing 1% casamino acids as nitrogen source with different levels of added calcium, the proteolytic activity of the cells increased in proportion to the calcium concentration [32]. The second factor responsible for the lower proteolytic activity of broth-grown cells is the repressive effect of free amino acids or peptides in the medium. In a recent study of the regulation of proteinase production in S. cremoris AM1 [49] it was shown that low molecular weight peptides present in caseinhydrolysate-containing media may exert a more potent repressive effect than free amino acids. From the results of this study a dual control of proteinase synthesis in starter bacteria was postulated. One control operates to inhibit proteinase-specific mrna synthesis when the endogenous amino acid pool exceeds the requirement for protein synthesis. However, since protein synthesis may be initiated during the repressed state by addition of the transcription inhibitor, rifamycin, a second level of control mediated by an inhibitor of translation of a stable proteinasespecific mrna was proposed. In this scheme, synthesis of the translational inhibitor is also regulated by the free amino acid pool level but at a different level from that triggering transcriptional inhibition. The existence of an accumulated pool of mrna specific for secreted proteinase has been postulated for other bacteria [50,51] but the evidence remains open to question [52] Peptidases Information on the peptidase activities of starter bacteria has been accumulating since the early 1970s and has been reviewed on several occasions [2,3,5,53]. The existence of a wide range of different types of peptidase - aminopeptidases, dipeptidases, tripeptidases and arylamidases - has been described. To date there appear to be no published reports of carboxypeptidase activity from starter streptococci although a highly specific carboxypeptidase from L. casei has been described [54]. Evidence has been produced for the existence of intracellular, cell membrane and cellwall-bound forms of one or more of these types of peptidase. It is not clear how many of these enzymes contribute to the catabolism of extracellular protein. In order to ascertain the significance of this wide array of enzymes it is necessary to establish how many different enzymes are present, their mode of action and bond specificity and their cellular location. Virtually all the information on peptidase activity in starter bacteria relates to various types of exopeptidase, i.e. enzymes catalysing the cleavage of one or two amino acid residues from the free end of the peptide chain. Apart from one early report [23] of endopeptidase activity (distinct from proteinase activity), the existence of starter peptidases capable of hydrolysing oligopeptide substrates at bonds distant from the N- or C-terminal ends has not been investigated.

9 I. Extracellular peptidases Considerable evidence has been accumulated for the existence of extracellular peptidases [27,55-58], i.e. peptidases capable of hydrolysing substrates prior to or during transport across the cell membrane, and has been reviewed previously [3]. Establishing the existence of such enzymes is important for defining the process involved in the utilization of exogenous protein. In view of the presence of a diverse array of highly active intracellular peptidases it is extremely difficult, using conventional cell fractionation procedures, to establish unequivocally that apparently extracellular activity is not due to the release of intracellular enzymes. Indeed the dipeptidase activity recovered from the growth medium [56] appears to be the same as that of one of the intracellular dipeptidases although the absence of other intracellular peptidase activities, e.g. aminopeptidase, in the growth medium raises the question of the basis for the apparently selective 'leakage' [53,58]. However, peptidases located in the cell wall or Table 3 bound to the cell membrane that can be released from these locations by lysozyme treatment appear to differ in their properties from intracellular peptidases [56]. The cell-wall-located dipeptidases from S. cremoris 1196 and S. lactis 763, in contrast to intracellular dipeptidases, were inhibited by mercaptoethanol. Inhibition of cell wall dipeptidase activity by EDTA was reversed by Mg 2 but not by Co 2 or Mn 2 whereas, with intracellular dipeptidases, Co 2 and Mn 2 were specifically required [56]. A comparison of the peptide specificity of cellwall-bound (lysozyme-released) and intracellular (lysis of spheroplasts) peptidases from S. lactis and S. cremoris [58] also provides some support for the existence of a distinct type of extracellular peptidase. Contamination of the cell wall fraction by cytoplasmic contents was assessed to be less than 3% by assay of aldolase and lactate dehydrogenase activities. The specificity profiles were assessed following separation of individual enzymes in the two fractions by polyacrylamide gel electro- Hydrolysis of peptides by soluble cell wall and intracellular fractions of S. lactis (S.1.) and S. cremoris (S.c.) (from Kolstad and Law [581) Substrate in zymogram Intracellular (R F values) S.1. S.c. S.1. S.c. S.1. S.c. Leu-GLy _ a q_ q_ jr_ q_ q_ q_ q_ q_ Pro-Phe - - Phe-Pro.... Ala-Phe.... Ala-Trp.... Leu-Ala.... Ala-Leu.... Pro-Leu..... Pro-Met.... Met-Pro.... His-Phe.... Gly-Phe.... ' Pro-Ile.... Val-Pro.... Trp-Ala..... Leu-Leu- Leu Ala-Leu- Gly Cell wall (R v values) S.1. S.c. S.I. S.c. S.1. S.c. - _ - _ - - _ - _ - _ _ _ a _ ~ no bands detected;,,, = band intensity from very weak to very strong, determined visually.

10 254 phoresis. The cell wall fraction contained a tripeptidase active against trileucine. An enzyme of similar specificity running with just sightly lower relative mobility was also present on gels from the intracellular fraction (see Table 3) raising the question of the distinctiveness of the cell wall tripeptidase. The cell wall fraction also contained a peptidase capable of hydrolysing a wide range of dipeptides. Again, a dipeptidase running with the same relative mobility was present on gels from the intracellular fraction. The cell wall enzyme from both S. lactis and S. cremoris showed a broader spectrum of activity toward the range of dipeptides tested but there were also evident similarities in the pattern of substrate preferences. Since the relative activities were recorded on the basis of visually determined band intensities the apparently narrower range of substrates hydrolysed may have been partly due to differences in the amount of enzyme present, although in the case of one dipeptide (alanyl leucine) the difference was very striking. An intracellular dipeptidase of lower mobility but similarly wide specificity was absent from the cell wall zymograms. The 'cell wall' zymograms from S. lactis and S. cremoris were very similar, a finding that is difficult to reconcile with earlier work [55] which demonstrated that whole cell suspensions of S. cremoris, in contrast to those of S. lactis, were unable to hydrolyse dipeptides while uptake competition experiments also indicated that S. cremoris is largely dependent on dipeptide transport systems and intracellular peptidases for its ability to utilize dipeptides. This same study [58] provided strong evidence for a distinct cell-membrane-located enzyme of very narrow specificity but, as the authors point out, its role in peptide catabolism is doubtful. Kolstad and Law [58] reported that no aminopeptidase activity was detectable in the cell wall fraction. On the other hand Exterkate [57] found that the p-nitroanilide derivatives of alanine, leucine and proline were hydrolysed more rapidly by suspensions of intact cells than by a disrupted cell suspension prepared from an equivalent concentration of cells. This was cited as evidence for an extraceuular location of aminopeptidases acting on these substrates but such a conclusion also requires information on the ability of the p-nitroanilides to penetrate the cell membrane and the possible instability or inhibition of enzymes following disruption. Exterkate questioned the effectiveness of lysozyme treatment for releasing cell-wall-bound enzymes suggesting that enzymes located at the cell wall-membrane interface may remain entrapped in residual polymeric structures not attacked by lysozyme. The purification and properties of an aminopeptidase released by incubation of early stationary phase cells in Ca2-free buffer has been described [27] but the release of intracellular marker enzymes was not monitored. A membrane-bound aminopeptidase specific for glutamyl or aspartyl peptides, and thus resembling aminopeptidase A from mammalian brush border membranes, has been purified from S. cremoris [59]. A role in ensuring an adequate supply of glutamate for microbial growth was suggested for this enzyme. The highly specific nature of this enzyme contrasts with the broad specificity aminopeptidases (presumably intracellular) found in most other studies (see Section ). Thus, while the balance of evidence obtained to date supports the existence of a distinct group of peptidases located external to the cell membrane, further work on the location and properties of individual enzymes of this group is required. Another question concerns the extent to which these enzymes are able to catalyse the breakdown of the large oligopeptide products of proteinase action (Section 4.1.4) to di- and tripeptides or free amino acids. Aminopeptidases of low specificity, if present, could degrade oligopeptides to a size capable of being transported across the cell membrane (Section 5). Alternatively, the exopeptidases could act in co-operation with other endopeptidases. Exterkate [23] reported the presence of two endopeptidases designated P37 and Ps0 which he subsequently [57] concluded were associated with the cell membrane. The endopeptidase nature of these enzymes was assumed from their ability to hydrolyse amino acyl nitroanilides with the N-terminal amino group blocked by a glutaryl group. However, hydrolysis of the C-N bond in aromatic amide derivatives of amino acids is diagnostic of an arylamidase but not necessarily an

11 255 endopeptidase [60]. Deductions on the endopeptidase nature of enzymes based on the use of substituted or derivatized substrates should be made with caution [61] Characterization of exopeptidases from starter bacteria Many of the published studies on peptidases in starter bacteria have involved disruption of cell suspensions by sonication [62-65], French press [66,67] or grinding [68] and investigation of peptidase activity in the cell-free supernatants following various purification procedures. The peptidase activity of such preparations would include intracellular peptidases plus those released from cell wall or cell membrane binding sites by the disruption and solubilization procedures used. Therefore, it is not possible to establish whether the peptidases described are intracellular or extracellular in origin. In a few cases, as described above, attempts have been made to establish the in vivo location of the peptidases under study [ Apart from the difficulty in establishing the cellular location of particular peptidase activities, the number of different peptidase enzymes in any one organism is not yet known. Several attempts have been made to investigate this question by fractionation of cell extracts by ion exchange chromatography, gel filtration or polyacrylamide gel electrophoresis and then assaying for peptidase activity using a variety of substrates. In many cases, the basis for selection of the assay substrates used is not stated - it is no doubt partly determined by substrate availability. The high proline content of the caseins has prompted several investigators to look specifically for peptidases acting on proline-containing substrates. At least five types of exopeptidase showing specificity for peptide bonds involving proline are known [69] (Table 4). Three of these types - aminopeptidase P, proline iminopeptidase (on the basis of hydrolysis of proline fl-naphthylamide) and an iminodipeptidase - were distinguished in cell-free extracts from S. cremoris SKll, S. lactis C2 and S. diacetylactis DRC1 [66]. Recently a X-prolyl dipeptidylpeptidase has been demonstrated in a wide range of Table 4 Exopeptidases Aminopeptidase P (EC ) Proline iminopeptidase (EC ) Iminodipeptidase (EC ) (prolinase) Imidodipeptidase (EC ) (prolidase) Dipeptidylpeptidase IV (EC ) Catalyses cleavage of: X-Pro-Y... Pro-X... Pro-X X-Pro X-Pro-Y... lactobacilli and lactic streptococci and in S. thermophilus [70]. Polyacrylamide gel electrophoresis showed this to be a distinct enzyme from prolyl iminopeptidase which was present at much lower levels of activity in nearly all of the 21 strains of lactic acid bacteria tested. An imidodipeptidase (prolidase) which is highly specific for X-prolyl dipeptides has been purified and characterized from S. cremoris H61 [65]. Proline iminopeptidase and aminopeptidase P activities have also been found in a wide range of lactobacilli [67]. Thus, examples of all five of the above types of peptidase have been reported from starter bacteria. This collection of exopeptidases would contribute significantly to the release of amino acids or dipeptides from the proline-rich oligopeptide products of proteinase action. Apart from exopeptidases showing specificity towards peptides containing proline, such evidence as is available on the specificity of other peptidases suggests the existence of a few enzymes of relatively broad specificity. A highly purified dipeptidase from S. cremoris H61 catalysed hydrolysis of a wide range of dipeptides except those containing proline or glycine as the N-terminal amino acid [63]. The substrates of this enzyme could be classified into three categories on the basis of the K m and Vma x values [64]. Those with the lowest K m (1-2.5 mm) possessed an aromatic amino acid in either the C- or the N-terminal position. A second group with a higher K m (4-6.5 mm) but also with a higher Vma ~ contained neutral hydrophobic amino acids, e.g. Leu or Ala. The third category containing polar charged amino

12 - an - a 256 acids had low Vma x and high g m values. Several of the dipeptides in the first two categories (e.g. Leu-Gly, Leu-Ala, Phe-Ala or Ala-Leu) were also the most readily hydrolysed substrates in other studies on dipeptidases from several other lactic streptococci [55,62] so it seems likely that the dipeptidase of the type characterized by Hwang et al. [63,64] may be widely distributed. Electrophoretic separation of the exopeptidases and the use of a range of dipeptide substrates for activity staining of the gels [58] also suggests the presence of only a small number of dipeptidases of broad specificity. In this study the most readily hydrolysed substrates again belong to the high affinity categories [64], but the dipeptidases resolved on the gels were also capable of hydrolysing dipeptides containing N-terminal proline or glycine (see Table 3) in marked contrast to the S. cremoris H61 dipeptidase. Thus the electrophoretic study [58] is not consistent with the idea that dipeptidases showing high activity toward-proline-containing dipeptides constitute a separate category. Further confirmation of the suggestion that starter bacteria contain a relatively small number of exopeptidases is provided by a recent analysis [71] of the peptidases in 11 strains of lactic streptococci using 53 different substrates including 34 dipeptides, 7 tripeptides, 2 tetrapeptides, 8 aminoacyl p-nitroanilides and 2 carbobenzoxydipeptides. On the basis of the profiles of activity toward this wide range of substrates, the 11 strains were divided into three clusters - A (containing five S. lactis strains and S. cremoris C13), B (containing four S. cremoris strains) and C (containing the single S. cremoris ntr strain). Cell-free extracts from representatives of each of the three clusters were fractionated on DEAE cellulose into three or four different peptidases which were then compared on the basis of substrate specificity, ph optimum and the effect of metal ions and inhibitors. The DEAE cellulose fractionation resolved peaks for: - an aminopeptidase catalysing hydrolysis of several aminoacyl p-nitroanilides, imidodipeptidase (prolidase) catalysing hydrolysis of X-Pro dipeptides, tripeptidase catalysing hydrolysis of all the tripeptides tested, - a dipeptidase with very broad bond specificity (Table 5). On the basis of the response to varying ph and to inhibitors, the 'general dipeptidase' peak (Table 5) was shown to contain at least two different enzymes: an iminodipeptidase (prolinase) acting on Pro-X dipeptides with an optimum ph of 6 and a dipeptidase with ph optimum of acting on all the other dipeptides tested. If these two different components of the dipeptidase peak ran with very similar mobility on polyacrylamide gels under the conditions used by Kolstad and Law [58] it would explain why a separate prolinase was not resolved in the latter study. S. cremoris ntr, the sole representative of cluster C, also had an unusual aminopeptidase fraction resolved on the DEAE cellulose column (Table 5). This fraction was active with a wide range of aminoacyl p-nitroanilides as well as certain dipeptides, tripeptides and tetrapeptides. While this fraction may not have been homogeneous it is also possible that it may contain an aminopeptidase of similar nature to a highly purified and characterized aminopeptidase isolated from L. acidophilus R26 [72]. This latter enzyme, which was shown to account for all the N-terminal exopeptidase activity present in crude extracts, was active against a wide range of dipeptides, a tripeptide and a tetrapeptide as well as aminoacyl p-nitroanilides. An apparently similar enzyme was purified from L. lactis [73]. It is therefore significant that the aminopeptidase activity found in S. lactis 527 [71] was not active against any of the dipeptides tested nor was the dipeptidase fraction able to use aminoacyl p-nitroanilides as substrates. Surveys of this kind [71] using a wide range of different substrates are important since many reports of enzymes designated as dipeptidases or aminopeptidases have not been adequately defined by testing against other possible substrates. A reasonably consistent picture of the exopeptidase complex of lactic streptococci is beginning to emerge from these various investigations. The complex appears to comprise perhaps five or six distinct enzymes - a general aminopeptidase, a wide specificity dipeptidase, two or more exopeptidases acting on bonds involving proline and

13 257 Table 5 Substrate specificities of active fractions (I-IV) of cell-free extract from lactic streptococci (from Kaminogawa et al. [71]) Substrates S. lactis 527 S. crernoris ML-14 S. cremoris ntr I II III IV I II III I II III IV Phe-Ala Val-Ala Tyr-Ala Ala-Ala Leu-Leu Lys-Leu Arg-Asp His-Ser Lys-Phe Glu-Ala Pro-Tyr Pro-Ala Leu-Pro Val-Pro Val-Gly-Gly Leu-Gly-Gly Phe-Gly-Gly Leu-Leu-Leu Ala-Ala-Ala-His Phe-Gly-Gly-Phe Leu-4-NA a AIa-4-NA Lys-4-NA Leu-2-NA b AIa-2-NA Arg-2-NA a 4-NA = 4-naphthylamide. b 2-NA = 2-nitroanilide. a distinct tripeptidase. Some of these may be localized outside the cell membrane possibly as isoenzymic forms of intracellular enzymes. Purification and characterization of each of these enzymes is necessary to establish whether they are all distinct proteins, the range of substrates that they are capable of hydrolysing and the contribution that each enzyme makes to the proteolytic degradation pathway. Elucidation of the complex would be greatly aided by the isolation of mutants deficient in specific peptidases such as have been obtained for enteric bacteria [74]. The extent to which the synthesis is regulated is also in need of investigation. Preliminary evidence [58] suggests that the exopeptidase activity is not affected by the composition of the medium to the same extent as proteinase activity. However, it is important to establish the constitutive nature of these enzymes more convincingly since the studies reviewed above have used starter bacteria grown on a wide range of different media which may account for some of the inconsistencies in the findings of different groups. 5. ROLE OF PROTEOLYTIC ENZYMES FOR GROWTH IN MILK Some growth of starter bacteria in milk can occur without protein hydrolysis since Prtvariants of lactic streptococci grow at low cell densities at almost the same rate as the (Prt ) parent strain [75-77, see Fig. 4a]. However, Prtcells stopped growing at cell densities that were from 5% (in pasteurized milk [77] (to 25% (in autoclaved milk [75,76]) of the maximum cell densities reached by the parent strain and, consequently, milk coagulation was markedly delayed.

14 D T b- :>, o) 10 o (a) S. cremoris 266 -, Prt- (or Prt*)./". ~ / 7/ td 55.~/7.~td 66 rain ~,/ o / / / / ~ / / ~ ~" Prt- (b) S. thermophilus 391 /// : 48 mino, ~ / hydrolysate ~/// I / i / / / ~ t d 73 rain (c) L. bulgaricus 9223 O_-<3 j-",o, - casein // /gontrol td 50 rain / ~ _ ~. I l I I I I I I I Hours Hours Hours Fig. 4. Growth (turbidity at 480 nm; [16,18]) of starter bacteria in low-heat skim milk (solid lines) and in the same milk with added enzyme-hydrolysed casein (broken lines, 10 mg Trypticase/ml). A 2% inoculum of freshly coagulated culture (in autoclaved milk, supplemented with Trypticase for the proteinase-negative variant and S. thermophilus) was added at time zero (t d indicates doubling time). The proteinase-positive parent strain of S. cremoris 266 and its proteinase-negative variant are designated Prt (O, ) and Prt (A, zx), respectively. Incubation temperatures for (a) S. cremoris 266, (b) S. thermophilus CNRZ 391 and (c) L. bulgaricus ATCC 9223 were 30 o C, 42 C and 42 C, respectively. From Thomas and Mills [2]. E El. 73 x 120 E O O- c- r-) >, > Q (a) is AM 2 S. cremoris E 8 (b) 100 E O_ o Va (D Q_ I I I 03 I I I Generations Generations Generations 801 = i I $ "6 >, > o U ~ E 8 Fig. 5. Specific radioactivity of bacterial protein during growth of S. cremoris strains AM 2 and E s in milk containing (a) 14C-labelled free amino acids, and (b) 14C-labelled milk protein. In (a), the addition of 14C-labelled valine (11), isoleucine (A) and glycine (O) increased the original level of free amino acids in the milk (Table 1) by 2.0%, 6.4% and 0.5%, respectively. In (b), the original milk protein concentration increased by 4%. A 2% inoculum was used so that growth stopped and cultures coagulated after about six generations. From Mills and Thomas [7].

15 259 This growth limitation is presumably due to depletion of essential amino acids initially present in the milk in low molecular weight form (Table 1). The specific growth rate of S. cremoris in milk increased 10-20% on addition of mixtures of essential amino acids and also when the casein concentration was increased from 3.0% to 3.5% [78]. After addition of enzyme-hydrolysed casein to milk the growth rates of Prt and Prt- cells of S. lactis C10 [76] and S. cremoris 266 (Fig. 4a) were indistinguishable. When S. cremoris strains were grown in milk containing a4c-labelled free amino acids the specific activity of bacterial protein reached a peak after 2-3 generations using a 2% inoculum (Fig. 5a). The specific activity of protein then declined indicating that, as the cell density reached 8-16% of the maximum, amino acids were supplied increasingly from other sources, consistent with the growth limitation of Prt- variants already discussed. During growth in milk containing 14C-labelled milk protein the specific activity of bacterial protein progressively increased (Fig. 5b) demonstrating that milk protein, and consequently starter proteinase activity, has an increasing role in supplying nitrogen as the cell density increases. Of the different milk proteins, r-casein is more readily hydrolysed by starter proteinases in vitro (Section 4.1.4), suggesting that it may be the principal nitrogen source for growth of Prt cells. Growth experiments with the different caseins as the only nitrogen source in a synthetic medium showed that r-casein in combination with a relatively low concentration of x-casein was the best substrate for growth of S. cremoris HP [79]. However, growth of S. cremoris AM2 and E8 in milk containing individual 14C-labelled proteins showed that all milk proteins acted as a nitrogen source for growth with the relative contribution decreasing in the order x-casein, /3-casein, /3-1actoglobulin, as-caseins and a-lactalbumin [7]. This may indicate that the accessibility of sensitive peptide bonds in milk is different from that in solutions of purified proteins. When r-casein is in self-association polymers, or complexes with asa-casein, its chymosin-sensitive bonds are not accessible to the enzyme [80]. In cheese milk, the polypeptides resulting from rennet action are a further potential source of nitrogen. M,LK CELL WALL ~"2~ OYTOP~AS~ ~;t- ~:~:~z~.;:,l ~, molecular s,eve ~ ~;~N~!~;I 1 CASEIN ~,oligopeptides ~j tr~aen#stp:et ~l~.e~tide s i ~ - pepbdes coupled... ~..., %.%;f, ~o.go -- T - I~'~;~t BACTERIAL I ~, " 1 PROTEIN Fig. 6. Utilization of casein for growth of lactic streptococci in milk. Previous reviewers [2,3] have postulated schemes by which lactic streptococci use casei;~ ;or growth in milk. From the information presently available there appears to be considerable variation in the detail (i.e. the number of different enzymes, their specifcity and their cellular location) of these pathways in different starter strains so a generalized scheme is presented (Fig. 6). Possession of proteinases near the cell wall surface allows cells to hydrolyse large molecules such as casein. Some of the resulting oligopeptides, possibly after further degradation by cell wall peptidases, will be small enough to diffuse into the cell wall - which acts as a molecular sieve. Monnet et al. [22] demonstrated cleavage of r-casein into a 166 residue N-terminal polypeptide and five oligopeptides consisting of 14, 11, 9, 7 and 2 amino acid residues (Fig. 3). It was not established whether prolonged digestion would lead to cleavage of other bonds nor is it known whether other proteinases, with different bond specificities, would degrade some of these to smaller oligopeptides. If, however, this degradation pattern [22] is typical of starter proteinases, and these results are consistent with the qualitative pattern found by others [17,26], then clearly further degradation prior to transport is essential since the size restriction for peptide transport through the membrane is four to five amino acid residues [81-83]. The obligatory involvement of peptide transport systems in the proteolysis pathway appears to differ in different groups of lactic streptococci [55]. They appear to be essential in S. cremoris and some strains of this species actually lack the ability to utilize dipeptides as a source of essential amino acids be-

16 260 cause of the absence of dipeptide transport systems. On the other hand S. lactis strains, while possessing active dipeptide transport systems, are less dependent on these because of the presence of cell-wall-bound peptidases. Distinct systems exist for amino acid, di- and oligopeptide transport in lactic streptococci [55,82,83; see review 53]. A recent study [84] involving S. cremoris Wg2 confirmed the energy-dependent utilization of dipeptides [83] and showed that the hydrolysis of leucyl-leucine was dependent on a ph gradient across the cell membrane. The rate of hydrolysis was apparently limited by the activity of the uptake system since hydrolysis was markedly accelerated on disruption of the permeability barrier but the results leave open the question as to whether the transport and hydrolysis steps are quite independent or are coupled via a membrane-bound dipeptidase system. Peptides may also be further degraded by membrane-bound peptidases (Section 4.2.1). Once inside the cell a wide range of cytoplasmic peptidases is present, apparently in all strains [66], to complete the obligatory hydrolysis of peptides to free amino acids. While our knowledge of peptide transport systems in lactic streptococci is still very limited and that in other starter bacteria is negligible, it is clear from these few studies that both extracellular and intracellular peptidases must contribute to protein catabolism and amino acid nutrition of these organisms. Whether the bulk of the amino acids used for starter growth in milk enter the cell in free form or as small peptides has yet to be defined. Although the level of proteinase in lactic streptococci is low, the overall proteolytic system (Fig. 6) is efficient enough to permit exponential growth in milk to high cell densities with a doubling time of -60 rain at 30 o C. Nevertheless, some step in the supply of nitrogen limits the growth rate in milk since the addition of peptides to milk stimulates the rates of growth and acid production by lactic streptococci [14,85,86; see Fig. 4]. Growth stimulation of lactic acid bacteria by peptides has long been known [see 87] and it appears that they can even be superior to free amino acids as a nitrogen source, in which case they must be taken up intact. It has been sug- gested [53] that peptide uptake can be beneficial over amino acid uptake if (i) peptide hydrolysis is linked to peptide transport; (ii) peptides are transported via a peptide carrier and hydrolysed internally; (iii) after uptake and hydrolysis of peptides some amino acid efflux, which can lead to the generation of a proton motive force, occurs. With S. thermophilus the stimulatory effect of peptide addition to milk [10,88,89] is much greater than with either lactic streptococci or L. bulgaricus (Fig. 4). After enzymatic hydrolysis of whole casein and fractionation of the peptides, it was shown that peptides in the M r range kda were the most stimulatory for S. thermophilus [89]. Stimulation of both lactic streptococci and S. thermophilus by adding/3-galactosidase to milk has been reported [90,91]. However, this effect was not due to lactose hydrolysis but rather to contamination of the enzyme by proteinase [92,93]. As already mentioned (Section 3), growth of S. thermophilus is also stimulated by elevated levels of indigenous milk proteinase. The proteolytic activity of S. thermophilus appears to be even lower than that of the lactic streptococci. Indeed, the growth of S. thermophilus in milk resembles the behaviour of Prt- variants of S. cremoris (Fig. 4). The highly active thermophilic starter cultures therefore contain S. thermophilus combined with more proteolytic lactobacilli [94] which make low molecular weight nitrogen, especially peptides, available for growth of the streptococci [see 95]. Similar stimulatory interactions involving nitrogen nutrition occur between Prt and Prtvariants in single strain cultures and between different strains in mixed cultures [96,97]. In the former case the interaction relates to differing levels of proteinase and the proportion of Prt cells can be reduced to 20-30% of the population without any important change in starter activity [46,86,98,99]. The concentration of low molecular weight nitrogen released in milk by different strains of lactic streptococci at comparable stages in acid development varied 15-fold [100] suggesting that strains differ in their ability to support a high proportions of Prt- variants [101]. In mixed cultures enzyme specificity may also be involved since the number of different proteinases in the cell walls varies between strains (Section 4.1.3).

17 261 Stimulatory interactions suggest that some of the low molecular weight compounds released as a result of proteolytic activity are free to diffuse away from the cell. The decrease in growth rate observed when milk cultures of S. lactis were agitated was thought to be due to the more rapid dispersal of proteolysis products from the cell surface [102]. In addition to the extracellular release of these peptides, there is presumably some efflux of the amino acids generated internally after peptide hydrolysis (Fig. 6). 6. ROLE OF STARTER PROTEOLYTIC EN- ZYMES IN CHEESE RIPENING Proteolysis is of fundamental importance in the ripening of cheese although this process can produce defects as well as contribute desirable changes. Various aspects of this subject have been extensively reviewed, e.g. flavour [103] and especially bitterness development [3, ], texture development [108] and accelerated ripening [109]. The role of the proteolytic enzymes of starter bacteria does not end with the cessation of starter growth. These enzymes, along with the rennet added as coagulant, the indigenous milk enzymes, and those on non-starter lactic acid bacteria, continue to act during cheese ripening with the contribution from the different components often varying with cheese type. With these numerous different proteolytic activities, sometimes acting synergistically, and a myriad of substrates the situation is very complex and interpretation of data difficult. However, the relative roles of the different proteolytic agents in Cheddar and Gouda cheese ripening can be partially defined [103,104, ]. The main role of starter enzymes appears to be the slow degradation of fl-casein and of the polypeptides that result from rennet action, especially on asrcasein. Investigations where both Cheddar and Gouda cheese were made using an aseptic vat technique showed that starter bacteria were responsible for increased levels of small peptides (MrS 1.4 kda) and free amino acids [ ] which are considered important in Cheddar cheese flavour [114,115]. Starter proteolysis thus contrib- utes to flavour development. Collectively, the peptidases of lactic streptococci are probably capable of complete hydrolysis of casein-derived peptides to free amino acids although only - 3% of the total nitrogen content of Cheddar cheese was present as free amino acids after 6 months [113]. The most abundant free amino acids are glutamic acid and leucine [113,115[. This may reflect their high content in casein, the presence of an amino peptidase specific for N-terminal glutamyl residues [59] and the ready hydrolysis of leucyl peptides (Table 3). Casein molecules contain a high proportion of hydrophobic residues (e.g. leucyl, prolyl, phenylalanyl) so that their hydrolysates ha,~c a marked propensity to bitterness, fl-casein, and particularly its C-terminal region, is likely to be the major source of bitter peptides formed in cheese [ ]. A cell wall proteinase preparation from S. lactis 763 released five peptides from this region (Fig. 3) indicating its potential to produce bitter peptides. Studies in model systems suggest that some S. cremoris strains are incapable of producing bitter peptides from whole casein [117]. These 'non-bitter' strains were missing one of the cell wall proteinases (PII, Table 2) present in 'bitter' strains suggesting that enzyme specificity was involved [26]. When the concentration of bitter peptides in cheese exceeds the taste threshold a bitterness defect is detected [see 118]. The level of bitter peptides depends on their rate of formation (which involves primarily rennet but also starter proteinases) relative to their rate of degradation (which mainly involves starter peptidases). Some of the factors relating to starter bacteria that affect these rates are shown in Fig. 7. Strains vary in the actual number of proteinases present (Section 4.1.3). For a given strain the effective concentration of starter proteinase in cheese depends on several factors. The maximum cell density (and hence the level of proteinase) attained in cheese varies with manufacturing conditions, especially the cooking temperatures which for some cheese varieties are at or near the limit for growth. Manipulation of the cooking temperature during Cheddar cheese manufacture so as to give maximum cell densities encouraged bitterness develop-

18 ~larter I(iltraceilular 262. ~ strata r e.~---cell denstty ~ stratn \ Stability of starter enzymes Cheese composition (especially salt-m-moisture) Ripening temperature Fig. 7. Starter-related aspects of proteolysis in cheese. Other enzymes are involved in this process, especially rennet which is a major contributor to the first stage. ment with all five strains tested [120]. The proportion of Prt to Prt- variants (Section 7) in starter cultures is also relevant. Manipulation of this ratio, which allowed the level of starter proteinase in cheese to be varied up to 4-fold while keeping the total concentration of starter cells constant, provided direct evidence that the level of starter proteinase is important in bitterness development in Cheddar cheese [99]. All strains of lactic streptococci have peptidases that are capable of degrading bitter peptides to non-bitter products [117,121] although the activity in 'non-bitter' strains was relatively low at ph 5 [121]. Although it was suggested that degradation of bitter peptides involved peptidases from the plasma membranes [117], it would appear from the cell fractionation procedure used in this study that soluble intracellular enzymes were more likely to be involved. Whereas surface-bound proteolytic enzymes will have access to substrate in cheese regardless of the structural integrity of cells, the access of peptides to the cytoplasmic peptidases is still open to question. As already discussed, peptide transport is energy-dependent. The sole energy source for lactic streptococci in cheese (lactose is not metabolized after the first few weeks from manufacture because of either lactose exhaustion or salt inhibition [122]. Therefore peptides are likely to be hydrolysed by cytoplasmic peptidases only after rupture of the plasma membrane. Electron-microscopic examination of 5-month-old Cheddar cheese showed that starter cell membranes remained remarkably intact even after extensive degradation of the cell wall by autolytic enzymes [123] (Fig. 8). These spheroplasts were maintained without bursting presumably because of the semi-solid gel structure in which they were embedded and the osmotic stability provided by the high solute concentrations in the moisture phase. Even when membranes do rupture, cytoplasmic peptidases are likely to remain localized in the cheese. Previous research suggested that intracellular dipeptidase was released into the cheese matrix as the starter died out reaching maximum concentrations in the cheese days after manufacture [113,124]. However, in these studies dipeptidase was 'extracted' by homogenizing cheese in hypotonic buffer. Under these conditions osmotically fragile spheroplasts will burst releasing intracellular enzymes into the soluble fraction and giving a false impression of their location in the cheese. It is thus possible that the action of intracellular peptidases in cheese is more restricted by substrate access than previously thought. This may explain the apparent conflict between the failure of lysozyme-treated cells to enhance cheese flavour [113] and the increased flavour intensity resulting from the artificial addition of cell-free peptidases from S. lactis [125]. Phage infection can result in a lower incidence of bitterness [126], possibly as a result of premature cell lysis [99]. Bitterness development may therefore be favoured, and the rate of flavour development reduced, when high densities of starter bacteria persist in cheese as intact cells. Strain differences could result partly from different rates of autolysis [127,128]. Other factors affecting the rate of starter proteolysis in cheese (Fig. 7) include the stability of starter enzymes [127], the salt-in-moisture level [107,108] and the ripening temperature [129]. In Cheddar and similar hard cheeses proteolysis is slow, being measured on a time scale of months, and limits the rate of maturation. Therefore to reduce ripening costs studies have been undertaken to accelerate proteolysis by addition of free enzymes that have specific roles [109]. A promising system, which can halve the ripening time, involves the use of liposome-encapsulated proteinase from B. subtilis combined with broad specificity peptidase activity derived from lactic streptococci [125]. The action of these cell-free

19 263 Fig. 8. Thin sections of starter bacteria in 5-month-old Cheddar cheese [123]. An increasing degree of cell wall degradation is evident from (a) to (c) with an intact protoplast shown in (c). The osmotically fragile cells have not burst except where marked ( ~ ) in (b). Even here, release of cell contents is limited. enzymes in the cheese will not be limited by substrate access. Alternative longer-term strategies for accelerated ripening may involve genetic manipulation of starter strains to increase the expression of rate-limiting proteolytic enzymes [130] and to produce strains that are more susceptible to autolysis in the cheese [131]. 7. PROTEINASE-NEGATIVE VARIANTS Early findings [132] indicate that cultures of S. lactis that coagulated milk rapidly (< 16 h at 22 C from 0.1% inoculum) contained cells that, on isolation, took several days to coagulate milk at 22 C. These slow-coagulating variants have been the focus of considerable recent research. Prt variants are distinguished from Prt cells on buffered milk agar [133] where colonies of the former cells are relatively small because of their limited growth (see Fig. 4a). An immunofluorescence method, using antibodies raised against the purified proteolytic system of S. crernoris, has been developed and is capable of distinguishing Prt and Prt- variants within 1 h [134]. The limited characterization of Prt- variants carried out so far suggests that while these cells are deficient in cell wall proteinase [20,24,76] they contain peptide transport systems [82,83] and peptidase activities [135] that are similar to those in the parent cells. Some Prt cultures of lactic streptococci segregate Prt- variants spontaneously because of plasmid loss [136]. In other strains the Prt phenotype is stable apparently because of chromosomal linkage. The mixed cultures used for lactic caseinmaking in New Zealand [86] and for cheesemaking in The Netherlands [97,129] and West Germany [46] often contain predominantly (70-80%) Prt- cells. The recent suggestions that Prt- cells grow faster than Prt cells at ph values above 6.0 [97,137], may have implications for the ph-controlled propagation of starter cultures which is now commonly used to increase culture cell density. The objective of lactic casein and cottage cheese manufacture is basically to recover casein by fermentation of skim milk with lactic streptococci. Any proteolysis results in loss of product thereby reducing the financial return. Some product loss is inevitable since even if the utilization of casein for bacterial protein synthesis during starter growth was 100% efficient then, from the data in Table 1,

20 264 at least 1% of the casein would be removed. Actual casein losses of 4-7% have been measured with Prt strains of S. cremoris [77]. The potential advantages of using rapid acid-producing starter cultures containing predominantly Prt- variants to increase product yield [86] have been proven for both lactic casein [77] and cottage cheese manufacture [138]. In these processes the contact time between substrate and enzyme is much longer than in the cheese vat and contact occurs at a more favourable (lower) ph for proteinase activity [26]. The yield increase claimed for the use of Prtvariants in Cheddar cheese manufacture [sse 139,140] has not been proven. Although the exclusive use of Prt- variants in cheesemaking has been advocated [ ], the other suggested advantages (decreased bitterness, reduced sensitivity to phage and antibiotics) would normally be offset by the need for different culture propagation and cheesemaking procedures. A likely consequence of using Prt- cultures would be a reduced ripening rate since gross proteolysis can be rate-limiting in the sequence of reactions leading to the accumulation of small peptides and amino acids [125]. 8. CONCLUDING REMARKS Much useful information resulting from the biochemical characterization of individual enzymic components of the proteolytic system has accumulated over the last two or three years. However, the interpretation of this information, in order to clarify the role of the system in milk fermentations, is fraught with difficulties. Knowledge of the genetic determinants of cell wall proteinase activity and the ability to manipulate these genes and their expression will enable rapid progress to be made in our understanding of the complexity, the mode of action and regulation of this group of enzymes. It may also help eventually in the isolation of useful new strains since many potential starters are deficient in proteolytic activity. Even less is known of the genetic basis of peptidase activity and peptide transport systems and the extent to which these components are subject to regulation by nutritional and other environmental factors. The proteolytic reactions of starter bacteria involve a balancing act in which a compromise must be achieved between just enough but not too much digestion. The developing commercial interest in proteinases and peptidases and their potential application in the food industry will inevitably impose restrictions on the free circulation of information on this complex of enzymes and its role in the nutrition and growth of starter bacteria. It would be very unfortunate if the study of this area, having reached a stage at which the problems are becoming clearly defined and the means for investigating them more readily available, now becomes increasingly a casualty of commercial secrecy. ACKNOWLEDGEMENTS We are grateful to our colleagues for helpful discussions and to many authors for supplying preprints of their work. REFERENCES [1] Lawrence, R.C. and Thomas, T.D. (1979) The fermentation of milk by lactic acid bacteria. In: Microbial Technology: Current State, Future Prospects (Soc. General Microbiol. Symposium Series, Vol. 29)(Bull, A.T., Ellwood, D.C. and Ratledge, C., Eds.), pp Cambridge University Press, Cambridge. [2] Thomas, T.D. and Mills, O.E. (1981) Proteolytic enzymes of starter bacteria. Neth. Milk Dairy J. 35, [3] Law, B.A. and Kolstad, J. (1983) Proteolytic systems in lactic acid bacteria. Antonie van Leeuwenhoek 49, [4] Desmazeaud, M. (1983) L'6tat des connaissances en mati~re de nutrition des bact6ries lactiques. Lait 63, [5] Marshall, V.M.E. and Law, B.A. (1984) The physiology and growth of dairy lactic-acid bacteria. In: Advances in the Microbiology and Biochemistry of Cheese and Fermented Milk (Davies, F.L. and Law, B.A., Eds.), pp Elsevier Applied Science Publishers, London. [6] Reiter, B. and Oram, J.D. (1962) Nutritional studies on cheese starters, 1. Vitamin and amino acid requirements of single strain starters. J. Dairy Res. 29, [7] Mills, O.E. and Thomas, T.D. (1981) Nitrogen sources for growth of lactic streptococci in milk. N.Z.J. Dairy Sci. Technol. 15, [8] Turner, K.W. and Thomas, T.D. (1975) Uncoupling of growth and acid production in lactic streptococci. N.Z.J. Dairy Sci. Technol. 10,

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