Peptidases of dairy propionic acid bacteria Valérie Gagnaire, Daniel Mollé, Terje Sorhaug, Joëlle Léonil

To cite this version:

Valérie Gagnaire, Daniel Mollé, Terje Sorhaug, Joëlle Léonil. Peptidases of dairy propionic acid bacteria. Le Lait, INRA Editions, 1999, 79 (1), pp.43-57. ￿hal-00929636￿

HAL Id: hal-00929636 https://hal.archives-ouvertes.fr/hal-00929636 Submitted on 1 Jan 1999

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Lait (1999) 79, 43-57 43 © Inra/Elsevier, Paris

Review

Peptidases of dairy propionic acid bacteria

Valérie Gagnaire-", Daniel Mollé-, Terje Serhaug", Joëlle Léonil"

a Laboratoire de recherches de technologie laitière, Inra, 65, rue de Saint-Brieuc, 35042 Rennes cedex, France b Department of Food Science, P.O. Box 5036 Agricultural University of Norway, N-1432 Âs, Norway

Abstract - Although propionic acid bacteria dominate Swiss-type cheeses, with populations reach- ing -109 colony forming units per g of cheese after the warm room ripening period, their role in proteolysis is far from being as weil established as that of the lactic acid bacteria. This review will focus on the localization and biochemical characteristics of the peptidases of dairy propionic acid bacteria. Using peptide substrates related to those encountered in cheese and electrospray ionization mass spectrometry analysis, the action of (s) in addition to that of (s), X-prolyl dipeptidyl aminopeptidase and (s) are c1early shown. Finally, the potential role of the propionic acid bacteria in Swiss-type chee se proteolysis is discussed. © InralElsevier, Paris.

propionic acid bacteria / proteolysis / peptidase / Swiss-type cheese / ripening

Résumé - Les peptidases des bactéries propioniques. Bien que les bactéries propioniques repré- sentent une flore dominante des fromages à pâte pressée cuite, avec une population pouvant atteindre environ 109 unités formant colonie par gramme de fromage pendant l'affinage en cave chaude, leur rôle dans la protéolyse au cours de l'affinage est encore loin d'être établi comparativement à celui des bactéries lactiques. Cette revue est consacrée aux peptidases des bactéries propioniques, à leur loca- lisation et à leurs caractéristiques biochimiques. L'utilisation de substrats peptidiques proches de ceux rencontrés dans le fromage et l'identification par spectrométrie de masse à source d'ionisation electrospray des produits issus de l'hydrolyse, par les peptidases de quatre souches de bactéries pro- pioniques, nous a permis de montrer clairement l'action de carboxypeptidase(s), en complément de celles d'aminopeptidase(s), X-prolyl dipeptidyl aminopeptidase et endopeptidase(s). Enfin, nous discuterons le rôle potentiel des bactéries propioniques dans la protéolyse des fromages à pâte pres- sée cuite. © InralElsevier, Paris.

bactérie propionique / protéolyse / peptidase / fromage à pâte pressée cuite / affinage

Oral communication at the 2nd Symposium on Propionibacteria, Cork, Ireland, June 25-27, 1998. * Correspondence and reprints. [email protected] 44 V. Gagnaire et al.

1. INTRODUCTION cheese after the warm room ripening period. However, their role in the proteolytic process Besides the propionic acid fermentation is not at ail as clear as that of the LAB ([ 15], which, in Swiss-type cheeses, leads to the [39]). production of typical flavour (acetate and The proteinases of PAB have been recen- propionate) and COz' responsible for the tly reviewed by Langsrud et al. [21], and formation of the eyes in the cheese, prote- appear to be of modest importance in the olysis is the main factor in ripening and breakdown of caseins. Indeed, dairy PAB flavour development [39]. A complex pro- grow slowly in milk [3], rendering their cess of casein breakdown into peptides and growth in cheese dependent on the primary amino acids occurs during ripening through hydrolysis of caseins by LAB during ripen- the catalytic action of several proteolytic ing. In contrast, dairy PAB have high pep- agents: rnilk coagulant, indigenous rnilk pro- tidase activities [34] suggesting they may teinase (plasmin), starter, non-starter and be involved in the hydrolysis of peptides secondary starter proteinases and peptidases. produced from caseins. In hard cooked cheeses, the indigenous The localization and biochemical char- milk proteinase, plasmin, is mainly respon- acteristics of peptidases of dairy PAB, sible for the primary hydrolysis of the including new results on their substrate caseins since the coagulant, rennet, is almost specificities, will be reviewed and compared completely inactivated during cooking and with LAB peptidases. has only a weak action on the caseins [3]. The main peptides identified in Parmigiano Reggiano [1, 2] and in Grana Padano [13] 2. PAB PEPTIDASES clearly showed the prominent action of plas- min on ~- and asz-caseins as weil as the 2.1. Detection of peptidase activity recurrent action of arninopeptidases and car- boxypeptidases of the microflora. Various peptidase activities have been detected in dairy PAB (table l). Sahlstrôm et Several studies have reported an essential al. [34] found that most of the peptidase role for the proteolytic agents from the activities present in two strains of P.freuden- starter lactic acid bacteria (LAB) in Swiss- reichii subsp. shennanii were mainly located type chee se [15, 39]. By comparing exper- intracellularly. Of the 6 to 7 peptidase bands imental cheeses manufactured with detected, one was associated with the cell aminopeptidase deficient mutant strains of wall, while two or three were associated Lb. helveticus or the parental strain as starter, with the membrane. Carboxypeptidase activ- Prost and Chamba [32] showed that the ity was reported by Sahlstrôm et al. [35] and enzymic system of this species can account two different isolated from the cell for one-third of the aminopeptidase activ- wall of two P.freudenreichii strains. El Soda ity found in cheese. Recently, general et al. [9, 10] also studied the intracellular aminopeptidase and X-prolyl system of Propionibacterium and arninopeptidase (PepX) activities from ther- found that the seven strains studied con- mophilic starters were shown to be promi- tained aminopeptidase, and nent in Emmental juice extracted before the caseinolytic activity. Floberghagen et al. secondary flora, which is mainly composed [14] showed that PAB strains have a wide of propionic acid bacteria (PAB), grows range of substrate specificities with high [16]. activity on di peptides and peptides con- Dairy PAB are the dominant flora in taining Pro or Phe as the N-terminal amino Swiss-type cheeses and reach numbers of acid. Such a high activity on N-terminal -109 colon y forming units (cfu) per g of aromatic amino acids, especially towards Table I. Peptidase activities detected in dairy PAB. Purified enzymes are shawn in table II. Tableau I. Activités peptidasiques détectées chez les bactéries propioniques laitières. Les enzymes purifiées sont présentées dans le tableau II.

Strains Substrate used Proteolytic activity tested Location References

P. shermanii a and FSCN 33 dipeptides including Pro-X dipeptidase activity and enzymes acting intracellular [14] P. pentosaceum ATCC487 5 and Phe-X peptides specifically on Pro-X peptides and others P. arabinosum ATCC 4965 on Phe at the amino end ;:p P. shermanii ATCC9614 ~ ~0.: ocr> P. freudenreichii subsp. 15 dipeptides and tripeptidases cell-wall, intracellular, [34] cr> o shermanii ATCC9614 membrane ...., 0- P.freudenreichii subsp. 3 tripeptides ~ shermanii INFa Q" "0 8 "0 P. freudenreichii subsp. Leu-pNA" and proline intracellular [30] ci' ::l shermanii ATCC13673 and E22 Pro-pNA iminopeptidase ri' P.freudenreichii subsp.freudenreichii GP6 f5 Tl 0.: P. acidipropionici ATCC4875 and 0" P. jensenii ATCC4869 f5 <> P. thoenii ATCC4874 ~:J.

P. shermanii NZ pNA derivatives aminopeptidase dipeptidyl aminopeptidase intracellular [9]

P. freudenreichii CNRZ23, CNRZ82, AU59 and AU722 intracellular P. acidipropionici CNRZ80 pNA derivatives dipeptides aminopeptidase [10] P. jensenii CNRZ87 whole casein caseinolytic activity

-l'o- Ut Table I. (continued) / Tableau I. (suite). """0'0

P. freudenreichii subsp. ~NAb derivatives aminopeptidase including activity extra, parietal and [7] [reudenreichii CIP103026 intracellular P. [reudenreichii subsp. specifically towards aromatic amino acids shermanii CIPI 03027 P. jensenii CIP 103028 P. thoenii CIP103029 carboxypeptidase and endopeptidase activity P. acidipropionici DSM4900 (not evidenced)

P. [reudenreichii subsp. shermanii N-Benzoyl-Gly-Phe (or Lys) carboxypeptidase cell-wall [35] ATCC9614 and INFa

P. [reudenreichii subsp. Leu-pNA, Pro-pNA, aminopeptidase intracellular [31] shermanii ATCC96!4 Gly-Pro-pNA derivatives PepX' intracellular and cell wall < Cl bradykinin and N-CBZ-Phe-Arg-AMCd intracellular ~ endopeptidase (JQ :l el..,. P. freudenreichii subsp. shermanii CIP! 0302 ~NAb and pNA derivatives specifie PepIe intracellular [33] (0 to proline PepX ~ dipeptides prolinase ~ prolidase carboxypeptidase P aminopeptidase P

P.frelldenreichii ISU P59 Pro-Gly PepI intracellular [28] P. acidipropionici ATCC4875 Gly-Pro other dipeptides containing or not Pro dipeptidases tripeptides tripeptidases

a pNA, or peptides para-nitroanilide derivatives / a pNA, dérivés para-nitroanilides de peptides ou d'acides aminés. b ~NA, amino acid or peptides j3-naphthylamides derivatives / h ~NA, dérivés ~-naphthylamides de peptides ou d'acides aminés.

C PepX, X-prolyl dipeptidyl aminopeptidase activity / C PepX, activité X-prolyl dipeptidyl aminopeptidase. dAMC, amid0-4-methyl-eoumarin / dAMC, amid0-4-methyl-eoumarine. e Pepl, proline iminopeptidase / e PepI, proline iminopeptidase. Peptidases of dairy propionic acid bacteria 47

Phe, was also found in two other P. freun- (molecular weight of 69 kDa, optimum denreichii strains [7]. Leucine-aminopepti- activity at pH 7.5 and 37 "C) were found dase activity was found in each of four dairy [7]. Both are metallo-enzymes which are PAB species, P. freudenreichii (including also inactivated by thiol inhibitors, indic at- subspecies freudenreichii and shermanii), ing probable requirement for a thiol group P. acidipropionici, P. jensenii and P. thoenii for activity. [30]. The Leu-aminopeptidase activity [30] Because of the high proline concentra- has been partially purified by El Soda et al. tion found in Swiss-type cheese, most atten- [9]. tion has been paid to activities cleaving pep- tide bonds involving proline. This has been 2.2.2. Dipeptidylaminopeptidase attributed to proteolytic activity of PAB by and dipeptidases Langsrud et al. [19,20]. Proline iminopep- tidase (PepI) and PepX activities were described by several authors [10, 12,27,30, It is only very recently that PepX from 31,33]. Quelen et al. [33] have reported P. freudenreichii subsp. shermanii NCDO prolinase and prolidase activities, but have 853 has been purified by a combination of not found carboxypeptidase or endopepti- diethyl-amino-ethyl (DEAE) chromatogra- dase activities specifie for proline. phy, hydrophobie interaction chromatogra- phy and chromatofocusing [11]. This intra- cellular , with a molecular mass of 2.2. Purification and characterization 84 kDa, is a serine inhibited by of the peptidases metals, such as Cu2+. It is active at acid pH values with 95 % of maximal activity at Table II summarizes the characteristics pH 5.5, whereas the PepX from most Lac- of the peptidases that have been purified tobacillus and Lactococcus species have a from dairy PAB. pH optimum close to neutrality. Sahlstrëm et al. [35] purified two cell- 2.2.1. wall peptidases, one from P. freudenreichii subsp. shermanii ATCC 9614 with a Rf Among the peptidases, PepI in cell-free value in PAGE of 0.57, and another from extracts of P. shermanii 13673 was the first P. freudenreichii subsp. shermanii INF-a enzyme to be purified [29]. The enzyme with a Rf of O. They could potentially rep- was inhibited by phenylmethylsulfonyl flu- resent the first breakdown step of casein oride suggesting it was a serine enzyme and peptides originating outside the cell [35]. by ethylene-diamine-tetra acetic acid Both are metallo-enzymes, with thiol groups (EDT A) indicating a requirement for metal at the . Their molecular masses ions. This peptidase hydrolysed specifically determined by gel filtration, were 82 and Pro-pNA and dipeptides, but only if they 134 kDa for Rf 0 and Rf 0.57, respectively. had a proline residue in the N-terminalposi- These cell wall peptidases hydrolyse tion (such as Pro-Met, Pro-Phe, Pro-Leu, Pro-Phe as weIl as N-terminal blocked pep- Pro-Ile and Pro-Gly) and the ~-casomor- tides (N-benzoyl-Gly-Phe and N-ben- phin des-Tyr fragment 7. The gene encod- zoyl-Gly-Lys) suggesting a carboxypepti- ing PepI has been cloned [46] and the intra- dase activity, but have little or no cellular localization of the protein aminopeptidase activity on Leu-pNA, Lys- confirmed. pNA and Ala-pNA. Carboxypeptidase In P.freudenreichii subsp. freudenreichii activity has been rarely described in LAB CIP103026, two fractions active towards except by El Soda et al. [8] for Lactobacil- Phe with closely related characteristics lus casei. -l'>- Table II. Characteristics and localization of the peptidases purified from dairy PAB. 00 Tableau II. Caractéristiques et localisation des peptidases purifiées chez les bactéries propioniques laitières.

Enzyme Substrate Strain Purification Mw Quatemary Type Optimum Location References kDa structure pH

Pepl Pro-!-X-(X)n P. shermanii 13673 homogeneity 61* Mono S 8.0 intra [29] cloned [46] PepX X-Pro-!-(X)n P. shermanii NCD0853 84*+ Mono S 5.5-7.5 intra? [II] Aminopeptidase Leu-!-X-(X)1l P. shermanii NZ partial ND ND M #7.0 intra [9] Lys-!-X-(X)n Dipeptidyl aminopeptidase Gly-Pro-!-(X\, P. shermanii NZ partial ND ND S #7.0 intra Phenylalanine aminopeptidase Phe--!-X-(X)n P. [reudenreichii subsp. homogeneity 69* Mono C 7.5 intra [7] < [reudenreichii CIPI 03026 ~Cl 134+ cell wall [35] (IQ Peptidases/ Pro-Phe and P. [reudenreichii subsp. partial ND M 8.6 ~;:l carbox ypeptidases N-Benzoyl-Gly-Phe (or Lys) shermanii ATCC9614 (Rf 0.57) §' P. [reudenreichii subsp. partial 82+ ND M 10.0 cell wall ~ shermanii INFu (Rf 0) ~ Endopeptidase bradykinin P.[reudenreichii ATCC9614 homogeneity 44+ Mono M 6.5 -8.0 intra [44] casein Met-enkephalin angiotensin 1 us1CN(l-23)

Oligopeptidase bradykinin Us{N(l-23) P. [reudenreichii ATCC9614 homogeneity 67' Mono M 6.7-7.5 intra [40] us1CN(l65-1 9) oxidized ~-chain insulin Oligopeptidase bradykinin, peptides < 17 aa P. [reudenreichii ATCC9614 homogeneity 33' Tetra M 6.7-7.5 intra [41]

Mw, Molecular weight determined by SOS-PAGE (*) or/and by gel filtration (+); intra, intracellular; S, ; M, metalloprotease; C, cystein protease; ND, not determined; J. hydrolysis site. Mw, masse moléculaire déterminée par SOS-PAGE (*) ou/et par gel filtration (+) ; S, sérine protease ; M, métalloprotease ; C, cystéine protease ; ND, non déterminé; J. site de coupure de l'enzyme. Peptidases of dairy propionic acid bacteria 49

2.2.3. retained at 7 "C and at pH 5.5, i.e. under conditions which simulate cheese. It is also One endopeptidase [44] and two active on other peptides, such as

3.2. Autolysis ofPAB and impact cheese during ripening requires an autolytic of the peptidases released mechanism, which is induced either physi- on the secondary proteolysis ologically or by environmental conditions in Swiss-type cheese [25]. The autolysis of PAB was studied by sev- As discussed aiready, PAB peptidases erai au th ors [19, 20, 23, 24, 27, 28]. are mainly located intracellularly. To have a Langsrud et al. [19,20] have observed a direct effect on proteolysis, the release of correlation between autolysis and the pro- these intracellular peptidase activities in duction of free proline in milk or modified

Figure 1. Identity of the peptides produced after 24 h hydrolysis of the tryptic/chymotryptic hydrolysate of f3-casein by the cell-free extracts of the four dairy propionibacteria species. A) Initial tryptic/chy- motryptic f3-casein hydrolysate. B, C, D and E) correspond to the peptides present after 24 h hydrol- ysis by the cell-free extracts of P. freudenreichii subsp. freudenreichii CIPI 03026, P. jensenii CIP103028, P. thoenii CIPI 03029 and P. acidipropionici TL249, respectively. ~-Casein (10 g-L-1 in sterile distiIled water) was first hydrolysed by a mixture of trypsin (5000K, Novo Industry A/S, Copenhagen, Denmark) and chymotrypsin (Sigma, Saint-Quentin Fallavier, France), both at an enzyme/substrate ratio of 1:1 000 (w/w). The pH was maintained at 7.2 by adding 0.5 mol-L:! NaOH for 3 h at 37 oc. Then, the enzymes were inactivated by heating at 80 "C for 20 min. The f3-casein hydrolysate was freeze-dried and stored at 4 "C until use. The hydrolysis of this tryptic/chymotryp- tic digest of f3-casein by the cell-free extract of PAB was performed at 30 "C in 50 mmol-L:' sodium phosphate buffer at pH 5.7 for 24 h containing 0.7 mg of freeze-dried ~-casein peptides-ml,"! and 137.5 ug intracellular protein. The reaction was stopped by heating at 100 "C in a water bath for 10 min. The samples were analysed by reverse-phase HPLC coupled on-line with electrospray ion- ization mass spectrometry and the molecular masses determined. Each peak was collected and sequenced by collision-induced dissociation using tandem mass spectrometry as described by Gag- naire et al. [17]. The conditions of elution on RP-HLPC were the same as described by Lemée et al. [22]. In the sequence of f3-casein U corresponds to phosphorylated serine residues. (0) corresponds to the peptides produced by the action of trypsin and chymotrypsin and (.) to those resulting from the action of the peptidases of PAB. V, chymotrypsin; T, trypsin action Figure 1. Identité des peptides produits après 24 h d'hydrolyse de l'hydrolysat trypsique/chymo- trypsique de caséine ~ par les extraits intracellulaires des quatre espèces laitières de bactéries pro- pioniques. A) Hydrolysat trypsique/chymotrypsique de caséine ~ initial; B, C, D et E) correspondent aux peptides présents après 24 h d'hydrolyse en présence respectivement d'extrait intracellulaire de P.freudenreichii subsp.freudenreichii CIP 103026, P. jensenii CIP 103028, P. thoenii CIP 103029 et de P. acidipropionici TL249. La caséine B, 10 g-L-1 d'eau distillée stérile, était initialement hydro- lysée par un mélange de trypsine (5 OOOK,Novo Industry A/S, Copenhague, Danemark) et de chy- motrypsine (Sigma, Saint-Quentin Fallavier, France), chacune avec un rapport enzyme/substrat de 1 :1()()O(p/p), à pH 7,2 maintenu constant par addition de NaOH 0,5 mol-L'" pendant 3 h à 37 oc. Puis les enzymes étaient inactivées par chauffage à 80 "C pendant 20 min. L'hydrolysat ainsi obtenu était lyophilisé et conservé à 4 "C jusqu'à utilisation. Les conditions d'hydrolyse de l'hydrolysat trypsique/chymotrypsique de caséine ~ par les extraits intracellulaires de bactéries propioniques étaient les suivantes: les extraits intracellulaires (137,5Ilg eq BSA·mL-I) étaient incubés à 30 "C en présence de 0,7 mg de peptides de caséine ~ lyophllisés-ml.vde tampon phosphate de sodium 50 mmol-L:' à pH 5,7 pendant 24 h. La réaction était arrêtée par chauffage dans un bain-marie à 100 "C pendant 10 min. Les échantillons étaient analysés par CLHP de phase inverse couplée au spectromètre de masse à source d'ionisation electrospray et les masses moléculaires étaient ainsi déterminées. Chaque pic était collecté puis séquencé par fragmentation au moyen du spectromètre de masse selon les conditions décrites par Gagnaire et al. [17]. Les conditions d'élution en CLHP de phase inverse étaient les mêmes que celles utilisées par Lemée et al. [22]. Dans la séquence de la caséine ~, la lettre U correspond à la sérine phosphorylée. (0) correspond aux peptides produits lors de l'hydrolyse par la trypsine et la chymotrypsine de la caséine ~ et (.) à ceux résultant de l'hydrolyse par les peptidases des bactéries propioniques. Action de la chymotrypsine (V); et de la trypsine (T). A 4 dl- 1LI_ 1il LIN 1vip 1!JI _ 1 ITVI _! IJ, Liu 1u 1u ?_Ol_ 1sil 1T ?ir liN 1Kh r3~1 _ 1Khi 0 13JI _ 1_ 10 10 l'l?I TI_ 1DI_ 1 270 1 DI KT1 15~1pl Ff

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160 170 175 180 205 209 FlplplolS zrvrrrrrvrt AlvlplvlP A\9.?\' L 1LI ViE ig~ plv 1LI G ~~O RIGlplrlfl1 Peptidases of dairy propionic acid bacteria 55

milk media. Furthermore, autolysis was the secondary proteolysis in cheese. This strongly strain dependent and varied largely was also observed in a non-Swiss-type from 10 to 90 % in both buffer and sodium cheese, Cheddar, with added PAB by Fer- lactate broth. In P. freudenreichii cells, nandez-Espla and Fox [12]. The addition of autolysis appeared to be strongly induced P. shermanii NCOO 853 at high levels to by depletion oflactate [23, 24, 27, 28]. How- Cheddar (between 107 and 109 cfu-g! of ever, in contrast to LAB in Cheddar [48] cheese) led to minor quantitative differences and in Saint Paulin [6], direct evidence of but no qualitative one in the peptide profile autolysis in Emmental cheese was not estab- of chee se made with and without PAB. lished until recently [47]. Using immunoblot- Recent data obtained in our laboratory ting analysis, Valence et al. [47] showed show that PAB utilize peptides produced that autolysis of P.freudenreichii occurred by the peptidases and proteinases of LAB very late during cold storage of Emmental as weil as most arnino acids except A1a, Pro, and that its extent was limited. In industrial Leu and Lys (unpublished results). This sug- French Emmental cheeses, neither deple- gests either transport of peptides into the tion of lactate nor loss of viability of PAB PAB cells and their subsequent hydrolysis after 60 d of ripening was observed, sug- by intracellular peptidases, or a turnover of gesting a low frequency of autolysis by PAB these amino acids during PAB growth. Pep- [43]. It is likely that in Swiss-type chee ses tide and amino acid utilization may induce with longer ripening times, the PAB could changes in the qualitative balance of pep- autolyse and release their enzyme pool. tides and amino acids potentially responsi- Thus, damaged cells of PAB were observed ble for the flavour characteristic of Swiss- in the ltalian cheese, Grana Padano, by scan- type cheeses. It will be interesting to study ning electron micrographs after eight months whether this impact on proteolysis is more of ripening [5]. pronounced in the case of Swiss-type To appreciate the real impact of PAB in cheeses with long ripening periods in which cheese is rather difficult because their the autolysis phenomenon of PAB cells may growth begins when that of LAB has ended. be more apparent, than in French industrial The impact of LAB seems to be prominent Emmental with its 60 d ripening time. since in most cases, the y have a higher autolytic [47] and proteolytic activity [16] than the PAB strains in cheese. Thus, 4. CONCLUSION throughout ripening, reverse phase HPLC peptide profiles of juice or aqueous water PAB appeared to be equipped with many extracts of Emmental cheeses showed a spe- of the enzymes necessary to produce amino cific 'fingerprint' which appeared from the acids and small peptides from caseins [21] cold room stage and can be related to the with II different peptidases purified and prominent action of proteolytic enzymes characterized. However, in contrast to LAB, from LAB starters (unpublished observa- it is not possible to find the complete prote- tions). The further modifications in the pep- olytic pathway of casein degradation into tide profile induced by PAB is compara- peptide and amino acids in PAB. Even if a tively low. Quantitatively, the nitrogenous proteinase and one or two peptidases are fractions produced by proteolysis (soluble N distributed in the cell wall and can initiate and non-protein N fractions) increased sig- casein hydrolysis, most of the peptidases nificantly during the warm room period, are intracellular. Consequently, for further when PAB have not yet reached a sufficient hydrolysis of casein derived peptides by population to participate significantly in pro- intracellular enzymes, peptide transport sys- teolysis in cheese « 107 cfu-g! of cheese) tems are necessary. This is especially crucial [43], confirming the low impact of PAB in since autolysis appears to be quite limited 56 V. Gagnaire et al. and the release of intracellular peptidases [9] El Soda M., Macedo A., OIson N.F., Aminopep- consequently weak. 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