Peptidases of Dairy Propionic Acid Bacteria Valérie Gagnaire, Daniel Mollé, Terje Sorhaug, Joëlle Léonil
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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 carboxypeptidase(s) in addition to that of aminopeptidase(s), X-prolyl dipeptidyl aminopeptidase and endopeptidase(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 enzymes 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 dipeptidyl peptidase 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, dipeptidase 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 dipeptidases 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" Leucyl aminopeptidase 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