Antimicrobial activity and stability of partially purified bacteriocins produced by Propionibacterium freudenreichii ssp. freudenreichii and ssp. shermanii Daniela Gwiazdowska, Krystyna Trojanowska To cite this version: Daniela Gwiazdowska, Krystyna Trojanowska. Antimicrobial activity and stability of partially purified bacteriocins produced by Propionibacterium freudenreichii ssp. freudenreichii and ssp. shermanii. Le Lait, INRA Editions, 2006, 86 (2), pp.141-154. hal-00895570 HAL Id: hal-00895570 https://hal.archives-ouvertes.fr/hal-00895570 Submitted on 1 Jan 2006 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 86 (2006) 141–154 © INRA, EDP Sciences, 2006 141 DOI: 10.1051/lait:2006001 Original article Antimicrobial activity and stability of partially purified bacteriocins produced by Propionibacterium freudenreichii ssp. freudenreichii and ssp. shermanii Daniela GWIAZDOWSKAa*, Krystyna TROJANOWSKAb a Department of Biochemistry and Microbiology, The Poznan University of Economics, Al. Niepodleglosci 10 60-679 Poznan, Poland b Department of Biotechnology and Food Microbiology, August Cieszkowski Agricultural University, ul. Wojska Polskiego 48 60-627 Poznan, Poland Received 19 April 2005 – Accepted 18 November 2005 Abstract – In the last decade a few bacteriocins synthesized by Propionibacterium were described. Almost all of them are produced by P. thoenii and P. jensenii species and only a limited number of reports is available on the antimicrobial activity of other species. In the present work bacteriocin preparations obtained from two strains of P. freudenreichii ssp. freudenreichii and one of P. freu- denreichii ssp. shermanii were partially characterized, including their antimicrobial spectrum and stability. All preparations were active against the examined bacteria of Lactobacillus (L. acidophi- lus, L. casei, L. delbrueckii and L. helveticus) and some strains of Propionibacterium. Crude bacte- riocins also showed bacteriostatic activity against Gram-negative bacteria and fungistatic activity against moulds belonging to the Alternaria and Fusarium genera. Bacteriocin preparations were heat-stable and did not lose their activity in the range of pH from 4.5 to 7.0. They were also stable in the presence of organic solvents such as ethanol, isopropanol, hexane and chloroform at the con- centration of 1 mg·mL–1; however, under the influence of some solvents an increase in bacteriostatic activity was observed. It could indicate a formation of aggregates by active proteins. The activity of all crude bacteriocins was significantly reduced when SDS at concentrations of 1 to 20 mg·mL–1 was used. bacteriocin / propionibacteria / antimicrobial activity / stability 摘要 – 由 Propionibacterium freudenreichii ssp. freudenreichii ssp. shermanii 产生的部分纯 化细菌素的抗菌活性和稳定性。据报道，在过去的十年里只有几种细菌素是由丙酸菌合成 的，并且这几种细菌素几乎都是由 P. thoenii 和 P. jensenii 产生的，而关于其它丙酸菌亚种 产生的具有抗菌活性的细菌素的报道非常少。本研究利用两株费氏丙酸菌费氏亚种 (P. freudenreichii ssp. freudenreichii ) 和一株费氏丙酸菌谢氏亚种 (P. freudenreichii ssp. shermanii) 菌株制备细菌素，并且对部分纯化细菌素的一些特性如抗菌谱和稳定性进行了研 究。实验结果表明所有部分纯化细菌素对所试验的乳杆菌 （嗜酸乳杆菌、干酪乳杆菌、德 氏乳杆菌和瑞士乳杆菌）和某些丙酸菌菌株具有明显的抗菌活性。粗制细菌素也具有抑制 细菌（革兰氏阴性菌）以及部分真菌如链格菌属 (Alternaria) 和镰孢霉菌属 (Fusarium) 的作 用。所有部分纯化的细菌素都具有热稳定，且在 pH 4.5~7.0 范围内能够保持活性。部分纯 * Corresponding author (通讯作者): [email protected] Article published by EDP Sciences and available at http://www.edpsciences.org/lait or http://dx.doi.org/10.1051/lait:2006001 142 D. Gwiazdowska, K. Trojanowska 化的细菌素在 1 mg·mL–1 乙醇、异丙醇、正己烷和氯仿等有机溶剂中具有很好的稳定，并 且有些溶剂能够提高细菌素的抗菌活性，这可能是由于形成活性蛋白聚集物的原因。但是 在 1~20 mg·mL–1 的十二烷基磺酸钠溶液中，所有粗制细菌素的活性都明显降低。 细菌素 / 丙酸菌 / 抗菌活性 / 稳定性 Résumé – Activité antimicrobienne et stabilité des bactériocines partiellement purifiées pro- duites par Propionibacterium freudenreichii ssp. freudenreichii et ssp. shermanii. Au cours de la dernière décennie, quelques bactériocines synthétisées par Propionibacterium ont été caractéri- sées. Presque toutes ces bactériocines sont produites par les souches: P. thoenii et P. jensenii et seul un nombre limité d’études concernent l’activité antimicrobienne d’autres souches. Ce travail porte sur des préparations de bactériocines obtenues de deux souches de P. freudenreichii ssp. freuden- reichii et une souche de P. freudenreichii ssp. shermanii qui ont été caractérisées partiellement pour leurs spectres antimicrobiens et leur stabilité. Toutes les préparations étaient actives envers les Lac- tobacillus examinés (L. acidophilus, L. casei, L. delbrueckii et L. helveticus) et certaines souches de Propionibacterium. Elles ont aussi montré une activité bacteriostatique envers les bactéries Gram- négatives et une activité fungistatique envers les moisissures appartenant au genre Alternaria et Fusarium. Les préparations de bactériocines étaient thermostables et elles ne perdaient pas leur acti- vité sur la plage de pH 4,5 à 7,0. Elles étaient aussi stables en présence de solvants tels que l’éthanol, l’isopropanol, l’hexane et le chloroforme à la concentration de 1 mg·mL–1, cependant sous l’influence de certains solvants une augmentation de l’activité antimicrobienne était observée, sug- gérant la formation d’agrégats par les protéines actives. L’activité de toutes les bactériocines natives était réduite considérablement par l’utilisation de SDS à des concentrations de 1 à 20 mg·mL–1. bactériocine / Propionibacterium / activité antimicrobienne / stabilité 1. INTRODUCTION from the cell and sometimes proteins taking part in posttranslation modification [23]. Bacteriocins are antibacterial peptides or Because of the presence of the protein proteins produced by a wide range of micro- component in the structure of bacteriocins, organisms. This is a group of metabolites they are inactivated by at least one, but usu- differing in their biochemical and physical ally by several different proteolytic enzymes, properties, molecular mass, mode of action, including pancreatic enzymes (trypsin and activity spectrum and genetic determinants. chymotrypsin) and stomach enzymes (pep- According to the definition of Tagg et al. sin) [28]. Bacteriocins containing lipid or [37] bacteriocins are bactericidal against carbohydrate components are also inacti- microorganisms closely related to the pro- vated by lipolytic and amylolytic enzymes ducer of bacteriocins, but many of them [11, 25, 35]. Enzymatic sensitivity is a very show antagonistic activity against bacteria important property because it indicates that belonging to genera different than the pro- bacteriocins will be decomposed in the ducer, including spoilage and pathogenic digestive tract. Therefore these metabolites bacteria. Research on the molecular level are safe for human health. Besides, some showed that bacteriocins are ribosomally synthesized peptides or proteins [13], gen- research has proved that bacteriocins added erally secreted outside the cell, although to food did not change its organoleptic that part of activity can stay within the biomass properties [28]. [28, 29]. The genes coding bacteriocins are Bacteriocins also have many physico- localized in plasmid or chromosomal DNA. chemical properties making them useful as Microorganisms which are capable of pro- food biopreservatives. The majority of ducing bacteriocins very often present them are heat-stable, resistant to pasteuri- immunity to this metabolite. The operon zation without losing their activity, active structure usually contains genes coding the in a wide range of pH and resistant to dif- active protein, immunity protein, proteins ferent organic and non-organic solvents [2, responsible for transporting bacteriocins 5, 15, 22, 24, 26, 31, 35, 36]. Bacteriocins from P. freudenreichii 143 Bacteriocins synthesized by bacteria strains belonging to P. thoenii or P. jensenii; naturally occurring in food are particularly data on the ability of other species to syn- interesting. The best studied are bacterioc- thesize bacteriocins is very limited. Only ins produced by lactic acid bacteria [6]. one bacteriocin produced by Propionibac- However, research carried out in the last terium freudenreichii, propionicin F, has two decades led to isolation and character- been described and characterized on the ization of new bacteriocins, produced by molecular level to date [4]. The aim of this bacteria belonging to other genera such as work was the isolation and partial purifica- Propionibacterium. tion of bacteriocins from Propionibacte- Propionic acid bacteria have been rium freudenreichii ssp. freudenreichii and known and used in the food industry for Propionibacterium freudenreichii ssp. many decades. They are especially impor- shermanii and determination of their anti- tant as a secondary starter culture in the pro- microbial activity and stability. duction of Swiss-type cheeses, where they are responsible for the formation of the characteristic flavor, texture and eyes. The 2. MATERIALS AND METHODS propionibacteria are also well known for their production of bactericidal and fungi- 2.1. Bacterial cultures static metabolites such as propionic acid, acetic acid, hydrogen peroxide and bacteri- ocins [8, 10, 14,