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Safety, probiotic and technological properties of Lactobacilli isolated from unpasteurised ovine and caprine cheeses

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Abstract

Eleven Lactobacillus plantarum from Slovak ovine and caprine lump and stored cheeses, and from four commercial probiotic and yogurt cultures (Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus acidophilus) identified using a Maldi-TOF MS analysis were screened in vitro for selected aspects correlated with safety (antibiotic susceptibility patterns, biochemical and haemolytic activity, presence of genes responsible for biogenic amines production), functional traits (including acid, bile tolerance and antimicrobial activity), ecological roles (ability to produce biofilms), and technological applications (acidification and milk coagulation capacity) for assurance of their quality and diversity. The antibiotic susceptibility showed two L. plantarum strains, 19l5 and 18l4, with the presence of the non-wild-type ECOFFs (epidemiological cut-off) for clindamycin and/or gentamicin. All these strains expressed a high acid tolerance at pH 2.5 after a 4 h exposure (bacteria viability varied between 60% and 91%), and bile resistance at 0.3% oxgall ranged from 60% to 99% with no haemolytic activity. Three wild L. plantarum strains, 17l1, 16l4, 18l2, had no harmful metabolic activities, and formed strong biofilms that were measured by a crystal violet assay. Simultaneously, the acid cell-free culture supernatant (ACFCS) from L. plantarum 18l2 had a marked inhibitory effect on the viability of the pathogens as evaluated by flow-cytometry, and also exhibited fast acidification and milk coagulation. As a result, we conclude that L. plantarum 18l2 can be included as part of the created lactobacilli collection that is useful as a starter, or starter adjunct, in the dairy industry, due to its desirable safety and probiotic characteristics, together with rapid acidification capacity compared with other investigated strains from commercially accessible products.

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References

  • Anderson RC, Cookson AL, McNabb WC, Kelly WJ, Roy NC (2010) Lactobacillus plantarum DSM 2648 is a potential probiotic that enhances intestinal barrier function. FEMS Microbiol Lett 309:184–192

    CAS  PubMed  Google Scholar 

  • Arena MP, Silvain A, Normanno G, Grieco F, Drider D, Spano G, Fiocco D (2016) Use of Lactobacillus plantarum strains as a bio-control strategy against food-borne pathogenic microorganisms. Front Microbiol 7:464. https://doi.org/10.3389/fmicb.2016.00464

    Article  PubMed  PubMed Central  Google Scholar 

  • Arena MP, Capozzi V, Spano G, Fiocco D (2017) The potential of lactic acid bacteria to colonize living and non-living surfaces and the investigation of their interactions and mechanisms. Appl Microbiol Biotechnol 101(7):2641–2657. https://doi.org/10.1007/s00253-017-8182-z

    Article  CAS  PubMed  Google Scholar 

  • Arora G, Lee BH, Lamoureux M (1990) Characteristics of enzyme profiles of Lactobacillus casei species by a rapid API–ZYM system. J Dairy Sci 73:264–273

    Article  CAS  Google Scholar 

  • Belicová A, Mikulášová M, Dušinský R (2013) Probiotic potential and safety properties of lactobacillus plantarum from Slovak Bryndza Cheese. BioMed Res Int Article ID 760298. doi:https://doi.org/10.1155/2013/760298

  • Berney M, Hammes F, Bosshard F, Eilenmann ET (2007) Assesment and interpretation of bacterial viability by using the live/dead baclight kit combination with flow Cytometry. Appl Environ Microbiol 73:3283–3290

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Berta G, Chebeňová V, Brežná B, Pangallo D, Valík Ľ, Kuchta T (2009) Identification of lactic acid bacteria in Slovakian bryndza cheese. J Food Nutr Res 48:65–71

    CAS  Google Scholar 

  • Bessede E, Angla-gre M, Delagarde Y, Sep Hieng S, Ménard A, Mégraud F (2011) Matrix-assisted laser-desorption/ionization BIOTYPER: experience in the routine of a University hospital. Clin Microbiol Infect 17:533–538

    Article  CAS  PubMed  Google Scholar 

  • Bizzini A, Jaton K, Romo D, Bille J, Prod’hom G, Greub G (2011) Matrix-assisted laser desorption ionization–time of flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial strains. J Clin Microbiol 49(2):693–696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blom H, Mørtvedt C (1991) Anti-microbial substances produced by food associated microorganisms. Biochem Soc Trans 19:694–698

    Article  CAS  PubMed  Google Scholar 

  • Borges S, Barbosa J, Silva J, Teixera P (2013) Evaluation of characteristics of Pediococcus spp. to be used as a vaginal probiotic. J Appl Microbiol 115:527–538

    Article  CAS  PubMed  Google Scholar 

  • Bujňáková D, Kmeť V (2002) Aggregation of animal lactobacilli with O157 enterohemorrhagic Escherichia coli. J Veterinary Med Ser B 49:152–154

    Article  Google Scholar 

  • Bujňáková D, Kmeť V (2012) Functional properties of Lactobacillus strains isolated from dairy products. Folia Microbiol 57:263–267

    Article  Google Scholar 

  • Bujňáková D, Vlková E, Rada V, Kmeť V (2004) Aggregation of lactobacilli and bifidobacteria with Escherichia coli O157. Folia Microbiol 49:143–146

    Article  Google Scholar 

  • Bujňáková D, Straková E, Kmeť V (2014) In vitro evaluation of the safety and probiotic properties of Lactobacilli isolated from chicken and calves. Anaerobe 29:118–127

    Article  PubMed  Google Scholar 

  • Cauwerts K, Pasmas F, Devriese LA, Martel A, Haesebrouck F, Decostere A (2006) Cloacal Lactobacillus isolates from broilers show high prevalence of resistance towards macrolide and lincosamide antibiotics. Avian Pathol 35:160–164

    Article  CAS  PubMed  Google Scholar 

  • Charteris WP, Kelly PM, Morelli L, Collins JK (1998) Antibiotic susceptibility of potentially probiotic Lactobacillus species. J Food Prot 61:1636–1643

    Article  CAS  PubMed  Google Scholar 

  • Coton M, Romano A, Spano G, Ziegler K, Vetrana C, Desmarais C, Lonvaud-Funel A, Lucas P, Coton E (2010) Occurrence of biogenic amine-forming lactic acid bacteria in wine and cider. Food Microbiol 27:1078–1085

    Article  CAS  PubMed  Google Scholar 

  • De-Keersmaecker SCJ, Verhoeven TLA, Desair J, Marchal K, Vanderleyden J, Nagy I (2006) Strong antimicrobial activity of Lactobacillus rhamnosus GG against Salmonella typhimurium is due to accumulation of lactic acid. FEMS Microbiol Lett 259:89–96

    Article  CAS  PubMed  Google Scholar 

  • Delgado S, O'Sullivan E, Fitzgerald G, Mayo B (2007) Subtractive screening for probiotic properties of Lactobacillus species from the human gastrointestinal tract in the search for new probiotics. J Food Sci 72:310–315. https://doi.org/10.1111/j.1750-3841.2007.00479.x

    Article  Google Scholar 

  • Delgado S, O'sullivan E, Fitzgerald G, Mayo B (2008) In vitro evaluation of the probiotic properties of human Bifidobacterium species and selection on new probiotic candidates. J Appl Microbiol 104:1119–1127

    Article  CAS  PubMed  Google Scholar 

  • Didienne R, Defargues C, Callon C, Meylheuc T, Hulin S, Montel MC (2012) Characteristics of microbial biofilm on wooden vats (‘gerles’) in PDO Salers cheese. Int J Food Microbiol 156:91–101

    Article  CAS  PubMed  Google Scholar 

  • Dubernet S, Desmasures N, Gueguen M (2002) A PCR-based method for identification of lactobacilli at the genus level. FEMS Microbiol Lett 214:271–275

    Article  CAS  PubMed  Google Scholar 

  • EFSA (2011) Scientific opinion on risk based control of biogenic amine formation in fermented foods. EFSA J 9(10):2393

    Article  Google Scholar 

  • EFSA (2012) Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importace. EFSA panel on additives and products or substances used in animal feed (FEEDAP). EFSA J 10(6):2740

    Google Scholar 

  • FAO/WHO (2002) Guidelines for the evaluation of probiotics in food. Report of a joint FAO/WHO working group on drafting guidelines for the evaluation of probiotics in food (30 April 30 and 1 May 2002). London, ON

  • Fazeli MR, Shahverdi AR, Sedaghat B, Jamalifar H, Samadi N (2004) Sourdough-isolated Lactobacillus fermentum as a potent anti-mould preservative of a traditional Iranian bread. Eur Food Res Technol 218:554–556

    Article  CAS  Google Scholar 

  • Fazeli MR, Toliyat T, Samadi N, Hajjaran S, Jamalifar H (2006) Viability of Lactobacillus acidophilus in various vaginal tablet formulations. Daru 14(4):172–177

    CAS  Google Scholar 

  • Flemming HC, Wingender J (2010) The biofilm matrix. Nat Rev Microbiol 8:623–633

    CAS  PubMed  Google Scholar 

  • Food and Agriculture Organization of the United Nations (FAO) (2006) Probiotics in food: health and nutritional properties and guidelines for evaluation. FAO, Food and Nutrition Pap 85

  • Hacin B, Rogelj I, Matijasic BB (2008) Lactobacillus isolates from weaned piglets’ mucosa with inhibitory activity against common porcine pathogens. Folia Microbiol 53:569–576

    Article  CAS  Google Scholar 

  • Hussain M, Khan MT, Wajid A, Rasool SA (2008) Technological characterization of indigenous enterococcal population for probiotic potential. Pak J Bot 40:867–875

    Google Scholar 

  • Hütt P, Shchepetova J, Lõivukene K, Kullisaar T, Mikelsaar M (2006) Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. J Appl Microbiol 100:1324–1332

    Article  PubMed  Google Scholar 

  • ISO 10932/IDF 223 (2010) Milk and milk products–Determination of minimal inhibitory concentration (MIC) of antibiotics applicable to bifidobacteria and non-enterococcal lactic acid bacteria (LAB). https://www.iso.org/standard/46434.html

  • Jamaly N, Benjouad A, Bouksaim M (2011) Probiotic potential of Lactobacillus strains isolated from known popular traditional Moroccan dairy products. Br Microbiol Res J 1(4):79–94

    Article  CAS  Google Scholar 

  • Jones SE, Versalovic J (2009) Probiotic Lactobacillus reuteri biofilms produce antimicrobial and anti-inflammatory factors. BMC Microbiol 9:35. https://doi.org/10.1186/1471-2180-9-35

    Article  PubMed  PubMed Central  Google Scholar 

  • Katz M, Medina R, Gonzales S, Guillermo O (2002) Esterolytic and Lipolytic activities of lactic acid bacteria isolated from Ewe's milk and cheese. J Food Prot 12:1997–2001

    Article  Google Scholar 

  • Klingberg TD, Axelsson L, Naterstad K, Elsser D, Budde BB (2005) Identification of potential probiotic starter cultures for Scandinavian-type fermented sausages. Int J Food Microbiol 105:419–431

    Article  CAS  PubMed  Google Scholar 

  • Kmeť V, Drugdová V (2012) Antibiotic susceptibility of microflora from ovine cheese. Folia Microbiol 57:291–293

    Article  Google Scholar 

  • Kmeť V, Lucchini F (1999) Aggregation of sow Lactobacilli with diarrheagenic Escherichia coli. J Veterinary Med Ser B 46:683–687

    Article  Google Scholar 

  • Kološta M, Slottová A, Drončovský M, Klapáčová L, Kmeť V, Bujňáková D, Lauková A, Greif G, Greifová M, Tomáška M (2014) Characterisation of lactobacilli from ewe’s and goat’s milk for their further processing re-utilisation. Potravinárstvo 8:130–134

    Google Scholar 

  • Kostinek M, Specht I, Edward VA, Pinto C, Egounlety M, Sossa C, Mbugua S, Dorti C (2007) Characterization and biochemical properties of predominant lactic acid bacteria from fermenting cassava for selection as starter cultures. Int J Food Microbiol 114:342–351

    Article  CAS  PubMed  Google Scholar 

  • Licitra G, Ogier JC, Parayre S, Pediliggieri C, Carnemolla TM, Falentin H, Madec MN, Carpino S, Lortal S (2007) Variability of bacterial biofilms of the “tina” wood vats used in the ragusano cheese-making process. Appl Environ Microbiol 73:6980–6987

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lina G, Quaglia A, Revedy ME, Leclercq R, Vadenesch F, Etienne J (1999) Distribution of genes encoding resistance to macrolides, lincosamides, and streptogramins among staphylococci. Antimicrob Agents Chemother 43:1062–1066

    CAS  PubMed  PubMed Central  Google Scholar 

  • Makras L, Triantafyllou V, Fayol-Messaoudi D, Adriany T, Zoumpopoulou G, Tsakalidou E, Servin A, De Vuyst L (2006) Kinetic analysis of the antibacterial activity of probiotic lactobacilli towards Salmonella enterica serovar Typhimurium reveals a role for lactic acid and other inhibitory compounds. Res Microbiol 157:241–247

    Article  CAS  PubMed  Google Scholar 

  • Maragkoudakis PA, Zoumpopoulou G, Miaris C, Kalantzopoulos G, Pot B, Tsakalidou E (2006) Probiotic potential of lactobacillus strains isolated from dairy products. Int Dairy J 16:189–199

    Article  CAS  Google Scholar 

  • Mariani C, Briandet R, Chamba JF, Notz E, Carnet-Pantiez A, Eyoug RN, Oulahal N (2007) Biofilm ecology of wooden shelves used in ripening the French raw milk smear cheese reblochon de Savoie. J Dairy Sci 90:1653–1661

    Article  CAS  PubMed  Google Scholar 

  • Martín R, Soberón N, Vaneechoutte M, Flórez AB, Vázquez F, Suárez JE (2008) Characterization of indigenous vaginal lactobacilli from healthy women as probiotic candidates. Int Microbiol 11:261–266

    PubMed  Google Scholar 

  • Mego M, Májek J, Končeková R, Ebringer L, Čierniková S, Rauko P, Kováč M, Trupl J, Slezák P, Zajac V (2005) Intramucosal bacteria in colon cancer and their elimination by probiotic strain Enterococcus faecium M-74 with organic selenium. Folia Microbiol 50:443–447

    Article  CAS  Google Scholar 

  • Mohammadi R, Sohrabvandi S, Motazavian AM (2012) The starter culture characteristics of probiotic microorganisms in fermented milks. Eng Life Sci 12:399–409

    Article  CAS  Google Scholar 

  • Monteagudo-Mera A, Caro I, Rodríguez-Aparicio LB, Rúa J, Ferrero MA, García-Armesto MR (2011) Characterization of certain bacterial strains for potential use as starter or Probiotic cultures in dairy products. J Food Prot 74:1379–1386

    Article  CAS  PubMed  Google Scholar 

  • Morelli L (2007) In vitro assessment of probiotic bacteria: from survival to functionality. Int Dairy J 17:1278–1283

    Article  Google Scholar 

  • Neelakantan S, Mohanty AK, Kauschik JK (1999) Production and use of microbial enzymes for dairy processing. Curr Sci 77:143–148

    CAS  Google Scholar 

  • Oguntoyinbo FA (2007) Identification and functional properties of dominant lactic acid bacteria isolated at different stages of solid state fermentation of cassava during Gari production. World J Microbiol Biotechnol 23:1425–1432

    Article  CAS  Google Scholar 

  • Ortu S, Felis GE, Marzotto M, Deriu A, Molicotti P, Sechi LA, Dellaglio F, Zanetti S (2007) Identification and functional characterization of Lactobacillus strains isolated from milk and Gioddu, a traditional Sardinian fermented milk. Int Dairy J 17:1312–1320

    Article  CAS  Google Scholar 

  • Pompilio A, Piccolomini R, Picciani C, Antonio DD, Savini V, Di Bonaventura G (2008) Factors associated with adherence to and biofilm formation on polystyrene by Stenotrophomonas maltophilia: the role of cell surface hydrophobicity and motility. FEMS Microbiol Lett 287:41–47

    Article  CAS  PubMed  Google Scholar 

  • Pompilio A, Crocetta V, Confalone P, Nicoletti M, Petrucca A, Guarnieri S, Fiscarelli E, Savini V, Piccolomini R, Di Bonaventura G (2010) Adhesion to and biofilm formation on IB3-1 bronchial cells by Stenotrophomonas maltophilia isolates from cystic fibrosis patients. BMC Microbiol 10:102–105

    Article  PubMed  PubMed Central  Google Scholar 

  • Russo P, Fernadéz de Palencia P, Romano A, Fernandéz M, Lucas P, Giuseppe S, Lopez P (2012) Biogenic amine production by the wine Lactobacillus brevis IOEB 9809 in systems that partially mimic the gastrointestinal tract stress. BMC Microbiol 12:1–10

    Article  Google Scholar 

  • Salvetti E, Orrù L, Capozzi V, Martina A, Lamontanara A, Keller D, Cash H, Felis GE, Cattivelli L, Torriani S, Spano G (2016) Integrate genome-based assessment of safety for probiotic strains: Bacillus coagulans GBI-30, 6086 as a case study. Appl Microbiol Biotechnol 100:4595–4605. https://doi.org/10.1007/s00253-016-7416-9

    Article  CAS  PubMed  Google Scholar 

  • Sauer S, Freiwald A, Maier T, Kube M, Reinhardt R, Kostrzewa M, Geider K (2008) Classification and identification of bacteria by mass spectrometry and computational analysis. PLoS One 3:2843–2853

    Article  Google Scholar 

  • Schoster A, Kokotovic B, Permin A, Pedersen PD, Dal Bello F, Guardabassi L (2013) In vitro inhibition of Clostridium Difficile and Clostridium Perfringens by commercial probiotic strains. Anaerobe 20:36–41

    Article  CAS  PubMed  Google Scholar 

  • Smetanková J, Hladíková Z, Zimanová M, Greif G, Greifová M (2014) Lactobacilli isolated from lump sheep’s cheeses ant their antimicrobial properties. Czech J Food Sci 2:152–157

    Google Scholar 

  • Song YL, Kato N, Liu CX, Matsumiya Y, Kato H, Watanabe K (2000) Rapid identification of 11 human intestinal lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA. FEMS Microbiol Lett 187:167–173

    CAS  PubMed  Google Scholar 

  • Tenorio C, Zarazaga M, Martinez C, Torres C (2001) Bifunctional enzyme 6′-N-aminoglycoside acetyltransferase-2′′-O-aminoglycoside phosphotransferase in Lactobacillus and Pediococcus isolates of animal origin. J Clin Microbiol 39:824–825

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Toledo-Arana A, Valle J, Solano C, Arrizubieta MJ, Cucarella C, Lamata M, Amorena B, Leiva J, Penadés JR, Lasa I (2001) The enterococcal surface protein, Esp, is involved in Enterocccus faecalis biofilm formation. Appl Environ Microbiol 67:4538–4545

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vakulenko SB, Donabedian SM, Voskresenskiy AM, Zervos MJ, Lerner S, Chow JW (2003) Multiplex PCR for detection of Aminoglycoside resistance genes in Enterococci. Antimicrob Agents Chemother 47:1423–1426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Van de Guchte M, Serror P, Chervaux C, Smovkina T, Ehrlich SD, Maguin E (2002) Stress responses in lactic acid bacteria. Antonie Van Leeuwenhoek 82:187–216

    Article  PubMed  Google Scholar 

  • Vrese M, Stegelmann A, Richter B, Fenselau S, Laue C, Schrezenmeir J (2001) Probiotics-compensation for lactase insufficiency. Am Clin Nutr 73:421–429

    Google Scholar 

  • Walencka E, Różalska S, Sadowska B, Różalska B (2008) The influence of Lactobacillus acidophilus-derived surfactants on staphylococcal adhesion and biofilm formation. Folia Microbiol 53:861–866

    Article  Google Scholar 

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Acknowledgement

This work is the result of the European Regional Development Fund project 26220220065 and VEGA project No. VEGA 2/0012/16.

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  1. Institute of Animal Physiology, Slovak Academy of Sciences, Soltesovej 4/6, 040 01, Kosice, Slovak Republic

    Dobroslava Bujnakova & Eva Strakova

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  1. Dobroslava Bujnakova
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Correspondence to Dobroslava Bujnakova.

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Bujnakova, D., Strakova, E. Safety, probiotic and technological properties of Lactobacilli isolated from unpasteurised ovine and caprine cheeses. Ann Microbiol 67, 813–826 (2017). https://doi.org/10.1007/s13213-017-1310-2

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