Skip to main content
SpringerLink
Log in

Lactobacillus casei and Lactobacillus fermentum Strains Isolated from Mozzarella Cheese: Probiotic Potential, Safety, Acidifying Kinetic Parameters and Viability under Gastrointestinal Tract Conditions

  • Published:
Probiotics and Antimicrobial Proteins Aims and scope Submit manuscript

Abstract

The objective of this study was to evaluate the probiotic properties of Lactobacillus casei and Lactobacillus fermentum strains, as well as to select novel and safe strains for future development of functional fermented products. The in vitro auto-aggregation, co-aggregation, hydrophobicity, β-galactosidase production, survival to gastrointestinal tract (GIT), and antibiotic susceptibility were evaluated. The selected strains were additionally tested by the presence of genes encoding adhesion, aggregation and colonization, virulence factors, antibiotic resistance, and biogenic amine production, followed by the evaluation of acidifying kinetic parameters in milk, and survival of the strains under simulated GIT conditions during refrigerated storage of fermented milk. Most strains of both species showed high auto-aggregation; some strains showed co-aggregation ability with other lactic acid bacteria (LAB) and/or pathogens, and both species showed low hydrophobicity values. Seven L. casei and six L. fermentum strains produced β-galactosidase enzymes, and ten strains survived well the simulation of the GIT stressful conditions evaluated in vitro. All strains were resistant to vancomycin, and almost all the strains were resistant to kanamycin. L. casei SJRP38 and L. fermentum SJRP43 were distinguished among the other LAB strains by their higher probiotic potential. L. fermentum SJRP43 presented fewer genes related to virulence factors and antibiotic resistance and needed more time to reach the maximum acidification rate (Vmax). The other kinetic parameters were similar. Both strains survived well (> 8 log10 CFU/mL) to the GIT-simulated conditions when incorporated in fermented milk. Therefore, these strains presented promising properties for further applications in fermented functional products.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Parker EA, Roy T, D’Adamo CR, Wieland LS (2018) Probiotics and gastrointestinal conditions: an overview of evidence from the Cochrane Collaboration. Nutr Rev 45:125–134

    Google Scholar 

  2. Sharma P, Tomar SK, Goswami P, Sangwan V, Singh R (2014) Antibiotic resistance among commercially available probiotics. Food Res Int 57:176–195

    Article  CAS  Google Scholar 

  3. Brazil (2000) Ministério da Agricultura, Pecuária e Abastecimento. Departamento de Inspeção de Produtos de Origem Animal. Resolução 05 de novembro de 2000

  4. FAO/WHO (2002) Guidelines for the evaluation of probiotics in food. Food and Agriculture Organization of the United Nations and World Health Organization Working Group Report. http: //who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf. Acesso em 10/04/2013

  5. Homayouni A, Azizi MR, Ehsani MS, Yarmand A, Razavi SH (2008) Effect of microencapsulation and resistant starch on the probiotic survival and sensory properties of synbiotic ice cream. Food Chem 111:50–55

    Article  CAS  Google Scholar 

  6. Bergillos-Meca T, Costabile A, Walton G, Moreno-Montoro M, Ruiz-Bravo A, Ruiz-López MD (2015) In vitro evaluation of the fermentation properties and potential probiotic activity of Lactobacillus plantarum C4 in batch culture systems. Food Sci Technol 60:420–426

    CAS  Google Scholar 

  7. Ejtahed HS, Mohtadi-Nia J, Homayouni-Rad A, Niafar M, Asghri-Jafarabadi M, Mofid V, Akbarian-Moghari A (2011) Effect of probiotic yogurt containing Lactobacillus acidophilus and Bifidobacterium lactis on lipid profile in individuals with type 2 diabetes mellitus. J Dairy Res 94:3288–3294

    Article  CAS  Google Scholar 

  8. Lollo PCB, Moura CS, Morato PN, Cruz AG, Castro WF, Betim CB, Nisishima L, Faria JAF, Maróstica M, Fernandes CO, Amayan-Farfan J (2013) Probiotic yogurt offers higher immune-protection than probiotic whey beverage. Food Res Int 54:118–124

    Article  CAS  Google Scholar 

  9. Ouwehand AC, Salminen S, Isolauri E (2002) Probiotics: an overview of beneficial effects. Antonie Van Leeuwenhoek 82:279–289

    Article  CAS  PubMed  Google Scholar 

  10. Silva LF (2015) Diversidade e evolução da microbiota lática autóctone em queijo Muçarela de búfala e aplicação tecnológica dos isolados. Ph. D. Thesis, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista “Julio Mesquita Filho”

  11. Von Mollendorff JW, Todorov SD, Dicks LM (2007) Factors affecting the adsorption of bacteriocins to Lactobacillus sakei and Enterococcus sp. Appl Biochem Biotechnol 142:209–220

    Article  CAS  Google Scholar 

  12. Charteris WP, Kelly PM, Morelli L, Collins JK (1998) Development and application of in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. J Appl Microbiol 84:759–768

    Article  CAS  PubMed  Google Scholar 

  13. EFSA European Food Safety Authority (2012) Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance. Eur Food Saf Authority 10:2740

    Google Scholar 

  14. Todorov SD, Furtado DN, Saad SM, Tome E, Franco BD (2011) Potential beneficial properties of bacteriocin-producing lactic acid bacteria isolated from smoked salmon. J Appl Microbiol 110:971–986

    Article  CAS  PubMed  Google Scholar 

  15. Todorov SD, Dicks LM (2008) Evaluation of lactic acid bacteria from kefir, molasses and olive brine as possible probiotics based on physiological properties. Ann Microbiol 58:661–670

    Article  Google Scholar 

  16. Vinderola CG, Reinheimer JA (2003) Lactic acid starter and probiotic bacteria: a comparative “in vitro” study of probiotic characteristics and biological barrier resistance. Food Res Int 36:895–904

    Article  CAS  Google Scholar 

  17. Bautista-Galego J, Arroyo-López FN, Rantsiou K, Jiménez-Diaz R, Garrido-Fernándes A, Cocolin L (2013) Screening of lactic acid bacteria isolated from fermented table olives with probiotic potential. Food Res Int 50:135–142

    Article  CAS  Google Scholar 

  18. Fortina MG, Ricci G, Borgo F, Manachini PL, Arends K, Schiwon K, Abajy MY, Grohmann E (2008) A survey on biotechnological potential and safety of the novel Enterococcus species of dairy origin, E. italicus. Int J Food Microbiol 123:204–211

    Article  CAS  PubMed  Google Scholar 

  19. Ramiah K, Van Reenen CA, Dicks LMT (2007) Expression of the mucus adhesion genes Mub and MapA, adhesion-like factor EF-Tu and bacteriocin gene plaA of Lactobacillus plantarum 423, monitored with real-time PCR. Int J Food Microbiol 116:405–409

    Article  CAS  PubMed  Google Scholar 

  20. Costa Y, Galimand M, Leclercq R, Duval J, Courvalin P (1993) Characterization of the chromosomal aac (6′)-Ii gene specific for Enterococcus faecium. Antimicrob Agents Chemother 37:1896–1903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Manson JM, Keis S, Smith JMB, Cook GM (2004) Acquired bacitracin resistance in enterococcus faecalis is mediated by an ABC transporter and a novel regulatory protein, BcrR. Antimicrob Agents Chemother 48:3743–3748

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Aarestrup FM, Agersù Y, Ahrens P, Christian J, Madsen M, Jensen LB (2000a) Antimicrobial susceptibility and presence of resistance genes in staphylococci from poultry. Vet Microbiol 74:353–364

    Article  CAS  PubMed  Google Scholar 

  23. Sutcliffe J, Grebe T, Tait-kamradt A, Wondrack L (1996) Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother 40:2562–2566

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gevers D, Danielsen M, Huys G, Swings J (2003) Molecular characterization of tet (M) genes in Lactobacillus isolates from different types of fermented dry sausage. Appl Environ Microbiol 69:1270–1275

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Aarestrup FM, Agerso Y, Smidt PG, Madsen M, Jensen LB (2000b) Comparison of antimicrobial resistance phenotypes and resistance genes in Enterococcus faecalis and Enterococcus faecium from humans in the community, broilers, and pigs in Denmark. Diagn Microbiol Infect Dis 37:127–137

    Article  CAS  PubMed  Google Scholar 

  26. Martín-Platero AM, Valdivia E, Maqueda M, Martínez-Bueno M (2009) Characterization and safety evaluation of enterococci isolated from Spanish goats’ milk cheeses. Int J Food Microbiol 132:24–32

    Article  CAS  PubMed  Google Scholar 

  27. Miele A, Bandera M, Goldstein BP (1995) Use of primers selective for vancomycin resistance genes to determine van genotype in enterococci and to study gene organization in vanA isolates. Antimicrob Agents Chemother 39:1772–1778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dutka-Malen S, Evers S, Courvalin P (1995) Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 33:24–27

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Robredo B, Singh KV, Torres C, Murray BE (2000) Streptogramin resistance and shared pulsed-field gel electrophoresis patterns in vanA-containing Enterococcus faecium and Enterococcus hirae isolated from humans and animals in Spain. Microb Drug Resist 6:305–311

    Article  CAS  PubMed  Google Scholar 

  30. Vankerckhoven V, Autgaerden T, Van Vael C, Lammens C, Chapelle S, Rossi R, Goossens H (2004) Development of a multiplex PCR for the detection of asa1, gelE, cylA, esp, and hyl genes in Enterococci and survey for virulence determinants among european hospital isolates of Enterococcus faecium. J Clin Microbiol 42:4473–4479

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Eaton TJ, Gasson MJ (2001) Molecular screening of Enterococcus virulence determinants and potential for genetic exchange between food and medical isolates. Appl Environ Microbiol 67:1628–1635

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lopes MDFS, Simões AP, Tenreiro R, Marques JJF, Crespo MTB (2006) Activity and expression of a virulence factor, gelatinase, in dairy enterococci. Int J Food Microbiol 112:208–214

    Article  CAS  Google Scholar 

  33. Arias CA, Robredo B, Singh KV, Torres C, Panesso D, Murray BE (2006) Rapid identification of Enterococcus hirae and Enterococcus durans by PCR and detection of a homologue of the E. hirae mur-2 gene in E. durans. J Clin Microbiol Infect Dis 44:1567–1570

    CAS  Google Scholar 

  34. de Las Rivas B, Marcobal A, Munoz R (2005) Improved multiplex-PCR method for the simultaneous detection of food bacteria producing biogenic amines. FEMS Microbiol Lett 244:367–372

    Article  CAS  PubMed  Google Scholar 

  35. Divya JB, Varsha KK, Nampoothiri KM (2012) Newly isolated lactic acid bacteria with probiotic features for potential application in food industry. Appl Biochem Biotechnol 167:1314–1324

    Article  CAS  PubMed  Google Scholar 

  36. Birri DJ, Brede DA, Nes IF (2012) Salivaricin D, a novel intrinsically tryosin-resistant lantibiotic from Streptococcus salivarius 5M6c isolated from a health infant. Appl Environ Microbiol 78:402–410

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Jeronymo-Ceneviva AB, Paula AT, Silva LF, Todorov SD, Franco BDGM, Penna ALB (2014) Probiotic properties of lactic acid bacteria isolated from water-buffalo mozzarella cheese. Probiotics Antimicro Prot 6:141–156

    Article  CAS  Google Scholar 

  38. Casarotti SN, Carneiro BM, Todorov SD, Nero LA, Rahal P, Penna ALB (2017) In vitro assessment of safety and probiotic potential characteristics of Lactobacillus strains isolated from water buffalo mozzarella cheese. Ann Microbial 67:289–301

    Article  CAS  Google Scholar 

  39. Reid G, Burton J (2002) Use of Lactobacillus to prevent infection by pathogenic bacteria. Microbes Infect 4:319–324

    Article  PubMed  Google Scholar 

  40. Galdeano C, De Moreno A, Vinderela G, Bonet ME, Perdigon GA (2007) Proposal model: mechanisms of immunomodulation induced by probiotic bacteria. Review. Clin Vaccine Immunol 15:485–492

    Article  CAS  Google Scholar 

  41. Todorov SD, Prévost H, Lebois M, Dousset X, Leblanc JG, Franco BDGM (2011) Bacteriocinogenic Lactobacillus plantarum ST16Pa isolated from papaya (Carica papaya)—from isolation to application: characterization of a bacteriocin. Food Res Int 44:1351–1363

    Article  CAS  Google Scholar 

  42. Nascimento LCS (2015) Probiótico e Substância de Produção culturas antimicrobianas potenciais para o ácido láctico e Aplicação em Leite Fermentado. Ph D. Thesis, Programa de Pós-Graduação em Ciência e Tecnologia de Alimentos, Instituto de Biociências, Letras e Ciências Exatas, Universidade Estadual Paulista

  43. Sánchez-Ortiz AC, Luna-González A, Campa-Córdova AI, Escamilla-Montes R, Flores-Miranda MDC, Mazón-Suástegui JM (2015) Isolation and characterization of potential probiotic bacteria from pustulose ark (Anadara tuberculosa) suitable for shrimp farming. Lat Am J Aquat Res 43:123–136

    Article  Google Scholar 

  44. Koss B, Suskovic J, Vukovic S, Simpraga M, Frece J, Matosic S (2003) Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J Ind Microbiol Biotechnol 94:981–987

    Google Scholar 

  45. Schar-Zammaretti P, Ubbink J (2003) The cell wall of lactic acid bacteria: surface constituents and macromolecular conformations. Biophys J 85:4076–4092

    Article  PubMed  PubMed Central  Google Scholar 

  46. Angmo K, Kumari AS, Bhalla TC (2016) Probiotic characterization of lactic acid bacteria isolated from fermented foods and beverage of Ladakh. Food Sci Technol 66:428–435

    CAS  Google Scholar 

  47. Todorov SD, Le Blanc JG, Franco BDGM (2012) Evaluation of the probiotic potential and effect of encapsulation on survival for Lactobacillus plantarum ST16Pa isolated from papaya. J Ind Microbiol Biotechnol 28:973–984

    CAS  Google Scholar 

  48. Sasaki K, Samant SK, Suzuki M, Toba T, Itoh T (2008) β-Galactosidase and 6-phospho-β-galactosidase activities in strains of the Lactobacillus acidophilus complex. Lett Appl Microbiol 16:97–100

    Article  Google Scholar 

  49. Zárate G, Chaia AP (2012) Influence of lactose and lactate on growth and β-galactosidase activity of potential probiotic Propionibacterium acidipropionici. Anaerobe 18:25–30

    Article  CAS  PubMed  Google Scholar 

  50. Meira SM, Helfer VE, Velho RV, Lopes FC, Brandelli A (2012) Probiotic potential of Lactobacillus spp. isolated from Brazilian regional ovine cheese. J Dairy Res 79:119–127

    Article  CAS  PubMed  Google Scholar 

  51. Paula AT, Jeronymo-Ceneviva AB, Silva LF, Todorov SD, Franco BDGM, Penna ALB (2015) Leuconostoc mesenteroides SJRP55: a potential probiotic strain isolated from Brazilian water buffalo mozzarella cheese. Ann Microbiol 65:899–910

    Article  CAS  Google Scholar 

  52. Wu C, He G, Zhang J (2014) Physiological and proteomic analysis of Lactobacillus casei in response to acid adaptation. J Ind Microbiol Biotechnol 41:1533–1540

    Article  CAS  PubMed  Google Scholar 

  53. García-Ruiz A, González de Llano D, Esteban-Fernández A, Requena T, Bartolomé B, Moreno-Arribas MV (2014) Assessment of probiotic properties in lactic acid bacteria isolated from wine. Food Microbiol 44:220–225

    Article  CAS  PubMed  Google Scholar 

  54. Begley M, Hill C, Gahan CGM (2006) Bile salt hydrolase activity in probiotics. Appl Environ Microbiol 72:1729–1738

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Kailasapathy K (2012) Microencapsulation of probiotic bacteria: technology and potential applications. Curr Issues Intest Microbiol 3:39–48

    Google Scholar 

  56. Watanabe M, Kinoshita H, Nitta M, Yukishita R, Kawai Y, Kimura K, Taketomo N, Yamazaki Y, Tateno Y, Miura K, Horii A, Kitazawa H, Saito T (2010) Identification of a new adhesin-like protein from Lactobacillus mucosae ME-340 with specific affinity to the human blood group A and B antigens. J Appl Microbiol 109:927–935

    Article  CAS  PubMed  Google Scholar 

  57. Perin LM, Miranda RO, Todorov SD, Franco BDGM, Nero LA (2014) Virulence, antibiotic resistance and biogenic amines of bacteriocinogenic lactococci and enterococci isolated from goat milk. Int J Food Microbiol 185:121–126

    Article  CAS  PubMed  Google Scholar 

  58. Guerrero-Ramos E, Molina-González D, Blanco-Morán S, Igrejas G, Poeta P, Alonso-Calleja C, Capita R (2016) Prevalence, antimicrobial resistance, and genotypic characterization of vancomycin-resistant Enterococci in meat preparations. J Food Protect 79:748–756

    Article  CAS  Google Scholar 

  59. Sattari M (2009) Prevalence of ant(4′)-Ia gene among clinical isolates of methicillin-resistant Staphylococcus aureus using multiplex-PCR method. Patho Res Pract 12:59–68

    Google Scholar 

  60. Jiménez E, Ladero V, Chico I, Maldonado-Barragán A, López M, Martín V, Fernández L, Fernández M, Álvarez MA, Torres C, Rodríguez JM (2013) Antibiotic resistance, virulence determinants and production of biogenic amines among enterococci from ovine, feline, canine, porcine and human milk. BMC Microbiol 13:1–12

    Article  CAS  Google Scholar 

  61. Oliveira RPS, Perego P, Converti A, Oliveira MN (2009) Growth and acidification performance of probiotics in pure culture and co-culture with Streptococcus thermophilus: the effect of inulin. LWT Food Sci Technol 42:1015–1021

    Article  CAS  Google Scholar 

  62. Casarotti SN, Monteiro DA, Moretti MMS, Penna ALB (2014) Influence of the combination of probiotic cultures during fermentation and storage of fermented milk. Food Res Int 69:67–75

    Article  CAS  Google Scholar 

  63. Casarotti SN, Carneiro BM, Penna ALB (2014) Evaluation of the effect of supplementing fermented milk with quinoa flour on probiotic activity. J Dairy Sci 97:6027–6035

    Article  CAS  PubMed  Google Scholar 

  64. Shori AB (2016) Influence of food matrix on the viability of probiotic bacteria: a review based on dairy and non-dairy beverages. Food Biosci 13:1–8

    Article  CAS  Google Scholar 

  65. Espirito Santo AP, Perego P, Converti A, Oliveira MN (2011) Influence of food matrices on probiotic viability—a review focusing on the fruity bases. Trends Food Sci Technol 22:377–385

    Article  CAS  Google Scholar 

  66. Ranadheera RDCS, Baines SK, Adams MC (2010) Importance of food in probiotic efficacy. Food Res Int 43:1–7

    Article  CAS  Google Scholar 

  67. Costa MGM, Ooki GN, Vieira ADS, Bedani R, Saad SMI (2017) Synbiotic Amazonian palm berry (açai, Euterpe oleracea Mart.) ice cream improved Lactobacillus rhamnosus GG survival to simulated gastrointestinal stress. Food Funct 8:731–740

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was sponsored by the São Paulo State Research Foundation (FAPESP—Projects 2014/02131-8 and 2014/02132-4) and The Brazilian National Council for Scientific and Technological Development (CNPq—Project 307155/2015-3).

Author information

Authors and Affiliations

  1. Department Food Engineering and Technology, Sao Paulo State University, Rua Cristóvão Colombo, 2265, Jardim Nazareth, São José Preto, SP, 15.0540-000, Brazil

    Bruna Maria Salotti de Souza, Taís Fernanda Borgonovi & Ana Lúcia Barretto Penna

  2. Faculty of Nutrition, Department of Food and Nutrition, UFMT - Mato Grosso Federal University, Cuiabá, MT, Brazil

    Sabrina Neves Casarotti

  3. Faculty of Pharmaceutical Sciences, Department of Food Science and Experimental Nutrition, USP - São Paulo University, São Paulo, SP, Brazil

    Svetoslav Dimitrov Todorov

Authors
  1. Bruna Maria Salotti de Souza
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Taís Fernanda Borgonovi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sabrina Neves Casarotti
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Svetoslav Dimitrov Todorov
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ana Lúcia Barretto Penna
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ana Lúcia Barretto Penna.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

de Souza, B.M.S., Borgonovi, T.F., Casarotti, S.N. et al. Lactobacillus casei and Lactobacillus fermentum Strains Isolated from Mozzarella Cheese: Probiotic Potential, Safety, Acidifying Kinetic Parameters and Viability under Gastrointestinal Tract Conditions. Probiotics & Antimicro. Prot. 11, 382–396 (2019). https://doi.org/10.1007/s12602-018-9406-y

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12602-018-9406-y

Keywords

Search

Navigation