Skip to main content
SpringerLink
Log in

Development of Thermotolerant Lactobacilli Cultures with Improved Probiotic Properties Using Adaptive Laboratory Evolution Method

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

Abstract

Probiotics play a significant role in functional foods. Heat stress and dehydration are the two principal mechanisms leading to inactivation and loss of probiotics viability in its production. There is a need to develop an industrial organism to withstand higher temperatures during its processing and storage. This current study aims to develop thermotolerant strains of Lacticaseibacillus casei N (N) and Lactobacillus helveticus NRRL B-4526 (H) by acclimatizing the wild-type strains to the higher temperature of 45 °C by adaptive laboratory evolution. A two-fold increase in biomass was observed in both acclimatized strains up to the 200th generation, which subsequently remained stable after 500 generations. The morphological change of these acclimatized strains was observed under scanning electron microscopy. Also, there was an increase in probiotic attributes of these acclimatized strains compared to their wild-types. Among two acclimatized strains, L. casei N-45 had shown higher tolerance in the acidic pH 3.0 (89.31%), the bile of 0.3% (84.45%), simulated gastric juice (79.12%), and simulated intestinal juice (73.86%). There was also an increase in salt tolerance (NaCl), radical scavenging activity, autoaggregation, coaggregation, and hydrophobicity of these adapted strains. The total protein profiling using 2D gel electrophoresis reveals the differences in protein expressions between wild-type and acclimatized strains. Specific protein spots from acclimatized strains of H-45 and N-45 were further subjected to MALDI-TOF MS/MS. Some of the identified proteins were recognized to play a role in RNA chaperones and protein synthesis during stress conditions.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Data Availability

Sequence data of an isolate Lacticaseibacillus casei N (N) is deposited with the primary accession number: CP077759 (https://www.ncbi.nlm.nih.gov/nuccore/CP077759). All data used or analyzed during this study are included in this article.

References

  1. Food and Agriculture Organization of the United Nations World Health Organization FAO, WHO (2002) Joint FAO/WHO working group report on drafting guidelines for the evaluation of probiotics in food. London, Ontario, Canada. https://www.who.int/foodsafety/fs_management/en/probiotic_guidelines.pdf

  2. Helland MH, Wicklund T, Narvhus JA (2004) Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley. Int J Food Microbiol 91:305–313. https://doi.org/10.1016/j.ijfoodmicro.2003.07.007

    Article  CAS  PubMed  Google Scholar 

  3. Parvez S, Malik KA, Ah Kang S, Kim HY (2006) Probiotics and their fermented food products are beneficial for health. J Appl Microbiol 100:1171–1185. https://doi.org/10.1111/j.1365-2672.2006.02963.x

    Article  CAS  PubMed  Google Scholar 

  4. Hussain MA, Hosseini Nezhad M, Sheng Y, Amoafo O (2013) Proteomics and the stressful life of lactobacilli. FEMS Microbiol Lett 349:1–8. https://doi.org/10.1111/1574-6968.12274

    Article  CAS  PubMed  Google Scholar 

  5. Dragosits M, Mattanovich D (2013) Adaptive laboratory evolution - principles and applications for biotechnology. Microb Cell Fact 12:1–17. https://doi.org/10.1186/1475-2859-12-64

    Article  Google Scholar 

  6. Kulkarni S, Haq SF, Samant S, Sukumaran S (2018) Adaptation of Lactobacillus acidophilus to thermal stress yields a thermotolerant variant which also exhibits improved survival at pH 2. Probiotics Antimicrob Proteins 10:717–727. https://doi.org/10.1007/s12602-017-9321-7

    Article  CAS  PubMed  Google Scholar 

  7. Mbye M, Baig MA, AbuQamar SF, El-Tarabily AK, Obaid RS, Osaili TM, Al-Nabulsi AA, Turner MS, Shah NP, Ayyash MM (2020) Updates on understanding of probiotic lactic acid bacteria responses to environmental stresses and highlights on proteomic analyses. Compr Rev Food Sci Food Saf 19:1110–1124. https://doi.org/10.1111/1541-4337.12554

    Article  PubMed  Google Scholar 

  8. Khalil AA (2006) Nutritional improvement of an Egyptian breed of mung bean by probiotic lactobacilli. African J Biotechnol 5:206–212

    CAS  Google Scholar 

  9. Erkus O (2007) Thesis: isolation, phenotypic and genotypic characterization of yoghurt starter bacteria. Msc Thesis 117

  10. Kenneth T (2020) Growth of bacterial populations. Todar's online textbook of bacteriology 3. http://textbookofbacteriology.net/growth_3.html

  11. Van Heerden JH, Kempe H, Doerr A, Maarleveld T, Nordholt N, Bruggeman FJ (2017) Statistics and simulation of growth of single bacterial cells: illustrations with B. subtilis and E. coli. Sci Rep 7:1–11. https://doi.org/10.1038/s41598-017-15895-4

    Article  CAS  Google Scholar 

  12. Wang J, Dong X, Shao Y, Guo H, Pan L, Hui W, Kwok LY, Zhang H, Zhang W (2017) Genome adaptive evolution of Lactobacillus casei under long-term antibiotic selection pressures. BMC Genomics 18:320. https://doi.org/10.1186/s12864-017-3710-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hippolyte MT, Augustin M, Hervé TM, Robert N, Somashekar D (2018) Application of response surface methodology to improve the production of antimicrobial biosurfactants by Lactobacillus paracasei subsp. tolerans N2 using sugar cane molasses as substrate. Bioresour Bioprocess 5:48. https://doi.org/10.1186/s40643-018-0234-4

  14. Archer AC, Halami PM (2015) Probiotic attributes of Lactobacillus fermentum isolated from human feces and dairy products. Appl Microbiol Biotechnol 99:8113–8123. https://doi.org/10.1007/s00253-015-6679-x

    Article  CAS  PubMed  Google Scholar 

  15. Yadav R, Puniya AK, Shukla P (2016) Probiotic properties of Lactobacillus plantarum RYPR1 from an indigenous fermented beverage Raabadi. Front Microbiol 7:1683. https://doi.org/10.3389/fmicb.2016.01683

    Article  PubMed  PubMed Central  Google Scholar 

  16. Zárate G, Chaia AP, González S, Oliver G (2000) Viability and β-galactosidase activity of dairy propionibacteria subjected to digestion by artificial gastric and intestinal fluids. J Food Prot 63:1214–1221. https://doi.org/10.4315/0362-028X-63.9.1214

    Article  PubMed  Google Scholar 

  17. Bao Y, Zhang Y, Zhang Y, Liu Y, Wang S, Dong X, Wang Y, ZhangH, (2010) Screening of potential probiotic properties of Lactobacillus fermentum isolated from traditional dairy products. Food Control 21:695–701. https://doi.org/10.1016/j.foodcont.2009.10.010

    Article  CAS  Google Scholar 

  18. Collado MC, Meriluoto J, Salminen S (2008) Adhesion and aggregation properties of probiotic and pathogen strains. Eur Food Res Technol 226:1065–1073. https://doi.org/10.1007/s00217-007-0632-x

    Article  CAS  Google Scholar 

  19. Kaushik JK, Kumar A, Duary RK, Mohanty AK, Grover S, Batish VK (2009) Functional and probiotic attributes of an indigenous isolate of Lactobacillus plantarum. PLoS One 4(12):e80999. https://doi.org/10.1371/journal.pone.0008099

    Article  CAS  Google Scholar 

  20. Sharma K, Mahajan R, Attri S, Goel G (2017) Selection of indigenous Lactobacillus paracasei CD4 and Lactobacillus gastricus BTM 7 as probiotic: assessment of traits combined with principal component analysis. J Appl Microbiol 122:1310–1320. https://doi.org/10.1111/jam.13426

    Article  CAS  PubMed  Google Scholar 

  21. Reuben RC, Roy PC, Sarkar SL, Alam RU, Jahid IK (2019) Isolation, characterization, and assessment of lactic acid bacteria toward their selection as poultry probiotics. BMC Microbiol 19:253. https://doi.org/10.1186/s12866-019-1626-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Du Toit M, Franz CMAP, Dicks LMT, Schillinger U, Haberer P, Warlies B, Ahrens F, Holzapfel WH (1998) Characterisation and selection of probiotic lactobacilli for a preliminary minipig feeding trial and their effect on serum cholesterol levels, faeces pH and faeces moisture content. Int J Food Microbiol 40:93–104. https://doi.org/10.1016/S0168-1605(98)00024-5

    Article  PubMed  Google Scholar 

  23. Perkins DN, Pappin DJC, Creasy DM, Cottrell JS (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20:3551–3567. https://doi.org/10.1002/(sici)1522-2683(19991201)20:18%3C3551::aid-elps3551%3E3.0.co;2-2

    Article  CAS  PubMed  Google Scholar 

  24. Paim DRSF, Costa SDO, Walter EHM, Tonon RV (2016) Microencapsulation of probiotic jussara (Euterpe edulis M.) juice by spray drying. LWT - Food Sci Technol 74:21–25. https://doi.org/10.1016/j.lwt.2016.07.022

    Article  CAS  Google Scholar 

  25. Haddaji N, Krifi B, Lagha R, Khouadja S, Bakhrouf A (2015) Effect of high temperature on viability of Lactobacillus casei and analysis of secreted and GroEL proteins profiles. African J Bacteriol Res 7:29–34

    Google Scholar 

  26. Brizuela MA, Serrano P, Ferez Y (2001) Studies on probiotics properties of two lactobacillus strains. Brazilian Arch Biol Technol 44:95–99. https://doi.org/10.1590/S1516-89132001000100013

    Article  CAS  Google Scholar 

  27. Tajabadi N, Mardan M, Saari N, Mustafa S, Bahreini R, Manap MYA (2013) Identification of Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus fermentum from honey stomach of honeybee. Brazilian J Microbiol 44:717–722. https://doi.org/10.1590/S1517-83822013000300008

    Article  CAS  Google Scholar 

  28. Capozzi V, Weidmann S, Fiocco D, Rieu A, Hols P, Guzzo J, Spano G (2011) Inactivation of a small heat shock protein affects cell morphology and membrane fluidity in Lactobacillus plantarum WCFS1. Res Microbiol 162:419–425. https://doi.org/10.1016/j.resmic.2011.02.010

    Article  CAS  PubMed  Google Scholar 

  29. De Angelis M, Di Cagno R, Huet C, Crecchio C, Fox PF, Gobbetti M (2004) Heat shock response in Lactobacillus plantarum. Appl Environ Microbiol 70:1336–1346. https://doi.org/10.1128/AEM.70.3.1336-1346.2004

    Article  CAS  Google Scholar 

  30. Ferrando V, Quiberoni A, Reinheimer J, Suárez V (2016) Functional properties of Lactobacillus plantarum strains: a study in vitro of heat stress influence. Food Microbiol 54:154–161. https://doi.org/10.1016/j.fm.2015.10.003

    Article  CAS  Google Scholar 

  31. Del Re B, Sgorbati B, Miglioli M, Palenzona D (2000) Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Lett Appl Microbiol 31:438–442. https://doi.org/10.1046/j.1365-2672.2000.00845.x

    Article  PubMed  Google Scholar 

  32. Moorman MA, Thelemann CA, Zhou S, Pestka JJ, Linz JE, Ryser ET (2008) Altered hydrophobicity and membrane composition in stress-adapted Listeria innocua. J Food Prot 71:182–185. https://doi.org/10.4315/0362-028X-71.1.182

    Article  CAS  PubMed  Google Scholar 

  33. de Souza BMS, Borgonovi TF, Casarotti SN, Todorov SD, Penna ALB (2019) Lactobacillus casei and Lactobacillus fermentum strains isolated from mozzarella Cheese: probiotic potential, safety, acidifying kinetic parameters and viability under gastrointestinal tract conditions. Probiotics Antimicrob Proteins 11:382–396. https://doi.org/10.1007/s12602-018-9406-y

    Article  CAS  PubMed  Google Scholar 

  34. Haddaji N, Mahdhi AK, Krifi B, Ismail MB, Bakhrouf A (2015) Change in cell surface properties of Lactobacillus casei under heat shock treatment. FEMS Microbiol Lett 362:1–7. https://doi.org/10.1093/femsle/fnv047

    Article  CAS  Google Scholar 

  35. Leyer GJ, Johnson EA (1993) Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium. Appl Environ Microbiol 59:1842–1847. https://doi.org/10.1128/aem.59.6.1842-1847.1993

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Mafu AA, Roy D, Goulet J, Savoie L (1991) Characterization of physicochemical forces involved in adhesion of Listeria monocytogenes to surfaces. Appl Environ Microbiol 57:1969–1973. https://doi.org/10.1128/aem.57.7.1969-1973.1991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Assefa E, Beyene F, Santhanam A (2008) Effect of temperature and pH on the antimicrobial activity of inhibitory substances produced by lactic acid bacteria isolated from Ergo, an Ethiopian traditional fermented milk. African J Microbiol Res 2:229–234

    Google Scholar 

  38. Wang AN, Yi XW, Yu HF, Dong B, Sy Q (2009) Free radical scavenging activity of Lactobacillus fermentum in vitro and its antioxidative effect on growing-finishing pigs. J Appl Microbiol 107:1140–1148. https://doi.org/10.1111/j.1365-2672.2009.04294.x

    Article  CAS  PubMed  Google Scholar 

  39. Grujović M, Mladenović KG, Nikodijević DD, Čomić LR (2019) Autochthonous lactic acid bacteria—presentation of potential probiotics application. Biotechnol Lett 41:1319–1331. https://doi.org/10.1007/s10529-019-02729-8

    Article  CAS  PubMed  Google Scholar 

  40. Begley M, Gahan CGM, Hill C (2005) The interaction between bacteria and bile. FEMS Microbiol Rev 29:625–651. https://doi.org/10.1016/j.femsre.2004.09.003

    Article  CAS  PubMed  Google Scholar 

  41. Bove P, Russo P, Capozzi V, Gallone A, Fiocco D (2013) Lactobacillus plantarum passage through an oro-gastro-intestinal tract simulator: carrier matrix effect and transcriptional analysis of genes associated to stress and probiosis. Microbiol Res 168:351–359. https://doi.org/10.1016/j.micres.2013.01.004

    Article  CAS  PubMed  Google Scholar 

  42. Chen MJ, Tang HY, Chiang ML (2017) Effects of heat, cold, acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1. Food Microbiol 66:20–27. https://doi.org/10.1016/j.fm.2017.03.020

    Article  CAS  PubMed  Google Scholar 

  43. Wu R, Zhang W, Sun T, Wu J, Yue X, He M, Zhang H (2011) Proteomic analysis of responses of a new probiotic bacterium Lactobacillus casei Zhang to low acid stress. Int J Food Microbiol 147:181–187. https://doi.org/10.1016/j.ijfoodmicro.2011.04.003

    Article  CAS  PubMed  Google Scholar 

  44. Hernández-Alcántara AM, Wacher C, Llamas MG, López P, Chabela MLP (2018) Probiotic properties and stress response of thermotolerant lactic acid bacteria isolated from cooked meat products. LWT - Food Sci Technol 91:249–257. https://doi.org/10.1016/j.lwt.2017.12.063

    Article  CAS  Google Scholar 

  45. Pan H, Agarwalla S, Moustakas DT, Moore JF, Stroud RM (2003) Structure of tRNA pseudouridine synthase TruB and its RNA complex: RNA recognition through a combination of rigid docking and induced fit. Proc Natl Acad Sci U S A 100:12648–12653. https://doi.org/10.1073/pnas.2135585100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Foster PG, Huang L, Santi DV, Stroud RM (2000) The structural basis for tRNA recognition and pseudouridine formation by pseudouridine synthase I. Nat Struct Biol 7:23–27. https://doi.org/10.1038/71219

    Article  CAS  PubMed  Google Scholar 

  47. Unciuleac MC, Goldgur Y, Shuman S (2015) Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase. Proc Natl Acad Sci USA 112:13868–13873. https://doi.org/10.1073/pnas.1516536112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Koistinen KM, Plumed-Ferrer C, Lehesranta SJ, Kärenlampi SO, Wright AV (2007) Comparison of growth-phase-dependent cytosolic proteomes of two Lactobacillus plantarum strains used in food and feed fermentations. FEMS Microbiol Lett 273:12–21. https://doi.org/10.1111/j.1574-6968.2007.00775.x

    Article  CAS  PubMed  Google Scholar 

  49. Feirer N, Fuqua C (2017) Pterin function in bacteria. Pteridines 28:23–36. https://doi.org/10.1515/pterid-2016-0012

    Article  CAS  Google Scholar 

  50. Jiang M, Chen X, Guo ZF, Cao Y, Chen M, Guo Z (2008) Identification and characterization of (1R,6R)-2-succinyl-6-hydroxy-2,4- cyclohexadiene-1-carboxylate synthase in the menaquinone biosynthesis of Escherichia coli. Biochemistry 47:3426–3434. https://doi.org/10.1021/bi7023755

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors gratefully thank the Director of CSIR-CFTRI, Mysore, for providing facilities to carry out this work. One of the authors, JB, gratefully acknowledges CSIR, New Delhi, for the award of Senior Research Fellowship.

Funding

This work was supported by the Council of Scientific Industrial Research-Senior research Fellowship (CSIR-SRF), New Delhi (File No: 31/5 (542)/2017-EMR-I).

Author information

Authors and Affiliations

  1. Microbiology and Fermentation Technology Department, CSIR-Central Food Technological Research Institute, Mysore, 570020, Karnataka, India

    Jyothna Bommasamudram & Somashekar Devappa

  2. CSIR-Institute of Microbial Technology, Chandigarh, 160036, India

    Pradeep Kumar, Sonal Kapur & Deepak Sharma

Authors
  1. Jyothna Bommasamudram
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pradeep Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sonal Kapur
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Deepak Sharma
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Somashekar Devappa
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JB: Investigation, writing, editing, and reviewing, PK, SK: Investigation of 2D gel electrophoresis, DS: Conceptualization of protein-related work, SD: Supervision, Conceptualization, editing, and reviewing. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Somashekar Devappa.

Ethics declarations

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bommasamudram, J., Kumar, P., Kapur, S. et al. Development of Thermotolerant Lactobacilli Cultures with Improved Probiotic Properties Using Adaptive Laboratory Evolution Method. Probiotics & Antimicro. Prot. 15, 832–843 (2023). https://doi.org/10.1007/s12602-021-09892-3

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12602-021-09892-3

Keywords

Search

Navigation