Skip to main content
Log in

The protective effect of L-theanine addition to a cryoprotectant on the storage viability of lactic acid bacteria

  • Research
  • Published:
International Microbiology Aims and scope Submit manuscript

Abstract

Lactic acid bacteria (LAB) that metabolize sugars to obtain energy and produce a large amount of lactate through the process are well known for their benefits. However, they can be used on a large scale only when good storage stability is guaranteed. The vitality and stability of several LAB strains were effectively protected in this investigation by L-theanine at 1% of the appropriate concentration (Lactiplantibacillus plantarum MG5023, Enterococcus faecium MG5232, Lactococcus lactis MG4668, Streptococcus thermophilus MG5140, and Bifidobacterium animalis subsp. lactis MG741). The inclusion of L-theanine as a protective agent significantly enhanced the viability of all strains throughout the freeze-drying process compared to that of the non-coated probiotics. The efficacy of L-theanine in improving bacterial stability and survivability was evaluated using accelerated stability tests, gastrointestinal (GI) tract survivability tests, and adhesion assays with intestinal epithelial cells. The cell surface was covered with substances including L-theanine, according to morphological findings, providing efficient defense against a variety of external stresses. Therefore, by exerting anti-freezing and anti-thawing properties, the adoption of L-theanine as a new and efficient protective agent may improve the stability and viability of a variety of probiotics.

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

Access this article

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

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Data availability

All data are presented in the paper.

References

  • Abadias M, Teixidó N, Usall J, Benabarre A, Vinas I (2001) Viability, efficacy, and storage stability of freeze-dried biocontrol agent Candida sake using different protective and rehydration media. J Food Prot 64(6):856–861

    Article  PubMed  CAS  Google Scholar 

  • Albadran HA, Chatzifragkou A, Khutoryanskiy VV, Charalampopoulos D (2015) Stability of probiotic Lactobacillus plantarum in dry microcapsules under accelerated storage conditions. Food Res Int 74:208–216

    Article  PubMed  CAS  Google Scholar 

  • Alp D, Kuleaşan H (2019) Adhesion mechanisms of lactic acid bacteria: conventional and novel approaches for testing. World J Microbiol Biotechnol 35:1–9

    Article  CAS  Google Scholar 

  • Baral KC, Bajracharya R, Lee SH, Han H-K (2021) Advancements in the pharmaceutical applications of probiotics: dosage forms and formulation technology. Int J Nanomed 16:7535

    Article  CAS  Google Scholar 

  • Bauer SAW, Schneider S, Behr J, Kulozik U, Foerst P (2012) Combined influence of fermentation and drying conditions on survival and metabolic activity of starter and probiotic cultures after low-temperature vacuum drying. J Biotechnol 159(4):351–357

    Article  PubMed  CAS  Google Scholar 

  • Chaffanel F, Charron-Bourgoin F, Soligot C, Kebouchi M, Bertin S, Payot S, Le Roux Y, Leblond-Bourget N (2018) Surface proteins involved in the adhesion of Streptococcus salivarius to human intestinal epithelial cells. Appl Microbiol Biotechnol 102:2851–2865

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Chen X, Li L, Yang F, Wu J, Wang S (2020) Effects of gelatin-based antifreeze peptides on cell viability and oxidant stress of Streptococcus thermophilus during cold stage. Food Chem Toxicol 136:111056

    Article  PubMed  CAS  Google Scholar 

  • Chen B, Wang X, Li P, Feng X, Mao Z, Wei J, Lin X, Li X, Wang L (2023) Exploring the protective effects of freeze-dried Lactobacillus rhamnosus under optimized cryoprotectants formulation. LWT 173:114295. https://doi.org/10.1016/j.lwt.2022.114295

    Article  CAS  Google Scholar 

  • Crittenden R, Weerakkody R, Sanguansri L, Augustin M (2006) Synbiotic microcapsules that enhance microbial viability during nonrefrigerated storage and gastrointestinal transit. Appl Environ Microbiol 72(3):2280–2282

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Cui S, Hu K, Qian Z, Mao B, Zhang Q, Zhao J, Tang X, Zhang H (2022) Improvement of freeze-dried survival of Lactiplantibacillus plantarum based on cell membrane regulation. Microorganisms 10(10):1985

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Hidese S, Ogawa S, Ota M, Ishida I, Yasukawa Z, Ozeki M, Kunugi H (2019) Effects of L-theanine administration on stress-related symptoms and cognitive functions in healthy adults: a randomized controlled trial. Nutrients 11(10):2362

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Jalali M, Abedi D, Varshosaz J, Najjarzadeh M, Mirlohi M, Tavakoli N (2011) Stability evaluation of freeze-dried Lactobacillus paracasei subsp. tolerance and Lactobacillus delbrueckii subsp. bulgaricus in oral capsules. Res Pharm Sci 7(1):31–36

    Google Scholar 

  • Kailasapathy K (2006) Survival of free and encapsulated probiotic bacteria and their effect on the sensory properties of yoghurt. LWT-Food Sci Technol 39(10):1221–1227

    Article  CAS  Google Scholar 

  • Kim JK, Shin EC, Park HG (2015) Fructooligosaccharides decreased the ability of probiotic Escherichia coli Nissle 1917 to adhere to co-cultures of human intestinal cell lines. J Korean Soc Appl Biol Chem 58(1):45–52

    Article  CAS  Google Scholar 

  • Kim S Il, Kim JW, Kim KT, Kang CH (2021) Survivability of collagen-peptide microencapsulated lactic acid bacteria during storage and simulated gastrointestinal conditions. Fermentation 7(3). https://doi.org/10.3390/fermentation7030177

  • Krausova G, Hyrslova I, Hynstova I (2019) In vitro evaluation of adhesion capacity, hydrophobicity, and auto-aggregation of newly isolated potential probiotic strains. Fermentation 5(4):100

    Article  CAS  Google Scholar 

  • Lee JY, Kang C-H (2022) Probiotics alleviate oxidative stress in H2O2-exposed hepatocytes and t-BHP-induced C57BL/6 mice. Microorganisms 10(2):234

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Lee WK, Kim TI, Park S, Park HK, Hong JT (2010) Anxiolytic effect of a combination of green tea extract and L-theanine. Lab Anim Res 26(1):63–68

    Article  Google Scholar 

  • Makinen K, Berger B, Bel-Rhlid R, Ananta E (2012) Science and technology for the mastership of probiotic applications in food products. J Biotechnol 162(4):356–365

    Article  PubMed  CAS  Google Scholar 

  • Menezes MF da SC de, Silva TM da, Etchepare M de A, Fonseca B de S, Sonza VP, Codevilla CF, Barin JS, Silva C de B da, Menezes CR de (2019) Improvement of the viability of probiotics (Lactobacillus acidophilus) by multilayer encapsulation. Ciência Rural 49:e20181020

  • Nguyen TH, Kim J-S, Kwon H-J, Kang C-H (2023) The Effect of a glutathione (GSH)-containing cryo-protectant on the viability of probiotic cells using a freeze-drying process. Fermentation 9(2):187

    Article  CAS  Google Scholar 

  • Nguyen TH, Kim Y, Kim JS, Jeong Y, Park HM, Kim JW, Kim JE, Kim H, Paek NS, Kang CH (2020) Evaluating the cryoprotective encapsulation of the lactic acid bacteria in simulated gastrointestinal conditions. Biotechnol Bioprocess Eng 25(2):287–292

    Article  CAS  Google Scholar 

  • Obradović N, Volić M, Nedović V, Rakin M, Bugarski B (2022) Microencapsulation of probiotic starter culture in protein–carbohydrate carriers using spray and freeze-drying processes: implementation in whey-based beverages. J Food Eng 321:110948

    Article  Google Scholar 

  • Oniszczuk A, Oniszczuk T, Gancarz M, Szymańska J (2021) Role of gut microbiota, probiotics and prebiotics in the cardiovascular diseases. Molecules 26(4):1172

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Palzer S (2009) Food structures for nutrition, health and wellness. Trends Food Sci Technol 20(5):194–200. https://doi.org/10.1016/J.TIFS.2009.02.005

    Article  CAS  Google Scholar 

  • Romyasamit C, Saengsuwan P, Boonserm P, Thamjarongwong B, Singkhamanan K (2022) Optimization of cryoprotectants for freeze-dried potential probiotic Enterococcus faecalis and evaluation of its storage stability. Drying Technol 40(11):2283–2292. https://doi.org/10.1080/07373937.2021.1931294

    Article  CAS  Google Scholar 

  • Saeed M, Naveed M, Arif M, Kakar MU, Manzoor R, Abd El-Hack ME, Alagawany M, Tiwari R, Khandia R, Munjal A (2017) Green tea (Camellia sinensis) and l-theanine: medicinal values and beneficial applications in humans—a comprehensive review. Biomed Pharmacother 95:1260–1275

    Article  PubMed  CAS  Google Scholar 

  • Saeed M, Xu Y, Zhang T, Ren Q, Sun C (2019) 16S ribosomal RNA sequencing reveals a modulation of intestinal microbiome and immune response by dietary L-theanine supplementation in broiler chickens. Poult Sci 98(2):842–854. https://doi.org/10.3382/ps/pey394

    Article  PubMed  CAS  Google Scholar 

  • Siaterlis A, Deepika G, Charalampopoulos D (2009) Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying. Lett Appl Microbiol 48(3):295–301

    Article  PubMed  CAS  Google Scholar 

  • Son SJ, Koh JH, Park MR, Ryu S, Lee WJ, Yun B, Lee JH, Oh S, Kim Y (2019) Effect of the Lactobacillus rhamnosus strain GG and tagatose as a synbiotic combination in a dextran sulfate sodium-induced colitis murine model. J Dairy Sci 102(4):2844–2853. https://doi.org/10.3168/jds.2018-15013

    Article  PubMed  CAS  Google Scholar 

  • Teixeira PC, Castro MH, Malcata FX, Kirby RM (1995) Survival of Lactobacillus delbrueckii subsp. bulgaricus following spray-drying. J Dairy Sci 78(5):1025–1031

    Article  CAS  Google Scholar 

  • Vanhee LME, Goemé F, Nelis HJ, Coenye T (2010) Quality control of fifteen probiotic products containing Saccharomyces boulardii. J Appl Microbiol 109(5):1745–1752. https://doi.org/10.1111/j.1365-2672.2010.04805.x

    Article  PubMed  CAS  Google Scholar 

  • Weinbreck F, Bodnár I, Marco ML (2010) Can encapsulation lengthen the shelf-life of probiotic bacteria in dry products? Int J Food Microbiol 136(3):364–367

    Article  PubMed  CAS  Google Scholar 

  • Williams J, Sergi D, McKune AJ, Georgousopoulou EN, Mellor DD, Naumovski N (2019) The beneficial health effects of green tea amino acid l-theanine in animal models: promises and prospects for human trials. Phytother Res 33(3):571–583

    Article  PubMed  Google Scholar 

  • Yuan Y, Yin M, Chen L, Liu F, Chen M, Zhong F (2022) Effect of calcium ions on the freeze-drying survival of probiotic encapsulated in sodium alginate. Food Hydrocolloids 130:107668. https://doi.org/10.1016/j.foodhyd.2022.107668

    Article  CAS  Google Scholar 

  • Yuste A, Arosemena EL, Calvo MÀ (2021) Study of the probiotic potential and evaluation of the survival rate of Lactiplantibacillus plantarum lyophilized as a function of cryoprotectant. Sci Rep 11(1):19078

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Zhao G, Zhang G (2005) Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying. J Appl Microbiol 99(2):333–338

    Article  PubMed  CAS  Google Scholar 

Download references

Funding

This research received no external funding

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization, C.-H.K.; methodology H.-J.K., T.H.N., and J.-S.K.; formal analysis, H.-J.K., T.H.N., and J.-S.K.; investigation, H.-J.K., T.H.N., and J.-S.K.; data curation H,-J.K. and T.H.N.; writing-original draft preparation, H.-J.K.; writing-review and editing, H.-J.K., T.H.N., and C.-H.K.; visualization, H.-J.K.; supervision, C.-H.K.; project administration, C.-H.K. All authors have read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Chang-Ho Kang.

Ethics declarations

Ethics approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

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

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kwon, HJ., Nguyen, T.H., Kim, JS. et al. The protective effect of L-theanine addition to a cryoprotectant on the storage viability of lactic acid bacteria. Int Microbiol (2023). https://doi.org/10.1007/s10123-023-00444-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s10123-023-00444-z

Keywords

Navigation