Skip to main content
SpringerLink
Log in

Safety Assessment of Potential Probiotic Lactobacillus fermentum MTCC-5898 in Murine Model after Repetitive Dose for 28 Days (Sub-Acute Exposure)

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

Abstract

Safety assessment of probiotic Lactobacillus fermentum MTCC-5898 (LF) with three doses (107, 109, and 1011 cfu/day/animal) was carried on Swiss albino mouse weanlings for 28 days using oral route. Health status of animals was monitored by physical assessment of body weight, organ indices, and histological appearances of liver and intestine along with measurement of hematological parameters (Hb, WBC, RBC count, MCHC, MCV, MCH), biochemical analytes in blood involving glucose, serum enzymes (ALT, AST and LDH), urea, creatinine, and lipid profile (total cholesterol, triglycerides, HDL, VLDL, LDL, and atherogenic index). LF showed no adverse effects on above parameters of general health status after continuous consumption for the experimental period. On the other hand, significant increase (p ≤ 0.05) in TGF-β (regulatory cytokine) and considerable decrease (p ≤ 0.05) in IFN-γ (pro-inflammatory cytokine) without any major changes in IL-4 and IL-12 in intestinal fluid on consumption of 109 cfu/animal/day confirmed its dose-specific response for immune homeostasis. Further, safety of LF was also confirmed by insignificant changes in release of FITC-dextran (4 kDa) in blood on its consumption than control group where only saline was given orally. Moreover, significantly (p ≤ 0.05) increased mRNA expression of claudin-1 and MUC-2 in intestinal epithelial cells on feeding L. fermentum further supported FITC-dextran permeability data which otherwise showed increased flux of FITC-dextran in blood on consumption of E. coli (109 cfu/animal/day) due to intestinal damage. Thus, in vivo results confirmed that Lactobacillus fermentum MTCC 5898 is safe and non-toxic to weanling mice and may be considered for functional food application after clinical testing.

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
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Sekirov I, Russell SL, Antunes LCM, Finlay BB (2010) Gut microbiota in health and disease. Physiol Rev 90:859–904

    Article  CAS  PubMed  Google Scholar 

  2. Fijan S (2014) Microorganisms with claimed probiotic properties: an overview of recent literature. Int J Environ Res Public Health 11:4745–4767

    Article  PubMed  PubMed Central  Google Scholar 

  3. Bernardo WM, Aires FT, Carneiro RM, de Sa FP, Rullo VE, Burns DA (2013) Effectiveness of probiotics in the prophylaxis of necrotizing enterocolitis in preterm neonates: a systematic review and meta-analysis. J Pediatr (Versão em Português) 89:18–24

    Article  Google Scholar 

  4. Khalesi S, Sun J, Buys N, Jayasinghe R (2014) Effect of probiotics on blood pressure: a systematic review and meta-analysis of randomized, controlled trials. Hypertension 64:897–903

    Article  CAS  PubMed  Google Scholar 

  5. Doron S, Snydman DR (2015) Risk and safety of probiotics. Clin Infect Dis 60:129–134

    Article  Google Scholar 

  6. Hanlon PR, Frestedt J, Magurany K (2017) GRAS from the ground up: review of the interim pilot program for GRAS notification. Food Chem Toxicol 105:140–150

    Article  CAS  Google Scholar 

  7. Hojsak I, Shamir R (2013) Safety of probiotics. In Probiotic bacteria and their effect on human health and well-being . Karger, Basel, pp 161–170

  8. Metchnikoff E, Metchnikoff II, Mitchell PC (1908) The prolongation of life: optimistic studies. GP Putnam's Sons, New York, USA

    Google Scholar 

  9. Ganguly NK, Bhattacharya SK, Sesikeran B, Nair GB, Ramakrishna BS, Sachdev HP, Batish VK, Kanagasabapathy AS, Muthuswamy V, Kathuria SC, Katoch VM (2011) ICMR-DBT guidelines for evaluation of probiotics in food. Indian J Med Res 134:22

    PubMed Central  Google Scholar 

  10. Sharma R, Kapila R, Kapasiya M, Saliganti V, Dass G, Kapila S (2014) Dietary supplementation of milk fermented with probiotic Lactobacillus fermentum enhances systemic immune response and antioxidant capacity in aging mice. Nutr Res 34:968–981

    Article  CAS  PubMed  Google Scholar 

  11. Yadav R, Khan SH, Mada SB, Meena S, Kapila R, Kapila S (2018) Consumption of probiotic Lactobacillus fermentum MTCC: 5898-fermented milk attenuates dyslipidemia, oxidative stress, and inflammation in male rats fed on cholesterol-enriched diet. Probiotics Antimicrob Proteins 13:1–10

    Google Scholar 

  12. Yadav R, Dey DK, Vij R, Meena S, Kapila R, Kapila S (2018) Evaluation of anti-diabetic attributes of Lactobacillus rhamnosus MTCC: 5957, Lactobacillus rhamnosus MTCC: 5897 and Lactobacillus fermentum MTCC: 5898 in streptozotocin induced diabetic rats. Microb Pathog 125:454–462

    Article  CAS  PubMed  Google Scholar 

  13. Lim TS, Messiha N, Watson RR (1981) Immune components of the intestinal mucosae of ageing and protein deficient mice. Immunology 43:401

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Yatzidis H (1960) Measurement of transaminases in serum. Nature 186:79–80

    Article  CAS  PubMed  Google Scholar 

  15. Bergmeyer HU, Gawehn K (1978) Principles of enzymatic analysis. Verlag Chemie, NewYork

    Google Scholar 

  16. Uchiyama M, Mihara M (1978) Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86:271–278

    Article  CAS  PubMed  Google Scholar 

  17. Friedewald WT, Levy RI, Fredrickson DS (1972) Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 18:499–502

    Article  CAS  PubMed  Google Scholar 

  18. Wang L, Llorente C, Hartmann P, Yang AM, Chen P, Schnabl B (2015) Methods to determine intestinal permeability and bacterial translocation during liver disease. J Immunol Methods 421:44–53

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25:402–408

    Article  CAS  PubMed  Google Scholar 

  20. Festing MF, Altman DG (2002) Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR J 43:244–258

    Article  CAS  PubMed  Google Scholar 

  21. Charan J, Kantharia ND (2013) How to calculate sample size in animal studies? J Pharmacol Pharmacother 4:303–306

    Article  PubMed  PubMed Central  Google Scholar 

  22. Basha S, Surendran N, Pichichero M (2014) Immune responses in neonates. Expert Rev Clin Immunol 10:1171–1184

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Perry P (2007) The ethics of animal research: a UK perspective. ILAR J48:42–46

    Article  Google Scholar 

  24. Stevenson RD, Woods WA Jr (2006) Condition indices for conservation: new uses for evolving tools. Integr Comp Biol 46:1169–1190

    Article  CAS  PubMed  Google Scholar 

  25. Kabeir BM, Yazid AM, Stephenie W, Nazrul Hakim M, Muhammad Anas O, Shuhaimi M (2008) Safety evaluation of Bifidobacterium pseudocatenulatum G4 as assessed in BALB/c mice. Lett Appl Microbiol 46:32–37

    CAS  PubMed  Google Scholar 

  26. Petterino C, Argentino-Storino A (2006) Clinical chemistry and haematology historical data in control Sprague-Dawley rats from pre-clinical toxicity studies. Exp Toxicol Pathol 57:213–219

    Article  CAS  PubMed  Google Scholar 

  27. Serfilippi LM, Stackhouse Pallman DR, Russell B, Spainhour CB (2003) Serum clinical chemistry and hematology reference values in outbred stocks of albino mice from three commonly used vendors and two inbred strains of albino mice. J Am Assoc Lab Anim 42:46–52

    CAS  Google Scholar 

  28. Ihedioha JI, Ugwuja JI, Noel-Uneke OA, Udeani IJ, Daniel-Igwe G (2012) Reference values for the haematology profile of conventional grade outbred albino mice (Mus musculus) in Nsukka, eastern Nigeria. Anim Res Int 9:1601–1612

    Google Scholar 

  29. Park JH, Lee YE, Moon EN, Seok SH, Baek MW, Lee HY, Kim DJ, Kim CH, Park JH (2005) Safety assessment of Lactobacillus fermentum PL9005, a potential probiotic lactic acid bacterium, in mice. J Microbiol Biotechnol 15:603–608

    CAS  Google Scholar 

  30. Lara-Villoslada F, Sierra S, Diaz-Ropero MP, Rodriguez JM, Xaus J, Olivares M (2009) Safety assessment of Lactobacillus fermentum CECT5716, a probiotic strain isolated from human milk. J Dairy Res 76:216–221

    Article  CAS  PubMed  Google Scholar 

  31. FAO/WHO (2001) Evaluation of health and nutritional properties of powder milk and live lactic acid bacteria; food and agriculture Organization of the United Nations and World Health Organization Expert Consultation Report, Rome

  32. Gowda S, Desai PB, Hull VV, Math AA, Vernekar SN, Kulkarni SS (2009) A review on laboratory liver function tests. Pan Afr Med J 3:17

    PubMed  PubMed Central  Google Scholar 

  33. Sher Y, Hung M (2013) Blood AST, ALT and UREA/BUN level analysis. Bio-Protocol 3:931

    Article  Google Scholar 

  34. Suckow MA, Brayton C, Danneman P (2001) The laboratory mouse. CRC, Boca Raton, Florida

    Google Scholar 

  35. Mazzaccara C, Labruna G, Cito G, Scarfo M, De Felice M, Pastore L, Sacchetti L (2008) Age-related reference intervals of the main biochemical and hematological parameters in C57BL/6J, 129SV/EV and C3H/HeJ mouse strains. PLoS One 3:3772

    Article  CAS  Google Scholar 

  36. Zhang Y, Li L, Guo C, Mu D, Feng B, Zuo X, Li Y (2016) Effects of probiotic type, dose and treatment duration on irritable bowel syndrome diagnosed by Rome III criteria: a meta-analysis. BMC Gastroenterol 16:62

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ouwehad AC (2016) A review of dose-responses of probiotics in human studies. Benefic Microbes 8:143–151

    Article  Google Scholar 

  38. Giannini EG, Testa R, Savarino V (2005) Liver enzyme alteration: a guide for clinicians. Can Med Assoc J 72:367–379

    Article  Google Scholar 

  39. Corbett JV (2008) Laboratory tests and diagnostic procedures with nursing diagnoses. San Francisco, California

  40. Gowda S, Desai PB, Kulkarni SS, Hull VV, Math AA, Vernekar SN (2010) Markers of renal function tests. N Am J Med Sci 2:170

    PubMed  PubMed Central  Google Scholar 

  41. Zhang H, Sun J, Wang QY, He YT, Gu HY, Guo HY, Ding QB, Yang JS, Ren FZ (2013) Safety assessment of Lactobacillus salivarius REN, a probiotic strain isolated from centenarian feces. J Food Sci Technol 19:1037–1043

    CAS  Google Scholar 

  42. Del Rio D, Stewart AJ, Pellegrini N (2005) A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis 15:316–328

    Article  PubMed  Google Scholar 

  43. Farmer EE, Davoine C (2007) Reactive electrophile species. Curr Opin Plant Biol 10:380–386

    Article  CAS  PubMed  Google Scholar 

  44. Kapila S, Sinha P (2006) Antioxidative and hypocholesterolemic effect of Lactobacillus casei ssp casei (biodefensive properties of lactobacilli). Indian J Med Sci 60:361–370

    Article  PubMed  Google Scholar 

  45. Aguilar EC, Queiroz MD, Oliveira DA, Oliveira NJ (2011) Serum lipid profile and hepatic evaluation in mice fed diet containing Pequi nut or pulp (Caryocar brasiliense Camb.). Food Sci Technol 31:879–883

    Article  Google Scholar 

  46. Santos EW, de Oliveira DC, Hastreiter A, da Silva GB, de Oliveira Beltran JS, Tsujita M, Crisma AR, Neves SM, Fock RA, Borelli P (2016) Values of hematological and biochemical reference for mice from the C57BL / 6, Swiss Webster and BALB/c lines. Braz J Vet Res Anim 53:38–145

    Google Scholar 

  47. Baroutkoub Baroutkoub A, Mehdi RZ, Beglarian R, Hassan J, Zahra S, Mohammad MS (2010) Effects of probiotic yoghurt consumption on the serum cholesterol levels in hypercholestromic cases in shiraz, southern Iran. Sci Res Essays 5:2206–2209

    Google Scholar 

  48. Shadnoush M, Hosseini RS, Mehrabi Y, Delpisheh A, Alipoor E, Faghfoori Z, Mohammadpour N, Moghadam JZ (2013) Probiotic yogurt affects pro-and anti-inflammatory factors in patients with inflammatory bowel disease. Iran J Pharm Res 12:929

    PubMed  PubMed Central  Google Scholar 

  49. Volynets V, Rings A, Bárdos G, Ostaff MJ, Wehkamp J, Bischoff SC (2016) Intestinal barrier analysis by assessment of mucins, tight junctions, and α-defensins in healthy C57BL/6J and BALB/cJ mice. Tissue Barriers 4:1208468

    Article  CAS  Google Scholar 

  50. Kumar M, Kissoon-Singh V, Coria AL, Moreau F, Chadee K (2016) Probiotic mixture VSL# 3 reduces colonic inflammation and improves intestinal barrier function in Muc2 mucin-deficient mice. Am J Physiol Gastrointest Liver Physiol 312:34–45

    Article  Google Scholar 

  51. Gotteland M, Cruchet S, Verbeke S (2001) Effect of Lactobacillus ingestion on the gastrointestinal mucosal barrier alterations induced by indometacin in humans. Aliment PharmacolTher 15:11–17

    Article  CAS  Google Scholar 

  52. Mennigen R, Nolte K, Rijcken E, Utech M, Loeffler B, Senninger N, Bruewer M (2009) Probiotic mixture VSL# 3 protects the epithelial barrier by maintaining tight junction protein expression and preventing apoptosis in a murine model of colitis. Am J Physiol Gastrointest Liver Physiol 296:1140–1149

    Article  CAS  Google Scholar 

  53. Hsieh CY, Osaka T, Moriyama E, Date Y, Kikuchi J, Tsuneda S (2015) Strengthening of the intestinal epithelial tight junction by Bifidobacterium bifidum. Phys Rep 3:12327

    Article  CAS  Google Scholar 

  54. Anderson RC, Cookson AL, McNabb WC, Park Z, McCann MJ, Kelly WJ, Roy NC (2010) Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by increasing the expression levels of genes involved in tight junction formation. BMC Microbiol 10:316

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors acknowledge ICAR-National Dairy Research Institute (NDRI) Karnal for providing laboratory facilities to carry out the present work.

Funding

The Department of BioTechnology, Ministry of Science and Technology, New Delhi provided funds (BT/PR15109/PFN/20/1174/2015).

Author information

Authors and Affiliations

  1. Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India

    Mrinal Samtiya, Mohd Iqbal Bhat, Taruna Gupta, Suman Kapila & Rajeev Kapila

Authors
  1. Mrinal Samtiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mohd Iqbal Bhat
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Taruna Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Suman Kapila
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rajeev Kapila
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rajeev Kapila.

Ethics declarations

The animal trials were conducted after the approval of the institutional animal ethics committee (ICAR-NDRI: Approval no. 41-IAEC-18-59 dated January 27, 2018).

Conflict of Interest

The authors declare that they have no conflict of interest.

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

Samtiya, M., Bhat, M.I., Gupta, T. et al. Safety Assessment of Potential Probiotic Lactobacillus fermentum MTCC-5898 in Murine Model after Repetitive Dose for 28 Days (Sub-Acute Exposure). Probiotics & Antimicro. Prot. 12, 259–270 (2020). https://doi.org/10.1007/s12602-019-09529-6

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12602-019-09529-6

Keywords

Search

Navigation