Keywords
How to Cite
Abstract
Many probiotic formulations are available in the market and are advertised for several preventive or curative roles. The aim of this study was the identification of microorganisms composing different lactic acid bacteria based pharmaceutical formulations and the ascertainment of their ability to survive gastro-intestinal (GI) stresses, the main requisite to produce beneficial effects. For this purpose, viable bacteria were enumerated by plate counts in different media. Denaturing Gradient Gel Electrophoresis-Polymerase Chain Reaction (PCR-DGGE) analysis was applied on pure isolates and on crude formulations to confirm the composition in species. Also, crude formulations were subjected to stresses characteristic of the GI tract (GIT) to assess cell survival. Results highlighted concentrations lower than those reported in the labels in almost all the formulations. Moreover, some discrepancies were observed between reported species and those ascertained through the identification, and the use of an erroneous nomenclature was highlighted. The GI stress test revealed that bacteria are strongly injured, and this fact was evidenced by a marked reduction in viable counts after the stress. In conclusion, a widespread number of lactic acid bacteria based formulations are sold as probiotics, but their probiotic requisites are not adequately observed.
References
Di Criscio T, Fratianni A, Mignogna R, et al. Production of functional probiotic, prebiotic and synbiotic ice creams. J Dairy Sci 2010; 93: 4555-4564. http://dx.doi.org/10.3168/jds.2010-3355
Regulation 178/2002 of the European Parliament and of the Council laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. Official Journal of the European Communities 2002; L31/1-L31/24.
Petrelli L. I probiotici: criteri per la qualificazione dei prodotti quali alimenti o medicinali nel diritto comunitario. Rivista di diritto agrario 2008; 4: 539-566. http://www.rivistadirittoalimentare. it/rivista/2011-03/PETRELLI.pdf
Iannitti T, Palmieri B. Therapeutical use of probiotic formulations in clinical practice. Clin Nutr 2010; 29: 701-725. http://dx.doi.org/10.1016/j.clnu.2010.05.004
FAO/WHO Expert Consultation on Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria. Cordoba, Argentina, 2001. (Accessed July 25, 2011, at http://www.who.int/foodsafety/ publications/fs_management/probiotics/en/)
Alakomi HL, Mättö J, Virkajärvi I, Saareal M. Application of a microplate scale fluorochrome staining assay for the assessment of viability of probiotic preparations. J Microbiol Methods 2005; 62: 25-35. http://dx.doi.org/10.1016/j.mimet.2005.01.005
Succi M, Tremonte P, Reale A, et al. Bile salt and acid tolerance of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese. FEMS Microbiol Lett 2005; 244: 129-137. http://dx.doi.org/10.1016/j.femsle.2005.01.037
Oozeer R, Leplingard A, Mater DD, et al. Survival of Lactobacillus casei in the human digestive tract after consumption of fermented milk. Appl Environ Microbiol 2006; 72: 5615-7. http://dx.doi.org/10.1128/AEM.00722-06
Ritter P, Kohler C, von Ah U. Evaluation of the passage of Lactobacillus gasseri K7 and bifidobacteria from the stomach to intestine using a single reactor model. BMC Microbiol 2009; 9: 87-95. http://dx.doi.org/10.1186/1471-2180-9-87
Tir Touil Meddah A, Yazourh A, Desmet I, Risbourg B, Verstraete W, Romond MB. The regulatory effects of whey retentate from Bifidobacteria fermented milk on the microbiota of the Simulator of the Human Intestinal Microbial Ecosystem (SHIME). J Appl Microbiol 2001; 91: 1110-1117. http://dx.doi.org/10.1046/j.1365-2672.2001.01482.x
de Palencia PF, Lopez P, Corbì AL, Pelàez C, Requena T. Probiotic strains: survival under simulated gastrointestinal conditions, in vitro adhesion to Caco-2 cells and effect on cytokine secretion. Eur Food Res Technol 2008; 227: 1475- 1484. http://dx.doi.org/10.1007/s00217-008-0870-6
Sumeri I, Arike L, Adamberg K, Paalme T. Single bioreactor gastrointestinal tract simulator for study of survival of probiotic bacteria. Appl Microbiol Biotechnol 2008; 80: 317- 324. http://dx.doi.org/10.1007/s00253-008-1553-8
Tharmaraj N, Shah NP. Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacteria, Lactobacillus casei, Lactobacillus rhamnosus, and Propionibacteria. J Dairy Sci 2003; 86: 2288-2296. http://dx.doi.org/10.3168/jds.S0022-0302(03)73821-1
Aureli P, Fiore A, Scalfaro C, Casale M, Franciosa G. National survey outcomes on commercial probiotic food supplements in Italy. Int J Food Microbiol 2010; 137: 265- 273. http://dx.doi.org/10.1016/j.ijfoodmicro.2009.12.016
Leuschener RGK, Bew J, Cruz A, et al. Enumeration of probiotic bacilli spores in animal feed: interlaboratory study. J AOAC Int 2003; 86: 568-575. http://lib3.dss.go.th/fulltext/ Journal/J.AOAC%201999-2003/J.AOAC2003/v86n3(mayjun)/ v86n3p568.pdf
Dave RI, Shah NP. Evaluation of media for selective enumeration of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus, and bifidobacteria. J Dairy Sci 1996; 79: 1529-36. http://dx.doi.org/10.3168/jds.S0022-0302(96)76513-X
Succi M, Tremonte P, Reale A, Sorrentino E, Coppola R. Preservation by freezing of potentially probiotic strains of Lactobacillus rhamnosus. Ann Microbiol 2007; 57: 537-544. http://dx.doi.org/10.1007/BF03175352
Querol A, Barrio E, Ramon D. A comparative study of different methods of yeast strains characterization. Syst Appl Microbiol 1992; 15: 439-446. http://dx.doi.org/10.1016/S0723-2020(11)80219-5
Klijn N, Weerkamp AH, de Vos WM. Identification of mesophilic lactic acid bacteria by using polymerase chain reaction-amplified variable regions of 16S rRNA and specific DNA probes. Appl Environ Microbiol 1991; 57: 3390-3393. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC183979/pdf/ae m00064-0344.pdf
Sheffield VC, Cox DR, Lerman LS, Myers RM. Attachment of a 40-base pairs G+C rich sequence (GC clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci 1989; 86: 297-303. http://dx.doi.org/10.1073/pnas.86.1.232
Reale A, Di Renzo T, Succi M, Tremonte P, Coppola R, Sorrentino E. Identification of lactobacilli isolated in traditional ripe wheat sourdoughs by using molecular methods. World J Microbiol Biotechnol 2011; 27: 237-244. http://dx.doi.org/10.1007/s11274-010-0448-x
Iacumin L, Cecchini F, Manzano M, et al. Description of the microflora of sourdoughs by culture-dependent and cultureindependent methods. Food Microbiol 2009; 26: 128-135. http://dx.doi.org/10.1016/j.fm.2008.10.010
Sanders ME. How do we know when something called “Probiotic” is really a probiotic? A guideline for consumers and health care professionals. Functional Food Rev 2009; 1: 3-12. http://dx.doi.org/10.2310/6180.2009.00002
Jijon H, Backer J, Diaz H, et al. DNA from probiotic bacteria modulates murine and human epithelial and immune function. Gastroenterol 2004; 126: 1358-1373. http://dx.doi.org/10.1053/j.gastro.2004.02.003
Zhang L, Li N, Caicedo R, Neu J. Alive and dead Lactobacillus rhamnosus GG decrease tumor necrosis factoralpha- induced interleukin-8 production in Caco-2 cells. J Nutr 2005; 135: 1752-6. http://jn.nutrition.org/content/135/7/1752. full.pdf+html
Adams CA. The probiotic paradox: live and dead cells are biological response modifiers. Nutr Res Rev 2010; 20: 1-10. http://dx.doi.org/10.1017/S0954422410000090
Mc Donald LC, Mc Feeters RF, Daeschel MA, Fleming HP. A differential medium for the enumeration of homofermentative and heterofermentative lactic acid bacteria. Appl Environ Microbiol 1987; 53: 1382-1384. http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC203874/pdf/aem00123-0186.pdf
Del Piano M, Morelli L, Strozzi GP, et al. Probiotics: from research to consumer. Dig Liver Dis 2006; 38: S248-S255. http://dx.doi.org/10.1016/S1590-8658(07)60004-8
Corcoran BM, Stanton C, Fitzgerald GF, Ross RP. Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Appl Environ Microbiol 2005; 71: 3060-3067. http://dx.doi.org/10.1128/AEM.71.6.3060-3067.2005
Sheehan WM, Sleator RD, Hill C, Fitzgerald GF. Improving gastric transit, gastrointestinal persistence and therapeutic efficacy of the probiotic strain Bifidobacterium breve UCC2003. Microbiol 2007; 153: 3563-3571. http://dx.doi.org/10.1099/mic.0.2007/006510-0
Watson D, Sleator RD, Hill C, Gahan CGM. Enhancing bile tolerance improves survival and persistence of Bifidobacterium and Lactococcus in the murine gastrointestinal tract. BMC Microbiol 2008; 8: 176-
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Copyright (c) 2014 Mariantonietta Succi, Elena Sorrentino, Tiziana Di Renzo, Patrizio Tremonte, Anna Reale, Luca Tipaldi, Gianfranco Pannella, Antonio Russo , Raffaele Coppola