Viability of the Lactobacillus rhamnosus IL1 Strain in Simulated...

[Full title: Viability of the Lactobacillus rhamnosus IL1 Strain in Simulated Gastrointestinal Conditions]

Life International Journal of Pharmacology ISSN 1811-7775 Life science alert ansinet Asian Network for Scientific Information

International Journal of Pharmacology 6 (5): 732-737, 2010 ISSN 1811-7775 2010 Asian Network for Scientific Information Viability of the Lactobacillus rhamnosus IL1 Strain in Simulated Gastrointestinal Conditions E. Vamanu and A. Vamanu University of Agronomic Sciences and Veterinary Medicine. Faculty of Biotechnology, Bd. Marasti No. 59, District 1, Bucharest, Romania Abstract: The aim of this study was to determine viability in the transit of the Lactobacillus rhamnosus ILI strain through the stomach and the small intestine. The preservation of the viability of the Lactobacillus rhamnosus 1 strain in gastrointestinal conditions is one of the main characteristics of the strain, in order to obtain probiotic products. The tests were performed with a cell suspension kept in NaCl 0.5%. The pepsin influence was determined al different. pH values, as well as the panercalin influence in the presence of bile salts. The influence of case in and mucin was also established. The results were read using the Colony Quant and they were registered in the log (CFU mL). Greater viability was preserved in case of mucin which was confirmed by the calculation of the mathematical parameters of viability and mortality, according to whether mucin is used or not. The main conclusion is that the tested strain maintains its viability, even at pII ranging between 1.8-2 and al even greater concentration of bile salts, of 2-3 mg ml. These results are confirmed by the cumulated effect of gastric and small intestinal juice, the Lactobacillus rhamnosus IL1 increasing its viability by an average of 20% in the presence of mucin. Key words: Mucin, pepsin, pancreatin, bile salts, colonyquant, CFU INTRODUCTION The development of probiotic use in the diet is a current theme on the functional products market. The viability and stability of probiotics in gastrointestinal conditions, during the transit, the resistance to antibiotics or the presence of other substances with antimicrobial effect have represented a significant challenge for all producers and researchers in the field. To be functional, probiotics must be viable and in sufficient number even after a longer period of time (De Vuyst, 2000). The synthesis of organic acids, antimicrobial peptides and polysaccharides, enhancing the installation of favorable microflora is added to all the above. The novelty in this field is the capacity of such strains to participate to the creation of biofilms, dependant upon the expolysaccharides synthesis in the intestine conditions. (Puangpronpitag et al., 2005; Philip et al., 2009). The production of probiotics as food supplements requires that the number of viable cells during the manufacture of the product and in the validity period should be of at least. 10°-10″ UFC g¯' (De Vuyst, 2000; Otles and Ozlem. 2003; Yateem et al., 2008). Before the probiotic strains could be used to obtain functional products, they must survive to the processing, to the gastrointestinal stress factors and they should be able to maintain their biological function in the host. All these. criteria must be taken into account when choosing the probiotic strain (Chichlowski et al., 2007). Lately, certain models to experiment in vitro the conditions in the human gastrointestinal tract were proposed. They have allowed the study of the lactic bacteria viability and of the influence of the products meant for the balancing of the disturbed intestinal microflora (Pacheco et al., 2010). These simulation systems range from simple ones where the lactic bacterial is treated in solutions of acid medium and solutions of hepatic bile (Favaro-Trindade and Grosso, 2002; Huang and Adams, 2001; Pacheco et al., 2010), to more complex systems that simulate the human gastrointestinal tract. To study the probiotic lactic bacteria interactions within the intestinal microbial environment or determine the effect of probiotic lactic bacteria and symbiotic products in the human intestinal microbiota (De Boever et al., 2000; Mainville et al., 2005; Pacheco et al., 2010). An intestinal human tract model that contained four chambers to simulate the stomach, duodenum, jejunum and ileum was proposed by Minekus et al. (1995) and Pacheco et al. (2010). Thus, this research will determine the viability in the Transit of the Lactobacillus rhamnosus IL1 strain through the stomach and the small intestine. The conditions at the Corresponding Author: Emanuel Vamami, University of Agronomic Sciences and Veterinary Medicine, Faculty of Biotechnology, Bd Marasti No. 59, District 1, Bucharest, Romania. Tel: +40742218240 Fax: +40215693492 732

Int. J. Pharmacol., 6 (5): 732-737, 2010 gastric level were simulated by using pepsin, at various pH values ranging between 1.5-3. The simulated pancreatic juice contained pancreatin and bile salts, in various concentrations, ranging between 1.5-5. Furthermore, there was tested the influence of casein and mucin on viability, as protectors of probiotic cells. Finally, there was determined the combined effect of the gastric juice and of the simulated small intestine action and there were calculated the mathematical parameters of cell viability and mortality. MATERIALS AND METHODS Biological material: The bacterial strain Lactobacillus rhamnosus 1 was maintained in glycerol 20% (Collection of Faculty of Biotechnology, Bucharest), at -82°C. The strain was revitalized by two successive cultures in MRS broth, at 37°C. The experiments were performed in the Industrial Biotechnology Laboratory of the Department of Biotechnology, in the first half of 2010. The gastric and small intestine juice were prepared according to the method described by Kos et al. (2000). In case of simulated gastric juice (pepsin 3 g 1.) There were used various pII values, of 1.5, 2, 2.5 and 3. The simulation of the small intestine juice (pancreatin 1 g L-) was made at various bile salts concentrations (1.5, 2, 3 and 5 mg mL). The mucin and casein influence on the strain viability was determined in the gastric and small intestine juice. A concentration of 1 g L 'in NaCl 0.5% was used and the determination was performed according to the method described by Kos et al. (2000). The cumulated effect of the simulated gastric and small intestine juice was determined at a pH of 2 and a bile salts quantity of 3 mg mL in the pancreatic juice. All tests were performed in Durham tubes, provided with silicone membrane meant for sampling (Kos et al, 2000; Sarahroodi et al., 2010; Puangpronpitag et al., 2009; Movsesyan et al., 2010). Furthermore, the effect of trypsin, chymotrypsin and pronase on viability was determined separately for each enzyme. Thus, in a Durham Tube, 1 mL of enzyme solution at a concentration of 1 mg ml., 0.3 mL NaC10.5% and cell suspension of 0.2 mL were added. In 2 h the viability was determined in the presence of mucin and casein (Kos et al., 2000; Sarahroodi et al., 2010; Philip et al.. 2009). The viability and the mortality were determined at various pH values according to the method described by Kos et al. (2000), in the presence of pepsin and respectively of pancreatin, together with various concentrations of bile salts. The same mathematical indices were calculated as well in the presence of mucin and casein according to the protection offered to the cell viability. The critical points were represented by the Crossing between the viability and mortality curves (Kos et al., 2000; Sarahroodi et al., 2010; Yateem et al., 2008). The viability was determined by insemination in double layer, in MRS broth, hourly. The plates were incubated for 48 h at 37°C and the results were read using the Colony Quant and they were registered in the log (CFU mL) (Kos et al., 2000, Sarahroodi et al., 2010; Otles and Ozlem, 2003). RESULTS In order to be used as probiotic, the tested strain must have good viability in the conditions of the gastric and intestinal transit. The effect of the gastrointestinal transit starting at the level of the stomach is caused by the pepsin, at a pH ranging between 1.5-3. The stationary time at this level doesn't exceed 2 h. Thus, Fig. I provides the viability of the II, 1 strain al gastric level. It is noted that the strain viability is directly influenced by the pH. At a pH of 1.5 it represents approximately 97% of the viability obtained for the other pH values, al. Oh of exposure. II. lowers to 17.5% within two hours of exposure at a pII value of 1.5. At a pH higher than 2, the strain maintains constant viability after one hour of exposure to the simulated gastric juice. In 2 h, as the pII increases from 1.5 to 2, the viability increases as well and continues to be constant at a pH of 2.5, being of 65.5% as to the initial one. According to the provided data, it results that the strani is resistant to low pH, which is extremely rare in the lactic probiotic bacteria strains. Mucin is a better protector than casein with respect to the viability of the Lactobacillus rhamnosus II strain exposed to the action of the simulated gastric juice. The viability depends upon the pH, but it is higher than when this substance is missing (Fig. 2). The viability values are Log (cfu mL) 400 8olh 7- ■2 h 6- 1.5 2.0 2.5 3.0 PH Fig. 1: Viability of the Lactobacillus rhamnosus ILI strain al simulated gastric juice exposure 733

Log (cuf mL) Log (caf mL) Int. J. Pharmacol., 6 (5): 732-737, 2010 --0b 1h -2b 1.5 2.0 2.5 3.0 pH Fig. 2: Casein effect on the viability of the Lactobacillus rhumnosus 1 strain in case of exposure lo simulated gastric juice Log (cfu mL) og (efu ml¯¯ 1.5 mg ml. bile salts 3 mg mL bile salts 2 mg mL bile salts 5 mg mL bile salts 1 2 3 4 Time (h) Fig. 1: Viability of the Lactobacillus rhamnosus ILI strain in case of exposure to the simulated small intestine juice 0 1 2 3 Time (h) 1.5 mg ml bile salts -2 mg mL bile salts 3 mg mL *5mg ml bile salts bile salts 0b 1b --2h 0- 1.5 2.0 2.5 3.0 pH Fig. 3: Mucin effect on the viability of the Lactobacillus rhamnosus IL strain in case of exposure to simulated gastric juice higher by 25 up to 50%, at a pII 1.5, both for casein and for mucin (Fig. 3). However, al pH 2, the viability value in the presence of mucin is by 12% higher than in the presence of casein. At a value of 2.5 or 3 of pH, the viability is relatively constant, notwithstanding the presence of casein or mucin. The differences in favor of the presence of mucin, at values of 2.5 and 3 of pII, are of approximately 5%, for an exposure of one or 2 h. Before testing the viability, in case of exposure to small intestine juice, the influence of other enzymes on the Lactobacillus rhamnosus strain was determined. Thus, a preservation of viability under the action of trypsin, pronase and chymotrypsin resulted, with an average of 6.58 log (CFU mL) as to the viability of the strain without enzymes. The value lowers in two hours, due to the action of the three above mentioned enzymes, below 10%. Fig. 5: Casein effect on the viability of the Lactobacillus rhamnosus 1 strain in case of exposure lo simulated small intestine juice In case of direct exposure to the simulated small intestine juice, the presence of bile salts causes the lowering of the viability, mainly due to the increase in the concentration thereof (Fig. 1). An increase of the bile salts of 3 or 5 mg mL determines in two hours a significant decrease of the viability, of 25% for 3 mg mL ´` of bile salts and 10% for the increase of the bile salts concentration to 5 mg ml. It must be noticed that for 2 mg mL of bile salls, the viability decreases below 10 CFU mL. ' merely after an exposure of 3 h. According to this figure, it is obvious that, with the increase of the stationary time in the presence of bile salts, the viability is directly influenced in a negative manner. By doubling the concentration of bile salts, the viability decreases by 40% after an exposure of 1 h In case of small intestine juice, the influence of cascin and mucin was determined. The two substances, but mainly mucin (Fig. 6), have a protective effect on the viability of the probiotic strain, as opposed with the pancreatin and bile salts effect. Although, the difference is small, the presence of casein (Fig. 5) determines a higher viability decrease. The decrease is directly 734

Log (cfu mL) 0Int. J. Pharmacol., 6 (5): 732-737, 2010 1.5 mg mL bile salts Specific cell viability 2 mg mL bile salts 3mg mL bile salts 5 mg mL bile salts 2 3 4 Time (h) Specific cell viability (%) Specific cell mortality Specifie cell viability in the presence of muein Specific cell mortality in the presence of mucin 80- 70- 60- 50- 40 -70 60 -50 40 30 -20 - 10 -0 1.5 2.0 2.5 Bile salts (mg mL) 3.0 Specific cell mortality (*) Fig. 6: Mucin effect on the viability of the Lactobacillus rhamnosus L1 strain in case of exposure to simulated small intestine juice Specific cell viability (*) 90 70 60 40 Specific cell viability Specific cell mortality Specific cell viability in the presence of mucin Specific cell mortality in the presence of murin 90 60 10- 10 0 0 1.5 2.0 2.5 3,0 pH Specific cell mortality (%) Fig. 7 Specific cell mortality and viability of the Lactobacillus rhamnosus 1 strain in case of exposure to simulated gastric juice correlated to the increase in the concentration of bile salts and in the stationary time. Within two hours from exposure, notwithstanding the concentration of bile salts. the viability decreases by an average of 35%. In two more hours, the viability decreases by an average of 6%. In The presence of protective agents, the viability doesn't. decrease below 10 CFU mL, notwithstanding the concentration of bile salts or the stationary time. The mathematical parameters of the viability and mortality were determined at various pll values in the presence of different bile salts concentrations. According to the data provided before, it results that mucin is a better protector than cascin. It must be noted that the mortality and viability lines don't cross in the presence of mucin, resulting appropriate protection at pH values lower than 2. According to the mathematical calculations, the viability, at pII 2, increases in its presence by 23.1% (Fig. 7). From the same figure, it results that the Lactobacillus rhamnosus IL1 strain has appropriate Fig. 8: Specific cell mortality and viability of the Lactobacillus rhamnosus 1 strain in case of exposure to simulated small intestine juice viability at pH of 1.8, according to the literature data, of at least 10 CFU mL for probiotics (Kos et al., 2000; De Vuyst, 2000). The same trend is noticeable in case of simulated small intestine juice (Fig. 8). In this situation the presence of mucin protects very well the cell viability, which is supported by the non-crossing of the viability and mortality curves. In the absence of mucin, the strain is strongly inhibited by the increase beyond 2 mg mL of bile salts concentration. Thus, at a bile salts concentration of 3 and 5 mg ml. 1, the presence of mucin determines an average viability increase of 10%. DISCUSSION The protector effect of mucin is noticeable in case of the cumulated action of gastric and small intestine juice on the viability of the IL1 strain. The viability is directly influenced by mucin, although in case of gastric juice action, it is high, of more than 50%, at pH 2. In this situation, the presence of mucin increases the viability value by more than 10%. If the simulated small intestine Juice acts on them as well, at a concentration of 2-3 mg ml. bile salts, the viability is kept at a percentage of 10%, when mucin is present. These data are supported by the previous researches of Kos et al. (2000), Patel et al. (2008) and Matijasic and Rogelj (2000). The results also represent added data to the findings of Nasrollah (2009) Homayony et al. (2008) and Trachoo et al. (2008). Although, it is a regular presence at the level of the gastric mucosa, it provides good protection for the lactic. bacteria strains in case of direct administration. The effect. of the mixture of mucin with various lactic bacteria freeze-dried strains merely determines an increase in viability. al the passage through the human 735

Int. J. Pharmacol., 6 (5): 732-737, 2010 gastrointestinal tract. This increase of the cell number at the stress exercised by pH 2 and a concentration of 2-3 mg mL bile salts contributes to finding new strains of extremely resistant lactic bacteria. Although regularly a viability of approximately 20% is maintained, after such transit, finding strains and conditions able to double the viability is a significant aspect. The researches of Kos et al. (2000), Sumeri et al. (2010) and Movsesyan et al. (2010) are in support of this result, with no disagreement values. To conclude, it was demonstrated that the Lactobacillus rhamnosus IL1 strain is able to survive to the gastrointestinal transit. The presence of mucin as compared to casein determines a viability increase of approximately 20%. The conditions in which the strain has maximal sensitivity were determined, namely pII below 2 and a bile salts concentration higher than 3 mg mL. which is significant in order to be able to use the strain in clinical studies. Knowing the protector and the cumulated gastric and intestinal effect on strain viability renders il. more competitive when used to create new probiotic products. ACKNOWLEDGMENT This study was supported by CNCSIS –UEFISCSU. project mimber 1119 PMII-IDEL 39/2008 (http://proiectidei.emanuelvamanu.ro/). REFERENCES code Chichlowski, M., J. Croom, B.W. McBride. G.B. Haverstein and M.D. Koei, 2007. Metabolic and physiological impact of probiotics or direct-fedmicrobials on poultry: A brief review of current knowledge. Int. J. Poult. Sci., 6: 694-701. De Boever, P., B. Deplancke and W. Verstraete, 2000. Fermentation by gut microbiota cultured in a simulator of the human intestinal microbial ecosystem is improved by supplementing a soygerm powder. J. Nutr., 130: 2599-2606. De Vuyst, L., 2000. Technology aspects related to the application of functional starter cultures. Food Technol. Biotechnol., 38: 105-112. Favaro-Trindade, C.S. and C.R.E. Grosso, 2002. Microencapsulation of L. acidophilus (La-05) and B. lactis (Bb-12) and evaluation of their survival at the pII values of stomach and in bile. J. Microencpasulat., 19: 185-194. Homayony, A., A. Ehsani, M.R. Azizi, A. Razavi, S.H. and M.S. Yarmand, 2008. Growth and survival of some probiotic strains in simulated ice cream conditions. J. Applied Sci. 8: 379-382. Huang, Y. and M. Adams, 2001. In vitro assessment of the upper gastrointestinal tolerance of potential probiotic dairy propionibacteria. Inter. J. Food Microbiol., 91: 253-260. Kos, B., J. Suskovic, J. Gorela and S. Malosie, 2000. Effect. of protectors on the viability of Lactobacillus acidophilus M92 in simulated gastrointestinal conditions. Food Technol. Biotechnol., 38: 121-127. Mainville, I., Y. Arcand and E.R. Farnworth, 2005. A dynamic model that simulates the human upper gastrointestinal tract for the study of probiotics. Inter. J. Food Microbiol., 99: 287-296. Matijasic, B.B. and I. Rogelj, 2000. Lactobacillus K7: A new candidate for a probiotic strain. Food Technol. Biotechnol., 38: 113-119. Minekus, M., P. Marteau, R. Havennar and J. Huis in't Veld, 1995. A multicompartmental stynamic computercontrolled model simulating the stomach and the small intestine. Allemal. Lab. Anim.. 23: 197-209. Movsesyan, I., N. Ahabekyan, I. Bazukyan, R. Madoyan and M. Dalgalarrondo et al., 2010. Properties and survival under simulated gastrointestinal conditions of lactic acid bacteria isolated from armenian cheeses and malsums. Biotechnol. Biotechnol. Eq.. 24: 444-449. Nasrollah, V., 2005. Probiotic in quail nutrition: A review. Int. J. Poult. Sci., 8: 1218-1222. Otles, S. and C. Ozlem, 2003. Kefir: A probiotic dairycomposition, nutritional and therapeutic aspects. Pak. J. Nulr, 2: 54-59. Pacheco, K.C. G.V. del Toro, F.R. Martinez and E. DuranParamo, 2010. Viability of Lactobacillus delbrueckii under human gastrointestinal conditions simulated in vitro. Am. J. Agric. Biol. Sci., 5: 37-42. Patel, P., T. Parekh and R. Subhash, 2008. Development of probiotic and symbiotic chocolate mousse: A functional food. Biotechnology, 7: 769-774. Philip, K., W.Y. Teoh, S. Muniandy and II. Yaakob, 2009. Pathogenic bacteria predominate in the oral cavity of Malaysian subjects. J. Biol. Sci., 9: 438-444. Puangpronpitag, D., N. Niamsa and C. Sittiwet, 2009. Antimicrobial properties of clove (Eugenia caryophyllum bullock and harrison) aqucous extract against food-borne pathogen bacteria. Int. J. Pharmacol., 5: 281-284. 736

Sarahroodi, S., A. Arzi, Int. J. Pharmacol., 6 (5): 732-737, 2010 A.F. Sawalha and A. Ashtarinezhad. 2010. Antibiotics self-medication among southern iramian university students. Int. J. Pharmacol., 6: 48-52. Sumeri I, L. Arike, J. Stekoltsikova, R. Lusna. S. Adamberg, K. Adamberg and T. Paalme, 2010. Effect of stress pretreatment, on survival of probiotic bacteria in gastrointestinal tract simulator. Applied Microbiol. Biotechnol., 86: 1925-1931. Trachoo, N., P. Wechakama, A. Moongngarm and M. Sullaji 2008. Stability of freeze-dried Lactobacillus acidophilus in banana, soybean and pearl barley powders. J. Boil. Sci., 8: 119-124. Yaleem, A., M.T. Balba, T. Al-Surayai, B. Al-Mutairi and R. Al-Daher, 2008. Isolation of lactic acid bacteria with probiotic potential from camel milk. Tut. J. Dairy. Sci., 3: 194-199. 737

Other Health Sciences Concepts:

[back to top]

Discover the significance of concepts within the article: ‘Viability of the Lactobacillus rhamnosus IL1 Strain in Simulated...’. Further sources in the context of Health Sciences might help you critically compare this page with similair documents:

Stomach, Protector, Gastric juice, Biological function, Critical point, Clinical studies, In vitro, PH value, Pathogenic bacteria, Oral cavity, Anti-microbial properties, Antimicrobial effect, Glycerol, Cell viability, Protective effect, Lactic acid bacteria, Food supplement, Bacterial strain, Functional food, Biological material, Processing, Mucin, Camel milk, Intestinal microbiota, MRS broth, Intestinal microflora, In vitro assessment, Low pH, Probiotic strain, Microencapsulation, Viable cells, Pepsin, Acid medium, Protective agent, Human gastrointestinal tract, Functional starter cultures, Enzyme solution, Small intestine action, Current knowledge, Kefir, Simulated gastric juice, Cell suspension, Direct administration, Duodenum, Jejunum, Ileum, Mathematical calculation, Trypsin, Poultry, Intestinal effect, Malaysian subjects, Casein, Bile salt, Gastrointestinal condition, Probiotic product, Probiotic potential, Probiotic use, Gastric level, Probiotic cells, Double layer, Stationary time, Lactic bacteria strains, Competitive, Technology aspects, Dynamic model.