Antimicrobial effect of Lactobacillus casei strain Shirota co-cultivated with Escherichia coli UAM0403

Efecto antimicrobiano de Lactobacillus casei variedad Shirota co-cultivado con Escherichia coli UAM0403

I. Figueroa-González¹, H. Hernández-Sánchez², G. Rodríguez-Serrano¹, L. Gómez-Ruiz¹, M. García-Garibay¹ and A. Cruz-Guerrero¹'*

¹ Departamento de Biotecnología Universidad Autónoma Metropolitana-Iztapalapa., Av. San Rafael Atlixco No. 186 Col. Vicentina, México D.F. 09340, México. *Corresponding author. E-mail: aec@xanum.uam.mx Tel. (55) 58044720; Fax: (55) 58044712

Received 1 of October 2009;
Accepted 22 of December 2009

Abstract

In order to assess the antimicrobial effect of L. casei Shirota and prebiotic effect of a galactoside (Oligomate 55), co-cultures of this probiotic bacterium and Escherichia coli were made in an in vitro fermentation with temporary and physicochemical characteristics of key regions of large bowel. Simulations of ascending colon, transverse colon and descending colon were used. With Oligomate 55 an enhancement of up to three-fold of the growth of L. casei Shirota was observed compared to glucose; then, our results confirmed that Oligomate 55 had a prebiotic effect on L. casei Shirota, stimulating its growth. In all studied regions L. casei Shirota had a bacteriostatic effect on E. coli UAM0403. Our results showed the prebiotic effect of Oligomate 55 on L. casei Shirota. Despite Oligomate 55 significantly stimulated the growth of L. casei Shirota, E. coli UAM0403 was able to grow with Oligomate 55 in the same amount as with glucose. These kinds of studies are important to be considered for an adequate selection of prebiotics as a critical step during synbiotic development.

Keywords: galactooligosaccharides, prebiotic; probiotic.

Resumen

Palabras clave: galactooligosacáridos, prebióticos, probióticos.

1. Introduction

Probiotics, prebiotics and synbiotics are based on the same idea: to create foodstuffs which after ingestion multiply healthy bacteria in the intestine (de Vrese and Schrezenmeir, 2008). Probiotics are living organisms that, when ingested in certain amounts, are capable of maintaining the balance of the intestinal microbiota, while prebiotics are carbohydrates that enhance the development of probiotics (Senok et al., 2005).

The ability of lactic acid bacteria to inhibit the growth of pathogenic bacteria is well known. According to Hua et al. (2007) lactobacilli are able to compete with pathogenic bacteria when they were incubated together, but the degree of inhibition was bacterial strain depended. The inhibition produced by lactic acid bacteria may be due to the production of organic acids such as lactic, propionic and acetic (Naaber et al., 2004), hydrogen peroxide, bacteriocins, bacteriocins-like substances and possibly biosurfactants which are active against certain pathogens and may be produced by different species of Lactobacillus (Millete et al., 2006).

Lactobacillus casei strain Shirota, one of the most intensively studied probiotics, has been used in the production of fermented milk products for more than 70 years, and has been proven as an important probiotic with many benefits, such as the improvement of the balance of intestinal microbiota and volatile fatty acids, antitumor action, stimulation of the immune system, and antimicrobial activity (Fujimoto et al., 2008).

Prebiotics are food ingredients that stimulate selectively the growth and activity of bifidobacteria and lactobacilli in the gut and thereby to promote health. Prebiotics are non-digestible oligosaccharides which reach the human colon without being hydrolyzed or absorbed in the upper part of the gastrointestinal tract (Chockchaisawasdee et al., 2004). Therefore, the crucial property of prebiotics is their effect on the microbiota of large bowel (Cummings and Macfarlane, 2002). It has been postulated that prebiotics are metabolized only by beneficial microorganisms; then, they are able to alter the composition of the gut micro-biota (Macfarlane et al., 2006). Among the wide variety of compounds tested as prebiotics, galactooligosaccharides and fructooligosaccharides are the most studied (Crittenden and Playne, 1996; Voragen, 1998). Oligosaccharides such as Oligomate 55 are currently industrially produced to be incorporated into food products. Oligomate 55 is the trade mark of a sugar formed by the transgalactosilation of β-galactosidase on lactose, and consists mainly of galactooligosaccharides which main component is 4'-galactosil-lactose and other components such as lactose and monosaccharides (Sar et al., 2004).

The aim of this study was to evaluate the inhibitory effect of Lactobacillus casei Shirota in co-culture with E. coli UAM0403 during fermentation in an in vitro simulation of large bowel, using the commercial prebiotic Oligomate 55 as selective agent to improve growth of probiotic bacteria.

2.1. Bacterial strains

Lactobacillus casei strain Shirota, isolated from Yakult (Yakult, México) and Escherichia coli UAM 0403 was obtained from the culture collection of the Universidad Autónoma Metropolitana. L. casei Shirota was maintained in Skim Milk medium (Difco, Detroit, USA) and E. coli UAM0403 in Nutritive Agar (B. D. Bioxon, México). Cultures were stored at 4°C in their respective media.

Lactobacillus casei Shirota isolation was performed as previously described (Tharmaraj and Shah, 2003) with minor modifications. Briefly, 1 ml of sample (Yakult) was 10-fold serially diluted (10³ to 10⁷) in sterile peptone water (casein peptone al 0.1%, pH 7.2) and stirred thoroughly. After, 100 μl of each dilution was spread on de Man Rogosa Sharpe (MRS) agar for lactic acid bacteria (Difco, Detroit, USA), MRS-NaCl (40 g/l NaCl) agar for Lactobacillus casei isolation. The agar plates were incubated at 37^° C for 72 h. Isolated bacteria were examined microscopically for cellular morphology and Gram stain phenotype.

2.2. Culture medium

Culture medium was prepared according to Mac-farlane et al. (1998). The medium contained (g/l): either glucose (J. T. Baker, Phillipsburg, NJ, USA) used as control, or Oligomate 55 (Yakult Pharmaceutical, Japan), 0.4; Yeast Extract (B. D. Bioxon, México), 3; Proteose-Peptone (B. D. Bioxon, México), 1; NaHCO₃, 0.4; NaCl , 0.08; K₂HPO₄, 0.04; KH₂PO₄, 0.04; CaCl₂, 0.008; MgSO₄-7H₂O, 0.008 (all salts were purchased by J. T. Baker, Phillipsburg, NJ, USA); and Tween-80, 1 ml/l (Sigma,St. Louis, MO, USA). Initial pH was adjusted according to every step of the fermentation with HCl 0.1 M.

2.3. Co-culture conditions

The co-culture growth of E. coli UAM0403 and L.casei Shirota was performed in 50 ml vessels containing 30 ml culture medium, reproducing significant physicochemical characteristics in large bowel (ascending colon, transverse colon and descending colon). Conditions were achieved by maintaining physiological temperature (37 °C), stirring (150 rpm) and residence time as occurring in humans (Macfarlane et al., 1998). Vessels were incubated in an orbital shaker with temperature control (Environ Shaker, New Brunswick, New Jersey, USA) under the following conditions of initial pH and residence time: ascending colon, 5.5 and 8 h; transverse colon, 6.2 and 12 h; descending colon, 6.8 and 10 h. Each region was simulated separately and continuity was kept by transferring 1 ml inoculum from the first vessel (region) to the next in the order presented above. At the end of the cultures the pH values of each region were measured.

2.4. Microbial growth quantification

The co-cultures were carried out in 50 ml vessels containing 30 ml culture medium starting with 10⁶ colony-forming units (CFU) of L. casei Shirota and E. coli UAM0403 (inoculated with 1 % v/v of an overnight culture obtained from a single colony). The control medium contained glucose and the test medium Oligomate 55 as carbon source. At the same time, control experiments for growth testing of both microorganisms in single cultures were made at same conditions. Samples were taken periodically in order to evaluate the concentration of each species during fermentation. Bacterial growth was followed by plate culture method on selective media. For E. coli UAM 0403 EMB Agar (Eosin Methylene Blue Agar, B. D. Bioxon, México) was used, while L. casei Shirota was pour-plated on Lactobacilli MRS Agar (Difco, Detroit, USA). All the dishes were incubated at 37^°C from 24 to 48 hours, and UFC/mL were counted.

The fermentation experiments were carried out in duplicate. For each replication, three samples were analyzed. Student-t test (p<0.05) was performed using the statistics software NCSS 2000 (NCSS, LLC. Utah, USA) in order to determine if there is a significant difference between the generation time of the E. coli UAM0403 in presence or absence of probiotic bacterium.

3. Results and discussion

Fig. 1 shows the growth of L. casei Shirota and E. coli UAM0403 in each studied region of large bowel. L. casei Shirota using both glucose and Oligomate 55 as carbon sources showed a significantly higher growth compared to E. coli UAM0403. The growth of L. casei Shirota also presented a significant increase with Oligomate 55 as carbon source compared to glucose. A maximum enhancement up to 3-fold (ascending colon, 4 h) was found.

Fig. 2 summarizes growth of both strains at the end of fermentation, growth of L. casei Shirota was similar in both co-culture with E. coli UAM0403 and single culture (control). On the other hand, the growth of E. coli decreased in presence of L. casei Shirota, therefore, the ability of L. casei Shirota to inhibit the growth of E. coli UAM0403 was established in these co-culture experiments. In presence of L. casei Shirota, growth of harmful microorganism decreased by a 4 log (descending colon, pH 6.8) compared with control. A small reduction of 2 log at transverse colon (pH 6.2) and 1 log in ascending colon (pH 5.5) in E. coli UAM0403 counts was also observed.

A bacteriostatic effect of L. casei Shirota was observed in all regions. The growth of E. coli was significantly lower compared to L. casei Shirota; however, E. coli UAM0403 was able to grow in both carbon sources without significant differences in practically all regions. This can be explained by the fact that Oligomate 55 contains free monosaccharides such as glucose and galactose in more than 18 % w/w (Sar et al., 2004). These results indicate that the inhibition of E. coli was due to the presence and metabolic activity of L. casei Shirota instead an effect of Oligomate 55.

In this study, was observed that the lowest pH value was reached in the ascending colon with Oligomate 55; cultures under these conditions showed the maximum pH reduction (from 5.5 to 4.1) as well as the maximum enhancement of L. casei Shirota growth. The minimum reduction of pH value (from 6.2 to 6.1) was found in the transverse colon with Oligomate 55, only under these conditions E. coli UAM0403 showed a significant growth compared to glucose. In all regions a slightly decrease in the pH value was observed, with a maximal decrease of 1.4 pH units (ascending colon with Oligomate 55). This result agrees with Fooks and Gibson (2002); who found that in 24-h cultures of L. plantarum 0407 and E. coli with fruc-tooligosaccharid¹e⁵s as carbon source, the pH value diminished only 1.15 units; however, inhibition of E. coli did occur. This suggests that besides organic acids production, other inhibitory metabolites could be produced during this kind of cultures. Brink et al. (2006) reported that lactic acid bacteria, such as L. casei LHS, produces a high level of antimicrobial activity when growing in MRS broth supplemented with glucose; they found that antimicrobial compounds with bacteriostatic effect were produced during cultures. Moreover, Millette et al. (2006) observed that a culture of L. acidophilus and L. casei inhibited or delayed the growth of pathogens such as E. coli; these authors suggested that antimicrobial activity could be due to production of organic acids and bacteriocins. Lactobacilli may exert their antibacterial activity through production of lactic acid and others metabolites such as hydrogen peroxide and short chain fatty acids. Also specific antibacterial compounds such as antibiotics or bacteriocins have been identified in the culture medium of several lactic acid bacteria. The mechanisms by which L. casei Shirota inhibited E. coli UAM0403 growth remain understood at present.

Generation times of E. coli UAM0403 are presented in Table 1. Data shown a significantly increase of bacteria generation time when grow in the presence of L. casei Shirota. E. coli UAM0403 had a generation time of 28.2 min when cultured alone in glucose media whereas the presence of the lactobacilli increased the generation time to 55.4 min. Also was observed decrease in generation time of E. coli UAM0403 in the simulation of the colon when it is in monoculture, indicating an adaptation of the microorganism to means. Whereas in co-culture, generation time is similar in the different regions from colon, this, suggests an effect of lactobacilli in growth of E. coli UAM0403. Neither, a prebiotic effect was observed in generation time of L. casei Shirota.

Conclusions

The results obtained indicate that the probiotic culture was able to inhibit the growth of a harmful bacterium. Moreover, results obtained in this work confirmed the effect prebiotic of Oligomate 55 during simulations of key regions of large bowel. Even though in all regions a bacteriostatic effect on E. coli UAM0403 was obtained, it was able to grow in both carbon sources, implying that this microorganism was capable to metabolize Oligomate 55. Further experiments must be done in order to determine which components of Oligomate 55 could be potentially consumed by other pathogens; this information may be useful in the design and optimization of synbiotics. Additional studies on antimicrobial compounds production must be done to a better understanding of the bacteriostatic effect of L. casei Shirota on E. coli UAM0403 and other microorganisms.

The authors acknowledge the financial support received from the National Council for Science and Technology (CONACyT), No. 0471-O.

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