Acid , Bile and Aggregation Abilities of Lactobacillus plantarum Strains Isolated from Akamu a Nigerian Fermented Maize Food

This study investigated the ability of two strains of Lactobacillus plantarum isolated from akamu a Nigerian fermented maize food to tolerate acid and bile condition. Auto-aggregation and co-aggregation with pathogens: Escherichia coli NCTC 11560 and Salmonella Enteritidis NCTC 5188 were also investigated. This was aimed at establishing preliminary probiotic potentials of these none intestinal L. plantarum isolates. Viability at pH 2 was significantly (p≤0.05) reduced from ≥8.26±0.05 to ≤4.94±0.49 Log10 CFU/mL after 3 h. Subsequent incubation in 0.3% ox gall bile media after 6 h enhanced growth to 5.73±0.13 and 7.93±0.12 Log10 CFU/mL for NGL5 and NGL7. The L. plantarum strains auto-aggregated but had no co-aggregation with the pathogens. After 5 h auto-aggregation at 37°C (>25%) was significantly (p≤0.05) greater than auto-aggregation at 22 24°C (<14%). The L. plantarum strains possessed abilities to survive passage through the GIT and auto-aggregated significantly at body temperature. This serves as a baseline data for further studies especially isolates that are not of intestinal origin.


Introduction
Akamu is a lactic acid fermented cereal-based food that constitutes a major infant complementary food and also serves as a component of adult main meals in most African countries.To be able to develop this product for more than its nutritional value to that of health enhancing benefits would require the characterization of the fermenting microorganism for probiotic potentials.Lactobacillus plantarum is one of the main Lactic acid bacteria associated with spontaneous fermentation of akamu [1].It is a versatile lactic acid bacterium, that is found in various indigenous fermented foods and is commonly associated with the human gastrointestinal tract (GIT) [2,3] and among the key species of the genera Lactobacillus that is being studied for their ability to confer health benefits.
In order to exert health-enhancing benefits, probiotics must possess certain potentials such as the ability to withstand passage through the GIT, adherence to mucus and/or human epithelial cells and cell lines, reduction of pathogen adhesion to surfaces through aggregation and inhibition of enteric pathogens [4,5].The acidity of the human GIT depending on the food substrate and the intervals of feeding vary from pH 1.5 to 4.5 [6].Bile in the human GIT is an important factor that affects bacteria cell viability and interaction with the environment as it is known to destroy the lipids and fatty acid components of bacteria cell membrane [7].Bile concentration is assumed to be higher in the upper part of the small intestine (jejunum).All bile depending on concentration are said to be inhibitory to most Gram-positive bacteria including the genera Lactobacillus.Viability at the site of action is presumed to be important although some effects may be mediated by cell components.Since the viability and activity of probiotics are needed at the lower digestive tract, it is expected that they should be resistant to acid and bile environment of the upper digestive tract [8].
Bacteria aggregation has been proposed to have both food preservation and therapeutic impact on intestinal microbiota [9].Auto-aggregation has been suggested to be necessary for adhesion to intestinal epithelial cells [10], [11], while co-aggregation form barrier that prevent pathogenic colonization of the GIT through the creation of different microenvironment around the pathogens and enhanced antimicrobial activities at close proximity [12,13].Aggregation phenomenon in many Lactobacillus strains are been investigated among others by spectrophotometric assays, in which bacteria aggregation are evaluated by the reduction of light absorbance in the bacterial suspended diluent using a spectrophotometer.Commonly used diluent include phosphate buffered saline (PBS) at different pH (pH 2-8) simulating both gastric and intestinal juice [11,14,15,16,17].
Although most studies had been with intestinal and dairy isolates [6,8,18], there is increasing interest not just in the development of food products containing beneficial American Journal of Food Science and Technology Lactobacillus strains but the characterisation of non-intestinal isolates for possible health-enhancing effects.This study, therefore, was aimed at investigating the acid and bile tolerance and the auto-and co-aggregation ability of L. plantatum strains: NGL5 and NGL7 isolated from akamua Nigerian fermented maize food as one of the important criteria for survival through the gastrointestinal track.

Microorganism and Inoculum Preparation
The two L. plantarum strains: NGL5 and NGL7 characterised in this study were previously isolated from akamu a Nigerian traditionally fermented maize food and identified using both conventional and molecular methods by [1].The commercial probiotic strain (LpTx) was isolated from a probiotic food supplement obtained from Health Food Shop, Rickard Lanes', Plymouth City Centre, UK, using the same method described by Obinna-Echem et al., [1].The pathogens: Escherichia coli NCTC 11560 and Salmonella enterica serovar Enteritidis NCTC 5188 obtained from stock cultures in the microbiological laboratory of Plymouth University, UK were the pathogens used in the aggregation assay.
The lactic acid bacteria (LAB) were cultivated on de Man, Rogosa and Sharpe (MRS) agar and the pathogens on Nutrient agar incubated at 37°C for 24 h.MRS and Nutrient broths were used for the LAB and pathogens broth cultures respectively.A distinct colony from the agar plate culture was inoculated into 10 mL of broth and incubated at 37°C without agitation for 18 -20 h.The cultures were harvested by centrifugation (Hettich Zentrifugen Rotina 46 S, Tuttlingen, Germany) at 4000 x g for 10 min and washed twice in phosphate buffered saline (PBS) (pH 7.3±0.2) and re-suspended in PBS such that 1 mL of inoculum produced 9 and 8 Log 10 CFU/mL for the Lab and the pathogens respectively.The media and the diluent used were obtained from Oxoid Limited (Basingstoke, Hampshire, UK).

Acid Tolerance
The ability of the L. plantarum strains to withstand acid condition was investigated using the methods described by Lin et al. [5] and Ding & Shah [8].One millilitre of washed L. plantarum cell suspension (c. 9 Log 10 CFU/mL CFU) was inoculated into 9 mL of modified MRS broth (pH 2.0 with 5 M HCl).pH 2 has been established as the appropriate pH for screening acid tolerance of probiotic LAB [5,6,19].The samples were incubated at 37°C for 3 h under anaerobic condition using anaerobic gas jackets (2.5 L AnaeroGen AN0025A, Oxoid, Basingstoke, England).Survival was measured by plating out serial dilutions on MRS agar plates at the beginning of the incubation time and every 1 h for 3 h.

Bile Tolerance
According to the method described Lin et al. [6] washed acid stressed cells were re-suspended in 10 mL MRS broth with or without 0.3% ox gall bile (Oxoid Ltd, Basingstoke, England) and incubated anaerobically at 37°C for 24 h.The viable microbial counts were determined on MRS agar after 0, 3, 6 and 24 h of exposure.The effect of the bile salt on the growth of the L. plantarum strains was then determined by comparing the viability in the MRS broth with those from the bile condition.

Bacterial Auto-aggregation
Aggregation abilities of the L. plantarum strains and the pathogens: E. coli NCTC 11560 and S. Enteritidis NCTC 5188, were evaluated spectrophotometrically using the method described by Del Re et al. [11].Briefly, 4 mL of the bacterial cell suspension (A 600nm of 0.5) were centrifuged (HARRIER 18/80 Refrigerated, MSE, Lower Sydenham, London, UK) at 6000 x g at 4°C for 10 min and re-suspended in same volume of their culture supernatant diluents and incubated at Room (22 -24°C) and Body (37°C) temperatures for 2, 5 and 24 h.Absorbance (A 600 ) of 1 mL of upper suspension was measured against same volume of PBS as blank.Auto-aggregation percentage was expressed as: (1 -A t /A i ) X 100, where A t and A i is the absorbance at the experimental time and before incubation respectively.

Bacterial Co-aggregation
Bacterial suspensions were prepared as in auto-aggregation.Equal volumes (2 mL) of the L. plantarum and the pathogen strains (1:1 v/v) were mixed and incubated at Room (22 -24°C) and Body (37°C) temperatures without agitation.Absorbance after mixing and after 2, 5 and 24 h of incubation was taken.Percentage Co-aggregation was expressed using the equation by Kos et al., [10]  represent the absorbance of the pathogen, L. plantarum and mixture of pathogen and L. plantarum respectively.

Statistical Analysis
Data obtained were statistically analysed using Minitab (Release 16.0) Statistical Software English (Minitab Ltd.Coventry, UK).Statistical differences and relationship among variables were evaluated by analysis of variance (ANOVA) under general linear model and Tukey pairwise comparisons at 95% confidence level.

Acid and Bile Tolerance Ability of Nigerian Fermented maize Food -L. plantarum strains
In Figure 1, viable counts of both strains of L. plantarum at pH 2 after 3 h were reduced from ≥8.26±0.05 to ≤4.94±0.49Log 10 CFU/mL.Subsequent incubation of the acid stressed cells in 0.3% ox gall bile media for 6 h resulted in growth enhancement to 5.73±0.13and 7.93±0.12Log 10 CFU/mL for NGL5 and NGL7, respectively.The viability of the acid stressed cells in MRS and MRS with 0.3% ox gall bile for NGL5 and NGL7 are presented in Figure 2 and Figure 3, respectively.There was significant (p<0.05)increase in viability with increase in time.The L. plantarum strains: NGL5 and NGL7 from Nigerian fermented maize study were able to withstand the acid and bile environment.Survival of approximately 5 Log 10 CFU/mL at the end of the 3 h of exposure to pH 2 was one log cycle less than 6 Log 10 CFU/mL reported by Ouwehand & Salminen [20] as the requirement for conferment of health benefit in the host.This however, could be dependent on the initial microbial inocula and the buffering effect of the substrates in the media.The food substance in which the LAB is relayed into the GIT may influence the intensity of the adverse effect that the acid and gastric juice may have on the LAB and thereby increase the survival and viability of the organism [21].The increase in the levels of the acid stressed L. plantarum cells in MRS was not significantly (p>0.05)different from that of the 0.3% ox gall bile condition.This was an indication that the L. plantarum strains tested in this study showed significant tolerance to 0.3% ox gall bile even after exposure to acid (pH 2.0).The ability of LAB to tolerate bile salt in-vitro could imply their capability to survive in the human small intestine.Although, the result indicated that some cells of the NGL5 were not able to recover within the first 3 h of exposure to bile.Similar viable cell counts for L. plantarum strains isolated from ikii were reported by Kalui et al. [19].Mathara et al. [22] reported high tolerance of L. plantarum strains to 0.5% ox gall after acid stress.The resistance to bile has been related to the activity of bile salt hydrolase which can hydrolyse combined bile salt and hence, reduce toxicity of the bile salt [15].

Aggregation Potential of Nigerian Fermented Maize Food Lactobacillus Plantarum Strains (NGL5 and NGL7)
The aggregation ability of the L. plantarum strains are shown in Table 1.They were able to auto-aggregate at both temperatures, although aggregation was significantly (p≤0.05)greater at 37°C than at room temperature.Auto-aggregation increased significantly (p≤0.05) with time.The auto-aggregation values obtained in this study (8 -17% and 11 -30% at room and body temperature respectively) were higher than the values reported for some strains of L. fermentum [15] but lower than that of Bifidobacterum longum [11].The auto-aggregation ability of the L. plantarum strains would be an advantage in achieving greater mass that is necessary for enhancing tolerance to the GIT system, exerting of certain health benefits and may aid the prevention of pathogen colonization [14,23].
The L. plantarum strains were unable to co-aggregate with the pathogens (<6%) at both temperatures as shown in Table 2. Co-aggregation of Lactobacillus spp. with pathogens enhances the prevention of pathogen colonization [15], however, the L. plantarum strains were unable to co-aggregate with the pathogens irrespective of the experimental conditions.This was comparable with the literature report that co-aggregation of enteropathogenic E. coli 3014 and S. Typhimurium with L. plantarum L4 was less than 5% [10].Although of the L. plantarum strains did not co-aggregated with the pathogens: E. coli NCTC 11560 and S. Enteritidis NCTC 5188, acid production by the L. plantarum strains during fermentation had proven to be capable of significantly inhibiting the pathogen in the food sample [24] which is of relevance in the consumption of safe product.Values with same superscript in the same column do not differ significantly (p≤0.05).N=3±SD NGL5 and NGL7 -L.plantarum strains isolated from akamu-a Nigerian fermented maize food LPTX -Commercial probiotic strain isolated from a probiotic food supplement.

Conclusion
The study revealed that the L. plantarum strains were able to tolerate acid and bile condition and exhibited good auto-aggregation potentials at 37°C This suggested that the organisms would be able to form adequate mass and survive passage through the GIT.

Figure 1 .
Figure 1.Effect of exposure to pH 2.0 on the viable count of two Lactobacillus plantarum strains (NGL5 and NGL7) isolated from Nigerian fermented maize food-akamu.

plantarum strains isolated from akamu-a Nigerian fermented maize food
LPTX -Commercial probiotic strain isolated from a probiotic food supplement.