Probiotic properties of Lactobacillus strains isolated from gizzard of local poultry.

BACKGROUND AND
OBJECTIVE: The aim of our study was to characterize gizzard Lactobacillus fermentum-group strains on the basis of their phenotypic profiles regarding characteristics of lactobacilli. In addition, their in vitro potential probiotic properties were evaluated.
MATERIALS AND METHODS: The lactic acid bacteria were isolated and identified from gizzard contents of Algerian local poultry using criteria of Bergey's Manual of Determinative Bacteriology and using methods and criteria of Sharpe. The strains were further characterized by tolerance to low pH and bile, coaggregation potential and adhesion to intestinal mucous. The antagonistic activity against some Enterobacteriaceae strains from poultry origin was also evaluated. RESULTS AND
CONCLUSION: Among the strains identified, both physiological and biochemical characteristics differed noticeably. The strains coded LP3 and LP10 survived simulated gastrointestinal conditions and were considered to be acid and bile tolerant. The majority of the strains exhibited antagonistic activity towards Escherichia coli, Klebsiella spp., Enterobacter spp., Shigella spp., Salmonella spp and Citrobacter spp. The best co-aggregation properties were obtained with two isolates. Lb. fermentum LP3 alone showed adherence specificity to the chicken intestinal epithelium.

INTRODUCTION

Due to concerns about residues in animal products and the development of bacterial resistance to antibiotics, the potential exists for the implementation of a complete ban on the use of antibiotics in animal feed. As a consequence, the development of alternatives to antibiotics is receiving considerable attention (1). The contemporary definition of a probiotic is “a microorganism which, when administered in adequate amounts, confers a health benefit on the host” (2). As living microorganisms, they produce no drug resistance or drug residues (3). The most common microorganisms found in the probiotic products currently available are lactic acid bacteria, especially Lactobacillus and Bifidobacterium species, which are resident microflora in the gastrointestinal tract of most animals (4).

To exert a beneficial effect, it is generally considered essential that probiotic cells remain viable during transit of the gastrointestinal tract (GIT) in sufficiently high numbers to either establish residence (i.e. to colonise) or to benefit the host (5). Required these characteristics include resistance to gastric acid and physiological concentrations of bile and adherence to intestinal epithelial cells (6).

In order to meet market and international health organization demands, the poultry industry is studying alternatives to antibiotics that could be both economically feasible, and maintain performance levels. Probiotics can be listed among these products. Isolation and study of new strains from the gizzard flora may lead to the development of novel probiotic products. The objective of the current study was to characterize and identify a number of lactobacilli isolated from gizzard of Algerian local poultry. The strains were further characterized by tolerance to low pH and bile, coaggregation potential and adhesion to intestinal mucous. The antagonistic activity towards some Enterobacteriaceae strains from poultry origin was also evaluated.

MATERIALS AND METHODS

Isolation and identification of lactobacilli

The lactobacilli strains were isolated from gizzard contents of Algerian local poultry. Decimal dilution of these samples were mixed with MRS medium and incubated at 37°C for 48 h under anaerobiosis (7). Selected colonies were picked from the higher dilutions and sub-cultured in MRS broth. The identification of the isolates was performed according to the criteria of Bergey’s Manual of Determinative Bacteriology and using the methods and criteria of Sharpe (8). The isolates were initially subjected to Gram staining and catalase test (3% H2O2). Only the Gram positive, catalase negative isolates were further identified. Growth at different temperatures was determined in MRS broths (10°C, 15°C, 40°C and 45°C). Hydrolysis of arginine was also recorded. The fermentative type was determined on agar.

The ability of the isolated strains to produce acid from different carbohydrates was determined by API 50 CHL test kits (BioMerieux, S.A., France). The results were loaded onto the API system software, which used the phenotypic data to predict a species identity (%) for each isolate.

Acid tolerance

Cells of overnight cultures (v/9v) were collected and inoculated by crop fluid content (v/4v). The cultures were incubated at 37°C for 3 h. Culture turbidity was measured at 620 nm and cfu number was determined at the end of incubation (9).

Bile salt tolerance

Overnight cultures (v/9v) were inoculated in MRS broth (control cultures) and MRS broth containing 0.3 (w/v) oxgall and incubated at 37°C for 4 h. Cultures turbidity was hourly monitored at OD650 nm, with determination of CFU number. The control comprised MRS broth without bile (9).

Inhibitory activity

The inhibitory activity was screened by the agar spot agar in MRS agar, under anaerobic conditions at 37°C (10). The indicator strains used were of poultry gizzard origin. The supernatant and neutral supernatant culture fluids were tested. The diameters of inhibition zones were measured after incubation overnight at 37°C.

Coaggregation assay

The bacterial cells were harvested by centrifugation at 5000 g for 15 min after incubation at 37°C for 18h, washed twice and resuspended in phosphate buffered saline (PBS) to give viable counts of approximately 108 CFU /ml. Equal volumes (2 ml) of each cell suspension were mixed together in pairs by vortexing. Control tubes were set up at the same time, containing 4 ml of each bacterial suspension on its own. The absorbances (A) at 600 nm of the suspensions were measured after mixing and after 5 h of incubation (6-9). The percentage of coaggregation was calculated using the equation

Where x and y represent each of the two strains in the control tubes, and (x + y) the mixture.

Adherence of LAB to epithelial cell of poultry

Segment of poultry ileum was washed with sterilized phosphate-buffer saline (PBS, pH 7.2). It was held at 4°C for 30 min and then washed three times with PBS. The epithelial cell concentration was adjusted to approximately 5×104 cells mL-1.

Briefly, cell pellet from overnight culture of LAB was resuspended to approximately 1×108 cells mL-1 in PBS (pH 7.2). One ml of such bacteria suspension was mixed with 1 mL of the cell suspension of epithelial cells. The mixture was incubated at 37°C for 30 min. The adhesion was observed using phase contrast microscopy (magnification fold, 200) after stained with 0.5% crystal violet for 5 min (9).

RESULTS

Isolation and identification of lactobacilli strains

Twenty (20) strains were isolated, purified and further identified from poultry gizzard. The isolates were found to be Gram-positive, catalase-negative rods, non motile, which phenotypically correspond to the genus Lactobacillus. According to the physiological and biochemical tests and to the carbohydrate fermentation pattern analyzed by API strip system software, 12 isolates strains were presumptively recognized as Lb. fermentum, 3 isolates as Lb.plantarum, 2 isolates as Lb.brevis and 3 isolates as Lb.casei ssp pseudoplantarum (Table 1). With respect to the sugar fermentation patterns, it was noted that all strains were able to ferment pentoses such as ribose. In contrast, some variations were also observed between the strains in particular to ferment raffinose and maltose.

Table 1

The identified isolates of lactobacilli.

Isolates	Identification using API strip system software	Identified as
LP1	98%
LP3	98%
LP6	98%
LP7	98%
LP8	97%
LP9	98%	Lb. fermentum
LP10	97%
LP11	98%
LP12	98%
LP20	93%
LP14	94%
LP17	94%
LP5	98%
LP16	97%	Lb. plantarum
LP2	97%
LP4	96%
LP13	95%	Lb. brevis
LP15	98%
LP18	94%	Lb. casei ssp.pseudoplantarum
LP19	95%

Sensitivity of isolates to several parameters

All isolates of Lb.fermentum were submitted to the study of their probiotic potential.

Tolerance to gastric juice

The degree of acid resistance exhibited by lactobacilli strains was determined and results (Table 2) showed that Lb.fermentum LP3 and LP10 seem to have better acid tolerance (100%) and the lowest acid tolerance was observed for Lb. fermentum LP8 (22.34%) after 3h of incubation. From the same table, it’s appearing that viable LAB counts decreased for a few strains. Results of this study were in accordance with those found by some authors.

Table 2

Effects of gastric juice on the growth of Lb. fermentum strains.

Lb. fermentum	Viable count (×105 CFUmL-1)
	Gastric juice of poultry extract (pH 2.7)
	h 0	h 3
LP1	70.4	50.3
LP3	63.2	92.0
LP6	95.4	60.8
LP7	98.4	63.2
LP8	188.8	42.2
LP9	84.8	50.4
LP10	82.4	94.2
LP11	88.0	84.0
LP12	87.2	37.6

All strains were tested for their ability to grow in presence of bile salts (Table 3). It appears that strains coded LP3 and LP10 exhibited good resistance to bile salt since the number of cells was important on MRS agar with 0.3% of bile salt. The viability of these isolates after 4 h of exposure to bile salts is not significantly affected. Some other strains of Lb.fermentum are found to be bile resistant, it’s the case of the isolates coded LP7 and LP6. In contrary some other strains, such as poultry isolates LP12 and LP11, were inhibited partially in the bile condition.

Table 3

Effects of bile salt on the growth of Lb.fermentum strains.

Lb.fermentum	OD620 nm
	Without bile salt (BS)	With 0.3% BS	of tolerance %
LP1	1.113	0.786	70.62
LP3	1.136	0.854	75.18
LP6	1.145	0.817	71.35
LP7	1.160	0.860	74.14
LP8	1.130	0.781	69.12
LP9	1.187	0.805	67.82
LP10	1.112	0.844	75.89
LP11	1.148	0.765	66.64
LP12	1.186	0.702	59.19

Antibacterial activity

The size of the inhibitory zone for the nine selected isolates against the five indicator enterobacteria is shown in (Table 4). All nine LAB strains were most effective against the Gram-negative bacteria. However, the strains coded LP3, LP8 and LP12 showed high inhibitory activity against the indicator bacteria and the inhibition zones are larger than 12mm.These bacteria are known as the main pathogens causing diarrhea in piglets. The supernatant from lactobacilli strains culture give zones of inhibition onto the indicator strains tested. The diameters of inhibition range from 5mm to 12mm.The best activity was obtained with supernatants of Lb.fermentum LP3, LP11 and LP12. In our study, supernatant broths were neutralized to pH 6.5; the inhibition activity to enterobacteria isolates became lower. The result obtained with neutral supernatant, showed that the inhibition was not related to lactic acid but might be due to other antimicrobial substances (H2O2, specific bacteriocin or nonbacteriocin).

Table 4

Growth inhibition zones of enterobacteria caused by some Lb.fermentum strains.

Enterobacteria Inhibition zone (mm)	LP1	LP8	LP6	LP7	LP3	LP9	LP12	LP11	LP10
Salmonella spp./	3	3	3	3	3	3	3	3	3
Supernatant	2	2	2	2	2	2	2	2	2
Neutral Supernatant	-	1	-	1	1	-	1	-	1
E. coli/	3	3	3	3	3	3	3	3	3
Supernatant	1	2	2	2	2	2	2	2	2
Neutral Supernatant	-	-	2	2	2	2	1	2	2
Enterobacter spp./	2	2	1	2	3	2	-	2	3
Supernatant	1	1	1	2	2	1	-	2	2
Neutral Supernatant	-	-	-	-	2	-	-	1	2
Klebsiella spp./	3	3	3	2	3	2	3	3	3
Supernatant	1	2	1	-	2	1	2	2	2
Neutral Supernatant	-	1	-	-	2	1	1	2	1
Shigella spp./	-	3	-	3	3	2	3	2	3
Supernatant	-	2	-	2	2	1	2	2	2
Neutral Supernatant	-	2	-	1	2	1	1	1	2
Citrobacter spp./	3	1	2	-	3	3	1	3	3
Supernatant	1	-	-	-	2	2	1	2	2
Neutral Supernatant	-	-	-	-	2	2	-	1	1

No inhibition;

inhibition zones smaller than 8mm;

inhibition zones between 8 and 12mm;

inhibition zones larger than 12mm

Coaggregation

Coaggregation of some Lb.fermentum with three enteropathogens bacteria Salmonella spp., Klebsiella spp and E. coli was examined (Table 5). Results are expressed as the percentage reduction after 5 h in the absorbance of a mixed suspension compared with the individual suspension. The best co-aggregation properties were obtained with Lb.fermentum LP3 and Lb.fermentum LP10.

Table 5

Coaggregation ability of some Lb. fermentum cells after 5h incubation.

Strain	Coaggregation (%)
	.Salmonella spp	.Klebsiella spp	E.coli
LP1	2.2	2.1	3.1
LP3	14.2	13.5	18.3
LP6	1.2	1.3	2.4
LP7	2.0	1.8	1.9
LP8	1.6	1.5	1.6
LP9	2.1	2.0	2.6
LP10	13.1	13.4	16.5
LP11	3.2	3.5	3.9
LP12	3.5	3.7	4.5

Assay of the adherence capability for LAB isolates

The ability to adhere to host intestinal mucosa is considered as an important selection criterion for LAB strains intended for probiotic use. It should be reminded that for LAB strain, only more than 15 LAB cells adhered on one epithelial cell, it could be considered as “positive” adherent strain. In our case, we found that Lb. fermentum LP3, LP10, LP11 and LP12 showed the adherence specificity to the chicken intestinal epithelium (Fig. 1).

Fig. 1

Adherence of isolated Lb. fermentum on the epithelium cells: (a) negative adhesion (control) (b) positive adhesion of Lb. fermentum LP3.

DISCUSSION

Lactobacilli occur among the normal intestinal flora of chicken and other animals from the first few days of their life. The capacity of lactobacilli to colonize the chicken crop by means of adherence has been well documented. Barnes (11) has reported that lactobacilli represent an important component of the intestinal flora of chickens, reaching 109g-1 of the caecal content. In poultry, the lactobacilli genus is sometimes represented by Lactobacillus acidophilus, Lb. fermentum, Lb. buchneri, Lb. ruminis, Lb. delbrueckii, Lb. coryniformis and Lb. viridescens (12). Fuller et al. (13) reported that Lb. fermentum was the dominant species found in the gastrointestinal tracts of the 2–10 day old piglets and the growing pigs. Fons et al. (14) also reported that Lb. fermentum was commonly found in the digestive tracts of pigs, rodents and humans.

The pH levels of gastric juice may vary from 2.0 to 3.5 depending on the feeding time, and the growing stage or the kind of animals (15). The results of study conducted by Idoui et al. (16) showed that Lb.plantarum BJ0021 was resistant to pH 3 and this strain shows a good resistance to rabbit gastric juice. The results found by Lin et al., (9) showed that Lb.acidophilus and Lb.bulgaricus from chicken were less stable in the pH 2.6 chicken gizzard extract, although Ashraf et al., (17) reported that lactobacilli strains isolated from chicken digestive tract (crop,gizzard, ileum and caecum) showed variability in viability at pH 2.

The bile in animal intestine is also an important factor which affects the viability of LAB cells (15). Jin et al., (18) demonstrated that twelve Lactobacillus strains isolated from chicken was slightly affected by bile salts (0.3%). The results of resistance against bile salt are supported by the findings of Gilliland (19), who reported that lactobacilli isolated from animal intestines showed high tolerance to biliary salts. Similar results were found in another study conducted by Patel et al. (20). In other study, Ashraf et al., (17) demonstrated that all tested culture of lactic acid bacteria showed resistance against different concentrations of oxgall.

Bacteria frequently used as probiotics in poultry production include species of Bacillus Bifidobacterium, Enterococcus, Escherichia, Lactobacillus, Lactococcus and Streptococcus. Strains of Lactobacillus have been selected as candidate probiotics on the basis of their in vitro inhibitory effect on avian pathoges. These avian pathogenic bacteria are known as the main pathogens causing diarrhea in piglets (21). Similar results were obtained by authors. Jin et al., (18) reported that 12 Lactobacillus isolated from chicken intestinal tract were able to inhibit five strains of Salmonella. An in vitro study by Bhatia et al., (22) proposed that lactic acid production by Lb. acidophilus is responsible for the inhibition of gastrointestinal pathogen.

Adherence to intestinal mucous is among the in vitro test that is frequently suggested for the evaluation of the probiotic potential of a bacterial strain (6). Rinkinen et al., (23) considered that the mucus adhesion properties are more dependent on the LAB strain than on the host. Attachment of probiotic strains to the epithelial cells and intestinal mucosal is prerequisite for the intestine colonization, as it influence the time of bacteria retention in the intestine and the functional activity of bacteria (24).

The pronounced disposition for aggregation of lactobacilli is well known and has previously been reported. For instance, Lb. fermentum LP3 and LP10 showed significant co-aggregation with pathogenic bacteria. In our study, the poultry epithelial cells were used as a substratum for the adhesion of lactobacilli strains. Within the same species large differences in the level of adhesion has been detected. This could be attributed to several factors such as the non-specific reaction by charge, non-specific reaction by hydrophobicity. Another suggested factor is the presence of the protenaceous components in the surface layered proteins of the strains that are involved in the adhesion process through their binding to carbohydrate portions of the colonic mucous layer (25).

CONCLUSION

From the results of our study, potential lactobacilli strains isolated from poultry gizzard able to be used as probiotics in poultry feed may be found. Strains of Lb. fermentum LP3 and LP10 were able to adhere to the intestinal epithelium of poultry. In addition, they were resistant to acid and were also bile tolerant. The same strains were able to inhibit the growth of enterobacteria isolates. Finally, for the confirmation of our in vitro results, in vivo study should be done.