MATERIALS AND METHODS

Microbial strains: Probiotic strain candidate (one strain of L. acidophilus P185) was identified by Mahrous (2006) which was isolated from healthy, breast- feeding infants (15 day old) and used after selection and identification according to Bergey’s Manual of Determinative Bacteriology, 9th edition (Holt et al., 1994) with confirm identification by SDS-PAGE technique and API System. The strain was tested for its probiotic characteristics i.e., gastric acid resistance, bile salt tolerance, antibacterial activity, adhesion to human mucus. Lactobacillus strain was cultivated in MRS (de Man Rogosa Sharpe) broth (Lab M, IDG, UK) and incubated at 37°C in BBL anaerobic jar (Becton Dickinson Microbiology Systems, Sparks, MD) provided with disposable BBL gas generating pack (CO₂ system envelopes, Oxoid, Ltd., West Heidelberg, Victoria, Canada).

The culture was preserved in reconstituted skim milk in eppindorf tubes, stored at -80°C with glycerol (20%, v/v). Prior to use, strain was a subcultured (1%, v/v) twice in MRS broth and adjusted at 1x10⁹ CFU mL^-1.

The E. coli (10536) strain obtained from ATCC was cultivated in nutrient broth (Lab M, IDG, UK) and incubated at 37°C for 24 h and preserved in reconstituted skim milk in eppindorf tubes.

Extraction of exopolysccharides from L. acidophilus: The EPS was isolated and purified according to Cerning et al. (1994) with some modification. The growth culture was heated at 100°C for 5 min to inactivate enzymes potentially capable of polymer degradation and the cells were removed by centrifugation at 8000 rpm for 5 min at 4°C. The EPS was precipitated using 2 vols of absolute ethanol. After standing overnights at 4°C, the resultant precipitate were collected by centrifugation at 8000 rpms for 20 min. The EPS was dissolved in deionized water, dialyzed against deionized water at 4°C for 24 h and freeze-dried. The freeze-dried powder was dissolved in 10% (w/v) trichloroacetic acid to remove proteins. The supernatant was dialyzed at 4°C against deionized water for 5 days and freeze-dried. These preparations were referred to as purified EPS and were stored at 4°C.

HPLC analysis: The EPS sample was analysed using a High Performance Liquid Chromatograph (HPLC) under the following conditions; mobile phase: 0.65 m msulfuric acid; columns: Aminex HPX-87H (30χ7.8 mm); temperature 75°C, flow rate, 0.7 mL min^-1, injection volume: 20 μL, detection at UV 205 nm by using refrence standard from sigma-addrich compony, D-(+) glucose (G8270), D-(+)glactose (G5388) and D-glucoronic acid (G5269).

Cell lysates and intact cells: Cells were harvested by centrifugation at 4°C for 30 min (5,000 g) after overnight incubation at 37°C and the pellet was washed twice with 20 mM sodium phosphate buffer (SPB, pH 7.4), then re-suspended in SPB. Washed cell suspension was disrupted with a ultrasonic cell disrupter (Brandson 4°C) and filtration (0.45 μm, Millipore). Cell debris was removed from centrifugation (10,000 g for 10 min and concentration was measured by the Bradford method (Bio-Rad Laboratories) and adjusted to 1 mg mL^-1. For the preparation of intact cells, cells were washed twice with SPB and re-suspended in SPB. The total cell number was adjusted to 10⁹ CFU mL^-1 (Kim et al., 2006).

Resistance to hydrogen peroxides and hydroxyl radical: Hydrogen peroxides and hydroxyl radical were observed by the method of Kullisaar et al. (2002). For the measurement of Lactobacilli resistance in the presence of hydrogen peroxide, cells were suspend at the level of 10⁷ CFU mL^-1 in Sodium Phosphate Buffer (SPB) and incubated with 1.0 mM hydrogen peroxide (30%) at 37°C. At 1 h time intervals, the number of viable cells was estimated at MRS agar plates. For resistance to hydroxyl radicals, cells (10⁷ CFU mL^-1) were incubated with the solution containing 10 mM THAs (terephthalic acid, Sigma) in SPB and 0.01 mM CuSO₄x5H₂O. The reaction was started by the addition of 1 mM hydrogen peroxide and the number of viable cells was estimated (Kim et al., 2006).

For blanks, the cells were suspended at the level of 10⁷ CFU mL^-1 in SPB and incubated at 37°C for 7 h. At 1 h time intervals, the number of viable cells was estimated on MRS agar plates and we examined that the number of viable cell did not affect for 7 h.

DPPH freed radicals scavenging assay: Evaluation of Antioxidant activity by in vitro technique with 2, 2^--diphenyl-1-picrylhydrazyl (DPPH) assay. DPPH (0.1 Mm) was prepared by dissolving 1.9 mg from DPPH to absolute methanol in 100 mL, then keeping in the dark place for 1 h and 1.0 mL of this solution was added to 3.0 mL of extract solution in methanol at different concentrations. Thirty minute later, the absorbance was measured at 517 nm. A blank was prepared for adding extract. Concentrations (1-18 μg mL^-1) were used as standard; lower absorbance of the reaction mixture indicates higher free radicals scavenging activity. The scavenging ability was defined as follows:

In vitro assessment of anti tumor activity of L. acidophillus bacteria and its EPS
Tumor cells: The initial inoculums of Ehrlich Ascites Carcinoma (EAC) were purchased from the National Cancer Institute, Cairo University, Egypt. EAC cells were propagated in NODCAR laboratories by weekly intraperitoneal injection of 0.2 of 1:5 mL^-1 saline solution of freshly drawn ascetic fluid (0.2x10⁶ EAC cells) from a donor mouse bearing 6-8 day old ascitic tumor, into three mice to ensure that the ascetic fluid will still propagated and can then be drawn from atleast on life mouse. Transplantation was carried out using sterile disposable syringes under aseptic conditions. The tumor growth was as rapid as to killing the mice within 18-20 day due to the accumulation of ascetic fluid and rarely the tumor showed distal metastasis or spontaneous regression. After making appropriates dilution, the non-viability was tested for trypan blue exclusion method, (Freshney, 2005), as shown follows:

By this method we can determine the effective dose of bacterial extracts induced more regression of tumors.

Effect of different doses from bacteria on the growth of solid tumors in male albino mice: Adult Male Swiss albino mice with an average body weight 20-25 g were used as experimental animals throughout the study. The mice were housed in specially designed cages and maintained in a thermostatically controlled room during the experimental period. They were fed an ordinary pellet diet and given up tap water ad-libitum in (NODCAR) these mice were divided into 4 groups having 5 animals each:

Statistical analysis: Data obtained was subjected to analysis of variance and the means were compared using the Least Significant Differences (LSD) tested for the 0.05 level, as recommended by Snedcor and Cochran (1982). In vitro experiments were conducted in triplicate.

RESULTS AND DISCUSSION

Resistance of L. acidophilus bacteria to hydroxyl radicals: Figure 1 and 2 show the resistance of L. acidophillus to hydrogen peroxide and hydroxyl radicals. Lactobacillus acidophilus was viable for 7 h in concentrations of 1.0 mM h ydrogen peroxides and 0.01 mM hydroxyl in the presence of highly damaging effect, both these intact cell and cell lysate, had strong anti-oxidatived activities with increasing inhibition percentage in cell lysate than intact cell. Anti-oxidatived activity of microorganisms is one of the reasons for their increased resistance to Reactive Oxygen Species (ROS). Kullisaar et al. (2002) reported that the antioxidant strain L. acidophilus had significantly high resistance to ROS. Kim et al. (2006) proved that L. acidophilus was the most active strain which hydroxyl radicals scavenging activity in cell lysates was 70 and 53% in intact cells. The scavenging of different types of ROS was thought to be one of the main antioxidant mechanisms of the antioxdant action exhibited by lactic acid bacteria (Namiki, 1990).

DPPH method: DPPH is a relatively stable organic radical, it has been widely used in the determination of antioxidant activities.

Fig. 1:	Perecantage of resistance of L. acidophilus bacteria to hydrogen peroxide. Bacteria was suspended at the level of 107 CFU mL-1 with conc. 1 mg mL-1 for both cell lysates and intact cells and incubated with 1.0 mM hydrogen peroxides

Fig. 2:	Perecantage of resistance of L. acidophilus to hydroxyl radical. Bacteria was suspended at the level of 107 CFU mL-1 with conc. 1 mg mL-1 for both cell lysates and intact cells and incubated with 0.01 mM hydroxyl radicals

Figure 3 shows the results of DPPH scavenging potentioal of different concentrations of L. acidophillis, cell lysates and intact cells. The maximum antioxidant activity was 69.41 and 65.215% with 80 μg mL^-1 of both cell lysates and intact cells. It is noticed that the antioxidant activaty of different concentrations of cell lysates was more effective than different concentrations of intact cells. Lin and Chang (2000) indicated that the radical scavenging ability of the intact cells and intracellular extracts of B. longum and L. acidophilus contribute good antioxidative effect on inhibiting linoleic acid peroxidation and scavenging the DPPH radical.

Chemical composition of exopolysaccharides (EPS): The results of exopolysaccharides content secreted by L. acidophilus are shown in Fig. 4, which shows different sugar content and concentrations. The sugar contents is glucose, galactose and D. glucoronic acid (71.9, 22.46 and 0.16 mg, respectively).

Fig. 3:	Antioxidant activities of different concentrations of L. acidophillis cell lysates and intact cells against DPPH radical

Fig. 4:	EPS by HPLC analyses containing D. glucoronic acid, D. glactose and D. glucose

Table 1:	In vitro cytotoxic effect of different concentrations of L. acidophilus and their exopolysaccharides on the non viability of Ehrlich Ascites Carcinoma (EAC) cells

Table 2:	Effect of tested Lactobacilli and its exopolysaccharides on the volume of solid tumor (mm3)
***Highly significant with p≤0.05

The chemical composition of exopolysaccharides from L. acidophilus has been investigated and there is agreement that EPSs from LABs is polysaccharides with D. glucose and D. galactose as main sugar constituents and the ratio of the two components varies (Laws et al., 2001).

In vitro assessment of the effective dose of L. acidophilus and its EPS against EAC cells: Different concentrations of L. acidophilus and its EPS (100, 200, 300, 400, 500, 600 and 700 μg mL^-1) was investigated on the non viability of Ehrlich Ascites Carcinoma (EAC) cell line as shown in Table 1. Results showed that different concentrations of L. acidophilus and its EPS inhibited the proliferation of EAC cells. The EPS showed more antiproliferatives effects against EAC cell line than L. acidophilus. The maximal precentage of inhabition of EAC cells line was (93 and 99%) with 700 μg mL^-1 of L. acidophilus and its EPS, respectively. This result is demonstrated by Doleyres and Lacroix (2005) which reported LAB EPS is provided beneficial physiological effects on human health, such as antitumours activity, immunomodulating bioactivity and antimutagenicity.

Effect of L. acidophilus and its exopolysaccharides on the volume of solid tumors: Further in vivo study for the highly toxic L. acidophilus and its exopolysaccharides has been done on solid tumor bearing mice. As show in Fig. 5 and Table 2, L. acidophilus and its exopolysaccharides caused significant reduction in the tumor volume as compared to that of the positive control group and effects of E. coli. The reduction of tumor volume (1.02±0.022 and 1.21±0.019 mm³) were observed when mice-bearing solid tumor was treated with exopolysaccharides extracted from L. acidophilus.

Fig. 5:	Effect of tested Lactobacilli and its exopolysaccharides on the volume of solid tumor (mm3)

The EPS from L. acidophilus showed powerful reduction of solid tumor volume than L. acidophilus itself while E. coli causing propagation of solid tumor volume than control mice. Baldwin et al. (2010) suggested that probiotics may be used as adjuvants in anticancer chemotherapy. Daniluk (2012) reported that the anticancer activity through induction into a poptosis of cancer cells seems to be promising to approach for use of some probiotic strains on support therapy or disease prevention. Probiotics also have a role in prevention of colon cancer (Dugas et al., 1999; Rafter, 2003). Different strains of probiotics had different effects on the intestinal luminal environment, epithelial and mucosal barrier function and the mucosal immune system (Hirayama and Rafter, 2000).

CONCLUSION

This study demonstrated that L. acidophilus having antioxidant activities for both intact cells and cell lysates. The L. acidophilus and its exopolysccharide (EPS) had antitumor activaty in vivo and in vitro. The exopolysccharide of L. acidophilus is more effective against Ehrlich Ascites Carcinoma (EAC) cells than L. acidophillus.