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Abstract
The present study was carried out to isolate and identify different strains of probiotic bacteria which will be tested on Immuno-dificient Western albino rats. Treatment of rats with (Bifidobacterium bifidium ATCC 20079, Egyptian bifidobacterium isolate, Lactobacillus acidophilus ATCC 20082 and Egyptian lactobacillus isolate) improved the humoral immune response (IgG and IgM).
The study includes:
1) Isolation of lactic acid bacteria (bifidobacteerium and lactobacillus)
2) Identification of the isolated strains using molecular genetics techniques.
3) Effect of probiotics on serum immunoglobulins (IgG and IgM) in Immuno-dificient rats.
To achieve this goal, four probiotic strains were used in this study, two strains were isolated from crude milk collected from Six October City, while the other two strains were obtained from MIRCEN, Ain Shams University, Cairo. After isolation using pouring plate technique on MRS and TPY media, Gram positive bacilli with catalase negative test isolates were selected for further identification. The biochemical results for bifidobacterium and lactobacillus showed the same pattern in gas production from glucose, growth at different temperatures and salt concentrations. On the other hand, lactobacillus was only able to grow in the presence of oxygen. Concerning the carbohydrate fermentation test, both strains showed the same pattern with glucose, lactose, fructose, galactose, sucrose and mannitol. Unlike lactobacillus, lactobacillusbacterium was only able to ferment rhamnose and xylose whereas lactobacillus showed positive fermentation pattern with mannose and sorbitol.
Furthermore, DNA was isolated for partial amplification of 16S rDNA using Polymerase Chain Reaction (PCR), primers were designed and were tested on primer designing tool on http://www.ncbi.nlh.nih.gov. The partial 16S rDNA give a rise of a band at 532 base pair for both Egyptian strains. Bands of interest were purified, and sequencing was performed for both samples. First 500 base were aligned using BLAST tool for both bifidobacterium and lactobacillus and dendogram analysis was carried out. Alignment of 16S rDNA of Lactobacillus showed homology of 88% with Lactobacillus kefiranofaciens but dendogram analysis showed more homology to Lactobacillus plantarum. While the other sample showed 91% homology and expected value of 3e-113 with uncultured bifidobacterium Clone R333 16S rRNA gene.
After isolation and identification of bifidobacterium and lactobacillus, the two isolates together with the reference strains (Bifidobacterium bifidum ATCC 20079 and Lactobacillus acidophilus ATCC 20082) were tested on fifty immune-deficient Western albino rats after administration of cyclosporine of a dose of 10 mg/kg/day for seven days. Initial serum immunoglobulins level (IgG and IgM) were determinded before and after immune-suppression for each rat, and then all of the fifty rats were equally divided into five groups of 10 rat in each according to the type of the administrated probiotic, while the last group was left as control group.
Serum IgG and IgM level were determined for all of the four groups after administration of probiotics for seven days, serum IgG and IgM level were also determined for Control.
The results of the present study revealed a high significant increase in serum IgG level in all of the four groups. Also it has been showed that Egyptian lactobacillus isolate provided the highest significant result which could be recommended for the use as booster supplements after vaccination.
In addition serum IgM level has been increased significantly in all of the four groups particularly the two Egyptian isolates bifidobacterium and lactobacillus.
According to this study, it could be concluded that:
• Probiotics could be used as an immunostimulant agents as they have highly significant effect on humoral immunity (IgG and IgM) in rats.
• It has been found that the Egyptian probiotic isolates have the highest significant effect in the activity of serum IgM level in comparison with the other reference strains.
• Finally it was observed that the Egyptian strain of lactobacillus have the highest immune-stimulant effect on both IgG and IgM and hence it could be use after vaccination as a booster dose supplements.
Recommendations
• Probiotics could be used as booster dose supplements after vaccination.
• Probiotics could be tested on human beings especially Immuno-dificient patients and elderly.