الفهرس 
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Abstract
Calcium kidney stone disease is a highly prevalent, painful and costly condition that is further aggravated by a high recurrence rate. Prevention of urinary calculi recurrence is one of the biggest challenges facing modern urology. Hyperoxaluria is one of the most prevalent metabolic abnormalities, which increases the risk of calcium stone formation. Urinary oxalate is derived from both the intestinal absorption of dietary oxalate and the endogenous metabolism of various precursors, mainly ascorbic acid and glyoxylate. The treatments for hyperoxaluria are mostly based on dietary restrictions, which may not be applicable to all participants. The absorption of dietary oxalate, however, could be affected by different factors; among them, the intestinal microbiota is currently receiving much attention. Thus, the objective of the present study was to isolate probiotic oxalate degrading lactobacilli and bifidobacteria and use them as starters to produce functional dairy product such as stirred yogurt for managing hyperoxaluria. To achieve this aim, the data of the present study can be summarized as follows:
I- Isolation, purification, identification of oxalate degrading lactobacilli, bifidobacteria and evaluating their probiotic properties.
In this part, thirty-eight different dairy products were collected from local markets, 495 isolates were isolated and characterized using
Gram’s stain, catalase test, glucose fermentation, and acid production test to select lactobacilli and bifidobacteria colonies. The results revealed that 116 isolates were Gram positive rods, catalase negative, able to ferment glucose and able to form clear zone in 0.5
% CaCO3 plate. Only 88 isolates were identified as lactobacilli and
28 isolates were identified as bifidobacteria. All the 116 isolates were examined, in vitro, for oxalate degradation. The results obtained could be summarized as follows:
• Bifidobacteria isolates were unable to degrade oxalate.
• 23 lactobacilli isolates were able to grow and produce a clear zone around the colonies, indicating that these isolates possessed the oxalate-degrading enzyme.
• The Enzymatic Activity Indices of C.O.L15 and R.O.L09 isolates had the highest Enzymatic Activity Index, indicating the highest oxalate degradation ability. While C.O.L01 and K2.O.L05 isolates had the lowest Enzymatic Activity Index.
• Z.O.L03 had the best growth in the presence of oxalate in
MRS-Ox broth.
• The combinations of the most potent isolates (i)-Z.O.L03and K2.O.L02 (ii)-K2.O.L02 and K3.O.L07 increased oxalate-degrading Enzymatic Activity Index.
• Genotype identification results of the most potent 7 isolates using 16S rDNA cleared that R.O.L09, K2.O.L02, C.O.LO6, K3.O.L07, and Y.O.L03 isolates were identified as Lactobacillus fermentum. Isolates C.O.L15 and Z.O.L03 were identified as Lactobacillus gastricus. The probiotic properties of these isolates were evaluated which are acid tolerance, bile salt tolerance,
antimicrobial activity, antibiotic susceptibility, exopolysaccharide (EPS) production, phenol resistance and fermentation of carbohydrates.
• All the seven Lactobacillus strains were able to save viable counts upon 6 log CFU/mL in pH 4 and 3 after 3 hours of incubation. At pH 2, all the isolated strains did not survive at this acidic pH and their viable numbers showed a severe reduction after
3 h of incubation. Lactobacillus fermentum NRAMJ5 was the only strain which exhibited high survival after 3 h of exposure to an acidic pH 2.
• All of the seven Lactobacillus strains were able to survive in the different concentrations 0.1, 0.2, 0.3, 0.5 and 1 % of bile salts and still produce viable counts of 6 Log CFU/mL, as required for them to be probiotic.
• The Lactobacillus strains had stronger antimicrobial activity against pathogenic Gram-negative bacteria than against Gram- positive bacteria. All strains had strong antifungal activity against A. niger, S. cerevisiae. Salmonella sp. and Yersinia sp. were more sensitive to the metabolites of Lactobacillus spp.
• All the strains were sensitive to cefuroxime, ampicillin\sulbactam, clindamycin, ampicillin, rifampin, gentamycin, chloramphenicol, tetracycline, amoxicillin, and cefoperazone. However, all of the isolates were resistant to ciprofloxacin, except for Lactobacillus gastricus NRAMJ2.
• All the tested strains were able to produce exopolysaccharides.
• None of the isolates were able to resist a concentration of phenol of 0.5%. However, all the tested strains could resist phenol concentrations of 0.1, 0.2, and 0.3 %.
• The ability of the seven Lactobacillus strains to ferment different carbohydrates was examined using the API 50 CH system. All isolates were able to ferment nine different carbohydrates: glucose, galactose, fructose, mannose, ribose, N-acetyl- glucosamine, salicin, sucrose and lactose.
• In conclusion, Lactobacillus fermentum NRAMJ5 and Lactobacillus gastricus NRAMJ2 were the most potent oxalate degrading strains and good candidates as probiotics for managing hyperoxaluria in vitro. These strains were selected to be a starter cultures for production functional stirred yoghurt and further investigation to elucidate their effect on hyperauxaluria induced rats in vivo.
II- Production of functional stirred yogurt.
1. Microbiological changes during storage period.
• Total viable counts in all yoghurt treatments increased till 7 days then decreased at the end of the storage period. Generally, Lactobacillus spp. tend to increase gradually in count till 7 days of storage period then tends to decrease. Also, Str. thermophilus continuously increased till the day 7 of storage in all treatments, followed by slight decrease till the day 14 of storage.
2. Chemical analysis
• Chemical composition for all treatments of fresh samples. The total solids ranged from 14.50 to 14.55 %. Also, the values of moisture ranged from 85.45 to 85.50 %. The values of fat and protein ranged from 3.70 to 3.75 % and 3.80 to 3.84 %, respectively.
• The pH values of all stirred yoghurt samples gradually decreased during the refrigeration storage. The lowest pH value recorded for treatment T 1 (Lactobacillus fermentum NRAMJ5).
• The changes in acidity values were opposite to pH changes. The acidity increased after two weeks of storage period.
III- Biological evaluation.
• In this part, the therapeutic potential of the isolated probiotic lactobacilli in reducing hyperoxaluria in vivo through intestinal oxalate degrading activity were evaluated in rats. The fecal, blood and urine samples of rats were analyzed to assess their role.
1- Microbiological Analysis.
• The mean total fecal microbial counts of the different experimental rat groups reduced after 21 days only in group 2 while higher counts of group 3 and 5 were seen in comparison with controls approximately increased 4 logs.
• On the day 7, all groups of rats showed a significant rise in the fecal count of Lactobacillus spp. After two weeks of feeding the counts of fecal labtobacilli showed a tendency to raise in the test groups in comparison with the controls. In 21 days, group 2 rats fed
high oxalate diet (diseased group) showed a fall in the mean of lactobacilli count. However, the Lactobacillus treatment groups showed an increase in the fecal lactobacilli count.
• The counts of mean coliforms exhibited a fall when compared the numbers in groups fed high oxalate diet only after two weeks of feeding.
2- Assessment of Biochemical Parameters.
• Urine pH values of the experimental rats showed a slight reduction after 7 days of feeding. In 14 days, rats fed high oxalate diet group (diseased group) exhibited a fall in the pH values (6.72) as compared with the control fed balanced diet (8.57).
• Urea levels in serum of all groups increased as compared to the control group. Urinary urea of group 4, 5 showed a significant reduction.
• All the rats treated with Lactobacillus spp. groups showed reduced urine excretion of proteins. While only group 3 which treated with Lactobacillus fermentum showed a reduction of serum protein.
• The aspartate aminotransferase (AST) values of all rat groups treated with Lactobacillus spp. and control group fed balanced diet were reduced as compared with the diseased group. While only group 5 which treated with mixed Lactobacillus spp. showed a sever reduction of acid phosphatase level. Only group 4 treated with Lactobacillus gastricus had the lowest alkaline phosphatase value at the end of experiment.
• Excretion of uric acid in urine of all group’s rats treated with Lactobacillus spp. showed a significant increase when compared to control group.
• The mean value of serum creatinine in all experimental groups 3, 4 and 5 showed a significantly lower than the diseased group. However, urinary excretion of creatinine (creatinine clearance) decreased in all rats received Lactobacillus spp.
• Calcium and magnesium serum excretion mean values in groups 3, 4 and 5 were lower than in the stone induced group. While serum phosphorous values were elevated in all rat groups except group 2.
3- Microscopic examination of oxalate crystals (CaOx) in urine samples of rats.
• The microscopic examination (40x) of calcium oxalate crystals in different experimental rat groups urine sediments. The results showed that there was a sever variation between the different groups. The control group fed balanced diet urine sample was devoid of oxalate crystals while group two fed high oxalate diet had high percentage (+6) of oxalate crystals in their urine samples. Additionally, Lactobacillus treatment groups low score (+1) of oxalate crystals.