الفهرس 
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Abstract
This study aimed to isolate different lactic acid bacteria from Rayeb milk, which were obtained from different locations, such in Minia and Beni suef governorates. The obtained bacterial isolates were then screened through probiotic selection criteria in a step by step procedures, which included about 10 subsequent tests (morphological, physiological and biochemical tests). Afterwards, the final objective was applying for production of a functional dairy product. (Rayeb milk). The obtained results could be summarized as follows:
1- Isolation and morphological properties of lactic acid bacteria:
Total of 78 isolates were obtained from 10 samples of Rayeb milk. Out of this number, 54 bacterial isolates were found to be Gram positive, catalase negative, which meets the requirements of lactobacilli characteristics. Those 54 isolates included 38 rod-shaped and 16 cocci-shaped isolates, and they were retained for further assessment for lactic acid bacteria possessing probiotic properties in order steps procedure, in which any group of isolates showing good performance for a tested property were again selected for the next step.
2-Acid tolerance (pH 4 and 3):
a) Tolerance to pH4:
Results of acid tolerance (growth at pH 4) were expressed as optical density (absorbance) at 660 nm and the growth increase was attributed the growth of the control. Among the (54) isolates (7 ) isolates from all samples gave rates of growth increase over than 10 fold comparing to the growth of the control, (19) isolates gave rates of growth increase between 5-10 fold, 25 isolates gave rates of growth increase between 1-5 fold ,and 3 isolates gave rates of growth increase lower than 1 fold.
b) Tolerance to pH3:
The acid tolerance at pH 3 was conducted only on 51 selected isolates. Among these (51) isolates (2) isolates from all samples gave rates of growth increase over than 10 fold, (10) isolates gave rates of growth increase between 5-10 fold, 28 isolates gave rates of growth increase between 1-5 fold , 8 isolates gave rates of growth increase lower than 1 fold ,and 3 isolates did not give any growth.
4- Bile salt tolerance (growth at 0.3% active ingredient):
Based on the previous test, only (40) isolates were selected. No isolates from all samples gave rates of growth increase over than 10 fold, (8) isolates gave rates of growth increase between 5-10 fold ,(28) isolates gave rates of growth increase between 1-5 fold,(2) isolates gave rates of growth increase lower than 1 fold ,and( 2) isolates did not grow.
5- Assay for NaCl tolerance:
Among the (36) tested isolates, (3) proved to be highly tolerant with optical density range > 1.2 followed by (10) isolates with optical density range >0.5 <1.0, then 19 isolates responded with >0.1<0.5 optical density, and 4 isolates proved to be very weakly tolerant.
6- Experiment Growth at Different Temperature
a) Growth at a temperature of 40 ºC
Among the (32) isolates, (20) isolates gave rates of growth increase between 5-10 fold, (12) isolates gave rates of growth increase between 1-5 fold.
b) Growth at a temperature of 45º C:
Among the (32) isolates (29) isolates gave rates of growth increases between 1-5 fold and (2) isolates gave rates of growth increases lower than 1 fold ,and( 1) isolates did not grow.
c) Growth at a temperature of 50º C:
Among the (29) tested isolates (2) (SIIB10 and MIIST1) proved to be highly tolerant to a temperature of 50º C.,(17) isolates gave rates of growth increase lower than1 fold, and(10) isolates were never gave growth.
7- Bacterial adhesion to intestinal epithelial cells test:
The adhesion test (in vitro) was carried out on the chosen (19) bacterial isolates (9 rod shaped, and 5 cocci shaped). Results indicate that bacterial isolates exhibited variable adhesion to epithelial cell of sheep intestine. Out of (19) isolates tested, (8) isolates showed strong adhesion, (4) isolates showed moderate adhesion, (2) isolates showed weak adhesion, and (5) isolates showed no adhesion.
8-Assay for coagulation of litmus skim milk:
Out of (14) isolates, only (3) isolates exhibited strong coagulation and (1) isolate gave moderate coagulation. During the 24hours period of incubation, coagulation took place in the test of isolates, and the most latest till 24 hours was (1) isolate.
9- Antimicrobial activity:
a) Activity against bacteria:
The selected isolates showed varied activity against bacteria with a superior effect of the two isolates SII ST4 and MI B6 against Bacillus cereus, the isolate MII ST6 against Pseudomonas sp, the two isolates SII ST4 and MII B5 against Pseudomonas aeruginosa, the two isolates SII B10 and SII ST4 against Staphylococcus aureus and the two isolates SII B5 and MII B5 against Listeria monocytogene
b) Against pathogenic fungi:
The selected isolates showed varied antifungal activity with a superior effect of the isolate MII LT against Fusarium oxysporum, and the isolate MII ST2 against Rhizoctonia solani
10- Rayeb milk preparation:
The mother culture was prepared using a skimmed milk by placing 100 ml milk in a sterile bottle and packing well, then
Pasteurized for 90 ºC for 30 minutes then cooled to 45- 42ºC. This milk was inoculated with 3%of the desired isolated strain and then incubated for 37ºC for 4 hours. The resulting culture called (the mother culture).In the next day, 3 ml of the mother culture was transferred to 100 ml pasteurized skimmed milk and incubated for 37º C for 4 hours and the resulting culture called (median culture).
Finally this median culture was used as inoculum at concentration of 3% to fresh pasteurized skimmed milk, then the inoculated milk was incubated at 37ºC for 24 hrs. the resulting Rayeb milk was stored at 4ºC.
Change in pH during storage:
The Rayeb milk prepared by the isolate MII ST1 showed the lowest pH value (3.66) after 21 days of storage. The isolates MII ST2 and SII b10 gave pH values 3.48 and 3.51 respectively after 28 days.
Sensory evaluation of fresh Rayeb milk:
The two prepared milk using MII ST2 and SII B10 isolates proved to be the most acceptable. They took 8.86/10 and 8.96/10 degrees respectively after 28 days of storage, while the isolate MII ST1 was the least acceptable one since it took 7/10 degrees. (average).
Bacterial culture viability in rayeb milk:
The number of viable bacteria in the control was 39x104 cfu/ml, while the samples taken from the Rayeb milk prepared by the isolates MII ST2 and SII B10 gave 200x105 and 180x105 cfu/ml respectively after 28 days. The isolate MII ST1 gave 191x105 cfu/ml after 21 days.