الفهرس 
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Abstract
SUMMARY
The aim of this thesis is to look forward for functional and biological properties of beverage from whey milk added with different ratios of Moringa oleifera leaves powder (5, 10 and 15g MOLP/ 100 ml sweet whey) as a means of naturally avoiding liver cancer using experimental rats.
6.1. Physicochemical analyses of Moringa oleifera leave powder (MOLP) and sweet whey beverage (SWB)
6.1.1. Proximate composition
The results revealed that moisture; carbohydrate, protein, fat, lactose, fibre and ash were 7.85, 31.44, 38.1, 4.17, 0.79, 7.45 and 6.80 %, respectively in MOLP. Fortification with MOLP showed an increase in protein, lactose, crude fibre, ash and fat with increase in the levels of MOLP and decrease in moisture content. Moisture, carbohydrate, protein, fibre, fat and ash contents varied in the range 92.73-89.96%, 1.14-3.40%, 0.98-3.20%, 0.02-0.58%, 0.23-0.27% and 0.23-0.27% from T1-T3, respectively. Data of whole whey was 95.73% water, 0.95% protein, 0.20% fat, 2.25% lactose, 0.18% ash.
6.1.2. Mineral (Macro and micro elements)
Calcium had the highest value of 945 mg/100g followed by potassium 692 mg/100g and phosphorus had the least value of 145 mg/100g among the macro-elements in MOLP. The fortified whey beverages showed a significant increase in a dose dependent manner in all macro and microelements as compared to control. The highest value among the micro-minerals was iron with 358 mg/100 g followed by manganese with 94.3 mg/100 g. MOLP contains a significant amount of both macro and micro elements, which explain the increase in fortified sweet whey.
6.1.3. Vitamins
All the ratios used for fortification sweet whey with MOLP showed an increase in vitamins concentrations especially folic acid as compare with
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SW which increase from 0.14 ppm in control sample to 117 ppm at T3 followed by B12 which exhibited 2.75 ppm and 119.54 ppm in control and T3, respectively. On the other hand, T2 was the higher in nicotinic and B12 (60.41 and 128.56 ppm) respectively, than T3 and T2. Moringa oleifera leaves powder (MOLP) had the highest ratio of Vitamins A and C (4352.16 μg/ml and 101.4259 ppm) respectively.
6.1.4. Phytochemicals
The obtained data showed that MOLP is a rich source of flavonoids 34.6 mg/ml and alkaloids 2.32 as well as total phenolic 1.14 g/ml. While, addition of MOLP showed a significant (P≤ 0.05) increase in all phytochemicals in comparison between control and fortified treatments, no significant had occurred between T2 and T3 in saponin, phenol and tannins. Moreover, T3 recorded high significant after MOLP was (31.3 mg/ml). 6.1.5. Antioxidant activity
The extent of DPPH radical scavenging at different ratios (50-150 ug/ml) of SWMOLP beverage was measured and compared with BHA and TBHQ as the standards . The radical scavenging effect was found to increase with increasing ratios of MOLP in sweet whey.
6.1.6. Amino acids
The highest amount of amino acids was Glutamic acid, which had a value of 3190mg/100gdry matter and the least content was Methionine with 490mg/100gdry matter. The same finding was noticed in treatment T1, T2 and T3 (248.82, 19.06), (250, 20) and (251.18, 20.94 mg/100 g dry matter) respectively. The dried Moringa leaves contained 17 amino acids, 7 were classified as essential amino acids, and 2 were semi-essential (histidine and Arginine) is essential for children and reasonable levels were present in the treatment samples. Methionine (490mg/100g dry matter) had the least values followed by cysteine (620mg/ 100g dry matter) of MOLP, which is
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commonly deficient in green leaves. Methionine and cysteine are powerful antioxidants that help in the detoxification of harmful compounds and protect the body from radiation,
The percentage ratio of total essential amino acid (TEAA) to the total amino acids (TAA) in MOLP was 38.11% , while in each treatment (T1, T2 and T3) were 38.67, 35.83 and 35.90% respectively, this mean T1 was the best sample because it contain the highest percentage of essential amino acids. Meanwhile T2 recorded the highest percentage of total non-essential amino acids (TNEAA) 58.33% followed T3 was 58.17%.
6.1.7. pH value
pH values decreased during storage. The increase in pH values within 14day after fortified with different ratios of MOLP depended mainly on temperature and was the most important at 5±1ºC, especially when SW was performed at pH 5.48.
The decrease in pH values within 14 days was influenced by the pH on treatments. However pH remained the lowest for 14 days after fortified at pH 5.48. pH of prepared beverage was decreased remarkably from 5.69 to 5.81 with the increase in MOLP ratios from 5 to 15g MOLP/100 ml sweet whey at zero time. The same finding was noticed in all storage periods. This indicated that the increase in the MOLP ratios decreased the active acidity in the prepared beverage.
6.1.8. Total soluble solid contents for treatments fortified with different ratios of MOLP
Storage period had statistically significant effect on the average TSS of the beverage (p≤0.05) up to 8 days of storage. The interaction effect between MOLP ratios of SWB and storage period was also significant (p≤0.05). The highest value of TSS was recorded to be 27.3 0Brix at the zero time (fresh) of T3 sample containing 15% MOLP. While the lowest value of TSS was 17.2 0Brix at the end of 14 days of storage for T1 containing 5% MOLP. Sweet whey has low lactic acid and high mineral contents. The
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highest pH of whey causes insolubility of some individual whey proteins, and hence whey is more difficult to process through membrane filtration.
6.2. Microbiological analyses of SWB fortified with different ratio of MOLP
The results of microbiological examination showed not detected in total plat count (log CFU/ml) of sweet whey (SW) and different treatment of SWB fortified with 5, 10 and 15g MOLP (less than 30 CFU/ml). Mould and yeast counts are considered indicative of the quality and shelf life of SWB fortified with different ratios of dry leaves of Moringa oleifera. In this regard, moulds and yeasts were not detected in fresh of all treatments and throughout the storage period. This may be due to Moringa oleifera leaves had antifungal and antimicrobial activities.
6.3. Sensory evaluation of SW fortified with different ratio of MOLP
As can be observed color and overall acceptance of T1 have gotten the highest degree as compared to others (scores of 16.18±2.79 and 16.20±1.53 respectively) at zero time (fresh) as seen it Fig (7). Sensory evaluation shows that blend T1 (5g MOLP/100 ml sweet whey) compared favorably with the other blends. The increase in ratios of MOLP significantly decreased the sensory properties of the beverage (P≤ 0.05) and the mean value of 16.18 for color for the T1 prepared beverage decreased to 13.73 at T3 at zero time. The same observations were noticed in storage periods in all treatments.
6.4. Volatile compounds
The volatile compounds of fresh sweet whey beverage (SWB) as well as fortification of SWB with MOLP at 5g (T1) were analyzed by GC and GC-MS because it was recorded the best result in sensory evaluation. A total of 26 volatiles were identified in SWB and selected fortification sample at 5%, including 7 aldehydes, 5 alcohols, 11 acids and 3 esters. The most abundant identified volatile compounds were acids which represent 34.48%
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and 25.77% in SWB and T1, respectively. Acetic acid was the most predominant acid in both investigated samples with 8.14% and 5.92% in SWB and T1, respectively.
6.5. Biological evaluation of sweet whey fortified with 10% Moringa oleifera leaves powder on experimental rats.
6.5.1. Body weight gain of experimental rats treated with sweet whey fortified with Moringa oleifera leaves powder
The initial body weights of all rats groups after one and two months of treatment were lower significantly different as compared to control(-) group 189.4±9.02 and 181.25±3.77 respectively, while final body weight of all rats groups were not significant different except control (+) group was decrease in significant (P≤0.05) after one and two months of treatment 229.8±11.76 and 270.4±16.2 respectively. On the other hand, protected, sweet whey and sweet whey MOLP beverage (10g MOLP/ 100 ml sweet whey) occurred higher increased in body weight gain 33.00±3.68, 32.24±4.3 and 36.67±3.89 respectively, compared to inducted group (20.63±2.89) after one month of treatment. These results hypothesized that the applied treatments (sweet whey and SWMOLP beverage 10g MOLP/ 100 ml sweet whey) may improve appetite and enhance weight gain. Also, it could be noticed that the weight gain of inducted was significantly lower (43.91±13.1) then in the other groups after two months of treatments followed by sweet whey groups (46.71±5.90). While protected and SWMOLP (10g MOLP/ 100 ml sweet whey) beverage groups were recorded higher significant (P≤0.05) after two months of treatment (65.27±6.06 and 62.10±10.78) respectively, as compared to the other groups in the same period of treatment.
6.5.2. Organs weight / body weight % of experimental rats treated with sweet whey and SWMOLP beverage (10g MOLP/ 100 ml sweet whey) for one and two months.
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A significant increase can be observed in the weight of liver in all the treatment groups (protected, sweet whey and SWMOLP beverage) except for the control (+) group was 2.10±0.22 after one month of treatment compared to control (-) group. While after two months of treatment noticed a significant decrease (P≤0.05) in all groups especially control (+) group was 2.03±0.42 compared with control (-) group 2.39±0.22. This results agree with results of Table (13) which have presented higher significant of liver enzyme (ALT and AST) in control (+) group and histopathology examination which illustrate liver damage.
A significant increase (P≤0.05) was noticed in kidney weight/body weight % after one month of treatment in protected, sweet whey and SWMOLP beverage groups 0.63±0.04, 0.65±0.04 and 0.67±0.06 respectively, as compared with control(-) group (0.57±0.05). On the other hand, after two months of treatment no significant change of kidney weight although control (+) group recorded less weight compared with control(-) group, while the other groups recorded high weight.
Spleen and brain were pale, and enlargement of this organ were also evident especially after one month of treatment except protected group in spleen was 0.36±0.09 and sweet whey group in brain (0.61±0.10) were less than control(-) group. After two months of treatment protected and SWMOLP beverage recorded the best results of spleen (0.28±0.01 and 0.36±0.04) and brain weight (0.48±0.02 and 0.46±0.09) respectively
6.5.3. Biochemical analysis of experimental rats treated with sweet whey and SWMOLP beverage (10g MOLP/ 100 ml sweet whey)
6.5.3. 1. Liver enzyme
The results of rats administered with diethylnitrosamine (control (+) group) showed a significant increase (P≤0.05) in the activities of the alanine aminotransferase (ALT) after one and two months of treatment (13.80±1.48 and 17.00±1.00) respectively as compared with control(-) group (7.40 ±1.52 and 8.00±1.83). The ALT values of the control (+) group were higher than
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those of other groups after one and two months of treatment. The increased value of the ALT at the end of experiment (after two months) was over two-fold when compared to the control(-) group. To clarify the direct effect of SWMOLP beverage on serum ALT. The rats were treated by SWMOLP beverage showed not significantly different after one and two months of treatment (7.20±1.30 and 7.80±24.67) respectively, as compared with control (-) group followed by protected then sweet whey groups.
Results of AST enzyme showed increase values of the control (+) group being near two fold after one and two months of treatment (29.40±2.97 and 28.60±2.70) respectively, when compared with the control(-) groups. Also protected and sweet whey groups showed an increase of AST after one and two months of treatment. The AST values of the SWMOLP beverage group did not show significant differences compared with the control (-) group (P≤0.05). The treatment of SWMOLP beverage significantly decreased the activities of AST at during the study period compared to the control (+) group (P≤0.05).
6.5.3. 2. Kidney functions
Data revealed that, serum urea level were not significantly changed in SWMOLP beverage group throughout the total experimental period after one month of treatment (42.30±1.82); as compared with the control (-) group (41.50±2.92), while after two months of treatment record significant decrease (40.90 ±1.75) as compared with control (-) group (44.88±2.29), while protected group came in the second level of values of urea. On the other hand, control (+) group recorded high significant increase in urea after one and two months of treatment (51.00±1.50 and 50.30± 5.27) as compared to the control (-) group and follow sweet whey group.
Creatinine, uric acid and urea reflect renal functions. It was noticed that increase in the mean values of urea in control (+) group then sweet whey compared to the control (-) group. The same trend was observed in serum creatinine. Treating rats with SWMOLP beverage showed that significant
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decrease in serum uric acid and creatinine after one month (1.86±0.11 and 0.72±0.10) respectively, as compared to the control (-) group. The same finding was noticed after two months of treatment of the same group. While control (+) group recorded high significant increase in uric acid and creatinine more than other groups.
6.5.3. 3. Immunoglobulin level
A significant (p≤0.05) reduction was recorded in the serum level of immunoglobulin G (IgG) for control (+) group after one and two months of treated (756.20±46.45 and 779.0±55.99) respectively, as compared with the control (-) group, followed by sweet whey and protected groups after one month of treated (851.0±86.1 and 847.67±73.08) respectively. There was a slight decrease in the serum level of immunoglobulin G (IgG), for the SWMOLP beverage, though not statistically significant as compared with control group after one and two months of treated.
A slight increase in the serum level of immunoglobulin IgM for the SWMOLP beverage group after one month of treated (7.77±0.76), but not statistically significant as compared with control (-) group (7.35±1.00). While a slight significant decrease in the same group in the serum level of immunoglobulin M (IgM) after two months of treated (7.23±1.31) as compared with control (-) group (7.82±1.19). On the other hand, control (+) group recorded high significant decrease of immunoglobulin M (IgM) as compared with control (-) group after one and two months of treated, followed by protected group was (5.69±0.39, 5.90±0.51) and sweet whey group was (5.88±0.41, 5.97 ± 0.62) respectively. So, the best result of immunoglobulin (IgM) was SWMOLP beverage group.
6.5.5. Histopathological examination
6.5.5.1. Immunopathological reaction in Hepatic tissue (Caspases 3)
Results showed severity of Immunopathorogical reaction in hepatic tissue of different experimental rats groups within the period of experiment. Caspas-3 of control group showed negative reaction. While control (+) group
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showed sever reaction (+++) after the second injection by DEN+CCL4 (two months of the start point of experiment) as compared with control (-) group), meanwhile after three months of the start point of experiment (one month of treatment) showed moderate reaction (++) and mild reaction (+) for sweet whey group as compared with others groups. All tested groups showed nil reaction (-) excepted control (+) group after four months of the start point of experiment (two months of treatment).
6.5.5.2 Histopathological findings of different Organs
6.5.5.2.1. Liver
6.5.5.2.1.a Control (-) and control (+) groups after two month of start point of the experiment:
The liver sections from control (-) group showed the normal histological structure of the central vein and surrounding hepatocytes in the parenchyma.
Fibroblastic cells proliferations with inflammatory cells infiltration were dividing the fatty change hepatocytes in the parenchyma into nodules in control (+) group.
6.5.5.2.1.b All experimental groups of rats on liver after one month of treatment:
In control (+) group’s focal area in the hepatic parenchyma showed dysplasia in the degenerated hepatocytes with diffuse kupffer cells proliferation after one month of treatment, Also the hepatocytes showed mitotic activity, karyomegaly and doublication in their nuclei.
The portal area showed oedema with few inflammatory cells infiltration associated with congestion in both portal and central veins in protected group.
Treated by sweet whey showed focal area in the parenchyma and showed karyomegally associated with fibrosis which was divide the parenchyma into nodules in sweet whey group. While, treated with SWMOLP beverage showed congestion in the central and portal veins
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associated with multiple numbers of newly formed bile ductless at the portal area.
6.5.5.2.1.c. All experimental rats of groups on liver after two month of treatment:
Sections of liver of control (+) rats group showed congestion in the central and portal veins associated with periductal inflammatory cells infiltration, as well as bile duct cystic dilatation in the portal area. Meanwhile mild congestion was observed in the portal veins in protected group.
On the other hand, there was focal area in the parenchyma had karyocytomegalic hepatocytes. While the portal area showed oedema with few inflammatory cells infiltration in sweet whey group. In SWMOLP beverage the portal area showed oedema with few inflammatory cells infiltration and hyperplasia in bile.
6.5.5.2.2. Kidney
6.5.5.2.2.a. Control (-) and Control (+) groups kidney after two month of the start point of the experiment:
The control (-) rats group showed that there were no histopathological alteration and the normal histological structure of the glomeruli and tubules at the cortex. While, control (+) group showed Sever congestion of the blood vessels with degeneration in the tubular lining epithelium were detected in the cortex. The corticomedullary portion showed focal haemorrhages between the degenerated tubules.
6.5.5.2.2.b. All experimental groups of rats on kidney after one month of treatment:
Results in control (+) group showed swelling and vacuolization in the lining endothelium of the glomerular tufts with tubular degeneration. Focal haemorrhage was detected in the corticomedullary portion between the degenerated tubules.
In protected group the cortex showed congestion in the blood vessels, while the corticomedullary junction had focal haemorrhage. Treated by sweet
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whey showed that, the cortical blood vessels were congested. While, there was no histopathological alteration as recorded in SWMOLP beverage group.
6.5.5.2.2.c. All experimental groups of rats on kidney after two months of treatment:
The glomeruli of control (+) group showed that vacuolization and swelling in the lining endothelium in association with tubular degeneration at the cortex. Focal haemorrhages were detected in the corticomnedullary portion. Protected group showed tubular degeneration and associated with tubular cystic dilatation in the corticomedullary junction.
Degeneration was detected in the tubular lining epithelium at the cortex of sweet whey group. Also, WMOLP beverage group showed that there was degeneration in the tubular epithelium and proliferation of the glomerular lining endothelium.
6.5.4.3. Spleen
6.5.4.3.a.Control (-) and Control (+) groups spleen after two months of the start point of the experiment:
Histopathological examination of the spleen sections of control (-) group showed that normal alteration and histological structure of the white and red pulps. While, there was lymphoid hyperplasia in the white pulps for control (+) group.
6.5.4.3.b. All experimental groups of rats on spleen after one month of treatment:
Congestion was observed in the red pulps of control (+) group. On the other hand, there was congestion in the red pulps while the white one had lymphoid depletion in protected group. Meanwhile, sweet whey group congestion was observed in the red pulps associated with lymphoid depletion in the white one. SWMOLP beverage group showed congestion in the red pulps, while the white one showed depletion in the lymphoid cells.
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6.5.4.3.c. All experimental groups of rats on spleen with after two month of treatment:
The white pulps showed lymphoid depletion associated with congestion and hemosiderosis in the red one for control (+) group. While hemosiderosis was detected in the red pulps for protected group. The red pulps showed congestion and hemosiderosis in sweet whey group. While, red pulps showed hemosiderosis in SWMOLP beverage group.
6.5.4.4. Brain:
6.5.4.4.a. Control (-) and Control (+) groups brain after two month of start point of the experiment
Control (-) group showed no histopathological alteration and the normal histological structure of the meninges, cerebral cortex, hippocampus, striatum, cerebellum and medulla oblongata. On contrary, neuronal degeneration was detected in the cerebral cortex, while the hippocampus showed nuclear pyknosis and degeneration in the neurons of the fascia dentate with atrophy in the subiculum. Also there was congestion in the blood vessels of striatum in control (+) group which injected twice by diethylnitrosamine (DEN) plus CCL4.
6.5.4.4.b. All experimental groups of rats on brain after one month of treatment:
There were neuronal degeneration and atrophy in the hippocampus at the area of subiculum of control (+) group as well as in the fascia dentate. Focal haemorrhage was noticed in striatum in the same group. While, congestion of protected group was detected in the blood vessels in striatum. On the other hand, Focal multiple eosinophilic plagues formation was detected in the striatum of sweet whey group. Meanwhile, SWMOLP beverage group showed nuclear pyknosis and degeneration in some neurons of the fascia dentate in hippocampus.
6.5.4.4.c. All experimental groups of rats after two month of treatment:
Control (+) group congestion was detected in the cerebral cortical bloodvessels with neuronal degeneration and nuclear pyknosis in the
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hippocampus. Also, the striatum showed congestion in the blood vessels, as well as eosinophilic plagues formation. Focal haemorrhage was noticed in the cerebellum. The brain focal haemorrhage in striatum of the protected group animal’s models. Nuclear pyknosis and degeneration in some neurons of the hippocampus were noticed in sweet whey group. Also, in SWMOLP beverage group the nuclear pyknosis was detected in some neurons of the fascia dentate of the hippocampus, associated with congestion in the blood vessels, as well as, focal haemorrhage