الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Plant-based milks are more affordable substitutes for cow’s milk, for people who cannot afford it due to its high price and restricted availability as well as for those who are allergic to cow’s milk. However, they do not offer the same nutritional benefits.In order to substitute plant-based milk with cow milk, it have to be fortified by bioactive material to reach high nutritional value like cow’s milk.
Soy milk one of the most important plant-based milk substitutes due to its 30% cholesterol-free oil content and 40% protein .In our study we fortified soy milk with calcium salts and vitamin E in order to obtain plant beverage with high nutritional value mimic the cow’s milk.The fortification took place in free form and in Nano-encapsulated form – a new technology - which musk the undesired flavors and keep the bioactive ingredients till they reach the stomach.
Materials and methods
Soybean seeds sample (100 g) were soaked, then coarsely grounded .The resulting slurry was filtered. Then, the raw filtered soybean milk preheated in a boiling water bath,then transferred to the electric stove for boiling.
Protein isolate extraction from soybean flour : One hundred g of soybean flour were weighted, adjusted to pH 8.5 using 2N NaOH , then centrifuged , take the supernatant was acidified by 2N HCL pH 4.5. The protein precipitated , refrigerated ,centrifuged, crude protein isolated neutralized then freeze dried.
Proximate analysis for ash, protein crude fat, crude fiber ,carbohydrates and moisture were determined in soy milk and flour .Phenolic content ,amino acid composition ,minerals content in soy milk ,flour and SPI ,fatty acid content of soy milk and flour .
Fortification of soybean milk by vitamin E or calcium salts (calcium chloride salt) or combined vitamin E and calcium salt in free and encapsulated form.
Calcium encapsulated by complexation, and Ca-nanoparticles were prepared by adding CaCl2 to the protein solutions at 10, 20, and 30 Mm concentrations/ 10 g SPI. In addition, Vitamin E dissolved in absolute ethyl alcohol (0.01%, w/v) and stirred for 30 min. Then, vitamin E added to encapsulate at 50, 100, and 150 mg/g SPI concentrations DROP by DROP to 100 Ml of the SPI solution at 25 ◦C. A pH of 7.5 achieved in the final suspension. We subjected the mixes to high-intensity ultrasound at an amplitude of 40% for 5 minutes in an ice bath to achieve homogeneity Nano capsule. Determination of encapsulation efficiency (EE) determined Particle size and ζ-potential and morphology of nanoparticles detected by transmission electron microscope (TEM). characterization of Nano capsules including encapsulation efficiency (EE %), poly disparity index (PDI), zeta-potential and Morphology of nanoparticles by transmission electron microscope (TEM) were detected.
Cytotoxicity of plain and encapsulated formulations of soybean milk on human cell line were detected.
Functional properties for soy bean milk formulations( free and nano-encapsulated), total phenolic, flavonoid and scavenging capacity of DPPH radical were determined.
Some physical properties were determined in plain soy milk and all formulations ( free and nano-encapsulated) : taste ,odor , color ,flavor and over all acceptability. Color in plain milk and all formulations were detected by Hunter device.
We can summarize the results as the following :
Chemical composition of soy milk and flour :
Significant differences in the amount of protein, fat, fiber, ash and carbohydrate between soy milk and soy flour based on a dry weight.The highest protein content was presented in soy flour (40.50 %).The highest percentage of fat was presented in soy flour (7.5%).The soy milk contains low percent of fiber compared to soy flour. The highest percentage of fiber was presented in soy flour( 3.75%), the highest percentage of ash was presented in soy flour (7.10%), the data of moisture content showed a significant difference between soy milk 88.5% and soy flour (9 %), The highest percentage of carbohydrate was reported in soy flour 20.88% followed by soy milk (0.03%).
Minerals in soybean milk, flour and SPI:
The level of S, Ca, K ,Fe, and P in soy milk, ,were 98,60,630,4.6,220 mg/L ,respectively which increased in soy flour to reach1675,2500,12500,100,3750 mg/Kg respectively, their concentration in SPI were 1750,1500,3750,65,2000 mg/Kg respectively. There were significant differences between soybean milk, flour and SPI in all the mentioned elements.
Amino acids content of soybean milk, flour and SPI:
The highest non-essential amino acid in SPI was glutamic acid (GLU) 137.02 mg/g which followed by aspartic acid (ASP) 68.69 mg/g, and proline 66.94 mg/g.Leucine and lysine have higher values of essential amino acids 42.45mg/g , 31.19 mg/g. All results recorded higher content of all essential and non- essential amino acids of SPI than soybean milk and flour.
Determination of fatty acids content of soybean milk, flour:
Soybean flour and soybean milk contain the most common fatty acids, In soybean milk and flour, there is more concentration of C18:2 linoleic Fatty acid. Followed by C18.1 Oleic fatty acid followed by Hexadecanoic acid C16.0 then Octadecanoic acid C18.0.
Determination of Phenolic content of soybean milk, flour and soy protein isolate:
We noticed that the highest concentration represented in Ferulic acid and found in soy flour (508.74 µg/g) and in SPI (491.78 µg/g). Cinnamic acid represented the lowest concentration and was 0.06 µg/ml in soybean milk, 2.66 µg/g in soy flour and 3.83 µg/g in SPI.
Particle size, Polydispersity index (PDI), zeta potential, and encapsulation efficiency (EE %) of SPI, SPI-E, and SPI-Ca:
The complexes containing entirely vitamin E exhibited encapsulation efficiency values of 76.24%, 75.25%, and 68.75% for 50, 100, and 150 mg concentrations, respectively. The study found that the complex embedded with CaCl2 demonstrated different amounts of EE for Ca when added with 10, 20, and 30 Mm, with values of 91.45%, 89.59%, and 52.35%, respectively. The phenomenon attributed to the ability of the protein and vitamin E or calcium complex to inhibit intermolecular interactions of proteins, thereby preventing the formation of aggregates. This improves the ability of proteins to serve as hydrophobic, physiologically active materials. The sample containing SPI with 50 mg of vitamin E exhibited a greater degree of encapsulation efficiency in comparison to the other two samples containing 100 and 100 mg of vitamin E. This trend observed with calcium 10Mm higher than 20 and 30Mm, which attributed to developing a more organized and condensed core-shell structure at a pH of 7.5. Sonic homogenization may alter the structure of SPI and enhance its interaction with VE or Ca.
Morphology of Nanoparticles
The nanoparticles that underwent treatment with calcium and vitamin E exhibited an effectively synthesized structure. The aggregation of protein molecules likely caused by calcium, which shields charged protein molecules repelling one another electrostatically
Viability assay in plain soybean milk and Nano-encapsulated formulations.
Cytotoxicity assessment on vero cell line of the fortified milk with the three nanoparticles preparations compared with the control plain milk, are exhibited in Table (4.7, 4.8), Figures (from 4.9 to 4.12). The main aim of cytotoxicity evaluation was to estimate the safe concentration for soybean milk fortification with these nanoparticles. Obtained results indicated that combination of calcium salts and vitamin E nanoparticles caused to decrease IC50 approximately to the half (202.0 ug/ml) compared with the individual Ca or vitamin E nanocapsules (439.25ug/ml, 400.63 ug/ml) respectively. Consequently, the applied fortification concentrations of the three nanocapsules preparations did not exceed the least IC50 value (202.0 ug/ml). These results are in agreement with El-Kholy et al., (161). These findings supported the use of vitamin E and calcium salt nanocapsules in food applications taking into consideration their safety
Determination of total phenolic content in soybean milk and all formulations
Our results showed that phenolic content in plain soy milk and all formulations (CSM,NEM,NCM,NECM,FEM,FCM,FECM) were 70.52, 70.83, 134.38, 149.49, 74.17, 83.13, 72.08 µg/ml respectively, fortified soy milk with vitamin E + calcium salt reported the highest concentration of phenolic compounds while plain soy milk showed the lowest concentration of phenolic compounds
The results recorded that soy milk capsulated with vitamin E +calcium salt and soy milk fortified with calcium salt Nano capsules had the best phenolic concentrations , while the plain soy milk and soy milk fortified with vitamin E Nano capsules had the lowest phenolic concentrations.
Determination of total flavonoid in soybean milk and all formulations
The results showed highest concentration of flavonoid compounds included in soy milk fortified with vitamin E Nano capsules (44.70g/ml) and soy milk fortified with free form calcium salt + vitamin E (44.70µg/ml) followed by soy milk fortified with free vitamin E (39.43g/ml).While flavonoid concentration in plain soy milk and other formulations (NCM,NECM,FCM) were 37.46, 18.12, 31.41, 35.88 µg/ml respectively
Determination of anti-oxidant activity (DPPH scavenging capacity)
The outcome demonstrated that different soybean milk formulations, including plain soybean milk, had various DPPH radical scavenging capacities .The DPPH scavenging capacity was the highest in soy milk fortified with nan encapsulated vitamin E (17.92%) and had the lowest IC50 (139.55).
While lowest DPPH scavenging capacity was reported in, soybean milk fortified with free form calcium salt (2.28%) with IC50 equal (1096.43).The results showed antioxidant activity in plain soy milk and other formulations (NCM, NECM, FEM, FECM) were 9.77, 5.86, 10.42, 11.40, 3.26 % respectively.
Color evaluation of control soybean milk and all the formulations:
FCM had the highest L*value followed by FECM and CSM 66.70, 66.63 and 65.81 respectively. FEM, NEM reported higher a* value than the other samples1.84, 1.75 respectively while NECM was the lowest 1.18.NECM recorded the highest b* value 5.36 followed by NCM 5.20 while NEM had the lowest value 2.95.
Hunter parameters of plain soybean milk and all the formulations indicate that plain soybean milk and all the formulations possessed yellowish green color.
Sensory evaluation of plain soybean milk and all formulations:
There were significant differences between control and all formulations of soybean milk in taste, texture and overall acceptability while data showed non- significant differences between control and all formulations of soybean milk in odor and color.
The color was higher in NCM than other formulations followed by CSM, while NECM and FECM had the same color .the odor reported high percentage in NEM followed by NCM .FCM was the lowest in odor evaluation. The test in CSM was the best followed by NCM and NEM, NECM, FEM and FCM, FECM had lower taste evaluation than other formulations. NCM and CSM followed by NEM reported higher texture evaluation while FCM and FECM had lower values .over all acceptability of control and encapsulated formulations of soy bean milk were higher than the over all acceptability of free fortified soy bean milk with vitamin E and calcium salt by the panelists .this indicates the importance of Nano-encapsulation in fortification of soy milk and recommends the fortification by encapsulation technique which is more acceptable by consumers with high quality sensorial aspects.
Taste evaluation in CSM, NEM, NCM, NECM, FEM, FCM, FECM were 6.90, 6.34, 6.60, 6.10, 6.04, 4.92, 5.88 respectively. While Color evaluation were 7.22, 7.02, 7.34, 7.00, 6.78, 6.86, 7.00 respectively, odor evaluation were 6.60, 6.64, 6.62, 6.48, 6.50, 6.20, 6.54 respectively, texture evaluation were 6.86, 6.78, 6.88, 6.70, 6.48, 6.00, 6.44 respectively and overall acceptability were 6.66, 6.56, 6.64, 6.54, 5.96, 5.60, 5.90.
5.2 Conclusion
As a result of analysing the nutritional value of soybean milk, flour, and soy protein isolate (SPI), it can be concluded that soybeans are a good source of protein due to the soybean flour’s high quality complete protein content especially threonine, leucine, lysine that provided 92.70, 90.81, 77.42 of AAS% requirement values respectively, taking into account that moisture content of soybean milk caused lower nutrients concentrations. Unsaturated fatty acids (USFs), particularly omega-6 and omega-9, account for 21.98 and 56.7%, respectively, of the total fat in flour. The primary phenolic component in soybean flour, milk, and SPI was ferulic acid. The synthesised calcium and vitamin E nanoparticles demonstrated their efficiency under TEM, stability, and safety up to the IC50 value (202 ug/mL) on vero cell line, which was taken into account in the application concentrations. Vit E and calcium salt fortification by nanocapsulation technique enhanced functional properties phenolic flavonoid content and antioxidant power also improved sensory attributes color, texture,odor,flavor and consumer overall acceptance. In light of their safety, research recommended the usage of calcium salt and vitamin E nanocapsules in food applications.
5.3 Recommendations
Based on the previous results its recommended to:
1- Substitute animal milk by plant-based beverages, for people who cannot afford it due to its high price and restricted availability as well as for those who are allergic to cow’s milk,which improves the puplic health, decrease the carbon foot print and help in obtaining sustainable environment.
2- Fortify the plant-based beverages with bioactive ingredients which raise its nutritional value to mimic the animal milk nutritional content.
3- Nanoencapsulation techniques , as new technology in fortification improve the physical propertise of the products and musking the undesired flavors .