الفهرس 
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Abstract
Natural pigments are highly unstable, very susceptible to degradation and also affected by several factors such as pH, storage temperature, light, oxygen, and the presence of enzymes, etc.
The main objectives of the present study were to extract anthocyanins pigment from hibiscus and black mulberry, evaluate their antioxidant activity and stability and utilize of nanotechnology technique to prepare natural pigment nanocomposites which can be used as safe and stable food colorants.
The obtained results can be summarized as follows:
1- Proximate composition and physicochemical analysis of hibiscus and black mulberry
Dried flowers of hibiscus contained 11.05 % moisture, 5.18 % protein, 1.70 % fat, 9.22 % ash, 14.67 % fiber and 72.85 % total carbohydrates .However, fresh black mulberry contained 81.86 % moisture, 2.08 % protein, 0.242% fat, 0.997% ash, and 1.07% fiber and
14.82% total carbohydrates.
The ascorbic acid, total phenolic and total anthocyanins contents of hibiscus were 46.47mg ascorbic acid, 128.60 mg gallic acid and 572.54 mg cyandin-g-3-glucoside / 100g sample of hibiscus respectively, while there were 14.8 mg ascorbic acid, 82.35 mg gallic acid and 453.30 mg cyandin g-3-glucoside / 100g sample of black mulberry. Results showed that hibiscus have a higher ascorbic acid, total phenolic content and total anthocyanins than black mulberry.
2- Effect of extraction media on anthocyanin contents and antioxidant activities of hibiscus and black mulberry extracts.
Different extraction media such as, ethanol (0 - 80 %), acidified water (1% HCl) with ethanol (0 - 80 %), acetic acid solution (1 - 2 %) and acidified (0.5 - 2 % lactic acid) 80 % ethanol were used to extract anthocyanins from hibiscus and black mulberry. The antioxidant capacity of different extracts were also evaluated using DPPH, ABTS and FRAP
assays. Among different tested media, acidified (2 %lactic) in 80 % ethanol, acidified (1%HCl) 50 % ethanol and 2 % acetic acid solution significantly exhibited higher anthocyanin content from hibiscus (being
725.91, 685.78 and 634.90 mg/100g, respectively).While, the maximum content of anthocyanins extracted from mulberry was significantly obtained by using 1 % HCl acidified water (1282.18 mg / 100 g). With regard to antioxidant activity, both the acidified (2 % lactic acid) 80 % ethanol and acidified (1% HCl) 50 % ethanol of hibiscus extracts showed strong antioxidant activity. On other hand, the strongest antioxidant capacity was obtained in 1 % HCl acidified water extract of mulberry (≈
71.0 mg ascorbic acid / g sample) which had the maximum amount of anthocyanins (1282.18 mg/100g).
3- Color characteristics of anthocyanins extracts from hibiscus and black mulberry
Color characteristics (L, a, b, Hue and chroma) of hibiscus and black mulberry extracts which exhibited the highest anthocyanin content were measured using a Hunter colorimeter.
The highest redness (a-value) and the lowest yellowness (b-value) are achieved by using 2 % acetic acid solution as a solvent (2.04 and 0.55 respectively), while the other hibiscus extracts showed lower a-value (1.21- 1.84) and higher b-value (0.55 to 0.74) for hibiscus extracts. On other side, acetic acid of hibiscus extracts showed significantly the lowest Hue-angle value (15.08) ° and the highest color saturation (chroma) of
2.11 compared with the other extracts. While, acidified 1 % HCl water of black mulberry extract showed low Hue-angle value (17.55) ° and the highest color saturation (chroma) of 4.94 compared with the other extracts, which indicate that acidified 1 % HCl water contains more components of red fractions of anthocyanin
4- Correlation between anthocyanin contents and antioxidant activity
One of the important aims of this study was to determine the correlation between antioxidant activity and anthocyanin content in hibiscus and black mulberry.
Strong correlation was observed between the anthocyanin levels and antioxidant activity (R2 = 0.980, 0984 and 0.981 measured by DPPH, ABTS and FRAP assays, respectively) of the hibiscus extracts. Additionally, the anthocyanin content was correlated to a good degree with antioxidant capacity values (R2 = 0.967 for both DPPH and ABTS and 0.963 for FRAP assay) of mulberry extract sample.
5- Synthesis of chitosan nanoparticles (CS-NP) and anthocyanin nanocomposites (AH-NC and AM-NC)
The aim of this part was to synthesis and characterization of
chitosan nanoparticles (CS-NP) and evaluation of their loading capacity with natural anthocyanins from hibiscus and black mulberry to produce anthocyanin nanocomposites (AH-NC and AM-NC).
The successful loading of anthocyanins into chitosan nanoparticles
(CS-NP) was confirmed by UV–Vis spectrophotometry. The solution of AH-NC and AM-NC gives a maximum absorption peak at 521 and 516 nm, respectively. A higher loading efficiency was observed when anthocyanin was loaded on chitosan nanoparticles, each gram of AH-NC and AM-NC contained 200 and 161 mg anthocyanin, respectively.
6- characterization of CS-NP, AH-NC and AM-NC
The CS-NP particles morphology was examined by Transmission Electron Microscopic (TEM) and it was found that chitosan particles have almost spherical shape with diameters ranged from 40 to 52 nm
For CS-NP, the determined particle size average by Zetasizer was
42 nm which agreed with TEM results. However, AH-NC and AM-NC showed greater particle size average than pure CS-NP. Both AM-NC and AH-NC showed higher zeta potential values than CS-NP. These results reflect the greater stability of AH-NC and AM-NC than CS-NP.
The results of FTIR suggested that the interaction between CS-NP and anthocyanin as AH and AM is physical in nature where no evidence for formation of new chemical bonds was appeared in the case of AM –NC and AH –NC. The results of XRD suggest that the presence of anthocyanin decreased the degree of crystallinity.
7- Antioxidant activity and color parameters of AH-NC and AM -NC
The antioxidant capacity of 2 % acetic acid extracts of AH and AM
was measured in comparison with their nanocomposites.
Results showed that AH-NC had significantly higher antioxidant activity values than that of AH. On contrary, AM –NC showed less AOA values than that of AM .
On the other hand, AH-NC showed significantly (p ≤ 0.05) high lightness (L*- value), redness (a- value), yellowness (b- value), hue angle and color saturation (Chroma) compared with AH. Also, AM –NC gave significantly high hue angle and chroma than AM.
8- Stability of AH, AM versus AH-NC and AM -NC
Kinetics and thermodynamics of degradation of AH, AM versus AH-NC and AM –NC were studied and the results showed that the half time values decreased significantly with the increase of temperature from
25 to 100 °C, which confirmed the sensitivity of the pigment to the thermal treatment. The half life values of the anthocyanin nanocomposite samples were greater than of anthocyanins extracts from hibiscus and black mulberry.
At different tested pH values AH-NC and AM-NC showed greater t1/2 values when compared to AH and AM. For example, at pH 2 the half life of AH-NC and AM-NC were 1.7 and 1.9 times less susceptible to degradation than AH and AM which indicate that both AH-NC and AM-NC achieved a greater stability at different values of pH than AH and AM
For all samples under investigation, the rate constant values at dark were lower than that at light for each sample which confirmed that the pigment is more stable when processed at dark where the light accelerated the degradation of anthocyanin pigment.
9- Color stability and sensory evaluation of gelatin gum and ice cream produced by AH, AM, AH-NC and AM –NC
The effect of incorporation of natural anthocyanins extracts from
hibiscus (AH) and black mulberry (AM) and their nanocomposities on
color stability and sensory characteristics of gelatin gum and ice cream during storage was studied
Storage of gelatin gum samples colored by pigment nanocomposite from hibiscus for 8 weeks at room temperature did not remarkably changed the color parameters (L, a, b, chroma and Hue – values ), while yellowness (b-values) and Hue values of the control gelatin gum samples were changed towards more orange color shade. However, no remarkable differences in color parameters (L, a, b, chroma, Hue-values) have been observed between the gelatin gum samples, prepared with AM or AM-NC.
Sensory evaluation of gelatin gum indicated that, the gelatin gum
prepared with AH-NC recorded high scores for all tested parameters than those of AH at zero time and during 8 weeks of storage. A significant decrease (p ≤ 0.05) in color, over all acceptability and total score values was observed for AM-gelatin gum after 8 weeks of storage On the contrary, non significant differences (p ≤ 0.05) were observed in all sensory parameters for AM-NC samples after 8 weeks of storage.
During storage, the ice cream samples prepared by anthocyanins
nanocomposites of hibiscus or mulberry (AH-NC and AM-NC) showed almost stable chroma and Hue values, while their control samples showed increasing change in chroma and increasing change towards orange and orange yellow Hue color shades.
Finally, ice cream prepared with hibiscus anthocyanins nanocomposites (AH-NC) was the most preferred by the panelists followed by those contained AM-NC. This result was in accordance with the results of high stability of AH-NC than AM-NC.
Results confirm the improvement in color parameters and sensory
evaluation of gelatin gum and ice cream samples induced by the use of pigment nanocomposites of hibiscus and mulberry as coloring agents. The improvement in color was based on better reflectance of surface light and equal distribution of color inside the matrix of two food items.
Conclusion
The present study confirmed that extraction of anthocyanins pigments from hibiscus and black mulberry and evaluation of health- promoting components in their extracts would increase the demand of these commodities by consumers and the food industry as added value natural pigments, and would open a new window of opportunities for use of these extracts in a variety of food applications. Also, loading of anthocyanins on chitosan nanoparticles (nanocomposites) may enhance the stability of anthocyanins for efficient utilization in some food products. In conclusion, nanotechnology technique was successfully implemented to load anthocyanins on chitosan nanoparticles which could potentially carry and stabilize natural anthocyanins pigment for use in food industries.