الفهرس 
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Abstract
SUMMARY AND CONCLUSION
This study was designed to produce and evaluate essential oil extracted from both of caraway fruitss and the biomass of caraway plantlets grown using one of the of the biotechnology methods (tissue culture) and using them as safe natural food additives in some food products. In-vitro the effect of five suitable media (MS, B5, MSB5, LS, and NN) and three nitrogen sources (tryptophan, glutamine and casein hydrolysate) at different concentrations on development and production of caraway biomass using plant tissue culture was studied. Also, enhancing the accumulation of the essential oil in the caraway plants biomass was investigated. The chemical composition and biological properties (i.e. antioxidant, antimicrobial and anticancer activities) of essential oil extracted from both caraway fruitss and caraway plants biomass were determined. The potential application of caraway essential oil on the quality and microbiological properties of some food products (i.e. cake and chicken breast meat) was also investigated.
5.1. Factors affecting on in-vitro micro-propagation of caraway:
5.1.1. Types of media:
- Results indicate that there are no significant differences between the use of any of the following media MS, B5, MSB5, and LS on micro-propagation of caraway, with the exception of the use of NN medium. On the other hand, there were significant differences between the use of all media on the number of shoots produced by each explant, with the exception of the use of B5 and LS. These results clearly indicate that the best medium for the growth and propagation of caraway plants is MSB5 medium.
5.1.2. Nitrogen sources:
- Results showed that a high concentration of glutamine (20, 30, or 40 mg/l) had a detrimental impact on the number of shoots produced by each explant. The best glutamine concentration was 10 mg/l, which led to the best increase in the number of shoots per explant (3.90 ± 0.10).
- As in the case of glutamine, the introduction of tryptophan did not result in an increase in the shoot response percentage at any of the tested concentrations. The medium that contained 20 mg/l tryptophan had the highest observed shoot response (100.00 ± 0.00%).
- No significant difference was observed in shoot response (%) as a result of using different concentrations of casein hydrolysate. While there were significant differences between the uses of each concentration in terms of the effect on the number of shoots produced by each explant, the best concentrations were 100 and 200 mg/L (4.60± 0.15, 4.63± 0.09 respectively).In conclusion casein hydrolysate at 200 mg/l is considered to be the best source of nitrogen from the sources under study when supplemented with the culture medium.
5.1.3. Elicitation treatment:
- The influence of varying quantities of methyl jasmonate (MJ) and salicylic acid (SA) alone or their combination on the quantitative and qualitative characteristics of the essential oil of caraway was investigated under in vitro circumstances.
- Supplement of basal tissue culture media with either SA or MJ at 50, 100 and 150 led to a decrease in biomass to 39.12, 36.63 and 44.64 % for SA respectively and to 20.88, 17.60 and 14.45 % for MJ respectively compared to control medium after 21 days of incubation.
- The same trend was observed when the cultivation medium was supplemented with different concentrations (µM) mixtures of SA25+MJ25, SA50 + MJ25, SA25 + MJ50,SA50 + MJ50 .
- The control treatment (without SA and MJ) produced the best results in terms of multiplication rate (4.42), but the lowest percentage of EO (0.7%). However, supplementation of basal medium with 50 µM SA, achieved the highest EO yield (1.63%) and multiplication rate (2.21).
- In the case of MJ the highest multiplication rate and percentage of essential oil yield was found at 100 µM (3.57, 1.81%, respectively).
- On the other hand, the highest multiplication rate (2.42) was observed when basal media was supplemented with elicitors mixtures of SA25 + MJ25. But, the best concentrations of elicitors mixtures in terms of %EO yield were SA25 + MJ50 which yielded the highest value (1.48%).
5.1.4. Extraction of caraway essential oil:
- The caraway EO yield through hydro-distillation (control) of fruitss (CS) was found to be (1.90%) on the dry weight. The essential oil yield in the elicited caraway plantlets produced by the tissue culture technique (ECPT) was almost close to the control (1.81%), while the non-elicited caraway plantlets produced by tissue culture technique (CPT) came in last place (0.7%).
5.2. Evaluation of caraway essential oil:
5.2.1. Chemical composition (GC-MS):
The chemical composition of caraway essential oil extracted from different explant types (CS, CPT and ECPT) were identified by GC-MS analysis.
- A 25, 23 and 23 components were identified from caraway oil for CS, CPT and ECPT, respectively.
- Constituents are divided into ten chemical groups in CS,CPT and ECPT; monocyclic terpenes (39.89,37.27 and 40.42%), bicyclic terpenes (0.7,2.51 and 0.85 %), aliphatic hydrocarbons (0.09,2.48 and 0.09%), aromatic hydrocarbons (0.10,1.36 and 0.09%), oxides (0.06,0.00 and 0.06 %)respectively, alcohols (0.57, 4.7 and 0.65%), esters (0.09,0.07 and 0.04%)respectively, aldehydes (0.09,0.09 and 0.07%), ketones (56.71,37.56 and 56.47%) and sesquiterpene (0.42,11.31 and 0.42%) respectively.
- Carvone and limonene were identified as the main compounds in caraway EO
- When comparing caraway essential oils extracted from different explant types, it is noted that the oils extracted from the plantlet are lower in carvone and limonene content (36.34, 35.76%, respectively), compared to the control essential oil extracted from the fruits of caraway (56.52, 39.49%, respectively).
5.2.2. In-vitro antimicrobial activity:
5.2.2.1. Inhibition zones using agar well diffusion method:
- No significant differences (P≥ 0.05) were observed in inhibition zone between the essential oils extracted from caraway fruits and those produced by tissue culture technique against all the tested strains.
- The CEO generated larger zone of inhibition against the Gram-positive bacteria strains (25.23 and 25.40 mm for S.aureus, and L. monocytogenes, respectively) compared to the Gram-negative strains (24.66 and 23.60 mm for E. coli. and Salmonella sp. respectively)
- It has been demonstrated that the Gram-negative bacteria are generally less sensitive than Gram- positive bacteria.
- The inhibition zone of CEO reached 21.63 and 22.80 mm for A.niger and Candida sp.
- The antimicrobial activity of CEO exhibited a stronger growth inhibiting effect against fungi than bacteria strains used in this study.
- According to these results the caraway essential oils showed effective inhibitory activity against all tested strains.
5.2.2.2. MIC and MBC or MFC sing the broth dilution method:
- No differences were observed for MIC and MBC of the two essential oils (C and T.C) against all tested bacterial strains.
- The antimicrobial activity of CEO exhibited a stronger growth inhibiting effect against Gram- positive bacteria (with exception B.cerus) than Gram- negative bacteria. The bactericidal activity of CEO against Gram- positive bacteria (S.aureus, and L. monocytogenes) was revealed at the concentration of 1.50 and 1.00 μl/ml compared to 2.00 μl/ml for both Gram-negative bacteria (E. coli. and Salmonella sp.)
- The MIC of the moulds strains was identical for the two oils under test against Candida sp (1.50μl/ml), while for A.niger, it was 2.00,1.50μl/ml for the C and T.C, respectively. While the MFC values for the both fungal strains were 2.50 μl/ml for (C) essential oil, and 2.00 and 2.50 μl/ml, respectively for (T.C) essential oil.
5.2.3. In-vitro antioxidant activity:
- In all antioxidant assays, caraway essential oils showed significant antioxidant activities equal or greater than BHA.
- Results showed that no significant (P ≤ 0.05) differences were observed between the DPPH values for both types of caraway EOs (C and T.C), however, they were marginally greater than those of the synthetic antioxidant BHA.
- The ABTS results followed a similar pattern of DPPH, although with better efficiency compared to the BHA.
- In the reducing power assay, caraway EOs proved that it can convert ferric ions (Fe3+) to ferrous ions (Fe2+), better than synthetic BHA.
5.2.4. In-vitro cytotoxicity:
- The cytotoxic activity was evaluated using the MTT (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide) assay. The proliferation rate of the liver cell line (HepG-2), Caucasian breast adenocarcinoma (MCF7) and Colon cell line (HCT116) were compared with normal kidney cells (VERO) by studying the IC50 value of Eos recovered from caraway fruits and its effect on cell viability.
- The colon cell line (HCT116) was observed to be more sensitive to treatment by caraway essential oil followed by liver cells, where it was IC50=390.12 and 589.75 µg/ml, respectively, while breast cells MCF7 appeared more resistant to treatment by caraway essential oil (IC50=843.52µg/ml) it was higher than the control cells (IC50=785.93 µg/ml), compared to untreated cells from each of the above cell types. These results show the potential of caraway EO in protective against cancer treatment.
5.3. Application of caraway essential oil in some food products:
5.3.1. Cake:
The effect of adding different concentrations of CEO as a natural antioxidants and antifungal sources on the quality of the cake was followed during storage.
5.3.1.1. Chemical composition of cake samples:
- No significant differences (P≥0.05%) were observed in chemical composition (i.e. moisture, protein, fat, ash, and total carbohydrate) between control cake samples and those contained different concentrations of CEO.
5.3.1.2. Sensory evaluation of cake samples:
- The cakes prepared from batters containing 0.200 and 0.250% CEO recorded significantly (P≤ 0.05%) the highest scores in terms of overall acceptability, while cakes made from batters containing 0.010 and 0.050% CEO received lower overall acceptability score values and were significantly equal to the control negative (C-) cake.
- The use of CEO in cake batter at concentrations of 0.100, 0.150, 0.200, and 0.250% (w/w of dough weight basis) did not affect or delay any of the quality characteristics of baked cakes.
- However, cake produced with 0.200% CEO obtained the highest score values and was the most accepted one, followed by the cake prepared with 0.250% CEO.
5.3.1.3. Effect of adding CEO on oil quality of cakes during storage:
- The influence of adding different concentrations of CEO on the acid value (AV), peroxide value (PV) and thiobarabitoric acid (TBA) of oil extracted from cake samples were determined during 30 days of storage at room temperature. The increment rate in the values were decreased with increasing of CEO concentrations from 0.010 to 0.250%.
- The increases were significantly higher in control negative cake sample compared to other samples in which synthetic antioxidants (BHA) or natural antioxidants (CEO) were incorporated.
- Also, it is observed that AV of oil samples containing CEO at 0.200 and 0.250% or BHA at 0.02% had the least AV in comparison with other samples or control.
- Addition of CEO at 0.200 and 0.250% could inhibit the rate of peroxide formation of the oil extracted from cake samples and their effect was almost less than BHA.
- Also, it can be observed that CEO at 0.150, 0.200 and 0.250% could inhibit the rate of formation of malonaldhyde in cake samples and their influence was almost equal or higher than0.02% BHA at the end of storage.
- It suggests that CEO has an effective antioxidant activity that is almost identical to synthetic antioxidants at these concentrations.
5.3.1.4. Antifungal activity of caraway essential oil in cake samples:
- Mould counts of cakes produced (with CEO) as compared to controls during a storage period of 30 days are displayed following:
- At zero time the moulds are not detected in all cake samples and after 5 days moulds started to be detected in the negative control cake and cakes containing low concentrations of CEO (0.010, 0.025 and 0.05%). In contrast, it did not appear in the positive control sample and those containing higher concentrations of essential oil.
- After 20 days moulds started to be visible to the naked eye on surfaces of negative control and cake samples containing low concentrations of CEO (0.010,0.025 and 0.05%).
- After 30 days moulds were visible to the naked eye on surfaces of all samples except positive control and cake samples containing concentrations of CEO (0.200 and 0.250%). Therefore, CEO had effective antifungal action in the cake samples, when applying the essential oil at high concentrations.
5.3.2. Chicken breast meat:
Poultry meat is a very popular food commodity due to its low production costs, low content of fat, high nutritional value and distinct flavor. Therefore, the aim of the present part was to monitor the microbiological quality of chicken breast after treatment by caraway essential oil during storage at 4 °C/21 days.
5.3.2.1. Proximate composition of the raw chicken breasts:
- The moisture, protein, fat, ash and total carbohydrate content were as follows: 72.73 ± 0.04, 22.46 ± 0.06, 1.84 ± 0.04, 1.12 ± 0.02 and 1.85 ± 0.00 % respectively.
5.3.2.2. Monitor the microbiological quality of chicken breast:
- The antimicrobial efficacy of different concentrations of caraway essential oil (0.00, 0.25, 0.5, and 0.75%) on the microbiological characteristics of chicken breast meat was evaluated during 21 days of cold storage at 4± 1 ºC.
- It could be noticed that with increasing the concentration of caraway essential oil increased the counts of the total microbial count, Salmonella sp., Lactic acid bacteria and Escherichia coli were significantly reduced.
- After 4 and 7 days of storage at 4± 1 ºC a significant decrease (P≤0.05%) was observed in the microbial count of chicken breast meat containing different concentrations of caraway essential oil in comparison with the control. The same trend was observed for Lactic acid bacteria and Escherichia coli. However, Salmonella spp. was not detected in any chicken breast meat samples containing caraway essential oil at 0.50 or 0.75 % after 7 days of storage at 4± 1 ºC while it was detected in one of five tested samples (20%) at the lowest concentration (0.25% ).
- After 21 days of storage at 4ºC a significant increase (P≤0.05%) was noticed in counts of the total microbial count, Lactic acid bacteria and Escherichia coli in samples containing different concentrations of caraway essential oil but at rates less than the control sample, while Salmonella sp. were detected in one of five samples containing essential oil at 0.5 and 0.75% (20% positive).
- In conclusion dipping chicken breast meat at 0.5 and 0.75% CEO has antimicrobial activity against a range of microorganisms particularly pathogenic bacteria during the storage.
Conclusion:
Due to climate change and an increasing world population means traditional farming methods may not be able to meet the anticipated growth in food demands. Therefore, alternative agricultural strategies should be considered. The present study confirmed that tissue cultures may present a possible solution, as they allow for controlled, closed, and sustainable manufacturing of healthy food ingredients today. Caraway (Carum carvi L.) is one of the most important medicinal plants and its essential oil is composed mainly of cyclic monoterpenes, i.e. carvone and limonene, which make up over 95% of the total essential oil. This study showed that this essential oil could be used as a natural antibacterial, antioxidant, and antifungal in foodstuffs, especially those containing lipids, in addition to their anticancer activity. The antioxidant, antibacterial, and anticancer effects of caraway essential oil along with its reputation as spice help the food industries to apply it as natural preservatives and antioxidant agents instead of synthetic ones.