الفهرس 
INTRODUCTION
AIM OF INVESTIGATIONOnly 14 pages are availabe for public view
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Abstract
This study investigated the ability of EO՚s as natural substances, in instead of chemical substances, to preserve fish and found an improvement in shelf-life and safety of product, in addition to the beneficial effect on consumer health. Four different groups of fish product treatments were investigated as follow:
Addition of EO՚s (coriander, cumin and parsley) under different levels 0.05%, 0.1% and 0.15% and a control sample (without any additions). All of these treatments were stored at - 18ºC for up to 6 months, and evaluated physically chemically, microbiologically and sensory at intervals periods of 0, 2, 4 and 6 months. The most important results obtained from this study are summarized as follows:
1- The chemical components of EO՚s (coriander, cumin and parsley) were fractionated and identified by GC-MS techniques:
A- The EO of coriander is particularly rich in linalool (79.94%), α-pinene (5.20%), camphor (4.14%), γ-terpinene (2.22%), geranyl acetate (1.59%), p-cymene (1.81%) and D-limonene (1.81%).
B- Analysis revealed that the major constituents of the EO from cumin were cuminaldehyde (23.13%), γ-terpinene (21.38%), 3-caren-lo-al (19.98%), 2-caren-lo-al (18.72%), sabinene (5.97%), neryl acetate (3.00%), O-cymene (2.98%) and phellandral (1.34%).
C- 20 essential compounds were identified in parsley EO. The major identified compounds were limonene (43.16%), α-pinene (15.34%), sabinene (12.71%), apiol (8.29%), myristcin (7.56%), β-myrcene (6.51%), p-cymene (2.37%) and 1,3,8-p-mentha triene (1.18%).
2- The antioxidant activity of EO՚s at different concentration by using DPPH radical scavenging was observed. It was noticed that coriander essential oil had the highest inhibition percentage of radical DPPH (78.64%). On the other hand, the inhibition percentage of BHT was (89.51%) under low concentration (10 µg).
3- All EO՚s showed antimicrobial activity against tested microorganisms:
A- Coriander EO was sensitive to Bacillus subtilis and Staphylococcus aureus. Meanwhile, the coriander EO had no inhibitory effect on Micrococcus roseus. Moreover, results revealed that moderate inhibition was observed against Candida albicans. Meanwhile, the coriander oil had highly inhibitory effect on Saccharomyces cervisiae. Furthermore, the coriander oil had no inhibitory effects on different types of fungi except Aspergillus niger which showed a weak inhibitory effect. The antimicrobial activity of coriander EO against bacteria was more effective than against fungi.
B- Cumin EO no inhibition zone was observed against all types of yeasts and fungi except Aspergillus niger. On the other hand, cumin EO inhibited Gram-positive bacteria to a higher degree than the Gram-negative bacteria in all bacterial strains except Micrococcus roseus which showed no inhibition zone with cumin oil.
C- Parsley EO showed antibacterial activities against Gram-positive bacteria but not against Micrococcus roseus. Additionally, the oil had antibacterial activities against Gram-negative bacteria tested but not against E.coli and Proteus vulgaris. Also, parsley EO was inactive against all tested strains of fungi except Aspergillus niger. Whereas, parsley EO had no inhibitory effects on Candida albicans it did showed moderate inhibitory effect on Saccharomyces cerevisiae.
4- According to the obtained results, of the EO՚s revealed that different cytotoxic activities toward the two human cancer cell lines. Also, the dead cells were increased by increasing the concentration of all plants՚ EO՚s. The highest MCF7 dead cell percentage was recorded by coriander EO as (10.37%) at a concentration of (0.00625%), while it was (58.44%) at (0.05%) concentration of coriander oil. Meanwhile, in cumin oil the MCF7 dead cell was (9.08%) at a concentration of (0.00625%), while it was (27.41%) at a (0.05%) concentration. The lowest MCF7 dead cell percentage was recorded by parsley EO at (4.12%) at a concentration of (0.00625%) and (25.99%) at a concentration of (0.05%). Meanwhile, the highest A-549 dead cells percentage was recorded by coriander EO at (10.65%) at a concentration of (0.00625), and for coriander EO it was (35.13%) at a (0.05%) concentration. Also, cumin oil recorded dead cell percentage at (9.40%) at a concentration of (0.00625%). Meanwhile it was (33.67%) for (0.05%). Furthermore, the lowest A-549 dead cell percentage was observed by parsley EO at (5.84%) and (29.27%) at concentration of (0.00625%) and (0.05%), respectively. Of all EO՚s investigated, coriander exhibited the strongest cytotoxicities toward cancer cells. Moreover, cumin and parsley EO՚s possessed less cytotoxic activities toward A549 and MCF7 cell lines.
5- The results indicated that different treatments of fish products had lower pH values than the control sample. Also, the addition of EO՚s at different levels led to slight decreases in pH values. Moreover, slight decreases in pH values were noticed in all treatments during storage at -18º C for 6 months, so the pH values in fish products containing EO՚s were ranged between (6.08 - 6.30) and (6.32 - 6.55) for fish patties and fish fingers at zero time. Meanwhile, at the end of the storage period the pH values ranged between (6.58 - 6.79) and (6.72 - 6.90) for fish patties and fish fingers, respectively.
6- Although water holding capacity (W.H.C) of all different treatments were convergent at zero time. Fish products containing EO՚s under level 0.15% showed an increase in (W.H.C). Also, the control sample displayed a slight decrease in (W.H.C) compared to other treatments. Meanwhile, (W.H.C) had a significant (p ˂ 0.05) decrease with increased storage periods.
7- The highest significant cooking loss was recorded for fish patties and fish fingers (36.95%) and (35.92%) (as a control) , when compared with other treatments. Also, it was noticed that the cooking loss significantly progressively increased as the period of frozen storage increased. Furthermore, the treatments containing EO՚s at a level 0.05% had significant higher cooking loss scores than those treatments containing EO՚s at level 0.1% and 0.15%. Also, the highest cooking loss was accounted for in the control sample, which at the end of storage period reached levels of (45.62%) and (45.24%) in fish patties and fish fingers, respectively.
8- Although, drip losses of all different fish product treatments were convergent at zero time, the control sample was recorded to have the highest significant percentage than the treatments. Also, it was found that treatments formulated with EO՚s at a level of 0.15% recorded the lowest significant percentages in drip loss when compared with treatments formulated with EO՚s at levels of 0.05% and 0.1%. The percentage of drip loss of all different fish product treatments progressively increased with a significant statistical difference by extending storage time.
9- Fish samples without any additions (control samples), which were used in preparation of different fish products contained; 78.02% moisture, 74.11% crude protein, 10.87% crude fat and 9.75% ash for fish patties. Meanwhile, fish fingers contained 74.49% moisture, 69.66% crude protein, 24.45% crude fat and 4.90% ash.
10- The addition of EO՚s to fish products caused a slight decrease in moisture content at zero time of storage. Moreover, moisture content of different fish products under investigation decreased during storage at -18ºC for 6 months compared with the control sample, which had a significant decrease (p ˂ 0.05) in moisture content.
11- The addition of EO՚s to fish products didn’t affect the protein content of different fish product treatments at zero time of storage. Moreover, protein content of different fish product treatments under investigation decreased during storage at -18ºC for 6 months compared with the control sample, which had a significant decrease (p ˂ 0.05) in protein content.
12- During storage at -18ºC for 6 months, the crude fat and ash contents recorded a slight increase in all fish product treatments under investigation. Crude fat of fish patties and fish fingers samples ranged between (10.87% and 11.93%) and (24.55% and 25.45%) (on dry-wet basis) at zero time of storage. Yet at the end of the storage period, crude fat increased to (15.06% and 16.87% for fish patty samples) and (25.80% and 26.87% for fish fingers samples). Meanwhile, ash content ranged between (9.81% and 10.55%) and (4.92% and 5.76%) at zero time of storage in all fish patties and fish finger samples. Ash content increased at the end of the storage period to (11.06% and 12.09% in fish patties) & (6.52% and 7.28% in fish fingers).
13- The total volatile bases nitrogen of untreated samples (control) sharply and significantly increased during frozen storage. Additionally, the addition of EO՚s to fish products reduced the increase in (T.V.B.N) content during frozen storage at -18 ºC for up to 6 months. Generally, the (T.V.B.N) for samples containing coriander EO under levels of 0.1% recorded the best treatments.
14- from the obtained results it is clearly noticed that there were continuous, significant sharp increases in the (T.M.A.N) amount in the control sample during frozen storage. Meanwhile, trimethylamine in EO-treated samples increased to a significant lower extent. Additionally, data showed that there was a significant difference increase (p ˂ 0.05) in (T.M.A.N) content of the control sample compared with samples treated with EO՚s during frozen storage at - 18ºC for up to 6 months. Generally, the (T.M.A.N) content significantly increased with the increasing of storage periods.
15- Results take the same trend for histamine contents as well as TVB-N and TMAN contents at the beginning and the end of storage periods (6 months) because histamine accrued by bacterial activity that the lowest histamine contents were noticed in samples containing coriander, followed by cumin and then parsley. Generally, histamine for samples containing coriander EO under a level of 0.1% recorded the best treatments. On the other hand, the lowest histamine content during and after storage periods (6 month) were noticed in samples containing EO at a level of 0.1%, followed by samples containing EO at level of 0.05%. Meanwhile, the production of histamine was higher in the samples treated with 0.15% of essential oils than in other treatments.
16- The results showed that there were significant differences (p ˂ 0.05) in (T.B.A) values between treated samples and the untreated sample (control sample) at zero time. Yet, there were no significant differences in (T.B.A) values among the treatments during storage periods. T.B.A values of the control sample showed continuous significant increases that reach a high of (2.239 mg malonaldehyde/kg sample) at the end of storage period. Although, the other treatments showed a slight significant incremental in (T.B.A) values during frozen storage at -18oC for 6 months. On the other hand, treatments containing EO at levels of 0.1% showed slight decreases in T.B.A values compared with control, treatments contained essential oil at level 0.05% and 0.15%. The samples formulated with coriander oil contained lower T.B.A values compared with samples containing cumin and parsley oil. Finally, it could be stated that the use of these EO՚s caused an extension of self-life of fish products during storage periods at -18ºC for 6 months.
17- The data revealed that saturated fatty acids of fish patties samples were C10:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0 C18:0, C20:0, C22:0andC24:0. The predominate, major monounsaturated fatty acids were C14:1, C15:1, C16:1, C17:1, C18:1, C20:1 and C24:1; and the major polyunsaturated fatty acids were C18:2, C18:2T, C20:2, C22:2, C18:3n6, C18:3n3, C20:3n6, C20:3n3, C22:3, C20:4, C22:4, C20:5, C22:5 and C22:6n3. The most abundant fatty acids found in control and other treatments were palmitic C16:0 and oleic acid C18:1. Also, considerable amounts of stearic acid C18:0, palmitolic acid C16:1 and linoleic acid C18:2 were also observed. It was also obvious that the sum of polyunsaturated fatty acids (PUFA) were considerably high. The dominant PUFA were the ω-6 series, which are found chiefly in C18:2 fatty acid. A moderate level of omega 3 fatty acids was observed in control and treated samples. Also, data showed a marked increase in the C18:1 content of all treated samples. The saturated fatty acids of fish fingers were C10:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0 C18:0, C20:0, C22:0 andC24:0. The predominated, major monounsaturated fatty acids were C14:1, C15:1, C16:1, C17:1, C18:1, C20:1 and C24:1; and the major polyunsaturated fatty acids were C18:2, C18:2T, C20:2, C22:2, C18:3n6, C18:3n3, C20:3n6, C20:3n3, C22:3, C20:4, C22:4, C20:5, C22:5 and C22:6n3. A dominant SFA in all the examined fish was palmitic acid (C16:0), The highest percentage of palmitic acid was assessed in fish fingers treated with cumin oil, while the lowest was in fish fingers treated with coriander oil. Among MUFA oleic acid C18:1 represented the highest relative percentage of all identified fatty acids. In addition, in fish fingers a share of palmitoleic acid (C16:1) were observed. from the results it could be observed that the frozen storage affected the fatty acids composition. Whereas some fatty acids were increased, some were decreased and some were absent in treated fish fingers. Generally, frozen storage at -18˚C for 6 months decreased unsaturated fatty acids in all samples. Results of experiments for fatty acids showed that the process of freezing and conserving fish for 6 months affected the amount of fatty acid in fish’s, thus reducing Omega-3 fatty acids and increasing Omega 6 fatty acids.
18- The addition of EO՚s reduced the total bacterial counts when compared with the control sample. Total bacterial counts for treated samples were lower than the control sample during storage periods. In general, it could be concluded that the addition of EO՚s (coriander, cumin and parsley) under a level of 0.1% caused decrement in total bacterial count in fish products compared with those that contained EO՚s at levels of 0.05% and 0.15% as well as the control sample. On the other hand, total aerobic count (T.C) gradually decreased during frozen storage at -18ºC in all treated samples but in different degrees.
19- The data indicated that fish product samples treated with EO՚s had lesser psychrotrophic bacterial count compared with the control sample. On the other hand, the decreasing rate of psychrotrophic bacterial count was higher in samples treated with EO՚s than the control sample during frozen storage. The addition of 0.15% EO՚s to fish products caused poor changes in psychrotrophic bacterial counts during storage compared with the control sample. Furthermore, the addition of EO՚s under a level of 0.05% caused a moderate reduction in psychrotophic bacterial counts compared with fish fingers containing the above mentioned essential oil at 0.15% and the untreated sample. In addition to that, these results showed that the addition of coriander oil at a level of 0.1% to the fish fingers sample was more effective on the psychrotophic bacterial counts than other treatments.
20- The obtained results observed that, there were no coliform group counts in all replicates of fish patties samples, neither before nor after frozen storage. Meanwhile, in fish fingers samples coliform group counts at zero time were higher in the control sample (5.5×104 log cfu/g) in comparison with other samples which ranged between (0.5×104 log cfu/g) and (3.5×104 log cfu/g) in fish fingers samples containing EO՚s. As storage periods progressed coliform group counts decreased to (1×103 log cfu/g) in control sample. Yet, their counts were reduced and disappeared in fish fingers samples containing parsley at a level of 0.1%, cumin at 0.1% and coriander at 0.05% and 0.1%, because of the inhibitory effects of these substances.
21- Staphylococcus aureus counts at zero time was higher in the control sample at (6×104 log cfu/g) and (9×104 log cfu/g) than fish patties and fish fingers in comparison with other samples, which ranged between (1.5 ×104 and 5 ×104 log cfu/g) in fish patties samples, while it was (3×104 and 7.5×104 log cfu/g) of fish fingers samples which contained EO՚s. As the storage period progressed, Staphylococcus aureus counts were decreased to (3×102 log cfu/g) and (1.5×103 log cfu/g) in the control group of fish patties and fish fingers. Yet, Staphylococcus aureus counts were disappear in other samples of fish patties. Also, Staphylococcus aureus counts were disappeared in tested samples except samples containing parsley under levels of 0.05% and 0.15% were (0.5×102 and 2×102) and cumin under level 0.15% was 1×102 log cfu/g.
22- Results showed that the yeast counts in the control sample were lower at zero time at 1.5×104 log cfu/g and disappeared at the end of the storage periods. Yet, the samples containing coriander, cumin and parsley under levels of 0.05%, 0.1% and 0.15% had the lowest number of yeast especially samples treated with coriander under levels of 0.1%. from these results it could be noticed that the gradual decrease in yeast counts might be attributed to the antimicrobial effect of these oils. Also, from the obtained data, it was observed that the control sample had higher yeast counts compared to the fish products treatments which were formulated with EO՚s under levels 0.05%, 0.1% and 0.15% at zero time and at the end of the storage periods. Coriander and cumin EO՚s under a level of 0.1% were more active at inhibiting yeast, which disappear after 2 month storage periods, compared with other EO՚s. Moreover, it could be noticed that the yeast disappear in most fish patties and fish fingers samples formulated with EO՚s under levels 0.05%, 0.1%and 0.15% after 6 months. These results indicated that the EO՚s at all concentrations inhibited the growth of yeast counts.
23- The data recorded that, at zero time of storage the mould counts in fish patties formulated with coriander, cumin and parsley at levels of 0.05%, 0.1% and 0.15% had counts of (1×103, 6×102 and 1x104); (2×103; 1×103 and 1.5x104); and (0.5x104, 1.5×103 and 1.5x104) log cfu/g, respectively. Mould counts in these samples were lower than the control sample (3x104 log cfu/g). Mould counts gradually decreased during storage periods in all treated samples but with variable degrees. Likewise, after storage for 6 months at -18˚C moulds disappeared in all treated samples of fish patties formula except the control sample, which had 2×102 log cfu/g. Also, the obtained results showed that mould counts increased in the control sample, its counts at zero time was 6 x104 log cfu/g and reached to 3.5×102 log cfu/g at the end of the storage periods. As for the fish fingers samples contained EO՚s under levels of 0.05%, 0.1% and 0.15% mould counts decreased as storage time progressed. Mould counts for these samples were less than the control sample. Furthermore, after storage for 4 months moulds disappeared in fish fingers samples containing coriander under levels of 0.05% and 0.1% as well as in those containing cumin at under 0.1%. After 6 months of the frozen storage, there were no mould cells in all tested samples except that which contained parsley oil at 0.15% and the control sample. The disappearing of moulds may be attributed to antimicrobial activity of these EO՚s.
24- Organoleptic evaluation of fish product samples:
A- The data revealed that the color score in all samples tended to significantly decrease throughout the storage periods. Also, during storage the best scores of color were given for the samples containing EO՚s at a level of 0.05%, followed by samples containing EO՚s at level 0.1%. This might be attributed to the color of EO՚s which had been added to the formula. The color score of the control sample was low with a significant difference in all cases when compared with other treatments.
B- from these data it could be noticed that, the best odor scores were given for the samples containing EO՚s at a level of 0.05%, followed by those containing EO՚s at a level of 0.1%. The lowest odor score was given for the samples containing EO՚s at a level of 0.15%. Generally, the odor score gradually decreased with a significant difference for all treatments as frozen storage period increase.
C- from the obtained results, it could be noticed that significant differences were found in the taste score of the different treated samples during frozen storage. The taste score for the control was significantly gradually decreased after the first month of storage until the end of storage.
D- from the data it could be observed that, the best texture scores were for the samples containing EO՚s at 0.15%, with no significant difference, followed by treatments which had EO՚s at 0.1%. But treatments which had EO՚s at a level of 0.05% had the lowest texture scores during frozen storage at -18ºC. Moreover, those treatments which had EO՚s recorded the highest significant texture score compared with the control sample. Generally, texture scores decreased with a significant difference with the progression of the storage period.
E- from data, it could be noticed that, the highest value of overall acceptability as assessed organoleptically was for samples containing EO՚s at levels of 0.05% at zero time through to the end of the storage period, followed by the samples containing EO՚s at a level of 0.1%, with no significant differences. As for the samples containing EO՚s at a level of 0.15%, their eating quality somewhat decreased. The lowest overall acceptability value was recorded for the untreated sample (control) at the beginning of frozen storage. Generally, significant differences of overall acceptability were observed for all spiced samples during frozen storage.