Abstract: Background and Objective: Myriophyllum spicatum, Ceratophyllum demersum and Eichhornia crassipes are economically important and widely distributed macrophytes species. The objective of the current study was to evaluate and compare the variations in proximate and fatty acid composition of these aquatic macrophytes with respect to macrophyte species and water characteristics of the sampling sites. Materials and Methods: Macrophytes were collected from five sites of River Nile Rayahs. A proximate analysis and high performance gas chromatography (HPGC) were used for evaluation of the nutritional components. Results: The results demonstrate a significance differences (p>0.05) between the three studied species regarding their nutritional components, in addition to their differential response to the different water variables. All the studied plants had a high organic matter content with the highest nitrogen free extract value (NFE) 77.55% in sample of E. crassipes from site 5. The highest protein, digestible crude protein, protein/lipid, protein/nitrogen free extracts, protein/energy contents, nitrogen and potassium contents were found in C. demersum from site 5. Moreover the highest metabolized energy, energy value of organic matter and Zn contents were recorded for M. spicatum from site 3. HPGC analysis of fatty acids indicate the presence of 10 saturated fatty acids (SFA), 6 monounsaturated (MUSFA) and 5 polyunsaturated (PUSFA), varied significantly in distribution with plant species and sampling site. Conclusion: This study reveals the economic potential of these macrophytes as a natural food resources for fish and animals especially C. demersum and M. spicatum from site 5.
INTRODUCTION
Aquatic macrophytes, besides serving as a base of aquatic food chain and important components of food web dynamics1,2, they are considered as one of the potential sources of food and fodder for humans, animals and fishes3-6. In addition to their used in medicine7,8 and as a source of some antimicrobial substances9-11. But not all aquatic macrophytes are suitable to be used as a potential food source as they are known to differ widely in their nutritional composition depending up on species, part of plant used, location and season5,12,13. So the knowledge of factors affecting their nutritional composition is very importance in order to evaluate their food potential14. Since fish is the most important source of essential fatty acids that originate from artificial food or phytoplankton and seaweeds in the food chain15 and required in humans diet16,17. Studies on plants lipids have been focused on due to its high essential fatty acids that cannot be synthesised by humans, fish and animals.
Nothing was found in literature concerning the fatty acids profiles of the Egyptian macrophytes species M. spicatum, C. demersum and E. crassipes, so in view of this and due to the known variation in nutritional composition of aquatic macrophytes in different habitats. The aim of this study was to evaluate and determine variations of nutritional components in M. spicatum, C. demersum and E. crassipes from River Nile Rayahs in terms of estimating their proximate and fatty acid composition. Regarding the negative impact of heavy metals on the food value18,19 measurement of some heavy metals was also involved.
MATERIALS AND METHODS
Study site: The Nile River bifurcates at El Kanater City to two main branches (Damietta and Rosetta branches) and four Rayahs (Rayah are the main irrigation Canals in Egypt). The Rayahs include El Rayah El-Tawfiki (T), El Rayah El-Menoufy (M), El Rayah El-Behery (B) and El Rayah El-Nasery (N). From the flora of these water courses three macrophytes species (Myriophyllum spicatum L., Eichhornia crassipes (Mart.) Solms and Ceratophyllum demersum L.) related to three families (Haloragaceae, Pontederiaceae and Ceratophyllaceae respectively) and representing different plants life forms were selected.
Collection and preparation of samples: The three selected macrophytes species were collected in August, 2018 (the time of their maximum growth period) from El Rayah El-Menoufy (M) and El Rayah El-Behery (B), however samples of M. spicatum were collected from the four Rayahs, in addition to the Nile River bifurcate point (RN). At each Rayah one site was only selected for samples collection (Fig. 1 and Table 1). Three samples were prepared for each macrophyte species. The collected macrophytes were kept with water in polyethylene bags and transferred to the laboratory. Where they were identefied20, cleaned, washed and additional moisture was removed before weighted. A part of each plant sample was dried at 100°C to constant weight for estimation of dry matter. The other part was dried separately at 50°C to constant weight, ground into fine powder and kept for chemical analysis.
Proximate analysis: Water content (WC) was considered as the loss in mass from wet sample at room temperature and drying at 100°C. Ash percentage was determined after Egyptian Pharmacopoeia21. Plant elements were extracted using sulphuric acid and perchloric acid following the method of Burgski22. Nitrogen (N) content was assessed by Kjeldahl method23. Total Protein (P) content was calculated by multiplying the nitrogen content by a factor24 of 6.25. Potassium (K) content was detected by flame photometry and total phosphorus (P) was determined following the method of Umoren et al.25. Heavy metals in all plants extracts were measured by atomic absorption spectrophotometer (Perkin Elmer 2100).
Calculated parameters: Nitrogen free extractives (NFE) was determined following the equation applied by Pádua et al.26:
NFE (DW %) = 100-(EE+CP+Ash)
where, EE is the ether extract (total lipids) and CP is the crude protein.
Digestible crude protein (DCP) was estimated following the equation of Demarquilly and Weiss27:
DCP (DW %) = 0.929 CP-3.52
Metabolized energy was determined according to Pantha28, using the values of 3.4 for carbohydrate, 8.1 for lipid and 4.2 for protein:
| Fig. 1: | Map of River Nile Rayahs with the selected sampling sites |
| Table 1: | Details, latitude and longitude of the sampling locations |
The energy content (EV) was determined on the basis of biochemical composition (g g–1 DW) using the standard conversion factors29 for lipids 9.45, carbohydrates 4.10 and protein 5.65 and expressed as Kcal g–1 DW where, cal is the calorie and DW is the dry weight.
Fatty acid analysis: Lipid contents in all plants species were extracted and estimated following Bligh and Dyer30. Fatty acid methyl esters (FAMEs) were prepared and analyzed following31 (BS EN ISO 5508. In a clean tube take 0.1-0.2 g of plant lipids, add 10 mL of 0.2 mol L–1 methanolic sulphuric acid and 2 mL of benzene, close the tube well and put it in a water bath at 90°C for 90 min. Cool, add 10 mL petroleum ether shake strongly and separate out the ethereal layer in a dry tube. Evaporate to dryness and the analysis was performed in a gas chromatograph HP (Hewlett Packard) 6890 GC under the following conditions.
GC condition:
| • | Detector: FID (Flame Ionization Detector) |
| • | Detector temperature: 250°C |
| • | Injection temperature: 220°C, injection volume 3 μL, split ratio 50 1 |
| • | Column: HP-INNOWax (polyethylene glycol), 60 and 0.25 mm ID, 0.2 μm film thickness |
| • | Carrier gas: Nitrogen, gas flow 2 mL min–1 |
| • | Oven program: Initial temp. 140°C for min |
| Table 2: | List of standard fatty acids used in the present investigation |
Fatty acids were identified by comparing the retention times of experimental samples to those of known standards (Table 2).
Statistical analysis: Different statistical techniques were used to assess the significance of difference of different macrophytes components in relation to plant species and water characteristics of sampling sites. Including, ANOVA test, Duncan’s multiple range test at p<0.05 (XL Stat version 2014) and the canonical correspondence analysis32 (CCA using CANOCO V. 4.0).
RESULTS
Main nutritional components: As shown in results (Table 3 and 4), the studied macrophytes species were characterized by their high water contents which ranged between 89.14-94.71% DW with the highest for E. crassipes. Among the studied samples M. spicatum from site 1 was shown to have the highest ash content 34.25%, whereas the highest organic matter content (91.55%) was recorded in the same species from site 3. All samples contained a moderate protein content with the highest value of protein, digestible crude protein, P/L, P/NFE, P/E, N, K (13.13, 8.67, 2.92, 0.20, 3.38, 2.1 and 4.8% DW, respectively) for C. demersum from site 5. Lipid contents ranged from 3.10-6.30% DW in M. spicatum from site 1 and from site 4 respectively, while NFE showed higher value (77.55% DW) in sample of E. crassipes from site 5. In addition, the highest value of ME (3.43 Kcal g–1 of DW), EV (4.18 Kcal g–1 of DW) were recorded for M. spicatum from site 3.
From the results (Table 5), it is obvious that both macro and micro-elements showed a significant difference between different plant samples. During this study both nitrogen and potassium were found within high level (2.10 and 4.85% DW respectively) in samples of C. demersum from site 5, meanwhile samples of M. spicatum from sites 4 showed the highest phosphorus contents (3.31×10–2 DW %). A wide pattern of variation could be observed with regard to the microelements, both iron and manganese occurred in relatively higher concentrations compared to zinc and copper. The highest iron and manganese level was detected in samples of M. spicatum from site 5 (8.11 and 1.99 mg g–1 DW respectively), however the highest Zn content 49.75 μg g–1 DW was found in M. spicatum from site 3 and the highest copper contents (21.25 μg g–1 DW) was recorded for E. crassipes from site 4.
Fatty acids profile: Results in Table 6-8 revealed the presence of different fatty acids, varied significantly in their numbers and concentrations with reference to the plant species and sampling site. As compared with the other studied species M. spicatum from site 5 was recorded to have the highest number and concentration of TFA (19 fatty acid, 9.51 g/100 g of lipid) in addition to the highest TUSFA (4.21 g/100 g of lipid) content, while the lowest numbers and the lowest fatty acids concentrations were recorded for samples of same species from site 1 and 2 (9 fatty acids and 1.53 g/100 g of lipid respectively). However, the highest concentrations of TSFA were found in C. demersum followed by M. spicatum from site 5 (5.41 and 5.30%, respectively).
Saturated FAs: Ten of saturated fatty acids were detected in the studied plants (Table 6).
| Table 3: | Mean values of the nutritional composition (% of DW) of the different studied plant species from different sites |
WC: Water contents, OM: Organic matter, NFE: Nitrogen free extracts, a-iSignificance differences (p<0.05) between different samples | |
| Table 4: | Mean values of digestible crude protein (DCP), metabolized energy (ME), energy value of organic matter (EV), P/L, P/E and P/NFE of the studied plant species expressed on dry weight basis |
a-hSignificance differences (p<0.05) between different samples | |
| Table 5: | Mean values of macro and microelements of the studied plant species expressed on dry weight basis |
a-iSignificance differences (p<0.05) between different samples | |
| Table 6: | List of saturated fatty acids detected in the studied macrophytes species |
nd: Not detected | |
| Table 7: | List of unsaturated fatty acids detected in the studied macrophytes species |
nd: Not detected | |
Myriophyllum spicatum from site 1 was shown to have the lowest number of SFA (5 fatty acids), however samples from site 5 were characterized by the highest numbers and concentrations of SFA. The most abundant FA in this group were palmitic acid and myristic acid. However, the long chain fatty acid arachidic acid was present only in all plant samples from site 5 in addition to samples of C. demersum from site 4.
Monounsaturated FAs: Six of monounsaturated fatty acids (MUFA) were detected (Table 7). In the studied plants the levels of oleic acid and myristoleic acid were found as the highest two fatty acids in this group within the range of 3.844-16.403 and 3.769-12.589%, respectively. In addition, M. spicatum from sites 3 and 5 was characterized by the presence of the MUSFA cis-11-Ecosenoic acid (C20:1).
Polyunsaturated FAs: The total polyunsaturated fatty acids ranged from 7.491% of FA in samples of E. crassipes from site 5-24.919% of FA in M. spicatum from site 5. All plant species from different sites contained linoleic acid (C18:2c an omega-6 fatty acid), however polyunsaturated fatty acids (C 18:3 αw3 an omega-3 fatty acid) linolenic acid and C20:3w3 were detected in M. spicatum from sites 4 and 5 and in samples of C. demersum, while absent in samples of E. crassipes (Table 7).
DISCUSSION
Results in Table 3-8 shows a-significant difference in all nutritional components with plant species and sampling site (p>0.05). Similar observations were recorded by Haroon et al.5 and Shaltout et al.33. All plant species were characterized by their high nutritional composition which represented by their high NFE contents ranged from 56.53-77.55%, that exceed the dietary requirements of shellfish and fish and the range of some rough fodder material for carbohydrates (from 10-30% and from 27.8-51.9%, respectively)34-35. In comparison with other aquatic plants and with the same plant species from different sites the values exceed the values recorded for Echinochloa stagnina 45.7 and 53.7%3,33, Nymphaea lotus and Pistia stratiotes 30.4-35.1%4 and that reported for Eichhornia crassipes 38.9, 54.1 and 40.89-49.24%3,33,13.
Protein have important function in all biological processes36 and the organism requirements of proteins were varied depending on age and species of the cultured organisms37 in addition to the rearing environment. The range of protein content of (5.69 and 13.13%) as recorded in this study is lower than that require for aquatic animals diet (32-52%)34,35, but lie in the proper level reported for terrestrial animal diet (6-12%) depending on the animal species38. The results of the present study are comparable to those reported by Haroon12 for Saccharum spontanum, Ceratophyllum demersum, Potamogeton pectinatus and Potamogeton crispus (7.8, 9.81, 10.13 and 14.50% DW respectively), Echinochloa stagnina, Eichhornia crassipes and Ceratophyllum demersum33 and Echinochloa stagnina, Eichhornia crassipes and Potamogeton tomentosum3, lower than that reported by Olele39, for Pistia stratiotes, Eichhornia crassipes and Ceratophyllum demersum (15.80±0.80, 21.65±0.65 and 19.65±0.65%, respectively) and Abdel Shafy et al.13 for Eichhornia crassipes 12.9-17.59% DW this may be related to the part of plant used, time and place of samples collection.
| Table 8: | Comparative total fatty acids, total saturated, total unsaturated and saturated to unsaturated fatty acids of the studied plant species from different sites |
TFA: Total fatty acids, TSFA: Total saturated fatty acids, TUSFA: Total unsaturated fatty acids, TSFA/TUSFA: Ratio of total saturated to total unsaturated fatty acids, results are expressed as g/100 g of lipid, a-iSignificance differences (p<0.05) between different samples | |
However, lipid was found relatively within low concentration (from 3.10-6.30% DW), these values are being higher than that reported for some rough fodder materials35 (0.5-3.1%). It was also higher than that reported for other hydrophytes from different water courses like: Potamogeton crispus, Potamogeton pectinatus, Polygonum tomentosum, C. demersum and Saccharum spontanum12 (2.40, 2.02, 2.0, 1.99 and 1.2% DW, respectively). Nymphea lotus and Pistia stratiotes8 (2.3-3.5%) and Eichhornia crassipes13 (2.27-4.79%).
Energy is essential for the maintenance of many life processes, so the ability of a food to supply energy is of a great importance in detecting its nutritional value to animals30. Metabolized energy results (2.43-3.43 Kcal g–1) in this study were found to be higher than that reported for P. stratiotus and N. lotus (2.04, 2.05 Kcal g–1, respectively)4.
As recorded in the literature the higher of protein to energy ratio (P/E), the better is the diet40. So, on the basis of high P/E values for C. demersum and M. spicatum from site 5 (3.38 and 3.21, respectively) it may be inferred that these two species are suitable for incorporation in fish and animal diets.
As recorded by Polisini and Boyed41 and Jobling42 adequate levels of essential mineral nutrients are one of the important aspects of plant nutritive quality but excessive concentration of these minerals in plant tissue lowers its nutritional value, moreover the highest concentrations of microelements are considered to be toxic43. From the result obtained, it is obvious that with the exception of M. spicatum from site 1, the ash content of the studied plant species (8.45-21.96%) lies in the range of some green roughages (from 8.6% in maize to 14.2% in cowpea) commonly used as livestock feed44. Depending on the results of Snow and Ghaly40 all investigated plants contain sufficient amount of P, Cu and Zn to meet the dietary requirements of aquatic animals and exceed that of K, Fe and Mn.
High number of different fatty acids groups (19 fatty acids) were detected in 2 species, M. spicatum and C. demersum in site 5 while 16 FA types were detected in E. crassipes, which was higher than that reported for other macrophytes from different water courses by Haroon et al.45, Haroon4 and Haroon and Abdel-Aal46 (6, 12 and 6 fatty acids respectively). Five of saturated fatty acid included tridecanoic, myristic, pentadecanoic, palmitic and stearic acid were detected in all plant species, in which palmitic and myristic acid were found in higher concentrations especially in C. demersum from site 5 and Eichhornia crassipes from site 4 respectively.
Concerning the USFA, Table 7 showed that three MUSFA were presence in all studied plants. Myristoleic and oleic acid were the highest concentration in E. crassipes from site 4, 5 respectively. Myriophyllum spicatium was characterized by the highest concentrations of TUSFA (4.21±0.01 g/100 g of lipid). Other previously studies of Haroon et al.45 and Haroon4 recorded arachidic acid as the major constituent of Inula erithmoides and Potamogeton pectinatus extracts, however palmitic acid represent the highest constituent of Halimione portulacoides fatty acids. In addition, myristic acid represents the highest amount of fatty acids separated from P. stratiotes shoots (28.3%) and N. lotus leaves (24.0%) extracts, while linolenic acid represents the major constituent of N. lotus stem (34.0%) fatty acids.
The polyunsaturated fatty acids, linoleic acid (C18:2c ω-6) and arachidonic (C20:4 α ω-6) acids were detected in all plant species but varied with the sampling sites, however α-linolenic acid (C18:3 α (ω-3) and Cis-11,14,17-Eicosatrienoic acid (C20:3 ω-3) were absence from E. crassipes and docosahexaenoic acid (DHA, 22:6 ω-3) was only detected in C. demersum from site 5. Although, C. demersum from site 5 was recorded to have the highest number (5) of PUSFA, M. spicatum was found to have the highest concentration (2.375% g of lipids).
Results in Table 9 and Fig. 2a shows the differential responses of different plant components to the different water variables. The highest protein values were found in all plant species from site 5 which was characterized by the highest water COD, NO3 and PO4 values compared with other sites.
| Table 9: | Simple linear correlation coefficient between the measured plants components and water physicochemical parameters |
| Fig. 2(a-b): | CCA-biplot ordination diagram of the macrophytes components in relation to the different water variables, (a) Axes F1 and F2: 58.03% and (b) Axes F1 and F2: 74.83% |
DCP: Digestible crude protein, NFE: Nitrogen free extracts, ME: Metabolized energy, EV: Energy value, N: Nitrogen, P: Phosphorus, K: Potassium, OM: Organic matter, WC: Water contents, P/L: Protein/lipid, P/E: Protein/energy value, P/NFE: Protein/nitrogen free extracts, Temp: Water temperature, TN: Total nitrogen, TP: Total phosphorus, EC: Electrical conductivity, TRANS: Transparency, DO: Dissolved oxygen, BOD: Biochemical oxygen demand, COD: Chemical oxygen demand, TFA: Total fatty acids, TSFA: Total saturated fatty acids, TUSFA: Total unsaturated fatty acids, TSFA/TUSFA: Ratio of total saturated to total unsaturated fatty acids |
|
| Table 10: | Simple linear correlation coefficient between the different fatty acids components and the water physicochemical parameters |
At the same time both lipid and NFE showed no clear relation with water physicochemical parameters. This is not in agreement with Tucker and Debusk47 they mentioned a seasonal variation in carbohydrates, protein and crude fibres contents of E. crassipes cultured outdoors with constant nutrient availability. In addition a significant positive correlation was observed between nitrogen (N) content in all plants tested and water NO3 and PO4 contents. However, Nichols and Keeney48 recorded lowest nitrogen content in summer samples of Myriophyllum spicatum, even though available nitrogen in the sediment was highest value at that time.
The correlation coefficient of different fatty acids and water physicochemical characteristics is presented in Table 10 that indicates the positive relationship between different fatty acid groups and water BOD, COD, NO3 and PO4. On the other hand the lowest TFA, TSFA and TUSFA were recorded for plant samples from site 2 which characterized by the highest water EC values. It was found that the three plant species were riche in their nutritional components, especially samples from site 5. Depending on the present results together with the previously recorded information the potential of these macrophytes as a natural food resources for fish and animals could be recommended, but some work concerning safety and toxicity is still needed.
CONCLUSION
The present work confirmed the nutritional importance of the three studied macrophytes species. All the studied species were found to be important sources of essential fatty acids (EFAs), in addition to their high organic matter, nitrogen free extract, potassium, ferrous and manganese contents that lay within the range of aquatic animal diet. The variation in their nutritional components with respect to species and location was observed. Ceratophyllum demersum and Myriophyllum spicatum from site 5 being the most suitable feeding items for animals and fish, as it contains high protein, lipid, P/E and fatty acids contents.
SIGNIFICANCE STATEMENT
This study discover the nutritional potential of M. spicatum, C. demersum and E. crassipes that can be beneficial for consumers to find a new natural food resources suitable to reduce the cost of commercial feeds. To support a recommendation for consumption of these aquatic plants as a natural food a full nutritional profile and screening for the presence of anti-nutritive factors, the presence of which could limit utilization prospects must be involved.
This study may help the researchers to determine the most suitable conditions that needed for cultivation of a good quality plants.
ACKNOWLEDGMENTS
The author would like to thank Prof. Dr. Mohamed E. Goher, Head of Chemistry Lab. for providing laboratory assistance in water quality analysis. Thanks was also extend to DR Abd-Ellatif Mohamed Hussian for his help in statistical analysis.