An Evaluation Of Date Seed Protein Biology Essay

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Date palm (Phoenix dactylifera L.) is considered the oldest fruit tree in the world. It is grown in dry and semidry terrains and has been an important foodstuff for the last 7000 years (Ahmed et al., 1995; A-Hooti et al, 1997; El-Sharnouby et al, 2009). Date palm belonging to the Arecaceae (prior Palmaceae or Palmae) family which almost involve 200 genera and more than 2000 species (Diaz et al, 2003 & Moore et al, 1973). Global production, industrialisation and utilisation of dates are increasing steadily; the worldwide production of date palm fruit has increased from 1.8 million tons in 1961 to 6.9 million tons in 2005 (Chao and Krueger, 2007). Date palm fruit(DPF) have been eaten fresh or used by many parts of the food industry to make products such as jam, bar, date syrup, date pies, alcohol, date chocolate and date confectionery etc. (Sablani et al, 2008). Date seeds powder and coffee substitute that have been recently introduced to the market (Rahman et al, 2007). Dates are richer than other fruits in protein (Ahmed et al, 1995); as well as date seeds could be an excellent source of functional foods because they contain a balance of fats, proteins, minerals and carbohydrates.

1.2 Protein concentrates:

Alibhai et al., 2006 & Wang et al., 2004, shown that the protein concentrate of soybean obtainable from defatted powder or flour by removing soluble constituents like sugars, oligosaccharides and other minor components from the defatted powder using acid as dilute solution in the pH ranges from 4.0-4.8 as shown in figure (1.1), Bouaziz et al., 2008 reported that date palm seed protein concentration obtain by useing 0.1 NaoH to extract the protein after stir for 40 min at room temperature, then Centrifuge and Freeze dried (as shown in fig. 1.2).

1.3 Protein isolates:

(L'hocine et al., 2006; Alibhai et al., 2006) used the dilute alkali to extract the protein from the defatted powder with subsequent centrifugation to produce the protein and remove the insoluble materials. Precipitate the protein at pH 4.5 using (HCl) as food grade acid to recovery the proteins due to their precipitation and concentration. Washing the pelt to remove the non protein soluble, neutralization at pH7 and freeze dryer produces the isolation of protein as shown in figure 1.3.

1.4 Functional properties of proteins:

Functional properties of proteins are those properties that determine behaviour of proteins in food system during production, processing, storage and consumption. Some functional properties of the proteins can be listed as follows:

1.4.1 Solubility:

The solubility of proteins are defined as an ability of protein to associate with water and is a affect of a various conditions such as pH, temperature, solvent, ionic strength and sugars(semenova et al, 2002)

1.4.2 Emulsion ability and stability:

Emulsions are two phase systems of liquids (water and oil) with one of the liquids dispersed in the other. there are two systems of emulsion, one of them are an oil droplets dispersed through an aqueous phase and known as an oil-in-water emulsion(o/w) such as milk, mayonnaise etc., whereas, the other are water-in-oil emulsions(w/o) called when the water droplets are dispersed in an oil such as butter and margarine(zhao et al, 2002).

1.4.3 Water holding capacity (WHC):

(WHC) of foods defined as the ability to hold or added the own water during the application of heating, pressing, forces or centrifugation (Zayas, 1997); whereas, Kenefel et al, (1991) defined the WHC as the ability of a food structure to prevent water from being released. Generally, there are two types of water held in a protein structure:1) the part trapped in the protein matrix or a corresponding co-matrix(fat, polysaccharide) and 2) the other part bound to the molecule that is no longer available as a solvent(Kneifel et al., 1991); water holding capacity is influenced by several conditions such as temperature, pH, shape, protein size, lipid and carbohydrate associated with protein(Han and Khan, 1990)

1.4.4 Gelation properties:

Gelation properties of proteins are applied in many food applications. A gel can be defined as steady state flow of continuous network of macroscopic dimensions in a liquid medium (Oakenfull et al., 1997). To form the gel requires thermal treatment (denaturation) molecules of the protein; thermal treatment is required to expose the reactive functional groups of the protein, followed by intermolecular interaction. These interactions involve physical interactions and chemical bonding. Gelation required minimum protein concentration; this concentration is a function of pH, temperature and ionic strength (Aussanasuwannakul, 2004 & Elssa, 2005).

1.4.5 Foaming ability and stability:

Foam production involve one of the proteins function as natural surfactants in many applications (Doucet, 2004) Foam considered colloidal dispersions as air phase disappeared in a solid or liquid phase (Yang and Baldwin, 1995). Liquid foams consist of discontinuous air phase dispersed in a continuum of liquid, whereas solid foams consist of discontinuous air phase dispersed within a continuous solid phase. In the food industry, cookies, breads and cakes are typical examples of solid foams; pure liquid can be form stable foams if their present an amount of surface active materials. To build the stabilising film of foam, protein is necessary as a surface active component to adsorb at gas liquid interface of the air bubbles (Raikos, 2006 & Foegeding et al., 2006).

2. Objectives of this Study:

This study considered the first study focusing on functional properties of date palm proteins. date seeds comprise approximately 10-15 % of the date's weight (Hussein et al, 1998; Hamada et al, 2002; Besbes et al, 2004) thus, from the global production of dates are almost 700.000 tonnes of date palm seeds are not currently used(El-Sharnouby et al, 2009) furthermore, In the date industry, date seeds are a problem of what to do with the waste date seed(Hamada et al, 2002&Besbes et al, 2004). The main objective of the current study was to convert waste of the date palm seed into useful protein powder and characterize that protein using a variety of techniques.

2.1 Program of study:

2.1.1 Study and investigate isolation, concentration and purification of proteins from date seed:

In this study we will attempt to recover a high proportion of the soluble protein from date seed. Previous studies by Hamada et al, 2002; suggest that at least 53% of the total protein can be recovered using simple aqueous extraction techniques. We will try to improve on this by using methodologies that are standard in the protein industry, and are commonly used for protein powders derived from other seed products (e.g. soya bean protein). We will investigate different purification methodologies in an attempt to prepare protein powders of a high protein content (80% or above). This will include methodologies such as dialysis and/or ultrafiltration to remove non-protein material from the extracted powder.

2.1.2 Investigate the characterize date seed protein using gel electrophoresis (SDS-PAGE) and Maldi-ToF mass spectrometry.

As far as we are aware the proteome (detailed protein composition) of the date seed, (nor for that matter the date flesh) has been determined and reported. We have access to a state of the art Maldi-ToF (Matrix-assisted laser desorption/ionization time of flight) mass spectrometer that will allow rapid identification of isolated proteins from date seed and flesh.

2.1.3 Study and investigate functional properties of seed protein:

If date seed protein is to be useful as a protein product in manufactured foods we need to confirm that it has acceptable functional properties. We will test the following functional properties of purified date seed and date flesh protein:

2.1.3.1 Emulsion ability and stability:

Stability of emulsions form is very important in the food industry, the important parameters for stability of emulsions are pH and temperature, Microscopes are the useful equipments in emulsions testing, and we will test the stability of emulsions by study the structure, sizes and shapes emulsions of purified date palm proteins.

2.1.3.2 Foaming ability and stability:

as far as we are knew the protein foams are an important components in food applications such as ice creams; the structure and physicochemical properties of the proteins are play an important function in foaming ability and stability, we will Investigate the role of date palm proteins in foaming ability and stability.

2.1.3.3 Gelation properties:

If the date palm protein is to be useful to form the gel, we need to Investigate that, we will study the affecting of heat treatment, pH, and protein concentration on gel formation.

2.1.3.4 Solubility:

Solubility of proteins is considering one of an important functionality in food system, so we will study effect of several conditions such as pH, heat treatment and protein concentration on date palm protein solubility.

2.1.3.5 Water holding capacity (WHC):

We will study the ability to hold the water under several parameters such as pH and temperature.

2.1.4 Study of compared date seed protein with other food proteins like milk, soybean and egg.

To confirm the usefulness of date seed protein as a functional ingredient we will compare date seed protein functional properties with those of proteins commonly used as food ingredients, i.e. milk, soybean and egg proteins.

2.1.5 Investigate and test functionality of date seed protein in model food systems:

To further confirm the usefulness of date seed protein as a functional ingredient we will test the functional properties in a range of food applications. These will include:

A. As a milk protein replacer and supplement in beverage

B. Yogurt.

C. Cheese.

2.1.6 Explanation of results based on literature review publication.

2.1.7 Aggregation of PhD thesis.

3. Materials and methods:-

3.1 Materials:

Date palm fruit (Phoenix dactylifera L.) at Tamr stage (complete maturity) of the commercially available Tunisian date palm Deglet Nour variety were purchased from the local supermarket in Edinburgh, UK.

3.2 Preparation of defatted seed powder:

Date palm seed powder (DPSP) was extracted with chloroform for 16h using a Soxhlet apparatus, then defatted powder was air dried at room temperature and kept in plastic containers at the refrigerator prior to use.

3.3 Physical analysis:

The weight of one hundred fruits and seeds was measured, and then the average of single fruit and seed, ratio of date palm seed or flesh was calculated and recorded.

3.4 Chemicals:

All chemicals and solvents that used in this study obtained from sigma-Aldrich ltd, UK & fisher scientific UK limited.

3.5 Analytical Methods:

Proximate analysis of date palm fruit and date palm seeds were carried out according to association of official analytical chemists (AOAC, 1995), all analytical determinations were carried out at triplicate and the final data were expressed on a dry weight basis, we determinate:(Moisture, total solids, Ash, Fat, Protein, Crude fibre and Total carbohydrates).

3.6 Extraction of protein:

All the extraction procedures were done in pH and salt conditions to concentrates, isolates the protein from date flesh and defatted date palm seed according to Ahmed et al, (1995); Wang et al, (2004); L'hocine etal, (2006); Bouaziz et al,(2008); and Elleuch et al, (2008) with some modifications as shown in table (3.1).

3.7 Electrophoresis (SDS-PAGE):

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of date palm seed protein was performed to determine molecular weights for proteins in 12% T polyacrylamide gel according to the method of Laemmili (1970).

Table 3.1 laboratory methods and their modification

method No.

Modification

Reference

1

Time of extraction (60 min).

Bouaziz et al, (2008)

2

Time of extraction (90 min).

Bouaziz et al, (2008)

3

Time of extraction (120 min).

Bouaziz et al, (2008)

4

Time of extraction (60 min), centrifuge speed and freeze-dried.

Wang et al., (2004)

5

Time of extraction (60 min), pH10 and centrifuge speed.

L'hocine et al., (2006)

6

Time of extraction (90 min), resolubilize the centrifuge supernatant at pH7and use UF.

Bouaziz et al, (2008)

7

Time of extraction (90 min), precipitate the protein at pH4.5, resolubilize the centrifuge supernatant at pH7.

Bouaziz et al, (2008)

8

Time of extraction(90 min), precipitate the protein at pH4.5, resolubilize the centrifuge supernatant at pH7 and use UF.

Bouaziz et al, (2008)

9

Time of extraction, pH 10, centrifuge speed and use UF.

L'hocine et al., (2006)

10

Time of extraction (90 min), pH 9, Precipitate at pH 4.5 and resolubilize at pH 7.

Bouaziz et al, (2008)

11

Use UF and freeze dried.

Ahmed et al, (1995)

12

Time of extraction (60 min) and centrifuge speed.

L'hocine et al., (2006)

13

Time of extraction (60 min), centrifuge speed and use UF.

L'hocine et al., (2006)

14

Time of extraction (60 min), centrifuge speed, the supernatant refrigerated before centrifuge and use UF.

L'hocine et al., (2006)

4. Results and discussion:

4.1 Some physical measurements of DPS and DPF:

The physical characteristics of date palm consider an important information as a data base in date industry that helps trading, processing and storage of dates, table 4.1 shows some physical measurements including: average weight of whole fruit, average weight of DPF, average weight of DPS, ratio PF to whole fruit, ratio DPS to whole fruit and number of fruit in Kg, the obtained data were; 9.51g, 8.53g, 0.98g, 89.70%, 10.30% and 106 fruit in Kg respectively, these results were higher than those reported by Al-Hooti etal, 1997, this might because of the different verity of dates, as well as different in agriculture treatments and environmental conditions.

4.2 proximate compositions of DPS and DPF:

Figure 4.1 presents the proximate compositions of date palm flesh and seed, the total solids, crude protein, crude fat and crude fibre of date seed were higher than those in date flesh, whereas, moisture, total ash and total carbohydrate were lower than those in date flesh; crude protein and crude fat were the major fractions in date seed, 5.64%, 8.14% respectively. the results of both (seed and flesh) contents were agreement with those reported by (Hamada etal, 2002; Al-Showiman&Baosman, 1992; Al-Hooti etal, 1997; Al-Hooti etal, 1998 and El-Shurafa etal, 1982), but higher than those reported by (Ellench etal, 2008 and Ahmed&Ahmed, 1995) and lower than those reported by (Besbes etal, 2004; Rahman, 2007; Besbes etal, 2009 and Sghaier, 2009). Those differences might be refers to the variability cultivars under studies, as well as, stages of maturity could be affect the work of enzymes activity (Hulm, 1971).

4.3 Chemical compositions of full fat date palm seed powder and defatted date palm seed powder:

Chemical composition of full fat date palm seed and defatted date palm seed is presented in figure 4.2, all the contents were increased after a defatting process, whereas, the fat content was decreased from 8.14% to 1.01% approximately 88% reduction; the colour of defatted date palm seed powder was lighter than the original date palm seed powder.

4.4 Determination of protein in date palm powder and residue after extraction with NaoH solution:

Figure 4.3 shows the percentage of protein in date palm seed powder and the residue after extraction with NaoH for 90 min, there is slight difference of protein contents in the residue after extraction with NaoH compare with protein content in seed powder, this results could be explained that the large fractions of protein in date palm seed after extraction with NaoH cannot be very digestible; Hamada et al, 2002 mention that the insoluble portions of date palm seed protein probably composed of high molecular weight polypeptides which have high aggregated.

4.5 Effect of pH on the extractability or protein from date palm (seed):

The effect of NaoH at different pH values on extractability of date palm seed protein is shown in figure 4.4. In the alkaline range, the highest extractability of protein was obtained at pH 10. Generally, as the pH increased from 7 to 11 protein extractability increased. After pH 11; protein extractability was significant decreased compared to the protein extracted at pH 10. In the acidic range, the highest protein extractability was observed at pH 1 whereas, the lowest protein extractability was remarked at between pH 3 and 5. Results in fig. 4.4 indicated that isoelectric points of most date palm seed powder proteins ranged between pH 3-5. These results were agree with those reported by Hang et al 1970; by Ekpenyong, 1986; and by Abu-Tarboush, 1995 in their studies of the bean proteins, conopher seeds, Karkade(Hibiscus sabdariffa) seed, respectively.

4.6 protein extraction of date palm seed:

Figure 4.5 illustrates experimental conditions of 9 methods prepared by some modification on standard methods, percentage of protein and percent yield of date palm seed were shown as well, the highest protein extraction (23.28%) was carried out with pH 10 and 60 min at 55°C, then using pH 4.5 to precipitate the protein as generally known that minimum solubility of protein at isoelectric point. On the other hand, the lower protein extraction (8.65%) was obtained by method 4. generally, whole the protein content increase the protein yield was decrease; the higher protein yield was 20.33% with protein content 8.93% whereas, the 23.28% of protein has only 4.20% as protein yield, these differences are likely to be that remains of polysaccharides or any other components weighted as a proteins or probably that proteins were conjugated with polysaccharides.

4.7 Protein extraction of defatted date palm seed powder (DDPSP) by different methods at several times:

Proteins extracted from DDPSP by three different methods are shown in Figure 4.6. Protein extractability increased slightly with increased time extraction. the higher percentage of protein extracted was at 90 min (9.26%) when using Bouaziz et al, (2008) method. in respect of Wang et al, (2004) and L'hocine et al, (2006) methods; the higher protein extracted was at 60 min compare with the time in standard methods.

4.8 Effect of pH on the extractability of protein from date palm fruit (flesh):

In acidic range, like date palm seed protein, date palm fruit (flesh) protein had the highest and the lowest extractability was observed at pH 1 and at pH 3-5, respectively. Whereas, in the alkaline range, the highest extractability of date palm fruit (flesh) protein was obtained at pH 9 (figure 4.7).

4.9 Protein extraction of date palm flesh:

Protein extracted from date palm flesh was presented in figure 4.8; lab method No. 14 shows the highest efficiency to extract date flesh protein compared with other lab. methods that we used in this process; 36.51% protein was the higher protein content we achieved.

4.10 SDS- PAGE of electrophoresis:

Date palm seed protein extracts from date powder was analysed by SDS-PAGE under reducing and non reducing conditions. There are two detectable bands which located at approximately around 62 and 55 kDa (figure 4.9, lane 2, 5, 6, and 7). These proteins might be albumins, globulins, glutelins or prolamins which are soluble in alkaline solutions (Hamada et al., 2002 and Bouaziz et al., 2008). Bands under reducing conditions appeared to be similar to that under non reducing condition since no distinct difference could be detected. Our results were nearly similar to those indicated by Sekhar et al., 1988 whose reported that date palm contained protein bands ranged between 45-66 kDa; but were slightly different to those reported by Ahmed et al., 1995 and Bouaziz et al., 2008; this difference could be refer to the differences of cultivars dates and/or Temperature resulting from grind the date palm seed.

5. Conclusion:

The increasing cost of foods particular food protein from animal source in the world has encouraged the food scientists to develop new source of plant proteins. thus, date palm (seed or flesh) could be one of these sources. Most of previous studies focused on the physical and chemical composition of date palm, recent study by Bouaziz et al, (2008) who extracted date palm seed proteins and they got only 16.95% of protein; our results showed higher percentage of date palm protein extracted so far, 23.28%, 36.51% protein for seed and flesh respectively.

6. Future plan for the second year:

A. Finish concentration, isolation and purification of proteins from date palm seed and flesh.

B. Characterization of date palm proteins (flesh and seed) using the gel electrophoresis (SDS-PAGE) and Maldi-ToF mass spectrometry.

C. Test of functionality and physico-chemical properties of purified date palm proteins, included:

1. Study of emulsion stability, ability and emulsion structure using Confocal laser scanning microscopy.

2. Study of solubility and rheological properties at different pH, temperatures and concentration.

3. Influence of sodium chloride and sugars on date palm protein solubility.

D. Apply date palm proteins with some food applications like yogurt, cheese and milk protein replacer and look at their functionality.

E. comparison the date palm proteins with other food proteins like milk, soybean and egg as a standards of proteins.

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