Vol:.(1234567890)

Food Measure (2018) 12:386–394DOI 10.1007/s11694-017-9651-x

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ORIGINAL PAPER

Extraction and functional characterization of isolated proteins from Aleppo pine seeds (Pinus halepensis Mill.)

Khalid Al‑Ismail1 · Nehaya Al‑Assoly1 · Mohammed Saleh1  

Received: 5 January 2017 / Accepted: 25 September 2017 / Published online: 27 September 2017 © Springer Science+Business Media, LLC 2017

and gelling properties that supply certain specific attractive characteristics to the final products.

Plant proteins play important roles in human nutrition, especially in countries with limited resources where average protein intake is less than that required [27].

The need to develop functional alternative sources of pro-tein has become mandatory due to the increased poverty and diversity of food applications [17]. In this regards, utilization of some legumes and oilseeds has settled to be viable alter-native for expanding protein sources such as soybean protein (SPI) [14], sunflower [12] and Moringa oleifera proteins [1].

Pinus halepensis Mill., commonly known as Aleppo Pine, is a pine native to the Mediterranean region [25]. In Jordan and Palestine, the seeds are often eaten as a snack either raw or roasted. Seeds are also used in several traditional foods including melban; a traditional food produced originally in Hebron, Palestine that is usually prepared from semolina-thickened concentrated grape juice. Moreover, P. halepensis Mill. seeds give a crunchy taste when added to the chewy fruit leather and are often added to porridge made from whole wheat [25]. The seeds are often ground to a powder and used as a thickener and flavoring in soups [25].

Raw or cooked P. halepensis Mill. seeds content of pro-tein may reach up to 29% [25]. Protein fractions; isolates and/or concentrates obtained from Aleppo pine seeds may provide an alternative source of functional and dietary sup-plements, in addition it is considered as essential ingredient in the food industry [25].

To the best of our knowledge, no information was found regarding the functional properties of APPI. Accordingly, the aim of this study was focused on characterizing the func-tional properties APPI.

Abstract The aim of this work was to extract and charac-terize Aleppo pine (Pinus halepensis mill.) proteins. Aleppo pine protein isolate (APPI) was isolated from defatted flour using alkaline solution and isoelectric precipitation. Protein solubility, emulsion capacity and stability, foaming capacity and stability, thermal properties, oil and water absorption capacity and gelling ability were evaluated. APPI was most soluble at pH 10 (47.36%) and pH 2 (44.38%), respectively and least soluble at pH 4 (10.21%). The highest emulsion capacity was detected at pH 2 with 220 mL of oil emulsified and emulsion was completely stable at pH 2, 8 and 10 for up to 48 h at 23.3 °C. APPI had a foaming capacity of 99 and 101% at pH 2 and 10, respectively and foaming stabil-ity of more than 120 min at 23.3 °C. Oil absorption capac-ity and water absorption capacity were 3.10 and 3.90 g/g, respectively.

Keywords Aleppo pine seed protein · Isolation · Functional properties

Introduction

Proteins are important ingredients in food industry because of their vital role in enriching, modification and improving the quality of foodstuffs, not only due to their high nutri-tive value, but because of other functional properties [7]. Among these functional properties: foaming, emulsifying

* Mohammed Saleh misaleh@ju.edu.jo

1 Department of Nutrition and Food Technology, Faculty of Agriculture, The University of Jordan, Amman, Jordan

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Materials and methods

Materials, sample collection and preparation

Aleppo pine seeds (about 6 kg) were purchased from a local market in Amman, Jordan. The seeds were washed and grinded manually using pistol and mortar. Egg white proteins were obtained from [Egg Albumen Powder, high whip instant extra, A8-051, B-6600 Bastogne (Belgium)], whey protein isolates were obtained from (DAVISCA, Bipro, USA) and SPI were obtained from (laboratory of The University of Jordan). Hexane anhydrous > 99% (Merck, Germany), NaOH (Food grade Sodium Hydroxide Pellets, India), HCl (Merck, Germany), Electrophoresis materials including ready gels, Coomassie brilliant blue R-250 and standard protein markers (Bio Rad, UAE).

Oil extraction

Lipids of the grinded Aleppo pine seeds (100  g) were cold extracted by hexane using a Soxhlet apparatus for 8 h following the AOAC [2] method and by adjusting the extraction temperature to 25 ± 5 °C. The oil was recovered by further evaporating the excess solvent using a rotary evaporator (25 ± 5oC) for 24 h to remove residual solvent and then grinded into flour using Waring blender, speed 5 (17,000 rpm). The defatted sample were then stored in airtight plastic container and kept in refrigerator until use. Lipid content was then calculated as the percentage of lipids left after the evaporation of hexane.

Preparation of APPI

The APPI was prepared according to the method of Sosulski et al. [24] with simple modification. In summary, the defat-ted Aleppo pine flour were dispersed in water (1 flour:10 water, w/v), pH was then adjusted to ten using 0.02 N NaOH and left at room temperature ± 5 °C for 2 h with continu-ous stirring. Samples were then centrifuged at 4000 rpm for 35 min. at 4 °C using a 5810R centrifuge, (Eppendorf, Germany). Extraction solution was collected and same supernatant was used to perform the extraction twice before extraction solution of the two sets were collected and mixed. pH of the combined extracted solutions was then adjusted to pH 4 (the PI of Aleppo pine protein) with a 0.02 N HCl and let stand at room temperature ± 5 °C for 15 min. (i.e., about 23 ± 5 °C) to aid precipitating proteins. Precipitated proteins were collected by subsequently centrifugation at 4000 rpm for 35 min. at 4 °C. The precipitates were then washed with distilled water, re-dissolved in water, neutralized to pH 7 with 0.02 N NaOH and then freeze-dried and grinded into flour. The percent to precipitate was then calculated as the percent of APPI.

Kjeldahl method according to AOAC [2] was used to measure Aleppo pine seeds protein content, moisture content was determined using the oven drying method [2] and car-bohydrate content was calculated by the difference method.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‑PAGE) of APPI

Proteins profiling of APPI was performed using SDS–poly-acrylamide gel Electrophoresis. Electrophoresis was per-formed on discontinues 4%-stacking and 12% resolving gels (Bio Rad). Gel was then fixed and stained with 0.2% Coomassie Brilliant blue R-250 in methanol:acetic acid:water (5:4:1, v/v/v) and de-stained before dry-ing and molecular weight calculations. Standard protein markers contained myosin (200,000 Da), β-galactosidase (116,250 Da), phosphorylase b (97,400 Da), serum albu-min (66,200 Da), ovalbumin (45,000 Da), carbonic anhy-drase (31,000 Da), trypsin inhibitor (21,500 Da), lysozyme (14,400 Da) and aprotinin (6500 Da) was used as a standard.

Thermal properties of APPI

Thermal properties were assessed by a PerkinElmer Pyris-1 differential scanning calorimeter (DSC) (Perkin-Elmer Co., Norwalk, CT). APPI samples (5.0 mg, dry basis) was weighed into an aluminum DSC pan and then moistened with 8 µL of deionized water using a microsyringe to make suspensions with a 20% concentration. Samples were her-metically sealed and allowed to stand for 1 h at room tem-perature ± 5 °C prior to heating in the DSC. Samples were then heated from 25 to 240 °C, with increments of 10 °C/min. From the curve obtained, the onset temperature (To), peak temperature (Tp) and enthalpy (ΔH) were calculated.

Protein solubility of APPI

Protein solubility was determined according to the modified methods of Rodriguez et al. [23]. In summary, 200 mg of APPI were dispersed in 20 mL of distilled water and pH of the mixture was adjusted to 2, 4, 6, 8, 10, with 1 N HCl and 1 N NaOH.

The mixture was then stirred at room temperature ± 5 °C for 30 min. and centrifuged at 4000 rpm for 30 min. Protein concentration in the sample was determined using Biuret method at test 540 nm wavelength. Results were expressed as mg protein/mL and were calculated from a standard curve of bovine serum albumin at concentrations range of 1–10 g/L. All analysis was performed in duplicate. Protein solubility was then calculated by the following Eq. (1):

(1)

Protein solubility % =Protein content in supernatant

Total protein content in sample× 100

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Emulsion capacity and emulsion stability of APPI

Emulsion capacity was evaluated according to Marshall et al. [18] method. One gram of proteins (APPI, SPI, EW and WPI) were whipped with 100 mL of distilled water at different pH ranges (2, 4, 6, 8, and 10) using 0.1 M HCL or 0.1 M NaOH and homogenized using kitchen blender at speed 2 (7000 rpm) to disperse the protein thoroughly. Refined corn oil was added at about 0.5 mL/s from a burette until the emulsion became thick and attained maximum vis-cosity. The rate of oil addition was reduced to 3–4 drops until the emulsion reached the breaking point, at which oil and water were separated into two phases. Emulsion capac-ity (EC) was expressed as mL of oil emulsified per gram of protein isolate before phase inversion. The emulsifica-tion process was carried out at room temperature (i.e., about 23 ± 5 °C). The emulsion was transferred into 250 mL gradu-ated cylinders and the emulsion stability was recorded after 0.25, 50, 1, 2, 3, 24 and 48 h at room temperature ± 5 °C by noting the amount of water separated from the oil [8]. All analyses were performed in duplicate.

Foaming capacity and stability of APPI

Foam capacity and foam stability of APPI were determined according to the method described by Dipak et al. [8]. In summary, 1 g of APPI was whipped with 100 mL of distilled water at different pH ranges (2, 4, 6, 8, and 10) for 5 min. using high speed blender (Waring blender, Model E8140) at speed 7 (22,000 rpm). The blend was immediately trans-ferred into a 250 mL graduated cylinder and the volume increase after whipping was measured.

Foam stability was evaluated based on volume change in a graduated cylinder after 120 min of storage. All analy-sis was performed in duplicate. Foam capacity (FC) was expressed as shown in Eq. (2):

Oil and water absorption capacity of APPI

Oil and water absorption capacity of APPI were determined following the procedure of Beuchat [4]. 1 g of the APPI was mixed with 10 mL of corn oil or distilled water for 30 s in a 25-mL centrifuge tube. The samples were allowed to stand at 25 °C for 30 min. and then were centrifuged at 2500 rpm for 30 min. The volume of the supernatant was measured in a 10 mL graduated cylinder. Results were expressed as grams of corn oil or water absorbed per gram of sample, taking the density of corn oil and water as 0.9 and 1 g/mL, respectively. All analyses were performed in duplicate.

(2)Foam capacity% =Volume after whipping − Volume befor whipping

Volume before whipping× 100

Minimum gelling concentration (MGC) of APPI

The gelling minimum concentration (GMC) of APPI was determined as follow [23]. In summary, solutions with vari-ous concentrations (1–20%) of APIP proteins were prepared in distilled water at acidic condition (pH was adjusted to two using 0.1 N HCl). The solutions with the prepared concen-tration (100 mL) were boiled for 30 min with constant stir-ring, followed by rapid cooling by immersion in tap water. Minimum gelling concentrations were calculated as the con-centration when a sample fails to slide down across the tube wall when tubes were inverted.

Experimental design and statistical analyses

Each treatment carried out in duplicate. The design followed in the experiment is CRD. Analysis of variance (ANOVA) was carried out on physical treatments data using JMP ver-sion 10.0 (SAS institute, Cary, NC). A level of 5% prob-ability between treatments was calculated based on the least significant differences (LSD). Aleppo pine seeds proteins characteristics were compared with egg, SPI and whey proteins.

Results and discussion

Proximate composition of Aleppo pine seeds

Aleppo pine seeds consist of 31.25% oil (dry matter) that is considered a very good source of oil. Results are in line with results reported by Tukan et al. [25]; while it was greater than that reported by Bagci et al. [3]; 21.1% and less than that reported by Cheikh-Rouhoua et al. [6]; 43.35%. The variation in oil content of Aleppo Pine Seeds was attributed to the differences in seeds source that may vary in its degree

of maturation, cone’s age as well as due to the variations in environmental and ecological conditions.

The protein content of Aleppo pine seed was (29.15%) and agrees with that reported by Tukan et al. [25]; while it was greater than those reported by Cheikh-Rouhoua et al. [6]; 22.7%. Variation in protein content of Aleppo pine was related to the variation in climatic condition, since Aleppo pine in Jordan exposes to elevated temperatures and water stress compared with that grown in Tunisia and Australia, these climatic stresses may cause elevation in protein con-tent of the seeds [25]. Aleppo pine seeds; 7.33%, which was comparable to those reported by Cheikh-Rouhoua et al. [6];

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8.3%. The moisture content of Aleppo pine seeds in this work was 7.57% and total carbohydrate was 24.7%.

Figure 1 shows the SDS-PAGE electrophoresis of APPI in comparison to standard proteins molecular weights (Kdal). Results show that APPI proteins molecular weights are smaller than 45 Kdals.

Thermal properties of APPI

The onset and peak gelatinization temperatures and gelatini-zation enthalpy of APPI and SPI were presented in Table 1. Gelatinization temperature was statistically lower for SPI than APPI but gelatinization enthalpy was lower for the lat-ter. However, the gelatinization enthalpy of APPI (277.2 J/g) was significantly lower than that of SPI (324.9 J/g), indicat-ing that APPI require less energy to melt. The lower enthalpy is probably explained by the weakening of hydrogen bonding by the hydrophobic alkenyl amino acids.

Protein solubility of APPI

Protein solubility is one of most important functional prop-erties that influence other functionalities such as emulsifica-tion, foaming and gelation [9]. The solubility of the APPI, EW, SPI and WPI at different pH values are presented in Table 2. Solubility was shown as U-shaped pattern, which are typical and similar to SPI [28].

APPI exhibited a minimum solubility at pH 4 (10.2%), which indicate that the isolectric point of the protein is

around pH 4. At isolectric point, proteins carry no net due the balance of between the positive and negative charges; i.e. the electrostatic repulsion is low and less water inter-acts with protein molecules promoting protein aggregation and precipitation via hydrophobic interactions [9]. Moving away from isolectric point, protein solubility increases on either side, which is attributed to electrostatic repulsion and hydration of the charged residues. The protein solubility of APPI at pH 2 (43.4%) and 8 (41.6%) were not significantly different (p ≥ 0.05). However, protein solubility 10 (48.4%) was significantly (p ≤ 0.05) greater than that at pH 2. In comparison between the solubility of APPI and SPI; results indicated that both proteins showed similar behavior. The isolectric points of the two proteins were between pH 4 and 4.5. Furthermore, APPI and SPI solubility at pH 8–10 were not significantly different (p ≥ 0.05). However, the solubility of APPI at pH 2 and up to pH 8 were significantly (p ≤ 0.05) greater than those observed for SPI. A result attributed to the greater content of hydrophilic amino acids at the sur-face of APPI. These results are in accordance with protein isolate solubility reported for SPI [13] and Fenugreek [9]. Moreover, our results of APPI solubility can be considered an indicator of good protein functionality such as emulsifi-cation, foaming and gelation. The low molecular weight of APPI (< 38 Kdal) is in line with the increased solubility of isolated proteins.

The results obtained for egg white protein were agreed with those of Gomes et al. [11] who reported that egg white

S APPI

200.0116.3

66.2

6.5

21.0

31.0

45.0

97.4

14.4

Fig. 1 SDS-PAGE electrophoresis of Aleppo pine protein isolates (APPI) and standard proteins molecular weights (S) (Kdal)

Table 1 Thermal properties of Aleppo pine protein isolates (APPI) and soybean protein isolate (SPI)

Protein isolate source

Gelatinization enthalpy (J/g)

Gelatinization tem-perature (oC)

Onset Peak

APPI 277.2 90.8 99.3SPI 324.9 81.5 93.2

Table 2 Solubility (%) of Aleppo pine protein isolates (APPI), egg white (EW), soybean protein isolate (SPI) and whey protein isolate (WPI) at various pH

*Means within the same column with different letters (smalls) are significantly different (p < 0.05) according to LSD. Means within the same row with different letters (capitals) are significantly different (p < 0.05) according to LSD

Protein source pH

2 4 6 8 10

APPI 44.4bA* 10.2dC 18.8cB 41.6bA 47.4bA

EW 86.9aBC 32.0bD 84.4aC 96.8aAB 100.0aA

SPI 32.0cB 14.9cC 33.6bB 43.9bA 46.8bA

WPI 82.1aB 66.0aC 80.2aB 91.4aAB 100.0aA

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protein solubility was affected by pH and most soluble above their isoelectric point than other proteins which is due to the fact that egg white is a mixture of different proteins [19]. The authors studied egg white solubility at different pH and NaCl concentrations. The authors reported the lowest solu-bility at pH 4.6 and maximum solubility at pH 9. Al-kahtani et al. [1] compared the functional properties of Moringa per-egrine and SPI and they found that the solubility of both protein isolates was the lowest between pH 4 and 4.5, (the pI region). The solubility increased at both sides of the iso-electric point region.

Pelegrine et al. [20], on the other hand, reported that solu-bility of whey proteins are affected by temperature and pH changes. The authors also observed an interaction between the temperature and pH with lowest solubility value at the isoelectric point. In general, the solubility of whey protein isolates and egg white proteins was not significantly different (p > 0.05) at acidic and basic pH. However, the solubility of whey protein isolates at isoelectric point was significantly greater (p < 0.05) than that of egg white protein. Results are might be due to the fact that whey protein isolates contain significant amounts of surface polar amino acids which enhance its solubility at isoelectric point.

Results clearly demonstrated dividing protein solubility into two groups; the animal proteins (whey protein isolates

and egg white protein) and the plant proteins (APPI and SPI) which show different solubility at different pH values. Animal sourced proteins solubility were significantly greater (p < 0.05) than those of plant proteins. These results might be related to the fact that animal proteins consist of several protein units and there complex interactions among them with wide-range of physiochemical properties of each unit making them ideal performed in multiple functions.

Emulsion capacity of APPI

The effect of pH on the emulsification capacity of APPI, EW, SPI and WPI at different pH values are presented in Table 3. In general the emulsion capacity versus pH of both proteins closely similar to their solubility indicating that solubility of protein is essential for protein to act as emulsifier. Minimum emulsion capacity (about 10 g oil/g protein) for both proteins occurred at or near isoelctric point (pH 4–4.5). The emul-sion capacity of Aleppo pine and SPI was not significantly different at pH 2 and 4. However, the emulsion capacity of SPI at pH greater than 4 and up to pH 10 was greater than those found for APPI. The emulsion capacity of the two proteins at pH 2 was about 217 g oil/ g protein. However, at pH 6, 8 and 10 the emulsion capacity of SPI was 2.7, 1.2 and 1.34 times greater than the corresponding values found for

Table 3 Emulsion stability (mL) and capacity (mL/g) of Aleppo pine protein isolates (APPI), egg white (EW), soybean protein isolate (SPI) and whey protein isolate (WPI) at various pH

*Values represent volumes in mL of water separated at room temperature**Means within the same pH of the column having different small letters are significantly different (p < 0.05) according to LSD

pH Protein source Time (h) Emulsion capacity (mL/g)0.25 0.5 1 2 3 24 48

2 APPI 0* 0 0 0 0 0 0 220.0cA**

EW 0 0 0 0 0 0 0 245.0bA

SPI 0 0 0 0 0 0 0 215.0cA

WPI 0 0 0 0 0 0 0 302.0aB

4 APPI 95 96 98 100 100 100 100 10.0cE

EW 0 0 0 0 0 0 0 215.0bB

SPI 90 98 98 100 100 100 100 10.0cD

WPI 0 0 0 0 0 0 0 262.5aC

6 APPI 50 78 78 90 94 96 96 55.0dD

EW 0 0 0 0 0 0 0 175.0bC

SPI 20 38 44 46 46 48 50 150.0c

WPI 0 0 0 0 0 0 0 310.0aB

8 APPI 0 0 0 0 0 0 0 145.0dC

EW 0 0 0 0 0 0 0 255.0bA

SPI 0 0 0 0 0 0 0 175.0cB

WPI 0 0 0 0 0 0 0 375.0aA

10 APPI 0 0 0 0 0 0 0 160.0dB

EW 0 0 0 0 0 0 0 265.0bA

SPI 0 0 0 0 0 0 0 215.0cA

WPI 0 0 0 0 0 0 0 362.0aA

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APPI. Although, the solubility of Aleppo pine seed protein isolate at pH 2, 8 and 10 were not significantly different, the emulsion capacity of this protein at pH 2 was significantly greater than those at pH 8 and 10. At pH 4 egg white protein and whey protein isolates records higher emulsion capacity than that of SPI and APPI.

The results obtained for SPI disagree with those of Al-Khatani et al. [1] who reported that the emulsion capacity of this protein was higher in the basic side than that in the acidic one. This indicates that there are other factors that affect the emulsion capacity. Similar relationship between emulsion capacity and pH has been reported by other work-ers [1, 9].

In general, the emulsion stability of SPI and of APPI was similar; stable emulsions were obtained at the acidic and basic pH. However, at pI regions the stability of these emul-sions was low; since they separated totally at pH (4) after 48 day of standing, while at pH 6 SPI provided more stable than that of APPI. The emulsion stability found in the pre-sent study for SPI at pH 8 was greater than that reported by Al-Kahatani et al. [1].

The emulsion capacity of egg white showed a minimum value at pH 6 which increased gradually with the increased or decreased of pH. Table 3 showed that the maximum capacity was at acidic pH (2) and alkaline pH (8, 10) with no significant differences (p < 0.05) between treatments. In contrast to that observed for the solubility the minimum emulsion capacity was at pH 6 compared to pH 4 for egg white solubility. Results were attributed to the fact that con-taining a mixture of different proteins could participate in different functionalities in formation and stabilization the emulsion. Similar observations on the relation of pH and

emulsification properties of sunflower, cashew and walnut proteins have been reported by Mao et al. [16].

Al-kahtani et al. [1] compared functional properties of M. peregrine and SPI and they found that emulsion capac-ity of both protein isolates was between pH 4 and 4.5, the pI region and increased at both sides of the pI region. However, M. peregrine protein isolates was reported to have higher emulsion capacity and stability than SPI.

Emulsion stability of APPI

Emulsion stability of APPI, EW, SPI and WPI at different pH values are presented in Table 3. Results showed similar trends of APPI and SPI emulsion stability. On the contrary, EW and WPI proteins showed stable emulsions at all pH value tested without any separation of water during 48 h of standing at room temperature ± 5 °C. APPI and SB emul-sions at pI were unstable since complete separation was observed after 2 h of storage. Emulsion stability of those proteins was stable at acidic and basic pH while at pI and regions around pI emulsions were totally separated after 48 day of standing for both proteins.

Foaming capacity and stability of APPI

Figure 2 shows that the foam capacity for both proteins was pH-dependent. One of the factors that affect foam capacity of protein is its flexibility which facilitates its migration to the water–air interface [9]. The ANOVA results showed that the foam capacities for both proteins (APPI and SBI) at pH 2 and 10 were the highest and they were not significantly different (p ≥ 0.05). This indicate that at these two pHs there

Fig. 2 Foaming capacity (%) APPI, EW, SPI and WPI at dif-ferent pH ranges

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was an appropriate balances between protein solubility and surface hydrophobicity which impart these proteins the high-est flexibility. However, foam capacity for APPI at pH 4, 6 and 8 was significantly (p ≤ 0.05) lower than that of SPI. One of the factors that affect foam capacity of protein is its flexibility which facilitates its migration to the water–air interface [9]. The lowest amount of foam capacity for APPI (20%) at pH4 and for SPI at pH 4.5 might be due to the strong interactions between protein molecules, indicating that they are at the lowest flexibility. The results in the pre-sent study were in agreement with those of Al-Kahtani et al. [1] and Fezyi et al. [9] who reported that foaming capacity and stability of proteins are pH-dependent.

Foam stability results of APPI at different pH values are shown in Table 4. Foam stability (FS) describes the abil-ity of the proteins to form strong cohesive film around air bubbles that withstands gravity and mechanical stress [7]. Table 4 shows that the highest foam stability of APPI and SPI after the 2 h of standing was at pH 2 and 10 followed by that at pH 6. The separated water from SPI and APPI foams at pH 2 was 17.6 and 36.8 mL; while that at pH 10 was 21.4 and 37 mL, respectively. The stability of foams in the acid and base sides might be due to formation of stable cohesive layers at the air–liquid interface that promotes texture and stability of the foam [1]. Table 4 also shows that the foam stability of SPI at pH 2, 8 and 10 was significantly (p ≤ 0.05) greater than the corresponding APPI foams. The volume of the separated water after the 2 h of sanding from APPI foams at pH 2 and 10 was 2.1 and 1.7 time, respectively than that of the corresponding SPI foams. However, APPI foam showed higher stability at pH 4 and 6. The SPI foam at pH 4 was completely collapsed after 2 h of standing. This could be due to the higher solubility, which may in thickener protein film at the water–air interface enhancing their foam capacity and stability.

Results also indicated a slight effect of pH on Whey protein isolate foaming capacity with minimum capacity

observed at pH 6 and 8 with foam capacity of 99% and 101% respectively. Maximum foam capacity was found at pH 10 (127%) and pH 2 (123%) (Fig. 2). Whey protein isolate pro-vide approximately stable foam at pH 6 and pH 10 with 44.49 and 44.85% total decrease of foam volume that created during the 120 min of standing (Table 4). Whereas, the foam at pH 4 and 8 showed significantly (p < 0.05) lower stabil-ity with 54.17 and 62.26% total decreased in foam volume (Table 4).

Our results are in agreement with those of Wong et al. [26]; Al-Kahtani [1] and Mao et al. [16] who reported that foam stability sharply decreases at the isoelectric point and increases by moving away from both sides of the pH scale.

Water and oil absorption capacity of APPI

The Water Absorption Capacity (WAC) and Oil Absorption Capacity (OAC) of APPI and SPI are represented in Table 5. The WAC for APPI was significantly (p ≤ 0.05) less than that of SPI. The WAC of SPI used in the present study was significantly greater than that reported by Al-Kahtani et al. [1]: 2.1 g/g SPI, while it is comparable to that reported by Fezi et al. [9]; 5.95 g/g SPI. Water adsorption capacity of proteins could be influenced by several factors such as amino acid composition, the ratio of surface polarity to hydropho-bicity, its content of oil [15]. On the other hand, WAC is an important factor in viscous food such as soup, spaghetti and in some bakery products such as bread and cake. In contrary that that observed for WAC, OAC for APPI was significantly greater than that found for SPI. The OAC for APPI was 1.7 times greater than that observed for SPI.

Oil absorption was attributed to the hydrophobic inter-action between the hydrocarbon chains of the fatty acids of the oil with hydrophobic amino acids. The lower WAC and the higher OAC of APPI indicate that this protein contain more hydrophobic amino acids than that of SPI. The importance of the capacity of proteins to absorb and retain oil and to interact with lipids is important in food

Table 4 Foam stability (%) of Aleppo pine protein isolates (APPI), egg white (EW), soybean protein isolate (SPI) and whey protein iso-late (WPI) at various pH

*Means within the same column with different letters (smalls) are significantly different (p < 0.05) according to LSD. Means within the same row with different letters (capitals) are significantly different (p < 0.05) according to LSD. Foaming stability measured as volume (mL) of water separated at room temperature

Protein source pH

2 4 6 8 10

APPI 36.8bC* 71.1bA 61.0cA 52.8cB 37.0bC

EW 41.8bC 100.0aA 100.0aA 70.5aB 73.2aB

SPI 17.6cD 100.0aA 78.6bB 37.8dC 21.4cD

WPI 100.0aA 54.2cBC 44.5dC 62.3bB 44.9bC

Table 5 Oil and water absorption capacity (g/g) of Aleppo pine pro-tein isolates (APPI), egg white (EW), soybean protein isolate (SPI) and whey protein isolate (WPI) at various pH

*Means within the same column with different letters are significantly different (p < 0.05) according to LSD. WPI and EW are completely soluble in water

Protein source Oil absorption capacity (OAC) (g/g)

Water absorption capacity (WAC) (g/g)

APPI 3.1 ± 0.14b* 3.9 ± 0.14c

EW 4.5 ± 0.71a 10.0a

SPI 1.8 ± 0.28c 5.6 ± 0.57b

WPI 3.0 ± 0.71b 10.0a

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formulations as formation of emulsions, fat entrapment in sausages, flavor absorption, and dough preparation. Determination of oil absorption capacity of certain pro-teins gives information whether selecting the protein as a raw material in food processing or not [5].

Minimum gelling concentration (MGC) of APPI

Minimum Gelling Concentration (MGC) is a qualitative parameter that expresses the minimum protein concentra-tion at which the gel does not slide along the test tube walls in inverted position, the lower the gelation concen-tration the better is the gelling ability of proteins [10]. It was observed that APPI didn’t show any ability to form gel at neutral pH. On the other hand, acidic solution of AP protein isolates (at pH 2) showed a good gelling capacity with MGC of 6%. Similar results were reported by Ragab et al. [22] who reported that the MGC of cowpea protein isolate was 6% and by Porras-Saavedra et al. [21] who found that the GMC of Lupinus albus was 7.5%. Since gelation is important in food industries because a protein with a low value of MGC can be a good thickening agent. Thus, AP protein isolate at concentration 6% could be considered a good thickening material and used in food industries.

Conclusion

Aleppo pine seeds contain 11% pure protein and pos-ses approximately similar functional properties, and in some cases even better, to those of SPI. Foam stability was better for APPI than those of SPI. The low gelling minimum concentration of APPI could make them suitable and applicable ingredient in wide range of food products. APPI is considered a good source of protein and can be introduced effectively in the food industry. Further studies are currently undergoing to measure sensory and consum-ers acceptability of food products made from Aleppo pine protein as a replacer of egg white protein.

Compliance with ethical standards

Conflict of interest The authors declare that they do not have any conflict of interest.

Ethical approval This study does not involve any human or animal testing.

Informed consent Written informed consent was obtained from all study participants.

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