INTRODUCTION:

Pumpkins belong to the genus Cucurbita, from Cucurbitaceae family, which consists of about 130 genera and 800 species. The popularity of pumpkin in many systems of traditional medication has led researchers turning their attention to this crop¹. Bioactive compounds in pumpkin seeds exhibit promising activities such as anthelmintic, antidiabetic, antidepressant, antioxidant, antitumor and cytoprotective². Pumpkin seeds are a rich source of iron, protein, manganese, magnesium, zinc, potassium, copper, phosphorous, unsaturated fatty acids, tocopherols and carotenoids³. There are several types of pumpkin grown around the world such as Cucurbita maxima, Cucurbita pepo, Cucurbita muschata, is used as food in the preparation of jam and candy or as fodder ^4,5.

Pumpkin has attracted increasing attention from scientists due to its nutritional profile⁶. Pumpkin Seed Protein Isolate is rich in antioxidants and effective in reducing the harmful effects of malnutrition. Many plants nutrients have many effects on several chronic and deadly diseases^7,8,9,10.

The use of plant proteins, especially from grains and oilseeds, has increased dramatically over the past decades as they have been used as a substitute for animal proteins in human nutrition, functional factors, and bioactive ingredients in food, cosmetics and pharmaceutical products¹¹. Usually, plant proteins are used as a protein isolate or protein concentrate, but their use may be limited due to some inappropriate functional properties such as low solubility, and to expand their field of use there are a number of alternative techniques to improve the functional properties of proteins by increasing the nutritional value and bioactivity^12,13. Plant proteins can be modified using chemicals, enzymatic modifications, or physical treatments using high temperature, pressure and ultrasound^14,15,16.

The objective of this study was to find the best substitution percentage of pumpkin seeds protein isolate in meat burger in order to reduce costs.

MATERIALS AND METHODS:

The local pumpkin seeds were used that were obtained from local markets.

Pumpkin seeds were cleaned, washed with distilled water, and dried with air at room temperature, and a certain amount of seeds were soaked with 1/5 distilled water (seeds /water) for 8 hours, and then they were also dried with air at room temperature¹⁷. Then it was ground with a laboratory mill, sieved with a 200-mesh sieve, then preserved in polyethylene bags at the refrigerator temperature until use.

Prepare defatted pumpkin seed powder:

The powder was prepared according to¹⁸ by treating ground pumpkin seed powder soaked, not soaked, and peeled (pulp only) with two types of solvents separately, Petroleum ether 40-60°C and a mixture of chloroform/ methanol in a ratio (3: 1 volume/volume). The ratio of the seed powder: solvent used is 1:10 (weight/volume) with continuous stirring by the magnetic stirrer for a period of 24hours, then the solvent was separated using a central centrifuge and the sediment was taken and dried at room temperature, and then stored in an airtight bottle at the refrigerator temperature until Usage.

A protein measurement test was performed for all the defatted samples (soaked, not soaked, peeled* pulp only *) in order to select the best solvent and form for use later in the study, using the standard Microkjeldal method¹⁹ and the protein percentage was extracted by multiplying the percentage of nitrogen in the sample by a conversion factor of 6.25.

Preparation of protein isolate:

The protein isolate of defatted pumpkin seed powder (peeled) was prepared according to the method mentioned by¹⁸, and stored until use.

Manufacture of burger:

Meat burger was made according to the method described by²⁰, using pure ground beef 85%, 1.5, 2, (2-5)% salt, spices (black pepper, crushed garlic) and filler, and cold water for the kneading, and the substitution was (0%, 10%, 20%) with protein isolate, which are named (A, B, and C) respectively, mixed well and formed with a weight of 100g per each, kept in the refrigerator for 4 hours, until it was grilled and had some physical tests done.

Physical Characteristics of burger during the manufacturing stages:

Measurement of Diameter reduction:

The aforementioned method was adopted by²¹ in measuring the percentage of shrinkage by diameter, and diameter was measured using vernier and in three specific locations in the Barker model with four replications per treatment, and the percentage of shrinkage per diameter was calculated:

R1- R2

Diameter reduction (%) = ----------- x 100

R₁= Diameter of burger sample before cooking.

R₂= Diameter of burger sample after cooking.

Water Holding Capacity WHC:

The water holding capacity of the protein isolate before adding it to the burger was estimated according to the method described by²¹ with some modification, as one mg of the sample was placed in a pre-weighed 10ml test tube, then 1ml of distilled water was added and stirred by a vortex magnetic stirrer for 10minutes at room temperature. The centrifugation was carried out at a speed of 5000g for 10minutes at room temperature, and the holding capacity of water was calculated:

W₁ - W₂

WHC = ----------

W₀

W₀ =Dry sample weight.

W₁ =Weight of the tube + weight of the precipitate after adding the water.

W₂ =Weigh the tube + the dry sample before adding water.

The water holding capacity of the burger sample (A, B, C) was estimated according to the method mentioned by²¹, as mentioned above.

Sensory evaluation:

The sensory evaluation of the burger sample was carried out by 10 people specialized in food processing. The method mentioned by²² in estimating the degrees of perceptual appreciation of the product was followed after grilling process. The characteristics included color, flavor, juiciness, freshness and the degree of general acceptance (Table 1).

Table 1. Scores of the evaluation questionnaire for the perceptual characteristics of burger

Scores	Color	Flavor	Juiciness	Freshness	General acceptance
7	Very acceptable	Strong flavor	Very juicy	Too mushy	Very acceptable
6	Acceptable	Medium flavor	Juicy	mushy	Acceptable
5	A little tolerable	Little flavor	A little juicy	Slightly tender	A little tolerable
4	Center	flavourless	Center	Center	Center
3	A little unacceptable	A little unacceptable flavor	Little dehydration	Slightly tough	A little unacceptable
2	Unacceptable	Medium unacceptable flavor	dry	Solid	Unacceptable
1	Very unacceptable	Very unacceptable flavor	very dry	Very solid	Very unacceptable

• Tenderness can be determined after five chewing and a feeling of chewing strength.

• Juiciness can be recognized after the first chew and the amount of moisture in the mouth after differentiating moisture from saliva.

Statistical analysis:

Statistical Analysis System -SAS (2012) was used in analyzing the data²³.

RESULTS AND DISCUSSION:

Defatted pumpkin seed powder:

The percentage of protein in defatted pumpkin seeds when using petroleum ether solvent was 37.12 and 34.12%, respectively in unsoaked and soaked seeds. The protein ratio for the two types of seeds above, but using chloroform/methanol mixture (1:3 volume/volume) instead of petroleum ether was 33.25 and 32.15% respectively. In peeled defatted not soaked seeds using petroleum ether outperformed the rest of the test sample in terms of protein content, (53.15%) and on this basis this type of seed was used in the preparation of protein isolate later (Table 2). Some researches indicated that the percentage of protein in defatted peeled pumpkin seeds (Cucurbita maxima) using petroleum ether (40-65° C) was 42.75%²⁴.

Table 2. Determination of protein in defatted pumpkin seeds soaked, unsoaked and peeled

Seed treatment	Solvent	Protein (%)
Whole soaked	Chloroform / Methanol(1:3v/v)	32.15
Whole unsoaked	Chloroform / Methanol(1:3v/v)	33.25
Whole soaked	Petroleum ether (40-60° C)	34.12
Whole unsoaked	Petroleum ether (40-60° C)	37.12
Peeled unsoaked	Petroleum ether (40-60° C)	53.15

Preparation of protein isolate:

In peeled pumpkin seeds after defatted, every 100 grams of dried peeled seeds gives approximately 59 grams dry weight of defatted seed powder,(Fig1) and this weight of the powder when used to prepare the protein isolate gives approximately (18 grams dry weight) protein isolate, meaning and the percentage of protein in this protein isolate is 63.87.

Figure 1 a: the protein isolate before drying b: the protein isolate after drying

Physical tests of the substituted burger:

Water holding capacity (WHC) of protein isolate:

The water holding capacity of the protein isolate was 4 mg/ml. A research indicated that the water absorption capacity of the defatted pumpkin seeds of Cucurbita moschata was 1.72mg/ml, while the water holding capacity of the decomposers increased and this may be due to polar groups such as –COOH and –NH2 increased during enzymatic hydrolysis²⁵. Among the four hydrolysates used, protamex hydrolysates had the highest water preservation capacity, (2.24 mg/ml). In alcalase hydrolysates, they were less water preserving, (1.68 mg/ml). This is due to the difference in the water holding capacity of the protein isolated from the defatted seeds, as the protein isolate increases the protein concentration, as well as to the difference in the variety used. A research mentioned that water holding capacity of defatted pumpkin seeds is 1.99 g water/g product⁴.

Shrinkage by diameter and water holding capacity (WHC) of the burger after grilling:

The results in Table (3) show the measurement of the shrinkage in the diameter of the burger after grilling, as the decrease in the diameter for the control sample (A) was 25.5%, while the decrease in the diameter for (B, C) samples in which (10, 20%) of the meat was substituted with protein isolate 16.6%, 16.6%. The highest water carrying capacity of sample B, which amounted to 54.52% compared to the control sample (A) and sample (C), was 28.0% and 23.2%, respectively. The increase in water absorption capacity depends on the large ability of the protein isolate to swell and open and then expose the sites of a new correlation, as for the decrease in the (C) sample, it may be due to the protein reaching the saturation point, and this was reflected in the characteristics of the sensory evaluation mentioned later^26,27,28.

In a study of substituting meat in chicken sausage with vegetable proteins, indicated that the use of plant proteins as a substitute for meat led to a significant change in the shrinkage of samples as no shrinkage was observed in the sample of the sausage without chicken, and this indicates the ability of the soy protein isolate²⁹. The user and the excellent gluten in preserving the mesh structures after cooking, and adding 20% of chicken led to a shrinkage of the diameter by 0.92% and increased to 10.93% when increasing the proportion of chicken to 60% and this may be due to the denaturation of proteins during cooking, which caused the release of liquids and change composition and shrinkage.

In a study of preparing a modification chicken burger by partial replacement of chicken meat with mushroom powder in ratio (0, 10, 15%) showed that the modification sample with 15% substitution was the best in terms of change in diameter compared to the control sample, as it had the highest value of reduction in diameter, for the control sample, which amounted to 18.04%, while this was decreased in the replacement sample to10, 15%, as the decline in diameter reached 8.38 and 5.95%, respectively²¹. It was also found that the replacement ratios had a significant effect in increasing the water holding capacity of 30.13, 52.25 and 58.03% in the control sample and the replacement sample 10 and 15% respectively.

Table 3. Measurement of shrinkage in diameter and water holding capacity WHC for burger samples after grilling

Sample	Shrinkage in Diameter (%)	WHC (%)
A	25.5	28 %
B	16.6	54.52%
C	16.6	23.2%

Sensory evaluation of burger sample after grilling:

Color: There was a significant difference in color between the B sample (6.0) compared to the C sample (5.3), while there were no significant differences between the B sample compared with the control sample A, and this indicates the proximity of the color characteristics in the two samples (Table 4).

Flavor: There was no significant difference in flavor for the control sample A (6.2) with sample B (5.8), while there was significant superiority for the control sample A compared with sample C (5.4). This indicates the proximity and acceptance of sample B in terms of flavor compared to sample C, which did not outperform significantly. The samples were slightly spicy due to the quality of the black pepper, which affected the flavor acceptance.

Juiciness: The sample B had a significant superiority in juiciness (5.5) over the sample C (4.2), and there was no significant superiority of sample B over the control sample A (5.2), and sample A was significantly superior to the sample C. This explains the reflection of the increased water holding capacity in sample B.

Tenderness:

There were no significant differences in the softness between the control sample A (5.5) compared with the replacement sample B (5.3). This indicates the proximity of the softness of the control sample with sample B, while both sample A and B were superior to the replacement sample C (4.5).

General acceptance:

The values of the control sample A and the 10% substitution sample B equaled the general acceptance (5.5), while the values of these two sample significantly exceeded the 20% replacement sample C (4.6). This indicates the acceptance of the replacement sample B.

Total:

The values of the control sample A and the substitution sample 10% B were equal in the final total (28.1), and thus significantly outperformed the 20% replacement sample C (24.0).

Sample B is the best sample, with a ratio of replacing 10% of the meat with the protein isolate from pumpkin seeds and closer to the control sample. The used plant protein in the experiment is a by-product after extracting oils from pumpkin seeds and does not require a high cost to be hydrolysis by enzymes.

In a research which observed that substituting meat in chicken sausage with vegetable proteins that chicken sausage was significantly superior in taste, and there was no significant difference in terms of color, smell, and general acceptance between chicken sausage, free and reduced chicken sausage and the proportion of chicken. The last two types have better texture and appearance in general compared to the chicken sausage sample²⁹.

A research indicated that preparing a chicken Burger by partial replacement of chicken meat with mushroom powder and in ratio (0, 10, 15%) that substituting chicken meat with mushroom powder improved the juiciness and freshness characteristics as the values increased by increasing the substitution rate compared to the control sample and this increase were significant²¹. Flavor scores and general acceptability increased significantly for the replacement sample.

Other research reported that the best treatment of substituted biscuit based on the trained and consumer panelists assessment was the biscuit treatment using 20% pumpkin seed flour³⁰.

Proteins could be used as drugs for many kinds of diseases beside their actions as nutrients for health therapy^31,32.

CONCLUSION:

The use pumpkin seeds protein in preparations especially in manufacturing substituted burger with plant proteins, for its nutritional values for containing glutamic acid and arginine.

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Received on 24.04.2024 Revised on 18.12.2024

Accepted on 06.05.2025 Published on 01.07.2025

Available online from July 05, 2025

Research J. Pharmacy and Technology. 2025;18(7):3089-3093.

DOI: 10.52711/0974-360X.2025.00443

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sample	Color	Flavor	Juiciness	Tenderness	General acceptance	Total
A	5.7	6.2	5.2	5.5	5.5	28.1
B	6.0	5.8	5.5	5.3	5.5	28.1
C	5.3	5.4	4.2	4.5	4.6	24.0
LSD (P≤0.05)^*	*0.559	*0.702	*0.784	*0.894	*0.819	*3.078