1. Introduction
Cucurbita moschata L. has great economic and social relevance, being a widely consumed fruit, with great acceptance for its sensorial and nutritional characteristics [
1]. The consumption of pumpkin is considered beneficial to health due to its composition of essential vitamins, being a source of minerals, proteins, as well as poly- and monounsaturated fatty acids, with a majority of linoleic, oleic, palmitic, and stearic acids, in addition to being a source of fiber [
2,
3]. Pumpkin seeds are rich in phenolic and antioxidant compounds, such as squalene, phytosterols, tocopherols (α, β, γ, and δ), tocotrienols, carotenoids, and flavonoids [
4,
5]. They are also a valuable source of proteins (24–40%) and lipids (37–46%), with a predominance of linoleic (42.6–46.3%), oleic (18–30%), stearic (9.5–18.7%), and palmitic (12.3–18.1%) fatty acids, minerals (magnesium, phosphorus, potassium, sodium, calcium, iron, copper, manganese, zinc, and selenium), and fiber (10–25%) [
4]. Based on their composition, pumpkin seeds demonstrate nutraceutical properties [
1] and can be used as a dietary supplement to help curb deficiency disorders, as well as to prevent cardiovascular diseases [
6,
7,
8].
A diet enriched with pumpkin seeds brings significant benefits, such as reduced serum triglyceride levels, increased fecal excretion of cholesterol, and decreased lipid accumulation in hepatocytes [
9]. Nonetheless, pumpkin seeds feature the same antinutritional factors that may restrict their application in food formulations, such as phytate, oxalate, hydrocyanic acid, and nitrate [
2]. These compounds can diminish protein digestibility and amino acid bioavailability, consequently impacting the protein’s overall quality. Furthermore, the elevated levels of phytic acid in pumpkin seeds may exacerbate these antinutritional effects by binding to minerals such as zinc, iron, and calcium, thereby reducing their absorption potential. However, the utilization of traditional methods, such as heat treatment, soaking, sprouting, and fermentation, can significantly reduce the percentage of these compounds [
4].
Pumpkin seed flour (PSF) is typically utilized for enriching soups, cookies, pancakes, and breads [
2]. There is currently a growing demand for healthier foods, particularly in the meat products sector. The presence of excessive amounts of sodium, saturated fats, cholesterol, and synthetic additives in meat products contributes to a higher risk of metabolic diseases. Thus, there is a need to produce functional and healthy foods capable of imbuing meat products with benefits that will positively impact consumer health [
3,
10].
Beef burgers are considered one of the most commercialized and consumed frozen meat products by all social classes because they are easy to prepare and have attractive sensory attributes [
10]. However, despite meat being rich in nutrients, a source of proteins, and containing all essential amino acids, there is evidence that high consumption of processed meat products contributes to the establishment of cardiovascular diseases, type 2 diabetes, obesity, and some types of cancer. In response, the consumption of meat products has been decreasing due to growing consumer concerns about their effect on human health and the environment. In order to associate meat products with functional claims that can promote health benefits, research has been developed to improve these products by associating them with vegetables. This association, in addition to contributing to the reduction in production costs, suggests the use of bioactive compounds that can improve the nutritional quality of such products [
3]. In this context, supplementing beef burgers with PSF could add nutritional, functional, and commercial value to the product.
Therefore, this research aims to develop and evaluate the nutritional and quality standard of beef burgers supplemented with Cucurbita moschata seeds added in different proportions. It is believed that PSF, due to the presence of bioactive compounds (already recognized in the literature), may add functional claims to the innovative product prepared, which may bring benefits to consumer health and assist in the promotion of technological and sustainable actions.
2. Materials and Methods
2.1. Reception of Raw Materials
Pumpkin seeds (C. moschata L.) were collected from merchants in the metropolitan region of Recife, Pernambuco, Brazil, and selected according to the species’ morphological characteristics, such as white color, flat, oval shapes, green color inside, and surfaces free of mechanical damage and visible fungal infections. The pumpkins (C. moschata L.) were grown in an integrated and sustainable agroecological production system by family farmers; the fruits were harvested 60 days after anthesis; and cooled ground beef (chuck) and the dry ingredients used to make the burgers were obtained from local commerce in Recife, Pernambuco, Brazil.
2.2. Obtaining the Flour (PSF)
In the production of PSF, the seeds with white outer hulls were washed in running water, sanitized in sodium hypochlorite solution (1%
w/
w) for 15 min, and then washed again in running water (
Figure 1). Afterward, the seeds were dried in a ventilated oven at 65 °C (±1 °C) for 24 h and cooled to room temperature. Subsequently, to obtain the whole flour, the dried seeds were crushed in an industrial blender; then, the flour was sieved (20 mesh) without residues. The flour was then packaged in polypropylene packaging and stored frozen in a conventional freezer at −18 °C (±1 °C) for subsequent analysis.
2.3. Characterization of PSF
For proximate composition, analyses were carried out in triplicate and expressed as percentages [
10]. The moisture was measured via oven (Stove for drying and sterilization-393/I, TECNAL, Rio Grande do Norte, Brazil) heating at 105 °C (±2 °C) for 24 h; the ash contents were determined via calcination in a muffle furnace at 550 °C for approximately 6 h until grayish-white ashes were obtained. Proteins were determined from total nitrogen using the Kjeldahl method, requiring the conversion of nitrogen in proteins by a factor of 5.75. Lipids were determined using the Soxhlet continuum extraction method, using hexane as a solvent. Carbohydrates were calculated as the difference—the sum of moisture, proteins, lipids, fibers, and ash content being subtracted from 100%. For the calculation of the total caloric value (TCV), the Atwater coefficient was used, which was 4.0 for proteins, 4.0 for carbohydrates, and 9.0 for lipids. Furthermore, the color was determined using a colorimeter (Colorimeter CR-400, Konica Minolta Sensing Americas, Inc., Ramsey, NJ, USA) to assess the
L*
a*
b* parameters [
11]. The enzymatic-gravimetric method was used with a previously degreased sample [
11] to determine the total dietary fiber content.
2.4. Preparation of Beef Burger
The burgers were divided into three samples: the standard S without addition of PSF (control group), F5 with the addition of 5% (
w/
w) of PSF, and F10 with the addition of 10% (
w/
w) of PFS. Chuck ground beef, onion paste, garlic paste, PSF, cold water, table salt, and black pepper were used as ingredients to formulate the burger patties, as described in
Table 1.
In the preparation of the burgers, all ingredients were separated and weighed. For the first homogenization, ground beef was mixed with the ingredients (onion, salt, black pepper, garlic). Following this initial mixture, PSF was added to the F5 and F10 samples; cold water was then incorporated, which was the second homogenization, aiding in uniformization of the other ingredients. The meat mass obtained was divided into three portions, the first being the standard one (S), with no addition of PSF (control group), and the other two samples having PSF added in the concentrations of 5% (F5) and 10% (F10).
The patties were shaped using a manual mold measuring 11 cm in diameter and 1.5 cm in height for standardization, weighing an average of 56 g, individually packed in polyethylene bags, and frozen in a freezer at −18 °C until the analyses were carried out.
2.5. Characterization of the Burgers
For the proximate composition, the analyses were carried out according to the methodology described in
Section 2.3.
To analyze the raw burgers’ water holding capacity (WHC), 5 g of each sample was weighed in test tubes and centrifuged at 3600 rpm for 15 min. The WHC was expressed as a percentage of water holding capacity. The WHC was calculated using the following Equation (1) [
12]:
where
Wac is the sample weight after centrifugation, and
Wbc is the sample weight before centrifugation.
The lipid oxidation determination analysis (TBARS) was carried out at 0, 30, and 60 days of production. Approximately 5 g of the cooled burger samples was placed in triplicate, mixed in 25 mL of trichloroacetic acid 7.5% (TCA), manually homogenized for 1 min, and filtered using qualitative filter paper. After filtration, to quantify malonaldehyde, 4 mL of the filtrate was collected and transferred to the test tubes with lids, to which 5 mL of the 2-thiobarbituric acid (TBA) solution and 1 mL of TCA 7.5% were added. All tubes were homogenized and kept in a hot water bath (90 °C) for 40 min; afterward, they were cooled to room temperature for absorbance reading. To this end, an aliquot was collected for absorbance reading at 538 nm for the spectrophotometer. The TBA values were expressed in mg MDA per kg of sample by means of the standard curve using 1,1,3,3-tetratoxypropane (TEP) [
13].
The burger extracts were evaluated for total phenolic content [
14] using 20 µL of the extract with 100 µL of the Folin–Ciocalteu reagent diluted in water (1:10). After 3 min, 80 µL of the sodium carbonate solution (75 g/L) was added. After 2 h at room temperature (25 °C), the samples’ absorbances were read in a spectrophotometer (BioTek μQuant Biospectro, Winooski, VT, USA) at 765 nm. A standard curve of gallic acid (10–60 mg/L) was used to calculate the results expressed in mg of gallic acid equivalent per 1 g of dry extract (mg GAE/g).
2.6. Characterization of the Burger after Cooking
For the determination of yield and weight loss, the burgers were defrosted at 4 °C for 12 h. The raw burgers were weighed and baked in an electric stove at 180 °C for 30 min until they reached an internal temperature of 72 °C, which was controlled using a thermometer. After baking, they were weighed again [
10]. The results were calculated according to Equation (2).
The shrinkage percentage was calculated with a manual caliper measurement of raw burgers and cooked burgers [
10]. The results were calculated according to Equation (3):
Next, a texture analysis (TPA) was performed [
12]. After cooking the burgers, the samples were cooled at room temperature. The analyses were carried out using a Texture Analyzer (Brookfield model CT3) for texture profile analysis at 70% (TPA70). To determine TPA70, the burgers were compressed to 70% of their original height using a cylindrical probe with a diameter of 25 mm (P/25) and a speed of 5 mm/s through a two-cycle sequence. The parameters of hardness, cohesiveness, elasticity, chewability, and adhesion were evaluated.
2.7. Microbiological Analysis of the Burgers
The microbiological analyses were carried out according to the ANVISA by Normative Instruction no. 161 from 1 July 2022, which establishes microbiological standards for food [
15]. Microbiological analyses of burger samples were carried out for treatments on days 0, 30, and 60 of below-freezing storage at −18 °C. Determinations were made using the rapid method 3M™ Petrifilm™ system (3M do Brasil Sumaré/SP).
The burger samples (10 g) were manually homogenized in a sterile bag in 90 mL of sterile peptone water. Then, dilutions were made up to 10−4 in sterile peptone solution for subsequent plating on 3M™ Petrifilm™ Count Plaque. Quantification (CFU/g) for Escherichia coli was performed on a 3M™ Petrifilm™ E. coli/Coliform Count Plate containing modified violet red bile medium and a glucuronidase activity indicator after incubation at 42 ± 1 °C for 24 ± 2 h with blue E. coli colonies. Quantification (CFU/g) for positive Staphylococcus-coagulase was performed in a 3M™ Petrifilm™ STX Staph Express containing Baird Parker’s modified chromogenic medium, which, after incubation at 37 °C for 24 h, produced red-violet colonies of coagulase-positive Staphylococcus. Quantification of mesophilic aerobic bacteria was carried out on 3M™ Petrifilm™ Aerobic Count Plates containing modified Standard Methods nutrients and an indicator for the enumeration of total aerobic bacteria (red colonies) visible after incubation at 35 °C for 48 h. For Salmonella sp. presumptive results (presence or absence), burger samples (25 g) were incubated at 41 °C for 24 h in 3M™ Salmonella enrichment base medium supplemented with 3M™ Salmonella enrichment supplement for subsequent plating on 3M™ Petrifilm™ Salmonella Express (SALX) system plates with a chromogenic culture medium system (selective and differential for Salmonella). After plate incubation at 41 °C for 48 h, Salmonella presented red colonies with yellow zones, sometimes associated with gas bubbles.
2.8. Consumer-Targeted Sensory Quality of the Burgers
This research was submitted to the University Federal de Pernambuco, Recife-PE, Brazil Research Ethics Committee, according to resolution CNS 466/2012 regulatory standards for research involving human beings, under opinion 6.002.731, and approved under CAAE 64793522.4.0000.5208. Consumer-targeted sensory quality assessment was carried out at the Department of Nutrition of the University Federal de Pernambuco. The experiment was conducted at the Food Experimentation and Analysis Laboratory (LEAAL), starting at 8:00 a.m. and ending at 5:00 p.m. The tasting was carried out in individual cabins at 20 °C under artificial light, according to [
16]. Burgers’ post-cooking temperature was controlled (kept above 70 °C) using an infrared thermometer. The burgers were made available to the consumers at a consumption temperature of approximately 45 °C and were served on disposable white plates, each coded with three random digits, accompanied by a glass of water, as well as water and salt crackers, which aimed to minimize possible interferences between the samples.
The sample population of volunteers participating in the sensory analysis comprised 100 students from the Federal University of Pernambuco (Recife, PE, Brazil) aged between 18 and 25 years old of both sexes (female and male); all were consumers of meat products. Participants were fully informed, in simple language, regarding the research protocol through a Term of Free and Informed Consent (TFIC) and were guided to fill out the form for sensory acceptability evaluation according to the burger’s overall acceptance, appearance, scent, taste, and texture. The individuals scored their degree of acceptance of the analyzed product using a nine-point hedonic scale, with responses ranging from “Really liked” to “Really disliked” [
12].
2.9. Evaluation of Total Phenolics and Antioxidants in PSF and Burgers
PSF extracts were evaluated in terms of total phenolic content [
14]. A gallic acid standard curve (10–60 mg/L) was used to calculate the results expressed in milligrams of gallic acid equivalent (GAE) per 1 gram of dry extract (mg GAE/g).
Total antioxidant capacity (TAC) was calculated using phosphomolybdenum reduction [
17], being expressed in relation to ascorbic acid and calculated using the following Equation (4).
In the DPPH method [
7], 1 mg of the extracts was used and diluted in 1 mL of methanol, of which a serial dilution was carried out at concentrations of 15.625–1000 µg/mL. A total volume of 40 µL of these concentrations was added to 250 µL of DPPH reagent in a 96-well plate. After 30 min of incubation in the dark at room temperature, absorbances were read at 517 nm. The percentage of DPPH radical elimination activity was calculated using Equation (5), where Ac = control absorbance, and Ae = sample absorbance (extract or oil):
For the ABTS method [
8], 20 µL of liquid extracts (10%) was mixed with 1 mL of the ABTS solution and left to rest for 6 min. Then, absorbance at 734 nm was read. The elimination of ABTS radicals was estimated as a percentage after the calculation of radical inhibition, calculated using Equation (6), where Ac = control absorbance, Ae = sample absorbance (extract or oil):
2.10. Statistical Analysis
Analyses were carried out in triplicate, and the results obtained were presented as mean ± deviation. Analysis of variance (ANOVA) was performed, and the means were compared using the Tukey test at a 5% significance level (p < 0.05).
For analyses of the burgers’ consumer-targeted sensory quality, statistical significance was determined using one-way ANCOVA with a significance level of 5% (p < 0.05); global impression was the main predictive effect, with texture, aroma, and color as covariates. All data were analyzed using Statistic 8.0.