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Article

Valorization of Pumpkin Seed Flour in Biscuit Production: Nutritional Enhancement and Sensory Acceptability

by
Claudia-Veronica Ungureanu
1,*,
Iana Morozova
1,
Georgiana Horincar
1 and
Dumitra Răducanu
2
1
Cross-Border Faculty, “Dunarea de Jos” University of Galati, 111 Domnească Street, 800201 Galati, Romania
2
Faculty of Science, “Vasile Alecsandri” University of Bacau, 157, Calea Mărăşeşti, 600115 Bacau, Romania
*
Author to whom correspondence should be addressed.
Sustainability 2025, 17(22), 10103; https://doi.org/10.3390/su172210103
Submission received: 7 October 2025 / Revised: 31 October 2025 / Accepted: 4 November 2025 / Published: 12 November 2025
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Abstract

In the context of increasing interest in healthy and sustainable nutrition, the food industry is challenged to develop innovative products that combine high nutritional quality with consumer acceptance. This study evaluated the potential of pumpkin seed flour (PSF) as a natural ingredient in biscuit formulations. PSF was analyzed for its proximate composition, and biscuits were formulated by replacing rice flour with various concentrations of 10%, 20%, and 30%. The products were analyzed for moisture, protein, fat, fiber, and ash content, subjected to microbiological testing, and evaluated sensorially using a 9-point hedonic scale. Results showed that PSF incorporation in biscuits significantly increased protein content from 6.20% in the control to 9.80% and fiber content from 2.10% to 5.90% in the formulation containing 30% PSF. Lipid content also increased proportionally with PSF addition. All samples complied with microbiological safety standards, and sensory evaluation indicated that biscuits with 10 and 20% PSF achieved the highest acceptability, particularly in terms of taste and texture. Overall, the use of PSF improves the nutritional density of biscuits, enhances their nutritional value, and supports sustainable food production by valorizing underutilized plant resources, in alignment with the United Nations Sustainable Development Goals.

1. Introduction

In recent years, the increasing interest in healthy and sustainable diets has driven the development of innovative food products that combine nutritional functionality, safety, and consumer acceptance. Among these, functional bakery products, such as biscuits, have attracted considerable attention due to their popularity, long shelf life, and potential to act as carriers for bioactive compounds [1]. One of the main strategies to improve the nutritional quality of bakery products involves partially replacing refined flour with plant-based ingredients rich in proteins, dietary fiber, and micronutrients [2]. Although conventional biscuit ingredients mainly provide energy-dense macronutrients, their reformulation with nutrient-rich plant materials offers opportunities for improved dietary value [3].
Recent innovations focus on increasing the nutritional and functional potential of bakery products through the incorporation of legumes, pseudocereals, oilseed flours, while also addressing health-related trends such as sugar and fat reduction or low-glycemic index formulations [4]. Moreover, fortification with functional additives such as probiotics, prebiotics, and natural extracts has been explored to improve gut health and overall well-being [5]. At the same time, sustainability concerns have encouraged the valorization of agro-industrial by-products, contributing to circular bioeconomy principles and reducing food waste [6].
In this context, pumpkin seeds represent a locally available yet underutilized plant resource with remarkable nutritional and functional potential. Although pumpkin pulp is commonly consumed as a vegetable, the seeds are often discarded during processing, even though they represent a valuable source of nutrients and bioactive compounds. The seeds contain substantial amounts of protein (25–37%), oil (37–45%), dietary fiber, essential minerals (magnesium, zinc, iron), and antioxidants such as phytosterols and tocopherols, which have been linked to cardiovascular health and oxidative stress reduction [5,6].
Pumpkin seed flour, obtained as a by-product after oil extraction, concentrates these valuable components and can effectively enhance the nutritional quality of bakery products. Previous studies have demonstrated that partial substitution of cereal flours with PSF improves protein and fiber content while maintaining acceptable sensory characteristics [6].
However, limited research has simultaneously addressed the nutritional improvement, technological performance, and sustainability dimension of biscuits enriched with PSF [7]. The present study aimed to comprehensively investigate the technological functionality, nutritional enhancement, and sensory performance of pumpkin seed flour (PSF) as a sustainable ingredient in biscuit production. Biscuit formulations were developed with three various concentrations of PSF (10%, 20%, and 30%) to assess their effects on physicochemical composition, microbiological safety, and sensory acceptability. Furthermore, the study sought to determine the optimal PSF level that enhances nutritional quality without compromising product texture or consumer preference. In addition, this research explored the contribution of PSF incorporation to sustainable food system development through the valorization of agro-industrial by-products and the promotion of circular bioeconomy principles, in line with the United Nations Sustainable Development Goal 12 (Responsible Consumption and Production).

2. Materials and Methods

2.1. Source of Materials

The pumpkin (Cucurbita pepo L.) fruits were harvested at full maturity from a certified farm located in Galati County, Romania (45°27′ N, 28°02′ E). Rice flour, powdered sugar, butter, baking powder, and fresh whole eggs were purchased from a local market (Galati, Romania). The biscuit samples were stored in high-density polyethylene (HDPE) bags. All the other chemicals, reagents and solvents used in the study were of analytical grade and obtained from Merck Pvt. Ltd. (Darmstadt, Germany).

2.2. Production of Pumpkin Seed Flour

PSF used in the biscuit formulations was produced from raw, hulled, food-grade pumpkin seeds (Cucurbita pepo L.). The seeds were manually sorted to remove mechanical impurities (husks, plant residues, foreign particles) and defective or moldy seeds, ensuring a clean and uniform raw material. The sorted seeds were dried in a hot-air oven (UN55, Memmert, GmbH, Schwabach, Germany) at 50 °C for 6 h to reduce the moisture content to a safe level of approximately 8%, suitable for storage and for preventing microbial growth and oxidative deterioration. The moderate temperature was selected to minimize nutrient losses and preserve heat-sensitive bioactive compounds, including unsaturated fatty acids, vitamin E, and phytosterols. After drying, the seeds were finely milled using a laboratory mill (Retsch ZM 200, Retsch GmbH, Haan, Germany) fitted with a 0.5 mm sieve, resulting in a homogeneous flour with fine granulometry. The PSF was then packed in airtight, food-grade polyethylene bags, flushed with air to reduce residual moisture condensation, and stored in a cool, dry place at 20 ± 2 °C until used in biscuit formulations. The processing procedure followed the methods previously described by [7], with minor modifications to adapt to local raw material conditions. All operations were performed in triplicate to ensure batch-to-batch consistency.

2.3. Formulation of Biscuits by Utilizing Pumpkin Seed Flour

To develop a biscuit with improved nutritional quality, three formulations were prepared according to the procedure described by [7], with slight modifications. In each formulation, PSF was incorporated as a partial substitute for rice flour at levels of 10%, 20%, and 30%. The control formulation (C), containing no PSF, served as the reference. The formulations were specifically designed to maintain the characteristic texture, desirable taste, and overall sensory quality of biscuits. These levels were selected to systematically investigate the effect of PSF addition on the nutritional composition, structure, sensory attributes, and color of the final product. The formulations are presented in Table 1.
Dough was prepared using the creaming method: butter and powdered sugar were mixed until creamy (using a KitchenAid Classic Stand Mixer, St. Joseph, MI, USA). The egg yolks were separated from the egg whites, and according to the recipe, only the yolks were used in the biscuit formulations. The yolks were then homogenized, after which rice flour, PSF, and baking powder were gradually incorporated. The dough was rolled to 5 mm thickness, cut into discs, and baked in a convection oven (UNOX XFT133, Cadoneghe, Italy) at 180 °C for 12 min. Biscuits were cooled to room temperature, packed in polyethylene bags, and stored at 20 ± 2 °C until analysis (Figure 1).

2.4. Sensory Properties of Biscuits

Sensory evaluation was conducted 24 h after baking, at room temperature (22 ± 2 °C) under natural diffuse light. The evaluation followed the ISO guidelines for sensory test room design) [8].
Biscuits were evaluated for appearance, visual color, flavor, sweetness, and overall acceptability. Samples were randomly presented to panelists on coded samples. A panel of 15 panelists (8 females and 7 males) aged 21–45 years who were regular consumers of bakery products participated in the evaluation. Each panelist assessed the samples using a 9-point hedonic scale (1 = “dislike extremely”; 9 = “like extremely”). Panelists rinsed their mouths with water between samples. Mean scores were calculated for each attribute, and overall acceptability scores were used to identify the optimal biscuit formulation.

2.5. Determination of Proximate Compositions of the Pumpkin Seed Flour and the Formulated Biscuits

The proximate composition of the pumpkin seed flour and the formulated biscuits was determined according to the official methods of the Association of Official Analytical Chemists (AOAC, 2023) [9]. The parameters analyzed included moisture content, total ash, crude protein, total fat, crude fiber, and total carbohydrate. All analyses were conducted in triplicate to ensure reproducibility and statistical reliability.
Moisture content was determined by using the standard oven-drying method (AOAC 925.10) [10], by heating samples in a forced-air oven at 103 ± 2 °C until a constant weight was achieved. The difference in weight before and after drying was expressed as 0% of moisture.
Total ash content was quantified as an indicator of total mineral content by incinerating samples in a muffle furnace at 550 ± 25 °C until all organic matter was completely oxidized (AOAC 923.03) [11]. The mineral residue was expressed as 0% dry matter.
Crude protein was quantified using the Kjeldahl method, with nitrogen content converted to protein by applying a factor of 6.25 (AOAC 2001.11/978.04) [12]. Total fat was measured using Soxhlet extraction with petroleum ether (boiling point 60–80 °C) as a solvent, employing a Socs Plus extraction system (Model T43-51-40-2L). Prior to extraction, the samples were subjected to moderate heat treatment (≤103 °C) to ensure dehydration and disruption of the protein matrix, thereby facilitating complete lipid release while minimizing oxidative degradation.
Crude fiber was determined by the Weende method (AOAC 985.29) [13], adapted to a filter bag system using an Ankom 220 Fiber Analyzer (Ankom Technology, Macedon, NY, USA). Samples underwent sequential acid (1.25% H2SO4) and alkaline (1.25% NaOH) digestion, simulating gastric and intestinal digestion. The remaining organic residue was dried, incinerated, and the loss in weight was expressed as crude fiber content.
Total carbohydrate content was calculated by difference, subtracting the sum of protein, fat, moisture, and ash from 100 g of sample weight. This indirect method is widely accepted in proximate analysis when direct carbohydrate quantification is not performed.
Acid and alkali hydrolysis followed by ignition of the residue as described by the method AOAC (2023) [11].
All determinations followed strict quality control measures, including the use of analytical blanks, calibration standards, and replicates. The use of AOAC-approved methods ensures comparability of results with previous studies and compliance with international standards.

2.6. Microbiological Analysis

Microbiological analysis was conducted to assess the safety of biscuits enriched with pumpkin seed flour. The tests were performed in accordance with current international standards for dry bakery products. The following microbiological indicators were evaluated:
  • Total viable count (ISO 4833-1:2013) [14]
  • Yeasts and molds (ISO 21527-2:2008) [15]
  • Escherichia coli (ISO 16649-2:2001) [16]
  • Salmonella spp. (ISO 6579-1:2017; absence in 25 g sample) [17]

2.6.1. Yeast and Mold

Ten grams of biscuit sample were homogenized in 90 mL of Buffered Peptone Water (BPW, Oxoid Ltd., Basingstoke, UK) and agitated for 2 min to obtain a uniform suspension. Serial dilutions were prepared by transferring aliquots from one tube to the next. From each dilution, 1 mL was aseptically pipetted onto triplicate Petri dishes. Approximately 15 mL of Potato Dextrose Agar (PDA; BBL™, 211929, BD Difco™, Franklin Lakes, NJ, USA) were then poured onto the plates and mixed gently. The plates were incubated under aerobic conditions at 25–28 °C for 5 days, after which yeast, and mold colonies were enumerated [18].

2.6.2. Total Plate Count

Plate count agar (PCA; Oxoid Ltd., Basingstoke, UK) was prepared according to the manufacturer’s guidelines. Subsequently, 1 mL of each respective serial dilution was carefully transferred onto the PCA using pipettes. The agar was then poured into Petri dishes and thoroughly mixed by rotating the dishes in both counterclockwise and clockwise directions. The Petri dishes were inverted and incubated under aerobic conditions at 30 °C for 24 h, as described by [19]. After the incubation period, the bacterial colonies were counted using a colony counter (Interscience Scan 1200, Interscience, Saint-Nom-la-Bretèche, France), and the results were expressed as colony-forming units per gram (CFU/g) of the sample.

2.6.3. Detection of Escherichia coli

Two grams of biscuit sample were homogenized in 90 mL of sterile distilled water to obtain a 10−1 dilution. Subsequently, 1 mL of this suspension was transferred into 9 mL of sterile distilled water to prepare a 10−2 dilution, and the procedure was repeated serially up to a 10−5 dilution. Enumeration was performed using the pour plate method: 1 mL of each dilution was pipetted into labeled Petri dishes, followed by the addition of approximately 20 mL of Violet Red Bile Agar (VRBA; Oxoid Ltd., Basingstoke, UK). The plates were gently mixed, allowed to solidify, and incubated under aerobic conditions at 37 °C for 24 h. Colony counts were recorded after the incubation period [18].

2.6.4. Detection of Salmonella spp.

Salmonella detection was performed on 25 g samples of each seed type, previously pre-enriched with 225 mL of tryptic soy broth (TSB; Bioxon, BD Difco™, Franklin Lakes, NJ, USA) and incubated under aerobic conditions at 35 °C for 24 h. Subsequently, 1 mL and 0.1 mL aliquots were transferred into tubes containing 9 mL of tetrathionate broth (TTB; Bioxon, BD) and Rappaport–Vassiliadis broth (RV; Bioxon, BD), respectively. Differential plating was carried out on Salmonella–Shigella (SS) Agar (Oxoid Ltd., Basingstoke, UK) to enumerate total Enterobacteriaceae and Salmonella–Shigella colonies. After incubation under aerobic conditions for 24 h at 37 °C, colonies exhibiting typical morphology ranging from pale yellow to red were counted as members of the Enterobacteriaceae family. Presumptive Salmonella colonies were further enumerated to determine the number of colony-forming units (CFU) per gram of sample [18,19,20].
Results were expressed as log CFU/g. All analyses were performed in duplicate to ensure reproducibility.
The microbiological criteria were compared with the limits established by the European Commission Regulation (EC) No. 2073/2005 on microbiological criteria for foodstuffs [20]. Compliance with these regulations is crucial for ensuring consumer safety and for supporting the marketability of novel functional products.

2.7. Statistical Analysis

All analyses were performed in triplicate. Results were expressed as mean ± standard deviation. One-way ANOVA was applied to determine the effect of PSF substitution level, and Tukey’s post hoc test was used to compare mean values (p < 0.05). Statistical analysis was performed with IBM SPSS Statistics v26 (IBM Corp., Armonk, NY, USA).

3. Results and Discussion

3.1. Proximate Analysis

Proximate analysis plays a crucial role in the evaluation of food products, as it provides information on their nutritional composition, including macronutrients such as proteins, fats, carbohydrates, fiber, and energy value. This analysis allows for the comparison of the nutritional value of raw ingredients and finished products, enabling the assessment of changes brought about by the novel ingredients.
The proximate composition of the pumpkin seed flour was determined experimentally in triplicate, and the results are expressed as mean ± standard deviation (n = 3, as-is basis). (Table 2). The PSF showed low moisture content, high protein levels, moderate fat content, and appreciable amounts of ash and crude fiber. These compositional characteristics indicate that pumpkin seed flour is a valuable source of nutrients and has considerable potential as an ingredient in the formulation of functional food products.
Compared with common cereal flours, PSF contains considerably higher amounts of protein, fiber, and minerals. Rice flour generally contains 7–9 g of protein and less than 1 g of fiber per 100 g [21]. It is mainly composed of starch, with relatively small quantities of dietary fiber and ash, which contribute to its light texture but limit its overall nutritional density. Therefore, the partial substitution of rice flour with PSF could enhance the nutritional and technological quality of bakery products by increasing their protein, fiber, and micronutrient contents.

3.1.1. Moisture Content

Moisture content was determined by the standard oven-drying method at 103 °C for 60 min until constant weight. As illustrated in Figure 2, moisture content exhibited a progressive decline with increasing levels of PSF, decreasing from 5.8% in the control sample (M) to 4.5% in the 30% PSF formulation (P3), corresponding to an approximate 22% reduction relative to the control. This reduction can be attributed to the higher protein and dietary fiber content of PSF, which increases water absorption during dough development but reduces water retention after baking, resulting in a drier product.
Comparable findings were reported by [22], who observed that incorporating PSF into rice flour-based formulations led to a consistent reduction in the moisture content of baked products. This effect can be attributed to the higher protein and fiber contents of PSF, which promote water absorption during dough development but limit moisture retention after baking. Similar trends have also been observed in bakery products fortified with other protein and fiber-rich flours, where lower post-baking moisture levels were attributed to reduced water-binding capacity following thermal processing [23,24]. The decrease in moisture content may also be linked to reduced availability of free water within the PSF-containing matrix, as stronger interactions between proteins, fibers, and starch molecules limit water retention during and after baking.
From a technological perspective, lower moisture content is advantageous, as it not only contributes to a crispier texture but also extends shelf life by reducing susceptibility to microbial spoilage.

3.1.2. Ash Content

Ash content represents the total amount of mineral substances (inorganic salts) remaining after complete combustion of organic matter in a food sample and is expressed as % of dry matter. In the case of biscuits, ash determination is an important indicator of nutritional value, the degree of flour refinement, and the contribution of mineral-rich ingredients such as seeds or alternative flour (e.g., pumpkin seed flour).
The ash content of the biscuit formulations increased progressively with the level of pumpkin seed flour (PSF) incorporation, ranging from 0.92% in the control sample to 1.8% in the 30% PSF formulation (Figure 3), corresponding to an approximate 96% increase relative to the control. This trend reflects the naturally high mineral content of PSF. According to previous studies, pumpkin seed flour is particularly rich in magnesium, zinc, iron, phosphorus, and potassium, which represent the predominant minerals contributing to its nutritional value [25]. The increase in ash content highlights an enhancement of the biscuits’ micronutrient profile, reinforcing their potential as functional foods.
These findings are consistent with previous research, where [26] reported a significant increase in ash content in biscuits fortified with pumpkin seed flour. Similarly, other studies [27,28] demonstrated that the use of oilseed-based flours improves the mineral density of baked products.
From a nutritional standpoint, the higher ash content indicates an enhanced supply of essential minerals, which may contribute to improved dietary mineral intake and overall nutritional quality. Moreover, the use of PSF represents a sustainable approach, valorizing an underutilized agro-industrial by-product, reducing food waste, and supporting circular economy principles. The increase in ash content thus highlights not only the enhancement of the micronutrient profile of the biscuits but also their potential as functional foods aligned with sustainable food system goals.

3.1.3. Titratable Acidity

As shown in Figure 4, the titratable acidity of the biscuits ranged from 2.1 °T in the control sample (0% PSF) to 2.7 °T in the formulation containing 30% PSF. A progressive increase in acidity values was observed with higher levels of PSF incorporation. This trend can be attributed to the presence of organic acids and phenolic compounds naturally occurring in pumpkin seeds, which contribute to the overall acidity of the product.
Although the differences between formulations were relatively small, they were consistent, indicating that PSF incorporation slightly enhances the acidity profile of biscuits. From a technological perspective, a moderate increase in acidity may improve microbial stability and extend shelf life, as slightly acidic environments are less favorable for spoilage microorganisms. From a sensory perspective, the values observed remain within the typical range for dry bakery products, where acidity is low and does not negatively affect acceptability.
Comparable results have been reported in bakery products enriched with seed flour or other plant-based ingredients, where small increases in titratable acidity were associated with higher concentrations of bioactive compounds and natural acids [29,30]. Thus, the incorporation of PSF not only improves the nutritional profile of biscuits but also contributes to their functional and preservative properties.

3.1.4. Protein Content

As shown in Figure 5, the protein content of the biscuits increased progressively with the higher proportion of PSF, from 6.20% in the control sample to 9.80% in the 30% PSF formulation (P3). This corresponds to an approximate 58% increase relative to the control, confirming the significant contribution of PSF to the protein enrichment of the biscuits.
This trend is consistent with the literature, as PSF is recognized for its high protein content (50–65%), which explains the observed increase [31,32]. The improvement in protein level represents a significant nutritional enhancement, particularly relevant for the development of functional bakery products targeted at athletes, vegetarians, or consumers with higher protein requirements.
In addition to its quantitative impact, the qualitative value of PSF proteins should also be highlighted. Pumpkin seed proteins are rich in essential amino acids, especially arginine and glutamic acid, which contribute to improved cardiovascular health and muscle recovery [33]. From a nutritional labeling perspective, formulations containing 20–30% PSF could be classified as a “source of protein” or even “high in protein”, depending on local and European regulations, as the protein contribution exceeds the thresholds established for such claims [34]. These findings indicate that the incorporation of PSF not only improves the nutritional density of biscuits but also supports the development of functional bakery products aligned with consumer demands for healthier and sustainable alternatives.

3.1.5. Lipid Content

Lipid content is an important parameter in evaluating the nutritional and energetic value of food products, as lipids strongly influence texture, flavor, and satiety. In this study, lipid determination was carried out using the Soxhlet extraction method with organic solvents (petroleum ether).
As presented in Figure 6, the lipid content of the biscuits increased progressively with higher levels of PSF substitution. This trend is expected, given that pumpkin seeds are naturally rich in lipids, with reported fat contents ranging from 37–45%, predominantly composed of unsaturated fatty acids [35]. The major fatty acids identified are linoleic acid (C18:2, omega-6) and oleic acid (C18:1, omega-9), which together represent more than 70% of the total fatty acid profile [36]. These unsaturated fatty acids are widely recognized for their health-promoting properties, including cardiovascular protection, improved lipid metabolism, and anti-inflammatory effects [37].
Compared with the control, the P3 formulation (30% PSF) showed an approximate 62% increase in lipid content, contributing to a higher energy value, improved tenderness, and richer flavor. However, since unsaturated fatty acids are highly susceptible to oxidation, the incorporation of natural antioxidants (such as tocopherols) or the use of active or oxygen-barrier packaging materials could be effective strategies to preserve product quality and extend shelf life [33]. Therefore, biscuits enriched with 20–30% PSF can be regarded as a valuable source of healthy fats and may be recommended within dietary patterns that emphasize unsaturated fatty acid intake, such as the Mediterranean diet. These findings are in line with previous studies reporting enhanced lipid content and improved nutritional quality in bakery products supplemented with pumpkin seed flour [34,36].

3.1.6. Carbohydrate Content

Figure 7 highlights the evolution of carbohydrate content (%) as a function of the progressive incorporation of PSF into biscuit formulations. A consistent decrease in carbohydrate levels is observed from the control sample (100% rice flour) to the enriched variants containing 10%, 20%, and 30% PSF (Figure 7). This trend is explained by the lower carbohydrate content of PSF compared with rice flour, combined with its higher levels of protein, lipids, and dietary fiber. Consequently, partial substitution of the base flour with PSF reduces the proportion of total carbohydrates in the final product, resulting in a nutritionally improved formulation with a lower glycemic potential and enhanced functional value.
The decrease in carbohydrate content may contribute to lowering the glycemic value of the biscuits, which is beneficial for individuals with diabetes or those seeking better glycemic control. These findings agree with previous studies, which have shown that pumpkin seed flour exhibits a significantly lower carbohydrate fraction compared to conventional cereal flours, while being a valuable source of proteins, unsaturated fatty acids, and minerals [38]. Similarly, [32] reported that the incorporation of pumpkin seed flour into bakery products reduced their glycemic index and improved their nutritional density. More recently, [39] emphasized that replacing refined flour with nutrient-dense alternatives such as seed-based flour not only enhances nutritional quality but also aligns with sustainable food innovation and consumer demand for functional products.

3.1.7. Crude Fiber Content of Biscuits Enriched with Pumpkin Seed Flour

Dietary fiber comprises nondigestible plant components that resist hydrolysis by human digestive enzymes yet are essential for gastrointestinal function and metabolic health. In this study, the crude fiber content of the biscuits enriched with pumpkin seed flour was quantified using a gravimetric procedure, whereby digestible constituents are removed by sequential acid–alkali treatment and the remaining fibrous residue is dried and weighed (AOAC, Official Methods) [13]. As shown in Figure 8, fiber content increases progressively with the level of PSF substitution, rising from 2.10 in the control sample to 5.90 in the 30% PSF formulation (P3). This represents an almost threefold increase compared with the control, indicating that partial replacement of rice flour with PSF is an effective strategy to enhance the fiber fraction of the final product. This improvement reflects the naturally high dietary fiber content of PSF, which contributes to improved nutritional quality and may support better digestive health.
These findings are consistent with reports that pumpkin seed-derived ingredients contain substantially less starch but are richer in proteins, lipids and fiber than conventional cereal flours, leading to higher total dietary fiber in bakery matrices when used as partial replacers.
The resulting fiber enrichment is relevant nutritionally, as higher fiber intakes are associated with improved laxation, greater satiety, attenuation of postprandial glycemia and favorable effects on blood lipids—attributes frequently targeted in the formulation of functional bakery products. In line with previous work on PSF-fortified baked goods, the present upward trend supports the positioning of PSF as a sustainable, nutrient-dense ingredient that improves the functional profile of biscuits without relying on synthetic additives [37,38,39].

3.2. Microbiological Analysis of Biscuits Enriched with Pumpkin Seed Flour

The microbiological evaluation was carried out to assess the hygienic and sanitary safety of biscuits enriched with different levels of pumpkin seed flour. Monitoring microbial contaminants is essential for compliance with legal safety standards and for ensuring consumer acceptability. Samples were stored at 25 ± 2 °C for 7 days in airtight polyethylene bags under ambient laboratory conditions before analysis, to simulate typical short-term storage prior to consumption. The results are presented in Table 3.
All analyzed microbiological parameters were within the legal limits established for dry bakery products. Total aerobic mesophilic bacteria ranged from 2.08 log CFU/g in the control to 2.26 log CFU/g in P3 (30% PSF). Yeast and mold counts followed a similar trend, ranging from 1.75 log CFU/g in control to 1.87 log CFU/g in P3. These values are low and typical for baked products, suggesting that PSF incorporation did not promote fungal growth during the storage period assessed. The modest increase observed with higher supplementation levels may be attributed to the additional availability of organic substrates (proteins, lipids, fibers) present in pumpkin seed flour, which could slightly favor microbial proliferation. However, these counts remain negligible from a food safety perspective [40].
No pathogenic bacteria such as Escherichia coli or Salmonella spp. were detected, confirming that the production process and the raw materials ensured adequate hygienic quality and compliance with international safety standards microbiological safety of all formulations. Overall, the results demonstrate that the incorporation of pumpkin seed flour does not compromise the microbiological safety of biscuits enriched with pumpkin seed flour. On the contrary, considering the relatively low microbial loads observed, PSF supplementation may contribute indirectly to product stability, possibly through the presence of bioactive compounds such as phenolics and antioxidants with antimicrobial potential, as previously reported in pumpkin seeds and their derivatives. These findings highlight the dual role of PSF: providing nutritional enrichment and potentially exerting a stabilizing effect on the microbial quality of bakery products.
All results complied with the microbiological safety criteria established for bakery products by EU Regulation (EC) No. 2073/2005 on microbiological criteria for foodstuffs [20] and were determined in accordance with ISO 4833-1:2013 (for total viable counts) and ISO 21527-2:2008 (for yeasts and molds). This confirms that all formulations met international standards for food quality and hygiene, ensuring the safety and stability of PSF-enriched biscuits [40,41]. These results support the use of pumpkin seed flour not only for its nutritional contribution but also as a natural stabilizing ingredient that may extend shelf-life and enhance food safety.

3.3. Sensory Evaluation

A sensory panel consisting of 15 trained panelists performed the sensory analysis of the baked biscuit samples. The following attributes were evaluated: external appearance, taste, aroma, color, texture, and overall acceptability. Each characteristic was scored on a hedonic scale from 1 (lowest) to 9 (highest). The cumulative scores obtained for each formulation are illustrated in Figure 9.
The radar chart (Figure 9) illustrates the sensory scores (1–9 scale) assigned by a trained panel (n = 15) for appearance, color, texture, aroma, taste, and overall acceptability. Among the tested formulations, sample P1 (10% PSF) achieved the highest scores, particularly for taste and aroma, while P3 (30% PSF) was rated lower in taste and texture, being described as slightly bitter and more compact. These findings suggest that moderate substitution levels (~10%) provide the best compromise between nutritional enhancement and consumer acceptability.
These observations are consistent with previous studies, which reported that bakery products enriched with pumpkin-derived ingredients are generally well accepted at moderate substitution levels, while higher incorporation tends to negatively affect sensory attributes, especially crispness and flavor. Similarly, research on seed-enriched baked goods has shown that moderate additions (5–15%) improve sensory appeal and consumer preference, whereas excessive replacement may lead to denser structures and bitter notes [42,43].
Thus, the present study confirms that pumpkin seed flour, when used at optimized levels (10%), can significantly improve the nutritional and functional value of biscuits without compromising sensory quality, thereby supporting its potential in sustainable food product development.

4. Conclusions

The present study demonstrated the successful development of biscuits enriched with pumpkin seed flour, confirming its value as a sustainable ingredient with high nutritional potential. PSF incorporation enhanced the nutritional profile of rice flour biscuits by increasing protein, fiber, and lipid contents while reducing carbohydrates, without negatively affecting sensory properties or microbiological safety.
PSF-based biscuits thus represent a promising alternative to conventional products, combining improved nutritional quality, consumer acceptance, and sustainability. This approach contributes to the valorization of underutilized agricultural by-products and supports circular bioeconomy principles. At the industrial level, process scale-up and cost–benefit analyses should be carried out to facilitate market introduction and to strengthen the role of PSF in sustainable food systems.
Future research should focus on optimizing formulations, assessing antioxidant stability, evaluating shelf-life under various packaging conditions, and conducting large-scale consumer studies to validate potential health benefits.

Author Contributions

Conceptualization, C.-V.U. and G.H.; methodology, C.-V.U.; software, I.M.; validation, C.-V.U., D.R. and G.H.; formal analysis, C.-V.U.; investigation, C.-V.U.; resources, I.M.; data curation, I.M.; writing—original draft preparation, C.-V.U.; writing—review and editing, C.-V.U. and G.H.; visualization, G.H.; supervision, D.R.; project administration, C.-V.U.; funding acquisition, C.-V.U. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Ethical review and approval were waived for this study by Institution Committee due to Legal Regulations (The research was based on anonymized questionnaires and secondary data, which, according to Romanian legislation and institutional rules, do not require prior approval according to Ordonanța nr. 57/2002 privind cercetarea științifică și dezvoltarea tehnologică, Legea nr. 183/2024 privind statutul personalului de cercetare-dezvoltare-inovare and Codul de etică și deontologie universitară al Universității “Dunărea de Jos” din Galați).

Informed Consent Statement

For sensory analysis, written informed consent was obtained from all the panelists.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors are grateful to the “Dunărea de Jos” University of Galati for providing the opportunity and support for this research work.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviation

The following abbreviation is used in this manuscript:
PSFPumpkin seed flour

References

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Figure 1. Biscuit formulations with different levels of pumpkin seed flour (PSF).
Figure 1. Biscuit formulations with different levels of pumpkin seed flour (PSF).
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Figure 2. Moisture content variation in biscuits with different levels of PSF. Values are means ± SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
Figure 2. Moisture content variation in biscuits with different levels of PSF. Values are means ± SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
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Figure 3. Ash content of biscuit formulations with different levels of PSF. Values are means ± SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
Figure 3. Ash content of biscuit formulations with different levels of PSF. Values are means ± SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
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Figure 4. Titratable acidity (°T) of biscuits with different levels of PSF incorporation. Values are means ± SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
Figure 4. Titratable acidity (°T) of biscuits with different levels of PSF incorporation. Values are means ± SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
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Figure 5. Effect of PSF in addition to the protein content of biscuits. Values are means ± SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
Figure 5. Effect of PSF in addition to the protein content of biscuits. Values are means ± SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
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Figure 6. Lipid content of biscuits with varying levels of PSF. Values means ±SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
Figure 6. Lipid content of biscuits with varying levels of PSF. Values means ±SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
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Figure 7. Carbohydrate content in biscuit formulations with PSF. Values are means ± SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
Figure 7. Carbohydrate content in biscuit formulations with PSF. Values are means ± SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
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Figure 8. Fiber content in biscuit formulations with PSF. Values are means ±SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
Figure 8. Fiber content in biscuit formulations with PSF. Values are means ±SD (n = 3). Bars with different letters indicate significant differences among samples (p < 0.05, Tukey’s test).
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Figure 9. Sensory profile of biscuits with different levels of PSF. Data represents sensory scores (n = 3). Statistical differences among formulations were determined by one-way ANOVA followed by Tukey’s post hoc test (p < 0.05).
Figure 9. Sensory profile of biscuits with different levels of PSF. Data represents sensory scores (n = 3). Statistical differences among formulations were determined by one-way ANOVA followed by Tukey’s post hoc test (p < 0.05).
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Table 1. Ingredients and their amount for nutritionally enriched biscuits.
Table 1. Ingredients and their amount for nutritionally enriched biscuits.
Ingredients (g)ControlP1 (10%)P2 (20%)P3 (30%)
Butter90909090
Rice flour150135120105
Powdered sugar50505050
Pumpkin seed flour-153045
Egg yolk25252525
Baking powder3333
Note: P1, P2, P3 represent formulations with 10%, 20%, and 30% PSF added, respectively.
Table 2. Proximate composition of pumpkin seed flour (g/100 g).
Table 2. Proximate composition of pumpkin seed flour (g/100 g).
ParametersPumpkin Seed Flour
Moisture content7.50 ± 0.10
Crude protein39.35 ± 0.02
Total fat7.26 ± 0.36
Total ash3.65 ± 0.09
Crude fiber11.29 ± 1.58
Total carbohydrate7.60 ± 0.66
Data expressed in mean ± SD.
Table 3. Microbiological analysis results of biscuit formulations.
Table 3. Microbiological analysis results of biscuit formulations.
SampleTotal Aerobic Mesophilic Bacteria (log CFU/g)Yeasts and Molds (log CFU/g)E. coli (log CFU/g)Salmonella spp. (log CFU/g)
Control2.081.75Not detectedAbsent
P1 (10%)2.151.79Not detectedAbsent
P2 (20%)2.201.83Not detectedAbsent
P3 (30%)2.261.87Not detectedAbsent
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Ungureanu, C.-V.; Morozova, I.; Horincar, G.; Răducanu, D. Valorization of Pumpkin Seed Flour in Biscuit Production: Nutritional Enhancement and Sensory Acceptability. Sustainability 2025, 17, 10103. https://doi.org/10.3390/su172210103

AMA Style

Ungureanu C-V, Morozova I, Horincar G, Răducanu D. Valorization of Pumpkin Seed Flour in Biscuit Production: Nutritional Enhancement and Sensory Acceptability. Sustainability. 2025; 17(22):10103. https://doi.org/10.3390/su172210103

Chicago/Turabian Style

Ungureanu, Claudia-Veronica, Iana Morozova, Georgiana Horincar, and Dumitra Răducanu. 2025. "Valorization of Pumpkin Seed Flour in Biscuit Production: Nutritional Enhancement and Sensory Acceptability" Sustainability 17, no. 22: 10103. https://doi.org/10.3390/su172210103

APA Style

Ungureanu, C.-V., Morozova, I., Horincar, G., & Răducanu, D. (2025). Valorization of Pumpkin Seed Flour in Biscuit Production: Nutritional Enhancement and Sensory Acceptability. Sustainability, 17(22), 10103. https://doi.org/10.3390/su172210103

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