foods

Article
Effect of Bioactive Compounds from Pumpkin Powder on the
Quality and Textural Properties of Shortbread Cookies
Aliona Ghendov-Mosanu 1 , Natalia Netreba 1, * , Greta Balan 2 , Daniela Cojocari 2 , Olga Boestean 1 ,
Viorica Bulgaru 1 , Angela Gurev 1 , Liliana Popescu 1 , Olga Deseatnicova 1 , Vladislav Resitca 1 ,
Carmen Socaciu 3 , Adela Pintea 3 , Tamar Sanikidze 4 and Rodica Sturza 1

1 Faculty of Food Technology, Technical University of Moldova, 9/9 Studentilor St.,

MD-2045 Chisinau, Moldova; aliona.mosanu@tpa.utm.md (A.G.-M.); olga.boestean@tpa.utm.md (O.B.);
viorica.bulgaru@tpa.utm.md (V.B.); angela.gurev@chim.utm.md (A.G.); liliana.popescu@tpa.utm.md (L.P.);
olga.deseatnicova@toap.utm.md (O.D.); vladislav.resitca@adm.utm.md (V.R.);
rodica.sturza@chim.utm.md (R.S.)
2 Department of Preventive Medicine, “Nicolae Testemitanu” State University of Medicine and Pharmacy,
165 Stefan cel Mare Blvd., MD-2004 Chisinau, Moldova; greta.balan@usmf.md (G.B.);
daniela.cojocari@usmf.md (D.C.)
3 Faculty of Veterinary Medicine, University of Agricultural Sciences and Veterinary Medicine,
3–5 Calea Manasturs St., 4003724 Cluj-Napoca, Romania; csocaciudac@gmail.com (C.S.);
apintea@usamvcluj.ro (A.P.)
4 Faculty of Exact and Natural Sciences, Javakhishvili Tbilisi State University, 1 Ilia Chavchavadze Ave.,
Tbilisi 00186, Georgia; tsanikidze@tsmu.edu
* Correspondence: natalia.netreba@tpa.utm.md

Abstract: The problem of food with functional ingredients, characterized by low energy intake
and a variety of phytonutrients with biological activity, is one of the concerns of the population.
The objectives of this study were to investigate the effect of pumpkin powder and its bioactive
components on the quality, color and textural properties of shortbread cookies. In the drying process
Citation: Ghendov-Mosanu, A.; of pumpkin powder (Cucurbita moschata) at 60 ± 2 ◦ C, the physicochemical parameters did not
Netreba, N.; Balan, G.; Cojocari, D.; change significantly in relation to fresh pulp. The chromatic parameters L*, a* and b* showed that the
Boestean, O.; Bulgaru, V.; Gurev, A.; pumpkin powder was brighter than the pulp, with a greater presence of yellow pigments. Pumpkin
Popescu, L.; Deseatnicova, O.; powder presented a rich source of bioactive compounds (polyphenols flavonoids, carotenoids) with
Resitca, V.; et al. Effect of Bioactive
an antioxidant potential of 161.52 mmol TE/100 g DW and 558.71 mg GAE/100 g DW. Antimicrobial
Compounds from Pumpkin Powder
activity against Gram-positive (Staphylococcus aureus, Bacillus cereus), Gram-negative (Escherichia coli,
on the Quality and Textural
Salmonella Abony and Pseudomonas aeruginosa) bacteria and high antifungal activity against Candida
Properties of Shortbread Cookies.
albicans were attested. The sensory, physicochemical, texture parameters and color indicators of
Foods 2023, 12, 3907. https://
doi.org/10.3390/foods12213907
shortbread cookies with yellow pumpkin powder (YPP) added in a proportion of 5–20% were
analyzed. The optimal score was given to the sample of 15% YPP. The use of 15–20% YPP contributed
Academic Editors: Vincenzo Lo Turco
to improved consistency due to the formation of complexes between starch and protein.
and Angela Giorgia Potorti

Received: 30 September 2023 Keywords: Cucurbita moschata; shortbread cookies; biologically active compounds; antioxidant
Revised: 21 October 2023 activity; antimicrobial activity; texture; sensory quality; physicochemical quality
Accepted: 23 October 2023
Published: 25 October 2023

1. Introduction
Copyright: © 2023 by the authors.
Pumpkin (Cucurbita moschata), widely cultivated in different climatic zones, famous for
Licensee MDPI, Basel, Switzerland. its nutritional value and health-promoting effects, is consumed in abundance as a functional
This article is an open access article food and as a medicine for the treatment of various health conditions [1–3]. Pumpkin seeds,
distributed under the terms and peel, pulp, flowers and leaves contain compounds characterized by high bioactivity, such as
conditions of the Creative Commons polysaccharides, proteins, polyphenols, carotenoids, phytosterols, vitamins and minerals,
Attribution (CC BY) license (https:// which positively affect human health [4–6].
creativecommons.org/licenses/by/ The qualitative and quantitative profile of biologically active compounds in pump-
4.0/). kin seeds, peels and cores depends on several factors: genotype [7], cultivation method,

Foods 2023, 12, 3907. https://doi.org/10.3390/foods12213907 https://www.mdpi.com/journal/foods

Foods 2023, 12, 3907 2 of 20

maturity degree, storage conditions and duration, processing method, drying and func-
tional compound extraction process [8,9]. Several studies have shown that the antioxidant
components contained in pumpkin inhibit free radicals and reduce the risk of cancer,
cardiovascular and neurodegenerative diseases [10,11].
Pumpkin pulp is a source of dietary fiber, especially pectin, with low energy value,
useful in lowering blood glucose levels [2]. The water content in pumpkin pulp varies
from 88 to 96%, and the caloric content per 100 g of fresh material varies, on average, from
15 kcal to 46 kcal [12]. Low molecular weight polysaccharides (3.5 kDa), extracted from
pumpkin pulp, inhibit free radicals and reduce oxidative stress [13]. Pumpkin pulp and
seed extracts showed antibacterial, fungicidal and antioxidant potential [14,15]. Research
has shown that pumpkin pulp flour had hypoglycemic and hypolipidemic effects [16]. The
antidiabetic activity of pumpkin powder was demonstrated by reducing blood glucose
with increased plasma insulin in alloxan-induced diabetic mice [17].
Despite its valuable phytonutrient content and health benefits, pumpkin is used little
in the Republic of Moldova’s food industry. One of the causes is the difficulty in processing
pumpkin and creating storage spaces with appropriate conditions. These impediments can
be reduced by using fruits and vegetables, including pumpkin, in dehydrated or powdered
form. The powders, obtained by the correct dehydration of fresh fruits and vegetables, as
well as byproducts resulting from their processing, are a concentrated form of biologically
active compounds that are bioaccessible, which can be applied in the production of foods
with increased biological potential [18,19]. Furthermore, it was determined that drying the
pumpkin at higher temperatures did not reduce the antioxidant activity of the pulp [20].
The β-carotene content of 3934.02 µg/100 g DW determined in pumpkin powder
was higher than in peels and seeds [14]. Pumpkin powder extracts showed increased
antioxidant effect due to the high content of biologically active compounds [4]. The to-
tal content of carotenoids determined by the researchers in the pumpkin pulp powder
was 35.2 mg/100 g DW, and of β-carotene, 6.18 mg/100 g DW. According to research [21],
pumpkin pulp extracts showed antioxidant, antiproliferative activities and stronger antibac-
terial potential than pumpkin peel extracts, against three bacterial species (Escherichia coli,
Staphylococcus aureus and Pasteurella multocida). The ones presented confirm that pumpkin
powder, due to its phytochemical content and functional properties, could be used as a
potential source of nutraceuticals in food production [22,23].
Nutraceutical is an umbrella term that includes foods, food parts or dietary sup-
plements that provide physical or protective benefits against chronic diseases. [24]. The
bioactive components of pumpkin pulp, such as polyphenols, fibers, polysaccharides,
proteins, lipids, amino acids, carotenoids (precursors of vitamin A), vitamins (vitamin C,
vitamin B2, vitamin E), minerals (potassium, calcium, magnesium, selenium) [4,25], have
been shown to be effective nutraceuticals in the treatment of oxidative stress-related healing
disorders, including allergies, Alzheimer’s disease, cardiovascular disease, cancer, diabetes,
ocular, immune, inflammatory and Parkinson’s diseases, as well as obesity [26].
Pumpkin powder is an exceptional raw material and natural colorant for nectars,
juices, fermented drinks, jams, marmalades, purees, teas, etc. Fortifying foods with pump-
kin powder gives them sweet taste, texture, color, volume and low-calorie content [12].
Recent research has shown that pumpkin powder, rich in minerals, vitamins, proteins
and antioxidants, can replace white flour in bakery products [27,28], which, in addition to
providing substantial nutrients, changes the texture and sensory properties of the fortified
product [29].
An excellent food matrix for the functional ingredients of pumpkin powder is cook-
ies [25], which are preferred by all categories of consumers, especially children.
Therefore, in addition to fresh consumption, it is important to implement technologies
to develop new nutraceuticals from the beneficial health constituents of pumpkin, as well
as to develop fortified food products that are attractive to consumers.
The objective of the present study was to investigate the effect of pumpkin powder and
its bioactive components on the quality, color and textural properties of shortbread cookies.
Foods 2023, 12, 3907 3 of 20

2. Materials and Methods

2.1. Chemical Materials
Folin–Ciocalteu reagent was purchased from Merck (Darmstadt, Germany). Gallic acid
(98%), quercetin (>95%), methanol (≥99.9%), tert-butyl methyl ether (≥99.9%), hydrochlo-
ric acid (37%), ethyl acetate (≥99.5%), petroleum ether, sodium hydroxide (97%), phe-
nolphthalein, sodium nitrite (≥97.0%), aluminum chloride, 2,2-diphenyl-1-picrylhydrazyl,
(±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, sodium citrate (≥99.0%),
trisodium citrate, silver nitrate (≥99.0%), hexane reagent (>99%), boric acid (≥99.5%), hy-
drochloric acid (37%), KjTabs VST, sulfuric acid (95.0–98.0%), potassium hydroxide (≥85%),
n-octanol (≥99%), anhydrous acetone (≥99.5%) were obtained from Sigma (Darmstadt,
Germany; Tokyo, Japan; Shanghai, China). Lutein and β-carotene were purchased from
Extrasynthese (Lyon, France). A Specord 200 Plus spectrophotometer (Jena, Germany) was
used for spectrophotometric measurements.

2.2. Biological Material

Fully ripe pumpkins of the variety butternut squash (species Cucurbita moschata), har-
vested in September, were procured from the company “S.C. S&S COM” S.R.L. (Costesti,
Moldova), located in the northern part of the Republic of Moldova (46.8677787780761700 lat-
itude, 28.7686119079589840 longitude and altitude of 80 m above sea level). The fruits were
washed and cut into two portions; the fruit matrix and seeds were scooped out.

2.3. Characterization of Pumpkin Fresh Pulp and Powder

Pumpkin halves were cut into slices. The slices were peeled and grated into shreds.
The shreds of pumpkin were dried in a laboratory drying oven, model SLW 115 SMART,
with forced air circulation at a constant temperature of 60 ± 2 ◦ C and relative humidity of
60–65%. The drying process was carried out for 24 h, until they reached a safe moisture
content level, under 8% (wet basis). The average water content was periodically measured
with a halogen moisture analyzer (model HG53, from Mettler Toledo, Columbus, OH,
USA), which was previously calibrated in terms of optimal operating parameters for
vegetable foods. The dry shreds of pumpkin were crushed to powder with a particle size
of 175 ± 15 µm and then sieved. The pumpkin powder was packed in a hermetic box for
subsequent use in this study and the preparation of cookies.

2.3.1. Color Analysis of Fresh Pumpkin Fruits, Pumpkin Powder and Shortbread Cookies
Using the CIELAB Method
The CIELab parameters, L*, a* and b*, were measured using a Chroma Meter CR-
400/410 colorimeter (Konica Minolta, Tokyo, Japan) according to the method in [30]. Each
sample was analyzed at three distinct points, measuring variations in brightness (L*),
red/green component (a*) and yellow/blue component (b*).
Equation (1) was used to determinate the overall difference in color—∆E∗ :

√ ∗
  
∆E∗ = Li − L0∗ )2 + ai∗ − a0∗ )2 + bi∗ − b0∗ )2 (1)

where
L*0 , a*0 and b0* ¯the values of the control sample;
L*i , a*i and bi* ¯the values of the samples with pumpkin powder.

2.3.2. Physicochemical Analysis of Fresh Pumpkin Fruits and Pumpkin Powder

The moisture content (MC) was determined by measuring the weight loss due to
evaporation of water (AOAC, 2012). The total soluble solids (TSS) of the pumpkin pulp
were measured using a handheld Brix refractometer at 0–32◦ (AOAC, 2012). The pH was
measured using a pH meter (TESTO 206-ph2, Pruszkow, Poland) calibrated with buffer
solutions pH 4.0 and 7.0, directly immersing the electrode in the beaker containing the
sample macerated with distilled water, according to the AOAC (2012) method [31]. The ash
Foods 2023, 12, 3907 4 of 20

content (AC) was estimated using the method described by Ranganna [32]. The titratable
acidity (TA) was estimated by titrating a known volume of sample against standard 0.1 N
NaOH using phenolphthalein as an indicator. The results were expressed in % citric
acid [32,33]. The fat content (FC), protein content (PC) and total fiber content (TFC) of the
samples were determined using the method reported by the AOAC (2012) [31]. The FC was
gravimetrically quantified after hexane extraction from the dehydrated sample using the
Soxhlet method in a SER148 Solvent Extraction Unit (VELP Scientifica, Monza, Italy) [33].
The PC was analytically estimated by determining the amount of total nitrogen content
with a conversion factor of 6.25, using the Kjeldahl method in a UDK129 (VELP Scientifica,
Italy). The method for determining TFC in FIWE Raw Fiber Extractors (VELP Scientifica,
Italy) involves a series of chemical treatments to remove all soluble and easily digestible
components from the sample. The remaining fiber components are then washed, dried
and weighed as the residual fiber component. The weight of the TFC is calculated as a
percentage of the initial weight of the food sample.

2.4. Extract Characterization

For sample extraction, 1 g of pumpkin powder was extracted by shaking the sample
1 min at 2000 rpm in a vortex mixer VX-200 (Labnet, Edison, NJ, USA) with 10 mL of acidi-
fied methanol (85:15 v/v, MeOH:HCl). Ultrasonic-assisted extraction (ISOLAB Laborgeräte
GmbH, Eschau, Germany) was performed at a frequency of 37 kHz for 30 min; then, the
mixture was centrifuged at 3000 rpm for 10 min. After filtration, the total polyphenol
content (TPC), total flavonoid content (TFC) and antioxidant activity by reaction with
DPPH radical were determined. The extract was stored in glass bottles at 4.0 ± 1.0 ◦ C, in
the dark.

2.4.1. Total Polyphenols Using Folin–Ciocalteu

Folin–Ciocalteu reagent was used to determine the total polyphenol content (TPC),
according to the method described by Waterman and Mole [34]. The results were calculated
from a calibration curve using gallic acid (0–10 mg/L, y = 0.5056x + 0.0650, R2 = 0.9977)
and expressed in equivalents of gallic acid per 100 g of dried weight (DW) of pumpkin
powder (mg GAE/100 g DW).

2.4.2. Total Flavonoids

Total flavonoid content (TFC) in pumpkin extract was determined spectrophotomet-
rically according to Zhishen et al. [35]. The flavonoid–aluminum complex was detected
at 510 nm. The quercetin calibration curve was used (0–0.250 mg/L, y = 0.7038x + 0.0004,
R2 = 0.9955). The results were expressed in quercetin equivalents per 100 g dry weight
(DW) of pumpkin powder (mg QE) /100 g DW).

2.4.3. Total Carotenoids

The total carotenoid content (TCC) was determined following the modified method
described by Ghendov-Mosanu et al. [36]. The plant material (3 g) was extracted with a
mixture of methanol/ethyl acetate/petroleum ether (1: 1: 1, v/v/v). After filtering the extract,
the residue was reextracted twice using the same solvent mixture. TCC was determined
using the spectrophotometric method. The absorption spectrum was plotted and the TCC
was measured at the maximum absorbance wavelength (450 nm).

2.4.4. Separation of Carotenoids Using Reverse-Phase High-Performance Liquid

Chromatography (RP-HPLC)
Carotenoids were separated using RP-HPLC, on an LC-20AT equipped with an SPD-
M20A diode array detector (DAD) (Shimadzu Corporation, Kyoto, Japan), according to
the method described by Ghendov-Mos, anu et al. [18]. Saponified carotenoid extracts
were used for this analysis. Carotenoids were identified based on their retention times
and absorption spectra, compared with those of available standards (violaxanthin, lutein,
Foods 2023, 12, 3907 5 of 20

β-cryptoxanthin, all-trans-β-carotene) and the available literature data. For the quantifi-
cation of β-carotene and lutein, external eight-point calibration curves were constructed
in the range 1–100 µg/mL. The regression equations were y = 481,115x − 43,279, correla-
tion coefficient: R2 = 0.9985 for β-carotene, and, respectively, y = 415,811x − 17,763 and
R2 = 0.9996 for lutein. The other compounds in the extract were determined using either
the curve for lutein (luteoxanthin and violaxanthin), or that of β-carotene (β-cryptoxanthin
and cis-β-carotene), Table 1.

Table 1. Spectral characteristics and retention times of carotenoids identified using RP-HPLC analysis.

Retention Max Absorption

Compound
Time (min) (nm)
Luteoxanthin 7.498 401, 420, 448
Violaxanthin 9.122 401, 420, 444
Lutein 10.121 421, 445, 473
β-cryptoxanthin 38.246 428, 451, 476
Cis-β-carotene 70.383 424, 446, 472
All-trans-β-carotene 75.511 421, 452, 478

2.4.5. Antioxidant Activity, DPPH Assay

The antioxidant activity of the pumpkin extract was determined using the DPPH free
radical method, according to Brand-Williams et al. [37]. The calibration curve with Trolox
(0–250 µmol/L) was used, and the results were expressed as mmol TE/100 g DW.

2.4.6. Antioxidant Activity by Silver Nanoparticle

To determine the antioxidant activity, the silver nanoparticle method was used accord-
ing to Pintea et al. [38]. This is a simple spectrophotometric method based on the reduction
of silver ion Ag+ (a solution of silver nanoparticles stabilized with sodium citrate and
starch) to metallic silver Ag0 nanoparticles, in the presence of antioxidants–polyphenols
extracted from pumpkin powder. The absorption of the samples is read at λ = 423 nm,
against the blank [39].
The pumpkin powder extract was obtained in the following way: 0.5 g of pumpkin
powder was extracted by shaking the sample for 1 min at 2000 rpm in a VX-200 vortex
mixer (Labnet, Edison, NJ, USA) with 5 mL of methanol. The extraction was carried
out using the ultrasound-assisted method (ISOLAB Laborgeräte GmbH, Germany) at a
frequency of 37 kHz for 30 min; then, the mixture was centrifuged at 3000 rpm for 10 min
and filtered. For quantification, we used the gallic acid calibration curve (0–0.05 g/L,
y = 20.646x + 0.0054, R2 = 0.9963). The results were expressed in gallic acid equivalents per
100 g dry weight of the product (mg GAE/100 g DW).

2.5. Antimicrobial Activity

2.5.1. Preparation of Inoculum
Antimicrobial properties of extracts (pumpkin powders were tested against Gram-
positive bacteria (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778), Gram-
negative bacteria (Escherichia coli ATCC 19433, Salmonella Abony ATCC BAA-2162, Pseu-
domonas aeruginosa ATCC 27853) and yeast (Candida albicans ATCC 10231). The strains
used were purchased from the American Type Culture Collection (ATCC). Gram-positive
and Gram-negative microorganisms were precultured in Müeller–Hinton broth at 37 ◦ C
for 18–24 h. Subsequently, a suspension was prepared from each microbial strain with a
concentration of 108 cells/mL according to the McFarland standard of 0.5 [40]. Candida
yeasts were cultivated for 48 h on potato dextrose agar at 37 ◦ C, and after that the inoculum
was prepared with a final concentration of 106 cells/mL [41].
Foods 2023, 12, 3907 6 of 20

2.5.2. Antimicrobial Activity Detection Using the Agar Well Diffusion Method
Antimicrobial activity was determined using the agar well diffusion method, as
displayed by Ben Hsouna et al. [42]. Wells with 8 mm diameter were made on Müeller–
Hinton agar plates using a sterile metallic cylinder. The previously prepared inoculum was
spread uniformly on the surface of the agar plates using a sterile swab. In each well, 100 µL
of dissolved powder (125 mg/mL dimethylsulfoxide (DMSO)) was introduced. The plates
were kept at +4 ◦ C for 2 h to ensure the diffusion of the extracts in the agar [43], subsequently
being incubated at 37 ◦ C for 24 h (bacteria) and 48 h (yeasts). The measurement of the total
zone of inhibition (including the diameter of the wells) after the incubation period allowed
for the detection of antimicrobial activity. DMSO was used as a negative control. All tests
were performed in triplicate.

2.5.3. Determination of MIC, MBC and MFC Using the Microdilution Well Method
The goal of our study was to determine whether the etiologic agent is resistant or
sensitive to the natural antimicrobial agents being tested. The minimum inhibitory concen-
tration (MIC) values were determined using the method described by Gulluce et al. [44]. In
a sterile 96-well microtiter plate with a total volume of 200 µL per well, initially or after
dispensing 100 µL of Müeller–Hinton peptone broth. Afterwards, we performed twofold
serial dilutions of pumpkin powder in the range of 0.25–150 mg/mL. A microbial suspen-
sion of 10µL was added to each well with a final inoculum concentration of 108 cells/mL
for bacteria and 106 cells/mL for yeasts.
The contents were homogenized; then, the plates were covered with sterile lids and in-
cubated for 24 h at 37 ◦ C (bacterial strains) and for 48 h at 37 ◦ C (fungal strains). The lowest
concentration of powder inhibiting the growth of microorganisms was established—MIC.
The minimum bactericidal concentration (MBC) values were interpreted as the smallest
amount of powder inhibiting the visible growth of the microorganisms. For determination
of MBC, each dilution was subcultured on Müeller–Hinton agar plates with subsequent
incubation of the plates and interpretation of the results. The MBC value is determined by
the lowest concentration of the tested compound that reduces the number of colonies on
the plate by up to 99.9% [45].
The minimum fungicidal concentration (MFC) was determined by serially subcultur-
ing 10 µL in potato dextrose agar plates and incubating for 48 h at 37 ◦ C. MFC represented
the lowest concentration that inhibited yeast growth [46].
All assays were caried out in triplicate and the results were expressed as a mean value
± standard deviation (SD).

2.6. Preparation and Characterization of Shortbread Cookies with Pumpkin Powder

2.6.1. Preparation Shortbread Cookies with Pumpkin Powder
Samples of shortbread dough were made using 5% to 20% yellow pumpkin powder
(5% YPP, 10% YPP, 15% YPP and 20% YPP) to determine its effects on the sensory and
physicochemical quality of the shortbread cookies. YPP was added directly to wheat flour.
For the control, a sample was prepared without the addition of YPP.
Premium quality wheat flour (moisture content: 13.7%, ash content 0.6%), powdered
sugar (average crystal size: 120 µm), butter (moisture content: 16.9%, fat content: 82.5%)
and potato starch (moisture content—18%) were used to make shortbread cookies.
Soft butter and powdered sugar were mixed for 6 ± 1 min. Afterwards, wheat flour
with potato starch, and with/without YPP was poured in slowly while stirring. The dough,
kneaded for 5–10 min, was left to rest for 30 min at 20 ± 1 ◦ C. The dough was rolled
on lightly floured surface to an even thickness of 5 mm. The biscuits were stamped out
using a round cookie cutter with a diameter of 60 mm, keeping cuts close together. The
stamped biscuits (20 ± 1 g) were baked in an electric convection oven (EASY EV-UME604-
LS, Luxstahl, PiggioTorriana, Italy) at 180 ± 2 ◦ C for 12 ± 1 min until cooked. After baking,
the biscuits were packed and stored in a dry place at room temperature.
The quantities of each ingredient used in the preparation are presented in Table 2.
Foods 2023, 12, 3907 7 of 20

Table 2. Formulation of the shortbread cookies.

Ingredients CS 5% YPP 10% YPP 15% YPP 20% YPP

Wheat flour, g 100 95 90 85 80
YPP, g 0 5 10 15 20
Powdered sugar, g 40 40 40 40 40
Butter, g 80 80 80 80 80
Potato starch, g 13 13 13 13 13
CS = 100% wheat flour; 5% YPP = 5% yellow pumpkin powder; 10% YPP = 10% yellow pumpkin powder; 15%
YPP = 15% yellow pumpkin powder; 20% YPP = 20% yellow pumpkin powder.

2.6.2. Texture Profile Analysis of Dough and Shortbread Cookies

A Stable Micro Systems TA.HD plus C analyzer, UK was used for texture profile
analysis of dough. The textural properties (hardness, cohesiveness, gumminess, resilience,
adhesiveness) of the dough samples were determined with a 40 mm cylindrical probe
through the double compression test, using P/75 stainless plate and respecting the follow-
ing parameters: pretest speed—100 m/s; test speed—5 m/s; posttest speed—5 m/s; cell
load—5 kg [47].
The texture parameters of shortbread cookies were made using a texture analyzer
(Stable Micro Systems TA. HD plus C, Godalming, UK) equipped with a 5 kg load cell. The
breaking force or hardness (g) of cookie samples was performed using the three-point bend
test with an HDP/3PB knife-edge probe. The biscuit samples were placed on base beams
with a distance of 4 cm between the two beams. The analyzer was set to a return-to-start
cycle with the following parameters: pretest speed 1 mm/sec; test speed 2 mm/s; posttest
speed 10 mm/s; trigger force—5 g; distance—5 mm [48].

2.6.3. Sensory Analysis of Shortbread Cookies

The sensory properties of the shortbread cookies samples were analyzed according to
ISO 6658:2017 [49]. Appearance, taste, odor, color and consistency of five cookie samples
were evaluated by a trained panel comprising academic staff and food industry experts
with necessary knowledge and experience consisting of 11 persons. Sensory analysis
was performed in individual booths at room temperature using white light. The panel
developed a list of attributes and evaluated the samples using a 5-point method: 5 = Like
extremely. Exceptional, ideal qualities, 4 = Like. Suitable qualities, 3 = Slightly. With slight
defects, 2 = Dislike. With obvious defects, 1 = Dislike extremely. With strongly pronounced
defects, 0 = Altered, with large changes. Each sample was tested in duplicate in sensory
laboratories that met the requirements of ISO 8589 [50]. The cookies were served in plastic
containers with lids, coded with three-digit random numbers, and presented to the raters
in randomized order. A rest period of 2–3 min between assessments was provided. Warm
water was used to rinse the mouth.

2.6.4. Physicochemical Analysis of Shortbread Cookies

The moisture content (MC) of the shortbread cookies was determined according to the
AACC standard (AACC, Method 44-15.02) [51]. The water swelling of shortbread cookies
was determined according to [18]. The method is based on the measurement of the mass
difference in the products immersed in water at a temperature of 20 ± 1 ◦ C for 2 min,
as recommended for flour confectionery products. The water activity (aw ) of the baked
cookies was measured at room temperature (25 ± 1 ◦ C) using an electronic dew-point water
activity meter LabMaster (Novasina AG, CH-8853 Lachen, Switzerland) [52]. Measurement
of aw was carried out until the value was concurrent.

2.7. Statistical Analysis

The test results are presented in this paper as mean values ± standard error of the
mean for three parallel measurements. The Microsoft Office Excel 2007 program (Microsoft,
Redmond, WA, USA) was used for statistical processing. One-way analysis of variance
Foods 2023, 12, 3907 8 of 20

(ANOVA) was performed according to the Tukey test, with a significance level of p ≤ 0.05.
Staturphics software Centurion XVI 16.1.17 (Statgraphics Technologies, Inc., The Plains,
VA, USA) was used.

3. Results and Discussion

3.1. Characterization of Pumpkin Fresh Pulp and Powder
The physicochemical indicators and color parameters determined for fresh pumpkin
pulp are presented in Table 3.

Table 3. Physicochemical indicators and color parameters (CIELab) of the pumpkin fresh pulp
and powder.

Parameters Fresh Pumpkin Pulp Pumpkin Powder

Physicochemical Parameters
Moisture content, g/100 g 89.6 ± 0.03 6.88 ± 0.05
pH 6.01 ± 0.30 n/d
Titratable acidity, % expressed in
n/d 1.05 ± 0.01
citric acid
Soluble solids content, g/100 g 8.97 ± 0.06 n/d
Fat content, g/100 g DW 2.13 ± 0.10 5.74 ± 0.21
Protein content, g/100 g DW 8.42 ± 0.21 11.68 ± 0.11
Total fiber content, g/100 g DW 7.53 ± 0.17 9.69 ± 0.35
Ash content, g/100 g DW 2.98 ± 0.01 4.07 ± 0.14
CIELab Chromatic Parameters
L* 59.84 ± 0.21 69.33 ± 0.13
a* 15.04 ± 0.06 6.67 ± 0.08
b* 69.16 ± 0.11 43.1 ± 0.05
Values represent means of three replicated trials, ± standard deviation; n/d = not determined; L* = luminosity;
a* = red/green component; b* = yellow/blue component.

Moisture values for butternut pumpkin samples (89.6%) were somewhat lower com-
pared to the MC of fresh pumpkin pulp, which, according to Adubofuor et al. and See et al.,
were 92.24–95.66% [53,54]. These values correlate with the data recorded for pumpkin
pulp of the cylindrical variety Cucurbita moschata—89.5–89.65% [55,56]. The rather high
MC of pumpkin fruit pulp indicates the susceptibility of the fruit to microorganisms and
spoilage [57].
There are differences in how fruits and vegetables are categorized based on pH.
Pumpkin fruit is generally classified as a medium-acidic product with a pH range of
4.99–5.50 [58]. The values obtained for the butternut variety study were above the range
4.90–5.50 reported for pumpkin fruits [58,59]. According to the values obtained in this
study, the fruits are within the pH range of 5.0 and above the pH range established for
low-acid products [60]. The pH value of the fresh pulp was 6.01 (Table 3). Malkanthi
et al. [26] recorded a pH of 5.75 for dry pumpkin powder (10 g powder in 100 mL water),
the acidity of which acidity was due to the dry form of the sample. Over the course of the
study, it was found that the content of dry matter in the studied variety of pumpkin was
8.97%, which agrees well with the result of organoleptic evaluation of the pulp. It should
be noted that the studied pumpkin variety contains an insignificant number of acids (this
fact explains the bland flavor of the pulp).
For the fresh pulp, the FC, PC, TFC and AC, expressed in g/100 g DW, were determined
as 2.13, 8.42, 7.53 and 2.98, respectively (Table 3). The values recorded by Kim et al. [29]
were 4.20, 11.3, 10.88 and 10.53 for lipid, protein, fiber and ash of pumpkin flesh, expressed
in g/kg raw weight. The AC in pulp of the studied variety (2.98%) was significantly higher
than that of pumpkin fruit pulp reported in the scientific works [53,54] and compares well
with the rates of 2.14–4.26% obtained in the scientific works [55,61]. Differences in AC may
be due to varietal and geographical differences in pumpkin fruit cultivation [62].
Foods 2023, 12, 3907 9 of 20

In the pumpkin pulp drying process, at the temperature of 60 ± 2 ◦ C, the physicochem-

ical parameters determined for the powder did not change significantly (Table 3). Other
authors [20] established that hot-air drying at temperatures of 70 ◦ C ensures very good
quality of pumpkin powder and increased antioxidant potential. The MC (6.88%) was lower
than in a previous study [63] (10.14%), but was within the safety limit, as Bothast et al. [64]
noted that pumpkin powder with more than 14% moisture is susceptible to fungus and
mold growth. In the pumpkin powder, the titratable acidity was 1.05%, expressed in citric
acid. These values correlate with Dhiman et al.—1.03% [65].
In pumpkin powder, higher values (g/100 g DW) for lipid, protein, fiber and ash
content of 5.74, 11.68, 9.69 and 4.07, respectively, were recorded. On the other hand, ash
(4.07%) in pumpkin powder was reported by Ptitchkina et al. [66]. This phenomenon
was probably due to the processes of splitting macromolecular complexes during the
procurement of the dry powder, which led to the release of lipids, proteins, and other
components from the vegetable product matrix.
See et al. [54] determined that in fresh pumpkin, the contents of moisture, fat, protein,
ash, fiber and carbohydrate were 92.24, 0.15, 0.98, 0.76, 0.56 and 5.31%, respectively, whereas
moisture, fat, protein, ash, fiber and carbohydrate in pumpkin powder were 10.96, 0.80, 9.65,
5.37, 0.81 and 72.41%, respectively. Bhat et al. [67] determined a lower β-carotene content
in fresh pumpkin (2.44 mg/100 g) compared to that in dried powder (7.30 mg/100 g).
The physicochemical parameters of the pumpkin powder also fall within the limits of
the values obtained by several researchers (Table 3). Malkanthi et al. [27] determined the
following contents (g/100 g) of moisture, protein, carbohydrates and ash in pumpkin flesh
powder: 14.8, 8.95, 67.00 and 5.56, respectively. Jabeen et al. [68] determined 9.9 g moisture,
2.3 g fat, 3.07 g protein, and a high amount of fiber (11.46 g) and ash (15.98 g/100 g) in
pumpkin flour.
Nor et al. [69] reported that pumpkin powder contained 7.10%, 3.10%, 1.80%, 5.70%,
and 82.30% of protein, fat, moisture, ash, and carbohydrates, respectively. Badr et al. [14]
determined the proximate composition of pumpkin flesh powder; the values for ash, fat,
fiber, moisture, protein and carbohydrates (g/100 g DW) were 6.64, 0.18, 11.25, 18.03, 15.50
and 48.40, respectively.
The values of the chromatic parameters L*, a* and b* were analyzed in pumpkin pulp
and powder (Table 3). The L* values show that the pumpkin powder (69.33) was brighter
than the pulp (59.84). The a* value of fresh pumpkin was higher (15.04) than powder (6.67),
indicating more redness in fresh pumpkin. The b* value of fresh pumpkin (69.16) was
also higher than for pumpkin powder (43.1), demonstrating a greater presence of yellow
pigments. This transformation can be explained by the isomerization of trans carotenes into
various cis isomers, recognized as one of the mechanisms for the degradation of carotenoids
in food [70]. Dehydration in hot air exposed the color pigments to the action of oxygen,
and the activity of the enzymes lipoxygenase and peroxidase, responsible for oxidative
degradation, also contribute to the reduction in pigment content [71].

3.2. Chemical Composition and Antioxidant Activity of the Pumpkin Powder

The research carried out in the present study showed that the biological value of
pumpkin powder increased due to the qualitative and quantitative content of antioxidants
determined using UV-VIS spectroscopy and RP-HPLC chromatography (Table 4).
The values determined for TPC of 363.15 mg GAE/100 g DW and TFC 77.83 mg
QE/100 g DW in the acidified methanolic extracts (85:15, v/v, MeOH:HCl) of pumpkin pow-
der variety butternut squash were higher, and the carotenoid content of 27.76 mg/100 g DW
was lower (Table 4) compared to the values determined by several researchers. Thus, Hus-
sain et al. [4] obtained powder from pumpkin flesh, dried first in the sun, then in a conven-
tional hot-air oven, at 60°C. For the 80% aqueous methanolic extract, these authors recorded
lower values for TPC (134.59 mg GAE/100 g powder) and TFC (77.11 mg CE/100 g,
catechin equivalent), but determined higher TCC content (35.2 mg/100 g) using the
HPLC method.
Foods 2023, 12, 3907 10 of 20

Table 4. The content of total polyphenols, flavonoids, total and individual carotenoids, and the
antioxidant activity of pumpkin powder used for experiments.

Indices Mean Value ± SD

Total polyphenols, mg GAE/100 g DW 363.15 ± 16.8
Total flavonoids, mg QE/100 g DW 77.83 ± 9.01
Total carotenoids, mg /100 g DW 27.76 ± 5.01
Luteoxanthin, mg/100 g DW 0.50 ± 0.16
Violaxanthin, mg/100 g DW 0.45 ± 0.07
Lutein, mg/100 g DW 2.19 ± 0.11
β-cryptoxanthin, mg/100 g DW 0.27 ± 0.04
Cis-β-carotene, mg/100 g DW 0.26 ± 0.09
All-trans-β-carotene, mg/100 g DW 0.66 ± 0.13
Antioxidant activity (DPPH), mmol TE/100 g DW 161.52 ± 21.6
Antioxidant activity by silver nanoparticles, mg
558.71 ± 11.3
GAE/100 g DW
Values represent means of three replicated trials, ± standard deviation. DPPH = 2,2 diphenyl-1-picrylhydrazyl-hydrate.

The content of biologically active substances in plant powders depends on several fac-
tors [8,9,29], including the method applied to obtain the extracts [72]. Thus, Asif et al. [21],
for methanolic extracts of 65%, 80%, 99.9% pumpkin puree, determined TPCs of 6.78, 5.15,
4.31 mg GAE/100 g and TFCs of 0.72, 0.65, 0.51 mg GAE/100 g, respectively.
Wang et al. [73] noted that the carotenoid content in Cucurbita maxima dried slices
extracted with 100% acetone was not influenced by the drying method. TCC determined in
hot-air-dried samples was 67.6 mg/100 g, and in freeze-dried samples 63.7 mg/100 g.
Research showed that the TPC, TFC and TCC determined in this study provide an
excellent antioxidant potential of 161.52 mmol TE/100 g DW for pumpkin powder applied
in shortcake manufacturing. Other research showed an antioxidant activity of pumpkin
pulp powder of 0.53 mmol AAE/100 g (ascorbic acid equivalents), a TPC of 192 mg
GAE/100 and a β-carotene content of 32.87 mg/100 g DW [27].
In pumpkin puree, it was found to be 23.6 mg/100 g of phenol content and 0.102 mmol
TE/100 g antioxidant activity [74]. Another experiment revealed that the phenolic content
of pumpkin pulp powder ranged from 159.69 to 35.94 mg GAE/L, whereas the antioxidant
activity ranged from 0.284 to 0.135 mmol AAE/L [75].
In the present study, the antioxidant activity of phenolic compounds from pumpkin
powder against the formation of silver nanoparticles was evaluated. The plasmonic re-
sponse of silver nanoparticles reduced with phenolic compounds was evaluated (558.71 mg
GAE/100 g DW) and correlated with the antioxidant capacity of gallic acid. The relation-
ship between the antioxidant capacity of phenolic acids and the corresponding optical
response of plasmons can be used as an innovative antioxidant detection test for samples
rich in phenolic compounds. Nanoparticle-based antioxidant assays have been shown
to be sensitive, and rapid methods for determining the antioxidant capacity of phenolic
compounds correlate well with classical antioxidant assays such as DPPH and ABTS [39].
According to the literature review, the TCC value is higher if minor carotenoids and
their esterified forms are considered. The main carotenoids identified in pumpkin were
β-carotene, α-carotene, lutein and zeaxanthin. β-carotene, the precursor of vitamin A, is
the major carotenoid in pumpkin species [76]. Table 4 and Figure 1 show the profile of
saponified carotenoids, identified in butternut squash powder, of which lutein has the
highest content (2.19 mg/100 g DW), followed by all-trans-β-carotene (0.66 mg/100 g DW).
Compared to other bibliographic results [76], the lutein content recorded in our research
was modest, although it was mentioned in the literature that in three pumpkin species, the
lutein concentration varied from 0 to 17 mg/100 g [77].
 carotene, α-carotene, lutein and zeaxanthin. β-carotene, the precursor of vitamin A, is the
major carotenoid in pumpkin species [76]. Table 4 and Figure 1 show the profile of sapon-
ified carotenoids, identified in butternut squash powder, of which lutein has the highest
content (2.19 mg/100 g DW), followed by all-trans-β-carotene (0.66 mg/100 g DW). Com-
pared to other bibliographic results [76], the lutein content recorded in our research was
Foods 2023, 12, 3907 modest, although it was mentioned in the literature that in three pumpkin species, 11 ofthe
20
lutein concentration varied from 0 to 17 mg/100 g [77].

Figure1.1.RP-HPLC
Figure RP-HPLCchromatograms
chromatograms ofof saponified
saponified carotenoid
carotenoid extracts
extracts of
of butternut
butternut squash
squash powder:
powder:
1-Luteoxanthin;2-Violaxanthin;
1-Luteoxanthin; 2-Violaxanthin; 3-Lutein;
3-Lutein; 4-β-cryptoxanthin;
4-β-cryptoxanthin; 5-Cis-β-carotene;
5-Cis-β-carotene; 6-all-trans-β-caro-
6-all-trans-β-carotene.
tene.
To determine the concentration of α- and β-carotenes and their isomers, Garvalho
et al. [78] applied HPLC
To determine to the pulp of two
the concentration of α-local
andspecies (A and
β-carotenes B) of
and fresh
their Cucurbita
isomers, moschata.
Garvalho et
The results
al. [78] showed
applied HPLC that
to (all-E)-β-carotene
the pulp of two local was the most
species abundant
(A and in both
B) of fresh samples,
Cucurbita with
moschata.
values of 244.22
The results showedand that
141.95 µg/g in sampleswas
(all-E)-β-carotene A and
the B,
mostrespectively.
abundant The values
in both for (9Z)-β-
samples, with
carotene
values ofwere 2.34and
244.22 µg/g (A) and
141.95 µg/g0.97 µg/g (B),Aand
in samples andthose for (13Z)-β-carotene
B, respectively. The valueswere
for3.67 (A)
(9Z)-β-
and 1.84 µg/g (B).
carotene were 2.34 µg/g (A) and 0.97 µg/g (B), and those for (13Z)-β-carotene were 3.67 (A)
and Kim et al. (B).
1.84 µg/g [29], using the HPLC method, determined the following concentrations of
Kim et al. Cucurbita
carotenoids in [29], usingmaxima
the HPLCpumpkin
method, pulp: α-tocopherol—2.31;
determined the following β-carotene—17.04;
concentrations of
β-cryptoxanthin—0.65
carotenoids in Cucurbita mg/kg
maxima raw weight. pulp:
pumpkin In ourα-tocopherol—2.31;
research, the contentβ-carotene—17.04;
of β-cryptoxanthin β-
was lower (0.27 mg/100 g DW).
cryptoxanthin—0.65 mg/kg raw weight. In our research, the content of β-cryptoxanthin
was The
lowerlower
(0.27 concentration
mg/100 g DW).of carotenoids in saponified form, identified in our re-
search, was probably influenced by the extraction method and the saponification process
of carotenoids.

3.3. Antibacterial Activity of the Pumpkin Powder

In this study, the antimicrobial activity of pumpkin powder against Staphylococcus
aureus ATCC 25923, Bacillus cereus ATCC 11778, Escherichia coli ATCC 19433, Salmonella
Abony ATCC BAA-2162, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC
10231 was evaluated (Table 5). Initially, the antimicrobial activity of the pumpkin powder
was qualitatively assessed by the presence or absence of microorganism growth inhibition
zones. The obtained results demonstrated that the pumpkin powder has a bactericidal
effect both on Gram-positive and Gram-negative bacteria, as well as on Candida yeasts.
Pumpkin powder demonstrated antibacterial activity with the maximum zone of
inhibition for Candida albicans (19.0 mm). For Pseudomonas aeruginosa, the zone of inhibition
was 14.0 mm, and for Bacillus cereus and Escherichia coli it was 12.0 mm. The smallest zone
of inhibition was recorded for the species Salmonella Abony (10.0 mm).
Foods 2023, 12, 3907 12 of 20

Table 5. Antimicrobial activity of pumpkin powder.

Powder Concentration, mg/mL

Test Strains Zone of Inhibition, mm
MIC MBC/MFC
Gram-positive bacteria
Staphylococcus aureus 11.0 ± 0.5 b 37.5 ± 1.5 b 75.0 ± 2.0 b
Bacillus cereus 12.0 ± 0.0 b 18.7 ± 1.0 a 37.5 ± 1.5 a
Gram-negative bacteria
Escherichia coli 12.0 ± 0.0 b 37.5 ± 1.3 b 75.0 ± 2.5 b
Salmonella Abony 10.0 ± 0.5 a 75.0 ± 1.5 c 150.0 ± 5.0 c
Pseudomonas aeruginosa 14.0 ± 0.5 c 37.5 ± 1.5 b 75.0 ± 2.5 b
Yeast
Candida albicans 19.0 ± 0.5 d 18.7 ± 0.6 a 37.5 ± 2.0 a
Different letters (a–d ) designate statistically different results (p ≤ 0.05). MIC = minimum inhibitory concentration;
MBC = minimum bactericidal concentration; MFC = minimum fungicidal concentration.

The results of the study on the MIC and MBC values of the pumpkin powder
are presented in Table 5. The MIC for Bacillus cereus and Candida albicans strains was
18.75 mg/mL, and for Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa it
was 37.5 mg/mL. Antimicrobial activity in higher concentrations was recorded on strains of
Salmonella Abony—75.0 mg/mL. Pumpkin powder also demonstrated a bactericidal effect
at a concentration of 37.5 mg/mL on Bacillus cereus and Candida albicans. The MBC for
strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa was 75.0 mg/mL,
and for Salmonella Abony it was150.0 mg/mL. The antimicrobial effect of pumpkin pow-
der is due to the high concentrations of polyphenols (363.15 mg GAE/100 g DW) and
flavonoids (77.83 mg QE/100 g DW) that it contains. These compounds are responsible for
the antimicrobial activity due to the polar isopropyl functionality of the phenolic compo-
nents. Polyphenols and flavonoids from pumpkin powder react with cellular components
of microorganisms and lead to leakage of nucleotides and proteinaceous material into
extracellular areas [10].
Asif et al. conducted research to investigate the antibacterial activity of 65%, 80%
and 99.9% methanolic extracts of pumpkin peel and flesh against Pasteurella multocida,
Escherichia coli, Staphylococcus aureus and Bacillus subtilis. The diameter of inhibition zone
of methanolic extracts of pumpkin peel and flesh against Pasteurella multocida was greater
than 15 mm, while the zone of inhibition against three other bacterial species was in the
range of 10 to 15 mm [21].
Dissanayake et al. determined the antimicrobial effect of pumpkin peel, seed and
leaf extracts using acetone, methanol and ethyl acetate solvents on Staphylococcus aureus,
Bacillus subtilis and Escherichia coli bacteria. Methanolic extract of pumpkin peel showed
the highest activity against Staphylococcus aureus with zones of inhibition of 7.58 mm and
4.83 mm for Bacillus subtilis, and no antimicrobial activity was found for Escherichia coli.
On the other hand, methanol extracts of pumpkin seeds showed no antimicrobial effect,
but ethyl acetate extract of pumpkin seeds showed an inhibition zone of 6.61 mm against
Staphylococcus aureus [79].
Tyan et al. [80] evaluated the antimicrobial activity of pumpkin extracts on seven
microbial strains and proved that Cucurbita moschata extracts possess antimicrobial activity
against all strains taken in the study.
Chonoko and Rufai [81] conducted research on the antimicrobial activity of pumpkin
and reported a zone of inhibition in the range of 7–10 mm of both ethanolic and methanolic
extracts of pumpkin peel against bacterial species Staphylococcus aureus and with the zone
of inhibition of 6–12 mm against Salmonella typhi.
Badr et al. [14] found that pumpkin seed oil exhibited strong antifungal activities
against the fungal species Saccharomyces cerevisiae. The antimicrobial effect exerted by
pumpkin powder is probably due to saponins, tannins, flavonoids, alkaloids and steroids
present in pumpkin [81].
Foods 2023, 12, 3907 13 of 20

Hussain et al. [10] tested the antimicrobial activity of three types of pumpkin methano-
lic extracts against various species of bacteria and fungi. Significantly different values
were obtained for the antibacterial activity of pumpkin peel, flesh and seed extracts against
the microbial species taken in the research. Against Salmonella typhi, all three types of
extracts showed significant antibacterial activity. Against Escherichia coli and Bacillus subtilis
species, pumpkin flesh extract showed a larger zone of inhibition compared to peel and
seed extracts. Against Staphylococcus aureus, pumpkin peel, flesh and seed extracts showed
nonsignificant activity.
The antimycotic activity of these extracts was also demonstrated. The highest activity
against the species Candida albicans was demonstrated by the methanolic extract from
seeds (9.34 mm), followed by the extract from the peel (8.69 mm) and flesh (7.80 mm) of
pumpkin [10].

3.4. Characterization of Shortbread Cookies

Sensory evaluation of the products showed that the highest average score was given
to 15% YPP (4.9 points), 20% PSC (4.55 points), and control samples (4.59 points) (Table 6).
The highest rating for the consistency of cookies was obtained with the use of YPP
15%—4.87 points. Replacing 20% of wheat flour with YPP slightly reduced the score
to 4.48 points. The control sample and cookies with the addition of 5–10% YPP had a
crumbly consistency. The use of 15–20% pumpkin powder contributed to firmer cookie
consistency. According to Seyhun et al. [82], the firmer cookie consistency with 15–20% YPP
can be attributed to amylose and amylopectin recrystallization, the formation of complexes
between starch and proteins, and to redistribution of water between the components of the
product, as well as other events that may occur in this type of cookie.
The highest rating for the taste of cookies was obtained in the samples with the
addition of 10 and 15% YPP—4.94 and 4.99 points, respectively. The use of 15% YPP
provided an odor at the highest level of 4.84 points. The use of a higher amount of pumpkin
powder slightly reduced this indicator to 4.45 points. Products with high YPP content
(15–20%) had a pronounced nutty flavor and odor.
The resulting shortbread cookies have a particular orange yellow color, which is typical
of the substitute ingredient, which is YPP. Assessed simultaneously regarding all attributes,
the result that received the highest rating was cookies with 15% pumpkin powder, with a
score of 4.83. The cookies with high concentration of YPP (15–20%) had a dark orange color,
which is the result of a more intensive Maillard reaction, which depends on the content
of sugars and proteins [83–85]. It should be noted that all products had an appropriate
shape, without bloating and dents, with a smooth surface. Taste and odor were typical of
the ingredients included in the product, without extraneous taste and odor.
Thus, the introduction of a natural component of raw plant materials—YPP—into the
developed biscuits recipe significantly improved organoleptic parameters, in particular,
the taste, odor and color of the products, which was noted by all tasters. In addition,
the biscuit samples obtained using the developed formulation had a better structure and
appearance, being denser and moister in texture compared to the control sample. The
introduction of YPP was found to compensate for the lack of flavor compounds compared
to the control. However, the addition of 20% YPP has a slight negative effect on taste
and odor (more pungent nutty taste). Overall, the addition of YPP in an amount not
exceeding 15% improves the overall consumer experience and has practical applications in
the development of a better and more presentable product.
The MC of cookies increased linearly from 3.37% to 3.87% with an increase in con-
centration of YPP, which can be explained by the biochemical composition of the YPP, in
particular, a fairly high content of proteins and dietary fiber, which account for the high
water-binding capacity of pumpkin powder, which retained more moisture in the finished
products. The results for moisture content of the shortbread cookies were similar to the
results obtained by Mishra and Kalpana [86], who incorporated potato flour in preparation
of the cookies, similar to the results obtained by Dziki et al. [87], who incorporated oat husk
Foods 2023, 12, 3907 14 of 20

in preparation of the cookies. The MC obtained for shortbread cookies with YPP compares
well with the rates obtained in the scientific works [88,89].

Table 6. Sensory, physicochemical, texture parameters and color indicators of pumpkin powder
shortbread cookies (results are presented as mean ± standard deviation).

Indicators/ Shortbread Cookies

Parameters CS 5% YPP 10% YPP 15% YPP 20% YPP
Sensory indicators
Average score of sensory
4.59 ± 0.04 a,b 4.68 ± 0.02 c 4.77 ± 0.01 d 4.90 ± 0.02 e 4.55 ± 0.05 a,b
profile
Consistency 4.64 ± 0.02 b 4.67 ± 0.03 b,c 4.72 ± 0.02 c 4.87 ± 0.01 d 4.48 ± 0.01 a
Taste 4.75 ± 0.01 b 4.84 ± 0.02 c 4.94 ± 0.02 d,e 4.99 ± 0.01 e 4.62 ± 0.02 a
Appearance 4.66 ± 0.01 a 4.69 ± 0.02 a 4.82 ± 0.0 b 4.96 ± 0.02 c 4.66 ± 0.01 a
Color 4.46 ± 0.03 a,b 4.63 ± 0.02 c 4.75 ± 0.0 d 4.83 ± 0.01 e 4.51 ± 0.02 b
Odor 4.45 ± 0.02 a 4.56 ± 0.02 b 4.61 ± 0.0 b 4.84 ± 0.02 c 4.45 ± 0.20 a
Physicochemical indicators
Moisture content, % 2.93 ± 0.053 a 3.37 ± 0.57 b 3.5 ± 0.21 c 3.67 ± 0.088 c,d 3.87 ± 0.082 e
Swelling in water, % 183.6 ± 1.4 c 166.8 ± 0.0 b 165.5 ± 1.4 b 163.6 ± 1.0 a,b 161.8 ± 0.5 a
Water activity (aw ), c. u. 0.177 ± 0.001 a 0.177 ± 0.001 a 0.176 ± 0.001 a 0.183 ± 0.001 b 0.199 ± 0.001 c
Texture parameters of cookie doughs
Hardness, g 1923.4 ± 45.7 c 893.2 ± 17.8 b 670.6 ± 12.4 a 630.0 ± 25.3 a 618.5 ± 31.5 a
Adhesiveness, g·s 1639.0 ± 26.2 c 742.2 ± 18.9 b 560.5 ± 21.3 b 514.1 ± 32.6 a,b 503.7 ± 17.4 a
Cohesiveness, % 0.200 ± 0.001 a 0.221 ± 0.001 b 0.221 ± 0.001 b 0.221 ± 0.001 b 0.221 ± 0.001 b
Resilience, % 0.102 ± 0.001 d 0.094 ± 0.001 c 0.093 ± 0.001 b,c 0.089 ± 0.0 a 0.088 ± 0.001 a
Gumminess, g 386.7 ± 8.2 c 196.5 ± 9.1 b 147.5 ± 14.1 a 138.6 ± 15.7 a 136.1 ± 11.3 a
Texture parameters of cookies
Hardness, g 1265.8 ± 31.4 a 1991.7 ± 26.5 b 2321.7 ± 19.4 c 2435.3 ± 22.7 c 2539.9 ± 28.4 d
CIELab chromatic parameters of shortbread cookie cores
L* 75.10 ± 0.45 d 67.17 ± 0.48 c 61.89 ± 0.03 b 56.56 ± 0.65 a 57.28 ± 0.28 a
a* 0.66 ± 0.15 a 0.11 ± 0.06 a 3.07 ± 0.12 b 5.60 ± 0.08 c 6.19 ± 0.12 c
b* 27.01 ± 0.18 a 31.21 ± 0.07 b 34.54 ± 0.08 b,c 35.39 ± 0.08 d 35.99 ± 0.07 d
∆E* - 8.99 ± 0.41 a 15.39 ± 0.58 b 20.94 ± 0.32 c,d 20.71 ± 0.67 c,d
CIELab chromatic parameters of shortbread cookie crusts
L* 75.39 ± 0.27 d 67.24 ± 0.46 c 60.91 ± 0.06 b 56.08 ± 0.84 a 55.23 ± 0.19 a
a* 1.42 ± 0.07 a,b 2.33 ± 0.04 b 4.16 ± 0.08 c 6.01 ± 0.05 d 8.71 ± 0.05 e
a b
b* 31.82 ± 0.23 34.54 ± 0.11 36.70 ± 0.12 c,d 37.73 ± 0.19 d,e 38.52 ± 0.14 d,e
∆E* - 8.64 ± 0.43 a 15.52 ± 0.31 b 20.71 ± 0.57 c 22.46 ± 0.64 c,d
Different letters (a–e ) designate statistically different results (p ≤ 0.05). L*—luminosity; a*—red/green component;
b*—yellow/blue component; ∆E*—overall difference of color; CS—control sample; YPP—shortbread cookies
with yellow pumpkin powder.

The swelling in water of the cookies decreased as the amount of YPP was increased.
This is due to the fact that the dough with increasing replacement of top-grade wheat flour
with YPP becomes more plastic, which further allows for obtaining a more loosened and
brittle cookie structure. It was found that with increasing quantity of YPP in the recipe of
shortbread cookies, wetness of the products decreases by 9.1% upon introduction of 15% of
YPP from the weight of flour and by 11.9% at 20%. This can be explained by the presence
of pectin substances in pumpkin powder, which retain a significant amount of water.
Cookie aw is very important to predict its stability and safety regarding microbial
growth and lipid oxidation rates. The aw of all measured cookies were in the range of
0.177 to 0.199 (Table 6). According to the reference, the aw for food spoilage by bacteria,
yeasts and molds are 0.90, 0.85–0.88 and 0.80, respectively [90]. The aw significantly
influences the shelf life of the product. The higher the aw value, the more likely the
development of microorganisms and product decomposition processes. aw values lower
than 0.60 prevent microbial spoilage and exhibit microbiological stability [91].
The use of pumpkin powder led to a decrease in the hardness of the cookie dough.
Pumpkin powder had a different chemical composition from wheat flour in terms of fat,
dietary fiber and gluten content. A drastic decrease of about two times was observed
between the CS and the sample where the wheat flour was substituted with only 5% YPP.
This dependence was also observed for the other samples, respectively, with a decrease in
Foods 2023, 12, 3907 15 of 20

hardness by 65–67%. The composite dough of wheat flour and pumpkin powder had much
lower hardness compared to the CS, possibly due to higher fat content in the pumpkin
powder. Similar results have been obtained by other authors [92] in the manufacture of cuts
with the addition of pumpkin flour. Also, Wongsagonsup et al. [93] obtained decreasing
results for bread dough hardness when made from a mixture of wheat flour and pumpkin
flour. This decrease in hardness results was explained by the weakening of the gluten
matrix, since pumpkin powder does not contain gluten.
The respective changes in the dough structure also influenced other texture parameters;
namely, for adhesiveness, decreasing values were recorded for all the dough samples with
the addition of pumpkin powder—a significant decrease of over 55% for 5% YPP compared
to the CS. Resilience also showed decreasing values, with a maximum of 13.7% for the
20% YPP and a minimum of 8% for 5% YPP. Cohesiveness, which represents the internal
links that hold together the structure of composite flour gels [94], remains practically
unchanged for all samples; the amount of pumpkin powder did not influence the results of
this parameter. The evolution of resilience and cohesiveness may be due to the weakening
of the dough matrix initiated by the increase in dietary fiber content, which also influences
the ability of the dough to retain gases [95].
Considering that gumminess is related to the evolution of hardness and cohesive-
ness [96], this parameter also showed decreasing values. The gumminess of the dough
samples with the addition of pumpkin powder compared to the control sample decreased
by almost 50% for the 5% YPP, by 61.8% for the 10% YPP, 64.2% for the 15% YPP, and by
almost 65% for the 20% YPP.
Similar results were obtained by Aljahani [94] for composite gels from wheat flour
and pumpkin powder (5%, 10% and 15%), a decrease in firmness and gumminess of a
maximum of 23.3% and, respectively, 25% for nonessential cohesiveness changes.
Hardness is a quality indicator specific to cookies. According to the data presented
in Table 6, the values for hardness increased compared to the CS. Increasing the amount
of pumpkin powder in the shortbread cookie recipe led to an increase in this texture
parameter, by approximately 36% for the 5% YPP, 45% for the 10% YPP, 48% for the 15%
YPP and 50% for the 20% YPP. The increase in shortbread cookie hardness values was due
to the weakening of the gluten network, due to the addition of pumpkin powder. Liubych
et al. [97] showed that the use of pumpkin powder in the manufacture of cookies led to an
increase in breaking strength: there was an increase from 1.05 kg for the control sample to
1.4–4.0 kg for samples with 5–20% pumpkin flour. The authors argued that the increase in
breaking strength values of the composite cookies was due to their small volume as well
as the reduction in the degree of swelling due to the reduced absorption capacity of the
pumpkin powder.
Table 6 shows the values of the color parameters L*, a* and b* for the core and crust of
the shortbread cookies. It was found that in both cases, for all cookie samples (CS and YPP),
the brightness values (L*) were over 50 and were in the clear zone [98]. It was found that
the L* of the cookies showed a decreasing trend with the increase in the level of substitution
of flour with pumpkin powder. Lower values of L* indicated that the cookies were more
closed at higher levels of substitution compared to the CS. This effect was caused by the
presence of natural pigments, such as carotenoids, which are naturally found in pumpkin
powder [76,77]. Also, crust L* values are lower for YPP due to the result of browning and
Maillard reactions, which depend on the content of reducing sugars and proteins on the
sample surface [85].
The values of the parameters a* and b* in both cases (core and crust) of CS and
YPP were positive, demonstrating the predominance of red color over green and a strong
predominance of yellow coloration, in disfavor of the blue, respectively. The resulting
color of the cookies was a dark yellow. It was also found that the values of parameters a*
and b* in the samples with pumpkin powder were higher than in the case of the CS. This
is probably due to the natural coloring pigments, carotenoids, in the pumpkin powder.
In general, the a* and b* parameter values of the shortbread cookies crust were higher
Foods 2023, 12, 3907 16 of 20

compared to their core. YPP samples contained more sugar, which facilitates browning and
Maillard reactions.
∆E* represents a dimensionless parameter, resulting from the combination of the
L*, a* and b* values of the pairs of samples, which indicates whether or not there are
differences in the colors perceived by the human eye, depending on the specific sensory
thresholds [99]. Lo Faro et al. showed the difference between colors: if ∆E < 0.2—an
imperceptible difference; if 0.2 < ∆E < 0.5—a very small difference; if 0.5 < ∆E < 1.5—a
small difference; if 2 < ∆E < 3—a barely distinguishable difference; if 3 < ∆E < 6—a very
distinguishable difference; if 6 < ∆E < 12—a large color difference; if ∆E > 12—completely
different colors. [99]. The CS was analyzed for shortbread cookies with different substitu-
tions of pumpkin powder. The values of ∆E* were found to be between 6 < ∆E* < 12, a
large color difference, and ∆E* > 12, completely different colors [100]. The ∆E* of the cookie
crust and core was also influenced by the presence of pumpkin powder, and the ∆E* values
increased with increasing replacement of pumpkin powder in the cookies. The pumpkin
composition significantly influenced color differences, since the baking conditions were the
same for all samples obtained.

4. Conclusions
In the process of drying pumpkin pulp at 60 ± 2 ◦ C, the physicochemical parameters
did not change significantly. Significant amounts for lipid, protein, fiber and mineral
content, respectively, of 5.47, 11.68, 9.69 and 4.07 g/100 g DW were recorded in pumpkin
powder. The chromatic parameters L*, a* and b* showed that the pumpkin powder was
brighter than the pulp, with the greater presence of yellow pigments.
The content of polyphenols, flavonoids, total and individual carotenoids of the pump-
kin powder was determined. It was found to be a rich source of bioactive compounds,
with an antioxidant potential of 161.52 mmol TE/100 g DW. The profile of saponified
carotenoids in pumpkin powder showed that lutein has the highest content (2.19 mg/100 g
DW), followed by all-trans-β-carotene (0.66 mg/100 g DW).
Pumpkin powder shows antimicrobial activity against both Gram-positive (Staphy-
lococcus aureus, Bacillus cereus) and Gram-negative (Escherichia coli, Salmonella Abony and
Pseudomonas aeruginosa) bacteria. High antifungal activity against Candida albicans species
was detected.
Sensory, physicochemical and texture parameters and color indicators of pumpkin
shortbread cookies with 5–20% YPP added, in relation to the CS, were analyzed. Sensory
evaluation showed that the highest average score was achieved by the 15% YPP sample.
The use of 15–20% YPP contributed to firmer cookie consistency due to the formation of
complexes between starch and proteins.
The dough with increasing replacement of top-grade wheat flour with YPP became
more plastic, which allowed for obtaining a more brittle cookie structure. As the amount of
pumpkin powder in the cookie recipe was increased, a constant increase in the breaking
strength values of the cookies was attested due to the weakening of the gluten network
and the reduced absorption capacity of the pumpkin powder.
The brightness of the cookies showed a decreasing trend with the level of replacement
of wheat flour with pumpkin powder; the resulting color of the cookies was dark yellow.
The values of parameters a* and b* in the samples with pumpkin powder were higher
than in the case of CS due to the natural coloring pigments, carotenoids. YPP samples had
higher sugar content, which facilitated browning and Maillard reactions. The difference
in colors perceived by the human eye of the cookie’s crust and core increased with the
replacement of pumpkin powder in the cookies.
Research results showed that pumpkin powder considerably improves the sensory
and textural characteristics of biscuits and is recommended for extending the range of
high-quality and food-safe flour products.

Author Contributions: Conceptualization, A.G.-M., N.N., G.B., D.C., O.B., V.B., A.G., L.P., O.D.,
V.R., C.S., A.P., T.S. and R.S.; methodology, A.G.-M., N.N., G.B., D.C., O.B., V.B., A.G., L.P. and
Foods 2023, 12, 3907 17 of 20

A.P.; software, A.G.-M., V.B. and V.R.; validation, A.G.-M., N.N., G.B., V.B., A.G., O.D., C.S., A.P.,
T.S. and R.S.; investigation, A.G.-M., N.N., G.B., D.C., O.B., V.B. and L.P.; resources, A.G.-M., N.N.,
G.B., O.D., V.R., C.S. and A.P.; data curation, A.G.-M., N.N., G.B., D.C., O.B., V.B., A.G., L.P., O.D.,
V.R., C.S., A.P., T.S. and R.S.; writing—original draft preparation, A.G.-M., N.N., G.B., D.C., O.B.,
V.B., A.G., L.P., O.D., V.R. and R.S.; writing—review and editing, A.G.-M., G.B., C.S., A.P., T.S. and
R.S.; supervision, A.G.-M., G.B., C.S., A.P., T.S. and R.S.; project administration, A.G.-M., G.B. and
T.S.; funding acquisition, A.G.-M. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Francophone University Agency (AUF) ECO 2023 DRECO-
7859, project “Extraction ‘verte’, stabilisation et valorisation des composants bioactifs de Ribes nidrigo-
laria et Cucurbita maxima”.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Acknowledgments: The authors would like to thank the Francophone University Agency (AUF) and
Cost Action CA 15136 EUROCAROTEN.
Conflicts of Interest: The authors declare no conflict of interest.

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